Methods for labeling, clearing, and imaging large tissues

Abstract

To provide an improved method for preparation and analysis of tissues including the whole animals and large mammalian brains.SOLUTION: The present invention relates to a method for preparing animal tissues for fluorescence microscopy for analyzing biodistribution of biopharmaceutical drugs and biodistribution of nanoparticles, animal tissues obtainable by this method, a method for analyzing these animal tissues, and a method for detecting metastases. The method for preparing animal tissues according to the present invention includes the whole body marker, transparency, and an imaging method. A method of the present invention is advantageous in that single cells in mammalian tissues including, for example, a pig and human brain, tumor metastasis at a single cell level as well as a distribution of biopharmaceutical drugs at a single cell level in the whole mouse (for example, a distribution of cancer targeted therapeutic antibodies in the whole animal such as an intact mouse) can be visualized.SELECTED DRAWING: Figure 1a

Description

Technical Field

[0001] The present invention relates to a method for preparing animal tissues for a fluorescence microscope for analyzing the biodistribution of biopharmaceuticals and for analyzing the biodistribution of nanoparticles, an animal tissue obtainable by this method, an analysis method for this animal tissue, and a method for detecting metastasis. The method for preparing animal tissues according to the present invention includes whole-body labeling, clearing, and imaging methods. The method of the present invention is advantageous in that it can visualize, for example, single cells in mammalian tissues including pig and human brains, tumor metastasis at the single-cell level, and the distribution of biopharmaceuticals at the single-cell level throughout a mouse (for example, the distribution of a cancer-targeting therapeutic antibody throughout an animal such as an intact mouse).

Background Art

[0002] Since metastases that occur at sites distal to the primary tumor often lead to the death of cancer patients, understanding the cytological details of tumor invasion and metastasis is important in cancer research. Metastasis is a complex process that affects various organs (Lambert et al., 2017; Massague and Obenauf, 2016), which is why the comprehensive tracking and visualization of single metastases on a whole-body scale has remained a major challenge to date. Currently available methods result in a biased and incomplete image of the amount of metastasis (for example, by examining tissue sections or detectable metastatic nodules on the surface in preselected organs) or have very poor spatial resolution, with little three-dimensional information and limited penetration in the deep parts of the body (for example, imaging based on bioluminescence) (de Jong et al., 2014).

[0003] Mammalian tissues are naturally opaque, hindering high-resolution imaging of any tissue deeper than several hundred micrometers (Tuchin et al., 2007). Recently developed tissue clearing methods have opened the way for studying intact organs such as neuronal projections in the entire mouse brain (Renier et al., 2016; Ye et al., 2016) and inflammatory processes in the intestine (Gabanyi et al., 2016) (Belle et al., 2014; Chung et al., 2013; Erturk et al., 2012; Erturk et al., 2011; Hama et al., 2011; Ke et al., 2013; Murray et al., 2015; Renier et al., 2014; Susaki et al., 2014; Yang et al., 2014). A few studies have demonstrated tissue clearing in human embryos (Belle et al., 2017) and even in entire adult mice (Pan et al., 2016; Tainaka et al., 2014; Yang et al., 2014). In particular, the ultimate DISCO (uDISCO) clearing method, developed by the inventors, enables both clearing and imaging of entire rodent tissues by shrinking them to one-third of their original size (Pan et al., 2016). This made 3D imaging and reconstruction of entire adult mice possible for the first time using a light-sheet microscope.

[0004] However, to date, there is no known method that allows for the visualization of target molecules (e.g., proteins present in cancer cells) in a complete mouse body at single-cell resolution without pre-cutting the mouse. For example, Yang et al. (2014) and Tainaka et al. (2014) claim to have achieved whole-body clearing of mice using known methods. However, these methods did not actually analyze the entire body using a fluorescence microscope, but only analyzed a section of the body. Therefore, there is no evidence in these previously published studies that fully intact adult mice were imaged at single-cell resolution.

[0005] Furthermore, to date, there is no technology to make tissue the size of a pig brain transparent. This is important for studying diseases and treatment options in mammalian brains using a holistic approach, as it will allow for an unbiased, holistic representation of disease conditions. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Lambert et al., 2017 [Non-Patent Document 2] Massague and Obenauf, 2016 [Non-Patent Document 3] de Jong et al., 2014 [Non-Patent Document 4] Tuchin and Tuchin, 2007 [Non-Patent Document 5] Belle et al., 2014 [Non-Patent Document 6] Chung et al., 2013 [Non-Patent Document 7] Erturk et al., 2012 [Non-Patent Document 8] Erturk et al., 2011 [Non-Patent Document 9] Hamara, 2011 [Non-Patent Document 10] Ke et al., 2013 [Non-Patent Document 11] Murray et al., 2015 [Non-Patent Document 12] Renier et al., 2014 [Non-Patent Document 13] Susaki et al., 2014 [Non-Patent Document 14] Yang et al., 2014 [Non-Patent Document 15] Renier et al., 2016 [Non-Patent Document 16] Ye et al., 2016 [Non-Patent Document 17] Gabanyi et al., 2016 [Non-Patent Document 18] Belle et al., 2017 [Non-Patent Document 19] Pan et al., 2016 [Non-Patent Document 20] Tainaka et al., 2014 [Non-Patent Document 21] Tuchin, 2016 [Non-Patent Document 22] Zipfel et al., 2003 [Non-Patent Document 23] Battke et al., 2011 A [Non-Patent Document 24] Gondi et al., 2013 [Non-Patent Document 25] Hong et al., 2017 [Non-Patent Document 26] Holliger and Hudson, 2005 [Non-Patent Document 27] Vick et al., 2015 [Non-Patent Document 28] Yoneda et al., 2001 [Non-Patent Document 29] Hama et al., 2015 [Non-Patent Document 30] Muyldermans, 2013 [Non-Patent Document 31] Gage et al., 2012 [Non-Patent Document Ghanavati et al., 2014 [Non-Patent Document 33] Lorns et al., 2012 [Non-Patent Document 34] Condeelis and Weissleder, 2010 [Non-Patent Document 35] Massoud and Gambhir, 2003 [Non-Patent Document 36] Massoud and Gambhir, 2007 [Non-Patent Document 37] Ntziachristos, 2010 [Non-Patent Document 38] Pichler et al., 2008 [Non-Patent Document 39] Timpson et al., 2011 [Non-Patent Document 40] Pandey and Mahadevan, 2014 [Non-Patent Document 41] Welti et al., 2013 [Non-Patent Document 42] Butler et al., 2010 [Non-Patent Document 43] Erturk et al., 2016 [Non-Patent Document 44] Wilson et al., 2010 [Non-Patent Document 45] Janeway et al., 1997 [Non-Patent Document 46] Ransohoff and Engelhardt, 2012. [Non-Patent Document 47] Louveau et al., 2015 [Non-Patent Document 48] Calvo et al., 2011 [Overview of the project]

[0007] Thus, there is a need for improved methods for preparing and analyzing tissues, including the brains of whole animals and large mammals.

[0008] The inventors believe that imaging optically transparent tissues can be a powerful preclinical technique for detecting, for example, fluorescently labeled cancer cells and / or therapeutic antibodies in intact bodies at cell resolution, including in mice.

[0009] Typically, in vitro or in vivo fluorescent labeling of cancer cells is achieved by the endogenous expression of fluorescent proteins such as GFP, YFP, and mCherry, which emit light in the visible spectrum. However, many tissues of the body also exhibit high autofluorescence in this region (Tuchin, 2016; Zipfel et al., 2003), which can hinder the reliable detection of single cancer cells through a centimeter-thick intact mouse body. According to a preferred embodiment of the present invention, labeling cells such as cancer cells using antibodies tagged with fluorescent dyes having emission peaks particularly in the far-red region is advantageous in overcoming such autofluorescence signals by resulting in a high signal-to-background ratio for reliable detection of single cells.

[0010] To this end, the inventors have developed a method for preparing animal tissue for fluorescence microscopy. Preferably, this method uses systemic labeling (e.g., immunolabeling) techniques based on antibody fragments such as nanoantibodies to specifically label endogenously expressed common fluorescent proteins (e.g., EGFP, mCherry), or endogenous cellular proteins having fluorescent dyes such as Alexa and Atto dyes, preferably with a far-red spectrum. The inventors then incorporated organic solvent-based clearing methods, such as the systemic DISCO clearing method (which is incorporated in its entirety by reference, see Pan et al., 2016), into the method of the present invention. The method of the present invention is advantageous in that it enables the visualization of cells such as cancer cells in intact see-through mice, even in tissues with high autofluorescence.

[0011] This novel pipeline according to the present invention is named LuCiD (whole-body labeling, u / 3Disco clearing, imaging, data visualization). The method of the present invention can be used, for example, to determine tumor metastasis and the in vivo distribution of cancer cell-targeting antibodies in mice. The inventors illustrated this finding by using mice transplanted with human breast cancer cells and directly injected with the monoclonal therapeutic antibody 6A10 against carbonic anhydrase XII (CA12) (for reference, the entire antibody is incorporated by reference for all purposes, see Battke et al., 2011; and Gondi et al., 2013). The method of the present invention offers several important advantages over other imaging techniques, including, but not limited to, the ability to detect spontaneous metastasis, monitor tumor drug-target interactions at the single-cell level in intact mice, and label antibodies after further determining the phenotype of the defined tumor microenvironment following rehydration of cleared tissue.

[0012] Unbiased detection of cancer metastasis and the distribution of tumor-targeted therapies in the body at the single-cell level has been a long-standing challenge in preclinical research.

[0013] Here, the inventors have developed a whole-body clearing method based on an organic solvent that can be used for analyzing micrometastases and the distribution of antitumor therapeutic antibodies at cell resolution in tissues such as the whole mouse. The method of the present invention is unbiased because it allows for the labeling and detection of target molecules at single-cell resolution in animal tissue (e.g., the whole mouse) without cutting the animal tissue before analysis. Advantageously, animal tissue prepared and analyzable at single-cell resolution according to the present invention without prior cutting is larger than that obtained by previously known methods. Therefore, bias caused by tissue cutting (and subsequent individual analysis of various cut portions of this animal tissue) is minimized by the method of the present invention. For example, bias caused by the analysis of selected organs or only portions of such organs can be minimized by the method of the present invention. In non-limiting embodiments, the organic solvent used in the method according to the present invention contributes to the advantageous effect of the present invention by shrinking the animal tissue to a smaller size, making it easier to approach the animal tissue with a fluorescence microscope and bringing the microscope objective to a given maximum working distance.

[0014] The method of the present invention is also advantageous compared to conventional methods in that it enables the clearing of tissues including skin, for example, the entire adult mouse including the skin.

[0015] The method of the present invention is also advantageous because, even with imaging using a commonly used epifluorescence microscope, it enables more detailed detection in intact see-through mice than can be visualized by bioluminescence measuring, and is therefore easily applicable to a variety of laboratories without requiring highly specialized equipment.

[0016] The method of the present invention can also reduce the time and cost required to investigate, for example, tumor micrometastases at the cellular level throughout the entire mouse body. In addition, researchers can easily evaluate the entire mouse body rather than selected tissues / organs, and because the method is highly sensitive (capable of identifying and quantifying single cells throughout the body), the number of mice used in research can also be significantly reduced by the method of the present invention.

[0017] Thus, the method of the present invention described herein can develop the transition of novel therapies to clinical practice more efficiently than conventional methods.

[0018] Furthermore, unlike known tissue clearing methods such as CUBIC and PACT, which weaken the tissue, the preparation method of animal tissue according to the present invention for fluorescence microscopy strengthens the animal tissue. Thus, advantageously, the animal tissue obtainable by the method of the present invention is suitable for dissection into various parts and further analysis of the dissected parts by fluorescence microscopy. According to the present invention, it can be understood that dissection of the animal tissue obtainable by the method of the present invention is often unnecessary because the animal tissue prepared according to the present invention and analyzable at single-cell resolution without prior dissection is larger than that obtained by previously known methods. However, even when dissection is desirable, the animal tissue obtainable by the present invention can be advantageously used. This can be particularly useful for further determining the characteristics of the micrometastases identified by the method of the present invention and their microenvironment after isolation.

[0019] Tissue labeling such as systemic immunohistochemistry using nanobodies Imaging endogenous proteins, such as endogenous fluorescent proteins, in thick biological tissue presents major challenges, including autofluorescence in the blue-green spectrum and fading during lengthy imaging and storage. In exemplary embodiments of the present invention, it is advantageous to label (e.g., immunolabel) endogenous fluorescent proteins, e.g., endogenous fluorescent proteins in cancer cells, with a stable far-red fluorescent dye, e.g., Atto or Alexa dye, to achieve the highest quality signal for detecting single tumor cells in an adult mouse. This technique is advantageous because it increases the signal-to-background ratio by up to 20-fold and enables visualization of single cells in tissue, particularly in a centimeter-thick mouse body. According to the present invention, it can be understood that the use of fluorescent dyes with even longer wavelength spectra, such as near-infrared fluorescent dyes, may be suitable for further improving image quality and potentially enabling the study of intracellular structures / molecules in the entire mouse body (see Hong et al., 2017, incorporating its entirety by reference for all purposes, as an example of suitable fluorescent dyes).

[0020] The method of the present invention uses labeling with a fluorescent dye-containing labeling agent having a molecular weight of 100 kDa or less (e.g., an antibody fragment complexed with a fluorescent dye). While a limited number of commercially available nanobodies currently exist as examples of such labeling agents, nano-booster (ChromoTek) is usable in the method of the present invention and can stain 21 diverse fluorescent proteins with a wide range of selection, such as EGFP, YFP, Venus, mCherry, and mRFP. Alternatively, the method of the present invention may also use other nanobodies or small fragments of conventional antibodies (e.g., scFv) (for example, see Holliger and Hudson, 2005, incorporating the whole antibody by reference for all purposes). Alternatively, novel nanobodies can be generated for the method of the present invention to study systemic diseases. For example, labeling agents (e.g., nanobodies) can be used as inflammatory or infection markers and may be useful in collecting unbiased readout information in whole mice for systemic inflammatory diseases such as multiple sclerosis or rheumatoid arthritis, or infections. Furthermore, in the embodiments of the present invention, it is demonstrated that animal-derived tissue imaged by the LuCiD method of the present invention can be subsequently rehydrated and stained with conventional antibodies using a standard protocol, thereby enabling the complete determination of the phenotypic characteristics of tumors / metastases and their microenvironment.

[0021] Detection of micrometastasis according to the present invention For example, unbiased, high-throughput mapping of tumor micrometastases at cell resolution throughout the body of rodents can be a useful tool for elucidating the biological background of tumor cell dissemination. In exemplary embodiments, the present invention includes the LuCiD method, which can be used for volumetric imaging of tumor micrometastases throughout the body of mice. The use of a single-plane laser scanning light-sheet microscope is the most preferred embodiment of the analytical method according to the present invention, for example, for detecting cancer cells in a see-through mouse, but even using a standard fluorescence microscope can provide new insights. For example, the inventors detected small micrometastases as small as 20 μm in diameter using epifluorescence microscopy. In addition, epifluorescence imaging helps to perform a direct scan of the body of a cleared mouse within minutes to determine the area of ​​interest, and then collect a large dataset by light-sheet microscopy. Subsequent light-sheet microscopy imaging can focus only on the organ / region of interest based on the epifluorescence data. This technique can significantly accelerate the research being conducted and reduce the amount of data to be analyzed. In the descriptive embodiments of the present invention shown below, light-sheet microscopy imaging generated approximately 4 TB of data for a single mouse body, compared to approximately 100 GB of data for a single organ, such as the lungs. In such descriptive embodiments, the inventors used Vision4D software (Arivis) to combine 3D imaging stacks acquired by ultramicroscopy. This allows for the combination and segmentation of TB-sized data acquired at cell resolution from a whole mouse. Other known computer programs for the analysis of microscopic images can also be used in accordance with the present invention.

[0022] The inventors have hereby demonstrated that the method of the present invention is advantageously suitable for the detection and mapping of cancer metastases at the cellular level throughout the mouse body and enables the precise identification of the location of a single disseminated cancer cell. Importantly, the inventors have also shown that the method of the present invention enables the re-detection of identified metastatic tissue by conventional antibody methods, such as gene expression profiling by RNA-seq and proteomics (mass spectrometry).

[0023] Thus, according to the present invention, the method for preparing animal tissue for fluorescence microscopy is advantageous in that it preserves proteins (functional epitopes) and DNA / RNA.

[0024] Therefore, the method of the present invention can further enable the characterization and molecular screening of micrometastases and single tumor cells identified in distal organs. According to the present invention, the use of molecular markers of specific subtypes of tumor cells, such as cancer stem cells, or inflammatory cells and extracellular matrix components derived from the tumor microenvironment, such as cancer-associated fibroblasts, T cells, and macrophages, can be useful in determining the precise spatiotemporal distribution of metastases in tissues, such as the entire body of rodents.

