Enzyme-Independent Photon Emission

Abstract

The invention relates to a new method for generating a luminescent signal, which is based on an enzyme-independent photon emission mechanism (EiPE), whereby luminescent light is generated when a bioluminescent substrate interact with a physical surface in the absence of any catalytic enzyme. In particular embodiments, the method is used to stimulate light emission by fluorescent molecules. The invention relates also to kits for implementing a method according to the present invention and to the use of a bioluminescent substrate, in the presence of a physical surface, to produce a luminescent signal in the absence of enzymatic catalyst.

Claims

1: A method of generating a luminescent signal, comprising the step of contacting a luciferin or reactive intermediate thereof with at least one physical surface in the absence of a luciferase, wherein the interaction of said luciferin with said physical surface generates a detectable luminescent signal.

2: The method according to claim 1, wherein said physical surface is the surface of a particle or biological matrix.

3: The method according to claim 2, wherein said particle comprises a metal, lipid, resin, and/or polymer microsphere or nanoparticle.

4: The method according to claim 2, wherein said biological matrix is chosen from the group consisting of actin matrix, collagen matrix, microtubule, microfilament and biofilm.

5: The method according to claim 1, wherein said luciferin is chosen from the group consisting of decanal, coelenterazine and coelenterazine analogs.

6: The method according to claim 1, wherein said at least one physical surface comprises a fluorescent molecule, and wherein said detectable luminescent signal is emitted by said fluorescent molecule.

7: The method according to claim 6, wherein said at least one physical surface is the surface of a fluorescent particle chosen from the group consisting of quantum dot, fluorescent polystyrene microsphere and fluorescent lipid nanoparticle.

8: The method according to claim 1, wherein said at least one physical surface is coated with a molecular probe.

9: The method according to claim 6, wherein said luciferin is contacted with different fluorescent molecules, each fluorescent molecule being bound to a separate physical surface.

10: The method according to claim 1, wherein said contacting step is performed in the presence of a detergent.

11: The method according to claim 1, wherein said luminescent signal is for imaging or sensing a biological target, in vitro or in vivo.

12: A kit for performing a method of generating a luminescent signal, comprising: at least one luciferin or reactive intermediate thereof, at least one physical surface, and instructions for the performance of the method according to claim 1, wherein the kit does not comprise any luciferase.

13: The kit according to claim 12, wherein the physical surface comprises a fluorescent molecule.

14. (canceled)

15: The method according to claim 2, wherein said at least one physical surface comprises a fluorescent molecule, and said detectable luminescent signal is produced by the fluorescent molecule.

16: The method according to claim 2, wherein said luciferin is chosen from the group consisting of decanal, coelenterazine and coelenterazine analogs.

17: The method according to claim 9, wherein the fluorescent molecules cover the optical and near infrared spectrum.

18: The kit according to claim 12, wherein said luciferin is chosen from the group consisting of decanal, coelenterazine and coelenterazine analogs.

19: The kit according to claim 12, wherein said physical surface is the surface of a particle or biological matrix.

Description

[0056] For a better understanding of the invention and to show how the same may be carried into effect, there will now be shown by way of example only, specific embodiments, methods and processes according to the present invention with reference to the accompanying drawings in which:

[0057] FIG. 1. Schematic depicting of the EiPE phenomenon. Upon the interaction of the substrate and an appropriate surface, a basal signal is generated (A). If a fluorophore is present, a wavelength-specific signal is observed (B). Non-limitative examples of interaction surfaces appropriate for EiPE (C).

[0058] FIG. 2. Initial Evidence of the Existence of EiPE. Using combinations of Listeria inoculate, decanal, and QD705 in PBS various solutions were formed as indicated and then dispensed into a black 96-well plate. The plate was then placed into an IVIS100 bioluminescence imaging system and observed under the 470 nm-490 nm, and 695 nm-770 nm filter sets. Luminescence was observed for the combinations of decanal+Listeria innocua, decanal+Listeria innocua+QD705, and for decanal+QD705. Blue-specific signal was found only when the Listeria inoculate were present. Further, QD705-specific signal was seen whenever the QD705 were mixed with the decanal, even in the absence of the Listeria inoculate. It is this final observation that led to the discovery of Enzyme-independent Photon Emission (EiPE).

[0059] FIG. 3. Wavelength-Specific Signal Generation Using EiPE with Various QDs. Solutions containing only the substrate, in this case Coelenterazine-h, or substrate and QD655, QD705 or QD800, were prepared in 1PBS and dispensed into Eppendorf tubes. The tubes were then placed into an IVIS100 bioluminescence imaging system and observed under the Total Light, 530-550 nm, 610-630 nm, 575-650 nm, 695-770 nm, and 810-870 nm emission filters. Wavelength-specific signal was observed for each solution that contained the QDs, with the maximum emission observed under the correctly corresponding emission filter. The signal of each solution was compared to the substrate control, and a massive increase in signal was observed. This increase approached nearly 300-fold when the QD705 and QD800 were used, indicating the massive specificity of the signal.

