A method for optically clearing tissue samples using an embedding medium.

Abstract

The present invention relates to a method for preparing cleared tissue samples of biological or human tissue for examination under an optical microscope, comprising the steps of: a) dehydrating the tissue sample using a dehydrating solvent; and b) transferring the dehydrated tissue sample to an embedding medium containing benzaldehyde anisol ether for clearing. Compared to the conventional embedding media, benzyl benzoate / benzyl alcohol mixtures and benzyl salicylate / methyl benzoate mixtures, benzaldehyde anisol ether has the advantage of being available as a pure substance, just like dibenzyl ether. Because the pure substance already has the desired refractive index, there is no need to mix the embedding medium to set the refractive index. Furthermore, the benzaldehyde anisol ether of the present invention penetrates dehydrated tissue more rapidly than conventional embedding media and can clear tissue more quickly.

Description

[[Technical Field]]

[0001] The present invention relates to a method according to the preamble of claim 1, a kit for preparing a biological tissue sample according to claim 10, and the use according to claim 13. [[Background Art]]

[0002] A transparent biological tissue sample is required to enable three-dimensional imaging of a tissue sample, for example by light sheet microscopy. In order to achieve transparency of a biological sample, it is particularly necessary to remove heme groups of the hemoglobin, which is a blood pigment, and lipids from the biological sample. In the "dehydration" process, the tissue is treated with various mixtures of a water-miscible organic solvent and water. This treatment is carried out with organic solvents in gradually increasing concentrations to completely remove water from the tissue. Many options exist here, for example, tetrahydrofuran, methanol, isopropanol, tert-butanol and ethanol. Ethanol is currently the most commonly used dehydration medium for clearing pathological tissues. The end result of all "dehydration" procedures is an anhydrous sample.

[0003] The final step in various tissue claritying methods is to adjust the refractive index to match that of the tissue to be observed under a microscope. According to Spalteholz ("On the transparency of human and animal preparations," S. Hirzel, 1911, Deutsches Reichs-Patent No.229044), the refractive index of dehydrated tissue is n=1.547 for bone. The refractive index of other tissues can be estimated to be approximately n=1.551, based on the optimal mixture he empirically determined. To obtain such high refractive indices, it is generally necessary to use aromatic compounds that are either immiscible or only slightly miscible with water. For his research, Spalteholz used a mixture of wintergreen oil (methyl salicylate) and benzyl benzoate or isosafrole in mixing ratios suitable for various tissues.

[0004] A variation of Spalteholz's method is known as “Ultramicroscopy: three-dimensional visualization of neuronal networks in the whole mouse brain” by Dodt, Leischner, Schierloh, Jahrling, Mauch, Deininger, Deussing, Eder, Zieglgansberger, and Becker (Nat Methods, 2007;4(4):331-6). Here, “Murray's clear” (a 2:1 mixture of benzyl benzoate and benzyl alcohol with a refractive index n=1.559) is used as the implantation medium. Another variation is known as “Chemical Clearing and Dehydration of GFP Expressing Mouse Brains” by Becker, Jahrling, Saghafi, Weiler, and Dodt H (2012)PLoS ONE 7(3):e33916, https: / / doi.org / 10.1371 / journal.pone.0033916). Spalteholz's embedding medium and many other aromatic compounds were also tested. Dibenzyl ether (DBE, refractive index n=1.562) proved to be the most suitable compound and has become the de facto representative standard in tissue clearing. The advantage of DBE is its ease of use, as there is no need to mix the solution and then check the refractive index, and it can be used directly as a pure substance.

[0005] WO2017 / 093323A1 discloses the use of ethyl cinnamate (ECi) and related cinnamic esters as a non-toxic alternative to previously used embedding media. It exhibits lower toxicity than other aromatic compounds used in tissue treatment, such as dibenzyl ether, benzyl alcohol, or benzyl benzoate. Since the desired refractive index is achieved by the pure substance, the mixing step can be eliminated, thereby eliminating a working step that could be a source of error.

