OPTICAL BIOPSY STAIN PANELS AND METHODS OF USE

Abstract

Optical biopsy staining panels for in vivo or in situ fluorescent staining of optical tissue (or other appropriate tissue), e.g., for the purpose of a direct biopsy such as an optical biopsy. The stain panels may feature a combination of a nuclear stain and a cytoplasmic stain, as a means of functioning as an invivo or in situ hematoxylin and eosin (H&E) stain. Examples of said stains may include anthracyclines such as Daunomcibin, acriflavines like Proflavine, anthracenediones such as Mitoxantrone, phenothiazines like Methylene Blue, and tri- and tetra-heterocyclic dyes like Fluorescein, Phloxine B, Phenol Red, Rose Bengal, Congo Red, and Indigo Carmine.

Claims

1. An optical biopsy stain panel comprising two or more agents, either of which fluorescently stains: a. nucleic acids; b. cytoplasm; c. cell or subcellular membranes; d. subcellular organelles; e. extracellular matrix components; or f. microbes.

2. The stain panel of claim 1, wherein one or more of the agents are clinical drugs or therapeutic drugs.

3.-4. (canceled)

5. The stain panel of claim 1, wherein the two or more agents are existing clinically-used agents, wherein at least one agent is a fluorescent nucleic acid stain selected from a group of nucleic acid-binding agents consisting of: aminoacridines, anthracyclines, anthracenediones, or phenothiazines.

6. The stain panel of claim 5, wherein the aminoacridines are 9-aminmoacridine or proflavine.

7. The stain panel of claim 5, wherein the anthracyclines are Daunorubicin, Doxorubicin, Epirubicin, Idarubicin, Nogalamycin, or Valrubicin.

8. The stain panel of claim 5, wherein the anthracenediones are Mitoxantrone or Pixantrone.

9. The stain panel of claim 5, wherein the phenothiazine is Methylene Blue.

10. The stain panel of claim 1, wherein the two or more agents are existing clinically-used agents, wherein at least one agent is a fluorescent cytoplasmic stain selected from a group of non-specific protein binding agents consisting of: heterotetracyclic and heteropentacyclic compounds; xanthylium dyes; azo dyes; indoles, or phthalein dyes.

11. The stain panel of claim 10, wherein the heterotetracyclic and heteropentacyclic compounds are fluorescein or Rose Bengal.

12. The stain panel of claim 10, wherein the xanthylium dye is phloxine B.

13. The stain panel of claim 10, wherein the azo dye is Congo Red.

14. The stain panel of claim 10, wherein the indole is Indigo Carmine.

15. The stain panel of claim 10, wherein the phthalein dye is phenol red.

16. The stain panel of claim 1, wherein the two or more agents are existing clinically-used agents, wherein at least one of the agents is a fluorescent cell stain or subcellular membrane stain selected from a group of lipid-binding agents consisting of (proto)porphyrins, ace inhibitors, adrenergics/dopaminergics, antihyperglycemics, antineoplastics, bacitracins, carbapenems, cefalosporins, cyclooxygenase inhibitors, indigotindisulfonic acid, kinase inhibitors, Na+ channel blockers, non-dihydropyridine calcium channel blockers, opioids, phosphodiesterase inhibitors, rifamycins, ristocetins, and tetracyclines.

17. The stain panel of claim 1, wherein the two or more agents are existing clinically-used agents, wherein at least one of the agents is a fluorescent organelle stain selected from a group of organelle binding agents consisting of antimitotics, opioids, restocins, rifamycins, porphyrins, atypical antihistamines, cephalosporins, anti-insomnia agents, anti-inflammatory agents, antineoplastics, antifungal agents, hematopoietic stem cell mobilizers, H1 antagonists, MAOIs, class 1 antiarrythmics, antiviral agents, topoisomerase inhibitors, cephalosporins, dopaminergics/adrenergics, non-steroidal anti-inflammatory agents, and broad-spectrum antimicrobial agents.

18. The stain panel of claim 1, wherein the two or more agents are existing clinically-used agents, wherein at least one of the agents is an extracellular matrix stain.

19. The stain panel of claim 1, wherein the two or more agents are existing clinically-used agents, wherein at least one of the agents is a microbial stain.

20. The stain panel of claim 1, wherein the two or more agents comprise a fluorescent nucleic acid stain and a fluorescent cytoplasmic stain, wherein the fluorescent nucleic acid stain is Daunorucibin Methylene Blue, Mitoxantrone, or Proflavine, and the fluorescent cytoplasmic stain is Fluorescein, Phenol Red, Phloxine B, or Rose Bengal.

21.-29. (canceled)

30. An apparatus where the illumination of an optical biopsy stain panel according to claim 1 is about a single wavelength, wherein observation occurs through color filtering means or amplification or conversion of the resultant fluorescence into observable color range.

31.-34. (canceled)

Description

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)

[0019] The features and advantages of the present invention will become apparent from a consideration of the following detailed description presented in connection with the accompanying drawings in which:

[0020] FIG. 1 shows proflavine, a topical antiseptic and wound irrigant, after being topically applied to a tissue sample (50 μl at 20 μM concentration) and imaged. Image field is 635.5 μm tall×wide. Inset is the structure of Proflavine.

[0021] FIG. 2A shows bovine lung tissue stained with Fluorescein (500 nM) and imaged using confocal microscopy excited with 488 nm wavelengths at various gains. Image field is 635.5 μm tall×wide.

[0022] FIG. 2B shows bovine lung tissue stained with Phenol Red (100 μM) and imaged using confocal microscopy excited with 488 nm wavelengths at various gains. Image field is 635.5 μm tall×wide.

[0023] FIG. 2C shows bovine lung tissue stained with Phloxine B (10 μM) and imaged using confocal microscopy excited with 488 nm wavelengths at various gains. Image field is 635.5 μm tall×wide.

[0024] FIG. 2D shows bovine lung tissue stained with Rose Bengal (100 μM) and imaged using confocal microscopy excited with 488 nm wavelengths at various gains. Image field is 635.5 μm tall×wide.

[0025] FIG. 2E shows the inset from FIG. 2A, which shows the fluorescence signal from fibers and variable cellular uptake.

[0026] FIG. 2F shows the inset from FIG. 2B, which shows the fluorescence signal from fibers and variable cellular uptake.

[0027] FIG. 2G shows the inset from FIG. 2C, which shows the fluorescence signal from fibers and variable cellular uptake.

[0028] FIG. 2H shows the inset from FIG. 2D, which shows the fluorescence signal from fibers and variable cellular uptake.

[0029] FIG. 2I shows an optimized autofluorescence signal, demonstrating prominent fibers but relative lack of cellular signal. Image field is 635.5 μm tall×wide.

[0030] FIG. 3 shows Daunorubicin and Fluorescein as DNA and protein/cytoplasm dyes, respectively. The combination of 25 μM Daunorubicin and 250 nM Fluorescein was added to bovine lung tissue samples and imaged using confocal microscopy with 488 nm excitation and 10 μm thick optical focal plane, with imaged fields being 635.5 μm.sup.2. Inset shows emission spectra of the combination at 488 nm excitation with the microscope channels indicated along the abscissa.

[0031] FIG. 4A shows Methylene Blue as a DNA dye combined with an off-label protein/cytoplasm dye (50 μM Methylene Blue with 250 nM Fluorescein). Imaging was with confocal microscopy, with a bovine lung sample thickness of 10 μm, and an image field of 635.5 μm.sup.2. Inset shows fluorescence emission spectra of the combination in similar concentration ratios also at 488 nm excitation, with the microscope detector channels along the abscissa.

[0032] FIG. 4B shows Methylene Blue as a DNA dye combined with an off-label protein/cytoplasm dye (100 μM Methylene Blue with 100 μM Phenol Red). Imaging was with confocal microscopy, with a bovine lung sample thickness of 10 μm, and an image field of 635.5 μm.sup.2. Inset shows fluorescence emission spectra of the combination in similar concentration ratios also at 488 nm excitation, with the microscope detector channels along the abscissa.

[0033] FIG. 4C shows Methylene Blue as a DNA dye combined with an off-label protein/cytoplasm dye (100 μM Methylene Blue with 10 μM Phloxine B). Imaging was with confocal microscopy, with a bovine lung sample thickness of 10 μm, and an image field of 635.5 μm.sup.2. Inset shows fluorescence emission spectra of the combination in similar concentration ratios also at 488 nm excitation, with the microscope detector channels along the abscissa.

[0034] FIG. 4D shows Methylene Blue as a DNA dye combined with an off-label protein/cytoplasm dye (100 μM Methylene Blue with 100 μM Rose Bengal). Imaging was with confocal microscopy, with a bovine lung sample thickness of 10 μm, and an image field of 635.5 μm.sup.2. Inset shows fluorescence emission spectra of the combination in similar concentration ratios also at 488 nm excitation, with the microscope detector channels along the abscissa.