[0025] Analysis of the in-vivo distribution of biopharmaceuticals according to the present invention Accurate determination of the in vivo distribution of biopharmaceuticals (e.g., antibody drugs) is crucial for evaluating their specificity and efficacy for therapies such as tumor treatment; however, no method exists that can provide such information at the cellular level in an intact living organism. Here, the inventors present the method of the present invention (also known as the “LuCiD” method in exemplary embodiments) as a novel tool that can be used to study not only the distribution of single tumor cells but also antibody-based therapeutics. Explanatory embodiments of the present invention demonstrate that the method of the present invention can enable the identification of antibody-targeted tumor cells, particularly for metastases in various organs, including the lungs, kidneys, brain, and liver. For example, the inventors surprisingly observed that many micrometastases in the lungs were targeted by the anti-CA12 therapeutic antibody 6A10, while some micrometastases distributed across the lungs and liver were not targeted by the antibody. Finally, the analytical method of the present invention is also advantageous in that it can be used to detect the binding of biopharmaceuticals (e.g., therapeutic antibodies) to non-target tissues (such as non-cancerous tissues in the case of cancer therapeutic antibodies) to point out potential off-target effects. This is illustrated in the following non-limiting examples in the entire mouse body, demonstrating quantification in organs.

[0026] Analysis of the distribution of nanoparticles in the body according to the present invention In an exemplary method for analyzing the in vivo distribution of nanoparticles, nanoparticles (DNA origami or carbon nanotubes) are complexed with polymers such as PEG to increase circulation time and stability. These can also be tagged with target moieties such as antibodies, peptides, or aptamers. For example, CpG peptides can be used to target immune cells. Finally, they can also be complexed with fluorescent dyes (e.g., Alexa or Atto dyes) and used according to the method of the present invention. The complexed nanoparticles can be dissolved in PBS at a concentration of 200 nM to 2 μM. Then, 100 to 200 μL of this solution is injected into mice either iv or ip. The mice are then perfused for at least 3 hours (or longer). The in vivo distribution of nanoparticles is determined according to the method of the present invention.

[0027] Thus, the present invention provides an advantageous labeling and analysis pipeline. This pipeline enables, for example, the visualization and analysis of tumor micrometastases and antibody-based therapies at single-cell resolution throughout the mouse body. Because the method of the present invention is time and cost-effective, it can be used to investigate various biomedical problems, such as biomedical problems related to various disease conditions or developmental processes affecting the entire body.

[0028] Therefore, the present invention encompasses the following preferred embodiments.

[0029] 1. Next step: a) A step of selectively decalcifying fixed animal tissue with a decalcification solution, b) A step of selectively decolorizing fixed animal tissue with a heme removal solution, c) A step of obtaining fixed animal tissue labeled with the fluorescent dye-containing labeling agent by labeling a target molecule in fixed animal tissue with a labeling solution containing a fluorescent dye-containing labeling agent having a molecular weight of 100 kDa or less and capable of binding to the target molecule, Prior to this labeling of the target molecule in step c), the fixed animal tissue is treated with a permeabilization solution, and the permeabilization solution and the labeling solution are different solutions. Alternatively, the animal tissue immobilized in this labeling of the target molecule in step c) is treated with a permeation solution, wherein the permeation solution and the labeling solution are the same solution. d) A step of clearing the fixed animal tissue labeled with this fluorescent dye-containing labeling agent with a clearing solution containing an organic solvent to obtain the animal tissue for use with a fluorescence microscope. A method for preparing animal tissue for use in a fluorescence microscope, including the preparation of animal tissue. 2. The method described in item 1, including step a). 3. The method according to item 1 or 2, wherein in step a), the decalcifying solution is selected from a solution containing EDTA and NaHCO3, a solution containing formic acid, a solution containing HNO3, or a solution containing HCl. 4. The method according to any one of items 1 to 3, wherein the fixed animal tissue can be obtained by fixing it with a fixation solution containing 4 wt% paraformaldehyde and optionally heparin. 5. The method described in any one of items 1 to 4, including step b). 6. The method according to any one of items 1 to 5, wherein step b) is carried out by perfusing the fixed animal tissue with the heme removal solution. 7. The method according to any one of items 1 to 6, wherein the heme removal solution is a heme chelate solution. 8. The method according to any one of items 1 to 7, wherein in step b), the heme removal solution comprises an amino alcohol suitable for heme removal and optionally a surfactant (detergent). 9. The method according to item 8, wherein the heme removal solution comprises a surfactant, the surfactant being an ionic surfactant, a nonionic surfactant, a zwitterionic surfactant, a chaotropic surfactant, or a combination thereof. 10. The method according to item 9, wherein the surfactant is an ionic surfactant and is sodium dodecyl sulfate or deoxycholate. 11. The method according to item 9, wherein the surfactant is a nonionic surfactant and is 4-(1,1,3,3-tetramethylbutyl)phenyl polyethylene glycol, t-octylphenoxypolyethoxyethanol, polyethylene glycol tert-octylphenyl ether, or polyoxyethylene (20) sorbitan monolaurate. 12. The method according to item 9, wherein the surfactant is a zwitterionic surfactant and is 3-[(3-colamidopropyl)dimethylammonio]-1-propanesulfonate hydrate. 13. The method according to item 9, wherein the surfactant is a chaotropic surfactant and is urea. 14. The method according to any one of items 8 to 13, wherein the amino alcohol is N,N,N',N'-tetrakis(2-hydroxypropyl)ethylenediamine, N-butyldiethanolamine, N-methyldiethanolamine, 4-(2-hydroxyethyl)morpholine, N-ethyldiethanolamine, 2-(diisopropylamino)ethanol, 4-methylmorpholine N-oxide, or 1-(2-hydroxyethyl)piperidine. 15. The method according to any one of items 8 to 14, wherein the amino alcohol is N,N,N',N'-tetrakis(2-hydroxypropyl)ethylenediamine. 16. The method according to item 15, wherein the heme removal solution is the following reagents in a 1:2 or 1:3 dilution, preferably in 0.1 M PBS: 25 wt% urea, 25 wt% N,N,N',N'-tetrakis(2-hydroxypropyl)ethylenediamine, and 15 wt% Triton X-100. 17. The method according to any one of items 1 to 16, wherein in step b), the heme removal solution comprises an oxidizing reagent for oxidizing heme. 18. The method according to item 17, wherein the oxidizing agent for oxidizing heme is benzyl peroxide, 3-chloroperoxybenzoic acid, or magnesium monoperoxyphthalate hexahydrate. 19. The method according to item 17, wherein the oxidizing agent for oxidizing heme is benzyl peroxide. 20. The method according to any one of items 1 to 19, wherein the fluorescent dye-containing labeling agent has a molecular weight of 60 kDa or less. 21. The method according to any one of items 1 to 20, wherein the fluorescent dye-containing labeling agent has a molecular weight of 50 kDa or less. 22. The method according to any one of items 1 to 21, wherein the fluorescent dye-containing labeling agent has a molecular weight of 40 kDa or less. 23. The method according to any one of items 1 to 22, wherein the fluorescent dye-containing labeling agent has a molecular weight of 30 kDa or less. 24. The method according to any one of items 1 to 23, wherein the fluorescent dye-containing labeling agent has a molecular weight of 20 kDa or less. 25. The method according to any one of items 1 to 24, wherein the fluorescent dye is capable of emitting infrared or red fluorescence. 26. The method according to any one of items 1 to 25, wherein the fluorescent dye is capable of emitting near-infrared or far-red fluorescence. 27. The method according to any one of items 1 to 26, wherein the maximum emission of the fluorescent dye occurs at wavelengths greater than 480 nm. 28. The method according to any one of items 1 to 26, wherein the maximum emission of the fluorescent dye occurs at wavelengths greater than 500 nm. 29. The method according to any one of items 1 to 26, wherein the maximum emission of the fluorescent dye occurs at wavelengths greater than 550 nm. 30. The method according to any one of items 1 to 26, wherein the maximum emission of the fluorescent dye occurs at wavelengths greater than 590 nm. 31. The method according to any one of items 1 to 26, wherein the maximum emission of the fluorescent dye occurs at wavelengths greater than 600 nm. 32. The method according to any one of items 1 to 26, wherein the maximum emission of the fluorescent dye occurs at wavelengths greater than 640 nm. 33. The method according to any one of items 1 to 26, wherein the maximum emission of the fluorescent dye occurs at wavelengths greater than 700 nm. 34. The method according to any one of items 1 to 26, wherein the maximum emission of the fluorescent dye occurs in the wavelength range of 640 nm to 700 nm. 35. The method according to any one of items 1 to 33, wherein the maximum emission of the fluorescent dye occurs at wavelengths less than 1000 nm or less than 900 nm. 36. The method according to any one of items 1 to 33, wherein the maximum emission of the fluorescent dye occurs at wavelengths smaller than 800 nm. 37. The method according to any one of items 1 to 36, wherein the fluorescent dye-containing labeling agent is an antibody fragment complexed with the fluorescent dye, and the antibody fragment is capable of binding to the target molecule. 38. The method according to any one of items 1 to 37, wherein the fluorescent dye-containing labeling agent is a nanobody complexed with the fluorescent dye, and the nanobody is capable of binding to the target molecule. 39. The method according to any one of items 1 to 38, wherein step c) is carried out by perfusing the fixed animal tissue with the labeled solution containing a fluorescent dye labeling agent. 40. The method according to any one of items 1 to 39, wherein the fluorescent dye-containing labeling agent is a fluorescent dye, and the fluorescent dye is capable of binding to the target molecule. 41. The method according to any one of items 1 to 40, wherein the fluorescent dye-containing labeling agent is a fluorescent dye, and the fluorescent dye is Nissl, propidium iodide, methoxy-x04, cresyl violet acetate, pyronin Y, thiazine red, lectin, Dil, Atto dye, and To-pro3. 42. The method according to any one of items 1 to 41, wherein the organic solvent has a refractive index that is deflected by 5% or less from the refractive index of the animal tissue. 43. The method according to any one of items 1 to 42, wherein the clearing solution containing an organic solvent has a refractive index that is deflected by 2% or less from the refractive index of the animal tissue. 44. The method according to any one of items 1 to 43, wherein the clearing solution containing an organic solvent has a refractive index of 1,500 to 1,600. 45. The method according to any one of items 1 to 44, wherein the clearing solution containing an organic solvent has a refractive index of 1.520 to 1.580. 46. ​​The method according to any one of items 1 to 45, wherein the organic solvent comprises benzyl alcohol, benzyl benzoate, dibenzyl ether, ethyl 3-phenyl-2-propenoate, allyl 3-phenyl acrylate, PEG (Mn=200-1000), PEGDA (Mn=200-1000), PEGMA (Mn=200-1000), 1-phenylnaphthalene and / or diphenyl ether. 47. The method according to any one of items 1 to 46, wherein the clearing solution containing an organic solvent further contains an antioxidant. 48. The method according to any one of items 1 to 47, wherein the clearing solution containing an organic solvent comprises benzyl alcohol, benzyl benzoate, and diphenyl ether in volume ratios from 4:8:3 to 10:20:3, and the antioxidant. 49. The method according to item 47 or 48, wherein the antioxidant is DL-alpha-tocopherol. 50. The method according to any one of items 47, 48, or 49, wherein the antioxidant is present in the clearing solution in an amount of 0.4 vol%. 51. The method according to any one of items 1 to 50, wherein step d) is carried out by perfusing the fixed animal tissue labeled with the fluorescent dye-containing labeling agent with the clearing solution containing an organic solvent. 52. The method according to item 51, wherein fixed animal tissue labeled with the fluorescent dye-containing labeling agent is perfused with the clearing solution containing an organic solvent for at least 6 hours. 53. The method according to item 51 or 52, wherein step d) further comprises perfusion with an increasing gradient of a decontaminated aqueous solution containing an additional 0 vol% to 100 vol% organic solvent prior to perfusion with the clarifying solution. 54. The method according to item 53, wherein after this perfusion, the gradient of this degreasing aqueous solution containing 0 vol% to 100 vol% of this further organic solvent is increased, and then the perfusion is performed with a degreasing solution containing another organic solvent. 55. The method according to item 54, wherein the additional organic solvent is tert-butanol, tetrahydrofuran (THF), methanol, ethanol, or 1,4-dioxane, and the perfusion with increasing gradient is carried out at a temperature above the melting temperature of the additional organic solvent. 56. The method according to item 54 or 55, wherein the other organic solvent is dichloromethane, chloroform, methanol, hexane, butanol, ethyl acetate, or tert-butyl methyl ether, and the perfusion with the other organic solvent is carried out at a temperature above the melting temperature of the other organic solvent. 57. The method according to any one of items 1 to 56, wherein the labeling solution, the permeation solution, and the clarification solution are actively delivered by pressurization, preferably by pressurization with a pump. 58. The method according to any one of items 1 to 57, wherein the labeling of the target molecule with the labeling solution and the treatment with the permeation solution are carried out by perfusing at a pressure higher than 80 mmHg, preferably higher than 150 mmHg. 59. The method according to any one of items 1 to 58, wherein the labeling of the target molecule with the labeling solution and the treatment with the permeation solution are carried out by perfusing at a pressure of 220 to 240 mmHg, preferably 230 mmHg. 60. The method according to any one of items 1 to 59, wherein the fixed and decolorized animal tissue is treated with a permeation solution prior to this labeling of the target molecule in step c), and the permeation solution and the labeling solution are different solutions. 61. The method according to item 60, wherein the permeate treatment solution is a dehydrated aqueous solution as specified in item 54 or 55. 62. The method according to item 60, wherein the permeation solution is a degreasing solution as specified in item 54 or 56. 63. The method according to item 60, wherein the permeation solution contains acetic acid. 64. The method according to item 60, wherein the permeation solution comprises guanidine hydrochloride and / or sodium acetate. 65. The method according to any one of items 1 to 59, wherein a fixed animal tissue is treated with a permeation solution in step c) for this labeling of the target molecule, and the permeation solution and the labeling solution are the same solution. 66. The method according to any one of items 1 to 65, wherein step b) is carried out by perfusing the fixed animal tissue with the heme removal solution at a pressure higher than 80 mmHg, preferably higher than 150 mmHg. 67. The method according to any one of items 1 to 66, wherein step b) is carried out by perfusing the fixed animal tissue with the heme removal solution at a pressure of 220 to 240 mmHg, preferably 230 mmHg. 68. The method according to any one of items 51 to 67, wherein the perfusion in step d) is carried out at a pressure higher than 80 mmHg, preferably higher than 150 mmHg. 69. The method according to any one of items 51 to 67, wherein the perfusion in step d) is carried out at a pressure of 220 to 240 mmHg, preferably 230 mmHg. 70. The method described in any one of items 1 to 69, wherein the animal tissue is of mammalian origin. 71. The method described in any one of items 1 to 70, wherein the animal tissue is of non-human mammalian or human origin. 72. The method described in any one of items 1 to 71, wherein the animal tissue is of rodent origin. 73. The method described in any one of items 1 to 72, wherein the animal tissue is derived from a mouse. 74. The method described in any one of items 1 to 73, wherein the animal tissue is the whole mouse. 75. The method described in any one of items 1 to 71, wherein the animal tissue is a pig brain. 76. The method described in any one of items 1 to 58, wherein the animal tissue is an entire organ or part thereof. 77. The method according to any one of items 1 to 76, wherein the target molecule labeled with the labeling agent in step c) is a structure present in the cells of the fixed animal tissue, preferably a protein, lipid, DNA, or RNA, more preferably a protein present in the cells of the fixed animal tissue. 78. The method according to any one of items 1 to 77, wherein the animal tissue comprises cancer, and the target molecule labeled with the labeling agent in step c) is a structure present in the cells of the cancer, preferably a protein, lipid, DNA or RNA, more preferably a protein present in the cells of the cancer. 79. The method according to any one of items 1 to 78, wherein the animal tissue includes cancer metastases, and the target molecule labeled with the labeling agent in step c) is a structure present in the cancer cells, preferably a protein, lipid, DNA or RNA, more preferably a protein present in the cancer cells. 80. The method according to any one of items 1 to 79, wherein the animal has been treated with a biopharmaceutical, the animal tissue comprises the biopharmaceutical, the biopharmaceutical is the target molecule labeled with the labeling agent in step c), or the biopharmaceutical is labeled with a further fluorescent dye in vitro, or the biopharmaceutical is fluorescence itself. 81. The method described in item 80, wherein the biopharmaceutical is a small molecule. 82. The method described in item 80, wherein the biopharmaceutical is a therapeutic protein. 83. The method described in item 80, wherein the biopharmaceutical is a therapeutic antibody. 84. A method described in any one of items 1 to 83, which is neither a surgical or therapeutic treatment of the human or animal body, nor a diagnostic method performed on the human or animal body. 85. An ex vivo method, as described in any one of items 1 to 84. 86. The method according to any one of items 1 to 85, wherein the animal tissue for fluorescence microscopy obtained in step d) is smaller in volume than the fixed animal tissue used in step b). 87. The method according to item 86, wherein the animal tissue for fluorescence microscopy obtained in step d) is 40% to 75% smaller in volume than the fixed animal tissue used in step b). 88. Animal tissue obtainable by any one of items 1 to 87 for preparing animal tissue for fluorescence microscopy, comprising the target molecule labeled with the fluorescent dye-containing labeling agent. 89. The animal tissue described in item 88, which is the whole rodent, preferably the whole mouse. 90. Animal tissue, either whole or in part, as described in item 88. 91. Animal tissue, as described in item 90, which is the entire organ of a mammal. 92. Animal tissue as described in item 88, consisting of a tissue mass measuring 2 x 2 x 2 cm. 93. The animal tissue described in any one of items 88-92, wherein the target molecule is labeled with the fluorescent dye-containing labeling agent, and all target molecules are detectable at single-cell resolution regardless of their location in the animal tissue when analyzed by light-sheet fluorescence microscopy. 94. i) A method for analyzing animal tissue according to any one of items 88 to 93, comprising the step of analyzing the tissue using a fluorescence microscope to detect the fluorescence of the fluorescent dye in the animal tissue. 95. ii) The analytical method according to item 94, further comprising the step of visualizing the detected fluorescence of the fluorescent dye to obtain an image, preferably a three-dimensional image, of the animal tissue. 96. The analysis method described in item 95, wherein the image is an image having single-cell resolution across the entire animal tissue. 97. The analytical method according to any one of items 94 to 96, wherein the animal tissue has a thickness of 20 cm or less, preferably 10 cm or less, and more preferably 5 cm or less. 98. The analytical method according to any one of items 94 to 97, wherein the animal tissue is 2 cm or less in thickness, preferably 1.5 to 2 cm in thickness. 99. The analytical method described in any one of items 94 to 98, further comprising the method described in any one of items 1 to 87, prior to step i). 100. The analytical method described in any one of items 94 to 99, wherein the fluorescence microscope is selected from the group consisting of a light-sheet fluorescence microscope, an epifluorescence microscope, a multiphoton microscope, and a confocal fluorescence microscope. 101. The analysis method described in any one of items 94-100, wherein the fluorescence microscope is a light-sheet fluorescence microscope. 102. An analytical method according to any one of items 94 to 101, further comprising the steps of i) iii) cutting the target tissue region, iv) rehydrating the cut target tissue region, and v) further analyzing the cut target tissue region. 103. The analytical method described in item 102, wherein in step v), the target tissue region cleaved is further analyzed by antibody-based immunostaining, gene profiling, preferably by RNA-seq gene profiling, or by proteomics, preferably by mass spectrometry proteomics. 104. The analytical method according to item 102 or 103, wherein the sectional tissue region of interest includes metastases, preferably metastases having a size of less than 200 tumor cells, more preferably metastases having a size of less than 100 tumor cells, even more preferably metastases having a size of less than 75 tumor cells, even more preferably metastases having a size of less than 50 tumor cells, and even more preferably metastases having a size of less than 25 tumor cells. 105. A method for detecting metastasis, including a method for analyzing animal tissue, as described in any one of items 94-104. 106. A method for detecting metastasis in the animal tissue at single-cell resolution throughout the animal tissue, as described in item 105. 107. A method for detecting metastasis according to item 105 or 106, wherein the animal tissue contains cancer metastases, and the target molecule to be labeled with the labeling agent is a structure, preferably a protein, present in the cells of the cancer. 108. A method for analyzing the in vivo distribution of a biopharmaceutical, comprising a method for analyzing animal tissue as described in any one of items 94-104, wherein the animal has been treated with the biopharmaceutical as provided for in item 80, the animal tissue contains the biopharmaceutical, and the biopharmaceutical is the target molecule labeled with the labeling agent. 109. The method described in item 108, wherein the biopharmaceutical is a therapeutic protein. 110. The method described in item 108, wherein the biopharmaceutical is a therapeutic antibody. 111. The method described in item 108, wherein the biopharmaceutical is a nanoparticle. 112. A method for analyzing the in vivo distribution of nanoparticles, comprising a method for analyzing animal tissue as described in any one of items 94 to 104, wherein the animal has been treated with nanoparticles, the animal tissue contains the nanoparticles, the nanoparticles are the target molecule labeled with the labeling agent, and / or nanoparticles selected from fluorescent dye complex-forming nanoparticles or nanoparticles that are fluorescence itself. 113. A method for analyzing the in vivo distribution of nanoparticles as described in item 112, wherein the nanoparticles are drug-carrying or are drugs themselves. 114. A method for testing neurodegeneration, comprising a method for analyzing animal tissue as described in any one of items 94-104, wherein the animal tissue contains neurons. 115. A method for testing neurodegeneration as described in item 114, wherein neurons in the animal tissue are fluorescently labeled, preferably by expressing a fluorescent protein. 116. A method for testing for neurodegeneration as described in item 114 or 115, wherein the test for neurodegeneration includes an analysis of vesicle formation on nerve axons. 117. A method for testing neuroinflammation, comprising a method for analyzing animal tissue as described in any one of items 94-104, wherein the animal tissue contains neurons. 118. A method for testing neuroinflammation as described in item 117, wherein immune cells in the animal tissue are preferably fluorescently labeled by expressing a fluorescent protein. 119. A method for testing neuroinflammation as described in item 117 or 118, comprising testing the activation of immune cells by analyzing the signal intensity and / or cell number of fluorescently labeled immune cells. 120. A method for testing meningeal lymphatic vessels, comprising a method for analyzing animal tissue as described in any one of items 94 to 104, wherein the animal tissue preferably includes an intact mouse head. 121. A method for testing meningeal lymphatic vessels as described in item 120, wherein the meningeal lymphatic vessels are preferably fluorescently labeled with a marker protein or with a tracer such as ovalbumin. 122. A method for testing meningeal lymphatic vessels according to item 120 or 121, wherein the meningeal lymphatic vessel contains this target molecule. [Brief explanation of the drawing]

