Supercontinuum microscope for resonance and non-resonance enhanced linear and nonlinear images and time resolved microscope for tissues and materials

Abstract

Supercontinuum (SC) (400 nm to 2500 nm) and a microscope produce enhanced microscopic images on sub-micron to cm scale of linear (.sub.1) and nonlinear (.sub.2, .sub.3, .sub.4 . . . ) processes via resonance including linear absorption, SHG, THG, SRG, SRL, SRS, 2PEF, 3PEF, 4PEF, and inverse Raman in a microscope for 2D and 3D imaging. Images and processes in 2D and 3D arise from electronic and vibrational resonances transitions in biological and medical tissues, cells, condensed matter applications. Resonant Stimulated Raman Scattering (RSRS) is proposed to improve vibrational imaging of biomaterials by using part of SC. Quantum mechanical processes from SC for 2 and 4 photons to improve resolution and imaging using entangled photons are described. The addition of time measuring instrument like a Streak camera and the scattering coefficient .sub.s can be mapped to create images of tissue and biomaterial in 5D: Space (3D), Time, and Wavelength.

Claims

1. Imaging apparatus comprising a microscope; a source of supercontinuum (SC) light, said microscope comprising an objective lens; optical narrow and wide band filters to provide SC pump and probe laser wavelengths configured to direct through said objective lens to a sample to be imaged having a scattering transport coefficient .sub.s; an optical detector; an optical splitter configured for directing said SC light towards a sample and directing the SC light reflected from the sample including linear and non-linear light components representative of the sample to said optical detector; imaging means for converting an output of said optical detector including said linear and non-linear components into signals corresponding to said linear and non-linear components utilizing at least one of electronic and vibrational resonances through said microscope; and a streak camera for time resolve data from a sample; and an XYZ scanner for scanning said SC light in selected ones of X, Y and Z directions prior to directing said SC light into said microscope for creation of 2D xy image slices at depth z and 3D by adding up z slices from 2D planes and time adds to 4D and wavelength from SC give 5D maps with .sub.s.

2. Imaging apparatus comprising a microscope; a source of supercontinuum (SC) light, said microscope comprising an objective lens; optical narrow and wide band filters to provide SC pump and probe laser wavelengths configured to direct through said objective lens to a sample to be imaged having a scattering transport coefficient .sub.s; an optical detector; an optical splitter configured for directing said SC light towards a sample and directing the SC light reflected from the sample including linear and non-linear light components representative of the sample to said optical detector; imaging means for converting an output of said optical detector including said linear and non-linear components into linear and non-linear signals utilizing at least one of electronic and vibrational resonances through said microscope, wherein said microscope is configured to image 5D tomography images: space (xyz), time (t) and wavelength () maps of tissue wherein time resolved the transport scattering length L.sub.tr=1/.sub.s at xy position at z for give wavelength, of SC for an image map of tissue obtained from I(t) scattered profile in time, where I(t) is the intensity of the signals as a function of time.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) The above and other aspects, features and advantages of the present invention will be more apparent from the following description when taken in conjunction with the accompanying drawings, in which:

(2) FIG. 1 illustrates Four (4)-Wave phase K matching triangle;

(3) FIG. 2 illustrates angular phase matching showing 4 photons coupling (2 Laser and Stokes and Anti-Stokes) angular emission vs wavelengths of the entangle 4 photons;

(4) FIG. 3 illustrates the angular emission 4 photon ring on slit of spectrometer;

(5) FIG. 4 illustrates linear and nonlinear optical effect second harmonic generation (SHG) and third harmonic generation (THG) processes energy level diagrams;

(6) FIG. 5 illustrates enhanced three photon emission in real states (v=real state);

(7) FIG. 6 illustrates intensity of SC for NLO resonance and non-resonances at second harmonic (SH), third harmonic (TH), Stimulated Raman, hyper-stimulated Raman. (2 photon), and (3 photon), Inverse Raman;

(8) FIG. 7 illustrates an SC microscope apparatus (major components) in accordance to the invention;

(9) FIG. 8 illustrates a time resolved SC streak camera microscope system combined with spectral imaging.

DETAILED DESCRIPTION

(10) Four Photon Entanglement Quantum Imaging for Deep Imaging

(11) The generation of light over the visible region in an angular pattern is another striking nonlinear optical effect observed when intense 532 nm picosecond laser pulses are passed through materials at different angles. This angular emission [FIGS. 1 and 2] is shown to result from the coupling of four photons via the nonlinear coefficient n.sub.2(.sup.3) to be entangled.

