Tissue treatment

Abstract

Histology and histopathology relate to the study of cells and tissues under microscopy to diagnose and monitor diseases, such as cancer. Differential staining and imaging of various (10) structures in tissue can provide detailed information at cellular and subcellular levels, which is essential in tissue analysis, including diagnostic and prognostic evaluations. The present invention relates to systems and methods for tissue treatment and imaging, including simultaneous dehydration and staining with a fluorescent nuclear dye and a fluorescent protein dye followed by optical slice microscopy.

Description

Technical Field

[0001] The present disclosure relates to systems and methods for tissue processing and imaging. Background Art

[0002] Histology and histopathology involve the study of cells and tissues under a microscope to diagnose and monitor diseases, such as cancer. Differential staining and imaging of various structures in tissues can provide detailed information at the cellular and subcellular levels, which is essential in tissue analysis, including diagnostic and prognostic evaluation.

[0003] While much of the field still relies on visual inspection by trained medical professionals, the development of new processing and imaging technologies has increased throughput and imaging quality on the front end. For example, processing with fluorescent dyes and the use of confocal and multiphoton microscopy provide high-quality images with fine cellular detail at various depths in the tissue. The ability to stain and image structures at depth in the tissue allows for three-dimensional analysis of tissue samples, which improves diagnostic accuracy. In contrast to diagnostic evaluations using visual inspection of physical thin sections of tissue, new technologies are digital in nature, allowing for convenient consultations, remote evaluations by expert pathologists, and the application of digital image analysis tools.

[0004] Fluorescence optical sectioning microscopy has not been widely used in high-throughput environments. Specifically, the processing techniques for available optical sectioning microscopy so far are not suitable for automation, time efficiency and cost control. It is not tuned yet to allow pathologists to perform routine analysis in the clinically relevant tissue of the entire scope. Therefore, other widely applicable, cost-effective and time-effective technology is needed to enable optical sectioning microscopy to be applied to an environment where efficiency is crucial.

[0005] In addition, short pulses of high intensity lasers can be used to produce second harmonic generation (SHG) mapping, which can be used to image protein structures such as collagen and amyloid. Collagen imaging can be used in the liver to quantify fibrosis, which is a hallmark of liver damage in a variety of liver conditions and is also an important clinical prognostic factor.

[0006] The evaluation of collagen fibrosis is a routine part of many types of histological evaluations due to the recognized correlation of the irrecoverable functional loss in collagen fibrosis and various tissue types. The evaluation of collagen is routinely implemented to evaluate the fibrosis in nephropathy, hepatopathy and pulmonary disease, as a key determinant of function and prognosis, and to describe the key structure in multiple other tissues (such as gastrointestinal tract, skin, breast, etc.). Typically, specific staining agents are used to highlight the presence of collagen in physical thin sections. This is done because other tissue features may be indistinguishable to a large extent in standard hematoxylin and eosin (H&E) staining agents. For example, in standard H&E staining, smooth muscle may look similar to collagen, and may be mischaracterized as the fibrosis caused by collagen, thereby causing confusion or even misdiagnosis.

[0007] One such stain specifically used for the characterization of fibrosis is called "trichrome". Several versions of trichrome stains exist, but all involve multiple dyes that are typically intended to achieve contrast for identifying collagen structures by selectively staining collagen, usually by controlling the pH at the time of staining in successive steps, or by using dyes with different selectivity for acidic or basic structures. Other techniques have also been developed involving antibodies to collagen (immunohistochemistry, IHC) or direct chemical stains such as Sirius Red that can be visualized under polarized light conditions to highlight the localization of specific collagen types with increased contrast.

[0008] Although these techniques represent a major advance in the field of histology and histopathology, expanding their use in daily clinical applications requires simplifying the process to reduce costs, increase throughput and provide consistent results. Many of the dyes used may be expensive and account for a significant portion of the reagent costs in the above techniques. In addition, dye concentrations are not standardized and providing consistent quality when imaging at a certain depth may be problematic.

[0009] In addition, common practice is to store tissue in 70% ethanol aqueous solution after formaldehyde fixation to some extent. Doing this is usually to stop the cross-linking of formaldehyde, and the tissue is kept in a relatively inert medium indefinitely, and the relatively inert medium can resist the growth of microorganisms, thus preventing tissue degradation. Notice by experiment that using ethanol solution to carry out tissue storage can have an adverse effect on the combination of some nucleic acid dyes (particularly fluorescent dyes). Some documents show that ethanol and nucleic acid are compounded in a mode that cannot be reversed quickly by being exposed to a solution based on methanol. Therefore, it is necessary to change the process of directly transferring the sample from the ethanol solution to the dyeing based on methanol, so as to achieve optimal dyeing in this case. Summary of the invention

[0010] Systems and methods for processing tissue samples are described. The systems and methods described herein allow for rapid and efficient processing and imaging of tissues at high resolution. The method comprises the following steps: obtaining a tissue sample and contacting the tissue sample with a fixing solution, the fixing solution comprising at least one fixing agent and at least one fluorescent dye. In one embodiment, the method further comprises the step of contacting the tissue sample with a clearing solution. In another embodiment, the method further comprises the step of imaging the tissue sample. In another embodiment, the at least one fluorescent dye is selected from the group consisting of eosin, 4′, 6-diamidino-2-phenylindole (DAPI), a nucleic acid stain sold by ThermoFisher Scientific (Waltham, Massachusetts) under the trademark SYTOX green, a compound sold by Molecular Probes under the name Alexa Fluor 594-NHS, acridine orange, rhodamine B, propidium iodide, and Hoechst dye. In some methods, sample can be colored with protein dye and nuclear dye simultaneously.Nuclear dye (particularly DAPI) can be used in collaboration with high-speed imaging at a concentration much higher than the concentration with surprising results previously recognized.For example, about 50 micromoles, about 100 micromoles, about 200 micromoles, about 300 micromoles, about 400 micromoles, about 500 micromoles, about 600 micromoles, about 700 micromoles, about 800 micromoles or larger micromoles of DAPI concentration can be used (for example, in many cases, for U.S. Patent Application Serial No. 14 / 324,019 and 14 / 790,917 reported in 10x or larger, the content of this U.S. Patent Application is incorporated herein by reference).In a preferred embodiment, DAPI concentration can be between 300 micromoles and 800 micromoles or 300 micromoles and 600 micromoles, and more preferably, for about 400 micromoles.

[0011] In certain embodiments, at least one fixative can be methacarn, while in some embodiments, formalin can be used as a fixation step, and tissue processing can be accomplished without the use of chloroform, which can contribute to reduced quality (e.g., speed and extent) of staining, particularly with DAPI. In another embodiment, the fixative solution further comprises a penetration enhancer, such as acetic acid.

[0012] Acetic acid can be used to counteract tissue shrinkage and enhance fluorescent dye performance, particularly in the case of nuclear and protein dyes used in combination with various methods of the invention. However, increasing the hydrogen ionization potential beyond a certain value can, for example, reduce eosin fluorescence beyond a desired level sufficient to identify cell features during laser imaging and / or place tissue samples at risk of degradation, and therefore, in certain embodiments, for the formulations of the invention, a pKa range (at room temperature) between 4.82 and 5.2 and preferably between 4.86 and 5 can be maintained.

[0013] In another embodiment, the step of contacting the tissue sample with a fixing solution is performed at between about 40°C and about 47°C and preferably at about 45°C. In certain embodiments, the fixing solution further comprises a red blood cell lysing agent. In some embodiments, the step of contacting the tissue sample with a fixing solution is performed over a period of about 1 hour. In another embodiment, the step of contacting the tissue sample with a fixing solution is performed over a period of less than 15 minutes. In another embodiment, the clearing solution comprises benzyl alcohol and benzyl benzoate. In another embodiment, the ratio of benzyl alcohol to benzyl benzoate is about 1:2. In another embodiment, the step of contacting the tissue sample with a clearing solution is performed over a period of about 10 minutes. In another embodiment, after imaging, the partially fixed and partially cleared tissue is placed in a fixative. In another embodiment, the steps of contacting the tissue sample with a fixing solution and contacting the tissue sample with a clearing solution are performed over a period of about 1.5 hours. In other embodiments, these steps occur over a period of about 4 hours. In other embodiments, the time period is about 8 hours, matching the incubation time periods established in existing tissue processing protocols, but with the incorporation of the unique reagent mixtures described herein. In another embodiment, the tissue sample has been fixed prior to obtaining the tissue sample. In some embodiments, the tissue has been previously fixed in formalin. In some embodiments, the tissue has been stored in 70% ethanol.

[0014] In certain embodiments, the combined dehydration / staining solution for tissue processing may include methanol and fluorescent dyes as dehydrating agents, the fluorescent dyes comprising DAPI at a concentration between about 300 μM and about 800 μM and eosin at a concentration between about 0.025 nM and about 2.5 nM, and the pKa of the solution is between about 4.84 and about 5. The solution may include about 10% acetic acid to obtain the desired pKa. The shelf life of this dehydration / staining solution may be shortened to about 3 days, after which the staining quality and / or speed may be significantly reduced when stored at room temperature. However, the method of the present invention may include storing the solution at low temperatures or at about -20°C or lower temperatures (e.g., about -80°C) to significantly extend the shelf life and reduce the degradation of the stain in the dehydration / staining solution.

[0015] The tissue sample may be fixed in formalin before being exposed to the dehydration / staining solution. The tissue sample may be incubated in formalin for about 5 minutes or longer. The tissue sample may be treated with the dehydration / staining solution at a temperature between about 40° C. and about 47° C.

[0016] In certain embodiments, tissue is placed in ethanol solution before obtaining these tissue samples.In this case, can overcome the inhibition that ethanol combines with nucleic acid dye by removing ethanol before painted.Can tissue be placed in aqueous solution, as water, saline or buffered saline or formalin solution, continue the time period proportional to tissue thickness, guarantee that water fully infiltrates thus, to substitute the ethanol that may interact with nucleic acid.Similarly, in other embodiments, can use the methanol that does not contain nucleic acid dye to substitute ethanol.For the sample of hundreds of microns thick, or for thicker sample, when target is to be no more than hundreds of microns to carry out imaging, the time that ethanol removes can be about 10 minutes.Thicker sample is carried out to need longer exposure to aqueous solution than deep layer imaging, this may need to spend more than 30 minutes, 1 hour, 2 hours, 4 hours, 8 hours, 16 hours or 24 hours.

[0017] A method for imaging a tissue sample is also described. The method comprises the following steps: obtaining a tissue sample; contacting the tissue sample with a fixing solution comprising at least one fluorescent dye; contacting the tissue sample with a clearing solution; and generating a tissue sample image by measuring the intensity values ​​of the fluorescence of the tissue sample, and optionally converting the intensity values ​​into effective optical densities so that the optical densities reproduce the color of the stain in the image of the generated tissue sample. In one embodiment, the tissue sample image is generated using an optical sectioning microscope. In various embodiments, the optical sectioning microscope is selected from the group consisting of: a multiphoton microscope (MPM), a confocal microscope, a structured illumination microscope, a super-resolution microscope, a selective plane illumination microscope (SPIM), a side illumination microscope, a spinning disk confocal microscope, and a deconvolution microscope. In another embodiment, the step of generating a tissue sample image further comprises second harmonic generation (SHG). In another embodiment, the sample image is a three-dimensional (3-D) sample image. In another embodiment, the sample image is obtained at a sample depth greater than 50 μμm. In another embodiment, the intensity values ​​are converted into effective optical densities using an exponential pseudo-color process.

[0018] In certain embodiments, the ability of eosin and other protein stains to fluoresce can be used in pre-imaging analysis to identify the target area for subsequent imaging. The advantage of doing so is to produce high-contrast images, which are specific to tissues and assist in identifying tissue locations. For example, the method can include extracting a standard image (non-microscopic) of a sample stained with eosin or another fluorescent stain. The image can be extracted under a specific wavelength excitation (such as red light, or green light, or white light) or using a filter, laser, or LED corresponding to such a wavelength range, and optionally can be collected using a filter that isolates a specific wavelength. The image can then be processed by isolating the relevant signal by an emission wavelength (such as a green wavelength (for eosin) or an orange / red wavelength (for Alexa-594 or Atto-594)). According to this analysis, a bounding box can be created around the area corresponding to the tissue, and image collection can be limited to the area. Therefore, microscopic imaging of a space without tissue can be avoided, thereby allowing more efficient use of imaging time.

[0019] Certain systems and methods of the present invention may include "macro" or microscopic imaging data (e.g., pre-processing or post-processing size, shape, or features) of a substantially fixed tissue sample as a digital fingerprint associated with patient, laboratory, or other identification information. "Macro" means any image of a tissue sample obtained by using a camera (e.g., a camera using a charge coupled device (CCD) or complementary metal oxide semiconductor (CMOS) or similar device). Thus, even in the absence of a standard label or barcode in the physical element (e.g., in the case where the identifier of a slide or tissue block or box is removed or destroyed in some way), an image comparison algorithm, for example, can then be used to identify the tissue sample. This method is applicable to any prepared tissue sample image, and can be specifically used to identify physical tissue microscope slides and samples prepared for imaging using optical sectioning microscopy (e.g., confocal microscopy, multiphoton microscopy, light sheet microscopy, deconvolution microscopy, or super-resolution microscopy). The image used for identification can be obtained in any of a variety of ways: it can be a microscopic image of the sample, or it can be a photograph obtained using standard white light illumination and standard red, green and blue sensor camera detection, or it can employ the use of specific illumination and detection filters described herein to improve isolation and characterization of relevant areas of interest in the biological material.