[0035] FIG. 5A shows Mitoxantrone as a DNA dye combined with a protein/cytoplasm dye (Mitoxantrone 50 μM and Fluorescein 250 nM). Imaging was with confocal microscopy settings at 488 nm excitation after topical application to the bovine lung specimen. Image field is 635.5 μm.sup.2 at 10 μm thickness. Inset shows fluorescence emission spectra of the combination in similar ratios also at 488 nm excitation.

[0036] FIG. 5B shows Mitoxantrone as a DNA dye combined with a protein/cytoplasm dye (Mitoxantrone at 100 μM and Phenol Red 100 μM). Imaging was with confocal microscopy settings at 488 nm excitation after topical application to the bovine lung specimen. Image field is 635.5 μm.sup.2 at 10 μm thickness. Inset shows fluorescence emission spectra of the combination in similar ratios also at 488 nm excitation.

[0037] FIG. 5C shows Mitoxantrone as a DNA dye combined with a protein/cytoplasm dye (Mitoxantrone 50 μM and Phloxine B 50 μM). Imaging was with confocal microscopy settings at 488 nm excitation after topical application to the bovine lung specimen. Image field is 635.5 μm.sup.2 at 10 μm thickness. Inset shows fluorescence emission spectra of the combination in similar ratios also at 488 nm excitation.

[0038] FIG. 5D shows Mitoxantrone as a DNA dye combined with a protein/cytoplasm dye (Mitoxantrone 100 μM and Rose Bengal 100 μM). Imaging was with confocal microscopy settings at 488 nm excitation after topical application to the bovine lung specimen. Image field is 635.5 μm.sup.2 at 10 μm thickness. Inset shows fluorescence emission spectra of the combination in similar ratios also at 488 nm excitation.

[0039] FIG. 6A shows Proflavine as a DNA drug-dye with Rose Bengal as a protein/cytoplasm drug-dye (50 μl of 100 μM Proflavine and 100 μM Rose Bengal). The dyes were pipetted in situ upon bovine tissue ex vivo and examined under confocal microscopy with a 488 nm laser with 10 μm optical thickness and imaged fields being 635.5 μm.sup.2. Inset shows the fluorescence emission spectrum of the combination at 488 nm excitation.

[0040] FIG. 6B shows bovine lung tissue fixed in 10% formalin, paraffin embedded, cut to 5 μm sections and stained with Hematoxylin and Eosin per routine protocol, then imaged at 20×.

[0041] FIG. 6C shows an inversion of the colors of FIG. 6B, providing a black background. Image field is 1400×1052 μm.

[0042] FIG. 7 shows human breast cancer metastasized to the lung and stained with Rose Bengal and Proflavine to outline the proteins and nucleus, respectively, at concentrations similar to that in FIG. 6A. Image field is 625 μm.sup.2.

[0043] FIG. 8A shows emission spectra of Mitoxantrone as a Hematoxylin-alternative candidate at 10 μM, with Phloxine B at 0.1 μM as an Eosin-alternative candidate drug-dye, excited at 375 nm.

[0044] FIG. 8B shows emission spectra of Mitoxantrone as a Hematoxylin-alternative candidate at 10 μM, with Phloxine B at 0.1 μM as an Eosin-alternative candidate drug-dye, excited at 405 nm.

[0045] FIG. 8C shows emission spectra of Mitoxantrone as a Hematoxylin-alternative candidate at 10 μM, with Phloxine B at 0.1 μM as an Eosin-alternative candidate drug-dye, excited at 455 nm.

[0046] FIG. 8D shows emission spectra of Mitoxantrone as a Hematoxylin-alternative candidate at 10 μM, with Phloxine B at 0.1 μM as an Eosin-alternative candidate drug-dye, excited at 488 nm.

[0047] FIG. 8E and FIG. 8F show concentration-dependent fluorescence of Mitoxantrone but not Phloxine B results in various concentration ratios of the two resulting in similar fluorescence intensity peaks, all excited at 455 nm. (Of note, smoothing after normalizing to peak intensity resulted in peaks not reaching exactly 1.0).

[0048] FIG. 9 shows an example of fluorescence incompatibility. At 488 nm excitation, increasing concentrations of Phloxine B (a proposed Eosin-like dye) resulted in progressive decrease of fluorescence emission from the Daunorubicin (a proposed Hematoxylin-like dye) fluorescence peak around 590 nm. Additional incompatibility results from overlapping of the component peak emissions.

[0049] FIG. 10 shows borderline compatibility of nucleic acid and cytoplasmic dyes. Actual (not normalized) fluorescence intensity of 10 μM Pyrvinium Pamoate decreased in the presence of 1 μM Rose Bengal, but not enough to obscure the Pyrvinium Pamoate component of the combined spectrum at 488 nm excitation.

[0050] FIG. 11 shows fluorescence emission spectra compatibility in vitro of the possible protein drug-dye Fluorescein with nucleic acid drug-dye Daunorubicin. Daunorubicin (1 μM), Fluorescein (1 nM) and the combination of the two were excited at 375 nm, with resultant fluorescence emission.

[0051] FIG. 12A shows fluorescence emission spectra compatibility in vitro of Methylene Blue as a DNA drug-dye with Fluorescein as a protein/cytoplasmic dye at 455 nm excitation.

[0052] FIG. 12B shows fluorescence emission spectra compatibility in vitro of Methylene Blue as a DNA drug-dye with 300 μM Phenol Red at pH 5 and 488 nm excitation.

[0053] FIG. 12C shows fluorescence emission spectra compatibility in vitro of Methylene Blue as a DNA drug-dye with Phloxine B at 2 μM at 520 nm excitation.

[0054] FIG. 12D shows fluorescence emission spectra compatibility in vitro of Methylene Blue as a DNA drug-dye with 1 μM Rose Bengal at 488 nm excitation.

[0055] FIG. 13A shows fluorescence emission spectra compatibility in vitro of the cytoplasmic drug-dye Fluorescein at 0.5 nM with nucleic acid drug-dye Mitoxantrone at 10 μM. The combination was excited at 375 nm, with the resultant emission spectrum shown.

[0056] FIG. 13B shows fluorescence emission spectra compatibility in vitro of the cytoplasmic drug-dye Phloxine B at 10 μM with nucleic acid drug-dye Mitoxantrone at 10 μM. The combination was excited at 405 nm, and the resultant spectrum displayed.

[0057] FIG. 13C shows fluorescence emission spectra compatibility in vitro of the cytoplasmic drug-dye Phenol Red at 100 μM with nucleic acid drug-dye Mitoxantrone at 10 μM. The combination of 10 μM Mitoxantrone and 10 μM Phenol Red at pH 5 was excited at 455 nm; the resultant spectrum is shown.

[0058] FIG. 13D shows fluorescence emission spectra compatibility in vitro of the cytoplasmic drug-dye Rose Bengal at 1 μM with nucleic acid drug-dye Mitoxantrone at 10 μM. The combination was excited at 455 nm and the resultant spectrum displayed.

[0059] FIG. 14 shows fluorescence emission spectra compatibility in vitro of Rose Bengal as a cytoplasmic drug-dye candidate with the nucleic acid drug-dye Proflavine. With 405 nm excitation, Proflavine at 0.25 μM, Rose Bengal at 10 μM, and the combination result in roughly the same order of magnitude peaks resulting from their respective fluorescence.

[0060] FIG. 15A shows fluorescence emission spectra compatibility in vitro of the cytoplasm-dye Fluorescein at 5 nM with nucleic acid drug-dye Pyrvinium Pamoate at 10 μM. The combination emission spectrum is shown with 455 nm excitation.

[0061] FIG. 15B shows fluorescence emission spectra compatibility in vitro of the cytoplasm-dye Phloxine B at 100 nM with nucleic acid drug-dye Pyrvinium Pamoate at 10 μM. The combination emission spectrum is shown with 455 nm excitation.

[0062] FIG. 15C shows fluorescence emission spectra compatibility in vitro of the cytoplasm-dye Phenol Red at 100 μM with nucleic acid drug-dye Pyrvinium Pamoate at 10 μM. The combination emission spectrum is shown with 488 nm excitation at approximately pH 5.

[0063] FIG. 15D shows fluorescence emission spectra compatibility in vitro of the cytoplasm-dye Rose Bengal at 1 μM with nucleic acid drug-dye Pyrvinium Pamoate at 10 μM. The combination emission spectrum is shown with 488 nm excitation.

DETAILED DESCRIPTION OF THE INVENTION

[0064] Unless otherwise explained, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which a disclosed invention belongs. The singular terms “a,” “an,” and “the” include plural referents unless context clearly indicates otherwise. Similarly, the word “or” is intended to include “and” unless the context clearly indicates otherwise. The term “comprising” means that other elements can also be present in addition to the defined elements presented. The use of “comprising” indicates inclusion rather than limitation. Stated another way, the term “comprising” means “including principally, but not necessary solely”. Furthermore, variation of the word “comprising”, such as “comprise” and “comprises”, have correspondingly the same meanings. In one respect, the technology described herein related to the herein described compositions, methods, and respective component(s) thereof, as essential to the invention, yet open to the inclusion of unspecified elements, essential or not (“comprising”).