[0006] Thus, currently, the only suitable pure liquid substances for embedding dehydrated samples are two compounds: dibenzyl ether and ethyl cinnamate. However, both of these substances have drawbacks. Dibenzyl ether is toxic. Ethyl cinnamate is less toxic, but its melting point is 6-9°C, so samples clarified with ethyl cinnamate cannot be stored in a refrigerator because the embedding medium crystallizes. In addition, ethyl cinnamate has a relatively high vapor pressure. [Overview of the Initiative]

[0007] The object of the present invention is to overcome the above-mentioned drawbacks and, in particular, to provide a method for preparing transparent tissue samples of biological tissue for examination by optical microscope, which requires less effort than producing a mixture as an embedding medium, avoids highly toxic embedding mediums, and allows for refrigerated storage of dehydrated tissue samples.

[0008] This objective is achieved by the method described in claim 1, in accordance with the present invention. This objective is further achieved by the kit described in claim 9 and the use described in claim 11. Further embodiments are the subject of the dependent claims or are described below.

[0009] The method according to the present invention for preparing transparent tissue samples of biological tissue for examination by optical microscope is: a) A step of dehydrating the tissue sample using a dehydrating solvent, b) A step of making the dehydrated tissue sample transparent by placing it in an embedding medium of a liquid having a refractive index matching that of the tissue, Includes. The embedding medium comprises benzaldehyde anithole ether, and is preferably selected from 3-methoxybenzaldehyde, 4-methoxybenzaldehyde, 2-hydroxy-5-methoxybenzaldehyde, and 4-ethoxybenzaldehyde.

[0010] The objective of the method according to the present invention is to achieve optical clarity of biological or human tissue samples for optical microscopy. Biological tissues include, for example, human or animal tissues.

[0011] The clearing step in the clearing method involves adjusting the refractive index to that of the tissue to be observed under a microscope. For this purpose, the tissue sample is transferred to an embedding medium, which is a solution for adjusting the refractive index. The embedding medium must be miscible with the solvent used in the dehydration step.

[0012] In one embodiment, the embedding medium comprises 10 to 100 volume percent of benzaldehyde ether and 0 to 90 volume percent of an optically suitable inert organic solvent having a refractive index of about 1.3, preferably about 1.5. For example, the embedding medium comprises 90 to 100 volume percent of benzaldehyde ether.

[0013] In other embodiments, the embedding medium comprises 10 to 100 volume percent of benzaldehyde ether and 0 to 90 volume percent of an optically suitable inert organic solvent having a refractive index of about 2.0, preferably about 1.65. For example, the embedding medium comprises 90 to 100 volume percent of benzaldehyde ether. All percentages according to this invention are in volume percent (vol%).

[0014] In a preferred embodiment, the embedding medium consists of the benzaldehyde nithole ether according to the present invention, i.e., the benzaldehyde nithole ether is used as a pure substance. In this specification, "pure substance" is understood to mean benzaldehyde nithole ether of technically available purity.

[0015] Benzaldehyde ether is benzaldehyde anisole ether. Preferably, the benzaldehyde anisole ether is selected from 3-methoxybenzaldehyde (methanisaldehyde; CAS number 591-31-1), 4-methoxybenzaldehyde (paraanisaldehyde; CAS number 123-11-5), 2-hydroxy-5-methoxybenzaldehyde (6-hydroxy-m-anisaldehyde; CAS number 672-13-9), and 4-ethoxybenzaldehyde (homoanisaldehyde; CAS number 10031-82-0). 3-methoxybenzaldehyde has a purity of about 97.0% as an industrial product. 4-methoxybenzaldehyde has a purity of about 97.0% as an industrial product. 2-Hydroxy-5-methoxybenzaldehyde has a purity of approximately 98.0% as an industrial product. 4-Ethoxybenzaldehyde has a purity of approximately 99% as an industrial product.