[0020] In various embodiments, treatment with the dehydration / staining solution of the present invention can be used to produce visualization of tissue containing acidic polysaccharides. Subsequent imaging methods can include converting dual channel fluorescence to H&E-like colors, thereby producing an image that looks similar to a combination of H&E staining and Alcian Blue staining. Although this combination is not typically performed with a physical slide, it provides the opportunity to perform a more complete assessment of the tissue (including both standard morphological assessment and immediate quantification of mucin) with fewer staining steps without adversely affecting the standard H&E assessment.

[0021] The present invention further provides tissue processing techniques that use tissue-specific fluorescent dye concentrations to achieve improved image quality and contrast at a depth and reduced waste compared to the prior art. The systems and methods of the present invention recognize that the quality of imaging at a depth depends on the tissue type and the associated protein content. Although it would be logical to assume that imaging of samples with higher protein content would benefit from higher fluorescent protein dye concentrations, the present methods observed that the image quality of such samples actually benefited from lower fluorescent protein dye concentrations during sample preparation.

[0022] Practical application of these principles, the method of the present invention includes assessing the protein content of tissue samples and changing the protein dye concentration for dyeing based on the protein content. The tissue-specific protein dye concentration of tissues with lower protein content (e.g., renal tissue) is usually higher than the tissue-specific protein dye concentration of tissues with higher protein content (e.g., epithelial tissue). In samples with relatively low protein content, relatively high concentrations can improve the contrast at a certain depth. However, similar protein dye concentrations in samples with higher protein content can increase the signal absorption of the shallower levels of the sample, thereby in optical sectioning microscopy methods, when the deeper levels in the sample are imaged (e.g., confocal imaging, multiphoton imaging and selective plane illumination), the contrast is reduced, and the image quality is degraded. Based on the relative emission and absorption spectrum of the specific dyes used, the increased absorption can affect both nuclear signals and protein fluorescence signals. However, since the concentration of protein in the tissue is generally higher than the concentration of nucleic acids, the interaction between protein-sensitive dye concentration and protein content is significantly greater on the impact of image quality at a certain depth.

[0023] An additional benefit of tailoring dye concentrations according to protein content is the efficient use of expensive reagents. Counterintuitively, using less dye to stain tissues with higher protein content saves costs, which can help promote the daily clinical use of the above-mentioned fluorescence imaging techniques.

[0024] The increased signal absorption caused by excessive dye concentration in protein-rich samples may also affect the ability to detect second harmonic generation at a certain depth. As mentioned above, SHG detection is highly correlated with fibrosis detection in kidney and liver samples. Therefore, customizing the dye concentration according to the protein content of the sample also supports more accurate SHG detection and the related diagnosis and prognostic evaluation provided by SHG detection in certain tissues.

[0025] Combined protein and nucleic acid staining can be used to accurately reproduce common histological stains interpreted by pathologists, such as hematoxylin and eosin (H&E) and so-called Trichrome stains. In such applications, the use of lower concentrations of protein-specific fluorescent dyes in high protein density samples still produces images of sufficient quality to reproduce standard H&E and Trichrome stains, thanks to the normalization of fluorescent signal intensity after signal acquisition and the adaptive nature of the back-end visual analysis.

[0026] Aspects of the present invention include methods for imaging a tissue sample by determining the protein dye concentration of the tissue based on the protein density or content of a given tissue type. The protein dye concentration is inversely correlated with the protein density. The tissue sample of the tissue type is then contacted with a solution containing at least one fluorescent protein dye of a determined concentration for the tissue type. An image of the tissue sample can then be generated by measuring the fluorescence intensity value of the tissue sample at a depth greater than about 200 μm. The solution can include a fluorescent nuclear dye, which can be selected from DAPI, SYTOX dye, SYTO dye, propidium iodide, acridine orange or Hoerst dye.

[0027] The at least one fluorescent protein dye may include eosin, rhodamine B (RhB), Alexa 594 NHS-ester and equivalents, or ANS. Generating an image of a tissue sample may include second harmonic generation (SHG). In certain embodiments, the tissue sample may include collagen. Typical examples of alcohol stock solutions of eosin are eosin Y, 1% alcohol solution, non-acidic (Polysciences, Inc, Warrington PA) or any solution consisting of 1% eosin by weight / volume. In some embodiments, the alcohol stock solution is eosin at 1% eosin by weight / volume in methanol. In other embodiments, the stock solution is 1% eosin by weight / volume in dimethyl sulfoxide (DMSO). The tissue sample may be derived from any biological source, and the at least one fluorescent protein dye may include eosin at about 0.4% of the stock solution. In some embodiments, the tissue sample may be a liver sample, and the at least one fluorescent protein dye may include eosin at about 0.02% of the stock solution. In certain embodiments, a tissue sample can be cleared prior to exposure to a nuclear fluorescent dye and then illuminated with a pulsed laser to produce images of axial sections of SHG molecules (e.g., collagen) to avoid interference from eosin or other fluorescent stains while providing valuable information about the collagen composition in the tissue sample.

[0028] In various embodiments, the tissue sample can be renal tissue, and the at least one fluorescent protein dye can include about 0.4% eosin of the stock solution. Alternatively, the tissue sample can be epithelial tissue, and the at least one fluorescent protein dye can include about 0.04% eosin of the alcohol stock solution. The sample image can be a three-dimensional (3-D) sample image. The tissue sample image can be generated using an optical sectioning microscope. The optical sectioning microscope can be a multiphoton microscope (MPM), a confocal microscope, a structured illumination microscope, a super-resolution microscope, a selective plane illumination microscope (SPIM), a side illumination microscope, a spinning disk confocal microscope, and a deconvolution microscope.

[0029] The solution may further comprise a fixative, such as methylsarcon. The method of the present invention may comprise contacting the tissue sample with a clearing solution prior to generating the tissue sample image. The clearing solution may comprise benzyl alcohol and benzyl benzoate. The solution may comprise a penetration enhancer. In certain embodiments, the method of the present invention may comprise converting the intensity values ​​into effective optical densities such that these optical densities reproduce the color of the stain in the tissue sample image. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 Mucin staining using a dehydration / staining solution according to various embodiments is shown. DETAILED DESCRIPTION

[0031] The present invention relates to methods for tissue preparation and image analysis, which realize the practical implementation of deep imaging of tissue samples. The methods described herein reduce the number of tissue processing steps, reduce the time required for processing tissue, and improve the clarity and contrast in the sample, thereby allowing deep tissue imaging of the sample. As shown herein, the method of the present invention provides complete visualization of biopsy-sized samples without time-consuming and labor-intensive post-clearing processing steps, thereby reducing the time between biopsy and morphological evaluation. In a non-limiting example, the cleared biopsy sample can be provided to a pathologist for direct visualization, or scanned for image distribution. In another non-limiting example, a preliminary diagnosis can be made based on the image and subsequent studies in sequence (if necessary). In another non-limiting example, the sample can be partially fixed and partially cleared for intraoperative evaluation or any other clinical scenario where rapid visual inspection is desired. In another example, the partially fixed sample can be completely fixed at a later time, and the partially cleared sample can be completely cleared at a later time.

[0032] As described in U.S. Patent Application Serial Nos. 14 / 324,019 and 14 / 790,917 and discussed in more detail below, methods for deep imaging of tissue samples stained with both nucleic acid-specific dyes and protein-specific dyes have been previously reported. Such techniques have been applied to reproduce common histological stains such as H&E and Trichrome stains for interpretation by pathologists. In certain embodiments, the methods of the present invention recognize the inverse relationship between the protein content in a tissue sample and the concentration of protein dye required to provide a suitable image at a certain depth. Certain systems and methods of the present invention recognize that the quality of imaging at a certain depth depends on the tissue type, wherein, counterintuitively, imaging of samples with higher protein content benefits from lower concentrations of fluorescent protein dyes, particularly certain protein fluorescent dyes (such as eosin) during sample preparation. At the same time, samples with relatively low protein content exhibit improved contrast at a certain depth when stained with high concentrations of fluorescent protein dyes (such as eosin).

[0033] Relative to the methods disclosed in U.S. Patent Application Serial Nos. 14 / 324,019, 14 / 790,917 and 17 / 000,994, the systems and methods herein can expand the capabilities to ensure that cells that are particularly susceptible to damage caused by certain treatment reagents can remain intact under the microscope, and the contents of each of these documents are incorporated herein. In certain embodiments, dehydration / staining formulations are described that allow for improved signals at a certain depth within a wider sample range. In certain embodiments, improvements in processing and imaging methods are ensured by minimizing the use of formaldehyde fixation (which itself is a function of time, tissue thickness and temperature) and by eliminating a component while controlling pKa within a relatively narrow range to change the staining formulation. The elimination of previously disclosed components of the dehydration / staining solution (i.e., chloroform) is due to a better understanding of the role played by chloroform and a reduced need for chloroform in the case of sufficient formalin fixation combined with the use of a specific pKa range.

[0034] Tissue fixation and chloroform-free processing methods

[0035] In some forms of traditional tissue processing, methanol serves as a dehydrating agent to replace tissue water, and because of its miscibility with organic solvents (such as xylene), it serves as an intermediate between these steps, which allows molten paraffin to penetrate. In other words, methanol is the first step in the goal of replacing tissue water with paraffin in current practice. Methanol itself can also serve as a fixative. Fixation is a process that retards the enzymatic activity of living cells, which will cause cell degradation. Formaldehyde retards enzymatic activity by causing a cross-linked chemical reaction of proteins (including enzymes), thereby retards its degradation activity. Methanol and other alcohols can achieve the same purpose by only replacing water. Enzymes need water to work, and alcohol dehydration can retard enzymatic activity. Therefore, methanol itself can also serve as a "fixative".

[0036] In earlier methods, the processing time was reduced by placing the tissue directly in methylsarconium. It has been noted that red blood cells treated according to earlier methods are more susceptible to lysis (fragmentation) than other cell types. Without wishing to be bound by a particular theory, it is believed that acid exposure of red blood cells whose membranes are not stabilized by the cross-linking provided by formalin can cause these red blood cells to swell and explode. In certain embodiments, this lysis can have potential benefits, understanding that the absorption of both the excitation laser and the emitted fluorescence by the red blood cells can be reduced, thereby improving imaging of more relevant cells.

[0037] Further studies have revealed that certain other cell types (such as renal tubular epithelial cells) are also extremely sensitive to direct exposure to methicillin. Damage to these other cell types can negatively affect histological interpretation, so it is desirable to avoid such direct exposure in certain embodiments and in the case of certain tissue types. In such cases, it is beneficial to perform some degree of formalin fixation (e.g., in a buffered solution at a neutral pH) prior to exposure of the tissue to an acid-containing dehydrating agent as described herein.

[0038] Formaldehyde solution or formalin fixation step can be used for any tissue type generally, although there is tissue that does not need this fixation step. Kidney morphology preservation can benefit from formalin exposure (standard 10% formaldehyde preparation). Liver can also benefit from formalin. Prostate, breast and lung usually do not necessarily obtain the same advantage, but also will not necessarily be affected by such exposure, and therefore, standardized warm formalin fixation before the combined staining agent / dehydration also performs well in these tissues. In certain embodiments, reducing or eliminating formalin exposure may be advantageous to avoid the degradation of nucleic acids for subsequent DNA and RNA analysis. Therefore, when determining whether to include formalin or other fixative exposure steps before dehydration / staining, tissue type (as discussed above) and the desired preference for morphology or nucleic acid analysis can be considered. As discussed above, because alcohol dehydration (such as methyl sacon exposure) can be used to fix tissue, it has not been used in history to use formaldehyde fixation before such acidic alcohol dehydration steps, and the advantage of this step recognized herein is a contribution of the present application.

[0039] In various embodiments, the key modification to the previous method is to eliminate chloroform. Chloroform is reported to play two different roles in the fixation of methyl sarconium. One is as a morphological preservative, which refers to counteracting tissue shrinkage that occurs during dehydration. The other is to potentially act as an adjuvant for rapid tissue penetration due to its low viscosity and dehydration. The latter effect may be minimal relative to methanol because methanol also has a very low viscosity. Certain methods of the present invention recognize that including chloroform in the dehydration / double staining process may reduce the quality of staining, especially the speed and degree of nuclear staining performed using DAPI. Another contribution of the present invention is the surprising discovery that the morphological preservation effect of chloroform is almost not significant in pathological applications when using optical sectioning microscopy. Although some evidence of tissue shrinkage has been observed, the extent of the collapse that occurs is not detectable in conventional microscopic assessments of images. It is possible that the acid itself has sufficient shrinkage-offsetting effects. Formalin fixation before dehydration / staining can also give cells a structure that is sufficient to reduce or avoid cell shrinkage or collapse.

[0040] In view of these observations and the fact that chloroform is toxic to humans and in addition it dissolves many plastics, some systems and methods of the present invention advantageously eliminate the use of chloroform and use additional methanol volumes to replace the chloroform. In some methods, this dehydration / staining preparation can include 86% methanol, 9.6% acetic acid, DMSO containing 3.8% 30mM DAPI, 0.0038g / L eosin. In other methods for dehydration / staining, the preparation includes 86% methanol, 9.6% acetic acid, DMSO containing 3.8% 30mM DAPI and 5.5uM Alexa 594-NHS ester or structural equivalents. Approximately 20% changes in each of these components are expected to achieve similar results.