[0030] [Figure 1a] This figure shows the advantages of the LuCiD pipeline and nano-level boosting for deep tissue fluorescence imaging. (Figure 1a) shows the LuCiD pipeline process in an adult mouse body. Specific regions containing target cancer cells can be cleaved, rehydrated, and further characterized by antibody labeling. [Figure 1b] This figure shows the advantages of the LuCiD pipeline and nano-level boosting for deep tissue fluorescence imaging. (Figure 1b) demonstrates light tissue transmission at various imaging wavelengths, and is of the same liver region (unlabeled) from a cleared mouse, imaged using a lightsheet microscope with green (excitation 470 nm) (left), red (excitation 561 nm) (center), and far-red channel (excitation 640 nm) (right). [Figure 1c] This figure shows the advantages of the LuCiD pipeline and nano-level boosting for deep tissue fluorescence imaging. Figure 1c is the fluorescence signal intensity profile normalized to the maximum intensity in the region indicated by the dashed line in Figure 1b. [Figure 1d]This figure shows the advantages of the LuCiD pipeline and nanoscale boosting for deep tissue fluorescence imaging. (Figure 1d) is a representative lightsheet image of tumor metastases expressing mCherry in the lungs of cleared, unlabeled mice. [Figure 1e] This figure shows the advantages of the LuCiD pipeline and nano-level boosting for deep tissue fluorescence imaging. (Figure 1e) shows a transposition boosted with anti-mCherry nanobodies complexed with Atto594. [Figure 1f] This figure shows the advantages of the LuCiD pipeline and nano-level boosting for deep tissue fluorescence imaging. (Figure 1f) shows a transposition boosted with anti-mCherry nanobodies complexed with Atto647N. [Figure 1g] This figure shows the advantages of the LuCiD pipeline and nano-level boosting for deep tissue fluorescence imaging. (Figure 1g) is a plot of signal intensity profiles along the dashed line in the panel (n=3 transpositions per mouse from each channel). [Figure 1h] This figure shows the advantages of the LuCiD pipeline and nanoscale boosting for deep tissue fluorescence imaging. (Figure 1h) is the fluorescence signal intensity profile normalized to the background of the data in (Figure 1g). [Figure 1i] This figure shows the advantages of the LuCiD pipeline and nanoscale boosting for deep tissue fluorescence imaging. (Figure 1i) is an example of deep tissue imaging of brain tumor metastases in the far-red spectrum in a see-through mouse after LuCiD. Tumor micrometastases (arrowheads) can be seen to a depth of several millimeters in the brain tissue. [Figure 2a] This figure shows the visualization of systemic metastasis in intact mice with high tumor load. (Figure 2a) is a normal bioluminescence image of a high tumor load mouse before LuCiD treatment. [Figure 2b]This figure shows the visualization of whole-body metastasis in intact mice with high tumor load. (Figure 2b) is a high-exposure bioluminescence image of a high-tumor-load mouse before LuCiD treatment. [Figure 2c] This figure shows the visualization of systemic metastasis in intact mice with high tumor load. In Figures 2c to 2h, the epifluorescence images of the same mouse after LuCiD treatment show further details of metastasis (dashed rectangles) compared with bioluminescence. [Figure 2d] This figure shows the visualization of systemic metastasis in intact mice with high tumor load. In Figures 2c to 2h, the epifluorescence images of the same mouse after LuCiD treatment show further details of metastasis (dashed rectangles) compared with bioluminescence, and in addition, Figure 2d shows major metastases visible in bioluminescence as bulk signals. [Figure 2e] This figure shows the visualization of systemic metastasis in intact mice with high tumor load. In Figures 2c to 2h, the epifluorescence images of the same mouse after LuCiD treatment show further details of metastasis (dashed rectangles) compared with bioluminescence, and in addition, Figure 2e shows the major metastases visible in bioluminescence as bulk signals. [Figure 2f] This figure shows the visualization of systemic metastasis in intact mice with high tumor load. In Figures 2c to 2h, the epifluorescence images of the same mouse after LuCiD treatment show further details of metastasis (dashed rectangles) compared with bioluminescence, and Figure 2f includes small micrometastases that can be easily detected in the lungs (arrowheads). [Figure 2g] This figure shows the visualization of systemic metastasis in intact mice with high tumor load. In Figures 2c to 2h, the epifluorescence images of the same mouse after LuCiD treatment show further details of metastasis (dashed rectangles) compared with bioluminescence, and in addition, Figure 2g shows the primary tumor. [Figure 2h]This figure shows the visualization of whole-body metastasis in intact mice with high tumor load. In Figures 2c to 2h, the epifluorescence images of the same mouse after LuCiD treatment show further details of metastasis (dashed rectangles) compared with bioluminescence, and Figure 2h includes small micrometastases that can be easily detected in the legs. [Figure 2i] This figure visualizes whole-body metastasis in intact mice with high tumor load. (Figure 2i) is a frontal view of 3D region segmentation obtained from light sheet imaging data corresponding to the dashed rectangular areas shown in (Figures 2a-2c). For simplification, only a few organs: the heart and lungs are segmented, and tumors are shown in black. [Figure 2j] This figure visualizes systemic metastasis in intact mice with high tumor volume. Figure 2j is a lateral view of 3D region segmentation acquired from optical sheet imaging data corresponding to the dashed rectangular areas shown in Figures 2a-2c. For simplification, only a few organs, the heart and lungs, are segmented, and tumors are shown in black. [Figure 2k] This figure shows the visualization of systemic metastasis in intact mice with high tumor volume. Figure 2k is the raw data (500 μm projection) from a light sheet microscope showing the tumor from the sagittal plane shown in Figure 2j. [Figure 2l] This figure shows the visualization of systemic metastasis in intact mice with high tumor volume. (Figure 2l) is the raw data (500 μm projection) from a light sheet microscope showing the tumor from the sagittal plane, as shown in (Figure 2j). [Figure 2m] This figure shows the visualization of systemic metastasis in intact mice with a high tumor load. Figure 2m is the raw data (500 μm projection) from a light sheet microscope showing the tumor from the sagittal plane shown in Figure 2j. [Figure 2n] This figure shows the visualization of systemic metastasis in intact mice with a high tumor load. Figure 2n is the raw data (500 μm projection) from a light sheet microscope showing the tumor from the sagittal plane shown in Figure 2j. [Figure 2o]This figure shows the visualization of systemic metastasis in intact mice with high tumor load. (Figure 2o) is a high-resolution light-sheet microscope image (single plane) showing a single tumor cell (enclosed area) detected by LuCiD. See also Figures 7-9. [Figure 2p] This figure shows the visualization of systemic metastasis in intact mice with high tumor load. (Figure 2p) is a high-resolution light-sheet microscope image (single plane) showing nuclei (labeled with propidium iodide (PI)) detected by LuCiD. See also Figures 7-9. [Figure 2q] This figure shows the visualization of systemic metastasis in intact mice with high tumor load. (Figure 2q) is a high-resolution light-sheet microscope image (single plane) showing a single tumor cell (circled area in Figure 2o) and nucleus (labeled with propidium iodide (PI) in Figure 2p) detected by LuCiD. See also Figures 7-9. [Figure 3a] This figure shows the visualization and quantification of systemic metastasis in intact mice with low tumor load. (Figure 3a) is a normal exposure bioluminescence image of a low tumor load mouse before LuCiD. Primary tumors and metastases in the axillary lymph nodes (ALNs) are marked with arrows in (Figure 3a). [Figure 3b] This figure shows the visualization and quantification of systemic metastasis in intact mice with low tumor load. (Figure 3b) is a high-exposure bioluminescence image of a low tumor load mouse before LuCiD. [Figure 3c] This figure shows the visualization and quantification of systemic metastasis in intact mice with low tumor load. (Figure 3c) is an epifluorescence image of LuCiD-treated mice showing further details of tumor metastasis compared to bioluminescence. [Figure 3d] This figure shows the visualization and quantification of systemic metastasis in intact mice with low tumor load. (Figure 3d) is an epifluorescence image of LuCiD-treated mice showing further details of tumor metastasis compared to bioluminescence. [Figure 3e]This figure shows the visualization and quantification of systemic metastasis in intact mice with low tumor load. (Figure 3e) is an epifluorescence image of LuCiD-treated mice showing further detail of tumor metastasis compared to bioluminescence, including several pulmonary micrometastases (arrowheads) smaller than 20 mm that are undetectable by bioluminescence. [Figure 3f] This figure shows the visualization and quantification of systemic metastasis in intact mice with low tumor load. (Figure 3f) is an epifluorescence image of LuCiD-treated mice showing further details of tumor metastasis compared to bioluminescence. [Figure 3g] This figure shows the visualization and quantification of whole-body metastases in intact mice with low tumor load. (Figure 3g) is a lateral view of the 3D reconstruction of the whole mouse after lightsheet microscopy imaging. All tumors / metastases in the mouse body are segmented (some are marked with arrows). [Figure 3h] This figure shows the visualization and quantification of whole-body metastases in intact mice with low tumor load. (Figure 3h) is an abdominal image of the whole mouse 3D reconstruction after light sheet microscopy imaging. All tumors / metastases in the mouse body are segmented (some are marked with arrows). [Figure 3i] This figure shows the visualization and quantification of systemic metastases in intact mice with low tumor load. (Figure 3i) shows the quantification of all metastases in the entire mouse based on their size (cell number) and quantity. [Figure 3j] This figure shows the visualization and quantification of systemic metastasis in intact mice with low tumor volume. (Figure 3j) is a 2D distribution map of tumor metastasis throughout the mouse body. The size of the spots represents the volume of tumor metastasis. The relative depth of the marked areas is shown on the right. [Figure 3k] This figure shows the visualization and quantification of systemic metastasis in intact mice with low tumor burden. (Figure 3k) is a 3D regional segmentation of tumor metastases detected in the lung, showing tumor cells (white dots) with an autofluorescence background. [Figure 3l]This figure shows the visualization and quantification of systemic metastases in intact mice with low tumor load. Figure 3l is a 3D scatter plot of all metastases in the lungs shown in Figure 3k, representing their size and location. [Figure 4a] This figure shows the visualization of whole-body metastases and therapeutic antibodies in intact mice. (Figure 4a) is a normal exposure bioluminescence image of low tumor-bearing mice injected with therapeutic antibody 6A10 conjugated with Alexa568 prior to LuCiD. Note that only tumors with luciferase signals (not therapeutic antibodies) can be seen. [Figure 4b] This figure shows the visualization of whole-body metastases and therapeutic antibodies in intact mice. (Figure 4b) is a high-exposure bioluminescence image of low-tumor-burden mice injected with therapeutic antibody 6A10 conjugated with Alexa568 prior to LuCiD. Note that only tumors with luciferase signaling (not the therapeutic antibody) can be seen. [Figure 4c] This figure shows the visualization of metastases and therapeutic antibodies throughout the entire body of intact mice. In Figures 4c-4g, the epifluorescence images of LuCiD-treated mice show details of both tumor metastases and antibody 6A10 distribution. Most micrometastases appear to be targeted by the antibody (arrow in Figure 4e), but some micrometastases appear not to be targeted by the antibody (arrow in Figure 4f). [Figure 4d] This figure shows the visualization of metastasis and therapeutic antibodies throughout the entire body of intact mice. In Figures 4c to 4g, the epifluorescence images of LuCiD-treated mice show details of both tumor metastasis and antibody 6A10 distribution. [Figure 4e] This figure shows the visualization of metastases and therapeutic antibodies throughout the entire body of intact mice. In Figures 4c-4g, the epifluorescence images of LuCiD-treated mice show details of both tumor metastases and antibody 6A10 distribution. Most micrometastases appear to be targeted by the antibody (arrow in Figure 4e), but some micrometastases appear not to be targeted by the antibody (arrow in Figure 4f). [Figure 4f]This figure shows the visualization of metastases and therapeutic antibodies throughout the entire body of intact mice. In Figures 4c-4g, the epifluorescence images of LuCiD-treated mice show details of both tumor metastases and antibody 6A10 distribution. Most micrometastases appear to be targeted by the antibody (arrow in Figure 4e), but some micrometastases appear not to be targeted by the antibody (arrow in Figure 4f). [Figure 4g] This figure shows the visualization of metastasis and therapeutic antibodies throughout the entire body of intact mice. In Figures 4c to 4g, the epifluorescence images of LuCiD-treated mice show details of both tumor metastasis and antibody 6A10 distribution. [Figure 4h] This figure shows the visualization of metastases and therapeutic antibodies throughout the entire body of an intact mouse. (Figure 4h) is an abdominal image of a 3D reconstruction of the whole mouse after lightsheet microscopy imaging. All tumors in the mouse body were segmented and overlaid with antibodies (shown as integrated white signals). [Figure 4i] This figure shows the visualization of metastases and therapeutic antibodies throughout an intact mouse body. (Figure 4i) is a lateral view of a 3D reconstruction of the entire mouse after lightsheet microscopy imaging. All tumors in the mouse body were segmented and overlaid with antibodies (shown as integrated white signals). [Figure 4j] This figure shows the visualization of whole-body metastases and therapeutic antibodies in intact mice. (Figure 4j) is a high-magnification image of the enclosed area in (Figure 4h), showing further detail of tumor micrometastases. [Figure 4k] This figure shows the visualization of metastasis and therapeutic antibodies throughout the body of intact mice. Figures 4k to 4m show details of antibody targeting in the enclosed region of Figure 4j, and Figure 4k shows tumor channels. See also Figures 10 and 11. [Figure 4l] This figure shows the visualization of metastasis and therapeutic antibodies throughout the entire body of intact mice. Figures 4k to 4m show details of antibody targeting in the enclosed region of Figure 4j, and Figure 4l shows the antibody channel. See also Figures 10 and 11. [Figure 4m]This figure shows the visualization of metastasis and therapeutic antibodies throughout the entire body of intact mice. Figures 4k to 4m show the details of antibody targeting in the enclosed region of Figure 4j, and Figure 4m shows the integration. See also Figures 10 and 11. [Figure 5a] This figure shows high-resolution light-sheet imaging of individual organs with metastases. (Figure 5a) shows 3D visualizations of the lungs, liver, kidneys, and brain. Segmented tumors are indicated by arrowheads (black), and tumors targeted by the therapeutic antibody (conjugated with Alexa568) are indicated by arrows (white). [Figure 5b] This figure shows high-resolution light-sheet imaging of individual organs with metastases. These are raw high-resolution light-sheet microscope images of single metastases in appropriate organs, showing overlaps of tumor cells and therapeutic antibodies (white) in the lungs, kidneys, and brain. [Figure 5c] This figure shows high-resolution optical sheet imaging of individual organs with metastasis. (Figure 5c) is a 2D distribution map. [Figure 5d] This figure shows high-resolution optical sheet imaging of individual organs with metastasis. (Figure 5d) quantifies untargeted metastasis in response to targeted therapeutic antibodies in various organs. Values ​​are mean ± sem, n=3 mice. [Figure 5e] This figure shows high-resolution light-sheet imaging of individual organs with metastasis. (Figure 5e) shows antibody distribution in organs: quantification of non-tumor host mouse tissue against tumor-binding therapeutic antibodies. Values ​​are mean ± sem, n=3 mice. [Figure 6a] This figure shows the visualization of tumor vascular structure and the re-detection of the tumor microenvironment. To examine the details of the vascular tumor microenvironment and therapeutic antibody distribution, vascular structures in low tumor-bearing mice were labeled by lectin perfusion. (Figures 6a and 6b) show small tumor populations that could not be seen by bioluminescence, as revealed by light-sheet microscopy scans through the mouse torso (dashed rectangles). [Figure 6b]This figure shows the visualization of tumor vascular structure and the re-detection of the tumor microenvironment. To examine the details of the vascular tumor microenvironment and therapeutic antibody distribution, vascular structures in low tumor-bearing mice were labeled by lectin perfusion. (Figures 6a and 6b) show small tumor populations that could not be seen by bioluminescence, as revealed by light-sheet microscopy scans through the mouse torso (dashed rectangles). [Figure 6c] This figure shows the visualization of tumor vascular structure and the re-detection of the tumor microenvironment. To examine the details of the vascular tumor microenvironment and therapeutic antibody distribution, the vascular structure of low tumor-bearing mice was labeled by lectin perfusion. Figures 6c to 6f are high-magnification light-sheet microscope images of the region shown in Figure 6b. Lectin-labeled blood vessels are shown in Figure 6c. [Figure 6d] This figure shows the visualization of tumor vascular structure and the re-detection of the tumor microenvironment. To examine the details of the vascular tumor microenvironment and therapeutic antibody distribution, the vascular structure of low tumor-bearing mice was labeled by lectin perfusion. Figures 6c to 6f are high-magnification light-sheet microscope images of the region shown in Figure 6b. The tumor is shown in Figure 6d. [Figure 6e] This figure shows the visualization of tumor vascular structure and the re-detection of the tumor microenvironment. To examine the details of the vascular tumor microenvironment and therapeutic antibody distribution, the vascular structure of low tumor-bearing mice was labeled by lectin perfusion. Figures 6c to 6f are high-magnification light-sheet microscope images of the region shown in Figure 6b. Therapeutic antibody 6A10 (injected 2 days before clearing) is shown in Figure 6e. [Figure 6f] This figure shows the visualization of tumor vascular structure and the re-detection of the tumor microenvironment. To examine the details of the vascular tumor microenvironment and therapeutic antibody distribution, the vascular structure of low tumor-bearing mice was labeled by lectin perfusion. Figures 6c-6f are high-magnification light-sheet microscopy images of the region shown in Figure 6b. Figure 6f is a 3D integration of the three channels shown in Figures 6c-6e. Note that most of the tumor is targeted by therapeutic antibodies in this highly vascularized tumor microenvironment. [Figure 6g]This figure shows the visualization of tumor vascular structure and the re-detection of the tumor microenvironment. To examine the details of the vascular tumor microenvironment and therapeutic antibody distribution, vascular structures in low tumor-burden mice were labeled by lectin perfusion. (Figures 6g-6n) are confocal microscopy images of lung metastatic tissue after systemic labeling with LuCiD, clearing, and imaging of tumor cells. The identified metastatic lung tissue was rehydrated, and (Figures 6g-6j) immunolabeling analysis of the tumor microenvironment was performed using tumor-associated fibroblast (α-smooth muscle actin; α-SMA) markers. [Figure 6h] This figure shows the visualization of tumor vascular structure and the re-detection of the tumor microenvironment. To examine the details of the vascular tumor microenvironment and therapeutic antibody distribution, vascular structures in low tumor-burden mice were labeled by lectin perfusion. (Figures 6g-6n) are confocal microscopy images of lung metastatic tissue after systemic labeling with LuCiD, clearing, and imaging of tumor cells. The identified metastatic lung tissue was rehydrated, and (Figures 6g-6j) immunolabeling analysis of the tumor microenvironment was performed using tumor-associated fibroblast (α-smooth muscle actin; α-SMA) markers. [Figure 6i] This figure shows the visualization of tumor vascular structure and the re-detection of the tumor microenvironment. To examine the details of the vascular tumor microenvironment and therapeutic antibody distribution, vascular structures in low tumor-burden mice were labeled by lectin perfusion. (Figures 6g-6n) are confocal microscopy images of lung metastatic tissue after systemic labeling with LuCiD, clearing, and imaging of tumor cells. The identified metastatic lung tissue was rehydrated, and (Figures 6g-6j) immunolabeling analysis of the tumor microenvironment was performed using tumor-associated fibroblast (α-smooth muscle actin; α-SMA) markers. [Figure 6j] This figure shows the visualization of tumor vascular structure and the re-detection of the tumor microenvironment. To examine the details of the vascular tumor microenvironment and therapeutic antibody distribution, vascular structures in low tumor-burden mice were labeled by lectin perfusion. (Figures 6g-6n) are confocal microscopy images of lung metastatic tissue after systemic labeling with LuCiD, clearing, and imaging of tumor cells. The identified metastatic lung tissue was rehydrated, and (Figures 6g-6j) immunolabeling analysis of the tumor microenvironment was performed using tumor-associated fibroblast (α-smooth muscle actin; α-SMA) markers. [Figure 