[0065] All embodiments disclosed herein can be combined with other embodiments unless the context clearly dictates otherwise.

[0066] Suitable methods and materials for the practice and/or testing of embodiments of the disclosure are described below. Such methods and materials are illustrative only and are not intended to be limiting. Other methods and materials similar or equivalent to those described herein can be used. For example, conventional methods well known in the art to which the disclosure pertains are described in various general and more specific references.

[0067] All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety for all purposes. In case of conflict, the present specification, including explanations of terms, will control.

[0068] The present invention features optical biopsy staining panels for in vivo or in situ staining of tissue, e.g., for the purpose of a direct biopsy such as an optical biopsy, or in conjunction with a physical biopsy followed by optical biopsy of the harvested tissue. While the compounds used in the optical biopsy staining panels may be FDA-approved chromogenic or fluorescent drugs, none have been used for fluorescent optical staining. Further, it was surprisingly discovered that combinations of said drugs at specific concentrations could be used as fluorescent stain panels.

[0069] Table 1 below shows examples of FDA-approved chromogenic/fluorescent drugs and their current uses

TABLE-US-00001 Chromogenic/fluorescent drug General in vivo Use/Treatment Brilliant Blue G (Acid Blue, Coomassie Ophthalmic contrast Brilliant Blue) Brilliant Green Topical antiseptic additive Congo Red Chromoendoscopy contrast agent Crystal/Gentian Violet Topical antiseptic additive, ophthalmic contrast Fluorescein Ophthalmic contrast, fluorescence angiography Indigo Carmine Neurosurgical, Gastrointestinal and Urological contrast Indocyanine Green Fluorescence angiography, lymphangiography Isosulfan Blue Lymphangiography Lugol's Iodine Solution Topical antiseptic, Stain for Schiller's Test Methylene Blue Antidote for methemoglobinemia, ophthalmic contrast Oftasceine Ophthalmic contrast Patent Blue (Sulfan Blue) Lymphangiography Phenol Red Chromoendoscopy Phloxine B Dental disclosing agent Porphyrinoids/Porphyrin precursors Neurosurgical and Urological surgery contrast (Hexview/Cysview, 5-ALA) Prussian Blue Antidote for cesium and thallium poisoning Rose Bengal Free Acid Ophthalmic contrast Toluidine Blue Visual diagnostic aid on mucosal lesions Trypan Blue Ophthalmic contrast

[0070] Table 2 below shows non-limiting examples of optical biopsy stain panels according to the present invention. The biopsy stain panels may, for example, be equivalent to a traditional H&E stain. For example, in certain embodiments, the compound in the “Dye 1” category represents a nuclear/DNA stain and the compounds in the “Dye 2” category represent a protein/cytoplasm stain.

TABLE-US-00002 Example Dye 1 Dye 2 1 Daunorucibin Fluorescein 2 Methylene Blue Fluorescein 3 Proflavine Fluorescein 4 Acridine Orange Fluorescein 5 Mitoxantrone Fluorescein 6 Pyrvinium Pamoate Fluorescein 7 Daunorucibin Phloxine B 8 Methylene Blue Phloxine B 9 Proflavine Phloxine B 10 Acridine Orange Phloxine B 11 Mitoxantrone Phloxine B 12 Pyrvinium Pamoate Phloxine B 13 Daunorucibin Phenol Red 14 Methylene Blue Phenol Red 15 Proflavine Phenol Red 16 Acridine Orange Phenol Red 17 Mitoxantrone Phenol Red 18 Pyrvinium Pamoate Phenol Red 19 Daunorucibin Rose Bengal 20 Methylene Blue Rose Bengal 21 Proflavine Rose Bengal 22 Acridine Orange Rose Bengal 23 Mitoxantrone Rose Bengal 24 Pyrvinium Pamoate Rose Bengal 25 Daunorucibin Congo Red 26 Methylene Blue Congo Red 27 Proflavine Congo Red 28 Acridine Orange Congo Red 29 Mitoxantrone Congo Red 30 Pyrvinium Pamoate Congo Red 31 Daunorucibin Indigo Carmine 32 Methylene Blue Indigo Carmine 33 Proflavine Indigo Carmine 34 Acridine Orange Indigo Carmine 35 Mitoxantrone Indigo Carmine 36 Pyrvinium Pamoate Indigo Carmine

[0071] Table 3 below shows non-limiting examples of clinically-used agents also working as lipid membrane stains based on similarity to membrane bilayer research dyes.

TABLE-US-00003 Research membrane dyes Specific Drugs with similarity to Heptamethine research dyes (>0.45 Drug Class Cy5 CY7 DiO/I cyanine dye-1 DrugBank.ca similarity score) (Proto)Porphyrins X X X X Hydroxocobalamin, Cyanocobalamin, Verteporfin Ace inhibitor X Benazepril Adrenergics/ X X X Ergotamine, Silodosin, Yohimbine dopaminergics Antihyperglycemic X Repaglinide Antineoplastic X X X X Vindesine, Bleomycin, Vinblastine Bacitracin X Bacitracin A Carbapenems X X X X Ertapenem Cefalosporins X X Cefpirome, Cefpiramide Cyclooxygenase X Meloxicam, Piroxicam inhibitor Indigotindisulfonic X Indigotindisulonic Acid acid Kinase inhibitors X X X Nintedanib Na+ channel blockers X X Ajmaline Non-dihydropyridine X Diltiazem calcium channel blockers Opioids X Codeine, Morphine Phosphodiesterase X Vardenafil, Sildenafil inhibitor Rifamycins X X X X Rifabutin, Rifapentine, Rifaximin Ristocetins X X Vancomycin, Oritovancin Tetracyclines X X X X Tigecycline

[0072] Table 4 below shows non-limiting examples of clinically-used agents with localization to mitochondria as the prototypical subcellular organelle.

TABLE-US-00004 Mitochondrial Dyes Mitotracker Mitotracker Mitotracker Mitotracker Drugs with similarity score >0.45 Orange Deep Red Red Green Antimitotic: Vinorelbine, Vinblasine, X X Vincristine, Vinflunine, Bleomycin Opioids: Pholcodine, X Benzhydrocodone, Oxycodone, Morphine, Codeine, Hydrocodone, Naltrexone, Methylnaltrexone, Naloxone, Oxymorphone Restocins: Vancomycin, Telavancin X Rifamycins: Rifaximin, Rifapentine X Porphyrins: Cyanocobalamin, X X Cobamamide Atypical Antihistamine: Tritoqualine X Cephalosporins: Ceftolozane, X Ceftaroline Fosamil, Ceftriaxone, Cefpirome Anti-Insomnia: Suvorexant X Anti-inflammatory: Benoxaprofen X Antineoplastic: Trabectedin X

[0073] Table 5 below shows non-limiting examples of clinically-used agents with localization to lysosomes and related organelles similar to research dyes.

TABLE-US-00005 Lysosome Dyes Lysotracker Lysotracker Lysotracker Lysotracker Lysosensor Lysosensor Lysosensor Drugs with similarity score >0.5 Red Yellow Green Blue Blue Yellow Green Antifungal: Butenafine, Naftifine, X X Terbinafine Hematopoietic Stem Cell Mobilizer: X X Plerixafor H1 Antagonist: Cyclizine, Hydroxyzine X X Maoi: Pargyline X Class 1 Antiarrythmics: Procainamide X Porphyrins: Cobamamide, X Methylcobalamin, Hydroxocobalamin, Cyanocobalamin Antimitotics: Vinorelbine, Vinflunine, X Vindesine, Vinblastine, Bleomycin Antiviral: Voxilaprevir, Grazoprevir, X Glecaprevir, Elbasvir, Simeprevir Topoisomerase Inhibitor: Irinotecan X Cephalosporins: Cefpirome, Cefpiramide, X Ceftriaxone, Ceftazidime, Cefixime, Cefotaxime, Cefmenoxime Dopaminergic/Adrenergic: Bromocriptine, X Ergotamine, Dihydroergotamine, Reserpine, Deserpine Rifamycins: Rifapentine, X Rifabutin, Rifampicin Opioid: Naldemedine X Non-Steroidal Antiinflammatory: X Oxaprozin Broad-Spectrum Antimicrobial: X Benzethonium

[0074] The present invention is not limited to the aforementioned examples of optical biopsy stain panels.