(12) This 4-wave process has been called four-photon parametric amplification or light by light scattering for quantum optical microscope for nm scale.

(13) Entanglement of 2 or more photons is based on quantum mechanisms and forms ways of different quantum information methods and imaging. Two photons are typically used on photon level requiring long processing and averaging times. The need for high intensity photon approach of coupled entangled photons using higher correlated photons is needed. Here we propose 4 wave approach from .sub.3 in angular and collinear generator arrangement. The coupled twins of Stokes and antiStokes with 2 pump and 2 laser photons are entangled at well defined angle of high photon levels to be used for various quantum information protocols such as cryptography and computation, loudly and non loudly in microscope or free space. Signal averaging times are reduced of 4-photon coupling.

(14) Nondegenerate four-photon stimulated emission (.sub.1.sub.2.sub.3.sub.4) in many materials originating from scale filaments created under high power 530 nm picosecond-pulse excitation [1]. The four-photon coupling process originates through the distortion of the atomic configuration inside the filaments in the materials. Positive and negative SPM frequency components are generated inside the filaments. The frequency-swept photons and laser photons are coupled to the laser field via the third-order susceptibility .sup.3 or the intensity-dependent dielectric refractive index coefficient n.sub.2. The four-photon process is of the type K.sub.L+K.sub.L={right arrow over (K)}.sub.A+{right arrow over (K)}.sub.S schematically depicted in FIG. 1 where K.sub.L, {right arrow over (K)}.sub.A, {right arrow over (K)}.sub.S are the wave vectors of the laser beam, Stokes-shifted photons, and anti-Stokes-shifted photons, respectively. The maximum amplification of the weak waves occurs along an angular direction governed by phase match among the four photons (See FIG. 2 and FIG. 3). These four photons (2 laser, Stokes, and anti-Stokes) are entangled over large number of wavelengths and angles in 4 wave triangle (FIG. 1) show 4 photons entanglement (2L, s and as). These 4 correlated photons form a new imaging tool for entangle images with intense beam of entangled photons.

(15) Resonance Linear and Nonlinear Effects

(16) There are no teachings on the use of SC source microscope applications for resonant effects in linear, SHG, THG, FHG (Fourth Harmonic generation), SRS (Stimulated Raman Scattering), 2PEF (2 Photon Excitation Fluorescence), 3PEF (3 Photon Excitation Fluorescence), 4PEF (4 Photon Excitation Fluorescence), and inverse Raman effect where frequencies in SC make transitions to electronic and vibrational states. Background theory for resonant and non-resonant SC can be used to enhance linear and non-linear optical effects via susceptibilities .sub.1, .sub.2, .sub.3, and .sub.4 when frequencies are close to transitions for enhanced microscope images. In the past non electronic resonant process for pumping and probing beams were used for imaging, such as CARS, SRS, and Multi photons in .sub.1, .sub.2, .sub.3, and .sub.4.

(17) SC can be used to probe enhanced linear and non-linear effects via the denominators in quantum mechanical description of 's via polarization P. Linear and nonlinear optical effect processes energy level diagrams are shown in FIG. 4 and FIG. 5.

(18) The polarization P is expanded in series of E, electric field of laser:
P=.sub.1E+.sub.2E.sup.2+.sub.3E.sup.3+.sub.4E.sup.4+(1)

(19) The susceptibilities .sub.1, .sub.2, .sub.3, .sub.4 . . . give size of optical effect, with the resonances appear in denominators for electronic and vibrational states. The SC covers resonance process for imaging.

(20) Using Quantum Mechanics Perturbation Theory:

(21) Linear Effects

(22) .sup.1 between ground g and excited states m:

(23) $\begin{array}{cc}{}^1\left({}_p\right)=\underset{m}{.Math.}\frac{{}_{\mathrm{gm}}{}_{mg}}{{}_{mg}-{}_p},& \left(2\right)\end{array}$
where .sub.mg=.sub.mg.sup.oe.sub.m, is dipole moment, and .sub.p is light frequency in SC. When .sub.mg.sup.o=.sub.p there is a resonance, .sup.1 is large and there is absorption.
Second Order Effects

(24) .sup.2such as SHG imaging. The equation .sup.2 is:

(25) $\begin{array}{cc}{}^2\left({}_p,{}_q\right)=\underset{\mathrm{mn}}{.Math.}\frac{{}_{\mathrm{gn}}{}_{nm}{}_{mg}}{\left({}_{\mathrm{ng}}-{}_p-{}_q\right)\left({}_{mg}-{}_p\right)},& \left(3\right)\end{array}$
where =.sub.p+.sub.q e, .sub.ng is dipole transition between grand g and excited state n (real or virtual).