6k] This figure shows the visualization of tumor vascular structure and the re-detection of the tumor microenvironment. To examine the details of the vascular tumor microenvironment and therapeutic antibody distribution, vascular structures in low tumor-burden mice were labeled by lectin perfusion. (Figures 6g to 6n) are confocal microscopy images of lung metastatic tissue after systemic labeling with LuCiD, clearing, and imaging of tumor cells. The identified metastatic lung tissue was rehydrated, and (Figures 6k to 6n) immunolabeling analysis of the tumor microenvironment was performed using vascular endothelial cell (MECA-32) markers. [Figure 6l] This figure shows the visualization of tumor vascular structure and the re-detection of the tumor microenvironment. To examine the details of the vascular tumor microenvironment and therapeutic antibody distribution, vascular structures in low tumor-burden mice were labeled by lectin perfusion. (Figures 6g to 6n) are confocal microscopy images of lung metastatic tissue after systemic labeling with LuCiD, clearing, and imaging of tumor cells. The identified metastatic lung tissue was rehydrated, and (Figures 6k to 6n) immunolabeling analysis of the tumor microenvironment was performed using vascular endothelial cell (MECA-32) markers. [Figure 6m] This figure shows the visualization of tumor vascular structure and the re-detection of the tumor microenvironment. To examine the details of the vascular tumor microenvironment and therapeutic antibody distribution, vascular structures in low tumor-burden mice were labeled by lectin perfusion. (Figures 6g to 6n) are confocal microscopy images of lung metastatic tissue after systemic labeling with LuCiD, clearing, and imaging of tumor cells. The identified metastatic lung tissue was rehydrated, and (Figures 6k to 6n) immunolabeling analysis of the tumor microenvironment was performed using vascular endothelial cell (MECA-32) markers. [Figure 6n] This figure shows the visualization of tumor vascular structure and the re-detection of the tumor microenvironment. To examine the details of the vascular tumor microenvironment and therapeutic antibody distribution, vascular structures in low tumor-burden mice were labeled by lectin perfusion. (Figures 6g to 6n) are confocal microscopy images of lung metastatic tissue after systemic labeling with LuCiD, clearing, and imaging of tumor cells. The identified metastatic lung tissue was rehydrated, and (Figures 6k to 6n) immunolabeling analysis of the tumor microenvironment was performed using vascular endothelial cell (MECA-32) markers. [Figure 7a]This figure shows the experimental design for tumor transplantation, the specificity of antibody labeling, and the morphological characteristics of the tumor. (Figure 7a) is an illustration of the experimental workflow for tumor transplantation. [Figure 7b] This figure shows the experimental design for tumor transplantation, the specificity of antibody labeling, and the tumor morphological characteristics. (Figure 7b) shows the whole mouse after the LuCiD pipeline. [Figure 7c] This figure shows the experimental design for tumor transplantation, the specificity of antibody labeling, and the morphological characteristics of the tumor. (Figure 7c) is a confocal image of the endogenous mCherry signaling of the tumor in the lung. [Figure 7d] This figure shows the experimental design for tumor transplantation, the specificity of antibody labeling, and the morphological characteristics of the tumor. (Figure 7d) is a confocal image of the endogenous mCherry signaling of tumors in the lung, labeled with an anti-mCherry nanobooster complexed with Atto647N. [Figure 7e] This figure shows the experimental design for tumor transplantation, the specificity of antibody labeling, and the tumor morphological characteristics. The integration is shown in Figure 7e. [Figure 7f] This figure shows the experimental design for tumor transplantation, the specificity of antibody labeling, and the tumor morphological characteristics. (Figure 7f) is a confocal image (arrowhead) of metastases in the lungs of animals labeled with an anti-mCherry nanobooster complexed with Atto647N. [Figure 7g] This figure shows the experimental design for tumor transplantation, the specificity of antibody labeling, and the morphological characteristics of the tumor. (Figure 7g) is a confocal image (arrowhead) of metastases in the lungs of animals labeled with propidium iodide (PI). [Figure 7h] This figure shows the experimental design for tumor transplantation, the specificity of antibody labeling, and the morphological characteristics of the tumor. (Figures 7f-7h) are confocal images of metastases in the lungs of animals labeled with anti-mCherry nanobooster complexed with Atto647N (Figure 7f, arrowhead) and propidium iodide (PI) (Figure 7g, arrowhead). Integration of the two channels is shown (Figure 7h, arrowhead). [Figure 8a]This figure shows the confirmation of nanobooster staining specificity in mCherry-expressing tumor mice. (Figure 8a) is a comparison between high tumor-bearing (mCherry-expressing) animals and C57BL / 6N control animals, both of which were boosted with anti-mCherry nanobodies complexed with Atto647N (white), showing the absence of nonspecific signals in organs derived from C57BL / 6N controls. This is a lightsheet microscopy image of the indicated region in organs derived from BL6 controls, showing the absence of specific signals. Note that the background (gray) has been enhanced to demonstrate the absence of signals in high-magnification images. [Figure 8b] This figure shows confirmation of nanobooster staining specificity in mCherry-expressing tumor mice. (Figure 8b) is a confocal image of tumor micrometastases boosted with 647 in lung tissue, which were immunolabeled with an anti-firefly luciferase antibody after rehydration of cleared tissue, and the cell nuclei were labeled with Hoechst. [Figure 9a] This figure shows a comparison between bioluminescence and epifluorescence imaging in low- and high-tumor-burden mice (Figures 9a-9d). It was found that bioluminescence imaging with normal exposure was not highly sensitive to detecting all metastases in low-tumor-burden mice. For example, the bioluminescence images with normal exposure were very similar in the mice in Figures 9a, 9b, 9c, and 9d. By applying LuCiD to such mice, the inventors found large metastases (arrowheads) in the axillary lymph nodes using a stereofluorescence microscope, although in some cases no tumor metastases were found (Figures 9a, 9b) (Figures 9c, 9d, boxed area 1). [Figure 9b]This figure shows a comparison between bioluminescence and epifluorescence imaging in low- and high-tumor-burden mice (Figures 9a-9d). It was found that bioluminescence imaging with normal exposure was not highly sensitive to detecting all metastases in low-tumor-burden mice. For example, the bioluminescence images with normal exposure were very similar in the mice in Figures 9a, 9b, 9c, and 9d. By applying LuCiD to such mice, the inventors found large metastases (arrowheads) in the axillary lymph nodes using a stereofluorescence microscope, although in some cases no tumor metastases were found (Figures 9a, 9b) (Figures 9c, 9d, boxed area 1). In the epifluorescence images, tumors (labeled with A647) are shown in white. [Figure 9c] This figure shows a comparison between bioluminescence and epifluorescence imaging in low- and high-tumor-burden mice (Figures 9a-9d). It was found that bioluminescence imaging with normal exposure was not highly sensitive to detecting all metastases in low-tumor-burden mice. For example, the bioluminescence images with normal exposure were very similar in the mice in Figures 9a, 9b, 9c, and 9d. By applying LuCiD to such mice, the inventors, using a stereofluorescence microscope, found large metastases (arrowheads) in the axillary lymph nodes in some cases, but not in others (Figures 9a, 9b) (Figures 9c, 9d, boxed area 1). Signals from the primary tumor were strong in both normal and high-exposure bioluminescence images (Figures 9c, 9d, boxed area 3), but metastases in the lungs (arrows) were not visible (Figures 9c, 9d, boxed area 2). [Figure 9d]This figure shows a comparison between bioluminescence and epifluorescence imaging in low- and high-tumor-burden mice (Figures 9a-9d). It was found that bioluminescence imaging with normal exposure was not highly sensitive to detecting all metastases in low-tumor-burden mice. For example, the bioluminescence images with normal exposure were very similar in the mice in Figures 9a, 9b, 9c, and 9d. By applying LuCiD to such mice, the inventors, using a stereofluorescence microscope, found large metastases (arrowheads) in the axillary lymph nodes in some cases, but not in others (Figures 9a, 9b) (Figures 9c, 9d, boxed area 1). Signals from the primary tumor were strong in both normal and high-exposure bioluminescence images (Figures 9c, 9d, boxed area 3), but metastases in the lungs (arrows) were not visible (Figures 9c, 9d, boxed area 2). In the epifluorescence image, the tumor (labeled with A647) is shown in white. [Figure 9e] This figure shows a comparison between bioluminescence and epifluorescence imaging in low- and high-tumor-burden mice. (Figures 9e and 9g) In high-tumor-burden mice, bioluminescence imaging provides a bulk heatmap of metastasis distribution. [Figure 9f] This figure (Figure 9f, Figure 9h) compares bioluminescence and epifluorescence imaging in low- and high-tumor-burden mice. In contrast to bioluminescence imaging, LuCiD resolved single metastases throughout the mouse body, even by stereofluorescence microscopy. In particular, in the lungs, even micrometastases with a diameter of less than 20 μm were resolvable in intact mice. [Figure 9g] This figure shows a comparison between bioluminescence and epifluorescence imaging in low- and high-tumor-burden mice. (Figures 9e and 9g) In high-tumor-burden mice, bioluminescence imaging provides a bulk heatmap of metastasis distribution. [Figure 9h]This figure (Figure 9f, Figure 9h) compares bioluminescence and epifluorescence imaging in low- and high-tumor-burden mice. In contrast to bioluminescence imaging, LuCiD resolved single metastases throughout the mouse body, even by stereofluorescence microscopy. In particular, in the lungs, even micrometastases with a diameter of less than 20 μm were resolvable in intact mice. [Figure 10a] This figure shows the disappearance of endogenously expressed mCherry signaling in tumors using the improved 3DISCO protocol: a combination of destaining, immunohistochemistry, and 3DISCO clearing. (Figure 10a) shows that tumor metastases in the lungs were imaged using a fluorescence stereomicroscope before and after the improved 3DISCO clearing, demonstrating the removal of endogenously expressed mCherry signaling. [Figure 10b] Figure 10b shows the disappearance of endogenously expressed mCherry signaling in tumors using an improved 3DISCO protocol: a combination of destaining, immunostaining, and 3DISCO clearing. The images are lightsheet microscopy images of a primary tumor with background (left) acquired in green (excitation 470 nm), the mCherry signal (center) acquired in red (excitation 561 nm), and a boosted signal (Atto647N) (right) acquired in a far-red channel (excitation 640 nm). [Figure 10c] The figure shows the disappearance of endogenous mCherry signaling in tumors using the improved 3DISCO protocol: destaining + immunostaining + 3DISCO clearing. (Figure 10c) is a plot of signal intensity profiles along the white line in panel b (n=3 mice). [Figure 10d] This figure shows the disappearance of endogenous mCherry signaling in tumors using the improved 3DISCO protocol: destaining + immunostaining + 3DISCO clearing. (Figure 10d) is the fluorescence signal profile normalized to the background of the data from Figure 10c, showing that the endogenous mCherry signal was depleted to a level similar to the background after improved 3DISCO clearing. [Figure 11a]This figure shows the co-localization of the antibody 6A10 signal with the nanobody signal as observed by confocal microscopy. (Figure 11a) is a confocal image of metastasis in the lungs of animals labeled with anti-mCherry nanobodies complexed with Atto647. [Figure 11b] This figure shows the co-localization of the antibody 6A10 signal with the nanobody signal using confocal microscopy. (Figure 11b) is a confocal image of metastases in the lungs of animals treated with therapeutic antibody 6A10 conjugated with Alexa568. [Figure 11c] This figure shows the co-localization of the antibody 6A10 signal with the nanobody signal using a confocal microscope. (Figure 11c) is a confocal image of metastasis in the lung of an animal. It shows the co-localization of metastasis with antibody 6A10 (arrowhead). [Figure 11d] This figure shows the co-localization of the antibody 6A10 signal with the nanobody signal using confocal microscopy. (Figure 11d) is a confocal image of metastasis in the kidney of an animal labeled with anti-mCherry nanobodies complexed with Atto647. [Figure 11e] This figure shows the co-localization of the antibody 6A10 signal with the nanobody signal using confocal microscopy. (Figure 11e) is a confocal image of metastases in the kidneys of animals treated with therapeutic antibody 6A10 conjugated with Alexa568. [Figure 11f] This figure shows the co-localization of the antibody 6A10 signal with the nanobody signal using a confocal microscope. (Figure 11f) is a confocal image of metastasis in the kidney of an animal. It shows the co-localization of metastasis with antibody 6A10 (arrowhead). [Figure 11g] This figure shows the co-localization of the antibody 6A10 signal with the nanobody signal as observed by confocal microscopy. (Figure 11g) is a confocal image of metastases in the liver of animals labeled with anti-mCherry nanobodies complexed with Atto647. [Figure 11h] This figure shows the co-localization of the antibody 6A10 signal with the nanobody signal as observed by confocal microscopy. (Figure 11h) is a confocal image of metastases in the liver of animals treated with therapeutic antibody 6A10 conjugated with Alexa568. [Figure 11i]This figure shows the co-localization of the antibody 6A10 signal with the nanobody signal using confocal microscopy. (Figure 11i) is a confocal image of metastasis in the liver of an animal. It shows the co-localization of metastasis with antibody 6A10 (arrowhead). [Figure 11j] This figure shows the co-localization of the antibody 6A10 signal with the nanobody signal using confocal microscopy. (Figure 11j) is a confocal image of a single cancer cell in the lung of an animal. Hoechst nucleus staining is shown in (Figure 11j). The enclosed areas are high-magnification images, i.e., regions indicated by dashed lines. [Figure 11k] This figure shows the co-localization of the antibody 6A10 signal with the nanobody signal using confocal microscopy. (Figure 11k) is a confocal image of a single cancer cell in the lung of an animal labeled with anti-mCherry nanobodies complexed with Atto594. The enclosed areas are high-magnification images, i.e., regions indicated by dashed lines. [Figure 11l] This figure shows the co-localization of the antibody 6A10 signal with the nanobody signal using confocal microscopy. (Figure 11l) is a confocal image of a single cancer cell in the lung of an animal treated with therapeutic antibody 6A10 conjugated with Alexa647. The enclosed areas are high-magnification images, i.e., regions indicated by dashed lines. [Figure 11m] This figure shows the co-localization of antibody 6A10 signaling with nanobody signals using confocal microscopy. (Figure 11m) is a confocal image of a single cancer cell in the lung of an animal. It shows co-localization with tumor nuclear staining. The enclosed areas are high-magnification images, i.e., regions indicated by dashed lines. [Figure 11n] This figure shows the co-localization of antibody 6A10 signaling with nanobody signals using confocal microscopy. (Figure 11n) is a confocal image of a single cancer cell in the lung of an animal. It shows the co-localization of the tumor with antibody 6A10. The enclosed areas are high-magnification images, i.e., regions indicated by dashed lines. [Figure 12a]This figure shows the analysis of lung metastasis in a tumor resection model. Primary tumors with a volume of less than 1.5 cm³ were resected 8 weeks after transplantation of MDA-231 cells into the mammary fat body, and mice were perfused 5 weeks later to introduce the LuCiD workflow. (Figure 12a) shows the appearance of nude mice before whole-body clearing. [Figure 12b] This figure shows the analysis of lung metastasis in a tumor resection model. Primary tumors with a volume of less than 1.5 cm³ were resected 8 weeks after transplantation of MDA-231 cells into the mammary fat body, and the mice were perfused 5 weeks later to introduce the LuCiD workflow. (Figure 12b) shows the appearance of nude mice after whole-body clearing. [Figure 12c] This figure shows the analysis of lung metastasis in a tumor resection model. Primary tumors with a volume of less than 1.5 cm³ were resected 8 weeks after transplantation of MDA-231 cells into the mammary fat body, and the mice were perfused 5 weeks later to introduce the LuCiD workflow. Figure 12c shows the appearance of nude mice after whole-body clearing. Note that in Figure 12c, the lungs become completely clear after clearing. [Figure 12d] This figure shows the analysis of lung metastasis in a tumor resection model. Primary tumors with a volume of less than 1.5 cm³ were resected 8 weeks after transplantation of MDA-231 cells into the mammary fat body, and the mice were perfused 5 weeks later to introduce the LuCiD workflow. Figure 12d shows the appearance of nude mice after whole-body clearing. Note that in Figure 12d, the skin becomes completely transparent after clearing. [Figure 12e] This figure shows the analysis of lung metastases in a tumor resection model. Primary tumors with a volume of less than 1.5 cm³ were resected 8 weeks after transplantation of MDA-231 cells into the mammary fat body, and mice were perfused 5 weeks later to introduce the LuCiD workflow. (Figure 12e) is a 3D regional segmentation of tumor metastases in the lungs of mice from which tumors were resected. [Figure 12f] This figure shows the analysis of lung metastases in a tumor resection model. Primary tumors with a volume of less than 1.5 cm³ were resected 8 weeks after transplantation of MDA-231 cells into the mammary fat body, and the LuCiD workflow was introduced by perfusing mice 5 weeks later. Figure 12f shows the quantification of all metastases in the lungs shown in Figure 12e, and their size distribution. [Figure 13] This figure shows the use of LuCiD to test neurodegeneration in the CNS of adult mice. In (Figure 13a), the entire CNS from a GFP-M mouse (2 months post-TBI) was cleared and imaged. Degeneration of the pyramidal axonal track is evident throughout the CNS (Figures 13b-13d). Note that morphological details of dystrophic neurons can be seen in all regions (Figures 13b-13d, arrowheads). (Figure 13e) is a non-lesion spinal cord image from a same-age control GFP-M mouse. Note that intact axons are descending (boxed). [Figure 14] This figure shows microglia / macrophage activation in CX3CR1-GFP mice (4 months post-TBI) after systemic anti-GFP nanobody labeling with LuCiD. Magnified images of the entire CNS (Figure 14a) and single cells (Figures 14b-14d) clearly show chronic activation of immune cells along the pyramidal axonal pathway. Note that details of cell morphology can be seen after nanoboost with LuCiD (Figure 14c, arrowhead). Figure 14e is a non-lesional spinal cord image from a same-age control CX3CR1-GFP mouse. Note the uniform distribution of microglia and the absence of central accumulation. [Figure 15]This figure shows the use of LuCiD to examine cerebral lymphatic vessels in the body of intact mice. (Figures 15a-15e) shows the perivascular distribution of lymphatic vessels labeled with ovalbumin (OV)-Alexa647 in the head (including the skull) of intact mice after whole-body clearing and imaging and endothelial cell labeling (lectin-FITC) in BL6 mice (Figures 15a, 15b) or VEGFR3-YFP gene-transformed mice (Figures 15c-15e). Note that the VEGFR3 signal (Figure 15d) and the ovalbumin tracer (Figure 15e) colocalize and both mark cerebral lymphatic vessels (Figures 15c-15e). These are imagings of immune cells in meningeal vessels in the intact head of a CX3CR1-GFP mouse (Figure 15f, arrowhead) and in a CX3CR1-GFP+CCR2-RFP dual-gene transgenic mouse (Figure 15g, arrowhead midway between dashed lines). Note that RFP (arrowhead midway between dashed lines) + macrophages / monocytes are removed from the brain (microglia marked with arrowheads in the brain region, to the right of the dashed line), and this infiltration can be tracked upon injury. MVs are meningeal vessels. [Figure 16]This figure shows the screening of decolorization with amino alcohols using mouse blood and spleen. Eleven different amino alcohols were mixed with PFA-fixed mouse blood and centrifuged. In (Figure 16a), the colorless pellet indicates that the red heme has been decolorized. In (Figure 16b), the decolorizing effect of eight good candidates was further examined using mouse spleen. All mouse spleen were decolorized. Images after 0 hours and 24 hours of incubation with the indicated amino alcohol are shown, respectively. The amino alcohols shown in the figure were as follows: 1. Quadrol, 2. N-butyldiethanolamine, 3. N-methyldiethanolamine, 4. N,N-dimethylmethyleneiminium chloride, 5. 1,3-bis(dimethylamino)-2-propanol, 6. 4-(2-hydroxyethyl)morpholine, 7. N-tert-butyldiethanolamine, 8. N-ethyldiethanolamine, 9. 2-(diisopropylamino)ethanol, 10. 4-methylmorpholine N-oxide, and 11. 2-(dibutylamino)ethanol. [Figure 17] This figure shows the process of clearing a pig's brain. (Figure 17a) is a fresh, dissected pig's brain. (Figure 17b) is a decolorized pig's brain after 24 hours of incubation with 2-(diisopropylamino)ethanol. (Figure 17c) shows the transparency of the pig's brain after clearing. [Figure 18] This figure shows the clearing and labeling of a large human brain sample. (Figure 18a) shows the transparency of a human brain measuring 3 cm × 3 cm × 1 cm after clearing. (Figure 18b) shows plaques and cells in the cleared human brain using fluorescence confocal imaging (arrows indicate methoxy-X04, stars indicate propidium iodide, and arrowheads indicate Nissl). [Modes for carrying out the invention]

