DEVICES, COMPOSITIONS AND METHODS FOR IMAGING WITH RAMAN SCATTERING AND FOR SUPER-MULTIPLEX VIBRATIONAL IMAGING

Abstract

Exemplary methods, systems and computer-accessible medium for detecting at least one condition of at least one disease. In particular, it is possible to administer at least one composition to at least one patient, where the at least one composition includes at least one bond-edited compound configured to target at least one of a disease tissue or a pathogen. Further, it is possible to receive information related to the administration of the composition to the patient. It is also possible to detect the at least one bond-edited compound using a Raman scattering arrangement.

Claims

1. A non-transitory computer-accessible medium having stored thereon computer-executable instructions for detecting at least one condition of at least one disease, wherein, when a computer arrangement executes the instructions, the computer arrangement is configured to perform procedures comprising: receiving information related to an administration of the at least one composition to the at least one patient, wherein the at least one composition includes at least one bond-edited compound configured to target at least one of a disease tissue or a pathogen; and detecting the at least one bond-edited compound using a Raman scattering arrangement.

2. The computer-accessible medium of claim 1, wherein the bond-edited compound comprises one, two, three, four, five, six, seven, eight, nine, ten or more vibrational tags.

3. The computer-accessible medium of claim 2, wherein the vibrational tag is at least one of: an alkyne tag, an azide tag, an isotope label, or a combination of an alkyne tag and a carbon-deuterium bond tag.

4. The computer-accessible medium of claim 3, wherein the isotope label is a carbon-deuterium bond tag.

5. The computer-accessible medium of claim 1, wherein the bond-edited compound comprises: at least one vibrational tag selected from the group consisting of CC, CN, NNN, CCCC, CCCN, C-D, and CC-D, at least one .sup.13C atom or one deuterium atom, an amino acid, a nucleoside or a nucleotide, a fatty acid, a monosaccharide or a disaccharide, glucose, a glucose derivative or propargyl glucose, or a cytokine or chemokine.

6. The computer-accessible medium of claim 5, wherein the amino acid is an essential amino acid.

7. The computer-accessible medium of claim 6, wherein the essential amino acid is selected from the group consisting of histidine, isoleucine, leucine, lysing, methionine, phenylalanine, threonine, tryptophan and valine.

8. The computer-accessible medium of claim 1, wherein the bond-edited compound is selected from the group consisting of anti-cancer agents, anti-inflammatory agents, anti-bacterial agents, anti-fungal agents and anti-viral agents.

9. The computer-accessible medium of claim 2, wherein the vibrational tag is transferred from the bond-edited compound to a down-stream metabolite of the bond-edited compound, and is detected in the down-stream metabolite.

10. A system for detecting at least one condition of at least one disease, comprising: a computer hardware arrangement configured to: receive information related to the administration of at least one composition to at least one patient, wherein the at least one composition includes at least one bond-edited compound configured to target at least one of a disease tissue or a pathogen; and detect the at least one bond-edited compound using a Raman scattering arrangement.

11. The system of claim 10, wherein the bond-edited compound comprises one, two, three, four, five, six, seven, eight, nine, ten or more vibrational tags.

12. The system of claim 11, wherein the vibrational tag is at least one of: an alkyne tag, an azide tag, an isotope label, or a combination of an alkyne tag and a carbon-deuterium bond tag.

13. The system of claim 12, wherein the isotope label is a carbon-deuterium bond tag.

14. The system of claim 10, wherein the bond-edited compound comprises: at least one vibrational tag selected from the group consisting of CC, CN, NNN, CCCC, CCCN, C-D, and CC-D, at least one .sup.13C atom or one deuterium atom, an amino acid, a nucleoside or a nucleotide, a fatty acid, a monosaccharide or a disaccharide, glucose, a glucose derivative or propargyl glucose, or a cytokine or chemokine.

15. The system of claim 14, wherein the amino acid is an essential amino acid.

16. The system of claim 15, wherein the essential amino acid is selected from the group consisting of histidine, isoleucine, leucine, lysing, methionine, phenylalanine, threonine, tryptophan and valine.

17. The system of claim 10, wherein the bond-edited compound is selected from the group consisting of anti-cancer agents, anti-inflammatory agents, anti-bacterial agents, anti-fungal agents and anti-viral agents.

18. The system of claim 10, wherein the vibrational tag is transferred from the bond-edited compound to a down-stream metabolite of the bond-edited compound, and is detected in the down-stream metabolite.

19. A method for detecting at least one condition of at least one disease, comprising: administering at least one composition to at least one patient, wherein the at least one composition includes at least one bond-edited compound configured to target at least one of a disease tissue or a pathogen; and detecting the at least one bond-edited compound using a Raman scattering arrangement.

20. The method of claim 19, wherein the bond-edited compound comprises one, two, three, four, five, six, seven, eight, nine, ten or more vibrational tags.

21. The method of claim 20, wherein the vibrational tag is at least one of: an alkyne tag, an azide tag, an isotope label, or a combination of an alkyne tag and a carbon-deuterium bond tag.

22. The method of claim 21, wherein the isotope label is a carbon-deuterium bond tag.

23. The method of claim 19, wherein the bond-edited compound comprises at least one of: at least one vibrational tag selected from the group consisting of CC, CN, NNN, CCCC, CCCN, C-D, and CC-D, at least one .sup.13C atom or one deuterium atom, an amino acid, a nucleoside or a nucleotide, a fatty acid, a monosaccharide or a disaccharide, glucose, a glucose derivative or propargyl glucose, or a cytokine or chemokine.

24. The method of claim 23 wherein the amino acid is an essential amino acid.

25. The method of claim 24, wherein the essential amino acid is selected from the group consisting of histidine, isoleucine, leucine, lysing, methionine, phenylalanine, threonine, tryptophan and valine.

26. The method of claim 19, wherein the bond-edited compound is selected from the group consisting of anti-cancer agents, anti-inflammatory agents, anti-bacterial agents, anti-fungal agents and anti-viral agents.

27. The method of claim 20, wherein the vibrational tag is transferred from the bond-edited compound to a down-stream metabolite of the bond-edited compound, and is detected in the down-stream metabolite.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0057] Further objects, features and advantages of the present disclosure will become apparent from the following detailed description taken in conjunction with the accompanying FIGS. showing illustrative exemplary embodiments of the present disclosure, in which:

[0058] FIGS. 1a and 1b are illustration of an imaging complex protein metabolism by stimulated Raman scattering (SRS) microscopy in live cells, tissues and animals. For example, FIG. 1a illustrates a cartoon for SRS imaging following metabolic labeling of deuterated amino acids (D-AAs) in live organisms (e.g. mice), which are first administered with D-AAs for certain period of time and then imaged by SRS to probe protein metabolism. FIG. 1b illustrates spontaneous Raman spectra from HeLa cells incubated with medium containing either regular amino acids (gray, dashed) or D-AAs illustrate three distinct ways to probe complex protein metabolism: imaging newly synthesized proteins by targeting 2133 cm.sup.1 from carbon-deuterium bonds (C-D), imaging degradation of pre-existing proteins by targeting the pure methyl group (CH.sub.3) distribution, and two-color pulse-chase protein imaging by labeling with two sub-groups of D-AAs (i.e., group I and group II).

[0059] FIGS. 2a-2e are illustration of a sensitivity optimization and time-lapse imaging of the de novo proteome synthesis dynamics. For example, FIG. 2a illustrates spontaneous Raman spectra of C-D peaks in HeLa cells incubated in optimized deuteration medium display a 50% increase when compared to the previously reported partial deuteration medium, and about 8 times higher than using leucine-d.sub.10 only. FIG. 2b illustrates SRS images of newly synthesized proteins in live HeLa cells confirm a 50% average signal increase. FIG. 2c illustrates SRS images of newly synthesized proteins in live neurons in optimized deuteration medium for 20 h. The zoom-in image highlights the fine dendritic structures (likely dendritic spines, arrow-headed). FIG. 2d illustrates SRS image of newly synthesized proteins in live HeLa cells with 1 h incubation of optimized deuteration medium. Control image with protein synthesis inhibition deprives most of the signal. FIG. 2e illustrates time-lapse SRS images of protein synthesis dynamics in a same set of live HeLa cells with continuous incubation in optimized deuteration medium. Scale bar, 10 m.

[0060] FIGS. 3a-3c are exemplary time-dependent SRS images of protein degradation. For example, FIG. 3a illustrates adopting a linear combination algorithm between 2940 cm.sup.1 and 2845 cm.sup.1 channels, the obtained SRS image exclusively from CH.sub.3 vibration display gradual degradation of pre-existing proteins in live HeLa cells cultured in optimized deuteration medium for 0 h, 24 h, 48 h and 96 h. FIG. 3b illustrates SRS images exclusively from CH.sub.2 vibration display the total lipid distribution at the corresponding time point. FIG. 3c illustrates a single exponential decay fitting from averaged image intensities from pre-existing protein in FIG. 3a, yielding a protein degradation time constant of 454 h. Error bars, standard deviation. Scale bar, 10 m.

[0061] FIGS. 4a-4d are exemplary pulse-chase SRS images of temporally defined proteins. For example, FIG. 4a illustrates structures and spontaneous Raman spectra of group I D-AAs (i.e. the branched chain amino acids). FIG. 4b illustrates structures and spontaneous Raman spectra of three examples of group II non-branched D-AAs. FIG. 4c illustrates spontaneous Raman spectra of HeLa cells cultured with group I D-AAs (element 405), showing multiple peaks with the first around 2067 cm.sup.1, and with group II D-AAs (element 410), showing a common peak around 2133 cm.sup.1. FIG. 4d illustrates two-color pulse-chase imaging by sequential labeling of group II and group I D-AAs in time with simultaneous expression of mutant huntingtin (mHtt94Q-mEos2) proteins. Cartoon displays experimental timeline of plasmid transfection and D-AA medium exchanges. The fluorescence image (overlaid with bright field) indicates the formation of a large aggregate (arrow-headed) of mHtt94Q-mEos2. The retrieved signals from linear combination of the original images at 2067 and 2133 channels display a large aggregation of mHtt proteins solely labeled by group II D-AAs during the first 22 h (pulse 415) and mHtt only labeled by group I D-AAs during the following 20 h (chase 420). The merged image, as well as the intensity profile, from the pulsed (element 415) and chased (element 420) images confirms with a yellow core and a green shell. Scale bar, 10 m.

[0062] FIGS. 5a and 5b are exemplary SRS images of newly synthesized proteins in live mouse brain tissues. For example, FIG. 5a illustrates SRS images at dentate gyrus of a live organotypic brain slice (400 m thick, from a P10 mouse) after culturing in D-AA medium for 30 hr. 2133 cm.sup.1 (C-D) image presents the distribution of newly synthesized proteins. The CH.sub.3 and CH.sub.2 images show the old protein pools and total lipids, respectively. FIG. 5b illustrates a 4-by-3 mm large field view overlay image of new proteins (C-D, element 505), old proteins (CH.sub.3, element 510) and total lipids (CH.sub.2, element 515) for a brain slice (400 m thick, from a P12 mouse) cultured in D-AA medium for 30 h. Scale bar, 100 m.

[0063] FIGS. 6a-6c are exemplary SRS images of newly synthesized proteins in vivo. For example, FIG. 6a illustrates SRS images of a 24-hpf (hpf: hours post fertilization) zebrafish. Wild-type zebrafish embryos were injected at 1-cell stage with 1 nL D-AA solution and allowed to develop normally for another 24 h before imaging. Bright field image shows the gross morphology of embryonic zebrafish at 24 hpf (dashed boxes). 2133 cm.sup.1 (C-D) image presents the distribution of newly synthesized proteins in the somites of an embryonic zebrafish tail. The CH.sub.3 image shows the old protein pool while the CH.sub.2 image depicts total lipid in the same fish. FIGS. 6b and 6c SRS images of live mouse liver are shown in FIG. 6b and intestine tissues FIG. 6c harvested from mice after administered with D-AA containing drinking water for 12 days. 2133 cm.sup.1 (C-D) channel shows newly synthesized proteins that resemble the distribution of total protein as shown in the 1655 cm.sup.1 image (Amide I). Scale bar, 10 m.

[0064] FIGS. 7a and 7b are exemplary SRS images at 2067 cm.sup.1 and 2133 cm.sup.1 channels of proteins labeled with group I D-AA shown in FIG. 7a, and group II D-AA shown in FIG. 7b.

[0065] FIGS. 8a-8c are exemplary raw C-D on-resonance (2133 cm.sup.1) and off-resonance (2000 cm.sup.1) SRS images of newly synthesized proteins in vivo in FIG. 6a, which illustrates SRS C-D on-resonance and off-resonance images of a 24 hpf embryonic zebrafish. The difference image between C-D on-resonance and off-resonance (pixel-by-pixel subtraction) shows pure C-D labeled protein distribution in the somites of an embryonic zebrafish tail, as in FIG. 6. FIGS. 8b and 8c illustrate SRS C-D on-resonance and off-resonance images of live mouse liver FIG. 6b and intestine FIG. 6c tissues harvested from the mice after administering with D-AA containing drinking water for 12 days. The difference image between C-D on-resonance and off-resonance (pixel-by-pixel subtraction) shows pure C-D labeled protein distribution in the liver and intestine tissues, shown in FIG. 6b and FIG. 6c, respectively. The residual signal presented in the off-resonance images mainly comes from cross-phase modulation induced by highly scattering tissue structures.

[0066] FIGS. 9a-9d are exemplary SRS images for newly synthesized proteins in vivo with intraperitoneal injection of mice with D-AA solutions. For example, FIGS. 9a and 9b illustrate SRS images of live mouse liver FIG. 9a and intestine FIG. 9b tissues harvested from mice after intraperitoneal injection injected with D-AAs solutions for 36 h. 2133 cm.sup.1 channel shows newly synthesized proteins (off-resonance image subtracted) that resemble the distribution of total proteins as shown in the 1655 cm.sup.1 image (Amide I). FIGS. 9c and 9d illustrate corresponding raw C-D on-resonance (2133 cm.sup.1) and off-resonance (2000 cm.sup.1) images are shown as references for liver FIG. 9c and intestine FIG. 9d tissues. Scale bar, 10 m.

[0067] FIGS. 10a-10c are exemplary bond-selective SRS images of alkynes as nonlinear vibrational tags. For example, FIG. 10a illustrates Spontaneous Raman spectra of HeLa cells and 10 mM EdU solution. Inset: the calculated SRS excitation profile (FWHM 6 cm.sup.1) is well fitted within the 2125 cm.sup.1 alkyne peak (FWHM 14 cm.sup.1, magenta). FIG. 10b illustrates linear dependence of stimulated Raman loss signals (2125 cm.sup.1) with EdU concentrations under a 100 s acquisition time. FIG. 10c illustrates the metabolic incorporation scheme for a broad spectrum of alkyne-tagged small precursors. a.u. arbitrary units.

[0068] FIG. 11 is a diagram of an exemplary system according to an exemplary embodiment of the present disclosure for providing a stimulated Raman scattering with A Pump beam (pulsed, pico-second) and an intensity-modulated Stokes beam (pulsed, pico-second). The Pump beam (pulsed, pico-second) and an intensity-modulated Stokes beam (pulsed, pico-second) are both temporally and spatially synchronized before focused onto cells that have been metabolically labeled with alkyne-tagged small molecules of interest. When the energy difference between the Pump photon and the Stokes photon matches the vibrational frequency (.sub.vib) of alkyne bonds, alkyne bonds are efficiently driven from their vibrational ground state to their vibrational excited state, passing through a virtual state. For each excited alkyne bond, a photon in the Pump beam is annihilated (Raman loss) and a photon in the Stokes beam is created (Raman gain). The detected pump laser intensity changes through a lock-in amplifier targeted at the same frequency as the modulation of Stokes beam serve as the contrast for alkyne distributions.

[0069] FIGS. 12a-12j are exemplary live SRS images of de novo synthesis of DNA, RNA, proteomes, phospholipids and triglycerides by metabolic incorporation of alkyne-tagged small precursors. For example, FIG. 12a illustrates Raman spectra of cells incubated with EdU, EU, Hpg, propargylcholine and 17-octadecynoic acid (17-ODYA). FIG. 12b illustrates live HeLa cells incubated with 100 M EdU alone (alkyne-on) and with 10 mM hydroxyurea (Control). FIG. 12c illustrates time-lapse images of a dividing cell incubated with 100 M EdU. FIG. 12d illustrates live HeLa cells incubated with 2 mM EU alone (alkyne-on) and with 200 nM actinomycin D (Control). FIG. 12e illustrates pulse-chase imaging of RNA turnover in HeLa cells incubated with 2 mM EU for 12 h followed by EU-free medium. FIG. 12f illustrates live HeLa cells incubated with 2 mM Hpg alone (alkyne-on) and with 2 mM methionine (Control). FIG. 12g illustrates live neurons incubated with 1 mM propargylcholine (alkyne-on). FIG. 12h illustrates live macrophages incubated with 400 M 17-ODYA (alkyne-on). FIG. 12i, illustrates C. elegans fed with 17-ODYA (alkyne-on). FIG. 12j, illustrates dual-color images of simultaneous EdU (2125 cm.sup.1) and propargylcholine (2142 cm.sup.1) incorporation. For FIG. 12b, FIG. d, and FIG. 12f, alkyne-off and amide images display the same set of cells as the alkyne-on images; lipid images capture the same cells as control images. Scale bars, 10 m. Representative images of 10-15 trials. a.u.=arbitrary units.

[0070] FIG. 13 is an exemplary SRS image of distal mitotic region of C. elegans germline incorporated with EdU. The composite image shows both the protein derived 1655 cm.sup.1 (amide) signal from all the germ cells, and the direct visualization of alkynes (2125 cm.sup.1 (EdU)) highlighting the proliferating germ cells. White circles show examples of EdU positive germ cells in the mitotic region of C. elegans germline. Scale bar, 5 m.

[0071] FIG. 14 are exemplary SRS images of fixed HeLa cells after incorporating with 2 mM Hpg. The alkyne-on image displays the Hpg distribution for the newly synthesized proteins. For the same set of cells, the off-resonant (alkyne-off) image shows vanishing signal, and the amide image shows total protein distribution. This result confirms that the detected signal is not from freely diffusive precursor Hpg itself (which is eliminated during the fixation process). Scale bar, 10 m.

[0072] FIGS. 15a-15c are exemplary click-chemistry illustrations based fluorescence staining of fixed HeLa cells. Fluorescence images of HeLa cells incorporated with FIG. 15a, EdU (for DNA); FIG. 15b EU (for RNA); FIG. 15c Hpg (for protein). Scale bars, 10 m.

[0073] FIGS. 16a-16c are exemplary SRS image in the same set of cells as in alkyne-on images, the alkyne-off images show a clear background, e.g., of propargylcholine incorporation in NIH3T3 cells and control experiments. For example, FIG. 16a illustrates fixed NIH3T3 cells after culturing with 0.5 mM propargylcholine for 48 hours. The alkyne-on image shows alkyne-tagged choline distribution. FIG. 16b illustrates treatment of fixed NIH3T3 cells with phospholipase C, which removes Choline head groups of phospholipids only in the presence of calcium. The alkyne-on image shows the strong decrease of incorporated propargylcholine signal, supporting its main incorporation into membrane phospholipids. FIG. 16c illustrates treatment of fixed NIH3T3 cells with phospholipase C in the presence of EDTA (chelating calcium). Propargylcholine signal is retained in the alkyne-on image. The amide images display total protein distribution. Scale bars, 10 m.

[0074] FIG. 17a-17e are exemplary illustration of in vivo delivery of an alkyne-bearing drug (TH in DMSO) into mouse ear. For example, FIG. 17a illustrates Raman spectra of a drug cream, Lamisil, containing 1% TH and mouse ear skin tissue. FIGS. 18b-18e illustrate SRS imaging of tissue layers from stratum corneum (z=4 m) to viable epidermis (z=24 m), sebaceous gland (z=48 m) and subcutaneous fat (z=88 m). To facilitate tissue penetration, DMSO solution containing 1% TH was applied onto the ears of an anesthetized live mouse for 30 min and the dissected ears are imaged afterwards. For all 4 layers: alkyne-on images display TH penetration; alkyne-off images show off-resonant background (The bright spots in FIG. 17d are due to two-photon absorption of red blood cells). The composite images show protein (1655 cm.sup.1) and lipid (2845 cm.sup.1) distributions. Scale bars, 20 m. a.u. arbitrary units.

[0075] FIGS. 18a and 18b are exemplary illustrations of in vivo delivery of an alkyne-bearing drug (TH in Lamisil cream, a FDA approved drug cream) into mouse ear. For example, FIGS. 18(a-b) illustrates SRS imaging of the viable epidermis layer (z=20 m) and the sebaceous gland layer (z=40 m). For both FIG. 18a and FIG. 18b: illustrates the alkyne-on images display the TH penetration into mouse ear tissues through lipid phase. The composite images show both protein (1655 cm.sup.1) and lipid (2845 cm.sup.1) distributions. Scale bars, 20 m.

[0076] FIG. 19 is an illustration of an exemplary synthesis route for a bond-edited compound.

[0077] FIG. 20a is an illustration of another exemplary synthesis route for a bond-edited compound (alkyne-D-glucose).

[0078] FIG. 20b is an exemplary illustration of the spectroscopic characterization of the bond-edited compound shown in FIG. 201a in PBS buffer and in mammalian cells.

[0079] FIG. 21 are exemplary illustrations of time-dependent alkyne-D-glucose (32 mM) uptake in live HeLa cells at 10 min, 30 min, 1 h, 2 h, 3 h and 4 h time points. The glucose signal inside mammalian cells is increasing over time.

[0080] FIG. 22 are exemplary illustrations of exemplary results of a competition experiment to confirm the uptake of alkyne-D-glucose. Regular D-glucose is added into cell medium for HeLa cells to compete with the uptake of alkyne-D-glucose. With the increasing concentration of regular D-glucose (10 mM, 50 mM and 100 mM), the alkyne-D-glucose signal decreases (as shown both in images and bar diagrams). When using L-glucose (which cells do not uptake) as competition for alkyne-glucose, the alkyne-D-glucose signal is retained.

[0081] FIG. 23a is an exemplary illustration of an alkyne-glucose uptake in neuronal culture.

[0082] FIG. 23b is an exemplary illustration of an alkyne-glucose uptake in brain slices.

[0083] FIG. 24 is an exemplary illustration of multicolor imaging of DNA synthesis with EdU (1), EdU-.sup.13C (2) and EdU-.sup.13C.sub.2 (3).

[0084] FIG. 25 is an exemplary illustration of pulse-chase imaging of DNA synthesis (EdU (1) for pulse and EdU-.sup.13C2 (3) for chase). The merged images show that the two compounds can label two temporally different cells populations for DNA synthesis.

[0085] FIGS. 26a-26c are exemplary illustrations of simultaneous three-color chemical imaging using alkyne probes for DNA synthesis (EdU-.sup.13C (2) at 2077 cm.sup.1) and RNA synthesis (EU-.sup.13C2 (13) at 2053 cm.sup.1 and 17-ODYA (12) at 2125 cm.sup.1).

[0086] FIG. 27 is a set of exemplary images of subcutaneous colon cancer. Subcutaneous colon cancer was grown for 15 days in mice, dissected out and cultured ex vivo in deuterated amino acids containing medium for 47 h (400 um thick). Live image of the tumors shows intensive protein synthesis activity.

[0087] FIGS. 28a and 28b are exemplary illustrations of active glucose metabolism in HeLa cells cultured in deuterated glucose medium. For example, FIG. 28a illustrates images after culturing in 0.1% D7-Glucose in EMEM for 48 hrs. FIG. 28b illustrates HeLa cell images after culturing 0.2% D7-Glucose in EMEM for 48 hrs.

[0088] FIG. 29 is a set of exemplary illustrations of an active glucose metabolism in tumor cell line U87MG cultured in deuterated glucose medium (0.1% D7-Glucose in EMEM) for 48 hrs.

[0089] FIG. 30 is a graph of an exemplary detection of D.sub.2O as a labeling reagent of the metabolism with stimulated Raman scattering.

[0090] FIG. 31 is a set of exemplary illustrations of exemplary SRS images of C-D formation using D.sub.2O as a metabolic reagent for various of live organisms.

[0091] FIGS. 32a-32c are exemplary illustration for imaging of .sup.13C-phenylalanine labeled proteins for protein turnover. For example, FIG. 32a shows an illustration of a spectroscopic characterization of Raman shift from 1004 cm.sup.1 to 968 cm.sup.1 with the labeling of .sup.13C-phenylalanine, and FIGS. 32(b) and 32c illustrate an exemplary time dependent .sup.13C-phenylalanine labeling, whereas FIG. 32b shows a spectrum and FIG. 32c illustrates exemplary SRS images, where the 968 cm.sup.1 signal for .sup.13C labeled proteins are increasing while the 1004 cm.sup.1 signal of old 1.sup.2C-proteins are decreasing.

[0092] FIG. 33a-33f are exemplary images based on SRS imaging of newly synthesized proteins by metabolic incorporation of deuterium-labeled all amino acids in live HeLa cells. For example, FIG. 33a illustrates an exemplary Spontaneous Raman spectrum of HeLa cells incubated with a medium containing deuterium-labeled all amino acids for 20 hrs, showing a 5 times stronger peak at 2133 cm.sup.1 than the spectrum in FIG. 2. FIG. 33b illustrates SRS image targeting the central 2133 cm.sup.1 vibrational peak of C-D shows a high-contrast image representing newly synthesized proteins. The same intensity scale bar is used herein as in FIGS. 2a-2e. Consistent with previous reports, nascent proteins are distributed with a higher percentage in nucleoli (indicated by arrows) which are the active sites for ribosome biogenesis involving rapid import and degradation of proteins. FIG. 33c illustrates SRS image of the same cells as in FIG. 33b at off-resonance frequency 2000 cm.sup.1 is background-free. FIGS. 33d-33f illustrate exemplary SRS images of same cells as in FIG. 33b at frequency 1655 cm.sup.1 (amide I stretching attributed primarily to proteins); 2845 cm.sup.1 (CH.sub.2 stretching attributed mainly to lipids) and 2940 cm.sup.1 (CH.sub.3 stretching attributed mainly to proteins) show the intrinsic distributions of total cellular lipids and proteins.

[0093] FIGS. 34a-33m are exemplary illustrations of a set of exemplary graphs and a set of exemplary images based on SRS imaging of time-dependent de novo protein synthesis and drug-induced protein synthesis inhibition effect in live HeLa cells incubated in deuterium-labeled all amino acid medium. For example, FIG. 34a-34f SRS image targeting the central 2133 cm.sup.1 vibrational peak of C-D displays a time-dependent signal increase (5 hrsFIG. 34a, 12 hrsFIG. 34b, 20 hrsFIG. 34c) of the newly synthesized proteins, with nucleoli being gradually highlighted. As a control, the amide I (1655 cm.sup.1) signal remains at a steady state over time (5 hrsFIG. 34d, 12 hrsFIG. 34e, 20 hrsFIG. 34f). FIGS. 34g-34i illustrate ratio images between the SRS image at 2133 cm.sup.1 (newly synthesized proteins) and the SRS image at 1655 cm.sup.1 (the amide I band from total proteins), representing the relative new protein fraction with subcellular resolution at each time point (5 hrsFIG. 34g, 12 hrsFIG. 34h, 20 hrsFIG. 34i). The bar represents the ratio ranging from low to high. FIG. 34j shows time-lapse SRS images of a live dividing HeLa cell during a 25 min time-course after 20-hour incubation with deuterated all amino acids medium. FIG. 34k illustrates a spontaneous Raman spectrum of HeLa cells incubated with both deuterium-labeled all amino acids and a protein synthesis inhibitor anisomycin (5 M) for 12 hrs shows the drastic attenuation of the C-D Raman peak at 2133 cm.sup.1. FIG. 31l shows an exemplary SRS image of the same sample displays near vanishing signal throughout the whole field of view. FIG. 34m shows, as a control, the image of the same cells at 2940 cm.sup.1 confirms that anisomycin does not influence the total protein level.