[0060] FIG. 4. Wavelength-Specific Signal Generation Using EiPE with Various Fluorophore-Embedded Polystyrene Microspheres. Solutions containing only the substrate, in this case Coelenterazine-h, or substrate and polystyrene microspheres embedded with various fluorophores, were mixed with 1PBS and dispensed into individual wells of a black 96-well plate. The fluorophore-embedded microspheres had the following excitation/emission maxima: 505 nm/550 nm, 580 nm/605 nm, 660 nm/680 nm, and 488/650 nm. The plate was then placed into an IVIS100 bioluminescence imaging system and observed under the Total Light, 470-490 nm, 530-550 nm, 610 Long Pass, 610-630 nm and 695-770 nm emission filters (A-F, respectively). Wavelength-specific signal was observed for each solution that contained the polystyrene microspheres, with the maximum emission observed under the correctly corresponding emission filter.

[0061] FIG. 5. The Effect of Different Substrates on Light Generation Using EiPE. The effect of different substrates on the ability to generate light using EiPE was investigated by mixing Coelenterazine or one of its derivatives with fluorophore-embedded microspheres. The light generated from two different fluorophore-embedded microspheres mixed with the commonly used coelenterazine derivatives h, hcp, and fcp were compared to the native form. The solutions were prepared in triplicate and dispensed into a black 96-well plate (A). The plate was placed into an IVIS100 bioluminescence imaging system and observed under the Total Light, 530-550 nm, and 575-650 nm emission filters (B-D, respectively). The microsphere location was confirmed using epifluorescence (E and F). As anticipated, the wavelength-specific signal is generated and confirmed to appear under the appropriate emission filters. Interestingly, the amount of signal generated varied from each derivative and for each fluorophore used. This indicates that for a given fluorophore used there exists an ideal derivative that promotes maximum signal generation.

[0062] FIG. 6. Analysis of EiPE Substrate Dependence. The resulting photons/s/cm.sup.2 from each acquisition as shown in FIG. 5 was normalized to the respective coelenterazine control, labeled as natural for each condition. Similar trends were observed for the 505/550 (A) and 580/605 (B) carboxylate functionalized fluorophore-embedded polystyrene beads. The greatest signal enhancement was observed when coelenterazine-h was used, and each derivative generated at least twice as much signal as the control. This result implies that further chemical modifications to the coelenterazine backbone may yield an increased in generated luminescence.

[0063] FIG. 7. Changing the Surface Area of ReSyn Beads and its Effect on EiPE Signal Generation. A ReSyn bead consists of a long polymeric chain that freely and naturally winds around itself generating a microsphere with a massive surface area to volume ratio. As such, the polymer generates a bead-like structure that is extremely porous and allows the free movement of liquids throughout its entirety. ReSyn beads were fabricated such that they had varying degrees of crosslinking, thus regulating the amount of surface area available for the EiPE reaction. In triplicate, solutions containing equal concentrations of the respective ReSyn microspheres and the coelenterazine-h substrate in 1PBS were prepared and dispensed into a black 96-well plate. The resulting luminescence was observed using an IVIS Spectrum under all the available filter sets (20 nm band pass filters which encapsulate the emission from 490 nm to 850 nm). The emission under the 490-510 nm filter is shown (inset). As is indicated, ReSyn A had the largest amount of available surface area, followed by ReSyn B and then ReSyn C. ReSyn M is a magnetic variant of ReSyn A, whereas Sub indicates a solution where no beads are present but only the substrate in 1PBS. While the resulting emission maxima for each ReSyn bead occurred at the same region (510-530 nm), the intensity of the emission is strongly correlated to the degree of crosslinking.

[0064] FIG. 8. Wavelength-Specific Signal Generation Using EiPE with QD705-Labelled ReSyn Beads. In triplicate, equal concentrations of ReSyn Beads labeled with various concentrations of QD705 and mixed with the coelenterazine-h substrate in 1PBS were distributed into a black 96-well plate and observed under the 710-730 nm filter set of an IVIS Spectrum (Top). The resulting p/s/cm.sup.2 were normalized to the average value of the beads that produced the most signal (0.152 M QD705, Bottom). As can be seen from the plotted data, the most intense QD705 signal occurred in the case where the concentration was at the highest. The resulting luminescence decreased in a linear manner.

[0065] FIG. 9. Wavelength Specific Signal Generation Using EiPE with Rhodamine6-Loaded Lipid Nanoparticles (LNPs) from Global Acorn. Three formations of lipid nanoparticles from Global Acorn were mixed with coelenterazine-h in 1PBS and distributed into a black 96-well plate. The three formations consisted of a control (ie, no fluorophore attached nor on the interior) LNP (C LNP), with rhodamine-6 both bound to the surface and loaded into the center of the LNP (R1), and with rhodamine-6 loaded into the center only (R2). The plate was placed into an IVIS100 bioluminescence imaging system and observed under the Total Light, 470-490 nm, 530-550 nm, 575-650 nm, and 610 long pass (LP) emission filters (Top images). The relative p/s/cm.sup.2 for each LNP under each filter set was normalized to the control LNP (Bottom). As can be seen, the LNP that was both labeled with and contained the rhodamine-6 produced the greatest amount of signal under the Total Light, 575-650 nm, and 610LP emission filters. This demonstrates that there exists the possibility to create biocompatible polymeric backbones to which a fluorophore or a fluorescent nanoparticle may be attached, and the wavelength of the luminescence predicted.