[0031] Definitions and General Technology Hereafter, unless otherwise defined, terms used in this invention shall be understood in accordance with their general meanings known to those skilled in the art.

[0032] All published documents, patents, and patent applications cited herein are incorporated by reference in their entirety for all purposes. Such published documents, patents, and patent applications referred to herein are identified by their first author name and year of publication. For each such identified reference, the corresponding reference, including the specific source of the published document (e.g., the name and volume of the scientific journal), can be found in the section titled “References.”

[0033] Materials, methods, and examples are for illustrative purposes only and are not intended to limit the scope unless otherwise specified.

[0034] The term "fluorescent dye" as used herein is not particularly limited. For example, a fluorescent dye may be a fluorescent protein or a synthetic compound such as a synthetic organic compound. Preferably, the fluorescent dyes used in accordance with the present invention are capable of emitting fluorescence in the red or infrared region, more preferably in the far-red or near-infrared region. The preferred maximum emission wavelength of the fluorescent dyes used in accordance with the present invention is shown in preferred embodiments of the present invention. Non-limiting examples of fluorescent dyes capable of emitting fluorescence in the far-red or near-infrared region and usable in accordance with the present invention are known in the art, for example, Hong et al. (2017), Near-infrared fluorophores for biomedical imaging. Nature Biomedical Engineering, Vol. 1, No. 0010, which is incorporated by reference in its entirety for all purposes. Fluorescent dyes capable of emitting fluorescence in the far-red or near-infrared region and usable according to the present invention are commercially available and preferably include, for example, ATTO dyes such as ATTO Rho13, ATTO 594, ATTO 610, ATTO 620, ATTO Rho14, ATTO 633, ATTO 647, ATTO 647N, ATTO 655, ATTO Oxa12, ATTO 665, ATTO 680, ATTO 700, ATTO 725 and ATTO 740, as well as Alexa Fluor® 568, Alexa Fluor® 594, Alexa Fluor® 610, Alexa Fluor® 633, Alexa Fluor® 635, Alexa Fluor® 647, Alexa Fluor® 660, Alexa Fluor® 680, Alexa Fluor® 700, Alexa Fluor® 750 and Alexa Fluor®. This includes Alexa Fluor® dyes such as Fluor® 790. Dyes having maximum emission at 488 nm, 555 nm, and 568 nm are also known in the art and can be used in the methods of the present invention.

[0035] The animal tissues usable in the methods of the present invention are not particularly limited. They may be derived from any animal species. In preferred embodiments of the present invention, the animal tissue may be derived from a non-human mammal or from a human. Preferably, the animal tissue derived from a non-human mammal is derived from a rodent, more preferably from a mouse. Even more preferably, the animal tissue is a whole mouse. In preferred embodiments of the present invention, the animal tissue may be a whole organ or a part thereof, preferably a human organ or a part thereof. In preferred embodiments according to all other embodiments of the present invention, the animal tissue may contain recombinantly expressed fluorescent proteins (e.g., GFP, YFP, and mCherry), which can be used as target molecules. For example, in preferred embodiments of the present invention, the animal from which the animal tissue is obtained may be an animal (e.g., a mouse) transplanted with cancer cells expressing such recombinant fluorescent proteins.

[0036] In the decolorization step of the preparation method of the present invention, fixed animal tissue is used. In a preferred embodiment of the present invention, the preparation method of the present invention begins with the decolorization step and does not involve the fixation of animal tissue. Therefore, in a preferred embodiment, all methods of the present invention may preferably be methods that are neither surgical or therapeutic methods for the human or animal body, nor diagnostic methods performed on the human or animal body. In all other related preferred embodiments of the present invention, the methods of the present invention are ex vivo methods. Thus, the methods of the present invention may preferably be performed outside of a living animal.

[0037] Suitable animal tissues for fixation in the method of the present invention can be easily identified by those skilled in the art. For example, in PFA fixation, mice are deeply anesthetized using a combination of midazolam, medetomidine, and fentanyl (MMF) (e.g., 1 mL / 100 g of mouse body weight; ip), and then the heart can be perfused with heparinized 0.1 M PBS at room temperature for 5-10 minutes until the blood is washed away (10 U / mL heparin, Ratiopharm; pressure 100-125 mmHg using a Leica Perfusion One system). After this procedure, the tissue can be fixed for 10-20 minutes with, for example, 4% paraformaldehyde (PFA) in 0.1 M PBS (pH 7.4) (Morphisto, 11762.01000). If vascular structure staining is desired, animal tissues such as those from a mouse (e.g., whole mouse) can be cardiac perfused with 20 ml of PBS (without heparin) containing 0.5 mg of FITC complex-forming lectin (EY Laboratories, F-2101-5), followed by fixation with PFA. Alternatively, for PaXgene fixation, mice can be deeply anesthetized using a combination of midazolam, medetomidine, and fentanyl (MMF) (e.g., 1 mL / 100 g of mouse body weight; ip), followed by cardiac perfusion with heparinized 0.1 M PBS at room temperature for 5-10 minutes until blood is washed away (10 U / mL heparin, Ratiopharm; pressure 100-125 mmHg using a Leica Perfusion One system). After this procedure, fixation can be performed by injection of 40-50 ml of PaXgene fixation solution. If preservation of PaXgene-fixed animals is required before further processing, the tissues can be maintained in PaXgene stabilizing solution. If vascular structure staining is desired, animal tissue such as that from a mouse (e.g., a whole mouse) can be cardiac perfused with 20 ml of PBS (without heparin) containing 0.5 mg of FITC complex-forming lectin (EY Laboratories, F-2101-5), followed by fixation with PFA.Subsequently, the skin can be carefully removed, or if the animal is nude (without fur), it can be left intact, and the body can be post-fixed in 4% PFA for 1 day at 4°C and then transferred to 0.1M PBS. The method of the present invention can be started immediately, or the whole mouse can be stored, preferably in PBS at 4°C for up to 4 weeks, or in PBS containing 0.05% sodium azide (Sigma, 71290) for up to 6 months.