[0041] The use of acetic acid provides a variety of benefits, including counteracting tissue shrinkage, improving cell permeability to reagents, and enhancing the performance of fluorescent dyes in certain dye combinations. In various embodiments, other acids can provide similar benefits. For example, more potent hydrochloric acid at lower concentrations can produce similar effects. However, the method of the present invention recognizes that increasing the hydrogen ionization potential (reducing pKa) to exceed a certain value will place tissue at risk of degradation in a time-dependent manner. Similarly, it can make the fluorescence of eosin decline, thereby reducing the ability to produce useful images using fluorescence-based imaging. Therefore, pKa levels are important considerations for achieving improved permeability and brighter nuclear signals in the combined dehydration / staining steps, balancing the risk of tissue damage caused by extremely acidic conditions. In certain embodiments, when used together with dehydration / staining solutions and methods described herein, a pKa range of about 4.82 to 5.2 and more narrowly 4.86 to 5 (at room temperature) is desired. The use of 5-15% acetic acid in methanol, and preferably 10% acetic acid in methanol, can achieve the desired color and deep penetration of the disclosed dye combinations in thick tissue specimens without causing unnecessary damage, especially after a controlled fixation step as described above. In addition, fixation in formalin can increase the permeability of cells and can reduce the need for the permeation effect of a low pKa. However, a low pKa may still be desirable to produce a good balance between nuclear staining and protein staining for simulating recognizable H&E staining using the imaging methods of the present invention and maximizing the information content of microscopic examinations.

[0042] Dye concentration

[0043] For both nuclear and protein stains, the ideal concentration may depend on a range of factors, including: tissue type and the corresponding density of nucleic acids / proteins, formalin fixation time, the absorption of one dye emission relative to another (e.g., eosin will reduce DAPI signal), excitation wavelength (single excitation wavelength saves time and instrument complexity / cost), excitation power, fluorescence emission separation (how much signal is collected for one dye relative to another for a given excitation power), signal integration time (the time spent collecting data at one point to improve signal-to-noise ratio), detector performance characteristics (such as sensitivity and dynamic range), and the extent of binding and signal enhancement / reduction caused by the staining preparation. Based on these considerations, it has been found that dehydration / staining solutions of the following composition can improve processing and imaging using the methods described herein.

[0044] DAPI is a preferred nuclear dye. However, the concentration of 10 micromolar used in early applications is too low to achieve fast optimal imaging. It has been found that the signal-to-noise ratio decreases rapidly as the total amount of dye excited at each position corresponding to the image pixel decreases, and the image pixel decreases due to faster imaging. Therefore, in the case of fast microscopy, a much higher DAPI concentration can be used relative to the DAPI concentration disclosed in the early applications discussed herein. In certain embodiments, it has been found that, specific to a two-photon excitation wavelength between about 740nm and 850nm, and preferably about 760nm to about 800nm, and more preferably 780nm, a concentration of about 1.1mM DAPI (about 110 times the concentration disclosed in the early reference applications and methods discussed below) produces an image of a nucleus without perceptible or almost imperceptible image degradation in a sample fixed for less than a few hours at high-speed imaging. While still achieving the desired imaging performance, the DAPI concentration should be minimized to reduce processing costs, because DAPI is a driving factor in the total cost of the solution described herein. Therefore, a concentration of about 550 micromolar can be used to produce acceptable images, but there may be perceptible degradation under high speed conditions. In some embodiments, about 800 micromolar is the desired concentration. In other embodiments, 1.1mM DAPI is the optimal concentration. In various embodiments, a concentration of up to about 2.2 millimolar can be used, beyond which the test cost may become prohibitively high and close to the binding limit of DAPI. In some embodiments, the dye concentration can vary based on the size of the tissue sample. Since the amount of the dye used rises in proportion to the sample size, the lowest possible concentration may be preferred in larger samples. Such larger samples may also not require more than one or two optical images, so the signal reduction that occurs with depth changes may not be a problem. Similarly, for rapid imaging of the surface required for intraoperative assessment of tissues (i.e., frozen sections), only shallow imaging may be required. For shallow imaging, a low concentration of 500 μM is sufficient. In contrast, for small samples such as biopsies (especially nuclear biopsies), it is desirable to image through 1 mm or larger tissues to obtain as much visual information as possible. Therefore, these smaller samples benefit the most from DAPI concentrations between 800 micromolar and 1200 micromolar, or preferably about 1000-1200 micromolar. There are various DAPI preparations, including DAPI dilactate and DAPI dihydrochloride, which are substantially equivalent in performance for this purpose. However, in some embodiments, DAPI dihydrochloride may require a slightly higher concentration relative to the concentration of other preparations to achieve equivalent effects in this method. Similarly, in some embodiments, Hoerst dye can replace DAPI with equivalent effects in its various preparations, which requires concentrations comparable to DAPI dihydrochloride and DAPI dilactate.

[0045] In some embodiments, high eosin concentrations may be a problem for protein-dense tissues, where eosin binds to proteins in the region between the objective and the imaging plane. Without being bound by a particular theory, this may be due to eosin absorbing the fluorescence emission of DAPI, thereby reducing the signal at a certain depth. These effects can be balanced between DAPI / Hoerst concentration / signal and eosin concentration to ensure that both channels are clearly displayed at the specific excitation wavelength and power used and weakened. In addition, relative to the previous methods described herein, when using an excitation wavelength of 780nm (which can be achieved using a less expensive laser), a lower concentration of eosin may be required because eosin excitation is close to its peak at this wavelength. As mentioned above, the dye concentration (nuclear and protein) should be balanced, and one concentration will affect the other. Therefore, when the DAPI concentration is about 1000μM (optimized as above), the complementary eosin concentration can be about 5.5uM. In a preferred embodiment, the eosin concentration is at least 3uM.

[0046] In various embodiments, the eosin concentration can be more flexible than the DAPI concentration and can be varied by up to a certain order of magnitude (0.3uM-60uM) in either direction while still being usable. Higher concentrations may reduce the depth detection of nuclear staining, but can be used for rapid staining and near-surface imaging. Alternatively, lower concentrations can still produce images that can be magnified enough to be detected with a high sensitivity detector.

[0047] Mucin detection

[0048] Detection of acidic polysaccharides, such as those comprising a major portion of mucins (mucus glycosylated proteins), can be used to evaluate tissue histologically. Examples include detection of "goblet cells" in the evaluation of esophageal reflux disease, characterization of the health of cells in the small and large intestines, and identification of subtypes of solid tumors from various organs.

[0049] The surprising advantage of the dehydration / staining solution described herein is that it can stain mucins strongly in a manner comparable to that of a standard specialty stain for such purposes, called alcian blue. In certain methods described herein, DAPI can be similar in its binding to hematoxylin, a standard nuclear stain used in physical slides. But hematoxylin is not fluorescent. DAPI binds to nucleic acids in a similar manner. Hematoxylin can also bind to mucins, but it is not usually stained at a detectable level under the recommended staining pH and dye concentration conditions unless a specific chemical type and pH conditions are used (e.g., Henwood, Anthony F. "Hematoxylin and eosin staining of mucins of the gastrointestinal tract". Journal of Histotechnology 40.1 (2017): 21-24).

[0050] Alcian blue is also non-fluorescent, so it cannot be used with available forms of laser-based microscopy. Specialty stains require additional work and specialized reagents, and the quality is technique-dependent. It also consumes tissue that could otherwise be used for other ancillary studies.

[0051] In contrast, some of the preparations described herein can produce unique visualization of tissues containing acidic polysaccharides because these preparations are strongly stained by the nuclear dye DAPI when reacting under acidic conditions in alcohol (such as the solution preparations described herein), in which case the pKa of the solution is about 4.88. Using the image processing techniques described herein, the dual-channel fluorescence is converted into H&E-like colors, resulting in a type of stain that visually looks similar to a combination of H&E and Alcian blue. Although this combination is not usually performed on a physical slide, it provides a more complete assessment of the tissue (including both standard morphological assessment and instant quantification of mucin) with fewer staining steps without adversely affecting the opportunity of standard H&E assessment. Figure 1 A comparison of a standard H&E stain method and the fluorescent nuclear stain method described herein is shown. The mucin in the epithelial cells shown is visible using the method of the present invention (upper panel) and is not visibly stained using the conventional method (lower panel).

[0052] Imaging analysis

[0053] Speed ​​is an important feature when digitally imaging microscopic tissue, and therefore, imaging dead space and other inefficiencies can be detrimental to image quality. However, missing tissue while scanning can lead to diagnostic errors, so it is important not to sacrifice relevant detail while trying to avoid dead space. Detecting tissue in whole slide imaging typically relies on taking low-power images and then using image analysis techniques to isolate tissue regions and then draw bounding boxes around the regions.

[0054] The techniques described herein recognize that tissues stained with eosin and other general protein binding dyes will fluoresce under excitation from specific wavelengths, that relevant portions of the tissue can be strongly stained with eosin, and that the fluorescent signal can be easily distinguished from the non-fluorescent signal. The difference between the extracted images of the stained tissue without excitation and then excited shows that the portion containing the fluorescence has a high signal-to-noise ratio, which can help separate dirt and other potential noise. Individual fluorescence imaging produces very specific wavelengths that can be isolated and produce sufficient signal-to-noise ratio for accurate and specific tissue detection.

[0055] Utilizing these findings, the methods of the present invention can include using a standard camera to extract images of stained tissues under light having a specific wavelength range and optionally under light having a second wavelength range (such as red light, or green light, or white light). For example, when using an orange / red fluorescent protein dye (such as Alexa 594 or Atto 594), yellow / orange light from a so-called light emitting diode (e.g., yellow, "sodium" yellow, amber, or orange LED) with a wavelength included in the range of 590nm can be used to excite the protein dye throughout the sample, and the camera can detect red light emission around 617nm (orange / red), thereby identifying the location where the tissue resides with high contrast. The desired excitation can also be achieved by other means known in the art, such as using filters that limit the emission wavelength. Similarly, the desired detection wavelength can be limited in the same manner. When using eosin as a protein dye, then ultraviolet, blue or green light (in the range of 440-560 nm, or preferably limited to about 527 nm, such as 520-530 nm) can be used to illuminate the tissue, and the light emission is collected between 510-640 nm and optimally in a range redder than the emission (such as 530-600 in the case of using a green LED) to improve the ability to isolate relevant signals by wavelength. In some versions, images can be extracted using green LED excitation. In other versions, images are extracted using amber LED excitation. Alternatively, an image for analysis can be generated by subtracting a first image extracted under one excitation and detection wavelength condition from a second image extracted under different excitation and detection wavelengths. For example, an image collected under green LED excitation produces a standard red / green / blue (RGB) image, and the desired signal can be isolated by subtracting one channel from another (such as subtracting a blue channel from a red channel), thereby improving the signal-to-background ratio and facilitating the determination of the area of ​​interest. The method can also include creating a thresholded mask from this processed image. In some embodiments, the area of ​​interest is determined using the range corresponding to the dye emission of the hue, saturation and value from the collected image. Optionally, for any of these methods, a median filter can be used to reduce the impact of noise on the accuracy of the tissue area. The median filter can also be used to set the minimum connection area to reduce the possibility of unnecessarily expanding the imaging area due to small dirt particles or noise being identified as tissue. A bounding box can then be created around the fluorescent area of ​​the tissue, and information can be provided to the imaging microscope system to limit image collection to the relevant area. Low-power imaging data (e.g., standard camera imaging) and bounding box information can be maintained in the metadata of the collected microscopic image.

[0056] In various embodiments, an electronic identifier for a microscopic sample can be created based on images collected during processing and pre-imaging or microscopic imaging itself. Image analysis can be used to identify unique features, such as the overall shape or size or the shape or size or features of a sub-area within a tissue sample. Such features can form a digital fingerprint associated with a sample, a patient's name, or a laboratory ID number and applied to its image. In certain embodiments, the original image file can be associated with the patient's name or ID number and stored in a database so that the tissue sample can then be compared with an image or a series of images to match the features, or with the aforementioned digital fingerprint of a pre-selected feature. Such analysis utilizes the systems and methods described herein, which keep the sample in a single orientation and configuration for processing and imaging steps, thereby allowing subsequent identification by these methods. It is noteworthy that this recognition technology is applicable to any image generated using any light wavelength and image detection method during processing, pre-imaging, or microscopic imaging (whether it is using fluorescent illumination, epi-illumination, or transillumination). In other words, it is not limited to images obtained using the blue light or UV light methods described in this application.

[0057] In a preferred embodiment, a substantially fixed non-microscopic image of a biological sample can serve as a unique identifier cross-referenced with a database of sample images. In certain embodiments, the biological sample can undergo a rigid transformation, such as translation or rotation within the field of view. The image can be obtained by a common camera (such as a CCD camera or a CMOS camera). The image can also include a fluorescent image. The database of sample images can include images collected by a pattern or by another pattern (such as a common camera or a fluorescent image) that is sufficient to enable the sample image to match the image present in the database. The database is used to link such images to other identification forms. Other identification forms can include digital identifiers, optical bar codes, patient names, names of personnel involved in the collection, processing or evaluation of tissue samples and images, or any other such information of alternative patterns comprising sample identification.

[0058] Images used for reference or stored in a database may be pre-processed to adjust image parameters such as contrast, brightness or color balance. Non-linear processing of the image may also be used, such as brightness thresholding. A portion of the image data may be used, such as a monochrome channel or a single region of the image.