[0066] FIG. 10. Signal Generation Using EiPE with Fluorophore-Embedded Microspheres in the Presence of Fixed J774A.1 Murine Macrophages in Suspension. An equal number of fixed J774A.1 murine macrophages were distributed into the wells of a black 96-well plate. Into the same wells, a decreasing number of 0.5 m fluorophore-embedded microspheres with maximum excitation and emission wavelengths of 580 and 605 nm, respectively, were distributed such that the microsphere-to-macrophage ratio ranged from 20788 to 20, with one well kept free from microspheres (noted as 0). After the microspheres were distributed, equal volumes of coelenterazine-h in 1PBS were added to each well and the plate placed immediately into an IVIS Spectrum bioluminescence imaging system. The plate was observed under all available emission filters (ranging from 490 nm-850 nm). The total photons/second/cm.sup.2 were determined for each well under each filter. The resulting total flux at 610-630 nm for each well was normalized by the flux found at 510-530 nm, and the resulting enhancement in red signal displayed. Each condition was repeated in triplicate. As can be seen, a substantial wavelength-specific signal was generated whenever the beads were present, with the magnitude of the signal dependent upon the microsphere-to-macrophage ratio. The microsphere-specific signal is clearly discernable from the background at as few as 20 microspheres per macrophage.

[0067] FIG. 11. The Existence of EiPE under in vivo Conditions. The dorsal side of four 6-week old female BALB/c mice was razed and the mice placed under general anesthesia. One solution containing only coelenterazine-h in 1PBS was prepared and used as the control (Coel). A second solution containing the coelenterazine-h in 1PBS along with QD705 was prepared (Coel+QD705). One mouse received one subcutaneous injection of the Coel control and was imaged under the Total Light, 470-490 nm, and 695-770 nm emission filters. The three other mice received identical volume injections of the Coel+QD705 solution and were also imaged under the same filter sets (A-C). The QD705 location was confirmed by epifluorescence (D). The resulting p/s/cm.sup.2 were compared under each filter set (E). As can be seen, nearly a three-fold increase in total signal is observed when the Coel+QD705 solution is present compared to the Coel control. This contrast is even stronger when the QD705-specific filter is used, providing a nearly 20-fold increase in signal.

[0068] FIG. 12. Using EiPE to Generate Fluorophore-Specific Signals from Multiple Luminescent Sources Covering Nearly the Entire Optical Spectrum. Coelenterazine-h in 1PBS was mixed with a multitude of fluorophores and fluorescent nanoparticles, including 505/550 nm, 580/605 nm, and 660/680 nm polystyrene microspheres (Yellow, Pink, and Far Red, respectively); QD655, QD705, and QD800; as well as unbound streptavidin-functionalized Alexa dyes (555, 633, and 700). The solutions were produced in triplicate and distributed into a black 96-well plate. The plate was observed in an IVIS Spectrum bioluminescence imaging system under all available filter sets (ranging from 490 nm-850 nm). The entire emission spectrum for each fluorophore was acquired and normalized to the peak emission wavelength. As can be seen, the appropriate fluorescence emission spectrum of each compound was achieved using EiPE.

[0069] FIG. 13. Multiplexed spectral deconvolution using EiPE. Polystyrene beads doped with one of three fluorophores (A,B,C) were mixed v/v (AB,AC,BC,ABC) in a 96 well plate in the presence of Coelenterazine-h and treated to reveal EiPE light. Multi-well plate was visualized first using fluorescence illumination (left panels) as a control, and next using EiPE light generation (right panels). Spectral deconvolution was performed using 8 filter sets covering the visual spectrum range of 490 to 730 nm with 20 nm bandwidths. The three component channels Spectral deconvolution revealed the presence of the beads both alone and mixed together identically for fluorescence and EiPE illumination.

[0070] FIG. 14. Addition of Triton X-100 increases EiPE signal. Solutions containing coelenterazine-h (5 g/mL), Triton X-100 at the indicated concentrations, and Green fluorosphere (A) or Far red fluorosphere (B) at the indicated concentrations were dispensed into individual wells of black 96-well plates. Control wells contained: (i) the substrate and the different concentrations of Triton X-100 and (ii) fluorospheres at different concentration without detergent, but with coelenterazine-h. The prepared plates were placed into an IVIS100 bioluminescence imaging system and observed under the Total light, 500 nm, 700 nm filter sets using an exposure time of 1 min. The resulting photons/second/cm.sup.2 (p/s/cm.sup.2) flux was then plotted. As can be seen, adding Triton X100 to low concentration of fluorospheres (0.025 or 0.05%) can improve by a factor 3-4 the signal produced by EiPE.