(26) For SHG =.sub.p+.sub.q=2.sub.q if p=q. When .sub.p, .sub.q, .sub.p+q match a real state, .sub.mg.sup.o or .sub.ng.sup.o, the denominator get small and close to 0 and .sup.2 get large and SHG increases, say for collagen .sub.ng340 nm, flavins .sub.ng440 nm, and Tryptophan .sub.ng290 nm, where

(27) ${}_{\mathrm{ng}}={}_{\mathrm{ng}}^0-i\frac{{}_m}{2}.$
The SC spectra for .sup.2 SHG can tell which molecule is activated, such as collagen, Flavin, Carotene, and others in skin and brain tissue. For SHG:

(28) $\begin{array}{cc}{}^2\left({}_p,{}_q\right)=\underset{\mathrm{mn}}{.Math.}\frac{{}_{\mathrm{gn}}{}_{nm}{}_{mg}}{\left({}_{\mathrm{ng}}-2{}_p\right)\left({}_{mg}-{}_p\right)},& \left(4\right)\end{array}$
SHG resonance for real states at .sub.ng=2.sub.qp in the SC for highlight regions in sample images under microscope.
Third Order Effects

(29) .sup.3 is THG microscopy, 2PEF, and SRS. .sup.3 is written in 4 terms:

(30) $\begin{array}{cc}{}^3\left({}_{},{}_r,{}_p,{}_q\right)\underset{\mathrm{mnv}}{.Math.}\frac{{}_{\mathrm{gv}}{}_{\mathrm{vn}}{}_{nm}{}_{\mathrm{ng}}}{\left({}_{\mathrm{vg}}-{}_{}\right)\left({}_{\mathrm{ng}}-{}_q-{}_p\right)\left({}_{\mathrm{ng}}-{}_p\right)},+\mathrm{where}{}_{\mathrm{eg}}={}_{\mathrm{eg}}^0-i\frac{{}_i}{2},& \left(5\right)\end{array}$
.sub.eg is dipole transition between i and g states and .sub.=.sub.p+.sub.q+.sub.r pumps. For .sub.p=.sub.q=.sub., .sub.=.sub.3.sub.p is THG term where .sub.p is pump portion of SC. So, when .sub.vq=3.sub.p=.sub.vq.sup.o then THG is enhanced in FIG. 5 in real states

(31) $\begin{array}{cc}{}_{\mathrm{vq}}={}_{\mathrm{vq}}^0-i\frac{{}_v}{2}={}_{}& \left(6\right)\end{array}$
Using SC there are resonance in real states, there will be enhance at SHG and THG to determine the type of 2P and 3P images in tissues, see FIG. 6 for resonant and non-resonant flat background. There may be peaks from Hyper Raman (2.sup.nd and 3.sup.rd order) and Hyper Rayleigh (2.sup.nd and 3.sup.rd orders) process from vibrations and index of refraction. There may be absorption in SC from excited state transitions in singlet Sn and triple Tn states NLO absorptions and absorption at antiStokes from vibrations in a sample to images as loss of SC at vibrations due to Inverse Raman events [2]. There may resonances from electronic states to enhance vibrational states for Resonant Stimulated Raman Scattering (RSRS) effect.

(32) Examples for images from key molecules of tissues, cells and smears from brain, breast, cervix and other cancers for:

(33) SHG collagen (380 nm)+SC (760 nm)=SHG380 nm increase (.sup.2)

(34) THG Tryptophan (296 nm)+SC (800 nm)=THG296 nm increase (.sup.3)

(35) 3 photon excitation fluorescence (3PEF) Tryptophan+SC=800 nm to 850 pump emission 340 nm; and

(36) 2 photon excitation fluorescence (2PEF) Flavins+SC=500 nm emission (.sup.3)

(37) Inverse Raman in SC from CH.sub.3 (Carbon-H Bond) and CH.sub.2 (Carbon-H Bond) from lipids and proteins in tissue sample.

(38) RSRS from Flavins and carotene from singlet states enhance SR Loss and Gain imaging.