[0094] FIGS. 35a-35f are exemplary illustrations of a set of exemplary graphs and a set of exemplary images based on SRS imaging of newly synthesized proteins by metabolic incorporation of deuterium-labeled all amino acids in live human embryonic kidney (HEK293T) cells. For example, FIG. 35a illustrates the spontaneous Raman spectrum of HEK293T cells incubated with deuterium-labeled all amino acids for 12 hrs shows a 2133 cm.sup.1 C-D peak nearly as high as the Amide I (1655 cm.sup.1) peak. FIG. 35b shows an exemplary SRS image targeting the central 2133 cm.sup.1 vibrational peak of C-D shows newly synthesized proteins in live HEK293T cells displaying a similar signal level as HeLa cells at 12 hrs (FIG. 35b). FIG. 35c shows, as a comparison, the off-resonant image is still background-free. FIGS. 35d and 35 (e) illustrate multicolor SRS images of intrinsic cell molecules: total proteins (1655 cm.sup.1 (FIG. 35d) and lipids (2845 cm.sup.1 (FIG. 35e). FIG. 35f illustrates the ratio image between new proteins (2133 cm.sup.1) and total proteins (1655 cm.sup.1) illustrates a spatial map for nascent protein distribution.

[0095] FIGS. 36a-36f are a set of exemplary images based on SRS imaging of newly synthesized proteins in both cell bodies and newly grown neurites of neuron-like differentiable mouse neuroblastoma (N2A) cells. During the cell differentiation process by serum-deprivation and 1 M retinoic acid, deuterium-labeled all amino acids medium is also supplied for 24 hrs. For example, FIG. 36a illustrates SRS images targeting the 2133 cm.sup.1 peak of C-D show newly synthesized proteins. FIG. 36 b illustrates SRS images targeting the 2940 cm.sup.1 CH.sub.3 show total proteins. FIGS. 36c and 36d illustrate zoomed-in images as indicated in the white dashed squares in FIG. 36a and FIG. 36b. FIG. 36e illustrates a ratio image between new protein FIG. 36c and total proteins FIG. 36d. While the starred neurites show high percentage of new proteins, the arrows indicate neurites displaying very low new protein percentage. FIG. 36f illustrates an exemplary merged image between new protein c (channel 3605) and total proteins shown in FIG. 36d (channel 3610). Similarly, starred regions show obvious new proteins; while arrows indicate regions that have undetectable new protein signal.

[0096] FIG. 37a is a prior art recipe for a mammalian cell culture.

[0097] FIG. 37b is an exemplary deuterium-labeled recipe based on the cell culture of FIG. 36;

[0098] FIG. 38 is an illustration of an exemplary block diagram of an exemplary system in accordance with certain exemplary embodiments of the present disclosure;

[0099] FIG. 39a is diagram of an exemplary SRS system according to an exemplary embodiment of the present disclosure;

[0100] FIGS. 39b-39e are exemplary illustrations of energy outputs according to an exemplary embodiment of the present disclosure;

[0101] FIGS. 40a-40d are exemplary graphs of signal sensitivities according to an exemplary embodiment of the present disclosure;

[0102] FIGS. 41a-41j are images taken with the exemplary SRS system according to an exemplary embodiment of the present disclosure;

[0103] FIG. 42a is an exemplary chart of pr-SRS spectra according to an exemplary embodiment of the present disclosure;

[0104] FIGS. 42b-42d are exemplary images taken using the exemplary pr-SRS system according to an exemplary embodiment of the present disclosure;

[0105] FIGS. 43a-43c are diagrams of exemplary dye molecules according to an exemplary embodiment of the present disclosure;

[0106] FIG. 43d is an exemplary graph of the wavenumbers versus normalized intensity of the exemplary dye molecules from FIGS. 43a-43c according to an exemplary embodiment of the present disclosure; and

[0107] FIG. 44 is a set of further images taken using the exemplary pr-SRS system according to an exemplary embodiment of the present disclosure.

[0108] FIGS. 45(a)-45(l) are exemplary illustrations of an electronic pre-resonance stimulated Raman scattering (e.g., epr-SRS) microscopy. FIG. 45(a) shows and illustration of an exemplary sspectroscopy of different SRS regimes, defined by the energy difference between molecular absorption peak (e.g., 0) and pump laser (e.g., pump). Representative SRS spectra at each regime: pure methanol at non-resonance; IR895 at rigorous resonance (e.g., abs 900 nm, methanol); Cy7.5 at pre-resonance (e.g., abs 800 nm, DMSO); ATTO740 at pre-resonance (e.g., abs 760 nm, DMSO). CO band in non-resonance and CC bands in pre-resonance are arrow-headed. Solvent bands are marked. is the homogeneous linewidth, 700 cm.sup.1. (b) Linear dependence of epr-SRS signals on ATTO740 concentrations (e.g., CC at 1642 cm.sup.1) under a 1-ms time constant. FIG. 45(c) shows Fast epr-SRS imaging of ATTO740 click-labeled 5-Ethynyl-2-deoxyuridine (EdU) for newly synthesized DNA in HeLa cells. FIG. 45(d) shows Off-resonance image and (e). The 100th frame image for the same set of cells shown in FIG. 45(c). FIGS. 45(f)-45(l) show Epr-SRS imaging of ATTO740 immuno-labeled -tubulin FIG. 45(f) shows Tom20 (g) in HeLa cells and Keratin 18 FIG. 45(h) in MCF7 cells; SiR SNAP-tagged genetically encoded H2B proteins FIG. 45(i) shows MitoTracker deep red FIG. 45(j) shows methylene blue; FIG. 45(k) in live HeLa cells; and oxidation product 4,4-dichloro-5,5-dibromoindigo from X-gal hydrolysis in E. coli FIG. 45(l) shows Scale bar: 10 m.

[0109] FIGS. 46(a)-46(d) are examples of multiplex epr-SRS imaging with commercial dyes in fixed and live mammalian cells. FIG. 46(a) shows Semi-log plot of the measured Raman cross-sections for conjugated CC of 28 organic dyes across a wide range of absorption peak energies (e.g., excited by pump=904-909 nm). Grey-shaded area indicates the defined epr-SRS region. FIG. 46(b) shows Resolvable epr-SRS spectra of 6 commercial dyes (e.g., dash-lined): CF640R (e.g., 1665 cm.sup.1), ATTO700 (e.g., 1657 cm.sup.1), ATTO740 (e.g., 1642 cm.sup.1), Cy5.5 (e.g., 1626 cm.sup.1), Alexa647 (e.g., 1606 cm.sup.1, 1359 cm.sup.1) and DyLight650 (e.g., 1606 cm.sup.1, 1370 cm.sup.1). Occasional residual backgrounds (e.g. DyLight650) are likely from two-photon absorption. FIG. 46(c) shows 8-color epr-SRS (e.g., channels arrowed in b) and fluorescence imaging in fixed HeLa cells. Epr-SRS: EdU (e.g., newly synthesized DNA, Cy5.5, red), -tubulin (e.g., bundles in cytokinesis, CF640R, green), Azidohomoalaine (AHA) (e.g., newly synthesized proteins, Alexa647, blue), Fibrillarin (e.g., nucleoli marker, ATTO740, yellow), Giantin (e.g., Golgi marker, ATTO700, cyan). Fluorescence: Nucblue (e.g., total DNA, gray), Wheat Germ Agglutinin (WGA) (e.g., Glycoproteins, Alexa488, orange); MitoTracker orange (e.g., mitochondria marker, magenta). FIG. 46(d) shows 8-color imaging in live Hela cells. Epr-SRS: Lysotracker (e.g., lysosome marker, blue), SYTO60 (e.g., nucleic acid stain, yellow), LipidTOX Deep Red (e.g., neutral lipid stain, cyan), WGA (e.g., Glycoproteins, ATTO740, orange), Rhodamine 800 (e.g., mitochondria marker, magenta). Fluorescence: Actin (e.g., RFP, red), endoplasmic reticulum (ER) (e.g., GFP, green), Nucblue (e.g., total DNA, gray). Scale bar: 10 m.

[0110] FIGS. 47(a) and 47(b) are examples of MAnhattan Raman Scattering (MARS) dyes bearing -conjugated, isotopically-edited and electronically fine-tuned triple bonds. FIG. 47(a) shows Design principles and structures for a library of epr-SRS nitrile and alkyne dyes. *Overwhelming background due to close electronic resonance. FIG. 47(b) shows two sets of MARS palettes and their normalized epr-SRS spectra in the cell-silent window. The upper panel contains 12 MARS dyes, whose vibrational frequencies are indicated by numbers after solid lines and the corresponding structures are solid-underlined in FIG. 47(a). The lower panel contains 10 MARS dyes, whose vibrational frequencies are indicated by numbers after dashed lines and the corresponding structures are dash-underlined in FIG. 47(a).

[0111] FIGS. 48(a)-48(e) are examples of super-multiplex optical microscopy and its applications for probing metabolic activity in nervous systems under physiological and pathological conditions. (a) 16-color live-cell imaging with 8 MARS dyes, 4 commercial vibrational dyes in the fingerprint and 4 additional fluorescent dyes (e.g., star indicated). Each pre-stained cell in the cell mix (e.g., gray image, left) is circled with the matching line color and shape (e.g., 16-color image, right) with the corresponding dye. FIGS. 48(b) and 48(c) show 8-color epr-SRS imaging of DNA replication and protein synthesis in hippocampal neuronal cultures (b1-b5) and organotypic cerebellar brain slices FIGS. 48(c1)-48(c5) and FIG. 48(d1)-48(d5) show Pulse (e.g., HPG)chase (e.g., AHA) imaging of proteome turnover dynamics in hippocampal neuronal cultures with proteasome inhibitor MG132 in the chase period, with the percentages of astrocyte and neuron cells that contain protein inclusions illustrated in FIG. 48(e). Error bars, mean+s.d based on imaging n=6 samples, with P<0.01 determined using two-sided Student's t-test. For labeling in b-c, Epr-SRS: III-tubulin (e.g., neurons, MARS2200, green in b-c; gray in d), Myelin basic protein (MBP) (e.g., oligodendrocytes, MARS2176, orange), Glial fibrillary acidic protein (GFAP) (e.g., astrocytes and neural stem cells, MARS2147, magenta), EdU (e.g., newly synthesized DNA, MARS2228, gray in b-c), HPG (e.g., proteins synthesized in pulse period, MARS2228, red in d), AHA (e.g., proteins synthesized in chase period, Alexa647, red in b-c; green in d). Fluorescence: Nucblue (e.g., total DNA, cyan), NeuN (e.g., neurons, Alexa568, blue), Nestin (e.g., neural stem cells and astrocytes, Alexa488, yellow). Scale bar: 10 m.

[0112] FIG. 49 shows an illustration of an exemplary block diagram of an exemplary system in accordance with certain exemplary embodiments of the present disclosure.

[0113] Throughout the drawings, the same reference numerals and characters, unless otherwise stated, are used to denote like features, elements, components or portions of the illustrated exemplary embodiments. Moreover, while the present disclosure will now be described in detail with reference to the figures it is done so in connection with the illustrative exemplary embodiments and is not limited by the particular exemplary embodiments illustrated in the figures, and provided in the appended claims.

DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS

[0114] The following detailed description is presented to enable any person skilled in the art to make and use the present disclosure. For purposes of explanation, specific nomenclature is set forth to provide a thorough understanding of the present invention. However, it will be apparent to one skilled in the art that these specific details are not required to practice the invention. Descriptions of specific applications are provided only as representative examples. The present disclosure is not intended to be limited to the exemplary embodiments shown, but is to be accorded the widest possible scope consistent with the principles and features disclosed herein.

[0115] As used herein, the term Raman scattering refers to a spectroscopic technique used to observe vibrational, rotational, and other low-frequency modes in a system. It relies on inelastic scattering, or Raman scattering, of monochromatic light, usually from a laser in the visible, near infrared, or near ultraviolet range. The laser light interacts with molecular vibrations, phonons or other excitations in the system, resulting in the energy of the laser photons being shifted up or down. The shift in energy gives information about the vibrational modes in the system. A variety of optical processes, both linear and nonlinear in light intensity dependence, are fundamentally related to Raman scattering. As used herein, the term Raman scattering includes, but is not limited to, stimulated Raman scattering (SRS), spontaneous Raman scattering, coherent anti-Stokes Raman scattering (CARS), surface-enhanced Raman scattering (SERS), Tip-enhanced Raman scattering (TERS) or vibrational photoacoustic tomography.

[0116] The exemplary system, method and computer accessible medium 4915, according to an exemplary embodiment of the present disclosure, can use alkyne as a vibrational tag coupled with narrow-band stimulated Raman scattering microscopy (SRS) for the detection of small molecules inside biological systems. The use of alkyne as a vibrational tag (e.g. a Raman tag) offers a large Raman cross-section enabling sensitive detection (See, e.g., References 13; 14). Additionally, the alkyne Raman peak can exhibit a narrow spectral width for the specific detection, which can reduce the probability of overlapping with other tags. Furthermore, the Raman peak of alkyne can lay exactly in the cell-silent region in the cell spontaneous Raman spectrum, bypassing the complex interference from vast pool of biomolecules in the fingerprint region. (See, e.g., FIG. Tb).

[0117] The exemplary SRS can be a sensitive vibrational imaging microscopy. By harnessing Einstein's stimulated emission process, the exemplary SRS can employ two-laser excitation (e.g., temporally and spatially overlapped Pump and Stokes lasers), boosting up the transition rate about 7 orders of magnitude as compared to the traditional spontaneous Raman microscopy, the transition process of which can be intrinsically weak (e.g., 10 to 12 orders of magnitude slower than fluorescence). (See, e.g., References 6; 8; 15). The exemplary SRS can be a bond-selective procedure with high specificity, in contrast with the spontaneous Raman imaging which can be a spectrum-based method. Instead of spreading the energy to the whole spectrum as in the spontaneous Raman imaging, the exemplary narrow-band SRS can focus its energy to the vibrational transition of a specific bond. A 6-ps pulse width can be chosen for both SRS pump and stokes lasers to achieve a spectral resolution of 5 cm.sup.1 for the detection of alkyne. The spectral width of the excitation profile from two combined lasers can be calculated to be 8 cm.sup.1, which can fit well within the spectral width of alkyne Raman peak that can be 14 cm.sup.1. (See, e.g., FIG. 1b). Hence, the exemplary laser pulse width can be long enough that all the laser energy can be used to specifically detect alkyne without energy waste, but short enough that the two-photon efficiency can be maintained since the exemplary SRS can depend on a nonlinear process.

[0118] The exemplary SRS signal can offer linear concentration dependence to the analyte without non-specific background. Compared to a previously known nonlinear vibrational imaging procedure such coherent anti-Stokes Raman scattering (CARS) microscopy, which suffers from spectral distortion, unwanted non-resonant background, non-straightforward concentration dependence and coherent image artifact, the exemplary SRS can exhibit straightforward image interpretation and quantification without complications from non-resonant background and phase-matching conditions (See, e.g., References 7; 8; 16). Besides the above-mentioned advantages, SRS can also have its own distinctive characters as an imaging procedure. For example, SRS can be immune to fluorescence background as compared to spontaneous Raman microscopy that can suffer from large fluorescence background. In addition, SRS, as a nonlinear process, can offer intrinsic 3D sectioning capability. Moreover, by adopting near-infrared excitation, SRS can offer deeper penetration depth and less photo-toxicity, which can be well suited for imaging live cells, tissues and animals. Recently, narrow-band SRS has achieved unprecedented sensitivity down to approximately 1000 retinoic acid molecules and up to video rate imaging speed in vivo. (See, e.g., Reference 17).

[0119] Alkyne can be a metabolic labeling tag in fluorescence microscopy utilizing click-chemistry with azide-linked fluorescent tags (See, e.g., References 18-23). Unfortunately, this type of click-chemistry based fluorescence detection usually requires non-physiological fixation and subsequent dye staining and washing. The exemplary Raman detection, in contrast, does not have such requirements, since it can directly image vibrational modes of alkyne, bypassing the subsequent additional processes.

[0120] All of the above applications can show the universal and distinct advantage of the exemplary SRS coupled with alkyne tags to image the small molecule metabolites dynamics and drug distributions in the live cells, organisms and animals with minimum perturbation and high specificity and sensitivity, extending the repertoire of reporters for biological imaging beyond fluorophores.

Method for Obtaining Biological Information in a Living Cell or a Living Organism with Bond-Edited Compounds

[0121] One aspect of the present disclosure relates to a method for obtaining biological information in a living cell or a living organism with bond-edited compounds using Raman scattering. The method comprises the steps of introducing an effective amount of one or more bond-edited compounds into a live cell or a living organism, and detecting a vibrational tag in the cell or organism with Raman scattering. In some exemplary embodiments, the Raman scattering is SRS.

[0122] The term biological information as used herein, refers to spatial distribution of the targeted molecules, such as one-dimensional line, or two-dimensional or three-dimensional images, and non-imaging information, such as a simple signal intensity or local spectrum on a single location or its time dependence.

[0123] As used herein, the term bond-edited compounds refers to compounds having one or more chemical bond that may serve as a vibrational tag for detection by Raman scattering. Examples of chemical bond that may serve as a vibrational tag include, but are not limited to, carbon-carbon triple bond, carbon-nitrogen triple bond, azide bond, carbon-deuterium bond, phenol ring, .sup.13C modified carbon-carbon triple bond, .sup.13C modified carbon-nitrogen triple bond, .sup.13C modified azide bond, .sup.13C modified carbon-deuterium bond, .sup.13C modified phenol ring and combinations thereof.

[0124] As used herein, the term effective amount refers to an amount that, when introduced into a live cell or organism, is sufficient to reach a working concentration needed for SRS imaging. The effective amount would vary based on the type of bond-edited compound, as well as the cells or organisms that the bond-edited compound is introduced into. In some embodiments, an effective amount of a bond-edited compound is the amount that is sufficient to reach an in vivo concentration of 1 M to 100 mM, 3 M to 30 mM, 10 M to 10 mM, 100 M to 1 mM, 10 M to 1 mM or 10 M to 100 M in a target cell or organ. In some embodiments, an effective amount of a bond-edited compound comprising a triple bond is the amount that is sufficient to reach an in vivo concentration of 1 M to 10 mM, 3 M to 3 mM, 1 M to 1 mM or 30 M to 300 M. In some embodiments, an effective amount of a bond-edited compound comprising a triple bond is the amount that is sufficient to reach an in vivo concentration of about 100 M. In other embodiments, an effective amount of a bond-edited compound comprising a C-D bond is the amount that is sufficient to reach an in vivo concentration of 10 M to 100 mM, 30 M to 30 mM, 100 M to 10 mM or 300 M to 3 mM. In some embodiments, an effective amount of a bond-edited compound comprising a C-D bond is the amount that is sufficient to reach an in vivo concentration of about 1 mM.

[0125] In some exemplary embodiments, the bond-edited compounds are small molecules. As used herein, the term small molecules refers to low molecular weight organic compound having a molecular weight of 1000 daltons or less. In some exemplary embodiments, the small molecules have a size on the order of 10.sup.9 m. Examples of small molecules include, but are not limited to, water, ribonucleosides, ribonucleotides, deoxyribonucleoside, deoxyribonucleotide, amino acids, peptides, choline, monosaccharides, disaccharides, fatty acids, glucose, adenosine triphosphate, adenosine diphosphate, cholesterol, neurotransmitters, secondary messengers, and chemical drugs.

[0126] In some exemplary embodiments, said bond-edited compound contains one, two, three, four, five, six, seven, eight, nine, ten or more vibrational tags. The vibrational tags may be the same type of tags or a mixture of one or more different tags.

[0127] In some exemplary embodiments, said vibrational tag is an alkyne tag. In other exemplary embodiments, said vibrational tag is an azide tag. In still other exemplary embodiments, said vibrational tag is an isotope label. In a further exemplary embodiment, said isotope label is a carbon-deuterium tag. In yet still other exemplary embodiments, said vibrational tag is a combination of an alkyne tag and a carbon-deuterium tag.

[0128] In particular exemplary embodiments, said at least one vibrational tag comprises at least one vibrational tag selected from the group consisting of CC, CN, NNN, CCCC, CCCN, C-D, and CC-D.

[0129] In a further exemplary embodiment, the vibrational comprises at least one .sup.13C atom or one deuterium atom.

[0130] In some exemplary embodiments, the bond-edited compound is an amino acid.

[0131] In further exemplary embodiments, the amino acid is an essential amino acid.

[0132] In a still further exemplary embodiment, the essential amino acid is selected from the group consisting of histidine, isoleucine, leucine, lysing, methionine, phenylalanine, threonine, tryptophan and valine.

[0133] In other exemplary embodiments, the bond-edited compound is a nucleoside or a nucleotide.

[0134] In still other exemplary embodiments, the bond-edited compound is a fatty acid.

[0135] In still other exemplary embodiments, the bond-edited compound is a monosaccharide or a disaccharide. In a further exemplary embodiment, the bond-edited compound is glucose, a glucose derivative or propargyl glucose.

[0136] In still other exemplary embodiments, the bond-edited compound is a pharmaceutical agent, such as an anti-cancer agent, anti-inflammatory agent, anti-bacterial agent, anti-fungal agent and anti-viral agent.

[0137] In still other exemplary embodiments, the bond-edited compound is a cytokine or chemokine.

[0138] In some exemplary embodiments, the bond-edited compound is EU-.sup.13C2 having a molecular structure of formula 13:

##STR00010##

[0139] In some exemplary embodiments, the bond-edited compound is EdU-.sup.13C2 having a molecular structure of formula 3:

##STR00011##

[0140] In some exemplary embodiments, the bond-edited compound is EdU-.sup.13C having a molecular structure of formula 2:

##STR00012##

[0141] In some exemplary embodiments, the bond-edited compound is EdU-.sup.13C having a molecular structure of formula 14:

##STR00013##

[0142] In some exemplary embodiments, the bond-edited compound is alkyne-D-glucose having a molecular structure of formula S3:

##STR00014##

[0143] In some exemplary embodiments, the bond-edited compound is metabolized in the living cell or organism and the vibrational tag is transferred from the bond-edited compound to a down-stream metabolite of the bond-edited compound (See, e.g., FIGS. 28-31).

[0144] In still other exemplary embodiments, the method comprises introducing into a live cell a mixture of bond-edited compounds that imaging with Raman scattering at two or more different wavelengths. In some related exemplary embodiments, the Raman scattering is SRS.

[0145] In still other exemplary embodiments, the method comprises introducing into a live cell a mixture of different bond-edited compounds that allow multiple color imaging with Raman scattering. In some related exemplary embodiments, the Raman scattering is SRS. In a particular exemplary embodiment, the mixture of different bond-edited compounds comprises EU-.sup.13C2, EdU-.sup.13C and 17-ODYA.

[0146] In some exemplary embodiments, the two or more bond-edited compounds target the same cellular component but at different time period (See, e.g., FIG. 25).

[0147] In still other exemplary embodiments, the method comprises introducing into a living cell a mixture of different bond-edited compounds that target different cellular components.

[0148] In still other exemplary embodiments, the method comprises introducing into a living organism a mixture of different bond-edited compounds that target different types of cells in the living organism.

[0149] In still other exemplary embodiments, the method comprises introducing into a living organism a mixture of different bond-edited compounds carrying different vibrational tags, and detecting the different vibrational tags with Raman scattering using a linear combination algorithm. In some related exemplary embodiments, the Raman scattering is SRS.

Exemplary Method for Making Bond-Edited Compounds

[0150] Another exemplary aspect of the present disclosure relates to a method for making a bond-edited compound.

[0151] In one exemplary embodiment, the bond-edited compound is synthesized by the route illustrated in FIG. 19. In another exemplary embodiment, the bond-edited compound is synthesized by the route illustrated in FIG. 20.

Exemplary Method of Detecting Disease Conditions

[0152] Another exemplary aspect of the present disclosure relates to a method for detecting a disease condition in a subject, comprising: administering to said subject a composition comprising a bond-edited compound targeting a disease tissue or pathogen, and detecting said bond-edited compound by Raman scattering.

[0153] In some exemplary embodiments, the subject is a mammal. Exemplary mammal subjects for use in accordance with the methods described herein include humans, monkeys, gorillas, baboons, zoo animals and domesticated animals, such as cows, pigs, horses, rabbits, dogs, cats, goats and the like.

[0154] In some exemplary embodiments, the disease condition is cancer.

[0155] In some exemplary embodiments, the disease condition is a neurodegenerative disease. In further exemplary embodiments, the neurodegenerative disease is selected from the group consisting of amyotrophic lateral sclerosis, Parkinson's, Alzheimer's and Huntington's.

[0156] In some exemplary embodiments, the disease condition is an inflammatory disease.

[0157] In some exemplary embodiments, the disease condition is a microbial infection.

[0158] In some exemplary embodiments, the disease condition is a bacterial infection.

[0159] In some exemplary embodiments, the disease condition is a viral infection.

[0160] In some exemplary embodiments, the disease condition is a fungal infection.

[0161] In some exemplary embodiments, the pathogen comprises bacteria.

Exemplary Method for Monitoring Treatment for a Disease Condition

[0162] Another exemplary aspect of the present disclosure relates to a method for monitoring treatment for a disease condition. The method comprises administering to said subject a composition comprising a bond-edited compound and detecting said bond-edited compound by SRS at a first time point, further administering to said subject said composition comprising a bond-edited compound and detecting said bond-edited compound by Raman scattering at a second time point, and comparing images obtained at the two time points.

[0163] In some exemplary embodiments, the first time point is a time point that is about or prior to the initiation of a treatment and the second time point is a time point that is after the initiation of the treatment.

[0164] In other exemplary embodiments, the first time point and the second time point are two time points during the course of a treatment.

[0165] In some exemplary embodiments, the treatment is a treatment for cancer.

[0166] In other exemplary embodiments, the treatment is a treatment for an inflammatory disease.

[0167] In other exemplary embodiments, the treatment is a treatment for a neurodegenerative disease.

Method for Screening an Agent

[0168] Another exemplary aspect of the present disclosure relates to a method for screening an agent. The method comprises administering said agent and at least one bond-edited compound to a live cell or organism, detecting the bond-edited compound in the live cell or organism using Raman scattering, and selecting a candidate agent based on one or more predetermined criteria, such as the uptake, accumulation, trafficking, or degradation of the said bond-edited compound in the said live cell or organism.

[0169] In some exemplary embodiments, the candidate agent is an anti-cancer drug.

[0170] In some exemplary embodiments, the bond-edited compound is selected from the group consisting of amino acid, nucleic acid, ribonucleic acid and glucose derivatives.

[0171] In some exemplary embodiments, the candidate agent is a skin regenerating agent.

[0172] In some exemplary embodiments, the candidate agent is a cosmetic agent.

Exemplary Method for Tracing a Cellular Process in a Live Cell with Raman Scattering

[0173] Another exemplary aspect of the present disclosure relates to a method for tracing a cellular process in a live cell with Raman scattering. The method comprises introducing into a live cell a bond-edited compound, and following the physical movement or the chemical reaction or the biological interaction of the bond-edited compound within the cell by SRS.

[0174] In some exemplary embodiments, the cellular processes are selected from the group consisting of DNA replication, RNA synthesis, protein synthesis, protein degradation, glucose uptake and drug uptake.

Exemplary Composition for Labeling Cells with Bond-Edited Compounds

[0175] Another exemplary aspect of the present disclosure relates to a composition for labeling a target cell with at least one bond-edited compound. In some exemplary embodiments, the composition is a culture medium comprising at least one bond-edited compound containing at least one vibrational tag. The at least one bond-edited compound may be selected based on the type of the target cell or a target component(s) within the target cell.