[0071] FIG. 15. Addition of Tween-20 increases EiPE signal. Solutions containing coelenterazine-h (5 g/mL), Tween-20 at the indicated concentrations, and Green fluorosphere (A) or Far red fluorosphere (B) at the indicated concentrations were dispensed into individual wells of black 96-well plates. Control wells contained: (i) the substrate and the different concentrations of Tween-20 and (ii) fluorospheres at different concentration without detergent, but with coelenterazine-h. The prepared plates were placed into an IVIS100 bioluminescence imaging system and observed under the Total light, 500 nm, 700 nm filter sets using an exposure time of 1 min. The resulting photons/second/cm.sup.2 (p/s/cm.sup.2) flux was then plotted. As can be seen, adding Tween-20 to low concentration of fluorospheres (0.025 or 0.05%) can improve by a factor 3-4 the signal produced by EiPE.

EXAMPLE 1: MATERIALS

Common Reagents

[0072] Coelenterazine-h was acquired from two different commercial sources (SigmaAldrich, France, and Zymera, Inc, USA). Coelenterazine (natural) and other coelenterazine derivatives (hcp, and fcp) were acquired from SigmaAldrich (France). Unless otherwise stated, the coelenterazine-h used was provided by Zymera, Inc. All the coelenterazines were immediately dissolved in 1,2-propanediol (SigmaAldrich, France) upon reception to a concentration of 0.5 mg/mL. The resulting solution was dispensed into 504 aliquots and kept at 20 C. until use. Prior to use, the coelenterazines were diluted 10-fold to a final stock concentration 0.05 mg/mL.

[0073] The decanal (SigmaAldrich, France) was stored in the dark until use.

[0074] The quantum dots (2 mM solutions) and the fluorescent polystyrene microspheres (2% solids) were acquired from Life Technologies (USA) and used as received. Custom polymeric microspheres were provided by ReSyn, LLC (South Africa), and custom lipid nanoparticles were provided by Global Acorn (United Kingdom).

TABLE-US-00001 TABLE I Summary of the commercial nanoparticles used within the respective figures Name of Material/ Catalog Comments/ Equipment Company Number Description Concentration Q-Tracker 655 Life Sciences Q21021MP 2 M Q-Tracker 705 Life Sciences Q21061MP 2 M Q-Tracker 800 Life Sciences Q21071MP 2 M Alexa 555 Life Sciences S21381 1 mg/mL Alexa 568 Life Sciences S11226 1 mg/mL Alexa 633 Life Sciences S21375 1 mg/mL Alexa 700 Life Sciences S21383 1 mg/mL Pink microspheres Life Sciences F8887 40 nm diameter 1% solids Yellow microspheres Life Sciences F8888 40 nm diameter 1% solids Far Red microspheres Life Sciences F8789 40 nm diameter 1% solids

Bioluminescence Imaging

[0075] An IVIS100 or an IVIS Spectrum, both provided by Perkin Elmer, was used to acquire all the luminescence images. The IVIS100 was equipped with multiple band pass filters as indicated in the figures. Total Light indicates the absence of an emission filter, thus allowing for all the emitted wavelengths to be detected. The IVIS Spectrum contains 18 band pass filters with each having a nominal bandwidth of 20 nm, ranging from 490 nm-850 nm. Each system provided the capability of standard epifluorescence imaging. The acquired data was analyzed using the provided software, LivingImage, versions 4.1 and 4.2.

Macrophage

[0076] J774A.1 murine macrophages (ATCC) were grown under standard conditions (DMEM with 10% fetal bovine serum).

Modified Bacteriophages

[0077] Modified bacteriophages that contained the genetic material to induce luxAB expression from infected Listeria were prepared using standard molecular biology techniques.

Mice

[0078] 6-week old female BALB/c mice were acquired from Janvier (France) and kept under standard growing conditions following all European Union rules and regulations concerning animal ethics. Immediately after experimentation, the mice were sacrificed and appropriately disposed of.

EXAMPLE 2: INITIAL EVIDENCE OF THE EXISTENCE OF EIPE

1. Methods

[0079] Aliquots from a fresh overnight culture of Listeria were exposed to modified bacteriophages that contained the genetic material to induce luxAB expression, for up to 2 hours. After exposure, three 100 L aliquots of the Listeria and bacteriophage mixture were dispensed into individual wells of a black 96 well plate. Of these wells, 10 L of decanal was added to one well while a second received 10 L of decanal and 10 L of QD705. As a control, nothing was added to the third well. A solution containing 100 L of 1PBS, 10 L of decanal, and 10 L of QD705 was added to a fourth well. The prepared plate was placed into an IVIS100 and observed under the Total Light, 470 nm-490 nm, and 695 nm-770 nm filter sets using an exposure time of 30 seconds. The location of the QD705 was verified using epifluorescence.