(39) Narrow-band (NB) and wide band (WB) filters are used to select pumping zones for enhanced signal images of molecules in tissues and cells in selected spectral areas of SC in FIG. 6.

(40) When the pump in SC matches real states at frequency in materialssolids, liquids, tissues and cells, chemical and/or condensed matter of microscope images, a 10 to 100 objectives are used. The invention provides a new ultrahigh resolution imaging method and device to use SC light as source in a microscope for medical, biological, and condensed matter to determine which molecule types are in resonance from SC spectrum. The major components of the SC microscope apparatus are shown in FIG. 7. The invention teaches the coupling a SC with optical microscope to obtain 2D and 3D images of molecule fingerprints for SHG, THG, 2PEF, 3PEF and linear absorption images of cells and inside cells for DNA and RNA and components inside cells using .sub.1, .sub.2, .sub.3, .sub.4 processes. Resonances are from electronic and vibrational states. The inverse anti stokes Raman will yield loss in the continuum at Raman AntiStokes wavelength from pump spectral line to give absorption images at vibration Raman lines in material: solids, liquids, tissue and biomedical media cells and smears.

(41) The SC microscope shown in FIG. 7 may be used as a pathology approach without using extrinsic dyes and stains but using instead multiphoton SC processes such as Second Harmonic generation (SHG), Third Harmonic generation (THG), 2PEF, 3PEF and SRS Loss and gain imaging of the key native label free molecules like the following: collagen, elastin, NADH, lipids, amino acids, and proteins in tissue and smears.

(42) In the past the gold standard was to use stains and dyes for pathology evaluation such as Eosin H&E, Hematoxylin, and chemical labeling for Histopathology evaluation of tissue slice or smear (PAP) in a clinical lab which took long time on the order of many hours to do. The tissues are fixed using formalin, paraffin embedded requiring many hours on the order of >8 hrs. The delay is not good while the optical SC NLO processes and images take less hour.

(43) The multiple photon SC microscope images with filters can reduce the expense and can analyze fresh unlabeled stain free tissue and smears. Native imaging using SC microscope can give pathology information without staining. The speed increases without the need for tissue preparation using dyes. The image contrast of 2PEF, 3PEF, SHG and THG and Stimulated Raman Scattering (SRS), Stimulated Raman Gain (SRG) and Stimulated Raman Loss (SRL) can be used for giving histopathology information from the images of local structure in the tissue and smears images using nonlinear optical methods from selected spectral parts of the SC. These form new microscope clinical tools for evaluating tissue without dyes to stain samples but using an optical pathology SC microscope to image the structure of tissue cells and components inside cells of tissue for cancer and clinical evaluation.

(44) The SC Microscope may be used for vibration state in tissue, cells and material for an ultra-broadband Stimulated Raman Application for chemical, biomarkers molecules in cells and tissues such as CH.sub.2 and CH.sub.3 vibrations from proteins and lipids about 1600 cm.sup.1 and 2900 cm.sup.1 and others are possible as would be evident to those skilled in the art.

(45) Resonant Stimulated Raman Scattering Via Anharmonic Interactions SC Microscope

(46) Raman scattering is one of the key optical spectroscopic processes arising from inelastic scattering of light with vibrations in materials. The scattered light has a characteristic frequency shift due to vibrations accompanied by generation of optical phonons in the material. The Raman effect has been an active field of research in various fields of science since its discovery in 1928 by Raman and Krishnan. Spontaneous Raman (SR), despite being the weakest form of scattering, has widely been used as a powerful technique to investigate complex molecular and solid-state systems. Raman investigations exploded in the sixties with the discovery of different types of lasers. The Raman process occurs when a photon is scattered from a vibrational mode having its energy difference from the incident beam by the vibrational frequencies. There are several different types of Raman processes that can occur depending on the types of interactions with laser, such as spontaneous, resonance, and stimulated Raman.