[0038] As used herein, terms such as "animal tissue for fluorescence microscopy" mean that the respective animal tissue is suitable for fluorescence microscopy.

[0039] Similarly, the term "heme removal" in relation to a solution refers to any solution suitable for heme removal. According to the present invention, heme removal is not limited to a specific mechanism, as long as the mechanism removes heme from tissue and / or decolorizes heme. For example, an amino alcohol suitable for heme removal, such as N,N,N',N'-tetrakis(2-hydroxypropyl)ethylenediamine, can be used, for example, as shown in a preferred embodiment. Such an amino alcohol competes with hemoglobin for heme binding and can therefore be used to remove heme from and from hemoglobin in tissue. Alternatively, benzyl peroxide can be used.

[0040] The term "fluorescent dye-containing labeling agent," when used in accordance with the present invention, is not particularly limited as long as it is suitable for labeling target molecules in decolorized fixed animal tissue, capable of binding to these target molecules, and has a molecular weight of 100 kDa or less. In preferred embodiments, the fluorescent dye-containing labeling agent is an antibody fragment complexed with the fluorescent dye, more preferably a nanobody (also known as a single-domain antibody) complexed with the fluorescent dye. Antibody fragments other than single-domain antibodies that can be used in accordance with the present invention are also known in the art and include, for example, Fab, F(ab'), monospecificity Fab2, bispecificity Fab2, scFv, bispecificity diabody and triplicity triabody, scFv-Fc, minibody, and HClG molecules.

[0041] As used herein, the term “target molecule” refers to any target molecule in a tissue. In a given application of the method of the present invention, such as a biomedical application, it is understood that an appropriate target molecule may be selected. Such a target molecule may be, for example, an endogenous molecule of an animal (e.g., a marker protein for a disease such as cancer) or a recombinant molecule, such as a recombinant protein. For example, in a preferred embodiment of the present invention, if the animal from which the animal tissue was obtained is an animal (e.g., a mouse) transplanted with cancer cells expressing such a recombinant fluorescent protein, then such a fluorescent protein may be a target molecule.

[0042] The term "labeling target molecules," as used herein, is also understood to include the possibility that two or more target molecules may be labeled by the method of the present invention. Thus, in preferred embodiments of the present invention, two or more target molecules, for example, two or three target molecules, are labeled. For example, in preferred embodiments of the present invention, if the animal from which animal tissue is obtained is an animal (e.g., a mouse) transplanted with cancer cells expressing such recombinant fluorescent protein, such fluorescent protein may be the first target molecule, and an anti-cancer biopharmaceutical (e.g., a therapeutic antibody against cancer) administered to this animal may be the second target molecule. Thus, in preferred embodiments such as this embodiment, the method for detecting metastasis and the method for analyzing the in vivo distribution of a biopharmaceutical according to the present invention can be performed together.

[0043] As used herein, the term “perfusion at pressure” refers to the pressure measurable at the tissue inlet. The pressure can be measured by any method known in the art. Preferably, the pressure is measured by a manometer, more preferably by a Kkmoon Digital Manometer Pressure Gauge Manometer (HT-1891). When using a Kkmoon Digital Manometer Pressure Gauge Manometer (HT-1891), a two-head connector (B. Braun Discofix® C Dreiwegehahn, 16494C) can be inserted into the pump infusion channel and connected to the manometer. The pump infusion channel can be set by a cardiac perfusion needle (Leica, 39471024), and the pressure can be measured at the pump infusion rate used in this method (provided the reading is stable).

[0044] Where used herein, “permeabilization solution” means a solution suitable for permeabilization of animal tissue. Such solutions are known in the art and can be readily selected by those skilled in the art, and include, for example, a surfactant suitable for permeabilization, such as Triton X-100. Preferably, the permeabilization solution used in accordance with the present invention further includes an agent suitable for extracting cholesterol from biological membranes. Such an agent suitable for extracting cholesterol from biological membranes includes, for example, methyl-β-cyclodextrin. Preferably, the permeabilization solution used in accordance with the present invention further includes an agent that loosens the collagen network, for example, trans-1-acetyl-4-hydroxy-L-proline. When a permeabilization solution is used in a particular step of the method of the present invention, it is understood that this does not exclude other solutions, for example, solutions used in a preceding step of the method, which may also contribute to and improve the permeabilization. For example, a heme removal solution may be a solution that contributes to permeabilization.

[0045] The tissue volume according to the present invention can be measured by any suitable method known in the art. Preferably, such volume can be measured by measuring the volume of liquid displaced by the tissue, for example, in a suitable cylinder.

[0046] Where used herein, "clearing solution" means a solution suitable for clearing animal tissue. Such solutions are not particularly limited, as long as they contain an organic solvent. It is understood that such organic solvents can be readily selected by those skilled in the art to be compatible with the method of the present invention, for example, based on their electromagnetic absorption / emission spectra (particularly that they lack fluorescence emission in the visible, red, and near-infrared regions). Preferably, the clearing solution containing an organic solvent has a refractive index similar to that of the animal tissue (e.g., bone), as reflected in the preferred embodiment. Such clearing solutions are particularly advantageous for clearing tissue. Examples of preferred organic solvents that can be used in such clearing solutions of the present invention include, for example, solvents containing benzyl alcohol, benzyl benzoate and diphenyl ether, solvents containing ethyl cinnamate, and solvents containing allyl cinnamate.

[0047] Methods for measuring refractive index are known in the art. The refractive index values ​​referred to herein are those measured at room temperature (i.e., 25°C) and standard atmospheric pressure (i.e., 760 mmHg).

[0048] "Further organic solvents" are not particularly limited when referring to the present invention. It is understood that such solvents can be selected by those skilled in the art so as to be suitable for dehydration. Examples of such solvents are THF, dichloromethane, and 1,4-dioxane. For example, according to the present invention, a preferred perfusion for increasing the gradient of further organic solvents from 0 vol% to 100 vol% may be perfusion with THF at a gradient of 0 vol% to 100 vol%, followed by incubation with dichloromethane. Alternatively, in all embodiments of the present invention, dichloromethane can be replaced with 1,4-dioxane.

[0049] Further improvements according to all other embodiments of the present invention can be achieved by adding a demineralization step. Such a step can further improve the transparency of the bone. The demineralization chemical is known and includes, for example, a solution containing EDTA, preferably further containing NaHCO3. Such a demineralization step is performed before the demineralization step, or, if the demineralization step is not performed, before the labeling step.

[0050] The term "present in a cell," when used herein in relation to a structure, refers to a structure present in that cell. This term does not mean that the structure must be present inside the cell, but also includes the possibility that the structure may be present on the surface of that cell.

[0051] The term "therapeutic antibody," as used herein, refers to any therapeutic antibody and therapeutic antibody fragment known in the art. Furthermore, this term is not limited to therapeutic antibodies and therapeutic antibody fragments themselves, but also includes complexes such as antibody-drug conjugates.

[0052] Where used herein, the term “low molecular weight” has meanings known in the art. Typically, the low molecular weights used in accordance with the present invention have a molecular weight of less than 900 daltons.

[0053] According to the present invention, whenever the term "contains" occurs, it may be optionally replaced with the term "consisting of".

[0054] The present invention will be described by the following non-limiting embodiments. [Examples]

[0055] Unless otherwise stated, the following methods will be used in the examples.

[0056] Mice, xenotransplantation experiments, and injection of therapeutic antibodies Female NSG (NOD / SCID / IL2 receptor gamma chain knockout) mice were obtained from Jackson Laboratory and reared at the Helmholtz Center Munich animal facility. All animal experiments were conducted in accordance with the facility guidelines of Ludwig Maximilian University of Munich and the Helmholtz Center Munich, after approval by the Ethical Review Board of the Government of Upper Bavaria (Regierung von Oberbayern, Munich, Germany). All data were reported according to the ARRIVE criteria. MDA-MB-231 breast cancer cells (Vick et al., 2015) transduced with a lentivirus expressing mCherry and enhanced with firefly luciferase were counted, filtered through a 100 μm filter, and resuspended in RPMI1640 medium. 2 × 10 cells per mouse 6 The cells were injected percutaneously into the left fourth mammary fat body at a dose of 50 μl. Tumor growth was monitored by whole-body bioluminescence measurement (photons / second) using the IVIS Lumina II Imaging System (Caliper Life Sciences) as described (Gondi et al., 2013). Briefly, mice were anesthetized with isoflurane, fixed in an imaging chamber, and imaged 15 minutes after luciferin injection (150 mg / kg; ip). Bioluminescence signals were quantified using Living Image software 4.2 (Caliper). Nine weeks after tumor cell injection, mice showing low, medium, or high metastatic signals by luminescence measurement (Figure 9) were assigned to various experimental procedures as described below, including injection of human carbonic anhydrase (CA) XII-specific antibody (6A10) (Battke et al., 2011), vascular structure staining, boosting of endogenous mCherry fluorescence, immunolabeling, and clearing. Forty-eight hours before perfusion, 20 μg of 6A10 antibody conjugated with Alexa-568 or Alexa-647 was injected into the tail vein of the mice.

[0057] Excision model NMRI nu / nu mice were obtained from the Janvier Institute, and animal experiments were conducted according to facility guidelines and approved by the Veterinary Department of the Darmstadt Regional Council in Germany. Before injecting tumor cells, the animals were anesthetized with 100 mg / kg of ketamine and 10 mg / kg of xylazine in 0.2 ml of 0.9% NaCl solution. For mammary fat body transplantation, anesthetized mice were placed supine under sterile conditions. A small incision (approximately 1 cm long) was made in the skin of the right lower abdomen to expose the mammary fat body. Then, 4 × 10⁶ MDA-231-Br cells were injected per animal. 6 A 50 μl suspension of tumor cells containing [number] cells was injected into the mammary fat body (Yoneda et al., 2001), and the wound was closed with a Michel clip. To estimate the tumor volume, it was measured using calipers, and the volume was given by the following formula: Vt = (a × b 2 Calculated according to ) / 2, where a represents the tumor length (longest measurement) and b represents the tumor width measured perpendicular to a. At the end of the experiment (tumor volume is 2 cm 3 If the condition reached a certain point, or if other symptoms requiring cessation of treatment were observed (e.g., cachexia, ulcer formation), the animals were sacrificed under deep anesthesia and perfused with 4% PFA. In the tumor excision experiment, the animals were anesthetized as described above, and under sterile conditions, the skin was incised approximately 2-3 mm away from the tumor. To further expose the tumor, the skin covering it was gently opened. The major arteries supplying the tumor were then identified and closed by thermal coagulation using a cauterization unit. To achieve complete removal of the tumor, the surrounding mammary fat pad was cut at least 2 mm away from the tumor. Any minor bleeding observed during tumor excision was stopped by thermal coagulation. The skin wound was then closed with a Michel clip. Postoperatively, the animals were administered an analgesic (Rimadyl) for one week and observed for the following weeks. At the end of the experiment, the animals were sacrificed under deep anesthesia and perfused with 4% PFA.

[0058] Perfusion and tissue treatment Mice were deeply anesthetized using a combination of midazolam, medetomidine, and fentanyl (MMF) (1 mL / 100 g of mouse body weight; ip). They were then cardiacly perfused with heparinized 0.1 M PBS at room temperature for 5–10 minutes until blood was washed away (10 U / mL heparin, Ratiopharm; pressure 100–125 mmHg using a Leica Perfusion One system) until the mice died. Afterward, they were fixed with 4% paraformaldehyde (PFA) in 0.1 M PBS (pH 7.4) (Morphisto, 11762.01000) for 10–20 minutes. For vascular structure staining, mice were cardiacly perfused with 20 ml of PBS (heparin-free) containing 0.5 mg of FITC complex-forming lectin (EY Laboratories, F-2101-5), followed by fixation with PFA. In NGS mice, the skin was carefully removed, and the body was post-fixed in 4% PFA for 1 day at 4°C before being transferred to 0.1M PBS. NMRI nu / nu mice were post-fixed in the same manner without skin removal. The LuCiD pipeline was started immediately, or the whole mouse body was stored in PBS at 4°C for up to 4 weeks, or in PBS containing 0.05% sodium azide (Sigma, 71290) for up to 6 months.

[0059] uDISCO whole body transparency The uDISCO protocol for clearing the entire body of a mouse was already described in detail in the reference (Pan et al., 2016). Briefly, a transcardiac circulatory system including a peristaltic pump (ISMATEC, REGLO Digital MS-4 / 8 ISM 834; reference tube, SC0266) was installed. Two channels were set up from the pump to circulate through the heart and into the vascular structure. Specifically, the first channel was used to pump the clearing solution into the mouse body, and the second channel was used to collect the solution exiting the mouse body and return the solution to the original bottle for recirculation. For the outflow tube of the first channel, which injected the solution into the heart, a perfusion needle (Leica, 39471024) was connected to the tube using the tip of a syringe (cut from a 1 ml syringe, Braun, 9166017V). Meanwhile, the inflow tube of the second channel, which recirculated the clearing solution, was fixed to a glass chamber containing the mouse body. The amount of circulating solution depended on the capacity of the clearing glass chamber. For example, if the maximum capacity of the glass chamber was 400 ml, 300 ml of solution was used for circulation.

[0060] All clearing procedures were performed in a fume hood. First, the mouse body was placed in a glass chamber, and a perfusion needle was inserted into the heart through the same hole used for PFA perfusion. Next, the chamber was covered with aluminum foil, and transcardiac circulation was started at a pressure of 230 mmHg (60 rpm with an ISMATEC pump). The mouse bodies were perfused twice for 6 hours with tert-butanol in the following gradients: 30 Vol%, 50 Vol%, 70 Vol%, 90 Vol% (in distilled water), and 100 Vol%. Finally, they were perfused for at least 6 hours with BABB-D4, a refractive index-matched solution containing 4 parts BABB (benzyl alcohol + benzyl benzoate 1:2, Sigma, 24122 and W213802), 1 part diphenyl ether (DPE) (Alfa Aesar, A15791), and 0.4% Vol vitamin E (DL-alpha-tocopherol, Alfa Aesar, A17039), until body clarity was achieved. Since the melting point of tert-butanol is 23-26°C, a heating mat set to 35-40°C was used for two rounds of circulation with 100% tert-butanol to prevent the solution from solidifying.

[0061] Decolorization and systemic immunohistochemistry The following nanobodies and dyes were used for whole-body immunostaining: Atto647N complex-forming anti-RFP / mCherry nanobooster (Chromotek, rba647n-100), Atto594 complex-forming anti-RFP / mCherry nanobooster (Chromotek, rba594-100), Hoechst 33342 (Thermo Fisher Scientific, 21492H), propidium iodide (PI, Sigma, P4864), and FITC complex-forming lectin (EY Laboratories, F-2101-5).

[0062] To remove residual blood and heme after PFA perfusion, animals were subjected to a round of perfusion with a destaining solution followed by immunostaining. The destaining solution was prepared by diluting CUBIC Reagent 1 in 0.1 M PBS at a 1:3 ratio (Susaki et al., 2014). CUBIC Reagent 1 was prepared by mixing 25 wt% urea (Roth, 3941.3), 25 wt% N,N,N',N'-tetrakis(2-hydroxypropyl)ethylenediamine (Sigma, 122262), and 15% Triton X-100 (AppliChem, A4975, 1000) in 0.1 M PBS. The same transcardiac circulation system, including the peristaltic pump used in uDISCO, was installed to perfuse mice during the destaining and immunostaining steps, except that a 0.20 μm syringe filter (Sartorius 16532) was connected to the tubing to efficiently prevent the aggregation of dye aggregates in the sample and maintain the high pressure of pump injection at approximately 230 mmHg. After post-fixation and washing with PBS, the mice were perfused overnight with 0.1 M PBS at room temperature, and then perfused with destaining solution at room temperature for 2 days, changing the solution every 12 hours. Subsequently, the animals were perfused at room temperature for 2 days with 300 mL of immunohistochemical solution containing 1.5% goat serum (Gibco, 16210072), 0.5% Triton X-100, 0.5 mM methyl-beta-cyclodextrin (Sigma, 332615), 0.2% trans-1-acetyl-4-hydroxy-L-proline (Sigma, 441562), 0.05% sodium azide (Sigma, 71290), 25 μL of nanobooster (stock concentration 0.5-1 mg / mL), 1 mg of lectin-FITC, and 350 μL of 10 μg / mL of Hoechst and / or propidium iodide (stock concentration 1 mg / mL) in 0.1 M PBS. Next, the mice were perfused twice for 12 hours with a washing solution (1.5% goat serum, 0.5% Triton X-100, and 0.05% sodium azide in 0.1M PBS) at room temperature, and finally perfused twice for 12 hours with 0.1M PBS at room temperature.

[0063] 3DISCO whole body transparency In whole-body passive clearing using 3DISCO, mice were incubated under dehydration conditions at room temperature, with the clearing solution gently circulated in a glass chamber and maintained at the top of a shaking oscillator (IKA, 2D digital) in a fume hood, thereby ensuring sufficient tissue clearing. For dehydration, the mouse bodies were incubated in 300 ml of tetrahydrofuran THF (Sigma, 186562) in distilled water with the following gradients: 50 Vol% THF, 70 Vol% THF, 80 Vol% THF, 100 Vol% THF, and again 100 Vol% THF (6-12 hours for each step). The mice were then incubated in dichloromethane (Sigma, 270997) for 1 hour, and finally in BABB. Throughout all incubation steps, the glass chamber was sealed with Parafilm and covered with aluminum foil.