[0176] In some exemplary embodiments, the culture medium comprises a plurality of amino acids, wherein over 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% of the amino acids are tagged with one or more vibrational tag. In other exemplary embodiments, the culture medium comprises a plurality of amino acids, wherein all amino acids are tagged with one or more vibrational tag.

[0177] In some exemplary embodiments, the culture medium comprises two, three, four, five, six, seven, eight, nine, ten or more different bond-edited compounds.

Exemplary Device for Imaging a Living Cell or a Living Organism with Bond-Edited Compounds

[0178] Another exemplary aspect of the present disclosure relates to a device for imaging bond-edited compounds by Raman scattering. The device comprises a first single-wavelength laser source that produces a pulse laser beam of a first wavelength, a second single-wavelength laser source that produces a pulse laser beam of a second wavelength, a modulator that modulates either the intensity or the frequency or the phase or the polarization or the combination of the above of the pulse laser beam of one of the first or second laser source, a photodetector that is capable of detecting SRS or CARS or spontaneous Raman scattering or the combination of the above from a biosample, and a computer.

[0179] In some exemplary embodiments, the energy difference between the photons produced by the first laser radiation and the photon produced by the second laser radiation matches with the energy of the vibrational transitions of the targeted vibrational tags. Photodetector of SRS detects part or all of the first laser beam or the second laser beam. The output of the photodetector (which could be a photodiode) is further processed by a lock-in amplifier or a resonant circuit.

[0180] Another exemplary aspect of the present disclosure relates to an apparatus for providing radiation to at least one structure, comprising: a radiation providing arrangement which is configured to provide a pump radiation and a stokes radiation, each at a fixed wavelength, whose energy difference is between about 2000 and 2500 wavenumbers.

[0181] In some exemplary embodiments, the radiation providing arrangement is a laser source.

Exemplary System and Method for Super-Multiplex Vibrational Imaging

[0182] For example, carrying out stimulated Raman scattering under electronic pre-resonance conditions (e.g., epr-SRS) can facilitate imaging with exquisite vibrational selectivity and sensitivity (e.g., down to 250 nM with 1-ms) in living cells. A palette of triple-bond-conjugated near-infrared dyes that each display a single epr-SRS peak in the cell-silent spectral window, and that with available fluorescent probes give 24 resolvable colors with potential for further expansion was created. Proof-of-principle experiments on neuronal co-cultures and brain tissues reveal cell-type dependent heterogeneities in DNA and protein metabolism under physiological and pathological conditions, underscoring the potential of this super-multiplex optical imaging approach for untangling intricate interactions in complex biological systems. SRS microscopy, developed for label-free imaging of chemical bonds, uses nonlinear Raman effects to accelerate weak vibrational transition by 108 times in the far field and thereby overcomes the sensitivity or biocompatibility limitations of other Raman imaging modalities. Coupling SRS with strong vibrational tags such as alkynes (e.g., CC) facilitates effective imaging of diverse biomolecules, but detection sensitivity can still be limited to about 15 mM for typical chemical bonds such as C H and 200 M for the stronger CC bond that leaves many targets (e.g., such as metabolites, proteins, RNA, organelles) out of reach.

[0183] The sensitivity limits can be associated with SRS microscopy having so far always operated in the non-resonance region, with the pump laser energy (e.g., pump) well below the molecular absorption peak energy (e.g., 0) (e.g., FIG. 45a). Electronic resonance can be known to significantly enhance Raman signals, and moving from non-resonance (e.g., 0>>pump) to rigorous resonance (e.g., 0=pump) indeed enhances the detected stimulated Raman loss signal (e.g., apparent pump intensity decrease) of IR895 by a factor of about 108 compared to the CO vibrational signal from methanol (e.g., purple and yellow spectra in FIG. 45a). However, this exemplary signal boost comes with a severe sacrifice in chemical specificity: the desired narrow-band vibrational signatures of IR895 are almost overwhelmed by the concurrent broad background that possibly originates from a combination of electronic-resonance four-wave mixing processes and competing pump-probe processes17. Judiciously adjusted detuning can retain signal enhancement and improve the contrast between vibrational signal and electronic background. Indeed, a moderate detuning to 0pump 2 (e.g., can be the homogeneous linewidth, typically 700 cm.sup.1) results in the electronic pre-resonance SRS (e.g., epr-SRS) spectrum of Cy7.5 starting to present an observable peak from the conjugated CC bonds with a vibrational-signal to electronic-background ratio (e.g., S/B) of 0.5 (e.g., cf. FIG. 45a, cyan spectrum). Further detuning to 3 of ATTO740 gives rise to a clean epr-SRS peak with a vanishing electronic background (e.g., cf. FIG. 45a, blue spectrum). A pre-resonance enhancement factor of 210.sup.5 can be achieved for this conjugated CC when compared to CO in methanol, a magnitude comparable to previous reports for resonance Raman spectroscopy. The epr-SRS detection limit of ATTO740 by targeting this peak can be determined to be 250 nM with 1-ms time constant (e.g., see FIG. 45b). This corresponds to about 3040 molecules within the focal volume, which can be about 1000 times more sensitive than the previous record of non-resonance SRS imaging and comparable to confocal fluorescence microscopy while retaining distinct vibrational contrast.

[0184] In the exemplary first proof-of-principle epr-SRS microscopy demonstration, ATTO740-labeled 5-Ethynyl-2-deoxyuridine (e.g., EdU, a metabolic target for newly synthesized DNA) in HeLa cells was imaged with a short pixel dwell time of 4 s (e.g., 0.4 s per 320-by-320 frame) (e.g., see FIG. 45(c)). The imaging pattern shows high resemblance to the corresponding fluorescence contrast pattern. Importantly, owing to the narrow vibrational resonance, the epr-SRS signal disappears when the pump laser wavelength k pump can be off resonance by only 2 nm (e.g., FIG. 45d)-demonstrating exquisite chemical selectivity unattainable with fluorescence. The epr-SRS signal retains 97% of its initial value even after 100 frames of continuous imaging (e.g., FIG. 45e), indicating satisfying photo-stability in the pre-resonance region.

[0185] To demonstrate the versatility of epr-SRS imaging, the ATTO740 immuno-labeled intracellular proteins were imaged, which were (e.g., FIG. 45f-g) alpha-tubulin with clearly resolved tubule structures (e.g., FIG. 45f) and Tom (e.g., a mitochondria receptor, FIG. 45g) and membrane receptors of tumor markers (e.g., keratin 18 in FIG. 45h). In addition to immuno-labeling, epr-SRS imaging of genetically encoded H2B proteins in live cells was also achieved through SNAP-tagging with far-red silicon-Rhodamine (SiR) dye (e.g., FIG. 45i). Organelle targets (e.g., MitoTracker deep red, FIG. 45j) and chemical drugs (e.g., methylene blue, a widely used drug and a photosensitizer in photodynamic therapy, FIG. 45k, likely enriched in lysosomes) can also be visualized in live cells. Finally, to demonstrate utility in a small-molecule based functional assay, the production of a non-fluorescent indigo product from a classic lacZ gene-expression assay in live E. coli was monitored (e.g., FIG. 45l).

[0186] An exemplary epr-SRS reporter dye palette was developed. Characterization of 28 commercial dyes across a wide range of 0 (e.g., FIG. 46a) confirmed that their Raman scattering cross-sections (e.g., (CC)) increase steeply as 0 approaches pump. This trend can be fitted quantitatively to the Albrecht A-term pre-resonance approximation, using a single fitting parameter K (e.g., Eq. 1) (e.g., FIG. 46a, red line):

[00001]$\begin{array}{cc}=K.Math..Math.{{}_0\left({}_0-{}_{\mathrm{vib}}\right)}^3.Math.{\left(\frac{\left({}_{\mathrm{pump}}^2+{}_0^2\right)}{{\left({}_0^2-{}_{\mathrm{pump}}^2\right)}^2}\right)}^2& \left(1\right)\end{array}$

where vib can be the vibrational transition energy and K can be a collection of frequency-independent factors of the dyes. An epr-SRS excitation region as 2<0pump<6 (e.g., FIG. 2a, grey shaded) was defined to ensure both chemical specificity (e.g., S/B>0.5) and sensitivity (e.g., <5 M with 1-ms). Within this near-infrared region approximately between 650 nm and 800 nm, 6 commercial dyes with largely overlapping fluorescence but mutually resolvable epr-SRS spectra (e.g., FIG. 46b, dash-lined) were identified as multiplexable reporters for the epr-SRS palette.

[0187] 5 CC peaks were selected to demonstrate the multiplex imaging capability (e.g., FIG. 46b, arrowed). Spectral cross-talks are minimized by a linear combination procedure (e.g., see methods). The epr-SRS imaging in the near-infrared can be orthogonal to fluorescence detection in the visible region (e.g., the non-resonance SRS region), so merging epr-SRS and fluorescent palettes enabled imaging with 8 different colors (e.g., 5 quantitative epr-SRS of CC bonds and 3 fluorescence channels) of the same set of fixed cells (e.g., FIG. 46c). Combining fluorescent proteins in the visible region with commercial organelle trackers in the epr-SRS region also enabled 8-color imaging of live cells (e.g., FIG. 46d), a feat difficult to achieve by other means.

[0188] The epr-SRS dye palette can be expanded further, especially when going beyond conjugated CC bonds and targeting triple bonds such as alkynes (e.g., CC) and nitriles (e.g., CN) that display a single sharp Raman peak in the wide silent window (e.g., from 1800 to 2800 cm.sup.1).sup.12-15,20. The challenge here can be that the triple bonds can be coupled with an electronic transition to enable pre-resonance enhancement, which can depend sensitively on the electronic-vibrational coupling strength.sup.16. Hence, simply installing a triple bond onto an arbitrary position of a dye can most often not yield satisfying signals. A new library of near-infrared dyes (e.g., FIG. 3a) was developed, where nitrile or alkyne triple bonds directly participate in the -conjugation systems of the molecule, with xanthene identified as a privileged scaffold.sup.21, 22 after initial screening. To generate more vibrational colors, three combined strategies guided by the classical .sub.vib{square root over (k/)} equation where k can be the spring constant reflecting the bond strength and were utilized, which can include, for example, =m.sub.1m.sub.2/(m.sub.1+m.sub.2)

can be the reduced mass. As illustrated by the x- and y-axes in FIG. 47a, substituting the 10-position of xanthene from O atom to C and Si 23 (e.g., FIG. 47a, green marked), gradually expanding the xanthene rings (e.g., FIG. 47a, yellowed marked), and modifying the terminal groups for alkyne dyes (e.g., FIG. 47a, purple marked) all fine tune the triple bond strength by affecting the electron-density of the -conjugation system. As indicated as z-axis, combinatorial isotopic editing of the two atoms involved in the triple bond (e.g., FIG. 47a, highlighted in red and blue) enables local modulation of their reduced mass into 4 possible combinations that further expand the vibrational palette.

[0189] Most of the dyes were synthesized (e.g., including their .sup.13C and .sup.15N isotopologues) in the exemplary conceived library (e.g., FIG. 47a), referring to promising structures with mutually resolvable epr-SRS peaks as Manhattan Raman Scattering (e.g., MARS) dyes. Two sets of MARS palettes (e.g., selected structures are underlined in FIG. 47a) were selected with up to 14 well-resolved epr-SRS colors in the cell-silent window (e.g., FIG. 47b), with all dyes displaying satisfying specificity (e.g., S/B>5) and sensitivity (e.g., <5 M with 1-ms). With the 6 commercial dyes in the fingerprint region, the exemplary expanded vibrational palette thus consists of 20 resolvable reporters in the near-infrared. Combining this vibrational palette with 4 more fluorescence channels typically obtainable in the visible region facilitates access to 24 colors for optical super-multiplexing.

[0190] The exemplary newly developed epr-SRS vibrational palette has biocompatibility that ensures sufficient cell viability, the utilized photostability and effective linear unmixing to enable proof-of-principle imaging on live HeLa cells. After being separately stained with individual dyes and then mixed together, these cells are subjected to 16-color live-cell imaging, which quadruples the number of color channels typically achieved by fluorescence alone. The resultant image shown in FIG. 4a was acquired through sequential imaging in 15 minutes, but further system development should enable simultaneous imaging. This exemplary super-multiplexing procedure was used to probe metabolic activity in complex nervous systems consisting of multiple cell types, under either physiological or pathological conditions. First, 8-color imaging was used to image DNA replication and protein synthesis activities, two crucial processes in neural development and function, in hippocampal neuronal cultures (e.g., FIG. 48b) and organotypic cerebellar tissues (e.g., FIG. 48c) and reveal highly heterogeneous DNA synthesis. Confirmed with labeling specificity (e.g., FIG. 48bl), two newly divided cells (e.g., EdU+, FIG. 48b2, arrowed) in neuronal co-cultures were identified. Multicolor cross-examination revealed that one was astrocytic (e.g., FIG. 48b3-b4, arrowed, III-tubulin-, NeuN and Nestin+, GFAP+), while the other oligodendrocytic (e.g., FIG. 48b3-b5 double-arrowed, III-tubulin-, NeuN, Nestin, GFAP but MBP+). Moving to imaging of organotypic cerebellar tissues of P11 mice (e.g., FIG. 48c1), a high number of EdU+ cells were observed, likely concentrated in the molecular layer (e.g., FIG. 48c2), which might be a progenitor-cell niche (e.g., FIG. 48c3-c5, Nestin+, MBP+ but NeuN, highlighted by arrows). These observations establish that the exemplary super-multiplex imaging platform can be well suited for probing molecular heterogeneity in nervous systems.

[0191] Neuronal systems were examined under proteasomal stress. Proteostasis, the balance between new protein synthesis and degradation of aberrant proteins, can be precisely regulated through cellular quality control, particularly the ubiquitin-proteasome system, with proteasome dysfunction implicated in many neurodegenerative diseases and aging processes. When studying individual cell types under proteasomal stress (e.g., modeled by applying proteasome inhibitor MG132), astrocytes were found to be more resistant than neurons and oligodendrocytes. Because such cell-type dependent vulnerability can be unexplored in more realistic environments where different cells can co-exist, 8-color imaging was used to examine the global proteostasis stress in neuronal co-cultures with largely preserved astrocyte-neuron coupling. As expected, applying MG132 (e.g., FIG. 48d1) retarded the proteome turnover. Interestingly, obvious cytosolic proteome aggregations (e.g., inclusions) were observed only in the chase channel (e.g., FIG. 48d2-d3), suggesting newly synthesized proteins are more susceptible to proteasomal impairment and prone to aggregation. This can be consistent with the fact that up to 30% of newly synthesized proteins are misfolded. Most importantly, subsequent cross-channel examination indicated that proteome inclusions appeared mostly in astrocytes instead of neurons (e.g., FIG. 48d4-d5, 48e). Considering that the formation of protein inclusions can exert a protective role by sequestering potentially harmful species from the cytosol, this result suggests that the astrocyte resistance observed before might be due to their ability to actively form protein inclusions that sequester the toxic misfolded species, whereas neurons are unable to do so.

[0192] While the above proof-of-principle observations demonstrate already the potential of the exemplary procedure, it can be improved further along multiple fronts. First, the MARS dye palette can be potentially expanded to 50 or more colors by filling the rather broad cell-silent window with individual sharp peaks from novel vibrational moieties. Second, genetically encoded infrared proteins could also be engineered to serve as vibrational reporters. Third, hyper-spectral imaging can be implemented to realize faster and simultaneous signal acquisition.

[0193] FIG. 49 shows a block diagram of an exemplary embodiment of a system according to the present disclosure. For example, exemplary procedures in accordance with the present disclosure described herein can be performed by a processing arrangement and/or a computing arrangement 4905. Such processing/computing arrangement 4905 can be, for example entirely or a part of, or include, but not limited to, a computer/processor 4910 that can include, for example one or more microprocessors, and use instructions stored on a computer-accessible medium (e.g., RAM, ROM, hard drive, or other storage device).

[0194] As shown in FIG. 49, for example a computer-accessible medium 4915 (e.g., as described herein above, a storage device such as a hard disk, floppy disk, memory stick, CD-ROM, RAM, ROM, etc., or a collection thereof) can be provided (e.g., in communication with the processing arrangement 4905). The computer-accessible medium 4915 can contain executable instructions 4920 thereon. In addition or alternatively, a storage arrangement 4925 can be provided separately from the computer-accessible medium 4915, which can provide the instructions to the processing arrangement 4905 so as to configure the processing arrangement to execute certain exemplary procedures, processes and methods, as described herein above, for example.

[0195] Further, the exemplary processing arrangement 4905 can be provided with or include an input/output arrangement 4935, which can include, for example a wired network, a wireless network, the internet, an intranet, a data collection probe, a sensor, etc. As shown in FIG. 49, the exemplary processing arrangement 4905 can be in communication with an exemplary display arrangement 4930, which, according to certain exemplary embodiments of the present disclosure, can be a touch-screen configured for inputting information to the processing arrangement in addition to outputting information from the processing arrangement, for example. Further, the exemplary display 4930 and/or a storage arrangement 4925 can be used to display and/or store data in a user-accessible format and/or user-readable format.

[0196] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to carry out the method of the present disclosure and is not intended to limit the scope of the invention. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc.), but some experimental error and deviation should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Centigrade and pressure is at or near atmospheric.

EXAMPLES

Example 1: In Vitro and In Vivo Labeling with Deuterium Tags

[0197] In the examples described below, three major technical advances are being implemented, together with a series of biological applications on complex tissues and model animals in vivo (FIG. Ta). First, we optimized the chemical composition of the deuterated culture medium that achieved much higher deuterium labeling efficiency, and improved imaging sensitivity and speed of our SRS instrumentation. These optimizations allow us to demonstrate time-lapse imaging of protein synthesis dynamics within single live cells. Second, we successfully imaged protein degradation in live HeLa cells by targeting Raman peak of methyl group (CH.sub.3) for the pre-existing protein pools and then employing a recently developed linear combination algorithm on measured SRS images at 2940 cm.sup.1 and 2845 cm.sup.1 channels. Third, inspired by the classic pulse-chase analysis of complex protein dynamics, two-color pulse-chase imaging was accomplished by rationally dividing D-AAs into two structurally different sub-sets that exhibit resolvable vibrational modes, as demonstrated by tracking aggregate formation of mutant huntingtin (mHtt) proteins. Finally, going beyond the cellular level to visualizing more complex tissues and animals in vivo, we imaged the spatial distribution of newly synthesized proteins inside live brain tissue slices and in both developmental embryonic zebrafish and mice (FIG. 1b). Taken together, these technical advances and biological applications demonstrate SRS microscopy coupled with metabolic labeling of D-AAs as a comprehensive and generally applicable imaging platform to evaluate complex protein metabolism with high sensitivity, resolution and biocompatibility in a broad spectrum of live cells, tissues and animals.

Exemplary Materials and Methods

[0198] Stimulated Raman scattering microscopy. An integrated laser (picoEMERALD with custom modification, Applied Physics & Electronics, Inc.) was used as the light source for both Pump and Stokes beams. Briefly, picoEMERALD provides an output pulse train at 1064 nm with 6 ps pulse width and 80 MHz repetition rate, which serves as the Stokes beam. The frequency-doubled beam at 532 nm is used to synchronously Seed a picosecond optical parametric oscillator (OPO) to produce a mode-locked pulse train (the idler beam of the OPO is blocked with an interferometric filter) with 5-6 ps pulse width. The wavelength of the OPO is tunable from 720 to 990 nm, which serves as the Pump beam. The intensity of the 1064 nm Stokes beam is modulated sinusoidally by a built-in electro-optic modulator (EOM) at 8 MHz with a modulation depth of more than 95%. The Pump beam is spatially overlapped with the Stokes beam with a dichroic mirror inside picoEMERALD. The temporal overlap between Pump and Stokes pulse trains is ensured with a built-in delay stage and optimized by the SRS signal of pure dodecane liquid.

[0199] Pump and Stokes beams are coupled into an inverted laser-scanning microscope (FV1200MPE, Olympus) optimized for near IR throughput. A 60 water objective (UPlanAPO/IR, 1.2 N.A., Olympus) with high near IR transmission is used for all cellular level imaging, and a 25 water objective (XLPlan N, 1.05 N.A., MP, Olympus) with both high near IR transmission and large field of view is used for brain tissue and in vivo imaging. The Pump/Stokes beam size is matched to fill the back-aperture of the objective. The forward going Pump and Stokes beams after passing through the sample are collected in transmission with a high N.A. condenser lens (oil immersion, 1.4 N.A., Olympus), which is aligned following Kohler illumination. A telescope is then used to image the scanning mirrors onto a large area (10 mm by 10 mm) Si photodiode (FDS1010, Thorlabs) to descan beam motion during laser scanning. The photodiode is reverse-biased by 64 V from a DC power supply to increase both the saturation threshold and response bandwidth. A high O.D. bandpass filter (890/220 CARS, Chroma Technology) is used to block the Stokes beam completely and transmit the Pump beam only. The output current of the photodiode is electronically pre-filtered by an 8-MHz band-pass filter (KR 2724, KR electronics) to suppress both the 80 MHz laser pulsing and the low-frequency contribution due to laser scanning across the scattering sample. It is then fed into a radio frequency lock-in amplifier (HF2LI, Zurich instrument) terminated with 50 to demodulate the stimulated Raman loss signal experienced by the Pump beam. The R-output of the lock-in amplifier is fed back into the analog interface box (FV10-ANALOG) of the microscope.

[0200] For HeLa cell imaging and brain tissue imaging, the time constant of the lock-in amplifier is set for 8 s, and the images are acquired by a 12.5 s pixel dwell time, corresponding to 3.3 s for a 512-by-512 pixel frame. For neurons and in vivo imaging of embryonic zebrafish and mice livers and intestines, the time constant is set to be 20 s, and the images are acquired by a 40 s of pixel dwell time, corresponding to 10.5 s for a 512-by-512 pixel frame. Laser powers after 60 IR objective used for cell imaging are: 100 mW for modulated Stokes beam and 112 mW for the Pump beam at 2133 cm.sup.1, 2000 cm.sup.1 and 1655 cm.sup.1 channels; 50 mW for modulated Stokes beam and 56 mW for Pump beam at 2940 cm.sup.1 and 2845 cm.sup.1 channels. Laser powers after 25 objective used for tissue and in vivo imaging are: 134 mW for modulated Stokes beam; 120 mW for the Pump beam of 2133 cm.sup.1, 2000 cm.sup.1 and 1655 cm.sup.1 channels; 67 mW for modulated Stokes beam and 60 mW for Pump beam at 2940 cm.sup.1 and 2845 cm.sup.1 channels.

[0201] Metabolic incorporation of deuterated amino acids. For HeLa cells: cells are Seeded on a coverslip in a petri-dish with 2 mL of regular medium for 20 h, and then replaced with D-AA medium (or group I and group II D-AA media) for designated amount of time. The coverslip is taken out to make an imaging chamber filled with PBS for SRS imaging. For hippocampal neurons, the dissociated neurons from newborn mice are Seeded for 10 days in regular Neurobasal A medium, and then replaced with the corresponding D-AA medium for designated amount of time before imaging. For organotypic brain slice, 400 m thick, P10 mouse brain slices are cultured on Millicell-CM inserts (PICM03050, millipore) in 1 mL CD-MEM culture medium for 2 h, and then change to in 1 mL CD-neurobasal a culture medium for another 28 h before imaging. For detailed recipe of D-AA media and in vivo labeling procedure in zebrafish and mice. (See Supporting Information). The experimental protocol for in vivo mice experiments (AC-AAAG2702) and zebrafish experiments (AC-AAAD6300) were approved by Institutional Animal Care and Use Committee at Columbia University.

[0202] Spontaneous Raman spectroscopy. The spontaneous Raman spectra were acquired using a laser Raman spectrometer (inVia Raman microscope, Ranishaw) at room temperature. A 27 mW (after objective) 532 nm diode laser was used to excite the sample through a 50, N.A. 0.75 objective (NPLAN EPI, Leica). The total data acquisition was performed during 60 seconds using the WiRE software. All the spontaneous Raman spectra have subtracted the PBS solution as background.

[0203] Image progressing. Images are acquired with FluoView scanning software and assigned color or overlaid by ImageJ. Linear combination was processed with Matlab. Graphs were assembled with Adobe Illustrator.

[0204] Culture medium. Regular HeLa cells medium was made of 90% DMEM medium (11965, Invitrogen), 10% FBS (10082, Invitrogen) and 1 penicillin/streptomycin (15140, Invitrogen); regular hippocampal neuron medium was made of Neurobasal A Medium (10888, Invitrogen), 1B27 serum free supplement (17504, Invitrogen) and 0.5 mM glutamine (25030, Invitrogen).

[0205] Htt-mEos2 plasmid construct and transfection. mHtt94Q-mEos2 plasmid was constructed by replacing CFP gene sequence in pTreTight-Htt94Q-CFP plasmid (Addgene, 23966) with mEos2 gene sequence from pRSETa-mEos2 plasmid (Addgene, 20341). For transfection of mHtt-mEos2 plasmid in HeLa cells, 4 g mHtt94Q-mEos2 plasmid was transfected using Transfection Reagent (FuGene, Promega).

[0206] Optimized Deuterium-Labeling Media 1) D-AA medium (CD-DMEM) for HeLa cells: adapted from regular recipe of DMEM medium (11965, Invitrogen). The D-AA culture medium for HeLa cells was made with 90% CD-DMEM, 10% FBS (10082, Invitrogen) and 1 penicillin/streptomycin (15140, Invitrogen).

TABLE-US-00001 Amino acids Concentration Product company components (mM) and catalog number Glycine-d.sub.5 0.4 DLM-280, Cambridge isotope L-ArginineHCl-d.sub.7 0.398 DLM-541, Cambridge isotope L-Cysteine2HCl 0.2 C6727, SIGMA (regular)* L-Glutamine-d.sub.5 4.0 DLM-1826, Cambridge isotope L-HistidineHClH.sub.2O 0.2 H5659, SIGMA (regular)* L-Isoleucine-d.sub.10 0.802 DLM-141, Cambridge isotope L-Leucine-d.sub.10 0.802 DLM-567, Cambridge isotope L-LysineHCl-d.sub.8 0.798 616214, ALDRICH (Isotech) L-Methionine-d.sub.3 0.201 DLM-431, Cambridge isotope L-Phenylalanine-d.sub.8 0.4 DLM-372, Cambridge isotope L-Serine-d.sub.3 0.4 DLM-582, Cambridge isotope L-Threonine 0.798 T8441, SIGMA (regular)* L-Tryptophan 0.078 T8941, SIGMA (regular)* L-Tyrosine-d.sub.2 0.398 DLM-2317, Cambridge isotope L-Valine-d.sub.8 0.803 DLM-488, Cambridge isotope [0207] Other components (vitamins, Inorganic Salts and glucose) are exactly the same as in the regular DMEM medium (11965, Invitrogen).

[0208] *The reasons these 4 amino acids are remain in their regular forms are because: first, their deuterated forms have limited number of side chain deuterium and are also relatively expensive; second, their occurrence (percentage) in mammalian cell proteins are small. Thus the lack of the deuterated version for these 4 amino acids would not influence the general deuterium labeling efficiency for CD-DMEM. Same reason applies to below media.

[0209] 2) D-AA medium (CD-Neurobasal A) for hippocampal neuron culture and organotypic brain slices: adapted from regular recipe of Neurobasal A medium (10888, Invitrogen). The D-AAs culture medium for hippocampal neurons was made of CD-Neurobasal A Medium, 1B27 serum free supplement (17504, Invitrogen) and 0.5 mM glutamine-d.sub.5 (DLM-1826, Cambridge isotope). The CD-Neurobasal A culture medium for organotypic brain slices was made of CD-Neurobasal A Medium, 1B27 serum free supplement (17504, Invitrogen), 0.5% glucose (15023, Invitrogen), 2 mM glutamine-d.sub.5 (DLM-1826, Cambridge isotope) and 1 penicillin/streptomycin (15140, Invitrogen).