[0075] Referring to FIG. 1, 50 μl at 20 μM of Proflavine was topically applied directly to ex vivo bovine lung tissue in situ and imaged immediately with confocal microscopy. The drug-dye Proflavin showed staining like those typical of a fluorescent DNA dye. Likewise, Fluorescein, Phenol Red, Phloxine B, and Rose Bengal were each added to ex vivo bovine lung tissue and imaged under confocal microscopy (see FIGS. 2A-2I). Each drug-dye resulted in variable staining of the proteinaceous connective tissue and cellular cytoplasm, granules or vesicles. Qualitatively at these concentrations, Fluorescein, Phloxine B and Rose Bengal drug-dyes appeared to localize to cell cytoplasm in addition to the connective tissue fibers, which fibers also demonstrated autofluorescence. Fluorescein was the only protein/cytoplasm dye without excessive overlap with the resultant fluorescence signal from Daunorubicin as a DNA-dye. This combination was then added topically to bovine lung tissue and imaged using confocal microscopy (FIG. 3).

[0076] Methylene Blue was tested as a fluorescent DNA drug-dye in combination with several protein/cytoplasm dyes. These combinations were applied topically to bovine lung samples and imaged using confocal microscopy at 488nm excitation (see FIGS. 4A-4D). Also, mitoxantrone was tested as a DNA dye in combination with several protein/cytoplasm drug-dyes. Again, the combinations were topically applied to bovine lung and examined with confocal microscopy at 488 nm excitation (see FIGS. 5A-5D).

[0077] Lastly, a combination of Proflavine and Rose Bengal was tested on bovine lung samples and evaluated with confocal microscopy (see FIG. 6A). Since H&E is the gold-standard to which these new optical stain panels are compared, bovine lung tissue was fixed in formalin, embedded in paraffin and sectioned at 5 μm thickness and finally stained with Hematoxylin and Eosin per routine protocol (see FIG. 6B). To provide a more intuitive comparison in making the background black, the color was inverted (see FIG. 6C).

[0078] FIGS. 8A-8F show emission spectra of various drug dye candidates. FIGS. 8A-8D show Mitoxantrone as a Hematoxylin-alternative candidate at 10 μM, with Phloxine B at 0.1 μM as an Eosin-alternative candidate drug-dye, and a combination of the two excited at different wavelengths: (FIG. 8A) 375 nm, (FIG. 8B) 405 nm, (FIG. 80) 455 nm, and (FIG. 8D) 488 nm. FIGS. 8E-8F show concentration-dependent fluorescence of Mitoxantrone but not Phloxine B results in various concentration ratios of the two resulting in similar fluorescence intensity peaks, all excited at 455 nm. The molar ratios varied primarily based on excitation wavelength, although some dyes demonstrated concentration-dependent change in fluorescence which resulted in unexpected changes in the molar ratio required for similar peak emission intensity. Specifically, Mitoxantrone as a DNA dye mixed with Phloxine B as a non-specific protein dye at 100:1 molar ratio resulted in primarily Mitoxantrone from the resultant fluorescence emission signal at 375 nm excitation (FIG. 8A), but nearly all the fluorescence emission signal from Phloxine B at 488 nm excitation (FIG. 8D). Additionally, there was overlap of emission spectra (FIG. 9) and the combination of certain DNA and protein dyes decreased the emission intensity relative to the same concentration of the component dye alone (FIG. 9 and FIG. 10).

[0079] FIGS. 11-15 demonstrate individual and combined fluorescence emission spectra of particular DNA dyes and protein/cytoplasm dyes. Of note, Pyrvinium Pamoate was not further evaluated based on substantial overlap of the emission spectra of all the protein/cytoplasm dyes.

[0080] The present invention also features methods of use of the optical biopsy stain panels of the present invention. In certain embodiments, the methods feature injecting one or a combination of stains (e.g., in series, at the same time) into the target tissue, introducing the microscope (e.g., microendocsope) to the tissue, and viewing the stain. In certain embodiments, the methods herein provide for a provisional diagnosis and allow for determining whether or not to proceed with a physical biopsy.

[0081] The present invention also features kits comprising one or a combination of dyes disclosed herein, e.g., the combinations disclosed in Table 2. For example, in certain embodiments, the kit comprises Daunorucibin and Fluorescein; in certain embodiments, the kit comprises Proflavine and Rose Bengal; etc.

[0082] Table 6 below shows non-limiting examples of optical biopsy stain panels and the parts of the cell that are fluorescently labeled.