2. Results

[0080] Using combinations of Listeria innocua, decanal, and QD705 in PBS (FIG. 2) various solutions were formed and then dispensed into a black 96-well plate. The plate was then placed into an IVIS100 bioluminescence imaging system and observed under the Total Light, 470 nm-490 nm (FIG. 2), and 695 nm-770 nm filter sets (FIG. 2). Luminescence was observed for the combinations of decanal+Listeria innocua, decanal+Listeria innocua+QD705, and for decanal+QD705. While signal was seen under the Total Light filter set for all three conditions, blue-specific signal was found only when the Listeria innocua were present. Further, QD705-specific signal was seen whenever the QD705 were mixed with the decanal, even in the absence of the Listeria innocua. It is this final observation that led to the discovery of Enzyme-independent Photon Emission (EiPE). The location of the QD705 was validated by epifluorescence.

EXAMPLE 3: WAVELENGTH-SPECIFIC SIGNAL GENERATION USING EIPE WITH VARIOUS QDS

[0081] Four different solutions were prepared and dispensed into individual eppendorf tubes. The first tube contained 80 l 1PBS and 20 l of coelenterazine-h. The remaining tubes contained 75 L of 1PBS, 20 L of coelenterazine-h, and 5 L of QD655, QD705, or QD800. Upon mixing, the tubes were placed into the IVIS100 and visualized under the Total Light, 530-550 nm, 610-630 nm, 575-650 nm, 695-770 nm, and 810-870 nm emission filters, with the exposure time set to 60 seconds. The location of the various QDs was verified using epifluorescence. The resulting photon flux (photons/second/cm.sup.2; p/s/cm.sup.2) from the enzyme-independent luminescence was normalized to tube 1, which was the PBS control.

[0082] Wavelength-specific signal was observed for each solution that contained the QDs, with the maximum emission observed under the correctly corresponding emission filter. The QD location was verified using epifluorescence. The signal of each solution was compared to the substrate control, and a massive increase in signal was observed (FIG. 3). This increase approached nearly 300-fold when the QD705 and QD800 were used, indicating the massive specificity of the signal.

EXAMPLE 4: WAVELENGTH-SPECIFIC SIGNAL GENERATION USING EIPE WITH VARIOUS FLUOROPHORE-EMBEDDED POLYSTYRENE MICROSPHERES

[0083] Five different solutions were prepared and dispensed into individual wells of a black 96 well plate. The first four contained 70 L of 1PBS, 20 L of coelenterazine-h, and 10 L of fluorescent polystyrene microspheres with the noted excitation/emission maxima (505 nm/550 nm, 580 nm/605 nm, 660 nm/680 nm, and 488/650 nm). The fifth well contained 80 L of 1PBS and 20 L of coelenterazine-h. The plate was placed into the IVIS100 and visualized under the Total Light, 470-490 nm, 530-550 nm, 610 Long Pass, 610-630 nm and 695-770 nm emission filters, using an exposure time of 60 seconds.

[0084] Wavelength-specific signal was observed for each solution that contained the polystyrene microspheres, with the maximum emission observed under the correctly corresponding emission filter (FIGS. 4A to 4F).

EXAMPLE 5: THE EFFECT OF DIFFERENT SUBSTRATES ON LIGHT GENERATION USING EIPE

[0085] Different derivatives of coelenterazine (h, natural, hcp, and fcp; SigmaAldrich) were used to investigate the substrate dependence on the observed EiPE effect using two different standard 0.5 m polystyrene microspheres (LifeTechnologies). The excitation/emission maxima of the microspheres were 580 nm/605 nm and 505 nm/515 nm, respectively. Each well contained 70 L of 1PBS, 20 L of the coelenterazine derivative, and 10 L of the fluorescent microspheres, and distributed into a black 96 well plate accordingly. The plate was then placed into an IVIS100 and visualized under the indicated filter sets using an exposure time of 60 seconds. The location of the microspheres was validated using epifluorescence. For the two types of fluorescent microsphere involved, the resulting EiPE-induced luminescence from each well was normalized to the average photon flux of the Natural coelenterazine derivative. The fold increase in signal was plotted versus the coelenterazine derivative used to induce the luminescent response.

[0086] As anticipated, the wavelength-specific signal is generated and confirmed to appear under the appropriate emission filters (FIG. 5). Interestingly, the amount of signal generated varied from each derivative and for each fluorophore used. This indicates that for a given fluorophore used there exists an ideal derivative that promotes maximum signal generation. Similar trends were observed for the 505/550 (A) and 580/605 (B) carboxylate functionalized fluorophore-embedded polystyrene beads (FIG. 6). The greatest signal enhancement was observed when coelenterazine-h was used, and each derivative generated at least twice as much signal as the control. This result implies that further chemical modifications to the coelenterazine backbone may yield an increased in generated luminescence.