[0059] Matching the input image to images contained in the database may be accomplished by any method known to one of ordinary skill in the art, and may include image cross-correlation, nearest neighbor mapping of anchor points or principal components, or machine learning algorithms.

[0060] In some embodiments, the biological sample is a whole sample. In alternative embodiments, the sample can be a slice of a biological sample mounted on a slide. In some embodiments, the sample can be embedded in a paraffin block.

[0061] Alternatively, the microscopic image of the biological sample that is substantially fixed can serve as a unique identifier cross-referenced with the database of the sample image. In some embodiments, the biological sample can undergo a rigid transformation, such as translation or rotation in the field of view. The image can be obtained by a microscope or other devices that can image the sample at high magnification, including optical microscopes, confocal microscopes and multiphoton microscopes, Raman microscopes, stimulated Raman scattering microscopes, coherent anti-Stokes scattering Raman microscopes, side illumination microscopes, total internal reflection microscopes, phase contrast microscopes or other phase-sensitive microscopes. The image can be based on transmitted light, reflected light, scattered light or dark field illumination, fluorescence, second or third harmonic light scattering, Raman light scattering, stimulated Raman scattering, coherent anti-Stokes Raman scattering.

[0062] Microscopic images can be extracted at a specified depth within the sample, such as by fluorescence multiphoton microscopy.

[0063] Microscopic images used for reference to or stored in a database may be pre-processed to adjust image parameters such as contrast, brightness or color balance. Non-linear processing of the image may also be used, such as brightness thresholding. A portion of the image data may be used, such as a monochrome channel or a single region of the image. The size of the microscopic image may be reduced prior to matching, for example as a thumbnail image.

[0064] The database of sample microscopic images may include images acquired by the same mode or by alternate modes sufficient to achieve image matching by algorithms known to those of ordinary skill in the art. Matching an input image to images contained in the database may be achieved by any method known to those of ordinary skill in the art, and may include image cross-correlation, nearest neighbor mapping of anchor points or principal component analysis, or machine learning algorithms.

[0065] In some embodiments, the biological sample can be a whole sample. In alternative embodiments, the sample can be a section of a biological sample mounted on a slide.

[0066] Combined collagen and nuclear / protein stain detection

[0067] Although there is a time-tested benefit in predicting and characterizing disease states based on the collagen assessment performed with trichrome staining, there is a significant limitation in this common procedure. One limitation is the poor reproducibility of visual estimation, and the high variability between and within observers. The attempt of quantitative image-based morphometric analysis performed based on the use of trichrome imaging, immunohistochemistry, and other collagen stains (such as Sirius red) has proven that its accuracy and correlation with other functional measures have improved, but additional time and manpower are required for use. This additional effort makes the preparation of tissue physics thin sections, which is already very laborious and time-consuming, more complicated. In addition, it is also routinely used only for single slice evaluation, which is expected to limit its accuracy from a statistical point of view, and thus limits the overall value of clinical management. Therefore, it is necessary to carry out a more comprehensive, accurate and quantifiable assessment of the collagen content in the tissue, ideally in a way that reduces overall effort.

[0068] Advances in confocal microscopy, multiphoton microscopy, and other optical sectioning techniques have expanded the ability to image three-dimensionally in tissue samples, generating 3D perspectives without the embedding, sectioning, scanning, and image processing associated with traditional serial sectioning, albeit at limited depth. Over the past decade, optical sectioning methods have been combined with chemical treatments to reduce light scatter in tissues, significantly improving deep tissue imaging and extending the depth of tissue reconstruction from a few microns to several millimeters using many of the techniques discussed herein.

[0069] Light scattering in tissues is due to differences in refractive index between membranes, proteins, and water, as a result of which light is redirected, reducing the ability to accurately determine the source. Clearing techniques used to reduce light scatter fall into two general categories: removal of lipid membranes and use of high refractive index fluids in place of water. The latter has the advantage of retaining stainable structures and requiring much less time and work. Among the clearing solutions, an inexpensive and safe mixture of compounds in a 1:2 ratio of benzyl alcohol / benzyl benzoate (BABB) is an improvement by Murray on the original clearing compound developed more than a century ago, with a refractive index that closely matches that of dehydrated proteins and membranes, about Nd 1.55. Ethyl cinnamate has similar properties and can be used for the same purpose.

[0070] Another characteristic called second harmonic generation (SHG) of multiphoton laser excitation has been used to selectively visualize the collagen content in tissues as described in U.S. Patent Application No. 17 / 000,994. Pulsed lasers utilize characteristics unique to non-centrosymmetric molecules. In tissue samples, such molecules are mainly present in collagen. The SHG process involves the use of a pulsed laser with a pulse short enough to make the instantaneous intensity sufficient to stimulate the collagen molecules simultaneously with two photons. It occurs with high probability only in a small volume determined by the focusing power of the objective lens, thereby achieving high spatial selectivity in three-dimensional space. When this process occurs, two photons combine and emit as a single photon (half the wavelength) twice the energy. According to the present invention, this specific non-fluorescent phenomenon can be combined with a transparent process to enhance the number of collagen data that can be obtained, and thereby enhance the accuracy of overall quantification, while also enabling the three-dimensional characterization of the collagen architecture in the sample.

[0071] However, there are specific challenges that cause difficulties in thick samples when SHG detection is routinely implemented simultaneously with H&E-like imaging (e.g., using fluorescent dyes described herein). The protein content of certain tissues (such as liver) is very high, which is related to the high metabolic activity of the liver as the main detoxification organ of the body. Ideally, SHG collagen visualization will be completed in parallel with protein and nucleic acid imaging, thereby reducing the overall work and processing time, while maximizing the information content obtained by imaging. However, when a pulsed laser using a wavelength between about 750nm and 900nm is used to excite this collagen signal, the SHG signal may be severely attenuated at a certain depth when the concentration of eosin used to stain the protein is high. This is a bit surprising because eosin is expected to neither absorb light of these excitation wavelengths nor absorb light of the generated SHG signal wavelength (just half of the excitation wavelength). However, observations have confirmed that high protein staining using eosin does produce attenuation of SHG signals, and absorption is a possible explanation for this attenuation. It is still helpful to visualize the anatomical structure around the SHG signal to think about whether the signal is from normal fibrous tissue or pathological reaction. Intrinsic fluorescence can be used to achieve this goal, but some protein fluorescent dyes facilitate this detection. Therefore, in certain embodiments, the eosin concentration can be minimized during staining to the lowest level that still allows visualization of microscopic protein-rich structures to increase the depth at which collagen signals can be simultaneously detected. For liver samples, using the staining methods, processing reagents, and multiphoton imaging systems described herein, a balance of protein signals fully reproducing the H&E stain while allowing imaging of collagen SHG at several hundred microns was achieved using a stock solution of eosin concentration of about 0.02% during the staining step.

[0072] The physical property of SHG is that the intensity of the signal is usually significantly higher in the transmission direction (parallel to the direction of the excitation light). In the case of trying to collect SHG signals propagating in the reverse direction (incident detection), such as using the same objective lens for excitation and detection, the SHG signal is usually attenuated. Unfortunately, the use of this geometry is usually varied with efforts designed to minimize the cost and complexity of the microscopy system, and in some cases, it may be determined by the design of the device holding the sample and the limitations of the mechanical elements of the functional multiphoton microscope. In such cases, any fluorescent dye that may emit a signal within the specific detection wavelength of the SHG signal may mask the signal of interest, thereby reducing the accuracy of SHG imaging. For example, when using DAPI or Hoechst for nuclear staining, the broad bright fluorescence of these dyes in the short visible wavelengths may easily mask the SHG signal, even when using a very narrow range of emission filters (meaning to minimize the contribution from these dyes). Furthermore, when a weak SHG signal is detected at a certain depth in epi-detection mode, eosin has a proportionally more relevant adverse effect on the excitation signal, and eosin can also absorb the emitted SHG signal, effectively creating an absorption filter for the relevant signal.

[0073] To avoid these problems, certain methods of the invention may include methods for processing a tissue sample in preparation for imaging using a multiphoton microscope under matched refractive index conditions to image at a depth with high resolution and contrast to provide optical sectioning information about collagen content and distribution, followed by a method for treating the tissue with a fluorescent dye and imaging in a manner that accurately reproduces the type of information obtained from thin tissue sections. Using the methods described herein, collagen information can be initially retrieved, followed by subsequent staining and imaging of the same sample (optionally in the same container) using an optical sectioning microscope, which optionally can be the same multiphoton microscope used to obtain the collagen information.

[0074] In view of the above-mentioned limitations of the standard method for collagen imaging and incorporating SHG signal detection in multiphoton microscopy, in certain embodiments, the sample can be processed to be transparentized without using any dye. This process can occur by any method known for chemical transparentization, which means replacing water content (including methods described herein) with a fluid having a high refractive index. When the fluid refractive index is about 1.47 or higher, this refractive index produces a clarity sufficient to usefully detect SHG at a depth of tens to hundreds of microns. Ideally, the refractive index of the fluid is 1.5 or higher. Most ideally, the refractive index of the fluid is 1.545, or about 1.55 when measured at 780nm. In a preferred embodiment, the process involves optionally fixing the tissue in formalin, then placing the sample in methanol, then placing the sample in BABB, and then subjecting it to SHG analysis for collagen characterization.

[0075] The sample can then be exposed to alcohol or other tissue treatment compounds known to those of ordinary skill in the art (such as xylene miscible with both organic solvents and alcohols) to remove the clearing compound, optionally in the container where the clearing treatment and imaging are performed. Exemplary containers for tissue sample processing are described in U.S. Patent No. 10,527,528, the contents of which are incorporated herein by reference. The sample can then be treated with a fluorescent dye in an alcohol solution (e.g., using any dye and / or dehydration / staining solution and method described herein), and cleared, processed, and subjected to optical sectioning fluorescence microscopy methods described herein. Therefore, in addition to the H&E type fluorescent staining analysis described herein, a clear SHG analysis of collagen content at a certain depth can also be obtained from the same tissue sample, thereby providing a more comprehensive overview for diagnostic and prognostic purposes.

[0076] As discussed above, the various systems and methods described herein relate to improvements and modifications to previously described image analysis methods for using fluorescent dyes and optical sectioning microscopy to produce images that are substantially indistinguishable from traditional histological stains. Various aspects of these methods are described below, and provide images of samples that simulate common pathological stains, thereby producing accurate and efficient interpretations of images. In contrast to standard color separation algorithms, these methods use the fluorescence of the sample (whether inherent or produced by fluorescent dyes) to invert the color separation technique to faithfully reproduce images containing the expected colors of tissues produced by common stains (such as hematoxylin / eosin and Wright / Giemsa), thereby allowing pathologists to easily interpret images. Unlike previous pseudo-color efforts, the methods described herein use exponential conversion equations to more closely match the optical quality of fluorescence emission with the quality of light absorption under traditional illumination of thin sections. As demonstrated herein, the methods of the present invention produce images having resolution and field of view similar to those produced using current histological methods, provide contrast similar to that of images obtained using commonly used histological stains, and allow subsequent conventional processing without significant adverse effects. The multichannel methods described herein provide direct pseudo-coloring of morphology in a manner similar to conventional stains, thereby allowing pathologists to easily identify prominent histological features.

[0077] In the case of biopsy, there is often a trade-off between retaining enough tissue for additional stains or molecular analysis and retaining enough hematoxylin and eosin (H&E) histology. The necessarily sparse sampling of traditional physical wax embedding and cutting histology techniques may miss important features. For example, colon polyps may be missed, small lesions of prostate cancer may not be diagnosed, and focal renal lesions may not be obvious. This problem is complicated by the need to discard the initial bulk shavings to obtain complete sections, especially in incompletely embedded samples. The methods described herein avoid these problems that occur when using current histological methods while allowing image reconstruction of the entire or deep portion of the biopsy sample.

[0078] In the case of rapid analysis, many artifacts and difficulties associated with tissue freezing and sectioning can be overcome by using the process presented herein to visualize unfrozen, uncut tissue. In contrast to other related efforts to visualize unfrozen, uncut fresh tissue using optical sectioning microscopy, the process described herein completely solves the challenges arising from poor refractive index matching and slow dye penetration that have prevented imaging with sufficient resolution and depth to make practical diagnosis using any other known techniques.

[0079] method

[0080] In one aspect, the present invention provides a method for processing and imaging a histological sample. In one embodiment, the method comprises the step of obtaining a sample. The sample can be any type of sample in general. For example, the sample can be a cell or cell group, an organism, a tissue, a cell lysate, a cell culture medium, a bioreactor sample, etc. In a preferred embodiment, the sample is a tissue sample. In another embodiment, the sample is a fluid sample in which the cell components have been concentrated, such as by centrifugation or filtration. Non-limiting examples of tissues include skin, muscle, small intestine, large intestine, breast, esophagus, heart, kidney, lung, liver, skin, placenta, prostate, pancreas, uterus, bone, bone marrow, brain, stomach, muscle, cartilage, lymph node, adipose tissue, tonsil, gallbladder and spleen and tumor tissue of any cell type and cerebrospinal fluid, pleural fluid, ascites or synovial fluid. In one embodiment, the tissue is liver tissue. In another embodiment, the tissue is kidney tissue. In another embodiment, the tissue is breast tissue. In another embodiment, the tissue is prostate tissue. The sample can be obtained by any method known in the art as understood by those skilled in the art. In some embodiments, the sample is obtained during surgery, biopsy, fine needle aspiration, culture, or autopsy. In one embodiment, the sample is a fresh sample. In another embodiment, the sample is a fixed sample. In one embodiment, the tissue sample is fixed before obtaining the tissue sample.