(47) An enhancement of the Raman signal, essential for studies at low concentrations or in low cross section compounds, is achieved by Resonant Raman Spectroscopy (RRS), in which the Raman excitation wavelength is tuned to match the energy of any electronic transitions of a system. Stimulated Raman scattering (SRS) was first discovered when a cell with nitrobenzene was introduced inside a ruby laser cavity. Woodbury and Ng observed a rather strong emission at the wavelength other than the fundamental wavelength (694.3 nm) of a ruby laser. The work of Stoicheff measured the various regions in Raman processes at different laser pump intensities of first Stokes in nitrogen and oxygen liquids, namely, SR, SRS, and saturation as the pump intensity grew. Several researchers have demonstrated different Raman gain from transient to transient depending on the pulse duration and vibrational lifetime under pico-second (ps) pulses. In the early 1970's, the white light continuum spanning the visible and part of NIR, now called supercontinuum (SC), was discovered by Alfano and Shapiro [1] in solids and liquids using ps pulses. The use of SR loss and gain is active for imaging biological materials such as brain for SRS microscopes.

(48) This part reports for the first time on novel nonlinear optical process on the observation of Resonant Stimulated Raman Scattering (RSRS) process for improving/enhancing imaging using resonant process which was found in a solution of -carotene in methanol using pump beam at the second harmonic generation (SHG) from a Q-switched Nd:YAG laser. RSRS combines both RRS and SRS nonlinear processes. The RSRS observed effect is attributed arising in part from cubic from quartic anharmonic vibrational interactions among solute carotene in resonance with solvent methanol vibrational modes.

(49) The discovery of RSRS is important towards improving over conventional SRS microscopy for imaging vibrational states of cancer, and the applications of this technique in the areas of neuroscience, cancer, and biomedicine.

(50) In SRS microscopy, the sample is coherently driven by two lasers: one is the pump beam with frequency .sub.L and the other is the Stokes beam with frequency .sub.s, where the difference is equal to a particular Raman-active molecular vibration of the sample. The SRS signals, including both stimulated Raman loss (SRL) at the laser pump beam and stimulated Raman gain (SRG) at the Stokes beam are generated due to the nonlinear interaction between the photons and the vibration of the molecules for imaging. The development of novel nonlinear vibrational spectroscopies has allowed broadband SRS to provide high intensity with low fluorescence background free coherent signal. In SRS, the sample is interrogated by a pair of overlapped narrowband ps Raman pulses and/or broadband femtosecond (fs) probe pulses. In SRS G/L process the vibrational spectra occurs with the incoherent fluorescence background and electronic susceptibility .sub.3 is efficiently suppressed.

(51) Background Theory on SRS SC Microscope

(52) The intensity of the spontaneous Raman (SR) is weak (10.sup.6 I.sub.L), where I.sub.L is the laser intensity. The power scattered is given by

(53) $\begin{array}{cc}{P}_s={N\left(\frac{}{}\right)}_R{I}_L=N{}_R{I}_L,& \left(7\right)\end{array}$
where cross-section .sub.R is given by:

(54) $\begin{array}{cc}{}_R={\left(\frac{}{}\right)}_{R}d,& \left(8\right)\end{array}$
and N is the number of molecules in the observed volume and

(55) $\left(\frac{}{}\right)$
is the differential Raman cross-section.

(56) When the excited laser wavelength approaches an electronic absorption in a material, the transitions among the states go from virtual to real. The Raman scattering signal becomes enhanced due to the resonant effect. Thus, enhancement arises from the cross section from the energy denominator of nonlinear susceptibility becoming small as the laser frequency matches the electronic energy states. The virtual transition of the intermediate state becomes real and Raman effect becomes larger by 10 to 1000 folds depending on how close the laser photon energy is in the transit from the ground state (i) to electronic state j. They are in resonances and out of resonances with the pump and Raman shifted light with the electronic states. This process is called Resonance Raman scattering (RRS).

(57) The Raman cross-section for single molecule is given by:

(58) 0$\begin{array}{cc}{}_R={.Math..Math.\frac{A_{\mathrm{ijjf}}}{\left({}_{\mathrm{ij}}-{}_L-i{}_j\right)}+\frac{A_{\mathrm{jijf}}}{\left({}_{\mathrm{jf}}-{}_L-i{}_j\right)}.Math.}^2& \left(9\right)\end{array}$
for in and out resonances, so when .sub.L approaches .sub.ij, the denominatorreduces and increases and Raman becomes resonant Raman scattering RRS. The frequency dependence cross section in Eq. 9 shows the salient resonance features between the pump and probe frequency with electronic absorption for enhancement.

(59) When an intense laser pulse (ns, ps, fs) enters a material, the Raman effect occurs. The light is first scattered over a large angle . As the Raman light travels with the pump laser in the forward and backward directions it can become larger than the Raman light traveling out of the beam at other angles as it propagates with laser pulse and over a length of more than 10 cm. Depending upon the intensity of the laser pump pulse the Raman light in the forward and backward directions can become so large that it is stimulated and becomes laser-like with high direction and coherence.