TABLE-US-00006 9- Profla- Dauno- Doxo- aminoacridine vine rubicin rubicin Epirubicin Idarubicin Fluorecein Nucleic Nucleic Nucleic Nucleic Nucleic Nucleic acids + acids + acids + acids + acids + acids + cytoplasm cytoplasm cytoplasm cytoplasm cytoplasm cytoplasm Rose Nucleic Nucleic Nucleic Nucleic Nucleic Nucleic Bengal acids + acids + acids + acids + acids + acids + cytoplasm cytoplasm cytoplasm cytoplasm cytoplasm cytoplasm Phloxine Nucleic Nucleic Nucleic Nucleic Nucleic Nucleic B acids + acids + acids + acids + acids + acids + cytoplasm cytoplasm cytoplasm cytoplasm cytoplasm cytoplasm Congo Nucleic Nucleic Nucleic Nucleic Nucleic Nucleic Red acids + acids + acids + acids + acids + acids + cytoplasm cytoplasm cytoplasm cytoplasm cytoplasm cytoplasm Indigo Nucleic Nucleic Nucleic Nucleic Nucleic Nucleic Carmine acids + acids + acids + acids + acids + acids + cytoplasm cytoplasm cytoplasm cytoplasm cytoplasm cytoplasm Phenol Nucleic Nucleic Nucleic Nucleic Nucleic Nucleic Red acids + acids + acids + acids + acids + acids + cytoplasm cytoplasm cytoplasm cytoplasm cytoplasm cytoplasm Hydroxycobalamin Nucleic Nucleic Nucleic Nucleic Nucleic Nucleic acids + acids + acids + acids + acids + acids + lipid lipid lipid lipid lipid lipid membranes membranes membranes membranes membranes membranes Cyanocobalamin Nucleic Nucleic Nucleic Nucleic Nucleic Nucleic acids + acids + acids + acids + acids + acids + lipid lipid lipid lipid lipid lipid membranes membranes membranes membranes membranes membranes Verteporfin Nucleic Nucleic Nucleic Nucleic Nucleic Nucleic acids + acids + acids + acids + acids + acids + lipid lipid lipid lipid lipid lipid membranes membranes membranes membranes membranes membranes Benazepril Nucleic Nucleic Nucleic Nucleic Nucleic Nucleic acids + acids + acids + acids + acids + acids + lipid lipid lipid lipid lipid lipid membranes membranes membranes membranes membranes membranes Ergotamine Nucleic Nucleic Nucleic Nucleic Nucleic Nucleic acids + acids + acids + acids + acids + acids + lipid lipid lipid lipid lipid lipid membranes membranes membranes membranes membranes membranes Siiodosin Nucleic Nucleic Nucleic Nucleic Nucleic Nucleic acids + acids + acids + acids + acids + acids + lipid lipid lipid lipid lipid lipid membranes membranes membranes membranes membranes membranes Yohimbine Nucleic Nucleic Nucleic Nucleic Nucleic Nucleic acids + acids + acids + acids + acids + acids + lipid lipid lipid lipid lipid lipid membranes membranes membranes membranes membranes membranes Repaglinide Nucleic Nucleic Nucleic Nucleic Nucleic Nucleic acids + acids + acids + acids + acids + acids + lipid lipid lipid lipid lipid lipid membranes membranes membranes membranes membranes membranes Vindesine Nucleic Nucleic Nucleic Nucleic Nucleic Nucleic acids + acids + acids + acids + acids + acids + lipid lipid lipid lipid lipid lipid membranes membranes membranes membranes membranes membranes Bleomycin Nucleic Nucleic Nucleic Nucleic Nucleic Nucleic acids + acids + acids + acids + acids + acids + lipid lipid lipid lipid lipid lipid membranes membranes membranes membranes membranes membranes Vinblastine Nucleic Nucleic Nucleic Nucleic Nucleic Nucleic acids + acids + acids + acids + acids + acids + lipid lipid lipid lipid lipid lipid membranes membranes membranes membranes membranes membranes Bacitracin Nucleic Nucleic Nucleic Nucleic Nucleic Nucleic A acids + acids + acids + acids + acids + acids + lipid lipid lipid lipid lipid lipid membranes membranes membranes membranes membranes membranes Ertapenem Nucleic Nucleic Nucleic Nucleic Nucleic Nucleic acids + acids + acids + acids + acids + acids + lipid lipid lipid lipid lipid lipid membranes membranes membranes membranes membranes membranes Cefpirome Nucleic Nucleic Nucleic Nucleic Nucleic Nucleic acids + acids + acids + acids + acids + acids + lipid lipid lipid lipid lipid lipid membranes membranes membranes membranes membranes membranes Cefpiramide Nucleic Nucleic Nucleic Nucleic Nucleic Nucleic acids + acids + acids + acids + acids + acids + lipid lipid lipid lipid lipid lipid membranes membranes membranes membranes membranes membranes Meloxicam Nucleic Nucleic Nucleic Nucleic Nucleic Nucleic acids + acids + acids + acids + acids + acids + lipid lipid lipid lipid lipid lipid membranes membranes membranes membranes membranes membranes Piroxicam Nucleic Nucleic Nucleic Nucleic Nucleic Nucleic acids + acids + acids + acids + acids + acids + lipid lipid lipid lipid lipid lipid membranes membranes membranes membranes membranes membranes Indigotindisulonic Nucleic Nucleic Nucleic Nucleic Nucleic Nucleic Acid acids + acids + acids + acids + acids + acids + lipid lipid lipid lipid lipid lipid membranes membranes membranes membranes membranes membranes Nintedanib Nucleic Nucleic Nucleic Nucleic Nucleic Nucleic acids + acids + acids + acids + acids + acids + lipid lipid lipid lipid lipid lipid membranes membranes membranes membranes membranes membranes Ajmaline Nucleic Nucleic Nucleic Nucleic Nucleic Nucleic acids + acids + acids + acids + acids + acids + lipid lipid lipid lipid lipid lipid membranes membranes membranes membranes membranes membranes Diltiazem Nucleic Nucleic Nucleic Nucleic Nucleic Nucleic acids + acids + acids + acids + acids + acids + lipid lipid lipid lipid lipid lipid membranes membranes membranes membranes membranes membranes Codeine Nucleic Nucleic Nucleic Nucleic Nucleic Nucleic acids + acids + acids + acids + acids + acids + lipid lipid lipid lipid lipid lipid membranes membranes membranes membranes membranes membranes or or or or or or mitochondria mitochondria mitochondria mitochondria mitochondria mitochondria Morphine Nucleic Nucleic Nucleic Nucleic Nucleic Nucleic acids + acids + acids + acids + acids + acids + lipid lipid lipid lipid lipid lipid membranes membranes membranes membranes membranes membranes or or or or or or mitochondria mitochondria mitochondria mitochondria mitochondria mitochondria Vardenafil Nucleic Nucleic Nucleic Nucleic Nucleic Nucleic acids + acids + acids + acids + acids + acids + lipid lipid lipid lipid lipid lipid membranes membranes membranes membranes membranes membranes Slidenafil Nucleic Nucleic Nucleic Nucleic Nucleic Nucleic acids + acids + acids + acids + acids + acids + lipid lipid lipid lipid lipid lipid membranes membranes membranes membranes membranes membranes Rifabutin Nucleic Nucleic Nucleic Nucleic Nucleic Nucleic acids + acids + acids + acids + acids + acids + lipid lipid lipid lipid lipid lipid membranes membranes membranes membranes membranes membranes Rifapentine Nucleic Nucleic Nucleic Nucleic Nucleic Nucleic acids + acids + acids + acids + acids + acids + lipid lipid lipid lipid lipid lipid membranes membranes membranes membranes membranes membranes or or or or or or mitochondria mitochondria mitochondria mitochondria mitochondria mitochondria Rifaximin Nucleic Nucleic Nucleic Nucleic Nucleic Nucleic acids + acids + acids + acids + acids + acids + lipid lipid lipid lipid lipid lipid membranes membranes membranes membranes membranes membranes or or or or or or mitochondria mitochondria mitochondria mitochondria mitochondria mitochondria Vaneomycin Nucleic Nucleic Nucleic Nucleic Nucleic Nucleic acids + acids + acids + acids + acids + acids + lipid lipid lipid lipid lipid lipid membranes membranes membranes membranes membranes membranes Oritovancin Nucleic Nucleic Nucleic Nucleic Nucleic Nucleic acids + acids + acids + acids + acids + acids + lipid lipid lipid lipid lipid lipid membranes membranes membranes membranes membranes membranes Tigecycline Nucleic Nucleic Nucleic Nucleic Nucleic Nucleic acids + acids + acids + acids + acids + acids + lipid lipid lipid lipid lipid lipid membranes membranes membranes membranes membranes membranes Vinorelbine Nucleic Nucleic Nucleic Nucleic Nucleic Nucleic acids + acids + acids + acids + acids + acids + mitochondria mitochondria mitochondria mitochondria mitochondria mitochondria or or or or or or lysosome lysosome lysosome lysosome lysosome lysosome Vinblastine Nucleic Nucleic Nucleic Nucleic Nucleic Nucleic acids + acids + acids + acids + acids + acids + mitochondria mitochondria mitochondria mitochondria mitochondria mitochondria or or or or or or lysosome lysosome lysosome lysosome lysosome lysosome Vincristine Nucleic Nucleic Nucleic Nucleic Nucleic Nucleic acids + acids + acids + acids + acids + acids + mitochondria mitochondria mitochondria mitochondria mitochondria mitochondria or or or or or or lysosome lysosome lysosome lysosome lysosome lysosome Vinflunine Nucleic Nucleic Nucleic Nucleic Nucleic Nucleic acids + acids + acids + acids + acids + acids + mitochondria mitochondria mitochondria mitochondria mitochondria mitochondria or or or or or or lysosome lysosome lysosome lysosome lysosome lysosome Bieomycin Nucleic Nucleic Nucleic Nucleic Nucleic Nucleic acids + acids + acids + acids + acids + acids + mitochondria mitochondria mitochondria mitochondria mitochondria mitochondria or or or or or or lysosome lysosome lysosome lysosome lysosome lysosome Pholcodine Nucleic Nucleic Nucleic Nucleic Nucleic Nucleic acids + acids + acids + acids + acids + acids + mitochondria mitochondria mitochondria mitochondria mitochondria mitochondria Benzhydrocodone Nucleic Nucleic Nucleic Nucleic Nucleic Nucleic acids + acids + acids + acids + acids + acids + mitochondria mitochondria mitochondria mitochondria mitochondria mitochondria Oxycodone Nucleic Nucleic Nucleic Nucleic Nucleic Nucleic acids + acids + acids + acids + acids + acids + mitochondria mitochondria mitochondria mitochondria mitochondria mitochondria Hydrocodone Nucleic Nucleic Nucleic Nucleic Nucleic Nucleic acids + acids + acids + acids + acids + acids + mitochondria mitochondria mitochondria mitochondria mitochondria mitochondria Naltrexone Nucleic Nucleic Nucleic Nucleic Nucleic Nucleic acids + acids + acids + acids + acids + acids + mitochondria mitochondria mitochondria mitochondria mitochondria mitochondria Methylnaltrexone Nucleic Nucleic Nucleic Nucleic Nucleic Nucleic acids + acids + acids + acids + acids + acids + mitochondria mitochondria mitochondria mitochondria mitochondria mitochondria Naloxone Nucleic Nucleic Nucleic Nucleic Nucleic Nucleic acids + acids + acids + acids + acids + acids + mitochondria mitochondria mitochondria mitochondria mitochondria mitochondria Oxymorphone Nucleic Nucleic Nucleic Nucleic Nucleic Nucleic acids + acids + acids + acids + acids + acids + mitochondria mitochondria mitochondria mitochondria mitochondria mitochondria Telavancin Nucleic Nucleic Nucleic Nucleic Nucleic Nucleic acids + acids + acids + acids + acids + acids + mitochondria mitochondria mitochondria mitochondria mitochondria mitochondria Cyanocobalamin Nucleic Nucleic Nucleic Nucleic Nucleic Nucleic acids + acids + acids + acids + acids + acids + mitochondria mitochondria mitochondria mitochondria mitochondria mitochondria or or or or or or lysosome lysosome lysosome lysosome lysosome lysosome Cobamamide Nucleic Nucleic Nucleic Nucleic Nucleic Nucleic acids + acids + acids + acids + acids + acids + mitochondria mitochondria mitochondria mitochondria mitochondria mitochondria or or or or or or lysosome lysosome lysosome lysosome lysosome lysosome Tritoqualine Nucleic Nucleic Nucleic Nucleic Nucleic Nucleic acids + acids + acids + acids + acids + acids + mitochondria mitochondria