[0081] The method of the present invention may include obtaining a sample and contacting the sample with a fixing solution. In a preferred embodiment, the sample is a tissue sample. In certain embodiments, the fixing solution comprises at least one dehydrating agent and at least one fluorescent dye. In some embodiments, the method may include transparentizing the sample by contacting the sample with a clearing solution so that the depth and clarity used to image the sample are increased. In some embodiments, the sample is fixed before contacting a solution comprising a fixing agent or a dehydrating agent and at least one fluorescent dye. In another embodiment, fresh tissue is directly placed in a combined fixing / dehydrating fluid with one or more dyes. In some embodiments, the step of imaging the sample is performed in combination with other imaging methods such as second harmonic generation (SHG). In certain embodiments, the sample can be partially fixed for intraoperative evaluation or any other clinical scenario where rapid visual inspection is desired. The partially fixed sample can be completely fixed at a later time. In another embodiment, the sample can be partially transparentized for intraoperative evaluation or any other clinical scenario where rapid visual inspection is desired. The partially transparentized sample can be completely transparentized at a later time.

[0082] In one aspect, the method of the present invention further comprises the step of dehydrating the sample. Dehydration helps to remove water from the sample so that a water-soluble, low-transparent agent can be used subsequently. It should be understood that a dehydrating agent or a dehydrating solution can also be used as a fixative or for fixing the sample. As used herein, the term "dehydrating agent" refers to a water-miscible anhydrous fluid. Non-limiting examples of dehydrating agents include alcohols, such as methanol, ethanol, and propanol. In one embodiment, the dehydrating agent is methyl methacrylate. In another embodiment, the dehydrating agent is methanol. In one embodiment, the dehydrating step acts as a fixative and occurs without fixing the sample in advance. In other embodiments, the dehydrating step is performed after fixing the sample. In one embodiment, the dehydrating step is performed after fixing the sample using a fixing solution comprising formalin.

[0083] The dehydration step can be performed, and continues any suitable time length. The time length can be generally any time length that is suitable for making the sample or a part of the sample miscible with the transparent agent. The time length can also be generally any time length that is suitable for preserving the sample or preserving a part of the sample. In certain embodiments, the time period can be from about 1 minute, about 5 minutes, about 10 minutes, about 15 minutes, about 30 minutes, about 1 hour, about 90 minutes, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 12 hours or about 24 hours. In one embodiment, the dehydration step is performed in about 1 hour of the time period. In another embodiment, the dehydration step is performed in about 12 hours to 16 hours.

[0084] In one aspect, the method of the present invention comprises the step of fixing the sample. The tissue sample can be fixed using any method known in the art as will be understood by those skilled in the art. In one embodiment, the sample is fixed by contacting the sample with a fixative. In another embodiment, the sample is fixed by contacting the sample with a fixing solution. In one embodiment, the fixing solution comprises at least one fixing agent. In one embodiment, the fixing agent is a dehydrating agent. In another embodiment, the fixing solution is a dehydrating agent. In another embodiment, the fixing solution comprises at least one fixing agent and at least one penetrant. Non-limiting examples of fixing agents include aldehydes (e.g., formaldehyde (paraformaldehyde, formalin), glutaraldehyde, acrolein (acrolein / acrylic aldehyde), glyoxal (glyoxal (ethanedial, diformyl), malonaldehyde (malonaldehyde / malonic dialdehyde), diacetyl (2,3-butanedione), and polyaldehydes; alcohols (i.e., protein denaturants; e.g., acetic acid, methanol, ethanol), polyvinyl alcohol, heavy metal oxidants (i.e., metal ions and metal complexes; e.g., osmium tetroxide, chromic acid); agents of unknown mechanism, such as chloro-s-triazide, cyanuric chloride, carbodiimides, diisocyanates, diimide esters, diethyl pyrocarbonate (diethyl oxydicarboxylate, anhydrous ethoxyformic acid), picric acid, mercuric chloride (mercuric chloride, dichloride of mercury) and other mercuric salts, and acetone. In one embodiment, a combination of fixatives is used. Such combinations result in formulations known in the art as Camoy's fixative, methylsarcon, Wolman's solution, Rossman's fluid, Gendre's fluid, Bouin's fluid, Zenker's fluid), Helly's fluid, B5 fixative, Susa fluid, Elftman's fixative, Swank and Davenport's fixative, Lillie's alcoholic lead nitrate, and cetylpyridinium chloride (CPC). Additives may include, but are not limited to, entities such as tannic acid, phenol, transition metal salts (zinc), lanthanum, lithium, potassium, etc. In one embodiment, the fixative is methadone. In another embodiment, the fixative is formalin. In another embodiment, the fixative is alcohol. In another embodiment, the fixative is methanol. In another embodiment, the fixative is polyvinyl alcohol. In another embodiment, the fixative is formaldehyde.In another embodiment, fixation of the sample occurs in vitro.

[0085] In some embodiments, at least one fluorescent dye is added to the sample during the fixing step, thereby producing simultaneous fixing and staining of the sample. In other embodiments, at least one fluorescent dye is added to the sample during the dehydration step, thereby producing simultaneous dehydration and staining of the sample. The incorporation of fluorescent dyes avoids the need for post-treatment staining, which is a time-consuming step in traditional sample preparation. Fluorescent dyes can be directly added to the sample during the fixing step. For example, fluorescent dyes can be added to the fixing solution. In another embodiment, fluorescent dyes are added to the sample after the fixing step is completed. In one embodiment, the fixing solution comprises a fixing agent and a fluorescent dye. In another embodiment, the fixing solution comprises at least one dehydrating agent and at least one fluorescent dye. In one embodiment, fluorescent dyes are directly added to the sample during the dehydration step. In one embodiment, the method for processing a tissue sample comprises the following steps: obtaining a tissue sample and contacting the tissue sample with a fixing solution, the fixing solution comprising at least one dehydrating agent and at least one fluorescent dye.

[0086] The skilled artisan will appreciate that the present invention contemplates the use of any fluorescent dye that is compatible with the fixation step. Examples of fluorescent dyes include, but are not limited to, nucleic acid stains sold under the trademarks POPO-1, TOTO-3, SYTOX, and SYTO by Thermo Fisher Scientific (Waltham, Mass.), TAMRA, BOXTO, BEBO, SYBR DX, dyes sold under the name Alexa Fluor dyes (e.g., Alexa Fluor 594, Alexa Fluor 594-NHS ester, Alexa Fluor 633, Alexa Fluor 633-NHS ester, Alexa Fluor 568, Alexa Fluor 568-NHS ester, Alexa Fluor 610, Alexa Fluor 610-NHS ester) and chemical equivalents sold under the names AZDye, XFD, Aurora Fluor, etc., fluorescein, rhodamine, rhodamine derivative dyes sold under the name ATTO (ATTO-TEC Siegen, Germany) (e.g., ATTO 590, ATTO 594, ATTO 610, ATTO 633, etc.), propidium iodide, Hoerst dye, tetramethylrhodamine, R-phycoerythrin, Cy-3, Cy-5, Cy-7, sulforhodamine 101 acid chloride, Phar red, allophycocyanin (APC), fluoresceinamine, eosin, dansyl, umbelliferone, 5-carboxyfluorescein (FAM), 2'7'-dimethoxy-4'5'-dichloro-6-carboxyfluorescein (JOE), 6-carboxyrhodamine (R6G), N,N,N',N'-tetramethyl-6-carboxyrhodamine (TAMRA), 6-carboxy-X-rhodamine (ROX), 4-(4'-dimethylaminophenylazo)benzoic acid (DABCYL), 5-(2'-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS), 8-anilino-1-naphthalenesulfonic acid ammonium salt (ANS), 4-acetamido- 4'-isothiocyanatodiphenylethylene-2,2'disulfonic acid, acridine, acridine isothiocyanate, acridine orange (N,N,N',N'-tetramethylacridine-3,6-diamine), R-amino-N-(3-vinylsulfonyl)phenylnaphthalimide-3,5, disulfonate (fluorescent yellow VS), N-(4-anilino-1-naphthyl)maleimide, o-aminobenzamide, brilliant yellow, coumarin, 7 -amino-4-methylcoumarin, 7-amino-4-trifluoromethylcoumarin (coumarin 151), cyanosine, 2-(4-amidinophenyl)-1H-indole-6-carboxamidine (DAPI), 5′,5′-dibromopyrogallol-sulfonphthalein (bromopyrogallol red), 7-diethylamino-3-(4′-isothiocyanatophenyl)-4-methylcoumarin diethyltriamine pentaacetate, 4,4′-diisothiocyanatodihydro-diphenylethylene-2,2′-disulfonic acid, 4,4′-diisothiocyanatodiphenylethylene-2,2′-disulfonic acid, 4-dimethylaminophenylazo-4′-isothiocyanate (DABITC), eosin isothiocyanate, erythrosine B, erythrosine isothiocyanate, ethidium, 5-(4,6-dichlorotriazine-2-yl)aminofluorescein (DTAF), QFITC ​​(XRITC), fluorescamine, 1R144, IR1446, malachite green isothiocyanate, 4c-methylumbelliferone, o-cresolphthalein, nitrotyrosine, pararosaniline, phenol red, B-phycoerythrin, o-phthalaldehyde, pyrene, pyrenebutyric acid, 1-pyrenebutyric acid succinimidyl ester, Reactive Red 4 (Cibacron.RTM. Brilliant Red 3B-A), lissamine rhodamine B sulfonyl chloride (lissamine rhodamine B sulfonylchloride), rhodamine B, rhodamine 123, rhodamine X, sulforhodamine B, sulforhodamine 101, sulfonyl chloride derivatives of sulforhodamine 101, tetramethylrhodamine, thiazole orange, riboflavin, rhodic acid, and terbium chelate derivatives. In one embodiment, the fluorescent dye is eosin. In another embodiment, the fluorescent dye is DAPI. In another embodiment, the fluorescent dye is a nucleic acid stain sold under the trademark SYTOX green by Thermo Fisher Scientific (Waltham, MA). In another embodiment, the fluorescent dye is acridine orange. In another embodiment, the fluorescent dye is rhodamine B. In another embodiment, the fluorescent dye is a nucleic acid stain sold under the trademark SYTO by Thermo Fisher Scientific (Waltham, MA). In another embodiment, the fluorescent dye is propidium iodide. In another embodiment, the fluorescent dye is Hoersted dye. ,

[0087] In certain embodiments, fluorescent dyes can selectively dye components of specific organelles or cells. In one embodiment, the fluorescent dye is a nuclear dye. Non-limiting examples of nuclear dyes include DAPI, nucleic acid stains sold by Thermo Fisher Scientific (Waltham, Massachusetts) under the trademark SYTOX, nucleic acid stains sold by Thermo Fisher Scientific (Waltham, Massachusetts) under the trademark SYTO, propidium iodide, acridine orange and Hoerst dye. In one embodiment, the nuclear dye is DAPI. In another embodiment, the fluorescent dye is a protein dye. Examples of protein dyes include but are not limited to eosin, rhodamine B (RhB) and ANS. In some embodiments, the fluorescent dye is an NHS ester of a rhodamine derivative used as a general protein dye. In some embodiments, the fluorescent dye is chemically equivalent to a compound sold with Alexa Fluor 594-NHS ester. In other embodiments, the protein dye is ATTO 594-NHS ester or its chemical equivalent. In some embodiments, the protein fluorescent dye is Alexa Fluor 610-NHS ester or its universal equivalent. In another embodiment, the fluorescent dye is a chemical equivalent of the compound sold under the name Alexa Fluor 633 NHS ester. In another embodiment, the intrinsic fluorescence of the cell is used to image the cellular proteins. In another embodiment, the fluorescence is produced by a combination of at least one nuclear dye and at least one protein dye. In another embodiment, the fluorescence is produced by a nuclear dye and intrinsic fluorescence. In one embodiment, the at least one protein dye is eosin. In another embodiment, the at least one protein dye is Alexa Fluor 594-NHS ester or a chemical equivalent thereof.

[0088] In certain embodiments, a morphological preservative is added to the sample during the fixing step and / or the dehydration step. The morphological preservative enhances the maintenance of the nuclear structure of the cell because it maintains the integrity of the cell membrane, for subsequent cytological staining, and / or reduces shrinkage or swelling during fixation or dehydration. As understood by those of ordinary skill in the art, any morphological preservative compatible with the fixing step can be used in the present invention. Non-limiting examples of morphological preservatives include acetic acid, trichloroacetic acid, formaldehyde, dioxane, chloroform, etc. In one embodiment, the morphological preservative is chloroform. The morphological preservative can be directly added to the sample during the fixing step. Alternatively, the morphological preservative can be added to the fixing solution. In one embodiment, the fixing solution comprises about 0% to about 50% of the morphological preservative. In another embodiment, the fixing solution comprises about 5% to about 15% of the morphological preservative. In another embodiment, the fixing solution comprises about 10% of the morphological preservative. In another embodiment, the fixing solution comprises about 20% to about 40% of the morphological preservative. In a preferred embodiment, the fixative solution comprises about 30% morphological preservative.