[0016] The purpose of dehydration step a) is to obtain an anhydrous tissue sample. In one embodiment, to remove water from the tissue, the tissue sample is preferably treated in multiple steps using various dehydration compositions in a descending water series, i.e., a mixture in which the proportion of water-miscible organic solvents gradually increases. Alcohols, ketones, or ethers are used as the dehydration medium. Suitable dehydration solvents include, for example, ethanol, methanol, isopropanol, tert-butanol (IUPAC: 2-methylpropan-2-ol), tetrahydrofuran, or acetone.

[0017] In this specification, the dehydrating medium has the following characteristics. 1. The fixative is completely miscible with water so that it can be gradually removed from the tissue in an increasing concentration series. 2. Completely miscible with an implantation medium whose refractive index matches that of dehydrated tissue.

[0018] In an alternative embodiment, dehydration step a) is carried out using 2,2-dimethoxypropane (DMP), which removes water from the tissue by a chemical reaction with the water present in the tissue.

[0019] In the method according to the present invention, in order to obtain an anhydrous tissue sample, dehydration step a), in one embodiment, - consists of aqueous ethanol with a gradually increasing ethanol concentration, wherein the ethanol concentration of the dehydration composition ranges from 30% by volume to 100% by volume, or - consists of aqueous tetrahydrofuran with a gradually increasing tetrahydrofuran concentration, wherein the tetrahydrofuran concentration of the dehydration composition ranges from 30% by volume to 100% by volume, or - consists of aqueous methanol with a gradually increasing methanol concentration, wherein the methanol concentration of the dehydration composition ranges from 30% by volume to 100% by volume, or - consists of an aqueous mixture of another alcohol, ketone or ether, wherein the solvent concentration of the dehydration composition ranges from 30% by volume to 100% by volume, comprises use of the dehydration composition.

[0020] In an alternative embodiment of the method according to the present invention, dehydration step a) for obtaining a tissue sample having a residual moisture content of >2%, - consists of aqueous ethanol with a gradually increasing ethanol concentration, wherein the ethanol concentration of the dehydration composition ranges from 50% by volume to 98% by volume, preferably from 70% by volume to 98% by volume, more preferably from 75% by volume to 98% by volume, or - consists of an aqueous mixture of another alcohol, ketone or ether, wherein the solvent concentration of the dehydration composition ranges from 50% by volume to 98% by volume, preferably from 70% by volume to 98% by volume, more preferably from 75% by volume to 98% by volume, is carried out using a dehydration composition.

[0021] The dehydration composition used preferably still contains 2 to 30% by volume of water even in the final stage of the dehydration step. In this embodiment, for example, denatured ethanol having a residual moisture content of 4 to 6% by volume can also be used.

[0022] In a further alternative embodiment of the method according to the present invention, the dehydration step a) is performed by a gradient mixer. For this purpose, the tissue sample is placed in a mixing vessel (gradient mixer). The dehydration solvent is introduced through an inlet until a residual moisture content of 5 to 20% by volume is reached. The dehydration solvent may have a solvent concentration of 100% by volume or a residual moisture content of 2 to 50% by volume. In a variation, the tissue sample in the gradient mixer is first directly introduced into a dehydration solvent with a solvent concentration of 50% by volume, after which the gradient is gradually increased.

[0023] Preferably, the tissue sample is transferred directly from the gradient mixer into the embedding medium without being pre-incubated in a high-purity solvent. Preferably, no incubation step in high-purity solvent is performed in step a) and step b).

[0024] It is surprising that clearing of tissue samples is possible by such a method. According to the prior art, the method in a gradient mixer is as follows. A tissue sample is introduced into a mixing vessel (gradient mixer). The mixing vessel has, for example, an inlet at the top and an outlet at a side. When a sample in 50% ethanol is placed in this mixing vessel and introduced through the inlet at a high concentration (95% by volume or more of solvent) using an infusion pump, a gradient that asymptotically approaches a value of 100% by volume is subsequently formed. This provides a sample in which the ethanol content increases steadily and advantageously. Since 100% cannot be reached in the mixture, the sample is usually subsequently incubated in high-purity ethanol. According to the prior art, this incubation step is repeated multiple times. Therefore, in practice, for cost reasons, the infusion pump can be regularly refilled with industrial grade ethanol.