mitochondria mitochondria mitochondria mitochondria Ceftoiozane Nucleic Nucleic Nucleic Nucleic Nucleic Nucleic acids + acids + acids + acids + acids + acids + mitochondria mitochondria mitochondria mitochondria mitochondria mitochondria or or or or or or lysosome lysosome lysosome lysosome lysosome lysosome Ceftaroline Nucleic Nucleic Nucleic Nucleic Nucleic Nucleic acids + acids + acids + acids + acids + acids + mitochondria mitochondria mitochondria mitochondria mitochondria mitochondria or or or or or or lysosome lysosome lysosome lysosome lysosome lysosome Fosamil Nucleic Nucleic Nucleic Nucleic Nucleic Nucleic acids + acids + acids + acids + acids + acids + mitochondria mitochondria mitochondria mitochondria mitochondria mitochondria Ceftriaxone Nucleic Nucleic Nucleic Nucleic Nucleic Nucleic acids + acids + acids + acids + acids + acids + mitochondria mitochondria mitochondria mitochondria mitochondria mitochondria or or or or or or lysosome lysosome lysosome lysosome lysosome lysosome Cefpirome Nucleic Nucleic Nucleic Nucleic Nucleic Nucleic acids + acids + acids + acids + acids + acids + mitochondria mitochondria mitochondria mitochondria mitochondria mitochondria or or or or or or lysosome lysosome lysosome lysosome lysosome lysosome Suvorexant Nucleic Nucleic Nucleic Nucleic Nucleic Nucleic acids + acids + acids + acids + acids + acids + mitochondria mitochondria mitochondria mitochondria mitochondria mitochondria Benoxaprofen Nucleic Nucleic Nucleic Nucleic Nucleic Nucleic acids + acids + acids + acids + acids + acids + mitochondria mitochondria mitochondria mitochondria mitochondria mitochondria Trabectedin Nucleic Nucleic Nucleic Nucleic Nucleic Nucleic acids + acids + acids + acids + acids + acids + mitochondria mitochondria mitochondria mitochondria mitochondria mitochondria Butenafine Nucleic Nucleic Nucleic Nucleic Nucleic Nucleic acids + acids + acids + acids + acids + acids + lysosome lysosome lysosome lysosome lysosome lysosome Naftifine Nucleic Nucleic Nucleic Nucleic Nucleic Nucleic acids + acids + acids + acids + acids + acids + lysosome lysosome lysosome lysosome lysosome lysosome Terbinafine Nucleic Nucleic Nucleic Nucleic Nucleic Nucleic acids + acids + acids + acids + acids + acids + lysosome lysosome lysosome lysosome lysosome lysosome Plerixafor Nucleic Nucleic Nucleic Nucleic Nucleic Nucleic acids + acids + acids + acids + acids + acids + lysosome lysosome lysosome lysosome lysosome lysosome Pargyline Nucleic Nucleic Nucleic Nucleic Nucleic Nucleic acids + acids + acids + acids + acids + acids + lysosome lysosome lysosome lysosome lysosome lysosome Procainamide Nucleic Nucleic Nucleic Nucleic Nucleic Nucleic acids + acids + acids + acids + acids + acids + lysosome lysosome lysosome lysosome lysosome lysosome Methylcobalamin Nucleic Nucleic Nucleic Nucleic Nucleic Nucleic acids + acids + acids + acids + acids + acids + lysosome lysosome lysosome lysosome lysosome lysosome Hydroxo- Nucleic Nucleic Nucleic Nucleic Nucleic Nucleic cobalamin acids + acids + acids + acids + acids + acids + lysosome lysosome lysosome lysosome lysosome lysosome Voxilaprevir Nucleic Nucleic Nucleic Nucleic Nucleic Nucleic acids + acids + acids + acids + acids + acids + lysosome lysosome lysosome lysosome lysosome lysosome Grazoprevir Nucleic Nucleic Nucleic Nucleic Nucleic Nucleic acids + acids + acids + acids + acids + acids + lysosome lysosome lysosome lysosome lysosome lysosome Glecaprevir Nucleic Nucleic Nucleic Nucleic Nucleic Nucleic acids + acids + acids + acids + acids + acids + lysosome lysosome lysosome lysosome lysosome lysosome Elbasvir Nucleic Nucleic Nucleic Nucleic Nucleic Nucleic acids + acids + acids + acids + acids + acids + lysosome lysosome lysosome lysosome lysosome lysosome Simeprevir Nucleic Nucleic Nucleic Nucleic Nucleic Nucleic acids + acids + acids + acids + acids + acids + lysosome lysosome lysosome lysosome lysosome lysosome Irinotecan Nucleic Nucleic Nucleic Nucleic Nucleic Nucleic acids + acids + acids + acids + acids + acids + lysosome lysosome lysosome lysosome lysosome lysosome Cefpiramide Nucleic Nucleic Nucleic Nucleic Nucleic Nucleic acids + acids + acids + acids + acids + acids + lysosome lysosome lysosome lysosome lysosome lysosome Ceftazidime Nucleic Nucleic Nucleic Nucleic Nucleic Nucleic acids + acids + acids + acids + acids + acids + lysosome lysosome lysosome lysosome lysosome lysosome Cefixime Nucleic Nucleic Nucleic Nucleic Nucleic Nucleic acids + acids + acids + acids + acids + acids + lysosome lysosome lysosome lysosome lysosome lysosome Cefotaxime Nucleic Nucleic Nucleic Nucleic Nucleic Nucleic acids + acids + acids + acids + acids + acids + lysosome lysosome lysosome lysosome lysosome lysosome Cefmenoxime Nucleic Nucleic Nucleic Nucleic Nucleic Nucleic acids + acids + acids + acids + acids + acids + lysosome lysosome lysosome lysosome lysosome lysosome Bromocriptine Nucleic Nucleic Nucleic Nucleic Nucleic Nucleic acids + acids + acids + acids + acids + acids + lysosome lysosome lysosome lysosome lysosome lysosome Ergotamine Nucleic Nucleic Nucleic Nucleic Nucleic Nucleic acids + acids + acids + acids + acids + acids + lysosome lysosome lysosome lysosome lysosome lysosome Dihydroer- Nucleic Nucleic Nucleic Nucleic Nucleic Nucleic gotamine acids + acids + acids + acids + acids + acids + lysosome lysosome lysosome lysosome lysosome lysosome Reserpine Nucleic Nucleic Nucleic Nucleic Nucleic Nucleic acids + acids + acids + acids + acids + acids + lysosome lysosome lysosome lysosome lysosome lysosome Deserpine Nucleic Nucleic Nucleic Nucleic Nucleic Nucleic acids + acids + acids + acids + acids + acids + lysosome lysosome lysosome lysosome lysosome lysosome Rifabutin Nucleic Nucleic Nucleic Nucleic Nucleic Nucleic acids + acids + acids + acids + acids + acids + lysosome lysosome lysosome lysosome lysosome lysosome Naldemedine Nucleic Nucleic Nucleic Nucleic Nucleic Nucleic acids + acids + acids + acids + acids + acids + lysosome lysosome lysosome lysosome lysosome lysosome Oxaprozin Nucleic Nucleic Nucleic Nucleic Nucleic Nucleic acids + acids + acids + acids + acids + acids + lysosome lysosome lysosome lysosome lysosome lysosome Benzethonium Nucleic Nucleic Nucleic Nucleic Nucleic Nucleic acids + acids + acids + acids + acids + acids + lysosome lysosome lysosome lysosome lysosome lysosome Rescinnamine Nucleic Nucleic Nucleic Nucleic Nucleic Nucleic acids + acids + acids + acids + acids + acids + lysosome lysosome lysosome lysosome lysosome lysosome Lymecycline Nucleic Nucleic Nucleic Nucleic Nucleic Nucleic acids + acids + acids + acids + acids + acids + lysosome lysosome lysosome lysosome lysosome lysosome Moxifloxacin Nucleic Nucleic Nucleic Nucleic Nucleic Nucleic acids + acids + acids + acids + acids + acids + lysosome lysosome lysosome lysosome lysosome lysosome Dactinomycin Nucleic Nucleic Nucleic Nucleic Nucleic Nucleic acids + acids + acids + acids + acids + acids + lysosome lysosome lysosome lysosome lysosome lysosome Ertapenem Nucleic Nucleic Nucleic Nucleic Nucleic Nucleic acids + acids + acids + acids + acids + acids + lysosome lysosome lysosome lysosome lysosome lysosome Mitoxan- Methylene Nogalamycin Valrubicin trone Pixantrone Blue Fluorecein Nucleic Nucleic Nucleic Nucleic Nucleic acids + acids + acids + acids + acids + cytoplasm cytoplasm cytoplasm cytoplasm cytoplasm Rose Nucleic Nucleic Nucleic Nucleic Nucleic Bengal acids + acids + acids + acids + acids + cytoplasm cytoplasm cytoplasm cytoplasm cytoplasm Phloxine Nucleic Nucleic Nucleic Nucleic Nucleic B acids + acids + acids + acids + acids + cytoplasm cytoplasm cytoplasm cytoplasm cytoplasm Congo Nucleic Nucleic Nucleic Nucleic Nucleic Red acids + acids + acids + acids + acids + cytoplasm cytoplasm cytoplasm cytoplasm cytoplasm Indigo Nucleic Nucleic Nucleic Nucleic Nucleic Carmine acids + acids + acids + acids + acids + cytoplasm cytoplasm cytoplasm cytoplasm cytoplasm Phenol Nucleic Nucleic Nucleic Nucleic Nucleic Red acids + acids + acids + acids + acids + cytoplasm cytoplasm cytoplasm cytoplasm cytoplasm Hydroxycobalamin Nucleic Nucleic Nucleic Nucleic Nucleic acids + acids + acids + acids + acids + lipid lipid lipid lipid lipid membranes membranes membranes membranes membranes Cyanocobalamin Nucleic Nucleic Nucleic Nucleic Nucleic acids + acids + acids + acids + acids + lipid lipid lipid lipid lipid membranes membranes membranes membranes membranes Verteporfin Nucleic Nucleic Nucleic Nucleic Nucleic acids + acids + acids + acids + acids + lipid lipid lipid lipid lipid membranes membranes membranes membranes membranes Benazepril Nucleic Nucleic Nucleic Nucleic Nucleic acids + acids + acids + acids + acids + lipid lipid lipid lipid lipid membranes membranes membranes membranes membranes Ergotamine Nucleic Nucleic Nucleic Nucleic Nucleic acids + acids + acids + acids + acids + lipid lipid lipid lipid lipid membranes membranes membranes membranes membranes Siiodosin Nucleic Nucleic Nucleic Nucleic Nucleic acids + acids + acids + acids + acids + lipid lipid lipid lipid lipid membranes membranes membranes membranes membranes Yohimbine Nucleic Nucleic Nucleic Nucleic Nucleic acids + acids + acids + acids + acids + lipid lipid lipid lipid lipid membranes membranes membranes membranes membranes Repaglinide Nucleic Nucleic Nucleic Nucleic Nucleic acids + acids + acids + acids + acids + lipid lipid lipid lipid lipid membranes membranes membranes membranes membranes Vindesine Nucleic Nucleic Nucleic Nucleic Nucleic acids + acids + acids + acids + acids + lipid lipid lipid lipid lipid membranes membranes membranes membranes membranes Bleomycin Nucleic Nucleic Nucleic Nucleic Nucleic acids + acids + acids + acids + acids + lipid lipid lipid lipid lipid membranes membranes membranes membranes membranes Vinblastine Nucleic Nucleic Nucleic Nucleic Nucleic acids + acids + acids + acids + acids + lipid lipid lipid lipid lipid membranes membranes membranes membranes membranes Bacitracin Nucleic Nucleic Nucleic Nucleic Nucleic A acids + acids + acids + acids + acids + lipid lipid lipid lipid lipid membranes membranes membranes membranes membranes Ertapenem Nucleic Nucleic Nucleic Nucleic Nucleic acids + acids + acids + acids + acids + lipid lipid lipid lipid lipid membranes membranes membranes membranes membranes Cefpirome Nucleic Nucleic Nucleic Nucleic Nucleic acids + acids + acids + acids + acids + lipid lipid lipid lipid lipid membranes membranes membranes membranes membranes Cefpiramide Nucleic Nucleic Nucleic Nucleic Nucleic acids + acids + acids + acids + acids + lipid lipid lipid lipid lipid membranes membranes membranes membranes membranes Meloxicam Nucleic Nucleic Nucleic Nucleic Nucleic acids + acids + acids + acids + acids + lipid lipid lipid lipid lipid membranes membranes membranes membranes membranes Piroxicam Nucleic Nucleic Nucleic Nucleic Nucleic acids + acids + acids + acids + acids + lipid lipid lipid lipid lipid membranes membranes membranes membranes membranes Indigotindisulonic Nucleic Nucleic Nucleic Nucleic Nucleic Acid acids + acids + acids + acids + acids + lipid lipid lipid lipid lipid membranes membranes membranes membranes membranes Nintedanib Nucleic Nucleic Nucleic Nucleic Nucleic acids + acids + acids + acids + acids + lipid lipid lipid lipid lipid membranes membranes membranes membranes membranes Ajmaline Nucleic Nucleic Nucleic Nucleic Nucleic acids + acids + acids + acids + acids + lipid lipid lipid lipid lipid membranes membranes membranes membranes membranes Diltiazem Nucleic Nucleic Nucleic Nucleic Nucleic acids + acids + acids + acids + acids + lipid lipid lipid lipid lipid membranes membranes membranes membranes membranes Codeine Nucleic Nucleic Nucleic Nucleic Nucleic acids + acids + acids + acids + acids + lipid lipid lipid lipid lipid membranes membranes membranes membranes membranes or or or or or mitochondria mitochondria mitochondria mitochondria mitochondria Morphine Nucleic Nucleic Nucleic Nucleic Nucleic acids + acids + acids + acids + acids + lipid lipid lipid lipid lipid membranes membranes membranes membranes membranes or or or or or mitochondria mitochondria mitochondria mitochondria mitochondria Vardenafil Nucleic Nucleic Nucleic Nucleic Nucleic acids + acids + acids + acids + acids + lipid lipid lipid lipid lipid membranes membranes membranes membranes membranes Slidenafil Nucleic Nucleic Nucleic Nucleic Nucleic acids + acids + acids + acids + acids + lipid lipid lipid lipid lipid membranes membranes membranes membranes membranes Rifabutin Nucleic Nucleic Nucleic Nucleic Nucleic acids + acids + acids + acids + acids + lipid lipid lipid lipid lipid membranes membranes membranes membranes membranes Rifapentine Nucleic Nucleic Nucleic Nucleic Nucleic acids + acids + acids + acids + acids + lipid lipid lipid lipid lipid membranes membranes membranes membranes membranes or or or or or mitochondria mitochondria mitochondria mitochondria mitochondria Rifaximin Nucleic Nucleic Nucleic Nucleic Nucleic acids + acids + acids + acids + acids + lipid lipid lipid lipid lipid membranes membranes membranes membranes membranes or or or or or mitochondria mitochondria mitochondria mitochondria mitochondria Vaneomycin Nucleic Nucleic Nucleic Nucleic Nucleic acids + acids + acids + acids + acids + lipid lipid lipid lipid lipid membranes membranes membranes membranes membranes Oritovancin Nucleic Nucleic Nucleic Nucleic Nucleic acids + acids + acids + acids + acids + lipid lipid lipid lipid lipid membranes membranes membranes membranes membranes Tigecycline Nucleic Nucleic Nucleic Nucleic Nucleic acids + acids + acids + acids + acids + lipid lipid lipid lipid lipid membranes membranes membranes membranes membranes Vinorelbine Nucleic Nucleic Nucleic Nucleic Nucleic acids + acids + acids + acids + acids + mitochondria mitochondria mitochondria mitochondria mitochondria or or or or or lysosome lysosome lysosome lysosome lysosome Vinblastine Nucleic Nucleic Nucleic Nucleic Nucleic acids + acids + acids + acids + acids + mitochondria mitochondria mitochondria mitochondria mitochondria or or or or or lysosome lysosome lysosome lysosome lysosome Vincristine Nucleic Nucleic Nucleic Nucleic Nucleic acids + acids + acids + acids + acids + mitochondria mitochondria mitochondria mitochondria mitochondria or or or or or lysosome lysosome lysosome lysosome lysosome Vinflunine Nucleic Nucleic Nucleic Nucleic Nucleic acids + acids + acids + acids + acids + mitochondria mitochondria mitochondria mitochondria mitochondria or or or or or lysosome lysosome lysosome lysosome lysosome Bieomycin Nucleic Nucleic Nucleic Nucleic Nucleic acids + acids + acids + acids + acids + mitochondria mitochondria mitochondria mitochondria mitochondria or or or or or lysosome lysosome lysosome lysosome lysosome Pholcodine Nucleic Nucleic Nucleic Nucleic Nucleic acids + acids + acids + acids + acids + mitochondria mitochondria mitochondria mitochondria mitochondria Benzhydrocodone Nucleic Nucleic Nucleic Nucleic Nucleic acids + acids + acids + acids + acids + mitochondria mitochondria mitochondria mitochondria mitochondria Oxycodone Nucleic Nucleic Nucleic Nucleic Nucleic acids + acids + acids + acids + acids + mitochondria mitochondria mitochondria mitochondria mitochondria Hydrocodone Nucleic Nucleic Nucleic Nucleic Nucleic acids + acids + acids + acids + acids + mitochondria mitochondria mitochondria mitochondria mitochondria Naltrexone Nucleic Nucleic Nucleic Nucleic Nucleic acids + acids + acids + acids + acids + mitochondria mitochondria mitochondria mitochondria mitochondria Methylnaltrexone Nucleic Nucleic Nucleic Nucleic Nucleic acids + acids + acids + acids + acids + mitochondria mitochondria mitochondria mitochondria mitochondria Naloxone Nucleic Nucleic Nucleic Nucleic Nucleic acids + acids + acids + acids + acids + mitochondria mitochondria mitochondria mitochondria mitochondria Oxymorphone Nucleic Nucleic Nucleic Nucleic Nucleic acids + acids + acids + acids + acids + mitochondria mitochondria mitochondria mitochondria mitochondria Telavancin Nucleic Nucleic Nucleic Nucleic Nucleic acids + acids + acids + acids + acids + mitochondria mitochondria mitochondria mitochondria mitochondria Cyanocobalamin Nucleic Nucleic Nucleic Nucleic Nucleic acids + acids + acids + acids + acids + mitochondria mitochondria mitochondria mitochondria mitochondria or or or or or lysosome lysosome lysosome lysosome lysosome Cobamamide Nucleic Nucleic Nucleic Nucleic Nucleic acids + acids + acids + acids + acids + mitochondria mitochondria mitochondria mitochondria mitochondria or or or or or lysosome lysosome lysosome lysosome lysosome Tritoqualine Nucleic Nucleic Nucleic Nucleic Nucleic acids + acids + acids + acids + acids + mitochondria mitochondria mitochondria mitochondria mitochondria Ceftoiozane Nucleic Nucleic Nucleic Nucleic Nucleic acids + acids + acids + acids + acids + mitochondria mitochondria mitochondria mitochondria mitochondria or or or or or lysosome lysosome lysosome lysosome lysosome Ceftaroline Nucleic Nucleic Nucleic Nucleic Nucleic acids + acids + acids + acids + acids + mitochondria mitochondria mitochondria mitochondria mitochondria or or or or or lysosome lysosome lysosome lysosome lysosome Fosamil Nucleic Nucleic Nucleic Nucleic Nucleic acids + acids + acids + acids + acids + mitochondria mitochondria mitochondria mitochondria mitochondria Ceftriaxone Nucleic Nucleic Nucleic Nucleic Nucleic acids + acids + acids + acids + acids + mitochondria mitochondria mitochondria mitochondria mitochondria or or or or or lysosome lysosome lysosome lysosome lysosome Cefpirome Nucleic Nucleic Nucleic Nucleic Nucleic acids + acids + acids + acids + acids + mitochondria mitochondria mitochondria mitochondria mitochondria or or or or or lysosome lysosome lysosome lysosome lysosome Suvorexant Nucleic Nucleic Nucleic Nucleic Nucleic acids + acids + acids + acids + acids + mitochondria mitochondria mitochondria mitochondria mitochondria Benoxaprofen Nucleic Nucleic Nucleic Nucleic Nucleic acids + acids + acids + acids + acids + mitochondria mitochondria mitochondria mitochondria mitochondria Trabectedin Nucleic Nucleic Nucleic Nucleic Nucleic acids + acids + acids + acids + acids + mitochondria mitochondria mitochondria mitochondria mitochondria Butenafine Nucleic Nucleic Nucleic Nucleic Nucleic acids + acids + acids + acids + acids + lysosome lysosome lysosome lysosome lysosome Naftifine Nucleic Nucleic Nucleic Nucleic Nucleic acids + acids + acids + acids + acids + lysosome lysosome lysosome lysosome lysosome Terbinafine Nucleic Nucleic Nucleic Nucleic Nucleic acids + acids + acids + acids + acids + lysosome lysosome lysosome lysosome lysosome Plerixafor Nucleic Nucleic Nucleic Nucleic Nucleic acids + acids + acids + acids + acids + lysosome lysosome lysosome lysosome lysosome Pargyline Nucleic Nucleic Nucleic Nucleic Nucleic acids + acids + acids + acids + acids + lysosome lysosome lysosome lysosome lysosome Procainamide Nucleic Nucleic Nucleic Nucleic Nucleic acids + acids + acids + acids + acids + lysosome lysosome lysosome lysosome lysosome Methylcobalamin Nucleic Nucleic Nucleic Nucleic Nucleic acids + acids + acids + acids + acids + lysosome lysosome lysosome lysosome lysosome Hydroxo- Nucleic Nucleic Nucleic Nucleic Nucleic cobalamin acids + acids + acids + acids + acids + lysosome lysosome lysosome lysosome lysosome Voxilaprevir Nucleic Nucleic Nucleic Nucleic Nucleic acids + acids + acids + acids + acids + lysosome lysosome lysosome lysosome lysosome Grazoprevir Nucleic Nucleic Nucleic Nucleic Nucleic acids + acids + acids + acids + acids + lysosome lysosome lysosome lysosome lysosome Glecaprevir Nucleic Nucleic Nucleic Nucleic Nucleic acids + acids + acids + acids + acids + lysosome lysosome lysosome lysosome lysosome Elbasvir Nucleic Nucleic Nucleic Nucleic Nucleic acids + acids + acids + acids + acids + lysosome lysosome lysosome lysosome lysosome Simeprevir Nucleic Nucleic Nucleic Nucleic Nucleic acids + acids + acids + acids + acids + lysosome lysosome lysosome lysosome lysosome Irinotecan Nucleic Nucleic Nucleic Nucleic Nucleic acids + acids + acids + acids + acids + lysosome lysosome lysosome lysosome lysosome Cefpiramide Nucleic Nucleic Nucleic Nucleic Nucleic acids + acids + acids + acids + acids + lysosome lysosome lysosome lysosome lysosome Ceftazidime Nucleic Nucleic Nucleic Nucleic Nucleic acids + acids + acids + acids + acids + lysosome lysosome lysosome lysosome lysosome Cefixime Nucleic Nucleic Nucleic Nucleic Nucleic acids + acids + acids + acids + acids + lysosome lysosome lysosome lysosome lysosome Cefotaxime Nucleic Nucleic Nucleic Nucleic Nucleic acids + acids + acids + acids + acids + lysosome lysosome lysosome lysosome lysosome Cefmenoxime Nucleic Nucleic Nucleic Nucleic Nucleic acids + acids + acids + acids + acids + lysosome lysosome lysosome lysosome lysosome Bromocriptine Nucleic Nucleic Nucleic Nucleic Nucleic acids + acids + acids + acids + acids + lysosome lysosome lysosome lysosome lysosome Ergotamine Nucleic Nucleic Nucleic Nucleic Nucleic acids + acids + acids + acids + acids + lysosome lysosome lysosome lysosome lysosome Dihydroer- Nucleic Nucleic Nucleic Nucleic Nucleic gotamine acids + acids + acids + acids + acids + lysosome lysosome lysosome lysosome lysosome Reserpine Nucleic Nucleic Nucleic Nucleic Nucleic acids + acids + acids + acids + acids + lysosome lysosome lysosome lysosome lysosome Deserpine Nucleic Nucleic Nucleic Nucleic Nucleic acids + acids + acids + acids + acids + lysosome lysosome lysosome lysosome lysosome Rifabutin Nucleic Nucleic Nucleic Nucleic Nucleic acids + acids + acids + acids + acids + lysosome lysosome lysosome lysosome lysosome Naldemedine Nucleic Nucleic Nucleic Nucleic Nucleic acids + acids + acids + acids + acids + lysosome lysosome lysosome lysosome lysosome Oxaprozin Nucleic Nucleic Nucleic Nucleic Nucleic acids + acids + acids + acids + acids + lysosome lysosome lysosome lysosome lysosome Benzethonium Nucleic Nucleic Nucleic Nucleic Nucleic acids + acids + acids + acids + acids + lysosome lysosome lysosome lysosome lysosome Rescinnamine Nucleic Nucleic Nucleic Nucleic Nucleic acids + acids + acids + acids + acids + lysosome lysosome lysosome lysosome lysosome Lymecycline Nucleic Nucleic Nucleic Nucleic Nucleic acids + acids + acids + acids + acids + lysosome lysosome lysosome lysosome lysosome Moxifloxacin Nucleic Nucleic Nucleic Nucleic Nucleic acids + acids + acids + acids + acids + lysosome lysosome lysosome lysosome lysosome Dactinomycin Nucleic Nucleic Nucleic Nucleic Nucleic acids + acids + acids + acids + acids + lysosome lysosome lysosome lysosome lysosome Ertapenem Nucleic Nucleic Nucleic Nucleic Nucleic acids + acids + acids + acids + acids + lysosome lysosome lysosome lysosome lysosome