[0089] In some embodiments, a penetration enhancer is added to the sample during the fixing step and / or the dehydration step. The penetration enhancer accelerates the entry of the dye into the darker part of the sample, while improving the coloring process as a whole. The penetration enhancer also accelerates the penetration of the fixative, dehydrating agent and / or transparentizing agent. Non-limiting examples of penetration enhancers include acids such as acetic acid, acetic acid-containing methacrylic acid, sulfoxides such as dimethyl sulfoxide (DMSO), azone, pyrrolidone, propylene glycol, fatty acids, essential oils, phospholipids, s-collidine and surfactants such as Tween. In one embodiment, the fixing solution contains about 0% to about 75% of the penetration enhancer. In another embodiment, the fixing solution contains about 0% to about 25% of the penetration enhancer. In another embodiment, the fixing solution contains about 5% to about 15% of the penetration enhancer. In a preferred embodiment, the fixing solution contains about 10% of the penetration enhancer.

[0090] In some embodiments, the penetration enhancer is at least one acid. In some embodiments, the acid is an organic acid. Non-limiting examples of organic acids include acetic acid, glacial acetic acid, lactic acid, propionic acid, butyric acid, succinic acid, citric acid, 3-hydroxypropionic acid, glycolic acid or formic acid. In one embodiment, the acid is acetic acid. Acetic acid can be used to increase the fixation speed, which is very important for rapid sample processing, while also significantly improving the quality and depth of images from transparently treated samples obtained using any type of slice image mode. Acetic acid can also be used to lyse red blood cells, which allows the removal of hemoglobin, which significantly destroys the clarity of the sample by virtue of its wide range of light absorption characteristics within the typical working wavelength range of conventional fluorescence imaging. In one embodiment, the step of fixing the sample further includes adding a lysing agent to the sample. In a specific embodiment, the step of fixing the sample further includes adding a red blood cell lysing agent to the sample. In another embodiment, the step of dehydrating the sample further includes adding a lysing agent to the sample. In another embodiment, the fixing solution further includes a red blood cell lysing agent. In a preferred embodiment, the red blood cell lysing agent is acetic acid. In other embodiments, the acid is an inorganic acid. The solution may further include at least one organic solvent. The non-limiting examples of organic solvents include methanol, absolute methanol, chloroform, methylene chloride, ethanol, isopropanol, acetone, ethyl acetate, acetonitrile, hexane, hexene, octane, pentane, cyclohexane, isooctane, xylene (ortho, meta or para) and 1-hexene. In one embodiment, the organic solvent is absolute methanol. Methanol can be used for tissue processing by blocking enzymatic function and degradation while retaining genetic and proteomics information to the maximum extent. In another embodiment, the organic solvent is chloroform. In one embodiment, the solution comprises two organic solvents and an acid. As a non-limiting example, the fixing solution comprises about 60% absolute methanol, about 30% chloroform and about 10% glacial acetic acid, which is also referred to as methylsarcon. When combined with fluorescent dyes and clearing treatment, methylsarcon can be used for deep fluorescent tissue section imaging of human samples for histological evaluation, and can be used to produce the contrast required for accurate histological evaluation.

[0091] The fixing step can be performed under any conditions that promote rapid tissue processing, such as conditions that increase the rate of chemical reactions and diffusion, as will be understood by those of ordinary skill in the art. In some embodiments, the fixing step is performed at an elevated temperature. As used herein, the term "elevated temperature" refers to a temperature higher than that experienced in the earth's atmosphere, preferably a temperature higher than 30°C. In one embodiment, the elevated temperature is in the range of about 20°C to about 75°C. In another embodiment, the elevated temperature is in the range of about 35°C to about 50°C. In another embodiment, the elevated temperature is about 45°C. In a non-limiting example, for the purpose of accelerating diffusion, chemical reactions or temperature, fixation, dehydration and / or staining are performed under microwave irradiation.

[0092] The fixing step can be performed any suitable time length. The time length can be any time length that is suitable for preserving sample generally. In certain embodiments, the time period can start from about 1 minute, about 5 minutes, about 10 minutes, about 15 minutes, about 30 minutes, about 1 hour, about 90 minutes, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 12 hours or about 24 hours. In one embodiment, the fixing step is performed in about 1 hour of the time period. In another embodiment, the fixing step is performed in about 12 hours to 16 hours. In another embodiment, the fixing step is performed in the process of several weeks to several years.

[0093] In one embodiment, the method of the present invention comprises the steps of simultaneously fixing, dehydrating and staining the sample by contacting the sample with a dehydrating solution comprising at least one dehydrating agent and at least one fluorescent dye. In another embodiment, the method of the present invention comprises the steps of simultaneously dehydrating and staining the sample by contacting the sample with a dehydrating solution comprising at least one dehydrating agent and at least one fluorescent dye.

[0094] In another aspect, the method of the present invention further comprises a step of transparentizing the sample. Transparentizing the sample increases the imaging depth and clarity of the sample. In one embodiment, the transparentizing step is performed in the absence of a fixing step. In some embodiments, the step comprises transparentizing the sample by contacting the sample with a transparentizing solution. As a non-limiting example, the sample is transparentized by replacing water with a transparentizing solution having a refractive index higher than that of water and more closely similar to the refractive index of proteins and organelles, thereby greatly reducing light scattering and enabling the imaging depth to reach several millimeters rather than several microns. In one embodiment, the transparentizing solution comprises at least one solvent. Any solvent can be used in the transparentizing solution, as long as the total refractive index of the transparentizing solution is higher than the refractive index of water, and the solvent does not destroy the morphology of the cellular components of the sample. In one embodiment, the refractive index of the transparentizing solution is in the range of about 1.4 to about 1.6. In another embodiment, the refractive index of the transparentizing solution is in the range of about 1.33 to about 1.49. In another embodiment, the refractive index of the transparentizing solution is greater than about 1.4. In another embodiment, the refractive index of the transparentizing solution is greater than about 1.5. In one embodiment, the clearing solution further comprises a pharmaceutical agent that is water-soluble and has a high refractive index, such as a high concentration sugar solution.

[0095] In some embodiments, the solvent is an organic solvent. Non-limiting examples of organic solvents that can be used as transparent agents include benzyl alcohol, benzyl benzoate, xylene, limonene, benzene, toluene, chloroform, petroleum ether, carbon disulfide, carbon tetrachloride, dioxane, glycerol, diphenyl ether, clove oil and cedar oil. In one embodiment, the solvent is benzyl alcohol. In another embodiment, the solvent is benzyl benzoate. In another embodiment, the solvent is xylene. In another embodiment, the solvent is glycerol. In another embodiment, the solvent is a sugar solution. In another embodiment, the solvent is dibenzyl ether. In another embodiment, the solvent is hexane.

[0096] In some embodiments, the clearing solution comprises a first solvent and a second solvent. In one embodiment, the ratio of the first solvent to the second solvent is in the range of about 100:1 to about 1:100. In another embodiment, the ratio of the first solvent to the second solvent is in the range of about 10:1 to about 1:10. In another embodiment, the ratio of the first solvent to the second solvent is in the range of about 5:1 to about 1:5. In a preferred embodiment, the ratio of the first solvent to the second solvent is about 1:2. In some embodiments, the solvent is an organic solvent. In a specific embodiment, the clearing solution comprises benzyl alcohol and benzyl benzoate. In one embodiment, the ratio of benzyl alcohol to benzyl benzoate is about 1:2.

[0097] The clearing step can be performed under any conditions that promote rapid clearing of the sample, as will be understood by one of ordinary skill in the art. In some embodiments, the clearing step is performed at an elevated temperature. In one embodiment, the elevated temperature is in the range of about 20° C. to about 75° C. In another embodiment, the elevated temperature is in the range of about 35° C. to about 50° C. In another embodiment, the temperature is about 22° C.

[0098] The clearing step can be performed in any suitable time length. The time length can generally be suitable for achieving sufficient reduction in light scattering, so that any time length that can image the desired depth in the sample. In certain embodiments, the time period can start from about 1 minute, about 5 minutes, about 10 minutes, about 15 minutes, about 30 minutes, about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 12 hours or about 24 hours. In one embodiment, the clearing step is performed in about 2 minutes to about 1 hour. In one embodiment, the clearing step is performed in about 2 minutes. In one embodiment, the clearing step is performed in about 30 minutes. In one embodiment, the clearing step is performed in about 12 hours.

[0099] In one embodiment, the transparent treatment step further includes the step of adding a solvent to the sample before adding the transparent solution. In some embodiments, the solvent is an organic solvent. In one embodiment, the solvent is an alcohol. Alcohol can be used to dehydrate the sample. Non-limiting examples of alcohol include methanol, ethanol, propanol, isopropanol, butanol, isobutanol, ethyl butanol, / -butanol, dioxane, ethylene glycol, acetone and amyl alcohol. In a preferred embodiment, the solvent is methanol. In one embodiment, the solvent is added in combination with a penetration enhancer. Non-limiting examples of penetration enhancers include acetic acid, polyethylene glycol (PEG), monomethyl glycol and dimethyl glycol, propylene glycol, polyvinyl pyrrolidone, etc., surfactants such as dimethyl sulfoxide (DMSO), polyoxyethylene sorbitol esters (e.g., TWEEN, such as TWEEN 80), sodium dimethyl sulfosuccinate, mild household detergents, etc. In one embodiment, the penetration enhancer is selected from the group consisting of acetic acid, DMSO and TWEEN. The addition of a solvent in combination with a penetration enhancer increases the rate of clearing with BABB by improving miscibility and permeability, and also eliminates the need for stepwise gradient steps of decreasing alcohol concentration.

[0100] Partly, the present invention provides a method for rapidly processing tissue samples. The time length can be generally suitable for fixing the sample and any time length that the sample is transparently processed. The time length can also be generally suitable for a part of the fixed sample and any time length that a part of the sample is transparently processed. In certain embodiments, the time period can start from about 1 minute, about 5 minutes, about 10 minutes, about 15 minutes, about 30 minutes, about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours or about 6 hours. In one embodiment, in about 1 hour to about 2 hours, the step of fixing the sample and transparently processing the sample is performed. In one embodiment, in about 1.5 hours of the time period, the step of fixing the sample and transparently processing the sample is performed. In another embodiment, in the time period of about 3 hours, the step of fixing the sample, coloring the sample and transparently processing the sample is performed.

[0101] In another aspect, the method of the present invention further comprises the step of imaging the sample. In one embodiment, the step of imaging the sample further comprises a visual image of the tissue sample. Any imaging method compatible with the sample processing method described herein can be used to image the sample. Preferred imaging methods include optical sectioning imaging methods based on fluorescence. Contrary to destructive 3-D histological methods such as colored plastic embedding systems and whole slide imaging (WSI), these methods based on fluorescence are non-destructive, thereby allowing the sample to be preserved for auxiliary research and molecular research such as immunostaining agents. Examples of sectioning imaging methods based on fluorescence include but are not limited to multiphoton microscopy (MPM), side illumination microscopy, traditional confocal microscopy, spinning disk confocal microscopy, structured illumination microscopy, etc. In animal tissues (Parra et al., 2010, J. Biomed. Opt. 15:036017-036017-5; Vesuna et al., 2011, J. Biomed. Opt. 16:106009-106009-6; Fu et al., 2009, Gastroenterology 137:453-465), the depth of imaging can be increased by combining MPM with optical clearing compared to the imaging depth of samples prepared using more traditional methods (such as formalin fixation). In one embodiment, the sample is imaged using multiphoton microscopy (MPM). In another embodiment, the sample is imaged using confocal microscopy. In another embodiment, the sample is imaged using structured illumination microscopy. In another embodiment, the sample is imaged using selective plane illumination microscopy. In another embodiment, the sample is imaged using deconvolution microscopy. In another embodiment, the sample is imaged using super-resolution microscopy. In another embodiment, the sample is imaged using side illumination microscopy. In another embodiment, the sample is imaged using spinning disk confocal microscopy. In one embodiment, the method for imaging a tissue sample comprises the steps of obtaining a tissue sample, contacting the tissue sample with a fixing solution comprising at least one fluorescent dye, contacting the tissue sample with a clearing solution, and generating an image of the tissue sample by measuring intensity values ​​of the fluorescence of the tissue sample and converting the intensity values ​​into effective optical densities such that the optical densities reproduce the color of the stain in the generated image of the tissue sample.

[0102] The imaging method of the present invention provides a method for image analysis that allows for the reproduction of images that are substantially indistinguishable from conventional histological stains. In one embodiment, the method involves a multi-channel method in which intensity values ​​of fluorescence from a sample are converted to optical density using an exponential pseudo-color process that is the inverse of a logarithmic pseudo-color process in which intensity values ​​of fluorescence are converted to optical density in red, green, and blue channels. In one embodiment, the step of imaging the sample further comprises the step of measuring intensity values ​​of the fluorescence of the sample and converting these intensity values ​​to effective optical density to reproduce the color of the stain in the sample image. In one embodiment, the intensity values ​​of one or more fluorescence channels are converted to effective optical density in one or more pseudo-color display channels using an exponential pseudo-color process, wherein the equation for generating the optical density includes a constant raised to the power of the intensity value of the fluorescence, as will be understood by one of ordinary skill in the art. Numerically:

[0103] Ch1=C1^(a1N+b1P)

[0104] Ch2=C2^(a2N+b2P)

[0105] Ch3=C3^(a3N+b3P)

[0106] Where Ch1, Ch2, Ch3 are color display channels such as red, green, blue, etc.; C1, C2, C3 are positive constants; a1, a2, a3, b1, b2, b3 are constants that can be positive or negative; and N and P are the fluorescence intensities recorded from different fluorescence channels. In one embodiment, an exponential pseudo-color process is used to convert the intensity values ​​to effective optical density.