[0064] Rehydration and immunohistochemical staining of cleared metastatic tissue after LuCiD. The following antibodies were used for tissue rehydration: anti-firefly luciferase (dilution 1:2000, Abcam, ab21176), anti-panendothelial cell antigen MECA-32 (dilution 1:25, BD Biosciences, 550563), anti-alpha smooth muscle actin (αSMA) (dilution 1:500, SIGMA, C6198), AlexaFluor 568 goat anti-rat IgG (H+L) (dilution 1:400, Life Technologies, A11077), AlexaFluor 647 goat anti-rabbit IgG (H+L) (dilution 1:400, Life Technologies, A21245), AlexaFluorPlus 555 goat anti-rabbit IgG (H+L) (dilution 1:400, A32732), AlexaFluor 488 goat anti-rabbit IgG (H+L) (dilution 1:400, Life Technologies, Inc. (A11034)

[0065] After identifying metastases in the lungs of LuCiD-treated mice, lung tissue was dissected and rehydrated by applying tert-butanol in a reverse gradient (6 hours each at 37°C with gentle shaking), as used for uDISCO clearing: twice with 100 Vol%, and twice with 90 Vol%, 70 Vol%, 50 Vol%, 30 Vol%, and 0.1 M PBS at room temperature. The rehydrated samples were incubated for 1 day at 37°C in 0.1 M PBS containing 0.2% gelatin, 0.5% Triton X-100, 0.05% sodium azide, and 5% normal goat serum (Belle et al., 2014). Subsequently, 1 mm sections were incubated overnight at 37°C with primary antibody diluted in the same solution, washed twice with PBS, and incubated for 4 hours at 37°C with secondary antibody.

[0066] Imaging using epifluorescence stereomicroscopy The entire body of the cleared mouse was fixed in the original clearing chamber and imaged using a Zeiss AxioZoom EMS3 / SyCoP3 fluorescence stereomicroscope with a 1× long working distance air objective lens (Plan Z 1×, 0.25NA, working distance (WD) = 56 mm). The magnification was set to 7×, and the imaging area was manually selected to target the entire mouse body. Images were acquired using GFP, RFP, and Cy5 filters, and the files were exported as RGB images, which were then combined using Adobe Photoshop CS6. For high-magnification images, scans were performed at up to 112× using the same microscope.

[0067] Light sheet microscopy imaging Single-plane illumination (light sheet) image stacks were acquired using an Ultramicroscope II (LaVision BioTec) with a 4 μm axial resolution. For whole-body screening of tumors and antibody signals at low magnification, an Olympus 1× air objective lens connected to an Olympus MVX10 zoom body was used. This resulted in a reduction and magnification range of 0.63× to a maximum of 6.3×. Using the 1× objective lens, a 2×2.5 cm field of view was imaged, targeting the entire width of the mouse body. Tile scans with 60% overlap along the longitudinal y-axis of the mouse body were acquired from the ventral and dorsal sides at a maximum depth of 13 mm, targeting the entire body volume using a 10 μm z-step. The exposure time was 150 minutes, the laser power was 3-4 mW (70%-95% power level), and the light sheet width was maintained at maximum. After tile imaging of the sample across the entire field of view, the scanned area was sectioned using a thin dental electric blade (0.2 mm) (Dremel, 8200) and further imaged. After low-magnification imaging of the whole body, individual organs (including lungs, liver, kidneys, brain, spleen, intestines, and bones) were sectioned and individually imaged using high-magnification objective lenses (Olympus 4× / 0.28NA [WD=10mm] and Zeiss 20× / 0.1NA [WD=4=mm]) connected to an Olympus rotating zoom body unit (U-TVCAC) maintained at 1×. High-magnification tile scans (4×4) with 20% overlap were acquired, and the light sheet width was reduced to obtain maximum illumination in the field of view while maintaining the same NA.

[0068] Confocal microscopy imaging Cleared samples, such as cut tissue, organ fragments, or entire organs, were placed on a 35 mm glass-bottomed Petri dish (MatTek, P35G-0-14-C), and then covered with one or two drops of refractive index-matching solution, such as BABB or BABB-D14. This mounting chamber did not need to be sealed. Samples were imaged using an inverted laser scanning confocal microscope system (Zeiss, LSM 880) with a 40× oil immersion lens (Zeiss, EC Plan-Neofluar 40× / 1.30 Oil DIC M27) ​​and a 25× water immersion length working distance objective lens (Leica, NA 0.95, WD=2.5 mm). The latter lens was mounted on a specially designed mounting thread. The z-step size was 1–2.50 μm.

[0069] Reconstruction of a full-body mouse scan Reflected fluorescence (2D composite image of the entire mouse body): The collected epifluorescence images were semi-automatically combined using Adobe Photoshop's photomerge function (File\automate\photomerge). Various channels were combined separately and then integrated in Adobe Photoshop to create a composite image.

[0070] Light sheet microscope (3D synthesis of the entire mouse body): A stack of light-sheet microscope images using ImSpector (LaVision BioTec GmbH) was acquired individually for each channel as 16-bit grayscale TIFF images. The stacks were initially aligned and then joined together using Vision4D (Arivis AG). Further image processing was performed mainly with Fiji (ImageJ2): first, the autofluorescence channel (imaging at excitation 488) was used to equalize the general outline of the mouse body. Organs were manually segmented by defining regions of interest (ROI). Data visualization was performed using Amira (FEI Visualization Sciences Group), Imaris (Bitplane AG), and Vision4D, for both volumetric measurements and maximum intensity projection color mapping.

[0071] Quantification Plots of fluorescence and normalized fluorescence signal profiles: By calculating fluorescence signal profiles plotted across the maximum peak, we were able to better compare deep tissue optical imaging between far-red channels beyond red and especially green channels due to less scattering, autofluorescence, and light absorption in the far-red channels. Identical z-stacks of liver samples were acquired by lightsheet microscopy at three different channels (excitation 470 nm, 561 nm, and 647 nm), and then the identical z-planes acquired from various channels were opened in Fiji software. A straight line was drawn across the organ from one side to the other in each channel, and the signal intensity profile defined by the line was measured. All values ​​in the plot were then normalized as a percentage of the maximum peak in the profile, and these are shown as representative line graphs (Figure 1D).

[0072] To compare the reduction in background and the improvement in signal relative to the background ratio (SBR) when the imaging channel is operated in the far-red or near-infrared range, lung metastases expressing mCherry were imaged at excitation 545 / 561 nm, lung metastases labeled with anti-mCherry nanobodies complexed with Atto594 were imaged at excitation 590 nm, and lung metastases labeled with anti-mCherry nanobodies complexed with Atto647N were imaged at excitation 640 nm (n=9 tumors per experimental group, consisting of 3 animals per imaging method). First, fluorescence signal profile plots were calculated for all such metastases. That is, tumor z-plane images were acquired by lightsheet microscopy, opened in Fiji, and a straight line of approximately 300-350 pixels was drawn across the tumor, including the surrounding tissue region considered as background. The signal profile was measured from the straight line defining the signal profile, and all values ​​from the plots obtained from representative animals for each experimental group are shown in a representative line graph (Figure 1G). Finally, the normalized plot shown in Graph Figure 1H was calculated by normalizing the lung metastasis plots obtained as described above against the mean signal intensity of each surrounding background.

[0073] In Figures 10C to 10D, to compare the signal-to-background ratio (SBR), samples were labeled with anti-mCherry nanobodies complexed with Atto647N, and primary tumors were imaged at excitation wavelengths of 470 nm, 561 nm, and 640 nm, respectively. Fluorescence signal intensity profiles and background-normalized profiles were plotted for each channel using the same strategy as described above.

[0074] Tumor size and binding of therapeutic antibodies: To quantify tumor size and number in large datasets, the inventors developed a custom-made Python script. This script divides the data into small 3D cubes (chunks) and processes them separately. Each separate chunk was then quantified using Fiji. Due to the nonlinear relationship between data size and processing speed (processing speed decreases significantly as data size increases), smaller datasets result in faster processing. Furthermore, various data packages can be distributed across multiple processors and managed simultaneously to further accelerate overall processing. The outputs of the parallel processors were integrated using the Python script. Among many, the final results included measurements of tumor volume, surface, radius, and coordinates. To quantify the number of cells in each tumor, the size of several isolated single cells (approximately 1700 μm) was measured. 3 The total number of metastases was estimated based on volumetric interpolation. The accuracy of the estimation was confirmed by the number of nuclei (labeled with PI or Hoechst) within the micrometastases. The tumor bar graph was created using Python (Matplotlib). This shows the frequency of metastasis by the same number of cancer cells. To analyze the in vivo distribution of therapeutic antibodies, the tumor channels, which were segmented into regions, were first applied as masks to the antibody channels, and specific signals were segmented into regions. This allowed the inventors to quickly determine the ratio of micrometastases targeted by therapeutic antibodies to those that were not targeted. To determine antibodies that did not target the tumor, signals that appeared only in antibody channels but not in tumor channels were segmented into regions.

[0075] Example 1: Systemic immunolabeling of adult mice The inventors aimed to develop an immunohistochemical staining method for labeling cancer cells expressing commonly used fluorescent proteins throughout the entire adult mouse body. The inventors determined that nanobodies, due to their small size (12-15 kDa compared to approximately 150 kDa for conventional antibodies), could be optimal for achieving complete immunolabeling throughout the adult mouse body (Muyldermans, 2013; Yang et al., 2014). To deliver nanobodies (referred to as nanoboosters) complexed with bright Atto dyes throughout the body, the inventors developed a permeabilization solution containing Triton X-100, methyl-β-cyclodextrin (for extracting cholesterol from biological membranes), and trans-1-acetyl-4-hydroxy-L-proline (for loosening the collagen network) (Hama et al., 2015). The inventors circulated this permeabilization solution throughout the entire body of mice using a high-pressure peristaltic pump (230 mmHg compared to standard mouse cardiac perfusion of 80–150 mmHg) (Gage et al., 2012; Ghanavati et al., 2014). This facilitated the delivery of the nanobooster to deep tissues via the cardiovascular system. To further reduce background in organs with high blood concentrations, such as the liver, the entire body of the mice was treated with amino alcohol before the systemic immunolabeling step (Tainaka et al., 2014). After clearing the mice, the intact bodies were imaged using standard epifluorescence and light-sheet microscopy. The collected images were combined to unbiasedly visualize the target cells in the intact see-through mice (Figure 1A).

[0076] The inventors chose to boost signals originating from cancer cells using nanobodies complexed with far-red dyes because light passing through biological tissue is not scattered as much in spectra such as far-red (Hong et al., 2017) and can reach the internal core of large organs such as the liver without being blocked on the surface (Figure 1B, Figure 1C). To utilize LuCiD in tumor metastasis, human breast cancer cells (MDA-MB-231 cells expressing mCherry and firefly luciferase) were transplanted into the mammary fat pads of NOD scid gamma (NSG) female mice, and tumors were allowed to grow and metastasize for more than 6–10 weeks (Figure 7A) (Gondi et al., 2013; lorns et al., 2012). The animals were then perfused transcardiacally using standard PFA fixation before application of the LuCiD protocol. First, endogenous mCherry signaling in tumor cells was boosted with anti-mCherry nanobodies complexed with Atto-594 or Atto-647N dyes, and the fluorescence signal was then evaluated in clear mice. The inventors found that the nanobooster specifically labeled tumor cells and enhanced the signal-to-background ratio by up to 20 times compared to the endogenous mCherry signal (Figures 1D-1H and 7B-7G). Due to the significant enhancement of signal intensity and the advantages of imaging in the far-red region, light penetrated deep tissues with little scattering, and tumor micrometastases were easily detected even in deep tissue regions such as the central brain (Figure 1I, arrowhead). To further confirm the specificity of LuCiD systemic immunolabeling, the following control experiment was performed: 1) Control mice were stained without tumor transplantation, resulting in no mCherry expression, and no labeling was found in any of the analyzed organs (Figure 8A). 2) Clear tissue derived from the primary tumor and lung metastases were rehydrated and stained again using a specific anti-luciferase antibody, confirming that endogenous mCherry fluorescence colocalized with both the nanobooster and the luciferase signal (Figures 7B-7D and 8B).

[0077] Example 2: Detection of cancer metastasis at the single-cell level in see-through mice Many preclinical studies on cancer metastasis utilize mouse models that observe cancer cell proliferation in the primary site and distal body regions using methods such as magnetic resonance imaging (MRI), computed tomography (CT), positron emission tomography (PET), single-photon emission computed tomography (SPECT), or ultrasound and bioluminescence imaging (Condeelis and Weissleder, 2010; Massoud and Gambhir, 2003, 2007; Ntziachristos, 2010; Pichler et al., 2008; Timpson et al., 2011). While such methods provide important long-term information on the size of primary tumors and large metastases, they typically resolve structures larger than 50 μm (approximately 75 cells) and therefore lack the resolution to detect smaller micrometastases consisting of fewer cells. The detection of smaller tumor cell populations is significant because they may represent dormant cancer cells or initial metastatic nodules; therefore, the inventors used LuCiD to test for cancer metastasis throughout the mouse body. To compare LuCiD with conventional imaging methods, bioluminescence images of mice transplanted with human breast cancer cells were obtained prior to LuCiD to determine the level of detectable tumor metastasis. Mice with low or high tumor loads were analyzed based on bioluminescence imaging as follows: Mice with no metastasis (Figure 9A) or metastasis limited to only one body region fell into the low tumor load category (Figure 9C), while mice with metastasis in two or more body regions fell into the high tumor load category (Figures 2A, 2B, and 9E, 9G). Consistent with previous findings using injection of MDA-MB-231 cells into the mammary fat body, the inventors consistently detected early, large metastases in the axillary lymph nodes by bioluminescence (Figure 9) (lorns et al., 2012). After bioluminescence determination, LuCiD was applied as follows: The entire mouse body was fixed with PFA, destained, immunolabeled with an anti-mCherry nanobooster complexed with Atto-647N, and cleared using the DISCO systemic method (Pan et al., 2016).First, the entire body of a clear mouse with high tumor load was visualized using a standard epifluorescence stereomicroscope (Figures 2C-2H). As expected, both the primary tumor (Figure 2G) and major metastases in the axillary lymph nodes (Figure 2E) were readily visible and could also be detected by bioluminescence imaging, although this lacked information about actual size and shape as a bulk signal (Figures 2A, 2B). In contrast, LuCiD also enabled visualization of several micrometastases in the lungs using conventional epifluorescence imaging, which could not be seen with bioluminescence (compare the areas marked with red arrowheads in Figure 2F and purple squares in Figures 2A and 2B). Thus, epifluorescence imaging can be completed within minutes and already provides greater detail and sensitivity compared to bioluminescence imaging (Figure 9).

[0078] Next, high-resolution 3D images were collected from intact mice using a single-plane laser scanning light-sheet microscope (Pan et al., 2016) to detect whole-body micrometastases in high-tumor-burden mice. The scanned tiles were joined, and the tumors were segmented. In the thoracic region, various metastases were observed not only in the lungs (segmented regions in Figures 2I and 2J) and lymph nodes, but also in the base of the neck and surrounding tissues (Figures 2I to 2N). Importantly, scanning with the light-sheet microscope allowed us to visualize micrometastases down to the single-cell level in the bodies of intact mice (Figures 2O to 2Q). Next, low-tumor-burden mice were imaged using LuCiD. Again, epifluorescence imaging has already shown further details of tumor metastases (Figures 3C to 3F) compared with bioluminescence imaging (Figures 3A and 3B). Using a light-sheet microscope in low-tumor-burden mice, large micrometastases were detected in the axillary lymph nodes, and hundreds of micrometastases of various sizes were detected throughout the body, particularly in the lungs (Figures 3G-3J). By magnifying the lungs of intact mice, the inventors were able to image the metastases at the cellular level and extract information about their size and spatial location (Figures 3K, 3L). For example, in the lungs shown in Figures 3I-3L, approximately 1490 micrometastases were detected, 62.2% of which were smaller than 75 cells and could not be imaged by standard methods including bioluminescence. Interestingly, the micrometastases, regardless of their size, were randomly distributed throughout the lungs, suggesting that they colonized independently in multiple locations. To demonstrate the applicability of the LuCiD pipeline in different tumor models, a primary mammary lipoma was used in a 1.5 cm² tumor. 3The tumors were resected after reaching a volume less than 75 cells, and the mice were observed for approximately 8 weeks. This resection model more closely resembles the clinical environment, is not limited by the growth of the primary tumor, and provides additional time for metastases to propagate and grow. In fact, the number of metastases in this model increased fourfold compared to animals that were not resected (Figure 12, Figure 3I). Interestingly, the inventors found that 88.5% of the micrometastases in the lungs were smaller than 75 cells (Figure 12), suggesting that the novel micrometastases persistently re-disseminated, possibly by circulating tumor cells. Thus, LuCiD reveals the cytological details of tumor metastases throughout the body in intact mice.