TABLE-US-00002 Amino acids Concentration Product company components (mM) and catalog number Glycine-d.sub.5 0.4 DLM-280, Cambridge isotope L-Alanine-d.sub.4 0.022 DLM-250, Cambridge isotope L-ArginineHCl-d.sub.7 0.398 DLM-541, Cambridge isotope L-Asparagine-d.sub.8 0.006 672947 ALDRICH (Isotech) L-Cysteine2HCl 0.26 C6727, SIGMA (regular)* L-HistidineHClH.sub.2O 0.2 H5659, SIGMA (regular)* L-Isoleucine-d.sub.10 0.802 DLM-141, Cambridge isotope L-Leucine-d.sub.10 0.802 DLM-567, Cambridge isotope L-LysineHCl-d.sub.8 0.798 616214, ALDRICH (Isotech) L-Methionine-d.sub.3 0.201 DLM-431, Cambridge isotope L-Phenylalanine-d.sub.8 0.4 DLM-372, Cambridge isotope L-Proline-d.sub.7 0.067 DLM-487, Cambridge isotope L-Serine-d.sub.3 0.4 DLM-582, Cambridge isotope L-Threonine 0.798 T8441, SIGMA (regular)* L-Tryptophan 0.078 T8941, SIGMA (regular)* L-Tyrosine-d.sub.2 0.398 DLM-2317, Cambridge isotope L-Valine-d.sub.8 0.803 DLM-488, Cambridge isotope [0210] Other components (vitamins, Inorganic Salts and glucose) are exactly the same as in the regular Neurobasal A medium (10888, Invitrogen).

[0211] 3) Group I D-AA medium for HeLa cells. The group I D-AA culture medium for HeLa cells was made with 90% group I D-AA medium, 10% FBS (10082, Invitrogen) and 1 penicillin/streptomycin (15140, Invitrogen).

TABLE-US-00003 Amino acids Concentration Product company components (mM) and catalog number Glycine 0.4 50046, SIGMA (regular) L-ArginineHCl 0.398 A6969, SIGMA (regular) L-Cysteine2HCl 0.2 C6727, SIGMA (regular) L-Glutamine 4.0 G8540, SIGMA (regular) L-HistidineHClH.sub.2O 0.2 H5659, SIGMA (regular) L-Isoleucine-d.sub.10 0.802 DLM-141, Cambridge isotope L-Leucine-d.sub.10 0.802 DLM-567, Cambridge isotope L-LysineHCl 0.798 L8662 SIGMA (regular) L-Methionine 0.201 M5308 SIGMA (regular) L-Phenylalanine 0.4 P5482 SIGMA (regular) L-Serine 0.4 S4311 SIGMA (regular) L-Threonine 0.798 T8441, SIGMA (regular) L-Tryptophan 0.078 T8941, SIGMA (regular) L-Tyrosine 0.398 T8566 SIGMA (regular) L-Valine-d.sub.8 0.803 DLM-488, Cambridge isotope [0212] Other components (vitamins, Inorganic Salts and glucose) are exactly the same as in the regular DMEM medium (11965, Invitrogen).

[0213] 4) Group II D-AA medium for HeLa cells. The group II D-AA culture medium for HeLa cells was made with 90% group II D-AA medium, 10% FBS (10082, Invitrogen) and 1 penicillin/streptomycin (15140, Invitrogen).

TABLE-US-00004 Amino acids Concentration Product company components (mM) and catalog number Glycine-d.sub.5 0.4 DLM-280, Cambridge isotope L-ArginineHCl-d.sub.7 0.398 DLM-541, Cambridge isotope L-Cysteine2HCl 0.2 C6727, SIGMA (regular) L-Glutamine-d.sub.5 4.0 DLM-1826, Cambridge isotope L-HistidineHClH.sub.2O 0.2 H5659, SIGMA (regular) L-Isoleucine 0.802 17403 SIGMA (regular) L-Leucine 0.802 L8912 SIGMA (regular) L-LysineHCl-d.sub.8 0.798 616214, ALDRICH (Isotech) L-Methionine-d.sub.3 0.201 DLM-431, Cambridge isotope L-Phenylalanine-d.sub.8 0.4 DLM-372, Cambridge isotope L-Serine-d.sub.3 0.4 DLM-582, Cambridge isotope L-Threonine 0.798 T8441, SIGMA (regular) L-Tryptophan 0.078 T8941, SIGMA (regular) L-Tyrosine-d.sub.2 0.398 DLM-2317, Cambridge isotope L-Valine 0.803 V0513 SIGMA (regular) [0214] Other components (vitamins, Inorganic Salts and glucose) are exactly the same as in the regular DMEM medium (11965, Invitrogen).

[0215] 5) D-AA medium (CD-MEM) for organotypic brain slice: adapted from regular recipe of MEM medium (11095, Invitrogen). The CD-MEM culture medium for organotypic brain slice was made with 90% CD-MEM, 10% FBS (10082, Invitrogen), 0.5% glucose (15023, Invitrogen) and 1 penicillin/streptomycin (15140, Invitrogen).

TABLE-US-00005 Amino acids Concentration Product company components (mM) and catalog number L-ArginineHCl-d.sub.7 0.597 DLM-541, Cambridge isotope L-Cysteine2HCl 0.1 C6727, SIGMA (regular)* L-Glutamine-d.sub.5 2.0 DLM-1826, Cambridge isotope L-HistidineHClH.sub.2O 0.2 H5659, SIGMA (regular)* L-Isoleucine-d.sub.10 0.397 DLM-141, Cambridge isotope L-Leucine-d.sub.10 0.397 DLM-567, Cambridge isotope L-LysineHCl-d.sub.8 0.399 616214, ALDRICH (Isotech) L-Methionine-d.sub.3 0.1 DLM-431, Cambridge isotope L-Phenylalanine-d.sub.8 0.19 DLM-372, Cambridge isotope L-Threonine 0.403 T8441, SIGMA (regular)* L-Tryptophan 0.049 T8941, SIGMA (regular)* L-Tyrosine-d.sub.2 0.199 DLM-2317, Cambridge isotope L-Valine-d.sub.8 0.393 DLM-488, Cambridge isotope [0216] Other components (vitamins, Inorganic Salts and glucose) are exactly the same as in the regular MEM medium (11095, Invitrogen).

[0217] 6) For zebrafish: Wild-type zebrafish embryos at the 1-cell stage were injected with 1 nL D-AA solution and allowed to develop normally for another 24 h. The zebrafish embryos at 24 hpf were manually dechorionated before imaging. D-AA solution was made of 150 mg uniformly deuterium-labeled amino acid mix (20 aa) (DLM-6819, Cambridge Isotope) dissolved in 1 mL PBS, with subsequent filtration using Millipore sterile syringe Filters (0.22 m, SLGV033RS).

[0218] 7) For mice: 1. Oral administration: 3-week-old mice were fed with D-AA containing drinking water for 12 days before harvesting the liver and intestine tissues. The drinking water was made of 500 mg uniformly deuterium-labeled amino acid mix (20 aa) (DLM-6819, Cambridge Isotope) dissolved in 200 ml PBS, with subsequent filtration using Millipore sterile syringe Filters (0.22 m, SLGV033RS). 2. Intraperitoneal injection: 3-week-old mice were injected with 500 l D-AAs solution at the 0.sup.th h, 12.sup.th h and 24.sup.th h. The tissues were then harvested at the 36.sup.th h after the first injection. D-AA solution was made of 500 mg uniformly deuterium-labeled amino acid mix (20 aa) (DLM-6819, Cambridge Isotope) dissolved in 2 ml PBS solutions, with subsequent filtration using Millipore sterile syringe Filters (0.22 m, SLGV033RS).

Example 1a: Sensitivity Optimization and Time-Lapse Imaging of the De Novo Proteome Synthesis Dynamics

[0219] The cell culture medium reported previously was prepared by supplying uniformly deuterium-labeled whole set of amino acids to a commercially available medium that is deficient of leucine, lysine and arginine (Wei L, Yu Y, Shen Y, Wang M C, Min W (2013) Vibrational imaging of newly synthesized proteins in live cells by stimulated Raman scattering microscopy. Proc. Natl. Acad. Sci. USA. 110:11226-11231). Due to the presence of other regular amino acids already in the commercial medium, the resulting partially deuterated medium has only about 60% deuteration efficiency. In the present paper, we custom prepared new media that replace nearly all the regular amino acids by the D-AA counterparts (details in Supporting Information). As shown in the spontaneous Raman spectra (FIG. 2a), the optimized medium (spectrum 205) displays a 50% signal increase compared with the partially deuterated medium (spectrum 210). Indeed, SRS images targeting C-D vibrational peak at 2133 cm.sup.1 confirms a 50% average intensity boost in live HeLa cells (FIG. 2b). The use of optimized D-AA medium now leads to an about 8 times higher signal than when using a single leucine-d.sub.10 (FIG. 2a, spectrum 205 vs. spectrum 210). In addition to improving labeling strategy, non-trivial instrumentation optimizations are also carried out to further improve SRS detection sensitivity and acquisition speed, including increasing the laser output and microscope system throughput for near-IR wavelengths, replacing the acousto-optic modulator (AOM) with an electro-optic modulator (EOM) for a 30% higher modulation depth, and employing a high-speed lock-in amplifier for faster image acquisition.

[0220] With much-improved sensitivity, protein synthesis can now be imaged with superb spatial and temporal resolutions. Spatially, we visualized newly synthesized proteins from fine structures (likely dendritic spines, indicated by arrow heads) of live neurons (FIG. 2c). Temporally, we could readily image newly synthesized proteins in live HeLa cells in less than one-hour incubation with the optimized deuteration medium (FIG. 2d). Control image with protein synthesis inhibitors only displays vague cell outlines which presumably come from the free D-AA pool (sub-mM concentration). Moreover, using a fast lock-in amplifier (details in Methods), our current imaging speed can be as fast as 3 s per frame (512512 pixels), nearly 10 times faster than before, which enables time-lapse imaging in live cells with minimum photo-toxicity to cell viabilities. FIG. 2(e) presents time-lapse SRS imaging of a same set of live HeLa cells gradually synthesizing new proteins over time from 10 min to 5 h incubation in optimized D-AA medium. The obvious cell migration and division prove their viability, supporting high bio-compatibility of our technique. To our best knowledge, this is the first time that long-term time-lapse imaging of proteome synthesis dynamics is demonstrated on single live mammalian cells.

Example 1b: SRS Imaging of Protein Degradation in Live HeLa Cells

[0221] Besides imaging protein synthesis, our imaging platform offers the ability to probe protein degradation simultaneously. Experimentally, we intend to probe the pre-existing protein pool by targeting the CH.sub.3 showing a strong peak at 2940 cm.sup.1, as newly synthesized proteins will be mostly carrying C-D peaked around 2133 cm.sup.1. However, the 2940 cm.sup.1 CH.sub.3 protein channel is known to suffer from undesired crosstalk from the CH.sub.2 lipid signal peaked at 2845 cm.sup.1. To obtain a clean protein component, we adopt two-color SRS imaging at both 2940 cm.sup.1 and 2845 cm.sup.1 channels followed by a linear combination algorithm which has been effectively applied in cells, tissues and animals. The subsequently obtained images show the pure distribution of old protein pools (exclusively from CH.sub.3) and the distribution of lipids (exclusively from CH.sub.2), respectively. Hence protein degradation could be tracked by imaging the old protein distributions over time when cells are growing in the D-AA medium.

[0222] FIG. 3a shows time-dependent SRS images of old protein distributions (CH.sub.3) in live HeLa cells when incubated with D-AAs from 0 h to 96 h. Clearly, the old protein pool is degrading, as shown by the decay of its average intensity. As a contrast, the total lipid images display no obvious intensity change (FIG. 3b). In addition, the spatial patterns of old proteins (FIG. 3a) reveal a faster decay in the nucleoli than the cytoplasm. This observation is consistent with the fact that neucleoli have active protein turnover and also with our previous report that C-D labeled newly synthesized proteins are more prominent in nucleoli (Wei L, Yu Y, Shen Y, Wang M C, Min W (2013). Vibrational imaging of newly synthesized proteins in live cells by stimulated Raman scattering microscopy. Proc. Natl. Acad. Sci. USA. 110:11226-11231). Single exponential decay fitting of the average intensities in FIG. 3a yields a decay time constant of 454 h (FIG. 3c), corresponding to a proteome half-live of 313 h which is very close to the data reported by mass spectrometry (35 h) (Cambridge S B et al. (2011) Systems-wide proteomic analysis in mammalian cells reveals conserved, functional protein turnover. J Proteome Res. 10:5275-5284). Therefore, our imaging platform is capable of observing both protein synthesis and degradation by imaging at C-D channel and CH.sub.3 channel, respectively, thus capturing proteomic metabolism dynamics in full-scope.

[0223] Retrieval of pure CH.sub.3 and CH.sub.2 signals by linear combination between 2940 cm.sup.1 and 2845 cm.sup.1 channels was conducted employing equations follow Lu F-K et al. (2012) Multicolor stimulated Raman scattering microscopy. Mol. Phys. 110:1927-1932; and Yu Z et al. (2012) Label-free chemical imaging in vivo: three-dimensional non-invasive microscopic observation of amphioxus notochord through stimulated Raman scattering (SRS). Chem. Sci. 3:2646-2654. Pure CH.sub.3 signal can be retrieved as [c].sub.protein 5.2*(2940 cm.sup.1 signal)-4.16*(2845 cm.sup.1 signal); Pure CH.sub.2 signal can be retrieved as [c].sub.lipid 1.2*(2845 cm.sup.1 signal)0.3*(2940 cm.sup.1 signal). This algorithm was tested with skin tissue samples, yielding similar results as reported in Lu F-K et al. (2012) Multicolor stimulated Raman scattering microscopy. Mol. Phys. 110:1927-1932; and Yu Z et al. (2012) Label-free chemical imaging in vivo: three-dimensional non-invasive microscopic observation of amphioxus notochord through stimulated Raman scattering (SRS). Chem. Sci. 3:2646-2654.

Example 1c: Two-Color Pulse-Chase SRS Imaging of Two Sets of Temporally Defined Proteins

[0224] Inspired by the popular pulse-chase analysis in classic autoradiography techniques and recent two-color BONCAT imaging (Beatty K E, Tirrell D A (2008) Two-color labeling of temporally defined protein populations in mammalian cells. Bioorg. Med. Chem. Lett. 18:5995-5999), we aim to exploit another dimension of probing dynamic protein metabolism with two-color pulse-chase imaging of proteins labeled at different times. To do so, we need to rationally divide total D-AAs into two sub-sets with distinct Raman spectra. We reasoned that Raman peaks of C-D stretching are closely related to their chemical environments, thus the structural difference between D-AAs should lead to diverse Raman peak positions and shapes. We then examined the spontaneous Raman spectra of each D-AA sequentially, and subsequently identified two subgroups. Group I contains three amino acids, leuine-d10, isoleucine-d10 and valine-d8, structurally known as branched-chain amino acids (FIG. 4a). All members of group I exhibits multiple distinct Raman peaks with the first one around 2067 cm.sup.1.

[0225] The rest of D-AAs without branched chains are then categorized into group II, all of which show a prominent Raman peak around 2133 cm.sup.1 (three examples shown in FIG. 4b). To test inside cells, Raman spectra of HeLa cells cultured in either group I D-AA medium only (element 405) or group II D-AA medium only (element 410) are shown in FIG. 4c. Based on the spectra, we choose to acquire two-color narrow-band SRS images at 2067 cm.sup.1 and at 2133 cm.sup.1. By constructing and utilizing a linear combination algorithm, similar to the one used for CH.sub.3 and CH.sub.2 above, pure signals of proteins labeled by group I D-AAs and by group II D-AAs can be successfully separated and quantitatively visualized (e.g. FIG. 7). In other exemplary embodiments, the images are obtained with a hyper-spectral imaging approach using broadband femtosecond lasers.

[0226] We now chose the mutant huntingtin (mHtt) protein in Huntington's disease as our model system for pulse-chase imaging demonstration. It is believed that Huntington's disease is caused by a mutation from normal huntingtin gene to mHtt gene expressing aggregation-prone mHtt proteins with poly-glutamine (polyQ) expansion (Walker FO (2007) Huntington's Disease. The Lancet. 369: 218-228). For easy visualization by fluorescence, we tagged mHtt (with 94Q) with a fluorescent protein marker, mEos2. As illustrated by the cartoon in FIG. 9d, HeLa cells were first transfected with mHtt94Q-mEos2 plasmid in regular medium for 4 h, and then replaced with group II D-AA medium for 22 h before changing to group I D-AA medium for another 20 h. SRS images are acquired at 2067 cm.sup.1 and 2133 cm.sup.1 channels, respectively, and subsequently processed with linear combination.

[0227] Fluorescence overlaid with bright field image informs us the formation of a large aggregate triggered by aggregation-prone polyQ expansion in mHtt94Q-mEos2 (FIG. 4d, fluorescence). Interestingly, proteins labeled with group II D-AAs during the initial pulse period mainly concentrate within the core of the aggregate (FIG. 4d, element 415), whereas proteins labeled with group I D-AAs during the subsequent chase period occupy the entire volume of the aggregate (FIG. 4d, element 420). The merged image between group I and group II images, as well as the intensity profiles across the aggregate, further confirm the observation of a yellow core inside and a green shell outside (FIG. 4d, merged). This two-color pulse-chase result suggests that the core is aggregated earlier in time and the later produced mHtt proteins are then recruited to and percolate through the aggregate to increases its overall size, in agreement with recently reported results by fluorescence (Schipper-Krom S et al. (2014)) Dynamic recruitment of active proteasomes into polyglutamine initiated inclusion bodies. FEBS Lett. 588:151-159). The demonstration here thus illustrates that our imaging platform using the two subgroups of D-AAs is readily applicable for performing pulse-chase imaging to probe the complex and dynamic aspects of proteome metabolism.

[0228] In order to achieve SRS imaging of pure group I D-AA labeled protein distribution and pure group II D-AA labeled protein distribution simultaneously, we construct a robust linear combination algorithm to retrieve the underlying pure concentration information for two-color pulse-chasing imaging similar to the one presented above from Lu F-K et al. (2012) Multicolor stimulated Raman scattering microscopy. Mol. Phys. 110:1927-1932; and Yu Z et al. (2012) Label-free chemical imaging in vivo: three-dimensional non-invasive microscopic observation of amphioxus notochord through stimulated Raman scattering (SRS). Chem. Sci. 3:2646-2654. Since SRS signals exhibit linear concentration dependence with analyte concentrations, two chemical species with different Raman spectra can be retrieved quantitatively with two-color SRS imaging. Hence, based on the spectra shown in FIG. 4c, we choose to acquire narrow-band SRS images at 2067 cm.sup.1 and 2133 cm.sup.1 channels, respectively, and perform subsequent linear combination algorithm to remove the spectral cross-talk.

[0229] The proper algorithm with the corresponding cross-talk coefficients is constructed with SRS images of standard reference samples, i.e., pure group I D-AA labeled protein and pure group II D-AA labeled protein. To do so, we labeled HeLa cells with only group I D-AA medium FIG. 7a and only group II D-AA medium FIG. 7b, respectively, and acquired a set of image pairs at 2067 cm.sup.1 and 2133 cm.sup.1 channels for each cell samples (e.g. FIG. 7).

[0230] For any sample labeled with both groups of D-AAs, the measured SRS signals at 2067 cm.sup.1 and 2133 cm.sup.1 channels can be written as the following, with linear relationship to group I D-AA and group II D-AA concentrations ([c]group I and [c]group II):

[00002]$\left[\begin{array}{c}2067.Math..Math.{\mathrm{cm}}^{-1}.Math..Math.\mathrm{signal}\\ 2133.Math..Math.{\mathrm{cm}}^{-1}.Math..Math.\mathrm{signal}\end{array}\right]=\left[\begin{array}{cc}{i}_{\mathrm{group}.Math..Math.I,2067.Math..Math.{\mathrm{cm}}^{-1}}& i_{\mathrm{group}.Math..Math.\mathrm{II},2067.Math.{\mathrm{cm}}^{-1}}\\ i_{\mathrm{group}.Math..Math.I,2133.Math..Math.{\mathrm{cm}}^{-1}}& i_{\mathrm{group}.Math..Math.\mathrm{II},2133.Math..Math.{\mathrm{cm}}^{-1}}\end{array}\right]\left[\begin{array}{c}{\left[c\right]}_{\mathrm{group}.Math..Math.I}\\ {\left[c\right]}_{\mathrm{group}.Math..Math.\mathrm{II}}\end{array}\right],$

where i.sub.groupI,2067 cm.sub.1, i.sub.groupI,2133 cm.sub.1, i.sub.groupII,2067 cm.sub.1, i.sub.groupII,2133 cm.sub.1 are the average pixel intensity recorded inside cells in FIG. 7a and FIG. 7b.

[0231] Thus group I D-AA and group II D-AA concentrations can then be easily solved as:

[00003]${\left[c\right]}_{\mathrm{group}.Math..Math.I}=\frac{\begin{array}{c}{i}_{\mathrm{group}.Math..Math.\mathrm{II},2133.Math..Math.{\mathrm{cm}}^{-1}}\left(2067.Math..Math.{\mathrm{cm}}^{-1}.Math.\mathrm{signal}\right)-\\ i_{\mathrm{group}.Math..Math.\mathrm{II},2067.Math.{\mathrm{cm}}^{-1}}\left(2133.Math..Math.{\mathrm{cm}}^{-1}.Math..Math.\mathrm{signal}\right)\end{array}}{i_{\mathrm{group}.Math..Math.\mathrm{II},2133.Math..Math.{\mathrm{cm}}^{-1}}.Math.i_{\mathrm{group}.Math..Math.I,2067.Math..Math.{\mathrm{cm}}^{-1}}-i_{\mathrm{group}.Math..Math.\mathrm{II},2067.Math.{\mathrm{cm}}^{-1}}.Math.i_{\mathrm{group}.Math..Math.I,2133.Math..Math.{\mathrm{cm}}^{-1}}},.Math.{\left[c\right]}_{\mathrm{group}.Math..Math.\mathrm{II}}=\frac{\begin{array}{c}{i}_{\mathrm{group}.Math..Math.I,2067.Math..Math.{\mathrm{cm}}^{-1}}\left(2133.Math..Math.{\mathrm{cm}}^{-1}.Math..Math.\mathrm{signal}\right)-\\ i_{\mathrm{group}.Math..Math.I,2133.Math..Math.{\mathrm{cm}}^{-1}}\left(2067.Math..Math.{\mathrm{cm}}^{-1}.Math.\mathrm{signal}\right)\end{array}}{i_{\mathrm{group}.Math..Math.I.Math..Math.I,2133.Math..Math.{\mathrm{cm}}^{-1}}.Math.i_{\mathrm{group}.Math..Math.I,2067.Math..Math.{\mathrm{cm}}^{-1}}-i_{\mathrm{group}.Math..Math.\mathrm{II},2067.Math.{\mathrm{cm}}^{-1}}.Math.i_{\mathrm{group}.Math..Math.I,2133.Math..Math.{\mathrm{cm}}^{-1}}}.$

[0232] Taking the average pixel intensity recording in FIG. 7a and FIG. 7b into the above equations, the final linear combination algorithm reads as:

[c]group I1.06*(2067 cm.sup.1 signal)0.0047*(2133 cm.sup.1 signal),(1)

[c]group II(2133 cm.sup.1 signal)1.15*(2067 cm.sup.1 signal).(2)

Example 1d: SRS Imaging of Newly Synthesized Proteins in Live Mouse Brain Tissues

[0233] Going above the cellular level, we now apply our imaging platform to a more complex level, organotypical brain tissues. In our study, we focus on the hippocampus because it is the key region in brains that involves extensive protein synthesis. As expected, active protein synthesis is found in the hippocampal region, particularly in the dentate gyrus, which is known for its significant role in both long-term memory formation and adult neurogenesis. SRS image at 2133 cm.sup.1 (FIGS. 5a, and 5c and 5d) of a live mouse organotypic brain slices cultured in D-AA medium for 30 h, reveals active protein synthesis from both the soma and the neurites of individual neurons in dentate gyrus. In addition, the old protein (CH.sub.3) and total lipids (CH.sub.2) images are presented simultaneously for multichannel analysis (see FIG. 5a).

[0234] In order to investigate spatial pattern of protein synthesis on a larger scale, we imaged the entire brain slice by acquiring large-area image mosaics. A 4-by-3 mm image (see FIG. 5b) of another organotypic slice displays overlayed patterns from new proteins (2133 cm.sup.1, element 505), old proteins (CH.sub.3, element 510) and lipids (CH.sub.2, element 515). Intriguing spatial variation is observed: while the distribution of old proteins are relatively homogenous across the field of view, newly synthesized proteins are either concentrated in dentate gyrus or scattered within individual neurons throughout the cortex, suggesting high activities in these two regions. Thus, we have demonstrated the ability to directly image protein synthesis dynamics on living brain tissues with subcellular resolution and multi-channel analysis, which was difficult to achieve with other existing methods. The intricate relationship between protein synthesis and neuronal plasticity is currently under investigation on this platform.

Example 1e: SRS Imaging of Newly Synthesized Proteins In Vivo

[0235] One prominent advantage of our labeling strategy is its non-toxicity and minimal invasiveness to animals. We thus move up to the physiological level to image protein metabolism in embryonic zebrafish and mice. Zebrafish are popular model organisms due to their well-understood genetics and transparent embryos, amenable to optical imaging. We injected 1 nL D-AA solution into zebrafish embryos at the 1-cell stage (150 ng D-AAs per embryo), and then allowed them to develop normally for 24 h (FIG. 6a, bright field) before imaging the whole animal. We found a high signal of newly synthesized proteins (FIG. 6a, 2133 cm.sup.1) in the somites at the embryonic zebrafish tail, consistent with the earlier BONCAT result (Hinz F I, Dieterich D C, Tirrell D A, Schuman E M (2012) Non-canonical amino acid labeling in vivo to visualize and affinity purify newly synthesized proteins in larval zebrafish. ACS Chem. Neurosci. 3:40-49). The spatial pattern of this signal appears similar to that of the old protein distribution (FIG. 6a, CH.sub.3), but almost complementary to the lipid distribution (FIG. 6a, CH.sub.2).

[0236] Finally we demonstrate on mammalsmice. We administered the drinking water containing D-AAs to 3-week-old mice for 12 days, and then harvested liver and intestine tissues for subsequent imaging. No toxicity was observed for the fed mice. The SRS images from both live liver tissues (FIG. 6b) and live intestine tissues (FIG. 6c) illustrate the distributions of newly synthesized proteins (2133 cm.sup.1, C-D) during the feeding period, which resemble the total protein distribution (1655 cm.sup.1, Amide I). On a faster incorporation timescale, live liver and intestine tissues obtained after intraperitoneal injection of D-AAs into mice for 36 h reveal spatial patterns (FIGS. 6b-6c) similar to the feeding results above as well as the click-chemistry based fluorescence staining (Liu J, Xu Y, Stoleru D, Salic A (2012) Imaging protein synthesis in cells and tissues with an alkyne analog of puromycin. Proc. Natl. Acad. Sci. USA 109:413-418). All these results support our imaging platform as a highly suitable technique for in vivo interrogation.

[0237] FIG. 8 shows raw C-D on-resonance (2133 cm.sup.1) and off-resonance (2000 cm.sup.1) SRS images of newly synthesized proteins in vivo in FIG. 6. FIG. 8a SRS C-D on-resonance and off-resonance images of a 24 hpf embryonic zebrafish. The difference image between C-D on-resonance and off-resonance (pixel-by-pixel subtraction) shows pure C-D labeled protein distribution in the somites of an embryonic zebrafish tail, as in FIG. 6a. FIGS. 8b-8c SRS C-D on-resonance and off-resonance images of live mouse liver FIG. 8b and intestine FIG. 8c tissues harvested from the mice after administering with D-AA containing drinking water for 12 days. The difference image between C-D on-resonance and off-resonance (pixel-by-pixel subtraction) shows pure C-D labeled protein distribution in the liver and intestine tissues, shown in FIG. 6b and FIG. 6c, respectively. The residual signal presented in the off-resonance images mainly comes from cross-phase modulation induced by highly scattering tissue structures.