[0025] In the method of the present invention, 1. the final (repeated if necessary) incubation step using absolute ethanol is eliminated, 2. for an ethanol content of about 80% or more, the endpoint reached using the gradient mixer is sufficient to further clear the tissue sample in step b).

[0026] In further alternative modifications, dehydration step a) is performed in a single pass. Instead of using a dehydration series of two or more passes, dehydration in step a) is performed in a single pass by placing the tissue sample in a dehydrating solvent having a solvent concentration of at least 70% by volume. This embodiment is particularly suitable for tissue samples with compact tissue that absorbs little water. In the next step b), the tissue sample is immediately placed in the embedding medium without incubation in a high-purity solvent for complete dehydration. This achieves a significant reduction in time and a substantial saving of reagents. The savings in reagents are, firstly, due to the smaller amount of solvent required. Secondly, there is no need to use high-purity anhydrous solvent; instead, industrial-grade solvents can be used. For example, in the case of ethanol, there is no need to use expensive 100% high-purity ethanol; instead, significantly cheaper denatured ethanol can be used. According to the prior art, incubation in a high-purity anhydrous solvent is repeated to completely remove any remaining moisture, and in many protocols, incubation in the embedding medium is repeated for the same reason. In both embodiments of the present invention, the final incubation to ensure the removal of residual moisture from the tissue is omitted, and reagents are saved by the method of the present invention.

[0027] In one embodiment of the method according to the present invention, the tissue sample is dehydrated in step a) and, before being optically cleared in step b), • Fixed and / or • Fixed with formaldehyde and / or • Washed and / or, • Washed with water and / or, • Incubated in an alkaline aqueous solution containing a nonionic detergent, and / or • Degreased with a detergent solution, and / or • Degreased with an organic solvent (degreased), and / or • Bleached with an oxidizing agent and / or, • It is decolorized by amino alcohol.

[0028] In one embodiment of the method according to the present invention, the tissue sample is fixed before being dehydrated in step a) and optically cleared in step b), The fixative is - Crosslinking fixatives such as formaldehyde, glutaraldehyde, acrolein, carbodiimide, diethyl pyrocarbonate, bisimide esters or glyoxal or mixtures thereof, and / or - Coagulation and fixing agents such as alcohols and other organic solvents, acids, potassium dichromate, lead nitrate, copper sulfate and mercury chloride, and mixtures thereof. Selected from.

[0029] The method according to the present invention is preferably carried out after the tissue preparation step and after the electrophoresis step. Therefore, the tissue sample to be processed in the method according to the present invention is already pre-treated. Pre-treatment by electrophoresis is preferably carried out according to the electrophoretic clearing method described in DE 10 2016 123 458 B3. For the performance of electrophoretic clearing, refer to the patent specification DE 10 2016 123 458 B3, the contents thereof shall be incorporated herein.

[0030] In the method according to the present invention, the optically cleared tissue sample is preferably examined under a microscope in a further step to obtain an image of the internal structure of the sample. The microscope is an optical microscope, preferably a light sheet microscope, a confocal microscope, a two-photon microscope, or an optical projection tomography microscope (OPT).

[0031] Dibenzyl ether (DBE), an embedding medium used to date, has a refractive index of n=1.562. Ethyl cinnamate, a second pure substance, has a refractive index of n=1.559. Among the benzaldehyde anisole ethers according to the present invention, for example, 3-methoxybenzaldehyde has a refractive index of n=1.552, and 2-hydroxy-5-methoxybenzaldehyde has a refractive index of n=1.580. By using the benzaldehyde-anisole ether according to the present invention as an embedding medium for tissue clearing of dehydrated tissue samples, transparency at least equivalent to, and in some cases even better than, that of tissue samples treated with methyl salicylate / benzyl benzoate and ethyl cinnamate was obtained.