[0083] Table 7 below shows non-limiting examples of optical biopsy stain panels according to the present invention. The stain panels listed below use a combination of nucleus dyes and protein/cytoplasm dyes that have been optimized for their compatibility.

TABLE-US-00007 Approximate Nucleus dyes peak Methylene Pyrvinium fluorescence Daunorubicin Blue Mitoxantrone Proflavine Pamoate (nm) 550, 590 695 685 508 700 Protein/ Fluorescein 505 X X X X cytoplasm Indigo 400, 450 X X X X X dyes Carmine Phloxine B 550 X X X Phenol Red 535 X X X Rose Bengal 565 X X X X Congo Red 425 X X X X X

[0084] Although there has been shown and described the preferred embodiment of the present invention, it will be readily apparent to those skilled in the art that modifications may be made thereto which do not exceed the scope of the appended claims. Therefore, the scope of the invention is only to be limited by the following claims. In some embodiments, the figures presented in this patent application are drawn to scale, including the angles, ratios of dimensions, etc. In some embodiments, the figures are representative only and the claims are not limited by the dimensions of the figures. In some embodiments, descriptions of the inventions described herein using the phrase “comprising” includes embodiments that could be described as “consisting essentially of” or “consisting of”, and as such the written description requirement for claiming one or more embodiments of the present invention using the phrase “consisting essentially of” or “consisting of” is met.