EXAMPLE 6: SURFACE FUNCTIONALIZATION AFFECTS THE RESULTING EIPE-INDUCED LUMINESCENCE

[0087] Fluorescent-embedded polystyrene microspheres with different surface modifications (Non-Reactive, Amine, and Streptavidin) were used to investigate the dependence of EiPE on the surface functionalization. Here, Non-Reactive indicates carboxylate functionalized, which is relatively inert in the presence of most chemicals. The wells containing the microspheres consisted of 70 L of 1PBS, 20 L of coelenterazine-h, and 10 L of the respective microsphere. The fourth well contained 80 L of 1PBS and 20 L of coelenterazine-h. Finally, another control was established using non-fluorescent carboxylate microspheres following the standard protocol of 70 L of 1PBS, 20 L of coelenterazine-h, and 10 L of the respective microsphere.

[0088] Solutions containing identical concentrations of the microspheres and Coelenterazine-h prepared in a black 96-well plate were observed under the Total Light filter set of an IVIS100 bioluminescence imaging system. The resulting photons/second/cm.sup.2 (p/s/cm.sup.2) flux is shown in Table II.

TABLE-US-00002 TABLE II Surface functionalization affects the resulting EiPE-induced luminescence Surface p/s/cm.sup.2/(10.sup.6) Non-Reactive 2.13 Amine 1.63 Streptavidin 3.45 Control 0.16 Blank Spheres 1.02

[0089] As can be seen (Table II) there exists a dependence on the surface functionalization.

EXAMPLE 7: CHANGING THE SURFACE AREA OF RESYN BEADS AND ITS EFFECT ON EIPE SIGNAL GENERATION

[0090] Non-fluorescent polymeric microspheres with varying degrees of crosslinking provided by ReSyn were used to investigate the surface area dependence of EiPE.

[0091] A ReSyn bead consists of a long polymeric chain that freely and naturally winds around itself generating a microsphere with a massive surface area to volume ratio. As such, the polymer generates a bead-like structure that is extremely porous and allows the free movement of liquids throughout its entirety. ReSyn beads were fabricated such that they had varying degrees of crosslinking, thus regulating the amount of surface area available for the EiPE reaction. Four types of microspheres were used: A, B, C, and M, where the degree of cross-linking increased, and the available surface area decreased, from A to B to C. The type M was a magnetized bead with an unspecified degree of crosslinking, but was anticipated to be similar to A. In triplicate, solutions containing 70 L of 1PBS, 20 L of coelenterazine-h, and 10 L of the respective microspheres were distributed into individual wells of a black 96 well plate. As a control, a solution consisting of 80 L of 1PBS and 20 L of coelenterazine-h was prepared and dispensed in triplicate into individual wells of a second black 96 well plate. The two plates were then placed into an IVIS Spectrum and observed under all the available filter sets 2 (20 nm band pass filters which encapsulate the emission from 490 nm to 850 nm) using an exposure time of 30 seconds. The average standard deviation of the photon flux (p/s/cm.sup.2) found for each type of bead and the control was then calculated and plotted against the central wavelength of the respective band pass filter.

[0092] As is indicated (FIG. 7), ReSyn A had the least amount of crosslinking, followed by ReSyn B and then ReSyn C. ReSyn M is a magnetic variant of ReSyn A, whereas Sub indicates a solution where no beads are present but only the substrate coelenterazine-h in 1PBS. While the resulting emission maxima for each ReSyn bead occurred at the same region (510-530 nm), the intensity of the emission is strongly correlated to the degree of crosslinking. The results indicate that more signal is generated if more surface area is available, suggesting that the surface of the bead strongly influences the EiPE effect.

EXAMPLE 8: WAVELENGTH-SPECIFIC SIGNAL GENERATION USING EIPE WITH QD705-LABELLED RESYN BEADS

[0093] Custom non-fluorescent polymeric microspheres labeled with varying concentrations of QD705 were provided by ReSyn and used to demonstrate the ability to create wavelength-dependent luminescent particles. The microspheres were labeled with four different concentrations of QD705: 0, 0.037, 0.081, and 0.152 M. In triplicate, solutions containing 70 L of 1PBS, 20 L of coelenterazine-h, and 10 L of the microspheres were prepared and dispensed into individual wells of a black 96 well plate. The plate was then placed into an IVIS Spectrum and observed under the 710 nm-730 nm filter set. The resulting photon flux was normalized by the average signal observed from the highest QD705 concentration.

[0094] As can be seen from the plotted data (FIG. 8), the most intense QD705 signal occurred in the case where the concentration was at the highest. The resulting luminescence decreased in a linear manner. This indicates that there exists the possibility to create biocompatible polymeric backbones to which a fluorophore or a fluorescent nanoparticle may be attached, and the wavelength of the luminescence predicted.

EXAMPLE 9: WAVELENGTH SPECIFIC SIGNAL GENERATION USING EIPE WITH RHODAMINE6-LOADED LIPID NANOPARTICLES (LNPS) FROM GLOBAL ACORN

[0095] Custom lipid nanoparticles (Global Acorn, UK) that contained no fluorophore (C LNP), were surface labeled and contained rhodamine-6 (R1), or that only contained rhodamine-6 (R2), were used to further demonstrate the ability to create wavelength-dependent luminescent particles that are highly biocompatible. Three different solutions consisting of 70 L of 1PBS, 20 L of coelenterazine-h, and 10 L of the respective lipid nanoparticles were prepared and dispensed into individual wells of a black 96 well plate. The plate was placed into an IVIS100 and visualized under the indicated filter sets at an exposure time of 60 seconds. The resulting photon flux (p/s/cm.sup.2) for each particle type were normalized to the value found for the C LNP.