[0238] FIG. 9 shows SRS imaging for newly synthesized proteins in vivo with intraperitoneal injection of mice with D-AA solutions. FIGS. 9a-9b SRS images of live mouse liver FIG. 9a and intestine 9b tissues harvested from mice after intraperitoneal injection injected with D-AAs solutions for 36 h. 2133 cm.sup.1 channel shows newly synthesized proteins (off-resonance image subtracted) that resemble the distribution of total proteins as shown in the 1655 cm.sup.1 image (Amide I). (c-d) Corresponding raw C-D on-resonance (2133 cm.sup.1) and off-resonance (2000 cm.sup.1) images are shown as references for liver c and intestine d tissues. Scale bar, 10 m.

Exemplary Physical Principle of Isotope-Based SRS Imaging

[0239] SRS microscopy can be a molecular-contrast, highly sensitive, imaging procedure with intrinsic 3D sectioning capability. It selectively images the distribution of molecules that carry a given type of chemical bonds through resonating with the specific vibrational frequency of the targeted bonds. (See, e.g., References 47, 54 and 65). As FIG. 5a illustrates, by focusing both temporally and spatially overlapped Pump and Stokes laser pulse trains into samples, the rate of vibrational transition can be greatly amplified by about 107 times when the energy difference of the two laser beams matches the particular chemical bond vibration, vib. (See, e.g., Reference 65). Accompanying such stimulated activation of one vibrational mode, one photon can be created into the Stokes beam, and simultaneously another photon can be annihilated from the Pump beam, a process called stimulated Raman gain and stimulated Raman loss, respectively. The energy difference between the Pump photon and the Stokes photon can be used to excite the vibrational mode, fulfilling energy conservation. FIG. 5b shows a high-frequency modulation procedure, where the intensity of the Stokes beam can be turned on and off at 10 MHz, and can be employed to achieve shot-noise-limited detection sensitivity by suppressing laser intensity fluctuations occurring at low frequencies. The transmitted Pump beam after the sample can be detected by a large-area photodiode, and the corresponding stimulated Raman loss signal, which also occurs at 10 MHz, can be demodulated by a lock-in amplifier. By scanning across the sample with a laser-scanning microscope, a quantitative map with chemical contrast can be produced from the targeted vibrating chemical bonds. As the SRS signal can be dependent on both Pump and Stokes laser beams, the nonlinear nature can provide a 3D optical sectioning ability.

[0240] The vibrational signal of C-D can be detected as an indicator for newly synthesized proteins that metabolically incorporate deuterium-labeled amino acids. (See, e.g., FIG. 5b). When hydrogen atoms can be replaced by deuterium, the chemical and biological activities of biomolecules remain largely unmodified. The C-D stretching motion can display a distinct vibrational frequency from all the other vibrations of biological molecules inside live cells. The reduced mass of the C-D oscillator can be increased by two folds when hydrogen can be replaced by deuterium. Based on the above Equation, vib can be reduced by a factor of 2. The experimentally measured stretching frequency can be shifted from 2950 cm.sup.1 of CH to 2100 cm.sup.1 of C-D. The vibrational frequency of 2100 cm.sup.1 can be located in a cell-silent spectral window in which no other Raman peaks exist, thus enabling detection of exogenous C-D with both high specificity and sensitivity.

[0241] Imaging optimization by metabolic incorporation of deuterium-labeled all amino acids in live HeLa cells with multicolor SRS imaging. Although leucine can be the most abundant essential amino acid, it only accounts for a small fraction of amino acids in proteins. Thus, the deuterium labeling of all the amino acids can lead to a substantial signal enhancement. Indeed, the spontaneous Raman spectrum (e.g., FIG. 33a) of HeLa cells incubated with deuterium-labeled all 20 amino acids (e.g., prepared by supplying uniformly deuterium-labeled whole set of amino acids to leucine, lysine and arginine deficient DMEM medium) can exhibit C-D vibrational peaks about five times higher than shown in FIG. 2 under the same condition. The corresponding SRS image at 2133 cm.sup.1 (e.g., FIG. 33b) can show a significantly more pronounced signal than that in FIG. 2 under the same intensity scale. In particular, nucleoli (e.g., indicated by arrows in FIG. 33b and verified by DIC visualization) can exhibit the highest signal. Nucleoli, the active sites for ribosomal biogenesis, have been reported to involve rapid nucleolar assembly and proteomic exchange (See, e.g., Reference 68-70). Such fast protein turnover can be reflected by the spatial enrichment of newly synthesized protein signals in those subcellular areas. (See, e.g., FIG. 33b). Note that SRS imaging here can be directly performed on live cells and hence free from potential complications due to fixation and dye conjugation. Again, the off-resonant image at 2000 cm.sup.1 can be clean and dark (e.g., FIG. 33c), proving the specificity of SRS imaging of C-D at 2133 cm.sup.1. In addition to imaging newly synthesized proteins, SRS can readily image intrinsic biomolecules in a label-free manner. By simply adjusting the energy difference between the Pump and the Stokes beams to match the vibrational frequency of amide I, lipids and total proteins respectively. FIGS. 33d-f show the SRS images of amide I band at 1655 cm.sup.1 primarily attributed to proteins, CH.sub.2 stretching at 2845 cm.sup.1 predominantly for lipids and CH.sub.3 stretching at 2940 cm.sup.1 mainly from proteins with minor contribution from lipids.

Exemplary Time-Dependent De Novo Protein Synthesis and Protein Synthesis Inhibition

[0242] Being linearly dependent on analyte concentration, SRS contrast can be well suited for quantification of de novo protein synthesis in live cells. Here the time-dependent protein synthesis images can be shown under the same intensity scale. (See, e.g., FIGS. 348a-c). As expected, the new protein signal (e.g., 2133 cm.sup.1) from 5-hour, 12-hour and 20-hour incubation can increase substantially over time (e.g., FIGS. 34a-c) while the amide I (e.g., 1655 cm.sup.1) signal can remain at a steady state. (See, e.g., FIGS. 34d-f). Since protein distribution can often be heterogeneous in biological systems, a more quantitative representation by acquiring ratio images can be shown between the newly synthesized proteins and the total proteome (e.g., from either amide I or CH.sub.3). FIGS. 34g-i depict the fraction of newly synthesized proteins (e.g., 2133 cm.sup.1) among the total proteome (e.g., 1655 cm.sup.1) and its spatial distribution. The fraction of newly synthesized proteins growing with time from 5 hours to 20 hours can highlight nucleoli as the subcellular compartments with fast protein turnover. (See, e.g., Reference 68-70). Such quantitative ratio imaging of new versus old proteomes can be very difficult to obtain using BONCAT or mass spectroscopy without the destruction of cells. Moreover, FIG. 34j shows time-lapse SRS images of a live dividing HeLa cell after 20-hour incubation in deuterium-labeled all amino acids medium, clearly proving the viability of cells under the imaging condition.

[0243] The effect of protein synthesis inhibition by chemical drugs can be further tested to validate that the detected C-D signal indeed derives from nascent proteins. HeLa cells incubated with deuterium-labeled all amino acids together with 5 M anisomycin, which can work as a protein synthesis inhibitor by inhibiting peptidyl transferase or the 80S ribosome system, show the absence of the C-D signal in the spontaneous Raman spectrum. (See, e.g., FIG. 34k). Furthermore, SRS imaging of the same samples (e.g., FIG. 34i) can exhibit drastically weaker signal (See, e.g., Reference 71) when compared to FIG. 34b without the protein synthesis inhibitor. As a control, the corresponding 2940 cm.sup.1 image (e.g., FIG. 34m) of total proteome remains at a similar level as the non-drug treated counterpart in FIG. 34f. Thus, the detected C-D SRS signal (e.g., FIGS. 34a-c) can originate from deuterium-labeled nascent proteins, which can vanish upon adding the protein synthesis inhibitor.

Exemplary Demonstration On HEK293T Cells And Neuron-Like Differentiable Neuroblastoma N2A Cells

[0244] Two additional mammalian cell lines can be chosen for further demonstration: human embryonic kidney HEK293T cells, and neuron-like neuroblastoma mouse N2A cells, which can be induced to differentiate with the growth of neurites (e.g., axons and dendrites). The spontaneous Raman spectrum (e.g., FIG. 35a) of HEK293T cells incubated with deuterium-labeled all amino acids for 12 hours can exhibit a 2133 cm.sup.1 C-D channel signal nearly as high as the 1655 cm.sup.1 amide channel signal. The resulting SRS image can show a bright signal for new proteins with an intense pattern residing in nucleoli. (See, e.g., FIG. 35b). As before, the off-resonant image (e.g., 2000 cm.sup.1) can display vanishing background (e.g., FIG. 35c); the amide I channel (e.g., 1655 cm.sup.1) image (e.g., FIG. 35d) can exhibit consistent overall proteome distributions similar to that in HeLa cells; CH.sub.2 channel (e.g., 2845 cm.sup.1) image (e.g., FIG. 35e) depicts a more diffusive lipid distribution in cytoplasm compared to that in HeLa cells. Consistent with the results obtained in HeLa cells above, the ratio image (e.g., FIG. 35f) between the newly synthesized proteins (e.g., FIG. 35b) and the total proteins (e.g., FIG. 35d) highlights nucleoli for active protein turnover in HEK293T cells as well. (See, e.g., References 44-46).

[0245] In addition to showing the ability to image newly synthesized proteins inside cell body, the exemplary SRS can also be applied to tackle more complex problems, such as de novo protein synthesis in neuronal systems. (See, e.g., Reference 26-28). Under differentiation condition, N2A cells massively grow new neurites from cell bodies and form connections with other cells. FIG. 10a shows the image of newly synthesized proteins after induction for differentiation, by simultaneously differentiating the N2A cells and supplying with the deuterium labeled all amino acids for 24 hours. Similar to HeLa and HEK293T cells, N2A cell bodies can be observed to display high-level protein synthesis. Newly synthesized proteins can also be observed in a subset of, but not all neurites (e.g., FIGS. 36a and 36b), which can imply that the observed neurites in FIG. 360a can be newly grown under the differentiation condition. For a detailed visualization, FIGS. 36c and 10d show the zoomed-in regions in the dashed squares in FIGS. 36a and 36b respectively. A more comprehensive examination is illustrated by both the ratio image (e.g., FIG. 36e) between FIGS. 36c and 36d and the merged image (e.g., FIG. 36f) with 3605 designating new protein signal from FIG. 36c and 3610 designating total protein signal from FIG. 36d. On one hand, both the ratio image and the merged image highlight the neurites with higher percentage of new proteins (e.g., indicated by stars), implying these neurites can be newly grown. On the other hand, from the merged image, there can be some neurites (e.g., indicated by arrows) showing obvious signals in the green channel (e.g., total proteins) only but with no detectable signal in the red channel (e.g., new proteins).

[0246] Hence, the neurites indicated by arrows can be most likely older than their starred counterparts. In addition, the transition from 3610 to 3605 in the merged image (e.g., FIG. 360f) can imply the growth direction by which new neurites form and grow. A more relevant system to study de novo protein synthesis and neuronal activities can be hippocampal neurons, which can be known to be involved in long-term memory formation (See, e.g., Reference 26-28). SRS image (e.g., 2133 cm.sup.1) of hippocampal neuron cells incubated with deuterium-labeled all amino acids can show a newly synthesized protein pattern in the neurites.

Example 2: In Vitro and In Vivo Labeling with Alkyne Tags

Exemplary Methods and Materials

[0247] Bond-selective stimulated Raman scattering (SRS) microscopy. FIG. 37b shows details of the microscopy setup. An integrated laser system (picoEMERALD, Applied Physics & Electronics, Inc.) was chosen as the light source for both pump and Stokes beams. Briefly, picoEMERALD provides an output pulse train at 1064 nm with 6 ps pulse width and 80 MHz repetition rate, which serves as the Stokes beam. The frequency doubled beam at 532 nm is used to synchronously Seed a picosecond optical parametric oscillator (OPO) to produce a mode-locked pulse train with 56 ps pulse width (the idler beam of the OPO is blocked with an interferometric filter). The output wavelength of the OPO is tunable from 720 to 990 nm, which serves as the pump beam. The intensity of the 1064 nm Stokes beam is modulated sinusoidally by a built-in electro-optic modulator at 8 MHz with a modulation depth of more than 95%. The pump beam is then spatially overlapped with the Stokes beam by using a dichroic mirror inside picoEMERALD. The temporal overlap between pump and Stokes pulse trains is ensured with a built-in delay stage and optimized by the SRS signal of pure dodecane liquid at the microscope.

[0248] Pump and Stokes beams are coupled into an inverted multiphoton laser-scanning microscope (FV1200MPE, Olympus) optimized for near-IR throughput. A 60 water objective (UPlanAPO/IR, 1.2 N.A., Olympus) with high near-IR transmission is used for all cell imaging. The pump/Stokes beam size is matched to fill the back-aperture of the objective. The forward going pump and Stokes beams after passing through the sample are collected in transmission with a high N.A. condenser lens (oil immersion, 1.4 N.A., Olympus) which is aligned following Kohler illumination. A telescope is then used to image the scanning mirrors onto a large area (10 by 10 mm) Si photodiode (FDS1010, Thorlabs) to descan beam motion during laser scanning. The photodiode is reverse biased by 64 V from a DC power supply to increase both the saturation threshold and response bandwidth. A high O.D. bandpass filter (890/220 CARS, Chroma Technology) is placed in front of the photodiode to block the Stokes beam completely and to transmit the pump beam only.

[0249] The output current of the photodiode is electronically pre-filtered by an 8-MHz band-pass filter (KR 2724, KR electronics) to suppress both the 80 MHz laser pulsing and the low-frequency fluctuations due to laser scanning cross the scattering sample. It is then fed into a radio frequency lock-in amplifier (SR844, Stanford Research Systems) terminated with 50 to demodulate the stimulated Raman loss signal experienced by the pump beam. The in-phase X-output of the lock-in amplifier is fed back into the analog interface box (FV10-ANALOG) of the microscope. The time constant is set for 10 s (the shortest available with no additional filter applied). The current SRS imaging speed is limited by the shortest time constant available from the lock-in amplifier (SR844). For all imaging, 512 by 512 pixels are acquired for one frame with a 100 s of pixel dwell time (26 s per frame) for laser scanning and 10 s of time constant from the lock-in amplifier. Laser powers after 60 IR objective used for imaging are: 130 mW for modulated Stokes beam; 120 mW for the pump beam in 2133 cm.sup.1, 2142 cm.sup.1, 2000 cm.sup.1 and 1655 cm.sup.1 channels, 85 mW for the pump beam in 2230 cm.sup.1 and 2300 cm.sup.1 channels, and 50 mW for pump beam in 2845 cm.sup.1 channels.

[0250] Spontaneous Raman Spectroscopy. The spontaneous Raman spectra were acquired using a laser confocal Raman microscope (Xplora, Horiba Jobin Yvon) at room temperature. A 12 mW (after the microscope objective), 532 nm diode laser was used to excite the sample through a 50, N.A.=0.75 air objective (MPlan N, Olympus). The total data acquisition time was 300 s using the LabSpec 6 software. All the spontaneous Raman spectra have subtracted the PBS solution background.

[0251] Materials. 5-Ethynyl-2-deoxyuridine (EdU) (T511285), 17-Octadecynoic acid (17-ODYA) (08382), DMEM medium without L-methionine, L-cysteine and L-glutamine (D0422), L-methionine (M5308), L-cysteine (C7602), 2-Mercaptoethanol (M3148) and Phorbol 12-myristate 13-acetate (P1585) were purchased from Sigma-Aldrich. 5-Ethynyl Uridine (EU) (E-10345), Homopropargylglycine (Hpg) (C10186), Alexa Fluor 488 Azide (A10266), Click-iT Cell Reaction Buffer Kit (C10269), DMEM medium (11965), FBS (10082), penicillin/streptomycin (15140), L-glutamine (25030), Neurobasal A Medium (10888) and B27 supplement (17504) were purchased from Invitrogen. RPMI-1640 Medium (30-2001) was purchased from ATCC. BCS (hyclone SH30072) was purchased from Fisher Scientific.

[0252] DMEM culture medium was made by adding 10% (vol/vol) FBS and 1% (vol/vol) penicillin/streptomycin to the DMEM medium. Methionine-deficient culture medium was made by supplying 4 mM L-glutamine, 0.2 mM L-cysteine, 10% FBS and 1% penicillin/streptomycin to the DMEM medium without L-methionine, L-cysteine and L-glutamine. RPMI-1640 culture medium was made of supplying the RPMI-1640 medium with 10% FBS, 1% penicillin/streptomycin and 50 M 2-Mercaptoethanol. Neuron culture medium was made of Neurobasal A Medium adding with 1B27 supplement and 0.5 mM glutamine. Culture medium for NIH3T3 cells was made by adding 10% (vol/vol) BCS and 1% (vol/vol) penicillin/streptomycin to the DMEM medium.

[0253] Propargylcholine synthesis. Propargylcholine was synthesized according to Jao, C. Y., Roth, M., Welti, R. & Salic, A. Proc. Natl. Acad. Sci. USA 106, 15332-15337 (2009). 3 mL propargyl bromide (80 wt. % solution in toluene) were added dropwise to 3 g 2-dimethylaminoethanol in 10 mL anhydrous THF on ice under argon gas protection and stirring. The ice bath was removed and the mixture was kept stirring at room temperature overnight. The white solids were filtered the next day and washed extensively with cold anhydrous THF to obtain 5 g pure propargylcholine bromide. All chemicals here are purchased from Sigma-Aldrich. NMR spectrum was recorded on a Bruker 400 (400 MHz) Fourier Transform (FT) NMR spectrometers at the Columbia University Chemistry Department. .sup.1H NMR spectra are tabulated in the following order: multiplicity (s, singlet; d, doublet; t, triplet; m, multiplet), number of protons. .sup.1H NMR (400 MHz, D.sub.2O) ppm: 4.37 (d, J=2.4 Hz, 2H); 4.10 (m, 2H); 3.66 (t, J=4.8 Hz, 2H); 3.28 (s, 6H); MS (APCI+) m/z Calcd. for C.sub.7H.sub.14NO [M].sup.+:128.19. Found: 128.26.

[0254] Sample preparation for SRS imaging of live cells and organisms. For all SRS imaging experiments of HeLa cells (e.g. FIG. 12), cells were first Seeded on coverslips with a density of 110.sup.5/mL in petri dishes with 2 mL DMEM culture medium for 20 h at 37 C. and 5% CO.sub.2. [0255] 1) EdU experiment, DMEM culture medium was then changed to DMEM medium (FBS-free) for 24 h for cell cycle synchronization. After synchronization, medium was replaced back to DMEM culture medium and EdU (10 mM stock in PBS) was simultaneously added to a concentration of 100 M for 15 h. [0256] 2) EU experiment, EU (100 mM stock in PBS) was added to the DMEM culture medium directly to a concentration of 2 mM for 7 h. [0257] 3) Hpg experiment, DMEM culture medium was then changed to methionine-deficient culture medium for 1 h, followed by supplying 2 mM Hpg (200 mM stock in PBS) in the medium for 24 h. [0258] 4) Propargylcholine and EdU dual-color experiment, DMEM culture medium was changed to DMEM medium (FBS-free) for synchronization. After synchronization, medium was replaced back to DMEM culture medium by simultaneously adding both propargylcholine (25 mM stock in PBS) and EdU (10 mM stock in PBS) to the culture medium to a concentration of 1 mM and 100 M, respectively, for 24 h.

[0259] For the propargylcholine experiment in neurons, hippocampal neurons were cultured on coverslips in 1 ml neuron culture medium for 14 d, and then propargylcholine (25 mM stock in PBS) is directly added into the medium to a final concentration of 1 mM for 24 h.

[0260] For the 17-ODYA experiment in macrophages, THP-1 cells were first Seeded on coverslips at a density of 2105/mL in 2 ml RPMI-1640 culture medium for 24 h, followed by 72 h induction of differentiation to macrophages by incubating with 100 ng/ml Phorbol 12-myristate 13-acetate (PMA) in the medium. Medium was then replaced with RPMI-1640 culture medium containing 400 M 17-ODYA (6:1 complexed to BSA) for 15 h.

[0261] For all of the above experiments, after incubation, the coverslip is taken out to make an imaging chamber filled with PBS for SRS imaging.

[0262] For the 17-ODYA experiment in C. elegans, OP50 bacterial culture was mixed well with 4 mM 17-ODYA (from 100 mM ethanol stock solution), and then Seeded onto nematode growth media (NGM) plates. After drying the plates in hood, wild type N2 day 1 adult C. elegans were placed onto the plates and fed for 40 h. C. elegans were then mounted on 2% agarose pads containing 0.1% NaN.sub.3 as anesthetic on glass microscope slides for SRS imaging.

[0263] SRS imaging of C. elegans germline after feeding with EdU. MG1693 (thymidine defective MG1655) E. Coli strain was cultured in 2 ml LB medium at 37 C. overnight, and transferred to 100 ml of M9 medium containing 400 M EdU for further growth at 37 C. for 24 h. The EdU-labeled MG1693 E. Coli was then Seeded on M9 agar plate. Synchronized day 1 adult worms developed in 20 C. were transferred to EdU-labeled bacterial plate for 3 h, and then were dissected to take out the germline for imaging (e.g. FIG. 13).

[0264] Cell preparation for click chemistry-based fluorescence microscopy. All experiments (e.g. FIG. 15) were carried out following the manufacturer's protocol from Invitrogen. HeLa cells were first incubated with 10 M EdU in DMEM culture medium for 24 h, or 1 mM EU in DMEM culture medium for 20 h, or 1 mM Hpg in methionine-deficient culture medium for 20 h, respectively. Cells were then fixed in 4% PFA for 15 min, washed twice with 3% BSA in PBS, permeabilized with 0.5% Triton PBS solution for 20 min, and performed click chemistry staining using Alexa Fluor 488 Azide in the Click-iT Cell Reaction Buffer Kit for 30 min. After washing with 3% BSA in PBS for three times, fluorescence images were obtained using an Olympus FV1200 confocal microscope with 488 nm laser excitation while the cells were immersed in PBS solution.

[0265] Enzymatic assays confirming propargylcholine incorporation into cellular choline phospholipids. We design our control experiments according to the click chemistry based assays reported in Jao, C. Y., Roth, M., Welti, R. & Salic, A. Proc. Natl. Acad. Sci. USA 106, 15332-15337 (2009) (e.g. FIG. 16). NIH 3T3 cells cultured with 0.5 M propargylcholine for 48 hours were fixed with 4% PFA for 15 minutes, rinsed with 1 mL TBS buffer twice and incubated with 1 mL 1 mg/mL BSA in TBS buffer for 1 hour at 37 C., with or without 0.02 U/mL phospholipase C (Type XIV from Clostridium perfringens, Sigma), in the presence of 10 mM CaCl.sub.2) (required for phospholipase C activity) (e.g. FIG. 16b) or 10 mM EDTA (e.g. FIG. 16c). The cells were then washed with TBS buffer and ready for SRS imaging.

[0266] Sample preparation for drug delivery into mouse ear tissues. Either DMSO solution or Drug cream (Lamisil, Novartis) containing 1% (w/w) active terbinafine hydrochloride (TH) was applied to the ears of an anesthetized live mouse (2-3 weeks old white mouse of either sex) for 30 min, and the dissected ears from the sacrificed mouse were then imaged by SRS (e.g. FIGS. 17 and 18). The amide (1655 cm.sup.1) and lipid (2845 cm.sup.1) images have been applied with linear spectral unmixing to eliminate cross talk before composition. The experimental protocol for drug delivery on mice (AC-AAAG4703) was approved by Institutional Animal Care and Use Committee at Columbia University.

[0267] Image progressing. Images are acquired with FluoView scanning software and assigned color or overlaid by ImageJ. Graphs were assembled with Adobe Illustrator.

Example 2a: Alkyne Tags

[0268] As an effective imaging modality for small biomolecules, we report a general strategy of using stimulated Raman scattering (SRS) microscopy to image alkynes (i.e., CC) as nonlinear vibrational tags, shown as bond-selective SRS in FIGS. 10a-c. As shown in FIG. 10a, Spontaneous Raman spectra of HeLa cells and 10 mM EdU solution. Inset: the calculated SRS excitation profile (FWHM 6 cm.sup.1, element 905) is well fitted within the 2125 cm.sup.1 alkyne peak (FWHM 14 cm.sup.1, magenta). FIG. 10b shows linear dependence of stimulated Raman loss signals (2125 cm.sup.1) with EdU concentrations under a 100 s acquisition time. FIG. 10c shows the metabolic incorporation scheme for a broad spectrum of alkyne-tagged small precursors. a.u. arbitrary units. Alkynes possess desirable chemical and spectroscopic features. Chemically, they are small (only two atoms), exogenous (nearly non-existent inside cells), and bioorthogonal (inert to reactions with endogenous biomolecules). These properties render alkynes key players in bioorthogonal chemistry, in which precursors labeled with alkyne tags form covalent bonds with azides fused to probes such as fluorophores for detection. However, such a click-chemistry approach prohibits live imaging, as it usually involves a copper-catalyzed reaction that requires cell fixation, while the copper-free version has slow kinetics and high background. Spectroscopically, the CC stretching motion exhibits a substantial change of polarizability, displaying a sharp Raman peak around 2125 cm.sup.1, which lies in a desirable cell-silent spectral region.sup.13 (FIG. 10a). Compared to the popular carbon-deuterium (C-D) Raman tag, alkynes produce about 40 times higher peaks. However the signal is still relatively weak and extremely long acquisition times (49 min per frame consisting of 127127 pixels) limit dynamic imaging in live systems.

[0269] The coupling of SRS microscopy to alkyne tags that we report offers sensitivity, specificity and biocompatibility for probing complex living systems. When the energy difference between incident photons from two lasers (pump and Stokes) matches with the 2125 cm.sup.1 mode of alkyne vibrations, their joint action will greatly accelerate the vibrational excitation of alkyne bonds. As a result of energy exchange between the input photons and alkynes, the output pump and Stokes beams will experience intensity loss and gain, respectively. Such intensity changes measured by SRS microscopy generate concentration-dependent alkyne distributions in three-dimensions (3D). FIG. 11 is an illustration showing a Pump beam (pulsed, pico-second) and an intensity-modulated Stokes beam (pulsed, pico-second) are both temporally and spatially synchronized before focused onto cells that have been metabolically labeled with alkyne-tagged small molecules of interest. When the energy difference between the Pump photon and the Stokes photon matches the vibrational frequency (vib) of alkyne bonds, alkyne bonds are efficiently driven from their vibrational ground state to their vibrational excited state, passing through a virtual state. For each excited alkyne bond, a photon in the Pump beam is annihilated (Raman loss) and a photon in the Stokes beam is created (Raman gain). The detected pump laser intensity changes through a lock-in amplifier targeted at the same frequency as the modulation of Stokes beam serve as the contrast for alkyne distributions.

[0270] SRS microscopy offers a number of advantages. First, SRS boosts vibrational excitation by a factor of 107, rendering a quantum leap of sensitivity (i.e., detectability and speed) over spontaneous Raman. Second, we use a 6-ps pulse width to match the excitation profile of alkyne (e.g. FIG. 10a), assuring efficient and selective nonlinear excitation. Third, SRS is free of background, whereas spontaneous Raman suffers from auto-fluorescence and coherent anti-stokes Raman scattering (CARS) suffers from non-resonant background3. Finally, we employ near-infrared laser wavelengths for enhanced tissue penetration, intrinsic 3D sectioning (due to nonlinear excitation) and minimal photo-toxicity.