[0107] For an example of a logarithmic color deconvolution process, see Ruifrok and Johnston, 2001, Anal. Quant. Cytol. Histol. 23: 291-299, which is incorporated herein by reference in its entirety.

[0108] In one embodiment, the fluorescence is the intrinsic fluorescence from the sample. In another embodiment, the fluorescence is the fluorescence from a fluorescent dye. In one embodiment, the fluorescent dye is a nuclear dye. In another embodiment, the fluorescent dye is a protein dye. The number of channels used can be varied as needed to obtain the desired image, as will be understood by those skilled in the art. In one embodiment, the number of channels is two channels. In a specific embodiment, the two channels are an intrinsic fluorescence channel and a fluorescent nucleic acid dye channel. In another embodiment, the two channels are a fluorescent nucleic acid dye channel and a fluorescent protein staining channel. In a non-limiting example, the intrinsic fluorescence mainly emitted by cross-linked proteins and corresponding to the staining usually achieved by protein stains such as eosin can be enhanced by using formalin as a fixative, which is a feature that promotes the reproduction of normal colors by improving the signal-to-noise ratio of this channel and promoting separation from nucleic acid fluorescence.

[0109] The imaging method of the present invention also provides images of samples that simulate common pathological stains so as to accurately and efficiently interpret the images. Examples of pathological stains that can be reproduced using the method of the present invention include, but are not limited to, hematoxylin, eosin, Wright, Giemsa, Masson's trichrome, Jones, trichrome, periodic acid Schiff (PAS) and reticulin stains. Combinations of pathological stains can also be reproduced using the method of the present invention. In one embodiment, the combination of pathological stains is hematoxylin and eosin (H&E). In another embodiment, the combination of pathological stains is Wright and Giemsa.

[0110] In some embodiments, the step of imaging the sample is performed in combination with another imaging method to produce multimodal imaging. In one embodiment, another imaging method is high harmonic generation. High harmonic generation allows the reproduction of other special histological stains, such as collagen stains (such as trichrome) and silver stains (such as Jones stains). In one embodiment, high harmonic generation is second harmonic generation (SHG). SHG is produced by multiphoton excitation of asymmetric repeat proteins (such as collagen) and can be combined with imaging methods (such as MPM) for simple identification and quantification of collagen fibrosis and amyloid. In one embodiment, another imaging method is fluorescence lifetime imaging. Fluorescence lifetime imaging can be used to provide additional contrast in MPM by distinguishing fluorophores with different lifetime characteristics or by distinguishing fluorescence from SHG. In another embodiment, another imaging method uses a variety of fluorescent antibodies. A variety of fluorescent antibodies can be used to perform immunohistochemistry in uncut samples with a variety of antigens that can be detected on the same cells. In another embodiment, the imaging method is used in combination with a diode laser. See Dechet et al., 2003, J. Urol. 169:71-74 and Durfee et al., 2012, Opt. Express 20:13677-13683, each of which is incorporated by reference in its entirety. Other techniques known in the art for increasing the scan rate of the sample image may be used in the imaging step, as will be understood by one of ordinary skill in the art. Non-limiting examples include multi-beam scanning systems, spatiotemporal multiplexing, and temporal focusing. See Bewersdorf et al., 1998, Optics Express 23:655-657; Amir et al., 2007, Optics Express 32:1731-1733; Oron et al., 2005, Optics Express 13:1468-1476; and Zhu et al., 2005, Optics Express 13:2153-2159, each of which is incorporated by reference in its entirety. In another embodiment, the imaging step is performed in real time using video imaging.

[0111] The methods of the present invention provide clear, high-quality images of samples obtained at greater sample depths than the sample depths of more traditional histological methods (such as samples fixed with formalin alone). In one embodiment, a sample image is obtained at a sample depth of about 100 nm to about 2 cm. In another embodiment, a sample image is obtained at a sample depth of about 100 nm to about 500 μm. In another embodiment, a sample image is obtained at a sample depth of about 100 nm to about 1 cm. In another embodiment, a sample image is obtained at a sample depth of about 50 μm to about 500 μm. In another embodiment, a sample image is obtained at a sample depth of about 100 nm to about 100 μm. In another embodiment, a sample image is obtained at a sample depth of about 200 μm. In another embodiment, a sample image is obtained at a sample depth of about 100 μm.

[0112] In one embodiment, imaging the sample provides digital sample data. This digital data can then be stored for later distribution, such as for consultation and health records, thereby increasing the accessibility of the image for further evaluation or re-evaluation. In addition, digital sample data can maintain the integrity of the data, which is contrary to physical samples that may be lost or destroyed and cannot be stored digitally.

[0113] In one embodiment, the sample image is a three-dimensional (3-D) sample image. The 3-D sample image can be generated using any method known in the art, as will be understood by those skilled in the art. In one embodiment, the 3-D sample image is generated by a complete biopsy. In another embodiment, the 3-D sample image generated by the entire biopsy provides a quantitative method for diagnosing a disease. In another embodiment, the 3-D sample image of the present invention provides an easy identification of subtle morphological findings in the imaged sample. For example, compared with other two-dimensional (2-D) histological methods currently known in the art, the 3-D sample image improves the quantitative and qualitative analysis of fibrosis observed in various conditions such as cirrhosis, hypertensive nephropathy, interstitial lung disease, and ovarian cancer. In another embodiment, the 3-D sample image of the present invention is used to diagnose malignant growth. In one embodiment, the method of the present invention provides full rotation control of the 3-D sample image. The diagnosis of malignant growth generally relies on visualizing the growth pattern, particularly in glandular-based conditions such as prostate cancer and breast cancer. This analysis is primarily based on two-dimensional orientation, which may require a pathologist to look back and forth (hopefully) in successive segments in order to make a diagnosis. In these 2D methods, visual inspection may be further complicated due to the poor embedding and orientation differences of the sample. In a non-limiting example, the 3-D sample image of the present invention can be used to diagnose metastatic colon cancer in the liver and diagnose endometrial abnormalities. In another non-limiting example, the 3-D reconstruction from the MPM imaging of the tissue processed through transparency can be used on the tissue sample of the complete biopsy size, and can also be used to produce the quantitative characterization of collagen fibrosis. Other non-limiting examples using 3-D sample images include identifying the low-grade abnormality of glandular cell growth (such as in the case of prostate neoplasms and breast neoplasms), evaluating the depth of tumor infiltration (such as for determining the depth of muscle infiltration in bladder biopsy) and more completely quantitative evaluation of fibrosis, which is particularly important in kidney and liver biopsies.

[0114] Microscope system

[0115] In another aspect, the invention relates to a microscope system for 2-D and 3-D imaging of a sample. In one embodiment, the microscope system is suitable for use in the methods of the invention described elsewhere herein.

[0116] An exemplary microscope system may include a laser, an optional beam shaper, a rotating polygon, a scan lens, a tube lens, a microscope objective, a dichroic mirror A, a dichroic mirror B, a plurality of emission filters, a plurality of detectors, and a translation stage. The microscope system of the present invention may further include any components commonly used in other similar microscope systems, such as a viewfinder, a power supply, a stage manipulation mechanism, an autofocus mechanism, an automation system, etc.

[0117] The laser comprises a laser source that provides multiphoton excitation of a dye, for example, for sample labeling. The laser may be any suitable laser, such as a femtosecond or picosecond pulsed fiber laser, or any other short pulse laser having a center wavelength that is adjustable for optimal excitation of the dye. Alternatively, the center wavelength may be non-tunable, but may be selected to correspond to a wavelength suitable for exciting the dye used to stain the sample. In one embodiment, the center wavelength is 780 nm.

[0118] In one embodiment, the laser beam emitted by the laser passes through a beam shaper. The beam shaper expands the laser beam to the width required to illuminate the back aperture of the microscope objective. In another embodiment, the beam shaper can transform the laser beam profile. For example, a laser beam with a Gaussian beam profile can be transformed to have a flat-top profile after passing through a beam shaper, which results in more efficient illumination of the back aperture of the microscope objective. In other embodiments, a beam shaper is not used.

[0119] The laser beam is rapidly scanned at an angle by a rotating polyhedron having a plurality of mirrored surfaces. In various alternative embodiments, the laser beam can be rapidly scanned at an angle by any of several resonant scanning mechanisms that are commonly used for rapid beam scanning. Non-limiting examples of resonant scanning mechanisms for rapid beam scanning include resonant galvanometers and digital micromirror devices.

[0120] The rapidly scanned laser beam is imaged to the back aperture of the microscope objective using a scan lens and a tube lens 40. In one embodiment, the scan lens is a telecentric lens and the scan lens 38 may be an F-theta lens. In various embodiments, the scan lens is any suitable lens that, when used in conjunction with the tube lens, images the angularly scanned beam from the surface of the rotating polygon to the back aperture of the microscope objective.

[0121] In one embodiment, the microscope system includes a dichroic mirror so that the fluorescence collected by the objective lens can be reflected by the dichroic mirror to be collected by one or more detectors. Alternatively, the beam path can be selected so that the dichroic mirror is used to reflect the excitation laser beam to the rear aperture of the microscope objective to transmit the collected fluorescence to the detector. In one embodiment, the fluorescence or SHG can be collected as transmitted light with or without the use of a collection lens (not shown) and a detector. The detector can be any detector sensitive to the appropriate wavelength. For example, in one embodiment, the detector includes a photomultiplier tube. The system can further include a dichroic mirror or any number of additional dichroic mirrors as needed to send fluorescence of different colors to different detectors. In one embodiment, the system can further include any number of emission filters as needed, wherein the emission filter placed in front of the detector excludes stray laser and other light interference.

[0122] In various embodiments, the microscope system of the present invention includes at least one microscope objective. In one embodiment, the microscope objective is compatible with a high refractive index immersion medium. In various embodiments, the objective is compatible with a high refractive index immersion medium with an index between 1.45 and 1.6. In one embodiment, the microscope objective is compatible with a high refractive index immersion medium with an index of 1.54. In another embodiment, the microscope objective is compatible with an immersion medium that is a BABB clearing agent disclosed elsewhere herein. Alternatively, the microscope system has an inverted microscope arrangement that includes a microscope objective having a relatively wide and flat aspect on the side facing the sample so that the immersion fluid can be easily retained between the objective and the sample box.

[0123] The microscope objective can include a numerical aperture for high quality optical axial sectioning with a short depth of field. In one embodiment, the numerical aperture is at least 0.8. In another embodiment, the numerical aperture is at least 1.0. Alternatively, the microscope objective 41 includes a large field of view for imaging the sample. In one embodiment, the objective has a field of view of about 500 microns. In another embodiment, the microscope objective has a field of view greater than about 500 microns.

[0124] The sample can be placed on a translation stage for imaging. The position of the translation stage relative to the microscope objective can be adjusted to any position to image different areas of the sample and for adjusting the focus. For example, in various embodiments, the translation stage can be adjusted so that the working distance between the microscope objective and the sample is between 100 μm and 2 mm. In one embodiment, the working distance can be adjusted to at least 100 μm to obtain sufficient depth so that a complete optical section can be obtained based on surface irregularities in a sample with distinct peaks and valleys.

[0125] Methods of using the microscope system

[0126] In another aspect, the present invention relates to a method of imaging a sample using the microscope system of the present invention. The method passes a laser beam through the system so that the microscope objective focuses the laser beam on a single point on the sample placed on a translation stage. In some embodiments, a rotating polyhedron causes the laser beam to quickly scan a line across the sample. In other embodiments, a resonant galvanometer directs the laser beam to quickly scan a line across the sample.

[0127] In one embodiment, the laser beam is rapidly scanned (raster scanned) in a stepped linear manner across the sample so that continuous scans produce rectangular 2-D images. In another embodiment, the translation stage can be moved along the imaging plane after the rectangular 2-D image is obtained so that a second rectangular 2-D image can be obtained by rapidly scanning the laser beam in a stepped linear manner across the sample. The translation stage can be moved along the imaging plane in additional incremental steps so that multiple rectangular 2-D images are obtained before interrogating a complete cross-section of the sample at a given imaging plane. Multiple rectangular 2-D images are stitched together using any method known in the art, such as using microscopy software, to create a complete cross-sectional image of the sample. In one embodiment, 2-D images with partial overlap facilitate automatic assembly of cross-sections of the sample.

[0128] In further embodiments, the sample can be translated axially relative to the objective lens so that the process of acquiring a 2-D image at a given image plane can be repeated, thereby representing different cross-sectional images of the sample. In one embodiment, successive rectangular 2-D image cross sections are acquired before interrogating the entire volume of the sample.

[0129] Kit of the present invention

[0130] The present invention also includes a kit comprising components and explanatory materials that can be used in the method of the present invention, which describe methods for processing tissue samples as described elsewhere herein. The kit can include reagents that can be used to perform the method of the present invention. For example, the kit can include reagents such as fixatives, dyes, and clearing solutions. The kit can further include devices that can be used to perform the method of the present invention. For example, the kit can include a sample holding device and a microscope system of the present invention, as described elsewhere herein.