(60) The intensity of Raman Stokes is given by a Beer-Lambert's law-like equation:
I.sub.RS(z)=I.sub.RS(0)exp(Gzz),(10)
where G is the gain, is the loss, and I.sub.RS(0) is initial Stokes from zero point fluctuation which has SR at z=0. In any SRS, the Raman gain must exceed the loss due to absorption in the media, where Gz>25 and the medium will experience an exponential growth of photon at Stokes frequency. The Raman light in the forward direction becomes much greater than spontaneous Raman and becomes SRS with about 1% to 10% of energy transferred from pump frequency.

(61) The Raman gain G is:

(62) $\begin{array}{cc}G=N\left(\frac{}{}\right)I_L& \left(11\right)\end{array}$
Carotene was selected as an ideal test solute to demonstrate RSRS in biomedical media in solution. Carotene is synthesized in plants and animals. It is a chromophore in carrots, tomatoes, and in skin, and is known for its orange color. In humans, carotene is involved in antioxidant processes and defense mechanisms. In this research observation, carotene provides the methanol solution with the necessary enhancement of cross-section in the visible where the absorption peaks at 450 nm extending out to 532 nm. The main absorption of carotene is from S.sub.2 state since S.sub.1 is dipole forbidden.

(63) The focus here is to state the first observation of Resonant Stimulated Raman scattering (RSRS) in a solution of -carotene in methanol using pump SHG from Q-switched Nd Laser of 5 ns at 532 nm laser beam. RSRS combines both RRS and SRS processes a first new non-linear optical (NLO) effect. The observation of RSRS is most important for new Stimulated Raman Loss (SRL) and Stimulated Raman Gain (SRG) microscopes in order to enhance signals of images from vibrations in biomedical tissues, cells and chemicals in samples. The selection of the pump or Stokes near an electronic resonance will improve the signal to noise ratio (i.e., S/N) of the SRS microscope image. Part of SC spectrum can be used to achieve resonance in the material spectra for RSRS, see FIG. 6.

(64) The key observation is that the carotene solute influences the vibrations of methanol. The solute-solvent system can have different interactions: vibrations between solute molecules, solvent molecules, or between solute and solvent. There is a coupling as shown in spontaneous Raman at 2834 cm.sup.1. Anharmonic coupling between solute and solvent from solvation of shells account for the relaxation of an excited solvent and solute molecules. The conservation of energy affect the relaxation of a vibration. If there is no energy match the vibration is long, and if the vibration matches among the vibrations decay is fast. In Fermi Golden rule the rate among states of interaction is from square of Hamiltonian from anharmonic terms from potential V.sub.n where n>3, and the density of final states is available. The latter term is main process to determine the system process from solutesolvent, solvent, and solute states. The anharmonic coupling allows for the flow of energy among the vibrational modes. A cubic anharmonicity allows for excitation of the solute and solvent vibrations modes to be exchange during interaction. A quartic anharmonicity would exchange correspond to vibration and bath phonon exchange, such as 2 vibrations from solute and solvent and a phonon bath.

(65) Vibrational energy processes in binary solvent A and solute B system can have cubic and quartic interactions. A possible quartic interaction to excite the 2834 cm.sup.1 in methanol solvent from resonance Raman of carotene is the 1525 cm.sup.1 and 1157 cm.sup.1 modes can generate 2834 cm.sup.1 and deactivate 150 cm.sup.1 methanol bath phonons such as 1525 cm.sup.1+1157 cm.sup.1.fwdarw.2834 cm.sup.1150 cm.sup.1 [A*A*B*Bgoes to AABB*]. A possible model where upon excitation by 532 nm, the carotene undergoes RR scattering at 1525 cm.sup.1 and 1157 cm.sup.1 than transfer energy to methanol with bath phonons from methanol to excite the 2834 cm.sup.1 of methanol mode.

(66) Kasier group investigated cubic interactions, one excited molecule say A* decays though resonant and non-resonance interaction in trinary collisions: A*AA, A*AB, and A*BB. To affect the vibration lifetime decay, Kaiser and coworkers observed the triple interaction of higher vibration CH.sub.3 with addition of another liquid of CCl.sub.4. The vibration lifetime of A* of CH.sub.3 increase with more of B. Therefore the Raman gain will become larger with addition of CCl.sub.4 going from transient gain to steady state gain. Raman gain will increase towards more steadystatelike when lifetime of the vibrations becomes longer. This effect will be more important using femtosecond and picosecond pulses, not nanosecond pump laser pulse. So in this study the resonance of B (i.e., carotene) to A (i.e., methanol) will be major cause for RSRS process.