[0271] We first detected the alkyne-tagged thymidine analogue 5-ethynyl-2-deoxyuridine (EdU) in solution (e.g. FIG. 10b). Under a fast imaging speed of 100 s, we determined its detection limit to be 200 M, corresponding to 12,000 alkynes within the laser focus. This approaches the shot-noise limit (Ip/Ip210-7) of the pump beam, which represents the maximum theoretical sensitivity of the system. To explore the general applicability of our approach, we went on to examine a broad spectrum of small biomolecules including alkyne-tagged deoxyribonucleoside, ribonucleoside, amino acid, choline and fatty acid (FIG. 10c), whose metabolic incorporation has been thoroughly tested in bioorthogonal chemistry studies.

[0272] We imaged the metabolic uptake of EdU during de novo DNA synthesis. HeLa cells grown in media with EdU show a sharp Raman peak at 2125 cm.sup.1 in the cell-silent region (e.g. FIG. 12a). Live-cell SRS imaging revealed EdU incorporation into the newly synthesized genomes of dividing cells (e.g. FIG. 12b, alkyne-on). Off-resonance images of the same cells, taken when the energy difference between pump and Stokes photons does not match vibrational peaks (alkyne-off), are background-free, confirming the purely chemical contrast of SRS. No EdU signal shows up in cells treated with the DNA synthesis inhibitor hydroxyurea, whereas lipids imaged at 2845 cm.sup.1 verify that these cells are normal based on morphology. Moreover, we tracked dividing cells every 5 min during mitosis (e.g. FIG. 12c), demonstrating acquisition speed and compatibility with live dynamics that are nearly impossible with spontaneous Raman. We also showed that our method is applicable to multicellular organisms. Actively proliferating cells can be clearly distinguished in C. elegans grown in the presence of EdU as exemplified in FIG. 13, where the composite image shows both the protein derived 1655 cm.sup.1 (amide) signal from all the germ cells, and the direct visualization of alkynes (2125 cm.sup.1 (EdU)) highlighting the proliferating germ cells. White circles show examples of EdU positive germ cells in the mitotic region of C. elegans germline. Scale bar, 5 m.

[0273] Next, we studied RNA transcription and turnover using the alkyne-tagged uridine analogue, 5-ethynyl uridine (EU).sup.8 in HeLa cells (e.g. FIG. 12a). The alkyne-on image (e.g. FIG. 12d) reveals localized EU inside the nucleus with higher abundance in the nucleoli, which are major compartments of rRNA-rich ribosomal assembly, and nearly disappears in the presence of the RNA synthesis inhibitor actinomycin D. Turnover dynamics are further demonstrated by pulse-chase SRS imaging (FIG. 12e), which indicates a short nuclear RNA lifetime (3 h) in live HeLa cells.

[0274] Many intricate biological processes such as long-term memory require protein synthesis in a spatiotemporal dependent manner. We imaged L-Homopropargylglycine (Hpg), an alkyne-tagged analogue of methionine, to visualize newly synthesized proteomes. HeLa cells grown in methionine-deficient media supplemented with Hpg display an alkyne peak (e.g. FIG. 12a) about 20 times lower than that of 10 mM EdU solution (e.g. FIG. 10a). The corresponding alkyne-on image (e.g. FIG. 12f) shows the distribution of newly synthesized proteins with spatial enrichment in the nucleoli (arrow indicated), which experience rapid protein exchange. Similar to EdU in solution, the detection limit of alkynes in mammalian cells approaches 200 M (with 100 s pixel dwell time) based on an average signal-to-noise ratio of 2 as we obtained in HeLa cells. The Hpg signal is well retained in fixed cells (e.g. FIG. 14), indicating little contribution from freely diffusing Hpg. The alkyne-on image displays the Hpg distribution for the newly synthesized proteins. For the same set of cells, the off-resonant (alkyne-off) image shows vanishing signal, and the amide image shows total protein distribution. This result confirms that the detected signal is not from freely diffusive precursor Hpg itself (which is eliminated during the fixation process). Scale bar, 10 m. Furthermore, adding methionine, which has a 500-fold-faster incorporation rate, to compete with Hpg causes the signal to disappear (e.g. FIG. 12f). Note that we verified the spatial patterns of EdU, EU and Hpg incorporation in live cells by performing click chemistry on fixed cells, with FIGS. 15A-C showing the fluorescence images of HeLa cells incorporated with a, EdU (for DNA); b, EU (for RNA); c, Hpg (for protein). Scale bars, 10 m.

[0275] Lipid metabolism is critical for many functions in healthy and diseased tissues, but few non-perturbative tags are available to monitor lipids in the cell. We thus monitored the metabolic incorporation of alkyne-tagged choline and fatty acids. Hippocampal neurons grown on propargylcholine present a clear 2142 cm.sup.1 Raman peak (e.g. FIG. 12a). Such a frequency shift from 2125 cm.sup.1 is due to the positive charge on the nitrogen near the alkyne (FIG. 10c). As revealed by enzymatic assays (e.g. FIGS. 16a-16c), the alkyne-on signal (FIG. 12g) mainly originates from newly synthesized choline phospholipids at membranes. To label fatty acids, we incubated 17-octadecynoic acid (17-ODYA) with THP-1 macrophages, which actively scavenge cholesterol and fatty acids. In FIG. 16a, fixed NIH3T3 cells are seen after culturing with 0.5 mM propargylcholine for 48 hours. The alkyne-on image shows alkyne-tagged choline distribution. In FIG. 16b, treatment of fixed NIH3T3 cells with phospholipase C, which removes Choline head groups of phospholipids only in the presence of calcium. The alkyne-on image shows the strong decrease of incorporated propargylcholine signal, supporting its main incorporation into membrane phospholipids. For FIG. 16c, treatment of fixed NIH3T3 cells with phospholipase C in the presence of EDTA (chelating calcium). Propargylcholine signal is retained in the alkyne-on image. For FIGS. 16a-c: in the same set of cells as in alkyne-on images, the alkyne-off images show a clear background. The amide images display total protein distribution. Scale bars, 10 m. The alkyne-on image (FIG. 12h) depicts the formation of numerous lipid droplets that indicates transformation into foam cells, a hallmark of early atherosclerosis. Multicellular organisms are also capable of taking up 17-ODYA for lipid imaging. New fatty acids in C. elegans appeared mainly inside lipid droplets upon SRS imaging, known to exist largely in the form of triglycerides (e.g. FIG. 12i). Such a fat accumulation process could serve as a useful model for studying obesity and diabetes. We were also able to perform dual-color imaging of propargylcholine (2142 cm.sup.1) and EdU (2125 cm.sup.1) incorporation due to the spectral sharpness and separation of their two alkyne peaks (FIG. 12j).

[0276] Finally, we tracked alkyne-bearing drug delivery (FIGS. 17a-e and 18a-b) in animal tissues by taking advantage of the intrinsic 3D sectioning property of SRS.

[0277] FIG. 17a depicts Raman spectra of a drug cream, Lamisil, containing 1% TH and mouse ear skin tissue. FIGS. 17b-e illustrate SRS imaging of tissue layers from stratum corneum (z=4 m) to viable epidermis (z=24 m), sebaceous gland (z=48 m) and subcutaneous fat (z=88 m). To facilitate tissue penetration, DMSO solution containing 1% TH was applied onto the ears of an anesthetized live mouse for 30 min and the dissected ears are imaged afterwards. For all 4 layers shown in FIGS. 17b-e, alkyne-on images display TH penetration; alkyne-off images show off-resonant background (The bright spots in d are due to two-photon absorption of red blood cells). The composite images show protein (1655 cm.sup.1) and lipid (2845 cm.sup.1) distributions. Scale bars, 20 m. a.u.=arbitrary units.

[0278] FIGS. 18a-b show SRS imaging of the viable epidermis layer (z=20 m) and the sebaceous gland layer (z=40 m). For both a and b: the alkyne-on images display the TH penetration into mouse ear tissues through lipid phase. The composite images show both protein (1655 cm.sup.1) and lipid (2845 cm.sup.1) distributions. Scale bars, 20 m.

[0279] Unlike bulky fluorophores, alkynes have little perturbation to pharmacokinetics and are common moieties in many pharmaceuticals. We chose terbinafine hydrochloride (TH), a US Federal Drug Administration approved alkyne-bearing antifungal skin drug, and imaged its drug delivery pathways inside mouse ear tissue to a depth of about 100 m by targeting its internal alkyne at 2230 cm.sup.1. TH images captured at various depths all exhibit patterns that highly resemble lipid distributions but not protein distributions, suggesting that TH penetrates into tissues through the lipid phase, consistent with its lipophilic nature. Our technique should be applicable to tracking other drugs after proper alkyne derivatization.

[0280] In conclusion, we report a general strategy to image small and biologically vital molecules in live cells by coupling SRS microscopy with alkyne vibrational tags. The major advantages of SRS lie in the superior sensitivity, specificity and compatibility with dynamics of live cells and animals. SRS imaging of alkynes may do for small biomolecules what fluorescence imaging of fluorophores has done for larger species.

Example 3: Synthesis of Bond-Edited Compounds

[0281] A. Synthesis of Alkyne-D-Glucose

##STR00015##

[0282] Sodium hydride (138 mg, 5.8 mmol) was added to a solution of 1,2:5,6-Di-O-isopropylidene--D-glucofuranose (Compound S1, 500 mg, 1.92 mmol, Aldrich D7600) in 10 mL dry DMF at 0 C. The solution was stirred at 0 C. for 30 min before propargyl bromide (80% in toluene, 0.43 mL, 3.84 mmol) was added dropwise. The reaction mixture was stirred at room temperature for 12 h before quenched with saturated ammonium chloride solution (10 mL). The mixture was extracted with ethyl acetate (225 mL), and the organic layer was combined, dried over anhydrous sodium sulfate, concentrated in vacuo, and purified by column chromatography on silica gel (0-50% Ethyl acetate in Hexanes) to give Compound S2 (518 mg, 90%) as a colorless oil. The .sup.1H NMR spectrum is in accordance with previously published values (A. Hausherr et al., Synthesis, 2001, 1377).

[0283] .sup.1H NMR (400 MHz, CDCl.sub.3) 5.88 (d, J=3.6 Hz, 1H), 4.30-4.24 (m, 3H), 4.14 (dd, J=7.6, 2.8 Hz, 1H), 4.11-4.06 (m, 2H), 3.99 (dd, J=8.8, 5.6 Hz, 1H), 2.47 (t, J=2.4 Hz, 1H), 1.50 (s, 3H), 1.42 (s, 3H), 1.35 (s, 3H), 1.31 (s, 3H).

[0284] HRMS (FAB+) m/z Calcd. for C.sub.15H.sub.23O.sub.6 [M+H].sup.+: 299.1495. Found: 299.1496

##STR00016##

[0285] Water (10 mL) and Dowex 50WX8 hydrogen form (600 mg, Sigma-Aldrich 217514) were added to Compound S2 (594 mg, 1.99 mmol). The mixture was heated to 80 C. for 20 h before filtered. The filtrate was concentrated in vacuo to give Compound S3 (416 mg, 1.91 mmol, 96%) as a white solid.

[0286] .sup.1H NMR (400 MHz, D.sub.2O) 5.13 (d, J=3.6 Hz, 1H), 4.44 (d, J=2.4 Hz, 2H), 3.79-3.73 (m, 2H), 3.70-3.61 (m, 2H), 3.51 (dd, J=9.8, 3.8 Hz 1H), 3.40 (t, J=9.6 Hz, 11H), 2.82 (s, 1H). .sup.13C NMR (101 MHz, D.sub.2O) 92.1, 80.8, 79.8, 75.9, 71.4, 71.2, 69.2, 60.4, 59.9.

[0287] HRMS (FAB+) m/z Calcd. for C.sub.9H.sub.14O.sub.6Na [M+Na]+: 241.0688. Found: 241.0683

[0288] B. Synthesis of EdU-.sup.13C (Compound 2) and EdU-.sup.13C.sub.2 (Compound 3)

##STR00017##

[0289] Synthesis of Compound 4:

##STR00018##

[0290] To a solution of 5-iodo-2-deoxyuridine (Compound 1, 150 mg, 0.42 mmol) in 1.5 ml of pyridine was added 0.4 ml (0.42 mmol) acetic anhydride at 0 C. The resulting mixture was warmed up to room temperature and stirred for 4 h, then poured into 5 ml of cold 1N NaHSO.sub.4 and extracted with ethyl acetate three times. The organic layer was washed with saturated NaHCO.sub.3 and brine, dried over anhydrous Na.sub.2SO.sub.4 and concentrated. The crude product was purified by column chromatography on silica gel (0-70% Ethyl acetate in Hexanes) to give Compound 4 (157.3 mg, 0.36 mmol, 85%) as a white solid.

[0291] .sup.1H NMR (400 MHz, CDCl.sub.3) ppm: 8.46 (s, 1H), 7.97 (s, 1H), 6.28 (dd, J=8.2, 5.7 Hz, 1H), 5.27-5.19 (m, 1H), 4.41 (dd, J=12.3, 3.2 Hz, 1H), 4.34 (dd, J=12.3, 2.9 Hz, 1H), 4.30 (q, J=2.9 Hz, 1H), 2.54 (ddd, J=14.3, 5.7, 2.1 Hz, 1H), 2.21 (s, 3H), 2.20-2.13 (m, 1H), 2.12 (s, 3H).

[0292] MS (APCI+) m/z Calcd. for C13H.sub.16IN.sub.2O.sub.7 [M+H].sup.+: 439.0. Found: 438.8

Synthesis of Compound 5:

[0293] ##STR00019##

[0294] To an oven-dried vial was added Compound 4 (72 mg, 164 mol), Pd(OAc).sub.2 (3.6 mg, 16 mol), PPh.sub.3 (8.6 mg, 33 mol), CuI (3.1 mg, 16 mol), DMF (2 ml), Et3N (50 mg, 69 l, 492 mol) and TMS.sup.13C.sup.13CH (25 mg, 250 mol) under Ar. The yellow mixture was stirred at RT for 15 h before concentrated in vacuo. The residue was purified by column chromatography on silica gel (0-70% Ethyl acetate in Hexanes) to give Compound 5 (48.4 mg, 118 mol, 72%) as a thin film.

[0295] .sup.1H NMR (400 MHz, Methanol-d4) 7.98 (d, J=5.0 Hz, 1H), 6.23 (dd, J=7.8, 6.0 Hz, 1H), 5.28 (dt, J=6.7, 2.6 Hz, 1H), 4.36 (t, J=3.1 Hz, 2H), 4.34-4.28 (m, 1H), 2.50 (ddd, J=14.5, 6.0, 2.5 Hz, 1H), 2.39 (ddd, J=14.5, 7.9, 6.6 Hz, 1H), 2.16 (s, 3H), 2.09 (s, 3H), 0.20 (d, J=2.5 Hz, 9H). .sup.13C NMR (101 MHz, MeOD) 99.54 (d, J=140.5 Hz), 96.95 (d, J=140.5 Hz).

[0296] MS (APCI+) m/z Calcd. for C.sub.16.sup.13C.sub.2H.sub.25N.sub.2O.sub.7Si [M+H]+: 411.2. Found: 411.0

Synthesis of Compound 3:

[0297] ##STR00020##

[0298] To a solution of Compound 5 (3.5 mg, 8.5 mol) in 0.9 ml MeOH and 0.1 ml H.sub.2O was added K.sub.2CO.sub.3 (6.0 mg, 43 mol) at RT. The reaction was stirred overnight before concentrated in vacuo. The residue was purified by reverse phase HPLC to give compound 3 (1.6 mg, 6.4 mol, 75%) as a thin film.

[0299] HPLC condition: 20 min gradient elution using H.sub.2O:MeCN starting from 100:0 to 85:15. Retention time: 15.4 min

[0300] .sup.1H NMR (400 MHz, MeOD) : 8.39 (d, J=5.6 Hz, 1H); 6.24 (t, J=6.4 Hz, 1H); 4.40 (m, 1H); 3.94 (dd, J=6.4, 3.2 Hz, 1H); 3.82 (dd, J=12, 3.2 Hz, 1H); 3.73 (dd, J=12, 3.6 Hz, 1H); 3.53 (dd, J=250.4, 54.8 Hz, 1H); 2.32 (ddd, J=13.6, 6, 3.6 Hz, 1H); 2.23 (m, 1H). .sup.13C NMR (101 MHz, MeOD) 82.87 (d, J=180.4 Hz), 75.85 (d, J=180.3 Hz).

[0301] MS (FAB+) m/z Calcd. for C.sub.9.sup.13C.sub.2H.sub.13N.sub.2O.sub.5 [M+H].sup.+: 255.09. Found: 255.11

Synthesis of Compound 9:

[0302] ##STR00021##

[0303] To a solution of ethynylmagnesium bromide in THF (5.0 ml, 0.5 M solution, 2.5 mmol) was added 15 ml THF under Ar. The solution was cooled to 78 C. and 2.4 ml n-BuLi in hexane (1.6 M, 3.8 mmol) was added dropwisely. After 30 min, chloro(dimethyl)octylsilane (1.21 ml, 1.06 g, 5.1 mmol) was added dropwisely. The reaction was then warmed to RT and stirred for another 3 h before filtered through a short pad of silica. The solvent was removed under reduced pressure and the residue was purified by column chromatography on silica gel (pure Hexanes) to give Compound 9 (885 mg, 2.4 mmol, 96%) as a colorless liquid.

[0304] .sup.1H NMR (400 MHz, Chloroform-d) 1.42-1.24 (m, 24H), 0.88 (t, J=6.6 Hz, 6H), 0.60 (dd, J=9.4, 6.2 Hz, 4H), 0.13 (s, 12H). .sup.13C NMR (101 MHz, Chloroform-d) 113.94, 33.37, 32.12, 29.49, 29.43, 23.92, 22.85, 16.26, 14.27, 1.55.

[0305] HRMS (EI+) m/z Calcd. for C.sub.22H.sub.46Si.sub.2 [M].sup.+:366.3138. Found: 366.3134

Synthesis of Compound 10:

[0306] ##STR00022##

[0307] In a glove box filled with Ar, catalyst 8 (36.5 mol, 5 eq.) was prepared in 0.5 mL dry CCl4 in situ according to the procedure documented by Jyothish and Zhang (Angew. Chem. Int. Ed. Engl. 50, 3435-8 (2011)). To the solution of catalyst 8 in CCl4 was added 9 (267 mg, 0.73 mmol) and a solution of Compound 5 (3.0 mg, 7.3 mol) in 0.5 mL dry CCl4. The mixture was heated to 70 C. for 8 h before concentrated in vacuo. The residue was purified by column chromatography on silica gel (0-70% Ethyl acetate in Hexanes) to recover Compound 5 (0.5 mg, 1.2 mol) and to give Compound 10 (1.0 mg, 2.0 mol, 27%, 33% B.R.S.M.) as a thin film.

[0308] .sup.1H NMR (400 MHz, Methanol-d4) 7.97 (d, J=5.6 Hz, 1H), 6.23 (dd, J=7.9, 5.9 Hz, 1H), 5.28 (dt, J=6.8, 2.4 Hz, 1H), 4.36 (dd, J=5.8, 3.4 Hz, 2H), 4.31 (dd, J=6.3, 3.2 Hz, 1H), 2.50 (ddd, J=14.5, 6.1, 2.5 Hz, 1H), 2.38 (ddd, J=20.2, 7.7, 6.1 Hz, 1H), 2.15 (s, 3H), 2.09 (s, 3H), 1.30 (s, 12H), 0.90 (t, J=6.9 Hz, 3H), 0.70-0.62 (m, 2H), 0.18 (s, 6H). .sup.13C NMR (101 MHz, MeOD) 97.56.

[0309] MS (FAB+) m/z Calcd. for C.sub.24.sup.13CH.sub.38N.sub.2NaO.sub.7Si [M+Na]+: 530.24. Found: 530.25

Synthesis of Compound 2:

[0310] ##STR00023##

[0311] To a solution of compound 10 (0.4 mg, 0.8 mol) in 0.5 ml MeOH and 0.05 ml H.sub.2O was added K.sub.2CO.sub.3 (2.0 mg, 14 mol) and TBAF (20 L, 1 M in THF) at RT. The reaction was stirred 7 h at RT before concentrated in vacuo. The residue was purified by reverse phase HPLC to give compound 2 (0.1 mg, 0.4 mol, 50%) as a thin film.

[0312] HPLC condition: 20 min gradient elution using H.sub.2O:MeCN starting from 100:0 to 85:15. Retention time: 15.4 min

[0313] The mass of the product is determined by UV-Vis (abs=288 nm, =12,000 cm.sup.1 M-1 in methanol).

[0314] .sup.1H NMR (500 MHz, Methanol-d4) 8.39 (d, J=5.7 Hz, 1H), 6.24 (t, J=6.5 Hz, 1H), 4.40 (dt, J=6.6, 3.6 Hz, 1H), 3.94 (q, J=3.3 Hz, 1H), 3.82 (dd, J=12.0, 3.1 Hz, 1H), 3.73 (dd, J=12.0, 3.4 Hz, 1H), 3.53 (d, J=51.3 Hz, 1H), 2.32 (ddd, J=13.6, 6.2, 3.7 Hz, 1H), 2.27-2.17 (m, 1H). .sup.13C NMR (101 MHz, MeOD) 76.00. MS (ESI+) m/z Calcd. for C.sub.10.sup.13CH.sub.13N.sub.2O.sub.5 [M+H]+: 254.09. Found: 254.70

Synthesis of EU-.SUP.13.C2 (Compound 13)

[0315] ##STR00024##

[0316] To an oven-dried vial was added compound S5 (15 mg, 50 mol), Pd(OAc)2 (1.1 mg, 5 mol), PPh3 (2.6 mg, 10 mol) CuI (1.0 mg, 5 mol), DMF (1 ml), Et3N (15 mg, 20.7 l, 150 umol) and TMS.sup.13C.sup.13CH (7.5 mg, 10.8 l, 75 mol) under Ar. The mixture was stirred at RT for 12 h before concentrated in vacuo. The residue was purified by column chromatography on silica gel (0-50% methanol in dichloromethane) to give compound S6 (9.0 mg, 26 mol, 52%) as a thin film.

[0317] .sup.1H NMR (400 MHz, Methanol-d4) 8.41 (d, J=4.9 Hz, 1H), 5.91-5.83 (m, 1H), 4.21-4.13 (m, 2H), 4.07-3.98 (m, 1H), 3.88 (dd, J=12.2, 2.6 Hz, 1H), 3.75 (dd, J=12.2, 2.8 Hz, 1H), 0.20 (d, J=2.3 Hz, 9H). .sup.13C NMR (101 MHz, MeOD) 99.24 (d, J=141.0 Hz), 96.95 (d, J=141.0 Hz).

[0318] MS (FAB+) m/z Calcd. for C.sub.12.sup.13C.sub.2H.sub.21N.sub.2O.sub.6Si [M+H]+: 343.12. Found: 343.17.

Synthesis of Compound 13:

[0319] ##STR00025##

[0320] To a solution of compound S6 (3.0 mg, 8.8 mol) in 0.6 ml MeOH and 0.1 ml H.sub.2O was added K.sub.2CO.sub.3 (5.0 mg, 36 mol) at RT. The reaction was stirred overnight before concentrated in vacuo. The residue was purified by reverse phase HPLC to give compound 13 (2.2 mg, 8.1 mol, 92%) as a thin film.

[0321] .sup.1H NMR (400 MHz, Methanol-d4) 8.47 (d, J=5.6 Hz, 1H), 5.93-5.83 (m, 1H), 4.21-4.13 (m, 2H), 4.06-3.98 (m, 1H), 3.88 (dd, J=12.2, 2.6 Hz, 1H), 3.75 (dd, J=12.2, 2.8 Hz, 1H), 3.54 (dd, J=250.4, 54.6 Hz). .sup.13C NMR (101 MHz, MeOD) 82.90 (d, J=180.2 Hz), 75.74 (d, J=180.2 Hz).

[0322] MS (ESI+) m/z Calcd. for C.sub.9.sup.13C.sub.2H.sub.13N.sub.2O.sub.6 [M+H]+: 271.08. Found: 271.51

[0323] FIG. 38 shows a block diagram of an exemplary embodiment of a system according to the present disclosure. For example, exemplary procedures in accordance with the present disclosure described herein can be performed by a processing arrangement and/or a computing arrangement 3802. Such processing/computing arrangement 3802 can be, for example entirely or a part of, or include, but not limited to, a computer/processor 3804 that can include, for example one or more microprocessors, and use instructions stored on a computer-accessible medium (e.g., RAM, ROM, hard drive, or other storage device).

[0324] As shown in FIG. 38, for example a computer-accessible medium 3806 (e.g., as described herein above, a storage device such as a hard disk, floppy disk, memory stick, CD-ROM, RAM, ROM, etc., or a collection thereof) can be provided (e.g., in communication with the processing arrangement 3802). The computer-accessible medium 3806 can contain executable instructions 3808 thereon. In addition or alternatively, a storage arrangement 3810 can be provided separately from the computer-accessible medium 3806, which can provide the instructions to the processing arrangement 3802 so as to configure the processing arrangement to execute certain exemplary procedures, processes and methods, as described herein above, for example.

[0325] Further, the exemplary processing arrangement 3802 can be provided with or include an input/output arrangement 3814, which can include, for example a wired network, a wireless network, the internet, an intranet, a data collection probe, a sensor, etc. As shown in FIG. 38, the exemplary processing arrangement 3802 can be in communication with an exemplary display arrangement 3812, which, according to certain exemplary embodiments of the present disclosure, can be a touch-screen configured for inputting information to the processing arrangement in addition to outputting information from the processing arrangement, for example. Further, the exemplary display 3812 and/or a storage arrangement 3810 can be used to display and/or store data in a user-accessible format and/or user-readable format.

Exemplary Imaging Intracellular Fluorophores with Sub-Micromolar Sensitivity Using Pre-Resonance Stimulated Raman Scattering

[0326] The exemplary system, method and computer-accessible medium, according to an exemplary embodiment of the present disclosure, can utilize pr-SRS microscopy and apply such exemplary procedure to image molecules in the pre-resonance Raman regime to achieve both superb sensitivity at sub-micromolar concentration, and chemical specificity for multiplex imaging (see, e.g., FIG. 39a). SRS microscopy can be a nonlinear two-laser pump-probe Raman technique, utilizing the stimulated emission quantum amplification from the Stokes beam photons in addition to pump beam photons to facilitate the intrinsically weak Raman transition. (See, e.g., References 99 and 100). The effective stimulated Raman cross section (.sub.st) can be defined compared to a spontaneous Raman cross section (.sub.sp) as, for example:

.sub.st=.sub.spG(p.sub.w.sub.s)(1a)

where G(p.sub.w.sub.s) can be the stimulated Raman gain factor depending on the Stokes beam power (p.sub.w.sub.s). The measured G(p.sub.w.sub.s) can be about 107-108 with a biologically compatible Stoke beam power. (See, e.g., Reference 100). With such powerful quantum amplification, SRS microscopy has been applied in a great deal of biological studies such as video-rate imaging in vivo, brain tumors detection and drug delivery tracking. (See, e.g., References 101-103). In addition, as a nonlinear microscopy that can utilize near-infrared excitation wavelength, SRS also offers both subcellular resolution and intrinsic 3D sectioning for deep tissue imaging.