[0096] As can be seen (FIG. 9), the LNP that was both labeled with and contained the rhodamine-6 produced the greatest amount of signal under the Total Light, 575-650 nm, and 610LP emission filters. This demonstrates that there exists the possibility to create biocompatible polymeric backbones to which a fluorophore or a fluorescent nanoparticle may be attached, and the wavelength of the luminescence predicted.

EXAMPLE 10: WAVELENGTH-SPECIFIC SIGNAL GENERATION USING EIPE WITH FLUOROPHORE-EMBEDDED MICROSPHERES IN THE PRESENCE OF FIXED J774A.1 MURINE MACROPHAGES IN SUSPENSION

[0097] J774A.1 murine macrophages were grown in standard DMEM medium with 10% fetal bovine serum. Starting from a fresh culture at approximately 70% confluence, the cell culture medium was removed and replaced with 5 mL of Trypsin. The cells were exposed for 5 minutes at 37 C. The resulting cell suspension was then placed into a 15 mL Falcon tube and centrifuged for 5 minutes at 1000 rpm. The cells were washed 3 times using 1PBS before being fixed in suspension using 1% PFA for 15 minutes. Upon completion, the fixed cells were again washed 3 times in 1PBS before being counted. The cell concentration was then adjusted such that a concentration of 70,000 cells per 100 L was achieved. Subsequently, 100 L of the cell suspension was aliquoted into 36 wells of a black 96 well plate.

[0098] Red fluorescent polystyrene microspheres (0.5 m diameter) with an excitation maximum at 580 nm and an emission maximum of 605 nm, with an initial concentration of 2% solids (Life Technologies), were serially diluted by 12 two-fold increments using 1PBS. From the dilutions, 10 L were added to each well with each dilution being added to three individual wells to create triplicates. As such, a bead to cell ratio ranging from 20,788 beads:cell down to 20 beads:cell was achieved. Further, 20 L of coelenterazine-h was added to each well. The completed plate was then placed into an IVIS Spectrum and visualized under all the available filter sets (ranging from 490 nm-850 nm) using an exposure time of 60 seconds. The resulting average and standard deviation of the total photon flux (p/s/cm.sup.2) for each well under each filter was plotted versus wavelength. Each condition was repeated in triplicate.

[0099] As can be seen (FIG. 10), a substantial wavelength-specific signal was generated whenever the beads were present, with the magnitude of the signal dependent upon the microsphere-to-macrophage ratio. The microsphere-specific signal is clearly discernable from the background at as few as 20 microspheres per macrophage (inset). This ratio is generally quite achievable suggesting that targeted EiPE could be developed for specific in vitro and in vivo applications.

EXAMPLE 11: THE EXISTENCE OF EIPE UNDER IN VIVO CONDITIONS

[0100] Two solutions consisting of either 50 L of coelenterazine-h with 150 IA of 1PBS or 50 L of coelenterazine-h, 50 L of QD705, and 100 L of 1PBS were prepared in eppendorf tubes and used to fill two different syringes. The dorsal side of four 6-week old female BALB/c mice was razed and the mice placed under general anesthesia (isofluorane) before being injected with either 50 L of the control (no QD705, 1 mouse) or 50 L of the solution containing the QD705 (3 mice). The mice were then placed into an IVIS100 and kept under anesthesia. They were then immediately visualized under the Total Light, 470-490 nm, and 695-770 nm filter sets with an exposure time of 60 seconds (FIGS. 11A to 11C). The location of the QD705 was verified using epifluorescence (FIG. 11D). The total photon flux (p/s/cm.sup.2) under each filter set was then plotted (FIG. 11E).

[0101] As can be seen (FIG. 11), nearly a three-fold increase in total signal is observed when the Coel+QD705 solution is present compared to the Coel control. This contrast is even stronger when the QD705-specific filter is used, providing a nearly 20-fold increase in signal. Also acquired was one mouse that received a QD705 injection without the presence of the coelenterazine-h substrate. As anticipated, minimal signal was observed in this case. The results shown here demonstrate the potential of EiPE as a tool to create in vivo luminescence generation. Given the previous evidence verifying the use of biocompatible markers, the EiPE phenomenon contains significant potential for in vivo and in vitro uses with the ability to generate wavelength-specific signals.