[0131] In one embodiment, the reagent is provided in a concentrated form so that the weight and size of the kit can be reduced and only a dilution solution is required for immediate use. In another embodiment, the kit further comprises a component (preferably sterile) of the reagent in a lyophilized form. For example, the component can be a pre-measured amount suitable for reconstitution and immediate use. The kit can further include one or more additional components, such as a reconstitution container, and additional reagents, such as deionized water, washing buffer, etc.

[0132] In certain embodiments, the kit includes explanatory materials. The explanatory materials may include publications, recordings, diagrams, or any other medium of expression that may be used to convey the usefulness of the device or kit described herein. The explanatory materials of the kit of the present invention may, for example, be attached to a package that contains instruments that may be necessary for one or more desired procedures. Alternatively, the explanatory materials may be sent separately from the package, or may be obtained electronically through a communication network (such as the Internet).

[0133] It has been found that high concentrations of certain protein dyes in protein-dense tissues significantly increase the absorption of fluorescent signals from deeper levels, which reduces contrast and degrades image quality. This phenomenon can be observed using any optical sectioning microscopy technique (such as confocal imaging, multiphoton imaging, and selective plane illumination) and supports the counter-intuitive inverse relationship that is the basis of certain methods of the present invention.

[0134] The absorption increase described above can affect nuclear specific fluorescent dye signal and protein specific fluorescent dye signal, depending on the relative emission and absorption spectrum of the dye adopted. However, the relative concentration of protein in tissue is much higher than the relative concentration of nucleic acid, and therefore, protein content and protein specific dye concentration are important driving factors for signal attenuation in the combined protein and nucleic acid staining assays. The reverse dye concentration and protein content method described herein can be applied to single stain assays and combined protein and nucleic acid stain assays. In the aforementioned combined stain assay, the protein specific stain concentration is reversely changed based on the sample protein content to benefit contrast and signal intensity not only in protein dye imaging but also in nuclear dye imaging.

[0135] Using fluorescent protein dye concentrations that are inversely related to the protein density of the tissue sample also provides the benefit of reducing waste and, therefore, reducing reagent costs by conserving dye when applied to imaging at any depth. However, when imaging beyond about 200 μm in protein-rich tissues, a signal reduction at certain depths caused by excessively high dye to protein content ratios associated with saturation can be observed. Therefore, additional image quality benefits may be observed in optical sectioning microscopy techniques that rely on imaging at such depths.

[0136] There may be a minimum level and a maximum level of dye concentration that can be applied regardless of protein concentration. At very low protein concentrations, there is a threshold level of dye concentration above which the protein is saturated and any additional stain is wasted. At very high protein densities, there is a minimum threshold of dye concentration required to achieve any dye penetration and fluorescent signal. Thus, this threshold may be in the middle range of tissue protein density, within which varying dye concentration inversely to protein density yields the greatest benefit.

[0137] As described in detail below, the methods of the present invention are particularly useful when processing and imaging tissue samples with relatively high (e.g., epithelial tissue) or low (e.g., renal tissue) protein content or in tissues where fibrosis is a diagnostic indicator (e.g., renal or liver tissue). The following table indicates examples of tissues and average protein content.

[0138] organize Approximate mass % of protein Fat 4 Connective tissue 37 Brain tissue 10 Heart tissue 17 lung 17 liver 18 kidney 18 Skeletal muscle 20 Pancreatic tissue 13 Cartilage 11 Yellow marrow 4 Red pulp 20 Bone tissue 22 skin 25 Ovarian tissue 14 Thyroid tissue 14

[0139] Although overall protein content can help determine dye concentration, protein density is of primary importance. For example, as mentioned in the examples, renal tissue is composed of tubular structures and is highly vascularized, which results in a protein density lower than that indicated by the level of its overall protein content. As discussed herein, deep tissue imaging of renal tissue accordingly tolerates dye concentrations higher than those in protein-dense tissues (such as epithelium).

[0140] Examples

[0141] Exemplary applications of the methods of the present invention include H&E-like images of kidney and skin at a certain depth obtained by multiphoton laser excitation after simultaneous fluorescent staining with the protein-specific fluorescent dye eosin and the nucleic acid-specific fluorescent dye DAPI. Although the two tissue types may appear to represent similar dense tissues on the surface, they differ significantly in terms of overall protein concentration. The kidney is composed of tubular structures and is highly vascular, which results in its overall protein content being significantly lower than that of the skin epithelium, which is composed of tightly packed cells, has relatively few blood vessels, and generally has an overlying layer composed of tightly packed keratin without vascularization. Relatively high concentrations of eosin dye (such as an alcohol stock solution of about 0.4%) in fluorescent kidney protein staining produce clear visualization of diagnostically relevant structures (such as the capillary walls of the blood filtering unit called the glomerulus), and images collected at depths exceeding about 1 mm are substantially unaffected by protein dyes located between the excited fluorophores and the fluorescence detector. The same high concentration of protein fluorescent dye causes both the eosin signal and the DAPI signal to be significantly dimmed in the image deeper than about 200 μm in the relatively protein-rich keratinized epithelial tissue. However, performing the same staining procedure as above but using eosin (about 0.04% alcohol stock solution) at a concentration of 1 / 10 of the above concentration allows sufficient contrast for protein structures at the surface, as well as more homogeneous bright and readable staining at depths of more than 300 μm. Therefore, it is preferred to change the concentration of the fluorescent dye used for protein staining based on the overall protein content of the sample.

[0142] The second exemplary application is the three-dimensional characterization of collagen in the liver. Although the liver is not an epithelial tissue, its protein content is relatively high, which is related to its high metabolic activity as a major detoxification organ. Collagen has the characteristics that can be used for imaging using multiphoton pulse laser excitation. When the intensity of the light pulse is high enough, such as when a femtosecond pulse laser is focused by a microscope objective, the energy of two photons that simultaneously arrive at the collagen fiber will be combined into a photon (half the wavelength) that is twice the energy. In the liver, the second harmonic generation (SHG) signal is almost solely derived from collagen, and can therefore be used as a quantitative measure of fibrosis. Fibrosis is a sign of liver damage in a variety of liver conditions, and is also an important clinical prognostic factor. Visualizing collagen in the entire liver sample is particularly desirable because it represents a more extensive and thus more accurate measure of sample collagen. Therefore, applying the method of the present invention to provide a detailed SHG analysis at a certain depth in liver tissue has noteworthy diagnostic and prognostic benefits.

[0143] Normalizing the refractive index of the entire tissue sample (such as by clearing) can further improve the collagen map of thick liver and other tissue samples. SHG analysis can be performed in parallel with protein and nucleic acid imaging, thereby reducing overall work and processing time, while maximizing the information content obtained by imaging. However, in this combined application, when using a wavelength between about 750nm and 900nm, the SHG signal can be severely attenuated at a certain depth when the concentration of eosin that stains proteins is high. When the SHG signal is collected in a transmission lens rather than by an excitation lens, its effect makes the surface excitation of SHG under a higher protein dye concentration cause the signal to decay more. In both cases, minimizing the eosin concentration during staining to a degree that still allows visualization of microscopic protein-rich structures helps to maximize the depth of the collagen signal detected simultaneously by SHG analysis.

[0144] For liver samples, using previously described staining methods, processing reagents, and multiphoton imaging systems, it was found that an alcoholic stock solution of eosin with a concentration of approximately 0.02% achieved a balance of protein signals that adequately reproduced the H&E stain while allowing imaging of collagen SHG at several hundred microns.

[0145] Incorporated by Reference

[0146] Other documents, such as patents, patent applications, patent publications, journals, books, articles, web page content, have been referenced and cited throughout this disclosure. All such documents are hereby incorporated herein by reference in their entirety for all purposes.

[0147] Equivalent form

[0148] Various modifications to the invention and many other embodiments of the invention in addition to the embodiments shown and described herein will become apparent to those skilled in the art based on the entire content of this document (including references to the scientific literature and patent literature cited herein). The subject matter of this article contains important information, illustrations, and guidance that can be adapted to practice the invention in its various embodiments and equivalent forms.

Claims

1. A method for processing a tissue sample, the method comprising: obtaining tissue samples; and The tissue sample is contacted with a solution comprising methanol, a fluorescent nuclear dye at a concentration between about 300 μM and about 800 μM, and a fluorescent protein dye at a concentration between about 0.025 nM and about 2.5 nM, the solution having a pKa between about 4.84 and about 5.

2. The method of claim 1, further comprising clearing the tissue sample with a clearing solution having a refractive index between about 1.5 and about 1.

57.

3. The method of claim 1, further comprising incubating the tissue sample in formalin prior to the contacting step.

4. The method of claim 3, wherein the tissue sample is incubated in formalin for at least 5 minutes.

5. The method of claim 1, further comprising generating an image of the tissue sample by measuring fluorescence of the tissue sample after the contacting step.

6. The method of claim 5, wherein the tissue sample is not exposed to chloroform prior to the generating step.

7. The method of claim 1, wherein the fluorescent nuclear dye comprises 2-(4-amidinophenyl)-1H-indole-6-carboxamidine (DAPI).

8. The method of claim 1, wherein the fluorescent nuclear dye comprises a Hoechst dye.

9. The method of claim 1, wherein the solution further comprises acetic acid.

10. The method of claim 1, wherein the clearing agent comprises benzyl alcohol / benzyl benzoate in a ratio of 1:

2.

11. The method of claim 1, wherein the contacting step is performed at a temperature between about 40°C and about 47°C.

12. The method of claim 3, wherein the incubating step is performed at a temperature between about 40°C and about 47°C.

13. The method of claim 1, further comprising storing the solution at a temperature between about -20°C prior to the contacting step.

14. The method of claim 1, wherein the fluorescent protein dye comprises eosin.

15. A method of imaging a tissue sample, the method comprising: Clearing unstained tissue samples with clearing agents; exposing the cleared unstained tissue sample with a pulsed laser to produce an image of second harmonic generation (SHG) molecules, the image being indicative of collagen content and distribution within the unstained tissue sample; contacting the clearing-treated unstained tissue sample with alcohol to remove the clearing agent; staining the unstained tissue sample with one or more fluorescent dyes in an alcohol solution; Clearing of stained tissue samples; The cleared stained tissue sample is imaged to produce an image of the fluorescent signal, the image being indicative of the amount and distribution of the stained molecules within the stained tissue sample.

16. The method of claim 15, wherein the cleared stained tissue is imaged using an optical sectioning microscope to obtain one or more fluorescence distribution level and intensity information.

17. The method of claim 15, wherein the clearing agent has a refractive index of about 1.47 or greater.

18. The method of claim 15, wherein the unstained tissue sample is fixed prior to the clearing step.

19. The method of claim 18, wherein the unstained tissue sample is fixed in formalin.

20. The method of claim 15, wherein the pulsed laser comprises a wavelength of approximately 780 nm.

21. The method of claim 15, wherein the cleared unstained tissue sample is exposed to the pulsed laser and the image of the second harmonic generation (SHG) molecules is produced through a single objective lens.

22. The method according to claim 15, wherein the transparentizing step comprises: incubating the unstained tissue sample in methanol for a period of time sufficient to substantially replace water in the sample with methanol; and The incubated unstained tissue sample is immersed in the clearing agent, wherein the clearing agent comprises a high refractive index clearing fluid.

23. The method of claim 22, wherein the high refractive index clearing fluid comprises benzyl alcohol / benzyl benzoate in a ratio of 1:

2.

24. The method of claim 22, wherein the high refractive index clearing fluid comprises ethyl cinnamate.

25. The method of claim 22, wherein the incubation step is between about 5 minutes and about 8 hours.

26. The method of claim 22, wherein the immersing step is between about 5 minutes and about 1 hour.

27. The method of claim 15, wherein the one or more fluorescent dyes comprise a fluorescent nuclear dye and a fluorescent protein dye.

28. The method of claim 27, wherein the fluorescent nuclear dye comprises 2-(4-amidinophenyl)-1H-indole-6-carboxamidine (DAPI).

29. The method of claim 27, wherein the fluorescent protein dye comprises eosin.

30. The method of claim 15, wherein the alcohol solution comprises methanol and acetic acid.

31. The method of claim 15, wherein the coloring and clearing steps are performed at between about 37°C and about 50°C.

32. The method of claim 16, wherein the optical sectioning microscope is selected from the group consisting of a confocal microscope, a selective plane illumination microscope, a deconvolution microscope, and a multiphoton microscope.

33. A method of non-microscopic imaging, the method comprising the steps of: substantially fixing a biological sample; obtaining an image of the sample; storing the image in a database along with non-image-based identification information; and The non-image based information is recalled from the database and the newly acquired non-microscopic image of the biological sample is matched to the image stored in the database.

34. The method of claim 33, wherein the image is obtained using a camera.

35. The method of claim 33, wherein the image is stored in the database in the form of a fluorescent image.

36. The method of claim 33, wherein the non-image-based identification information is selected from the group consisting of a digital identifier, an optical barcode, a patient's name, and a collector's name.

37. The method of claim 33, wherein the newly acquired non-microscopic image is of the same type as the image stored in the database.

38. The method of claim 33, wherein the newly acquired non-microscopic image is of a different type than the type of the images stored in the database.

39. The method of claim 33, wherein the image of the sample is pre-processed to adjust a property selected from the group consisting of brightness, contrast, and color balance.

40. The method of claim 33, wherein the biological sample is a core needle biopsy, a surgical resection, or a section of tissue mounted on a microscope slide.

41. The method of claim 33, wherein one or more newly acquired images are matched to images stored in the database by using image cross-correlation.

Images