[0327] However, current SRS sensitivity can still be far from that of fluorescence microscopy. The measured SRS detection limit of a typical chemical bond such as carbon-hydrogen bond (CH, .sub.sp10.sup.30 cm.sup.2) can be about 15 mM. (See, e.g., Reference 99). Even for chemical bonds with exceptional strong Raman polarizability, such as alkynes (CC), the reported detection limit can be about 200 M. (See, e.g., Reference 103). Efforts have been devoted to synthesize molecules with stronger Raman vibration, however, only a few molecules present such property, and the improvements can typically only be about 27 times. (See, e.g., Reference 104). Presently, all the SRS applications focus on probing molecules in the non-resonance region where the absorption peak energy (0) of the molecules can be much larger than the pump laser energy (pump). (See, e.g., FIG. 39b). One way to further improve SRS sensitivity can be by moving from non-resonance Raman to resonance Raman, where 0pump< (e.g., can be the homogeneous line width for a molecule, typically, 800 cm.sup.1), thus achieving Raman intensity gain benefiting from the coupling of electronic transition. In this exemplary case, the resonance enhanced spontaneous Raman cross-section .sub.sp,PR could be shown as, for example:

.sub.sp,RE=.sub.spG.sub.RE(2a)

where, G.sub.RE can be the resonance enhanced Raman gain factor, which can increase when the energy difference between 0 and pump can decrease. Furthermore, the effective resonance enhanced SRS cross-section can be expressed as, for example:

.sub.st,RE=.sub.spG.sub.REG(p.sub.w.sub.s)(3a)

[0328] To illustrate the resonance Raman enhancement in the nonlinear SRS microscopy, for example, a non-resonance SRS spectrum of Coumarin 153 can be measured, which can show an absorption peak wavelength (max) at about 422 nm (e.g., 23697 cm.sup.1, 0>>pump) (see, e.g., FIG. 39b) and a rigorous resonance SRS spectrum of IR895 with max=895 nm (e.g., about 11173 cm.sup.1, 0pump=0) (see, e.g., FIG. 39c) by sweeping the pump laser wavelength across the range of about 895-940 nm (e.g., 11173-10638 cm.sup.1) with a fixed Stokes laser wavelength at about 1064.2 nm (e.g., 9398 cm.sup.1). The measured Raman cross-section for the conjugated carbon-carbon double bond (CC) for IR895 (see, e.g., FIG. 39c, element 3905) can approach about 104 as compared to that of Coumarin 153, while CC of Coumarin 153 may only be about 1.5 times of CC (e.g., FIG. 39b, element 3910). However, with the increasing of the Raman intensity, a large background can appear possibly due to the competing pump-probe process of simultaneous excitation saturation, and stimulated emission of the electronic transition for IR895. Moreover, the measured Raman peak for IR895 can present an inverse Lorentzian shape due to the rigorous resonance Raman measurement for the stimulated Raman loss signal. (See, e.g., Reference 105). For a spectroscopy measurement, these complications may not be an issue with post-acquisition processing. For example, femtosecond SRS spectra of rigorous resonant fluorophores have been heavily documented and the appearance of such an intense background is also well known. (See, e.g., References 106 and 107). Nevertheless, such a background can be extremely undesirable for imaging applications, which can benefit from direct visualization of spatial information.

[0329] Close inspection for both the non-resonance SRS spectrum of Coumarin 153 (see, e.g., FIG. 39b) and rigorous resonance SRS spectrum of IR895 (see, e.g., FIG. 39c) can indicate that a rigorous resonance can bring background more than vibrational signals. Therefore, an exemplary detuning from rigorous resonance can reduce the electronically related background faster than the vibrational signal. For example, this can be rationalized since the electronically related background can benefit from the excitation of the real population, which can vanish if the pump laser energy does not match with the excitation energy, while the vibrational signal can be mediated by virtual state. Indeed, this rationalization can be supported by the SRS spectrum of Cy7.5, which can show a ratio of approximately 0.3 between on-resonance Raman signal and off-resonance Raman signal with a detuning of 0pump 2 (see, e.g., FIG. 39c) compared to that of 0.03 for IR895 when 0pump 0. (See, e.g., FIG. 39b). A further detuning to 3 pinpointed an ATTO740 dye (max=740 nm) with both a Raman intensity gain of 1000 times compared to CC and a clean off-resonance background (see, e.g., FIG. 39d) can be performed. This can be the so-called pre-resonance Raman region, which has been revealed, and can be the most suitable regime for SRS imaging for both high sensitivity and chemical specificity. This can be called the pr-SRS region when 0pump can fall within the range of 3-5.

[0330] An exemplary configuration and principle for pre-resonance SRS microscopy according to an exemplary embodiment of the present disclosure, is shown in FIGS. 39a-39e. For example, FIG. 39a illustrates a diagram of an exemplary SRS system. FIG. 39b illustrates an exemplary energy diagram and the corresponding non-resonance SRS spectrum of 4 mM Coumarin 153 in DMSO under power of Ppump=24 mW, Pstokes=24 mW. FIG. 39c shows an exemplary energy diagram and the corresponding rigorous resonance SRS spectrum of 10 M IR895 in methanol under power of Ppump=1.2 mW, Pstokes=9.6 mW. FIG. 39d provides an exemplary energy diagram and the corresponding resonance SRS spectrum of 100 M Cy7.5 in DMSO under power of Ppump=12 mW, Pstokes=9.6 mW. FIG. 39e shows an exemplary energy diagram and the corresponding pre-resonance SRS spectrum of 100 M ATTO740 in DMSO under power of Ppump=24 mW, Pstokes=24 mW. For example, elements 3905, 3910, 3915 and 3920 can indicate the highest Raman peaks in the molecules that attribute to the CC bond vibration.

[0331] To illustrate further the pr-SRS enhancement between the experiment results and theory, FIG. 40a shows measured .sub.sp(CC) for 25 different fluorophores over a wide range of 0. Assuming the molecular structure variation can be negligible in the consideration for pre-resonance Raman enhancement factor, the graph of FIG. 40a was over-plotted with the theoretically approximated pr-Raman cross-section30 over the same 0 range of, for example:

[00004]$\begin{array}{cc}{}_{R.Math.E}=K.Math.{{}_0\left({}_0-{}_{\mathrm{vib}}\right)}^3.Math.{\left(\frac{\left({}_{p.Math.u.Math.m.Math.p}^2+{}_0^2\right)}{{\left({}_0^2-{}_{p.Math.u.Math.m.Math.p}^2\right)}^2}\right)}^2& \left(4.Math.a\right)\end{array}$

where vib can be the vibrational transition energy. Based on the above, it was determined that the pre-resonance enhancement from theoretical calculations can match very well with the experimental results. The grey shaded area 4005 from FIG. 40a indicates the selected fluorophores most suitable for pr-SRS imaging with both high enough pre-resonant enhancement and chemical specificity. For the dyes in the shaded area 4005 from FIG. 40a, ATTO740 can be the best dye presenting a pre-resonance enhancement gain (G.sub.pRE) of 105 when compared with .sub.sp(CH), thus .sub.st,pRE(ATTO740) can reach 10-18 cm.sup.2 with an experimental demonstrated G(p.sub.w.sub.s)108, approaching that of single molecule absorption cross-section at 10-16 cm.sup.2.

[0332] With such sensitivity, both high sensitivity and chemical specificity for the exemplary pr-SRS technique in solutions can be analyzed. By appropriately selecting the imaging condition with excitation power that can be low enough not to damage the fluorophore but high enough to mildly saturate the Raman transition of ATTO740, the measured detection limit with shot-noise limited sensitivity for ATTO740 can be about 0.7 M with about a 1 ms time constant and about a 2 M with about a 100 s time constant that can be suitable for live-cell imaging (see, e.g., FIG. 40b). For pr-SRS, which can still probe the virtual-state mediated Raman transition benefiting from pre-resonance enhancement, the pump laser may not directly excite the fluorophore to the real electronic state. Therefore the photobleaching damage can be minimum. To demonstrate the high chemical specificity in pr-SRS, 3 dyes (Cy5, sulfo-Cy5, ATTO655) that absorb similarly around 650 nm can be selected. While the three dyes (e.g., Cy5 (e.g., element 4025), Sulfo-Cy5 (e.g., element 4030) and ATTO 655 (e.g., element 4035)) present almost indistinguishable absorption and emission spectra (see, e.g., FIG. 40c), ATTO655, as an oxazine dye, shows a CC Raman peak at 1664 cm.sup.1 (see, e.g., FIG. 40d element 4010) that can be completely distinguishable from the CC peak of cyanine dyes (Cy5, sulfo-Cy5) at 1606 cm.sup.1 (e.g., FIG. 40d, element 4010). In addition, sulfo-Cy5 can also be distinguishable from Cy5 between the 1372 cm.sup.1 peak and the 1359 cm.sup.1 peak (see, e.g., FIG. 40d, elements 4015 and 4020) even with a minor chemical modification of aromatic sulfonation. Thus, it can be shown spectroscopically that fluorophores with inseparable electronic spectroscopy can be easily differentiated in Raman spectrum because of the chemical selectivity from vibrational spectroscopy.

[0333] FIGS. 40a-40d show exemplary graphs of an exemplary pr-SRS signal with superb sensitivity and chemical specificity. For example, FIG. 40a illustrates a semi-log plot of the measured Raman cross-section for 25 fluorophores with various absorption peak energies. Grey shaded-area 4005 of FIG. 40a indicates the selected pre-resonance SRS region. FIG. 40b illustrates the linear dependence of stimulated Raman loss signals at 1642 cm.sup.1 with ATTO740 concentrations under an about 100-s time constant. FIG. 40c illustrates the overlapping absorption and emission spectra of Cy5, sulfo-Cy5 and ATTO655. FIG. 40d illustrates the SRS spectra of Cy5, sulfo-Cy5 and ATTO655 with discernible Raman peaks.

[0334] Pr-SRS imaging on intracellular fluorophores can be provided which have achieved exceptional image contrast with a panel of immuno-labeled specific types of intracellular proteins including tubulin, Tom20 (e.g., mitochondria marker), giantin (e.g., Golgi marker) and neurofilament heavy proteins (e.g., Neuronal Marker) with ATTO740 and Dylight650 dyes in either cultured hippocampal neurons or HeLa cells. (See, e.g., FIGS. 41a-41e). Such high quality SRS images can be obtained for an individual type of proteins labeled with fluorophores. Besides the heightened imaging sensitivity, the specific chemical selectivity using the exemplary system, method, and computer-accessible medium according to an exemplary embodiment of the present disclosure can also be attained, where the on-resonant image at 1642 cm.sup.1 (pump=905.9 nm) for ATTO740-labeled tubulin can be clearly shown, but the corresponding off-resonance signal at 1702 cm.sup.1 (pump=901 nm) can vanish when the energy difference between the pump and Stokes photons does not match the vib for the CC vibrational transition of ATTO740. Such important chemical specificity can extend beyond an achievement for standard fluorescence-based detection systems as illustrated in FIGS. 41g-41j, in which the Alexa647 labeled 5-Ethynyl-2-deoxyuridine (EdU) for DNA detection shows a clear Raman vibrational on-off resonance contrast by tuning pump just to about 2 nm away from the peak. (See, e.g., FIGS. 41g and 41h). Further, the two-photon fluorescence signal remains the same by tuning the excitation laser wavelength by about 2 nm off the peak. (See, e.g., FIGS. 41i and 3j).

[0335] Indeed, FIGS. 41a-41j show exemplary pr-SRS images with superb sensitivity and chemical specificity. For example, FIG. 41a illustrates an ATTO740 immuno-labeled tubulin in hippocampal neurons targeting the 1642 cm.sup.1 peak. FIG. 41b illustrates an ATTO740 immuno-labeled atom.sup.20 in HeLa cells at 1642 cm.sup.1. FIG. 41c illustrates an ATTO740 immuno-labeled gaintin in HeLa cells at 1642 cm.sup.1. FIG. 41d illustrates a Dylight650 immuno-labeled neurofilament heavy protein in hippocampal neurons at 1606 cm.sup.1. FIG. 41e illustrates an on-resonance ATTO740 immuno-labeled tubulin in HeLa cells at 1642 cm.sup.1. FIG. 41f illustrates an off-resonance signal at 1702 cm.sup.1 on the same HeLa cells as in FIG. 41e. FIG. 41g illustrates an on-resonance Alexa647 labeled 5-Ethynyl-2-deoxyuridine for newly synthesized DNA in HeLa cells at 1606 cm.sup.1. FIG. 41h illustrates an off-resonance image at 1580 cm.sup.1 on the same HeLa cells as in FIG. 41g. FIGS. 41i and 41j illustrate two-photon fluorescence images of the same HeLa cells as in FIG. 41g at about 810 nm (see e.g., FIG. 41i) and about 812 nm (see e.g., FIG. 41j) of the two-photon excitation peak of Alexa647.

[0336] Thus, from both the spectroscopy and imaging perspectives, the superb sensitivity and the distinct chemical specificity for the exemplary pr-SRS of fluorophores is shown. With such sensitivity and specificity, the uses of the exemplary system, method and computer-accessible medium for biomedical researches, among which large number multi-color imaging can be important. For example, FIG. 42a illustrates an exemplary graph of a 6-color channel pr-SRS multiplex imaging possibility with 6 different fluorophores which include 80 m Atto740 (e.g., element 4205), 100 m Atto 700 (e.g., element 4210), 250 m Cy5.5 (e.g., element 4215), 500 m Alexa647 (e.g., element 4220), 200 m Atto665 (e.g., element 4225) and 500 m Dylight650 (e.g., element 4230). Among the different fluorophores shown, each of the three dyes shows an almost overlapping absorption and emission spectra. By specifically selecting the target, FIG. 42b demonstrates the 4-color pr-SRS imaging for intracellular fluorophores with distinct and quantitative signal separation of Cy5.5 (max=673 nm) labeled newly synthesized DNA, ATTO740 (max=740 nm) labeled nucleoli fibrillarin proteins, Atto700 (max=700 nm) labeled -tubulin and Alexa647 labeled newly synthesized proteins after linear combinational procedure. The 4-color overlay image (see, e.g., FIG. 42c) presents clearly defined spatial relationships between the targeted four types of molecules. Moreover, since SRS signal can be orthogonal with fluorescence signal, besides the multiplex pr-SRS imaging alone, tandem imaging with the fluorescence could further expand the numbers of simultaneously detected colors. To demonstrate such configuration, a DAPI (max=350 nm) that stains DNA, dylight488 (max=488 nm) staining calnexin (e.g., ER marker) and mito-tracker orange (e.g., 543 nm) staining mitochondria for three-color fluorescence imaging, in addition to the above 4-color multiplex pr-SRS imaging in the same set of cells, can be selected (see, e.g., exemplary images of FIG. 42d). Since, for example, the 4 dyes for pr-SRS imaging can all be with an absorption peak at about 650-740 nm with far-red emission, and the 3 dyes for fluorescence imaging can be around 350-543 nm that can be in the non-resonance SRS region with minimum resonance enhancement, both the fluorescence signal and the pr-SRS signal can be free of interference from each other. Thus, the exemplary system, method and computer-accessible medium can achieve at least 7-color imaging.

[0337] In particular, FIGS. 42a-42d illustrate simultaneous 7-color quantitative pr-SRS and fluorescence tandem imaging. For example, FIG. 42a illustrates pr-SRS spectra with 6 possible colors for 6 fluorophores of ATTO740 (1642 cm.sup.1), ATTO700 (1657 cm.sup.1), Cy5.5 (1626 cm.sup.1), alexa647 (1606 cm.sup.1, 1358 cm.sup.1), ATTO655 (1665 cm.sup.1) and Dylight650 (1606 cm.sup.1, 1358 cm.sup.1). FIG. 42b illustrates a 4-color quantitative pr-SRS imaging of Cy5.5 labeled EdU for newly synthesized DNA; ATTO740 immuno-labeled Fibrillarin protein of nucleolar marker; ATTO740 immuno-labeled alpha-tubulin; Alexa657 labeled AHA for newly synthesized proteins in the same HeLa cells. FIG. 42c illustrates an overlay of 4-color pr-SRS images in FIG. 42b. FIG. 42d illustrates tandem fluorescence imaging of DAPI labeled DNA; Dylight488 immuno-labeled Calnexin protein of Endoplasmic reticulum marker; Mitotracker orange labeled mitochondria.

[0338] Additional exemplary labels can be created for pr-SRS palette using various other vibrational moieties. Unlike CC, which can exhibit multiple peaks in the crowded fingerprint region, triple bonds, including alkyne or nitrile, can display a single sharp Raman peak in the wide silent window (e.g., from about 1800 to about 2800 cm.sup.1) free from cellular background. Thus pr-SRS imaging of triple bonds can greatly expand a vibrational palette with minimum cross talks. This can be non-trivial because triple bonds may need to be coupled with an electronic transition in order to gain resonance enhancement. Thus, described herein is a new family of vibrational dyes in which triple bonds can directly participate in the -conjugation systems. For example, general dye scaffolds with optimal conjugation position of triple bonds were determined, and then their absorption peaks were tuned into the pr-SRS region, ensuring both intensity and chemical specificity. To generate more vibrational colors, an isotopic edition on the triple bonds was utilized in conjunction with exquisite electron-density tuning on the -conjugation system to shift the peak frequency. The resulting 10 exemplary reporters, termed Manhattan Raman scattering (MARS) dyes (see e.g., FIGS. 43a-43d: e.g., MARS2242 element 4305, MARS2228 element 4310, MARS2215 element 4315, MARS2200 element 4320, MARS2186 element 4325, MARS2176 element 4330, MARS2159 element 4335, MARS2148 element 4340, MARS2114 element 4345 and MARS2061 element 4350), display well-resolved individual pr-SRS peaks in a cell-silent window. Except for MARS2124 whose pr-SRS spectrum can exhibit some background likely from two-photon absorption, the other 9 exemplary MARS dyes have negligible (e.g., about <15% on average) spectral cross-talk under the narrow-band (e.g., about 6 cm.sup.1) laser excitation.

[0339] Additionally, the exemplary system, method and computer-accessible medium, according to an exemplary embodiment of the present disclosure, can utilize pr-SRS that can achieve an increased detection sensitivity down to sub-micromole and high chemical specificity for multicolor imaging. For example, a narrow region of the absorption peaks of dyes can be selected that can be suitable for pr-SRS imaging, which can benefit from pre-resonance Raman enhancement, but does not suffer from any other competing pump-probe signal contributing to a large off-resonance background. With such sensitivity and specificity, 4-color imaging can be achieved by commercially available dyes with pr-SRS alone, which can already be comparable to the typical number limit in multi-color fluorescence imaging. In tandem with fluorescence microscopy, simultaneous imaging of 3 more colors can be obtained, thus, almost doubling the number limit of multicolor fluorescence imaging. This number can be expanded furthermore with custom synthesized molecules leading to more resolvable pr-SRS colors.

Exemplary SI Methods and Materials

[0340] An integrated laser (e.g., picoEMERALD with custom modification, Applied Physics & Electronics, Inc.) can be used as the light source for both pump and Stokes beams. picoEMERALD can provide an output pulse train at 1064 nm with 6 ps pulse width and 80 MHz repetition rate, which serves as the Stokes beam. The frequency-doubled beam at 532 nm can be used to synchronously seed a picosecond optical parametric oscillator (OPO) to produce a mode-locked pulse train (e.g., the idler beam of the OPO can be blocked with an interferometric filter) with 56 ps pulse width. The wavelength of the OPO can be tunable from about 720 to about 990 nm, which can serve as the pump beam. The intensity of the about 1064 nm Stokes beam can be modulated sinusoidally by a built-in electro-optic modulator (EOM) at about 8 MHz with a modulation depth of more than about 95%. The pump beam can be spatially overlapped with the Stokes beam with a dichroic mirror inside picoEMERALD. The temporal overlap between pump and Stokes pulse trains can be ensured with a built-in delay stage and optimized by the SRS signal of pure dodecane liquid.

[0341] Pump and Stokes beams can be coupled into an inverted laser-scanning microscope (e.g., FV1200MPE, Olympus) optimized for near IR throughput. An about 60 water objective (UPlanAPO/IR, 1.2 N.A., Olympus) with high near IR transmission can be used for all cellular level imaging, and a 25 water objective (XLPlan N, 1.05 N.A., MP, Olympus) with both high near IR transmission and a large field of view can be used for brain tissue and in vivo imaging. The Pump/Stokes beam size can be matched to fill the back-aperture of the objective. The forward going Pump and Stokes beams, after passing through the sample, can be collected in transmission with a high N.A. condenser lens (e.g., oil immersion, 1.4 N.A., Olympus), which can be aligned following Kohler illumination. A telescope can then be used to image the scanning mirrors onto a large area (e.g., about 10 mm by about 10 mm) Si photodiode (e.g., FDS1010, Thorlabs) to descan beam motion during laser scanning. The photodiode can be reverse-biased by about 64 V from a DC power supply to increase both the saturation threshold and response bandwidth.

[0342] A high optical density (O.D.) bandpass filter (e.g., 890/220 CARS, Chroma Technology) can be used to block the Stokes beam completely, and transmit the Pump beam only. The output current of the photodiode can be electronically pre-filtered by an about 8-MHz band-pass filter (e.g., KR 2724, KR electronics) to suppress both the 80 MHz laser pulsing and the low-frequency contribution due to laser scanning across the scattering sample. It can then be fed into a radio frequency lock-in amplifier (e.g., SR844, Stanford Research Systems) terminated with about 50 to demodulate the stimulated Raman loss signal experienced by the pump beam. The in-phase X-output of the lock-in amplifier can be fed back into the analog interface box (e.g., FV10-ANALOG) of the microscope. For all imaging, about 256 by 256 pixels can be acquired for one frame with an about 200 s of pixel dwell time (e.g., 13 s per frame) for laser scanning and about 100 s of time constant (e.g., 6 db filter) from the lock-in amplifier. For FIGS. 41a-41f and 4-color pr-SRS imaging at channels 1606 cm.sup.1 and 1626 cm.sup.1, laser powers can be Ppump=24 mW, Pstokes=48 mW. For FIGS. 41g and 41h, laser powers can be Ppump=24 mW, Pstokes=12 mW. For FIGS. 41i and 41j, laser power can be 24 mW for two-photon imaging. For 4-color pr-SRS imaging at channels 1642 cm.sup.1, 1657 cm.sup.1 and 2940 cm.sup.1 (e.g., cellular background) laser powers can be Ppump=24 mW, Pstokes=48 mW.

Exemplary SRS Spectra

[0343] Stimulated Raman scattering spectra for all fluorophores can be acquired by fixing the stokes beam laser at about 1064.2 nm, and scanning the pump laser through a designated wavelength range point by point.

Exemplary Materials

[0344] Fluorophores can include, for example: 5-Ethynyl-2-deoxyuridine (T511285, Aldrich); L-Azidohomoalanine (AHA) (C10102, Invitrogen), Click-iT Cell Reaction Buffer Kit (C10269, Invitrogen).

[0345] Fluorophores for SRS in FIG. 40a include: Benzotriazole (B11400, Sigma, max270 nm); Coumarin 153 (546186, Sigma, max422 nm); Rhodamine 6G (83697 Sigma, max528 nm); Rhodamine B (83689 Sigma, max553 nm); Sulforhodamine 101 (S7635 Sigma, max580 nm); Alexa Fluor 633 NHS Ester (A-20105, Invitrogen, max633 nm); CF640R-azide (92085, Biotium, max640 nm); Cy5-azide (A3030, Lumiprobe, max645 nm); sulfo-cy5-azide (777323 Sigma, max646 nm); Alexa647-azide (A10277, Invitrogen, max650 nm); Dylight 650 NHS Ester (62265, Thermo Scientific, max650 nm); Atto 655 azide (11774 Sigma, max660 nm); Cy5.5-azide (178, AAT-bioquest, max678 nm); ATTO680 (94875 Sigma, max680 nm); Rhodamine 800 (83701 Sigma, max680 nm); Alexa 680 NHS Ester (A-20008, Invitrogen, max680 nm); Alexa700 NHS Ester (A-20010, Invitrogen, max700 nm); ATTO700 (30674 Sigma, max700 nm); ATTO725 (47156 Sigma, max725 nm); ATTO740 (91394 Sigma, max740 nm); Alexa750 NHS Ester (A-20011, Invitrogen); Cy7-azide (A5030, Lumiprobe, max749 nm); 3,3-Diethylthiatricarbocyanine iodide (381306 Sigma, max765 nm); Cy7.5-azide (A6030, Lumiprobe, max788 nm); IR820 (543365 Sigma, max820 nm).

[0346] Other Fluorophores can include: IR895 (392375 Sigma, max895 nm); MitoTracker Orange CMTMRos (M-7510, Invitrogen); NucBlue Fixed Cell ReadyProbes Reagent (DAPI) (R37606, Invitrogen).

[0347] Primary antibodies can include: Anti-Fibrillarin antibodyNucleolar Marker (ab5821, Abcam); Anti-200 kD Neurofilament Heavy antibody (ab4680, Abcam); Anti-a-Tubulin antibody (T9026, Sigma); Anti-Giantin antibody (ab24586, Abcam); Anti-Tom20 Antibody (sc-11415, Santa Cruz Biotechnology); Anti-Calnexin antibodyER Membrane Marker (ab140818, Abcam).

[0348] Secondary antibodies conjugated with fluorophores can include, e.g.: Goat-anti-Rabbit IgG-Atto 740 antibody (49559, Sigma); Goat-anti-Mouse IgG-Atto 700 antibody (2110, Hypermol); Goat anti-Chicken IgY DyLight 488 antibody (SA5-10070, Thermo Scientific).

Exemplary Sample Preparation for Intracellular Cell Imaging

[0349] For immuno-staining cells can be fixed in methanol for about 28 min or first in about 4% PFA for about 8 min and then replaced with methanol for about 20 min more. Cells can then be washed with about 10% goat serum/1% BSA/0.3 M glycine solution twice before permealization in about 0.01% triton PBS for about 45 min. Primary antibody can then be added with about 1:200 dilution in about 3% BSA in 4 C overnight. After blocking with about 10% goat serum for about 30 min, secondary antibody conjugated with fluorophores can be added with about 1:100 dilution in about 10% goat serum in 4 C overnight.

[0350] For 7-color pr-SRS and fluorescence tandem imaging, HeLa cells can be seeded on a coverslip in a petri-dish with about 2 mL of DMEM for about 20 h, and then replaced with Methionine-deficient medium for about 30 min. Then about 1 mM AHA and about 100 M EdU can be added in to medium for about 18 hr. An about 400 nM MitoTracker Orange can be added into medium for about 30 min before fixation of the cell with about 4% PFA for about 8 min and then replaced with methanol for about 20 min more. Immuno-staining follows the procedure above. After immuno-staining, about 4 M Cy5.5-azide can be added to the cells with click-it Cell Reaction Buffer for the reaction with EdU following the manual from Invitrogen. After washing with PBS, about 4 M alexa647-alkyne can be added to the cells with click-it Cell Reaction Buffer for the reaction with AHA. At last, DAPI can be added to cells for 20 min following the instruction from manual.

[0351] DMEM was made of about 90% DMEM medium (e.g., 11965, Invitrogen), about 10% FBS (e.g., 10082, Invitrogen) and about 1 penicillin/streptomycin (e.g., 15140, Invitrogen); Methionine-deficient medium was made by supplying about 4 mM L-glutamine, about 0.2 mM L-cysteine, about 10% FBS and about 1% penicillin/streptomycin to the DMEM medium without L-methionine, L-cysteine and L-glutamine.