EXAMPLE 12: USING EIPE TO GENERATE FLUOROPHORE-SPECIFIC SIGNALS FROM MULTIPLE LUMINESCENT SOURCES COVERING NEARLY THE ENTIRE OPTICAL SPECTRUM

[0102] Coelenterazine-h in 1PBS was mixed with a multitude of fluorophores and fluorescent nanoparticles, including 505/550 nm, 580/605 nm, and 660/680 nm polystyrene microspheres (Yellow, Pink, and Far Red, respectively); QD655, QD705, and QD800; as well as unbound streptavidin-functionalized Alexa dyes (555, 633, and 700). The solutions were produced in triplicate and distributed into a black 96-well plate. The contents of the wells are defined in the following Table III (please note that each solution was repeated three times though it is only listed once):

TABLE-US-00003 TABLE III Contents of the wells .sub.max em (nm) Flourophore.sup.a 1x PBS.sup.a Coelenterazine-h.sup.a Yellow spheres 515 10 70 20 Alexa555 555 5 75 20 Pink spheres 605 10 70 20 Alexa633 633 5 75 20 QD655 655 5 75 20 Far Red spheres 680 10 70 20 QD705 705 5 75 20 Alexa700 700 5 75 20 QD800 800 5 75 20 .sup.athe values represent the distributed volume in L

[0103] The prepared plate was then placed into an IVIS Spectrum and viewed under all available filter sets (ranging from 490 nm-850 nm). The observed photon flux for each bead was then normalized to the average photon flux of its maximum emission wavelength. The resulting normalized values were then averaged and the standard deviations calculated, and the data plotted against the filter set used.

[0104] As can be seen (FIG. 12), the appropriate fluorescence emission spectrum of each compound was achieved using EiPE. This demonstrates that EiPE can be applied to a wide range of fluorophores nearly regardless of their emission wavelength. Furthermore, EiPE can be used to determine spectral fingerprints and multiplexing, allowing for unparalleled spectral precision.

EXAMPLE 13: MULTIPLEXED SPECTRAL DECONVOLUTION USING EIPE

[0105] Polystyrene beads doped with one of three fluorophores (A,B,C) were mixed v/v (AB,AC,BC,ABC) in a 96 well plate in the presence of Coelenterazine-h and treated to reveal EiPE light. Multi-well plate was visualized first using fluorescence illumination (FIG. 13; left panels) as a control, and next using EiPE light generation (FIG. 13; right panels). Spectral deconvolution was performed using 8 filter sets covering the visual spectrum range of 490 to 730 nm with 20 nm bandwidths. The three component channels Spectral deconvolution revealed the presence of the beads both alone and mixed together identically for fluorescence and EiPE illumination thereby validating the utility of the latter as a novel alternative luminescence based method for multiplexed spectral deconvolution of mixed fluorophores.

EXAMPLE 14. DETERGENT ADDITION INCREASES THE EIPE SIGNAL

[0106] Fluorescent polystyrene microspheres were used to investigate the effect of detergent addition on EiPE signal.

[0107] Green fluorosphere, carboxylate modified (0.1 m; 505/515 nm; 2% solid solution; MOLECULAR PROBES/LIFE TECHNOLOGIES) and far red fluorosphere (0.2 m; 660/685 nm; 2% solid solution; MOLECULAR PROBES/LIFE TECHNOLOGIES) were prepared as 2 stock solutions in H.sub.2O and used at final concentrations of 0.025, 0.05, 0.25 and 0.5%, respectively.

[0108] Triton X100 and Tween-20 were prepared as 10 stock solutions in H.sub.2O and used at final concentrations of 0.001, 0.005, 0.01, 0.05, 0.1, 0.25 and 0.5%, respectively.

[0109] Combinations of 100 L of the different concentrations of the respective microspheres, 20 L of the different concentrations of the respective detergents, 20 L of coelenterazine-h (5 g/mL) and 60 L of H.sub.2O were dispensed into individual wells of five black 96 well plates. Control wells contained: (i) coelenterazine-h and different concentrations of the respective detergents (no fluorospheres) and (ii) fluorospheres at different concentration without detergent, but with coelenterazine-h. The prepared plates were placed into an IVIS Spectrum bioluminescence imaging system and observed under the Total light, 500 nm, 700 nm filter sets using an exposure time of 1 min. The microsphere location was confirmed using epifluorescence (Excitation: 500 nm/Emission: 540 nm and Excitation: 675 nm/Emission: 720 nm). The resulting photons/second/cm.sup.2 (p/s/cm.sup.2) flux in the presence of Triton X-100 or Tween-20 was then plotted (FIGS. 14 and 15).

[0110] As can be seen (FIGS. 14 and 15), adding detergent (either Triton-X100 or Tween-20) to low concentration of fluorospheres (0.025 or 0.05%) can improve by a factor 3-4 the signal produced by EiPE.

[0111] 0.01% detergent is optimal to increase the signal of green fluorospheres present at 0.025 or 0.05% in the solution. This is seen in the open configuration or with the 500 nm filter, but not at 700 nm, as expected.

[0112] Similar observation is made using the far red fluorospheres, with the difference that light is emitted at 700 nm instead of 500 nm, and that increased EiPE is seen with the high concentration of beads.

[0113] Altogether, these observations show that detergent can be used as an additive to increase EiPE light output per particle, allowing a better EiPE detection of fewer particles.