(67) The solute carotene affects the vibrations (1525 cm.sup.1+1152 cm.sup.) transfer of the resonance to solvent methanol (M) (2834 cm.sup.1 and phonon bath) in a quartic interaction (C1*C2*M1*M2), thereby enhancing the cross section. A theoretical analysis following on the underlying physics is needed to explain the RSRS process observed vibrations of solute carotene and solvent methanol. Time resolved femtosecond pump probe is in order to test and determine the energy transfer speculative mechanism.

(68) Streak Camera Time Resolved Microscope

(69) Using the short pulse associated with SC allows for Time resolved imaging to yield temporal properties of biomaterials and condensed matter imaging. Light propagating in turbid media such as tissue undergoes scattering which can blur images. The signal (light intensity) is governed and defined by key parameters for tissue: the scattering coefficient .sub.s, the transport coefficient .sub.s, the absorption coefficient .sub.a, and the mean cosine scattering angle parameter g. These are a function of wavelength (). From .sub.s and .sub.s, the mean cosine scattering angle parameter g can be obtained directly from time resolved measurements of the transmission and backscatter using a Streak Camera to measure transmitted or reflected signal form a point xyz in the sample 1 (t, xy).

(70) One can create a map on sample at xyz and extract the .sub.s from the tail in time. The light transport is made up of ballistic and diffusive components, see Ref 4. The ballistic light can provide high quality images, reveal hidden objects in turbid media and is represented by .sub.s and .sub.a. The transport theory of diffuse light intensity is represented by the diffuse equation

(71) $\begin{array}{cc}\frac{I\left(r,t\right)}{t}=\left(DI\left(r,t\right)\right)-v{}_{a}I\left(r,t\right)+q^{\left(0\right)}\left(r,t\right)& \left(12\right)\end{array}$
where q.sup.(0)(r,t)=(r)(t) is the incident source, r is the position, v is the speed of light, and D is the diffusion coefficient, given by

(72) $\begin{array}{cc}D=\frac{v}{3{}_s\left(1-g\right)}& \left(13\right)\end{array}$
where .sub.s=.sub.s(1g) and

(73) ${}_s^{}=\frac{1}{\mathrm{Ltr}.}$

(74) The value of .sub.s is extracted from D in Eq. 14. Similarly, as shown by Yoo [4] in the case of a slab sample, the transport theory of diffuse light intensity can be described by

(75) $\begin{array}{cc}{I}_z\left(t\right)=\frac{1}{4d^{2}t}\underset{m=1}{\overset{}{.Math.}}m\sin \left(\frac{mz}{d}\right)\exp \left[-{\mathrm{Dt}\left(\frac{m}{d}\right)}^2\right]\exp \left(\frac{-\mathrm{vt}}{I_a}\right)& \left(14\right)\end{array}$
where d=z+2z.sub.0, z.sub.0=0.71/t and where z.sub.0 is the extrapolation length and z is the thickness of the sample. Thus, the transport length 1/.sub.s and the absorption length L.sub.a can be approximated, along with temporal information, using these equations (see Ref 4) at each point in the tissue to map out these values for different parts of brain and for cancer and non cancer region.

(76) The streak camera, the heart of the time-resolved SC microscope, allows for direct measurement of .sub.s() from the temporal profile. Using a 100 fs pulsed Ti: Sapphire laser or SC laser at select wavelengths around the first NIR optical window (from 700 nm to 1100 nm) and a microscope objective of >10 to 20 X, .sub.s() can be obtained in space at different sites in the brain, skin and breast tissues. 3D maps of .sub.s from tissue slices will be acquired. One can develop 5D image maps of tissue using space (3D), time t, and wavelength .

(77) The full potential of SC time-resolved measurements is taught. One can form a Streak Cameras with 5 to 10 ps resolution imaging system to get .sub.s() at xyz of tissues and biomaterials. The spatial location of an abnormality in a scattering medium such as the brain or the breast using the time behavior of the scattered light through turbid media may be a valuable noninvasive tool for Streak Camera microscope.

REFERENCES

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