Exemplary Linear Combination Procedure

[0352] Because the exemplary SRS signal can be linearly dependent on the analyte concentration, the 4-channel pr-SRS signal for the concentrations of the ATTO740 labeled nucleoli Fibrillarin protein, ATTO700 labeled u-tubulin, Cy5.5 labeled EdU and Alexa647 labeled AHA can subtract the cellular background contribution calibrated from 2940 cm.sup.1 channel and can be expressed in the following the exemplary matrix:

[00005]$.Math.\left[\begin{array}{c}\mathrm{cross}.Math.\text{-}.Math.\mathrm{section}\\ \mathrm{matrix}\end{array}\right]\left[\begin{array}{c}{C}_{\mathrm{nucleoli}}\\ C_{\mathrm{tubulin}}\\ C_{\mathrm{EdU}}\\ C_{\mathrm{AHA}}\end{array}\right]=\left[\begin{array}{c}{S}_{1657}-\left(S_{2940}/1.63\right)\\ S_{1626}-\left(S_{2940}/2.45\right)\\ S_{1642}-\left(S_{2940}/5.56\right)\\ S_{1606}-\left(S_{2940}/11.46\right)\end{array}\right],\mathrm{and}.Math.\left[\begin{array}{c}\mathrm{cross}.Math.\text{-}.Math.\mathrm{section}\\ \mathrm{matrix}\end{array}\right]=\left[\begin{array}{cccc}{}_{\mathrm{ATTO}.Math..Math.740,1657}& {}_{\mathrm{ATTO}.Math..Math.700,1657}& {}_{\mathrm{Cy}.Math..Math.5.5,1657}& {}_{\mathrm{alexa}.Math..Math.647,1657}\\ {}_{\mathrm{ATTO}.Math..Math.740,1642}& {}_{\mathrm{ATTO}.Math..Math.700,1642}& {}_{\mathrm{Cy}.Math..Math.5.5,1642}& {}_{\mathrm{alexa}.Math..Math.647,1642}\\ {}_{\mathrm{ATTO}.Math..Math.740,1626}& {}_{\mathrm{ATTO}.Math..Math.700,1626}& {}_{\mathrm{Cy}.Math..Math.5.5,1626}& {}_{\mathrm{alexa}.Math..Math.647,1626}\\ {}_{\mathrm{ATTO}.Math..Math.740,1606}& {}_{\mathrm{ATTO}.Math..Math.700,1606}& {}_{\mathrm{Cy}.Math..Math.5.5,1696}& {}_{\mathrm{alexa}.Math..Math.647,1606}\end{array}\right]$

[0353] Therefore, concentration of each labeled molecules can be solved by:

[00006]$\left[\begin{array}{c}{C}_{\mathrm{nucleoli}}\\ C_{\mathrm{tubulin}}\\ C_{\mathrm{EdU}}\\ C_{\mathrm{AHA}}\end{array}\right]={\left[\begin{array}{c}\mathrm{cross}.Math.\text{-}.Math.\mathrm{section}\\ \mathrm{matrix}\end{array}\right]}^{-1}\left[\begin{array}{c}{S}_{1657}-\left(S_{2940}/1.63\right)\\ S_{1626}-\left(S_{2940}/2.45\right)\\ S_{1642}-\left(S_{2940}/5.56\right)\\ S_{1606}-\left(S_{2940}/11.46\right)\end{array}\right]$

[0354] Each fluorophore cross section number can be measured using about a 500 M solution in each channel by SRS under the same power and acquisition time as in final the cellular imaging condition. Therefore, the solved molecule concentrations can be in the unit(s) of M.

[0355] FIG. 44 illustrates exemplary raw images for 4-color pr-SRS microscopy at channels of 1606 cm.sup.1, 1626 cm.sup.1, 1642 cm.sup.1, 1657 cm.sup.1 and 2940 cm.sup.1 before linear combination algorithm retrieval.

Example 4: Super-Vibrational Multiplex Imaging

Exemplary Stimulated Raman Scattering (SRS) Microscopy.

[0356] An integrated laser (e.g., picoEMERALD with custom modification, Applied Physics & Electronics, Inc.) was used as light source for both pump and Stokes beams. Briefly, picoEMERALD provided an output pulse train at 1064 nm with 6-s pulse width and 80 MHz repetition rate, which served as the Stokes beam. The frequency-doubled beam at 532 nm was used to synchronously seed a picosecond optical parametric oscillator (OPO) to produce a mode-locked pulse train (e.g., the idler beam of the OPO was blocked with an interferometric filter) with 56 ps pulse width. The wavelength of the OPO was tunable from 720 to 990 nm, which served as the pump beam. The intensity of the 1064 nm Stokes beam was modulated sinusoidally by a built-in electro-optic modulator (EOM) at 8 MHz with a modulation depth of more than 95%. The pump beam was spatially overlapped with the Stokes beam with a dichroic mirror inside picoEMERALD. The temporal overlap between pump and Stokes pulse trains was ensured with a built-in delay stage and optimized by the SRS signal of deuterium oxide (e.g., 99.9 atom % D, 151882 ALDRICH).

[0357] Pump and Stokes beams were coupled into an inverted laser-scanning microscope (e.g., FV1200MPE, Olympus) optimized for near IR throughput. A 25 water objective (e.g., XLPlan N, 1.05 N.A., MP, Olympus) with both high near IR transmission and large field of view was used for measurements of all solutions, cells and tissues unless specified. A 60 water objective (e.g., UPlanAPO/IR, 1.2

[0358] N.A., Olympus) with high near IR transmission was used for X-gal assay and live-cell 8-color imaging. The pump/Stokes beam size was matched to fill the back-aperture of the corresponding objectives for imaging. The forward going pump and Stokes beams after passing through the samples were collected in transmission with a high N.A. condenser lens (e.g., oil immersion, 1.4 N.A., Olympus), which was aligned following Kohler illumination. A telescope was then used to image the scanning mirrors onto a large area (e.g., 10 mm by 10 mm) Si photodiode (e.g., FDS1010, Thorlabs) to descan beam motion during laser scanning. The photodiode was reverse-biased by 64 V from a DC power supply to increase both the saturation threshold and response bandwidth. A high O.D. bandpass filter (e.g., 890/220 CARS, Chroma Technology) was used to block the Stokes beam completely and transmit the pump beam only. The output current of the photodiode was electronically pre-filtered by an 8 MHz band-pass filter (e.g., KR 2724, KR electronics) to suppress both the 80 MHz laser pulsing and the low-frequency contribution due to laser scanning across the scattering sample. It was then fed into a radio frequency lock-in amplifier (e.g., SR844, Stanford Research Systems or HF2LI, Zurich instrument) terminated with 50 to demodulate the stimulated Raman loss signal experienced by the pump beam. The in-phase X-output of the lock-in amplifier was fed back into the analog interface box (e.g., FV10-ANALOG) of the microscope. All laser powers were measured after objective lens. For spectroscopy measurement in FIG. 45, PStokes=18.4 mW. For detection sensitivity measurement, laser powers were Ppump=24 mW, PStokes=184 mW. For cellular and tissue imaging, the range of laser powers used were Ppump=7-24 mW, PStokes=46-120 mW. Pixel dwell times for all images were ranging from the fastest 4 s (e.g., 0.4 s per 320-by-320 frame, FIG. 45c-e) to the slowest 200 s (e.g., 20.5 s per 320-by-320 frame, FIG. 46c-d, FIG. 48b-d). Time constants of lock-in amplifier were chosen to be about two times shorter than the pixel dwell time. All fluorescence images were acquired by commercial Olympus FV1200MPE microscope equipped with 488 nm, 543 nm and 633 nm CW lasers. Two-photon fluorescence was excited by the tunable pump laser at designated wavelengths and detected by Non-descanned PMTs. For two-photon fluorescence imaging of Nucblue (e.g., FIGS. 2 and 4), it can be excited by OPO laser at 780 nm, and the fluorescence was detected after passing through a 690 nm short pass filter, a 570 nm short pass dichroic and a 450/40 nm band-pass filter (e.g., Thorlabs, FB450-40). The detections for two-photon fluorescence signals of Nucblue in the UV/blue and of epr-SRS in the near IR are still orthogonal to each other with no issue of signal overlapping. For two-photon fluorescence imaging of Alexa647, it can be excited by OPO laser around 810 nm, and the fluorescence was detected after passing through a 690 nm short pass filter, a 570 nm short pass dichroic.

Exemplary Stimulated Raman Scattering (SRS) Spectra Acquisition

[0359] Stimulated Raman scattering spectra for all dyes were acquired by fixing the Stokes beam at 1064 nm, and scanning the pump beam through the designated wavelength range point by point.

Exemplary Linear Combination Procedure

[0360] Since epr-SRS signals are linearly dependent on analyte concentrations, quantitatively distinguishing different dyes with overlapped Raman bands could be performed through a simple and robust linear combination procedure. The measured N-channel epr-SRS signals (S) for N dyes could be expressed as the products of a NN dye Raman cross-section matrix (M) and dye concentrations (C), with addition of the cellular amide backgrounds in each channels (B):

S=M.Math.C+B

[0361] Dye concentrations were thus solved by, for example:

[00007]$.Math.C=M^{-1}.Math.\left(S-B\right)$$.Math.C.Math.\text{?}:S.Math.\text{?}:B.Math.\text{?}:M=\text{?}..Math.\text{?}.Math.\text{indicates text missing or illegible when filed}$

For FIG. 2c-d of epr-SRS in fingerprint region, B was achieved by acquiring a reference image at the label-free channel of 2940 cm.sup.1 for total proteins and then scaling it as backgrounds at individual epr-SRS channels. The scaling factors were determined in reference HeLa cells without labels under the corresponding imaging conditions. For FIG. 48 with MARS dyes, B was vanishing and the equation becomes:

C=M.sup.1.Math.S

[0362] The procedure was implemented by a Matlab program.

[0363] Protein Labeling with Dye NHS Esters.

[0364] 1) Conjugation to secondary antibodies: Dye NHS esters were dissolved in DMSO to a concentration of 2 mg/mL. Secondary antibodies were dissolved/diluted to 2 mg/mL in PBS buffer of a pH of 8.3, adjusted with sodium bicarbonate buffer. For antibodies purchased with concentrations lower than 2 mg/mL, proteins were first concentrated with Amicon Ultra Centrifugal Filters (e.g., UFC501096, EMD, Millipore). A 50 L dye-NHS solution was slowly added to a 0.5 mL protein solution. Reaction was incubated at RT for 60 min under constant but slow stirring. The labeled proteins could be separated from unreacted dye NHS esters by gel permeation chromatography with Sephadex G-25 (e.g., G25150 SIGMA) with a column of diameter around 1 cm and length longer than 12 cm. Sephadex G-25 was first swelled in PBS buffer at 90 C. water bath for 1 h. After gel settling down in room temperature, buffer was exchange to fresh PBS buffer and gels were stored at 4 C. For gel chromatography, column was packed with swelled gels, and then equilibriumed with PBS. The labeled protein solution was then loaded and eluted with PBS buffer. First band with light color for dye conjugated proteins was collected. This solution was then centrifuged and supernatant was collected and concentrated with Amicon Ultra Centrifugal Filters (e.g., UFC501096, EMD, Millipore) into a final concentration of 1-2 mg/mL in PBS with 30% glycerol and 5 mM sodium azide and stored at 20 C.

[0365] 2) Conjugation to Wheat Germ Agglutinin (e.g., WGA, L0636 SIGMA): All procedures were the same as described above except the dye-protein ratio: a 17.5 L 2 mg/ml dye-NHS solution was slowly added to a 0.5 mL 2 mg/ml WGA solution.

[0366] Sample information (e.g., cell, tissues, antibodies and chemical reagents).

[0367] All samples were made into imaging chambers using an imaging spacer (e.g., GBL654008 SIGMA) between a coverslip and a microscope slide. The chamber was filled with PBS for imaging. All imaging results were experimentally replicated for more than 5 times.

[0368] Cell lines: Cell lines were purchased from ATCC: HeLa (e.g., ATCC CCL-2); MCF7 (e.g., ATCC HTB-22); HEK 293T/17 (e.g., ATCC CRL-11268). These cell lines were authenticated and mycoplasma negative. Cell medium: DMEM medium was made of 90% DMEM (e.g., 11965, Invitrogen), 10% FBS (e.g., 10082, Invitrogen) and 1 penicillin/streptomycin (15140, Invitrogen). Methionine-deficient DMEM medium was made by supplying 4 mM L-glutamine, 0.2 mM L-cysteine, 10% FBS and 1% penicillin/streptomycin to DMEM without L-methionine, L-cysteine and L-glutamine (e.g., D0422 SIGMA).

[0369] Mice and tissues: The animal experimental protocol (e.g., AC-AAAQ2457) was approved by the Institutional Animal Care and Use Committee at Columbia University. 400 m thick cerebellar brain slices from P11 wild-type (e.g., C57BL/6) mice of either sex were cultured with Methionine-deficient brain slice culture medium containing 1 mM AHA and 100 M EdU for 17 h. Organotypic slices were then fixed with 4% PFA for 30 min, followed with 0.5% Triton permeabilization for overnight at 4 C. No statistical method was used to predetermine sample size. All experimental procedures were performed in a non-blinded manner and no randomization was applied.

[0370] Antibodies: Primary antibodies for single-color labeling: Anti-Fibrillarin antibodyNucleolar Marker in rabbit (e.g., ab5821,Abcam); Anti-200 kD Neurofilament Heavy antibody in rabbit (e.g., ab8135, Abcam); Anti-a-Tubulin antibody in rabbit (e.g., ab18251, Abcam); Anti-Tom20 Antibody in rabbit (e.g., sc-11415, Santa Cruz Biotechnology); Anti-Giantin antibody in rabbit (e.g., ab24586, Abcam); Anti-Cytokeratin 18 antibody in mouse (e.g., ab7797, Abcam); Anti-CD44 antibody in mouse (e.g., ab6124, Abcam); Anti-EpCAM antibody in rabbit (e.g., ab71916, Abcam); Anti-IGF1 Receptor beta subunit antibody in mouse (e.g., ab80547, Abcam). For multicolor labeling: Anti-Fibrillarin antibodyNucleolar Marker in mouse (e.g., ab4566, Abcam); Anti-a-Tubulin antibody in chicken (e.g., ab89984, Abcam); Anti-Giantin antibody in rabbit (e.g., ab24586, Abcam); Anti-III-tubulin in chicken (e.g., ab107216, Abcam); Anti-Myelin Basic protein in mouse (e.g., ab62631, Abcam); Anti-GFAP in goat (e.g., ab53554, Abcam); Anti-Nestin in rat (e.g., ab81462, Abcam); Anti-NeuN in rabbit (e.g., ab177487, Abcam). Secondary antibodies: Goat-anti-rabbit ATTO740 antibody (e.g., 49559, Sigma); Goat-anti-mouse ATTO740 antibody (e.g., 2111, Hypermol); Goat-anti-rabbit ATTO700 antibody (e.g., 2310, Hypermol); Goat anti-chicken CF640R antibody (e.g., 20084, Biotium); Donkey-anti-rabbit Alexa568 (e.g., ab175692, Abcam); Donkey-anti-rat Alexa488 (e.g., abl50153, Abcam). Donkey-anti-mouse antibody (e.g., Sigma, SAB3701101), Donkey-anti-chicken (e.g., Invitrogen, SA172002) and Donkey-anti-goat antibody (e.g., Sigma, G6638) were conjugated with MARS dyes.

Fluorophores: ATTO740 NHS Ester (e.g., 59808 SIGMA), IR895 (e.g., 392375 SIGMA).

[0371] Experimental Procedures for Epr-SRS Imaging

[0372] Imaging newly synthesized DNA in HeLa cells by metabolic incorporation of 5-Ethynyl-2-deoxyuridine (e.g., EdU, T511285, Aldrich) and detection with ATTO740-azide (e.g., FIG. 45c-e):

[0373] HeLa cells were seeded on a coverslip in a petri-dish with DMEM for 20 h, and then replaced with DMEM medium without FBS for another 20 h for synchronization. 10 M EdU in fresh DMEM medium were then added to cells for 15 h. Cells were fixed with 4% PFA for 20 min, permeabilized with 0.5% Triton permeabilization for 10 min. 1 M ATTO740-azide (e.g., AD 740-101, ATTO-TEC) in Click-iT Cell Reaction Buffer (e.g., C10269, Invitrogen) was then added to cells to react with EdU for 20 min. Cells were washed twice with PBS before imaging.

[0374] Immuno-Staining for Single-Color Epr-SRS Imaging of Fixed HeLa or MCF7 Cells (e.g., FIG. 45f-h):

[0375] HeLa/MCF7 cells were seeded on a coverslip in a petri-dish with 2 mL of DMEM for 20 h, and then fixed with 4% PFA for 20 min or methanol for 25 min. After fixation, cells were washed with 10% goat serum/1% BSA/0.3 M glycine solution twice before permeabilization in 0.1% Tween PBS for 40 min. Primary antibody was subsequently added by 1:200 dilution in 3% BSA at 4 C. overnight. After blocking with 10% goat serum for 30 min, secondary antibody conjugated with fluorophores was added by 1:100 dilution in 10% goat serum at 4 C. overnight. The samples were blocked with 10% goat serum for 30 min before imaging.

[0376] Epr-SRS Imaging of SiR-SNAP-Tagged H2B Proteins (e.g., FIG. 45i):

[0377] HEK293T cells were seeded on a coverslip in a petri-dish with DMEM for 20 h before transfected with 250 ng SNAP-H2B plasmid (e.g., New England BioLabs) for 48 h. 10 M SNAP-SiR (e.g., S9102S, New England BioLabs) were then added to medium for 45 min at 37 C. Cells were washed 3 times with PBS before imaging.

[0378] Epr-SRS Imaging of Mitotracker Deep Red (e.g., FIG. 45j):

[0379] 500 nM MitoTracker deep red (e.g., M22426, Invitrogen) in HBSS was added to cells for 20 min at 37 C. Cells were washed twice with PBS before imaging.

[0380] Epr-SRS Imaging of Methylene Blue (e.g., FIG. 45k):

[0381] 10 M methylene blue (e.g., M9140 SIGMA-ALDRICH) in PBS was added to cells for 20 min at 37 C. Cells were washed twice with PBS before imaging.

[0382] Epr-SRS imaging of the indigo product from X-gal gene expression assay (e.g., FIG. 45l): BL21 E. Coli were grown in LB to log phase before adding 100 M X-gal (e.g., XGAL-RO ROCHE) and 1 mM IPTG (e.g., 16758 SIGMA-ALDRICH) for 20 min at 37 C. E. coli were then concentrated and washed with PBS before imaging.

[0383] 8-Color Epr-SRS and Fluorescence Imaging of Fixed HeLa Cells (e.g., FIG. 46c):

[0384] HeLa cells were seeded on a coverslip in a petri-dish with DMEM for 20 h, and then replaced with methionine-deficient DMEM medium for 30 min. 1 mM L-Azidohomoalanine (e.g., AHA, C10102, Invitrogen) and 100 M EdU were then added into medium for 18 h. 400 nM MitoTracker Orange CMTMRos (e.g., M-7510, Invitrogen) was added into medium for 30 min and 2 M Alexa 488-WGA (e.g., W11261, Invitrogen) was added together for the last 15 min before fixation of the cells with 4% PFA for 8 min followed with methanol for 20 min. The following immuno-staining procedures were same as described above. After secondary-antibody incubation, samples were blocked with 10% goat serum for 30 min. Then 5 M Cy5.5-azide (e.g., 178, AAT-Bioquest) with Click-iT Cell Reaction Buffer was added to cells to react with EdU for 20 min. After washing with PBS, 2.5 M Alexa 647-alkyne (e.g., A10278, Invitrogen) with Click-iT Cell Reaction Buffer was added to cells to react with AHA for 20 min. Lastly, NucBlue Fixed Cell ReadyProbes Reagent (e.g., R37606, Invitrogen) was added to cells for 10 min. Cells were washed with PBS before imaging.

[0385] 8-Color Epr-SRS and Fluorescence Imaging of Live HeLa Cells (e.g., FIG. 46d):

[0386] HeLa cells were first seeded on coverslips in petri dishes with DMEM culture medium at 37 C. for 24 h. ER-GFP (e.g., C10590, Invitrogen) and Actin-RFP (e.g., C10583, Invitrogen) plasmids were transfected into cells for 48 h following the protocol from Invitrogen. 500 M oleic acid (e.g., 01383 SIGMA) coupled with BSA in DMEM culture medium was then added to cells for 7 h to induce the formation of lipid droplets. Before imaging, cells were first incubated with 6 M SYTO60 (e.g., S11342, Invitrogen), 120 nM LysoTracker Deep Red (e.g., L12492, Invitrogen) and 400 nM Rhodamine 800 (e.g., 83701 SIGMA) in HBSS simultaneously for 30 min at 37 C., followed by staining with NucBlue Live ReadyProbes Reagent (e.g., R37605, Invitrogen) in HBSS for 20 min at 37 C. Then cells were incubated with ATTO 740-conjugated WGA in HBSS for 30 min at 37 C., followed by staining with LipidTOX Deep Red (e.g., H34477, Invitrogen) in HBSS with a dilution of 1:20 for 30 min at room temperature before imaging.

[0387] 16-Color Epr-SRS and Fluorescence Imaging of Live HeLa Cells (e.g., FIG. 48a):

[0388] HeLa cells were first seeded in 24-well dish for 20 h. Each well of cells were labeled with a single color of epr-SRS or fluorescent dyes for 30 min in PBS: MARS2237 (e.g., 1 M), MARS2228 (e.g., 4 M), MARS2209 (e.g., 1 M), MARS2200 (e.g., 4 M), MARS2183 (e.g., 1 M), MARS2176 (e.g., 4 M), MARS2154 (e.g., 1 M), MARS2147 (e.g., 4 M), ATTO OXA12 (e.g., 10 M), Cy5.5 (e.g., 2 M), MitoTracker deep red (e.g., 2.5 M), SYTO60 (e.g., 10 M). FM 4-64 (e.g., T13320, Invitrogen, 20 g/ml), MitoTracker orange (e.g., M7510, Invitrogen, 400 nM), Alexa488-WGA (e.g., W11261, Invitrogen, 2 g/ml), Nucblue (e.g., R37605, Invitrogen). Cells were then washed with PBS and detached from each well by trypsin treatment for 2 min; then mixed together in fresh DMEM medium before gently centrifuge (e.g., 1000 rpm, 1.5 min) to pellet. PBS was added to the pellet and after gentle mix; cells were sandwiched in imaging chamber. After settling down for 15-20 min on imaging stage to reduce cell movement, images were acquired. Single dye staining and 2-color mix were first examined and no cross staining between cells was found during the imaging period (e.g., i.e. each cell was maintained with a single pre-stained color). Obvious stage drift was not observed during 16-color image acquisitions. FIG. 48a was acquired through sequential imaging in 15 minutes.

[0389] 8-Color Epr-SRS and Fluorescence Imaging of Hippocampal Neuronal Cultures and Cerebellar Brain Slices (e.g., FIG. 48b-c):

[0390] Regular hippocampal neuron medium was made of Neurobasal A Medium (e.g., 10888, Invitrogen), 1B27 serum free supplement (e.g., 17504, Invitrogen) and 0.5 mM glutamine (e.g., 25030, Invitrogen). Methionine-deficient neuron medium was custom made from regular recipe of Neurobasal A medium (e.g., 10888, Invitrogen) without methionine, and supplied with 1B27 serum free supplement and 0.5 mM glutamine. Methionine-deficient medium for organotypic cerebellar brain slices was made by supplying methionine-deficient neuron medium with 1B27 serum free supplement, 2 mM glutamine, 0.5% glucose and 1% penicillin/streptomycin. Regular medium in D9 hippocampal neuronal cultures was first replaced by methionine-deficient medium containing 1 mM AHA and 100 M EdU for 17 h. Cells were then fixed with 4% PFA for 20 min, followed with 0.5% Triton permeabilization for 15 min. Primary antibodies were then simultaneously added with 1:120 dilution in 3% BSA for overnight at 4 C. After blocking with 10% donkey serum for 30 min, secondary antibodies conjugated with fluorophores were added with 1:80 dilution in 10% donkey serum for overnight at 4 C. Samples were blocked with 10% donkey serum for 30 min with NucBlue Fixed Cell ReadyProbes Reagent added for the last 10 min. Then 1 M MARS2228-azide with Click-iT Cell Reaction Buffer was added to cells/slices to react with EdU for 20 min. After washing with PBS, 12 M Alexa 647-alkyne with Click-iT Cell Reaction Buffer was added to react with AHA for 20 min. Cells were washed with PBS before imaging. 8-color epr-SRS and fluorescence imaging in neuronal cultures (e.g., FIG. 48d):

[0391] Regular Medium of D12 hippocampal neuronal cultures was replaced by methionine-deficient medium containing 1 mM L-Homopropargylglycine (e.g., HPG, C10186, Invitrogen) for 12 h. The medium was then exchanged to fresh methionine-deficient medium containing 1 mM AHA with or without 10 M MG132 (e.g., M7449 SIGMA) for another 10 h. Cells were fixed with 4% PFA for 20 min, permeabilized with 0.5% Triton for 15 min. The immuno-staining procedures were the same as described above. Then 1.25 M MARS2228-azide with Click-iT Cell Reaction Buffer was added to cells/slices to react with HPG for 20 min. After washing with PBS, 4 M Alexa 647-alkyne with Click-iT Cell Reaction Buffer was added to react with AHA for 20 min. Cells were washed with PBS before imaging.

[0392] Image Statistics for FIG. 48e.

[0393] 6 technical replicates of 8-colour images were acquired from 3 independent neuronal co-cultures (e.g., the same condition as described above for FIG. 48d) for statistical presentation. For each neuronal co-culture, 2 fields of view were selected close to the center of the culture dish to maintain consistency. All images were taken under the same imaging parameters. Cross-checking 8-colour staining, the percentages of astrocytes with aggregations (e.g., the number of astrocytes with aggregations divided by the total number of astrocytes) and the percentages of neurons with aggregations (e.g., the number of neurons with aggregations divided by the total number of neurons) were calculated for each image sets. Their means and standard deviations are then presented in FIG. 48e, together with a P value from two-sided Student's t-test.

[0394] Live/dead cell viability assay: It can be performed using the LIVE/DEAD viability/cytotoxicity kit for mammalian cells (e.g., Molecular Probes L-3224). HeLa cell standards and HeLa cells with dye staining or SRS pre-exposure were incubated with 2 M calcein AM and 4 M EthD-1 working solution for 20 minutes at 37 C. before imaging.

[0395] Data and code availability All data supporting this study are available from the corresponding author on request.

[0396] As shown in FIG. 49, for example, a computer-accessible medium 4915 (e.g., as described herein above, a storage device such as a hard disk, floppy disk, memory stick, CD-ROM, RAM, ROM, etc., or a collection thereof) can be provided (e.g., in communication with the processing arrangement 4905) to facilitate the various exemplary embodiments of the present disclosure, including for detecting at least one condition of at least one disease. The computer-accessible medium 4915 can contain executable instructions 4920 thereon. In addition or alternatively, a storage arrangement 4925 can be provided separately from the computer-accessible medium 4915, which can provide the instructions to the processing arrangement 4905 so as to configure the processing arrangement to execute certain exemplary procedures, processes and methods, as described herein above, for example.

[0397] Further, the exemplary processing arrangement 4905 can be provided with or include an input/output arrangement 4935, which can include, for example a wired network, a wireless network, the internet, an intranet, a data collection probe, a sensor, etc. As shown in FIG. 49, the exemplary processing arrangement 4905 can be in communication with an exemplary display arrangement 4930, which, according to certain exemplary embodiments of the present disclosure, can be a touch-screen configured for inputting information to the processing arrangement in addition to outputting information from the processing arrangement, for example. Further, the exemplary display arrangement 4930 and/or a storage arrangement 4925 can be used to display and/or store data in a user-accessible format and/or user-readable format.

[0398] The foregoing merely illustrates the principles of the disclosure. Various modifications and alterations to the described exemplary embodiments will be apparent to those skilled in the art in view of the teachings herein. It will thus be appreciated that those skilled in the art will be able to devise numerous systems, arrangements, and procedures which, although not explicitly shown or described herein, embody the principles of the disclosure and can be thus within the spirit and scope of the disclosure. Various different exemplary embodiments can be used together with one another, as well as interchangeably therewith, as should be understood by those having ordinary skill in the art. In addition, certain terms used in the present disclosure, including the specification, drawings and claims thereof, can be used synonymously in certain instances, including, but not limited to, e.g., data and information. It should be understood that, while these words, and/or other words that can be synonymous to one another, can be used synonymously herein, that there can be instances when such words can be intended to not be used synonymously. Further, to the extent that the prior art knowledge has not been explicitly incorporated by reference herein above, it is explicitly incorporated herein in its entirety. All publications referenced are incorporated herein by reference in their entireties.

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