[0079] Example 3: Determination of therapeutic antibodies using LuCiD Monoclonal antibodies targeting key tumor cell antigens represent some of the most promising oncologies to emerge in the last 20 years. Numerous tumor-targeted monoclonal antibodies have become part of the standard of care for various solid and hematological malignancies, and many more are in early or late clinical development (Pandey and Mahadevan, 2014). Typically, therapeutic antibodies are produced against tumor-associated antigens overexpressed by cancer cells. When such antibodies are injected, they distribute throughout the body and target cancer cells. However, to date, there is no method to determine the distribution of therapeutic antibodies throughout the body at cell resolution. Here, the inventors used LuCiD to determine the in vivo distribution of the monoclonal therapeutic antibody 6A10 against human CA12 (Battke et al., 2011). CA12 is overexpressed in various types of cancer, and blocking its activity with antibody 6A10 reduces tumor growth (Gondi et al., 2013). 6A10 complexed with Alexa-568 (which has a tumor signal boosted by Atto-647N) or 6A10 complexed with Atto-647N (which has a tumor signal boosted by Atto-594) was injected intravenously at 20 μg 9 weeks after transplantation of MDA-MB-231 cells, and mice were perfused 2 days after antibody injection for LuCiD analysis. Because the excitation / emission spectrum of Alexa-568 overlaps with the endogenous mCherry signal of cancer cells, the inventors completely quenched the endogenous mCherry signal in mice injected with 6A10 antibody complexed with Alexa-568 using an improved 3DISCO protocol, namely a combination of destaining + immunostaining + 3DISCO clearing (Figure 10) (Pan et al., 2016). To reiterate, endpoint bioluminescence imaging in mice showed detectable signals only in the primary tumor and axillary lymph nodes of low tumor-burden mice (Figures 4A and 4B). To determine whether LuCiD could simultaneously image both tumor cells and the in vivo distribution of the therapeutic antibody 6A10, the inventors first used epifluorescence microscopy to observe the accumulation of antibody 6A10 in the primary tumor (Figures 4C and 4G) and metastases in the axillary lymph nodes (Figures 4C and 4D).Focusing on the lungs, it was possible to observe micrometastases targeted by antibody 6A10 (Figure 4E, white arrows) and other micrometastases not targeted by antibody 6A10 (Figure 4F, arrows). Next, high-resolution light-sheet microscopy images of tumor metastases and the complex-forming antibody 6A10 were acquired throughout the body of intact see-through mice to determine the complete distribution of targeted tumors (Figures 4H-4M). The inventors found that in the kidneys and brain, all tumor micrometastases were targeted by 6A10, while in the liver and lungs, only 75% and 81% were targeted, respectively (Figures 5A-5D, Figure 12). In addition, 5% of antibody binding sites in the kidneys, 27% in the liver, and approximately 40% in the lungs and brain were nonspecific (non-tumor-carrying host tissue) (Figure 5E). Overall, this analysis demonstrates that the method of the present invention provides a robust platform for tracking the in vivo distribution of therapeutic antibodies and micrometastases in the bodies of intact mice.

[0080] Example 4: 3D visualization of tumor vascular structure and determination of tumor microenvironment phenotype. Both primary tumors and metastases rely on blood vessels for oxygen and metabolite supply. In addition, tumor vascular structures provide conduits for disseminating tumor cells distally, for influxing immune cells into the tumor, and for delivering therapeutic agents (Welti et al., 2013). Furthermore, vascular angiocrine signaling can form specialized vascular niches that control diverse aspects of tumor biology, including proliferation, invasion, self-renewal, or treatment resistance (Butler et al., 2010). Using LuCiD, the inventors characterized metastatic tumor vascular structures by simultaneous visualization of tumor cells, therapeutic antibodies, and blood vessels in intact mice. Whole-body imaging of low tumor-bearing mice using light-sheet microscopy identified primary tumors and distal micrometastases from major metastatic sites (Figures 6A, 6B). The inventors then collected high-resolution light-sheet microscopy images from intact mice to visualize vascular structures in metastatic regions (Figures 6C-6F). The inventors also found that the therapeutic antibody reached the majority of tumor micrometastases in this vascularized region (Figures 6D-6F).

[0081] To further characterize the small micrometastases detected throughout the body, it may be important to perform immunolabeling on the target region defined after the LuCiD pipeline. To achieve this objective, metastatic lung tissue imaged by LuCiD was rehydrated by reversing the uDISCO protocol. Subsequently, these tissues were immunostained using alpha-smooth muscle actin (αSMA) (Figures 6G-6J), a cancer-associated fibroblast marker, and MECA-32 (Figures 6K-6N), a vascular endothelial cell marker. These results demonstrate that with LuCiD, further phenotypic determination of selected tissues is possible through antibody labeling.

[0082] Example 5: The method of the present invention can be used to determine the details of neurodegeneration throughout the CNS of a mouse. To test chronic neurodegeneration throughout the CNS in mice, the inventors induced traumatic brain injury (TBI) in a Thy-1 GFP-M mouse strain in which a subset of neurons express GFP. The inventors used a closed-head (intact skull) TBI mouse model to better mimic human trauma cases compared to an open-skull model. TBI was induced in the somatosensory-motor cortex of mice using a controlled cortical impact (CCI) model ("Impact One" device, Leica). This allows for the induction of highly reproducible, mild and moderate TBI, as the inventors recently described (Erturk et al., 2016). The following parameters were used: pistol velocity 6.5 m / s, impact duration 350 milliseconds, and impact depth 2.0 mm. This TBI fractures the skull and induces holes visible on MRI within 2-3 weeks. Two months after TBI, the inventors perfused mice and applied a LuCiD pipeline to boost GFP signaling throughout the mouse body using anti-GFP nanobodies complexed with Atto647N. Subsequently, the intact central nervous system of the mice was imaged using a light-sheet microscope. The inventors discovered degeneration of pyramidal motor axons descending from the brain to the spinal cord (Figure 13). Axonal degeneration was evident in the fragmentation (vesicle formation) in the enclosed region compared to the spinal cord of non-lesion mice (Figure 13E) (Figures 13B-13D). While similar experiments can be performed by visualizing endogenous GFP without boosting (Pan et al., 2016), it should be noted that boosting GFP provides: 1) more detail compared to endogenous GFP due to the brighter dye; 2) a more stable GFP signal (without fading over time) that provides detail in high-resolution images that can be acquired by long-term high-magnification scans; and 3) boosting in the far-red channel using anti-GFP nanobodies tagged with A647N facilitates imaging of samples in the far-red channel, which exhibits less autofluorescence compared to the green (488 nm) channel.

[0083] Example 6: The present invention can be used to analyze the presence of chronic neuroinflammation throughout the CNS of mice. Inflammation is thought to have multiple manifestations, involving both repair and destruction, depending on the time elapsed since injury, the types of immune cells involved, and the location of the tissue. In addition to knowing the target inflammatory cell types and their course (acute, chronic, or both), it is generally necessary to know the precise locations where they should be targeted. The latter challenge remains largely unclear.

[0084] Our data using LuCiD demonstrate the development of neuroinflammation not only in the brain but throughout the entire spinal cord (Figure 14). Here, we induced closed head TBI in CX3CR1-GFP mice as described above (Figure 13), in which case macrophages / microglia and some monocytes were labeled with GFP. Four months after TBI, the animals underwent systemic LuCiD using anti-GFP nanobody labeling to enhance the GFP signal. Note that the activation of immune cells along the descending motor axons is evident, as seen in the increased signal intensity and cell number related to motor pathway degeneration (Figure 13). It is also worth mentioning that we were able to label immune cells using nanobodies against endogenous proteins of immune cells. These findings suggest that TBI induces a widespread chronic inflammatory response, which is thought to encompass not only the damaged brain region but also the spinal cord and potentially peripheral organs. The study suggests that infiltration of blood-derived immune cells exacerbates disease progression (Wilson et al., 2010). Our own data shows that lymphocytes are chronically present in the brains of injured mice, even several months after the injury (Erturk et al., 2016). How these lymphocytes find their way to the brain (i.e., to persist there) after such a long period remains a mystery.

[0085] Example 7: The method of the present invention can be used to test meningeal lymphatic vessels in a whole transparent mouse. The lymphatic system, associated with various lymphatic organs in the body, is crucial for the immune response (Janeway et al., 1997). It circulates lymphocytes and other leukocytes throughout the body. Until recently, the brain was thought to lack any lymphatic connections (Ransohoff and Engelhardt, 2012). Recently, meningeal lymphatic vessels lining the dural sinuses between the mouse skull and brain were discovered (Louveau et al., 2015). Such meningeal vessels transport immune cells (Louveau et al., 2015), which potentially have a tendency to invade the brain. However, it is unclear how such meningeal lymphatic vessels in the brain may contribute to the pathogenesis of TBI (or other diseases). Because cerebral lymphatic vessels are located directly between the skull and brain, they are destroyed when the brain is retrieved for standard histological diagnosis. Therefore, they can only be tested in intact heads, such as when using LuCiD technology. Using various labeling methods, the inventors demonstrated that LuCiD enables imaging of meningeal lymphatic vessels in the heads of intact mice (Figure 14). Generally, meningeal lymphatic vessels can be labeled by 1) a tracer such as ovalbumin-Alexa647 injected into the cisterna magna or nasal cavity (then discharged into the lymphatic vessels to specifically label them) (Figure 14A), 2) the use of a gene-transformed reporter mouse such as VEGFR3-YFP mouse (Calvo et al., 2011) (Figures 14B-14E), and 3) antibody labeling of the heads of intact mice using a specific marker such as Lyve-1 (Louveau et al., 2015). Subsequently, the target cells / molecules in the heads of intact mice can generally be imaged. For example, the inventors successfully imaged immune cell populations in CX3CR1-EGFP mice (Figure 14F) and CX3CR1-EGFP × CCR2-RFP dual-gene transgenic mice (Figure 14G).

[0086] Example 8: Screening of decolorizing amino alcohols using mouse blood and spleen To identify additional solutions for decolorization, we first tested whether amino alcohols could directly decolorize blood. Eleven different amino alcohols were mixed with blood from PFA-fixed mice and centrifuged. A colorless pellet indicates that the red heme has been decolorized (Figure 16, panel (a)). Therefore, we further examined the decolorizing effect of eight good candidates using mouse spleens. All mouse spleens were decolorized, as shown in Figure 16, panel (b). Images after 0 hours and 24 hours of incubation with the indicating amino alcohol are shown, respectively. The amino alcohols shown in the figure were as follows: 1. Quadrol, 2. N-butyldiethanolamine, 3. N-methyldiethanolamine, 4. N,N-dimethylmethyleneiminium chloride, 5. 1,3-bis(dimethylamino)-2-propanol, 6. 4-(2-hydroxyethyl)morpholine, 7. N-tert-butyldiethanolamine, 8. N-ethyldiethanolamine, 9. 2-(diisopropylamino)ethanol, 10. 4-methylmorpholine N-oxide, and 11. 2-(dibutylamino)ethanol.

[0087] Example 9: Clearing of porcine brain and clearing and labeling of large human brain samples. The method of the present invention has also been applied to the entire organs of large animals, including pigs and humans.

[0088] In particular, the method according to the present invention was used for decolorizing and clearing porcine brains. Figure 17(a) shows a dissected fresh porcine brain, and Figure 17(b) shows a decolorized porcine brain after 24 hours of incubation with 2-(diisopropylamino)ethanol. Finally, Figure 17(c) shows the transparency of the porcine brain after clearing. These results demonstrate that the decolorization and clearing steps of the method of the present invention can be applied to large animals, including pigs.

[0089] Next, the method according to the present invention was also used to clear and label a large sample of human brain. In particular, Figure 18(a) shows the transparency of a human brain measuring 3 cm × 3 cm × 1 cm after clearing. Figure 18(b) shows a fluorescence confocal image showing plaques and cells in the cleared human brain. These results demonstrate that clearing and labeling by the method of the present invention is applicable to large samples of human brain, and that such cleared and labeled samples can be analyzed by fluorescence microscopy. The inventors also experimentally found that it is possible to decolorize and clear the entire human brain. Furthermore, it is expected that fluorescence imaging can be applied to imaging the entire human brain prepared according to the present invention. [Industrial applicability]

[0090] The methods and products of the present invention are industrially applicable and can be used, for example, in the testing of biopharmaceuticals such as therapeutic antibodies.

[0091] References [Table 1] [Table 2] [Table 3] [Table 4]

Claims

1. A method for analyzing animal tissue, wherein the animal tissue is obtained by a method for preparing animal tissue for use with a fluorescence microscope, and the method for preparing the animal tissue comprises the following steps: a) A step of decalcifying fixed animal tissue with a decalcification solution, b) A step of decolorizing the fixed animal tissue with a heme removal solution, c) A step of obtaining fixed animal tissue labeled with the fluorescent dye-containing labeling agent by labeling a target molecule in the fixed animal tissue with a labeling solution containing a fluorescent dye-containing labeling agent capable of binding to the target molecule, wherein the labeling agent has a molecular weight of 100 kDa or less, The fixed animal tissue is treated with a permeation solution before the labeling of the target molecule in step c), and the permeation solution and the labeling solution are different solutions. Alternatively, the fixed animal tissue is treated with a permeation solution during the labeling of the target molecule in step c), and the permeation solution and the labeling solution are the same solution, and d) A step of clearing the fixed animal tissue labeled with the fluorescent dye-containing labeling agent with a clearing solution containing an organic solvent to obtain the animal tissue for use with a fluorescence microscope. Includes, The animal tissue contains the target molecule labeled with the fluorescent dye-containing labeling agent, The method for analyzing the animal tissue includes the step of analyzing the tissue using a fluorescence microscope in order to detect the fluorescence of the fluorescent dye in the animal tissue, The method for analyzing the animal tissue further comprises, after step i), iii) a step of cleaving a tissue region of interest, iv) a step of rehydrating the cleaved tissue region of interest, and v) a step of further analyzing the cleaved tissue region of interest, wherein in step v), the cleaved tissue region of interest is further analyzed by antibody-based immunostaining, gene profiling, or proteomics. method.

2. The method according to claim 1, wherein the gene profiling is gene profiling by RNA sequencing, or the proteomics is proteomics by mass spectrometry.

3. The method according to claim 1 or 2, wherein the fluorescent dye-containing labeling agent has a molecular weight of 60 kDa or less, 50 kDa or less, 40 kDa or less, 30 kDa or less, or 20 kDa or less.

4. The method according to any one of claims 1 to 3, wherein the fluorescent dye-containing labeling agent is an antibody fragment complexed with the fluorescent dye, and the antibody fragment is capable of binding to the target molecule.

5. The method according to any one of claims 1 to 4, wherein the animal tissue is A) derived from a mammal, B) derived from a non-human mammal or human, C) derived from a rodent, D) derived from a mouse, E) pig brain, F) an entire organ or a part thereof, or G) a tissue mass measuring 2 × 2 × 2 cm.

6. The analytical method according to any one of claims 1 to 5, further comprising the step of ii) visualizing the detected fluorescence of the fluorescent dye to obtain an image of the animal tissue.

7. The method of analysis according to any one of claims 1 to 6, wherein the animal tissue is 20 cm or less in thickness, or the animal tissue is 2 cm or less in thickness.

8. The analytical method according to any one of claims 1 to 7, wherein the analytical method further comprises, prior to step i), a method for preparing animal tissue as defined in claim 1.

9. The analytical method according to any one of claims 1 to 8, wherein the fluorescence microscope is selected from the group consisting of a light sheet fluorescence microscope, an epifluorescence microscope, a multiphoton microscope, and a confocal fluorescence microscope.

10. The method of analysis according to any one of claims 1 to 9, wherein the fluorescence microscope is a light sheet fluorescence microscope.

11. The method of analysis according to any one of claims 1 to 10, wherein the cleaved target tissue region includes metastasis.

12. A method for detecting metastasis, wherein the method comprises a method for analyzing animal tissue according to any one of claims 1 to 11, wherein the animal tissue contains cancer metastasis, and the target molecule labeled by the labeling agent is a structure present in the cancer cells.

13. A method for analyzing the in vivo distribution of a biopharmaceutical, wherein the method comprises a method for analyzing animal tissue according to any one of claims 1 to 11, wherein the animal is treated with the biopharmaceutical, the biopharmaceutical is the target molecule labeled by the labeling agent in step c), or the biopharmaceutical is labeled with a further fluorescent dye in vitro, or the biopharmaceutical is fluorescence itself, the animal tissue contains the biopharmaceutical, the biopharmaceutical is a therapeutic protein, or the biopharmaceutical is a nanoparticle.

14. A method for analyzing the in vivo distribution of nanoparticles, wherein the method comprises a method for analyzing animal tissue according to any one of claims 1 to 11, wherein the animal is treated with the nanoparticles, the animal tissue contains the nanoparticles, and the nanoparticles are selected from nanoparticles that are the target molecule labeled by the labeling agent and / or fluorescent dye complex-forming nanoparticles or fluorescence itself.

15. A method for testing neurodegeneration or neuroinflammation, wherein the method comprises a method for analyzing animal tissue as described in any one of claims 1 to 11, the animal tissue containing neurons.

16. A method for testing meningeal lymphatic vessels, wherein the method comprises a method for analyzing animal tissue as described in any one of claims 1 to 11.

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