[0032] Spalteholz's research has already suggested that the refractive indices of different tissues differ slightly from one another. In his original work published in 1911, he describes a series of human tissues in descending order of refractive index, empirically determined based on the optimal mixing ratio of methyl salicylate and BB: young embryo (5:1-3:1 depending on weight) < adult demineralized bone (5:3) n=1.547 < adult muscle tissue (2:1) which is almost the same as that of an older embryo < brain and spinal cord (1:1). This variability directly affects tissue examination using clarity and microscopic examination of different tissues, and in particular, quantitative spectroscopic determination that can be performed using a microscope. The method according to the present invention offers the advantage of using various pure substances, which can be selected depending on the tissue to approximate the refractive index of the tissue, as the implantation medium.

[0033] Compared to benzyl benzoate / benzyl alcohol mixtures (BABB) and methyl salicylate / benzyl benzoate mixtures, which have been commonly used as embedding media, benzyl aldehyde anisol ether, like dibenzyl ether, has the advantage of being usable as a pure substance. Because it is a pure substance, the desired refractive index is already achieved, so there is no need to mix the embedding media to adjust the refractive index. This eliminates the mixing step and, therefore, eliminates extra work steps and the potential for errors.

[0034] Remarkably, the benzaldehyde anisol ether according to the present invention penetrates dehydrated tissue much faster than conventionally known embedding media, rapidly clearing the tissue. This saves time in preparing the sample and allows for faster examination of the sample with an optical microscope. Consequently, the results of the optical microscope examination can also be obtained more quickly. The density of the dehydrated tissue is determined by the density of the dehydrating solvent remaining in the tissue, such as ethanol. Generally, embedding media have a higher density, so the complete penetration of the embedding media into the tissue sample is easily monitored by the tissue sample settling in the embedding media. For example, in the case of 2-hydroxy-5-methoxybenzaldehyde, the embedding media achieves complete penetration into the tissue sample after 30-45 minutes, i.e., the tissue sample lies at the bottom of the container. On the other hand, when using conventional embedding media such as dibenzyl ether or methyl salicylate-benzyl benzoate mixtures, this step typically requires several hours to overnight. Therefore, the method according to the present invention offers the advantage that the embedding medium diffuses more rapidly into the tissue of the tissue sample, resulting in a significant reduction in sample preparation time. Consequently, the tissue sample can be examined more quickly using an optical microscope.

[0035] When a tissue sample containing residual moisture is transferred to an organic solvent used as an embedding medium, in which this moisture can no longer be dissolved, the moisture becomes trapped within the tissue, leading to turbidity that negatively affects clarity due to incomplete mixing. Therefore, tissue samples have traditionally been incubated multiple times in, for example, high-purity ethanol, and then often repeatedly incubated in the embedding medium, in order to prevent adverse effects on the clarity of the sample. According to prior art, as already described, incubation in high-purity anhydrous solvent was repeated to completely remove any remaining moisture, and in many protocols, incubation in the embedding medium was repeated for the same reason.

[0036] According to the present invention, tissue clearing is performed using benzaldehyde anisol ether. Surprisingly, it was found that good clarity could be obtained even with tissue samples containing residual moisture. At small residual moisture levels of 2-5 vol%, no adverse effect on clarity was observed, and even at high moisture levels of up to 20 or 30 vol%, depending on the type of tissue treated, only very slight turbidity was observed, making optical microscopy of the sample still possible.

[0037] Therefore, unlike all conventionally known embedding media, the method according to the present invention provides a particularly quick and simple method for dehydrating tissue samples, as low residual moisture content of up to 10% by volume does not impair the results of optical microscopy, and even higher residual moisture content results in acceptable transparency of the tissue sample.

[0038] Furthermore, the method according to the present invention simplifies the procedure compared to conventional clearing methods using embedding media. According to the prior art, tissue samples are transferred from container to container during dehydration, while increasing the concentration of the dehydrating solvent, for example, by increasing the ethanol concentration. Therefore, a considerable number of individual steps are required, and these steps are discrete. Moreover, the use of a high-purity solvent, for example, high-purity ethanol, is always required in the last one or more steps.

[0039] In the method according to the present invention, it is also possible to use a dehydrating solvent containing residual water. For example, instead of high-purity ethanol, modified 95-97% industrial-grade ethanol can be used. Therefore, the method according to the present invention also leads to a reduction in the cost of the solvent used.

[0040] Furthermore, when using a gradient mixer, the method according to the present invention does not require subsequent complete dehydration with a high-purity solvent, thus simplifying the procedure.

[0041] Benzaldehyde anisol ether also has low toxicity. This is advantageous when used as an implantation medium, as the tissue sample in the microscope is located directly below the user's airway and the user may inhale vapors rising from the sample. According to the European Chemicals Agency (ECHA), the toxicity of para-anisaldehyde is not toxicly problematic, unlike dibenzyl ether or benzyl benzoate / benzyl alcohol mixtures.

[0042] The kit according to the present invention for the preparation of biological tissue samples for optical microscopy is - A dehydrating solvent for dehydrating tissue samples, - comprising an embedding medium for making a dehydrated tissue sample transparent by placing it in the embedding medium, The embedding medium is the benzaldehyde anithole ether selected from 3-methoxybenzaldehyde, 4-methoxybenzaldehyde, 2-hydroxy-5-methoxybenzaldehyde, and 4-ethoxybenzaldehyde. The benzaldehyde anithole ether is preferably used at the concentrations and purities described above.

[0043] The dehydrating solvent in this kit is an ether, ketone, or alcohol, and preferably the solvent is selected from ethanol, methanol, isopropanol, tert-butanol, 2,2'-thiodiethanol, trichloroethanol, tetrahydrofuran, and acetone.

[0044] According to the present invention, a benzaldehyde anithole ether selected from 3-methoxybenzaldehyde, 4-methoxybenzaldehyde, 2-hydroxy-5-methoxybenzaldehyde, and 4-ethoxybenzaldehyde is used as an implantation medium for the preparation of biological, particularly human, tissue samples for examination by optical microscope. The benzaldehyde anisol ether used in accordance with the present invention is preferably used at the concentrations and purities described above. [Examples]

[0045] (Example 1) Approximately 0.25ml (0.5×0.5×1cm 3 Several tissue samples (pork lungs) with the following volume were dehydrated with anhydrous ethanol and photographed. The tissue samples dehydrated with ethanol are shown on the left side of Figure 1.

[0046] Subsequently, the dehydrated tissue samples were placed in 5 ml of each embedding medium for 3 hours. The embedding mediums used were 2-hydroxy-5-methoxybenzaldehyde according to the present invention, and, as a comparative example, a mixture of methyl salicylate and benzyl benzoate adjusted to the same refractive index as 2-hydroxy-5-methoxybenzaldehyde. The results of the clearing step were also photographed in the same manner. The results are shown on the right side of Figure 1. Clearing using methyl salicylate / benzyl benzoate already shows good transparency of the tissue sample. However, the transparency achieved using 2-hydroxy-5-methoxybenzaldehyde of the present invention was even better.

[0047] (Example 2) As the embedding medium, 2-hydroxy-5-methoxybenzaldehyde, ethyl cinnamate, and dibenzyl ether were used, in approximately 0.25 ml (0.5 × 0.5 × 1 cm). 3 Several tissue samples (pork lungs) having the volume of ) were prepared as described in Example 1.

[0048] Next, the tissue samples were immersed overnight in anhydrous ethanol, after which the embedding medium was washed out of the tissue samples. The tissue samples were transferred to ethanol with a specified residual moisture content to achieve a specified residual moisture content in the tissue, incubated for 2 hours, and then returned to each embedding medium for imaging. The tissue samples were placed in ethanol with ethanol content of 95 vol%, 90 vol%, and 80 vol%. The results were imaged and are shown in Figure 2. The first row shows the tissue sample in the 100% dehydrated embedding medium, the second row shows the tissue sample in the embedding medium incubated in ethanol with a moisture content of 5 vol%, the third row shows the tissue sample in the embedding medium incubated in ethanol with a moisture content of 10 vol%, and the fourth row shows the tissue sample in the embedding medium incubated in ethanol with a moisture content of 20%. The bottom row shows the same tissue sample in anhydrous ethanol, i.e., before being placed in each embedding medium. When completely dehydrated in ethanol, all samples become equally opaque.

[0049] This indicates that the embedding media known from prior art, dibenzyl ether and ethyl cinnamate, achieve the desired transparency only when there is no residual moisture or only a very small amount of residual moisture. In the case of DBE, at a residual moisture content of 5 vol%, the sample is no longer transparent and therefore cannot be examined under a microscope. With ECi, when the residual moisture content reaches 95%, cloudiness clearly occurs, and the tissue sample is no longer completely transparent. On the other hand, tissue samples cleared with 2-hydroxy-5-methoxybenzaldehyde remained completely transparent even at a residual moisture content of 10 vol% (90 vol% ethanol), and showed only slight cloudiness at a residual moisture content of 20 vol% (80 vol% ethanol).

[0050] (Example 3) Approximately 0.25ml (0.5×0.5×1cm 3A tissue sample having a volume of ) was dehydrated with anhydrous ethanol. The tissue sample was then transferred to an embedding medium in a sample container. The embedding mediums used were 2-hydroxy-5-methoxybenzaldehyde according to the present invention, and, as comparative examples, dibenzyl ether and a mixture of methyl salicylate and benzyl benzoate. In the case of 2-hydroxy-5-methoxybenzaldehyde, the tissue sample clearly sank below the liquid surface in just 15 minutes, and depending on the type of tissue, it was observed to lie at the bottom of the container after 30-45 minutes. With dibenzyl ether, it took several hours for the tissue sample to sink to the bottom, which was about the same time required as for the mixture of methyl salicylate / benzyl benzoate and DBE.

[0051] In further experiments, the rate at which the dehydrating solvent ethanol in tissue samples was replaced by the embedding medium was measured. The embedding mediums used were 2-hydroxy-5-methoxybenzaldehyde according to the present invention, and, as comparative examples, dibenzyl ether and ethyl cinnamate. Tissue samples were placed in the embedding medium for 15 minutes, 30 minutes, and 70 minutes, and then photographed. The results are shown in Figure 3. The top row shows the tissue samples after clearing for 15 minutes. The middle row shows the tissue samples after clearing for 30 minutes. The bottom row shows the tissue samples after clearing for 70 minutes. It can be seen that the tissue samples treated with 2-hydroxy-5-methoxybenzaldehyde were already clear after 70 minutes, the samples treated with ECi were only slightly clear, and the tissue samples treated with DBE were not yet clear at all. Thus, the clearing treatment according to the present invention achieves remarkably rapid clearing of tissue samples.

[0052] The present invention is not limited to one of the embodiments described above, and can be modified in various ways. All features and advantages, including processing steps, that emerge from the claims, description, and drawings may be essential to the present invention, both individually and in a wide variety of combinations. [Brief explanation of the drawing]

[0053] [Figure 1]This is a photograph of the tissue sample taken in Example 1. [Figure 2] This is a photograph of the tissue sample taken in Example 2. [Figure 3] This is a photograph of the tissue sample taken in Example 3.

Claims

1. A method for preparing a transparent tissue sample of biological tissue for examination by optical microscope, a) A step of dehydrating the tissue sample using a dehydrating solvent, b) A method comprising the step of clearing the dehydrated tissue sample by placing it in an embedding medium containing a benzaldehyde anithole ether selected from 3-methoxybenzaldehyde, 4-methoxybenzaldehyde, 2-hydroxy-5-methoxybenzaldehyde, and 4-ethoxybenzaldehyde.

2. The method according to claim 1, wherein the embedding medium comprises 10 to 100 volume percent of benzaldehyde nisol ether and 0 to 90 volume percent of an optically suitable inert organic solvent having a refractive index of about 1.3, preferably 1.

5.

3. The method according to claim 1, wherein the embedding medium comprises 10 to 100 volume percent of benzaldehyde nisol ether and 0 to 90 volume percent of an optically suitable inert organic solvent having a refractive index of about 2.0, preferably 1.

65.

4. The method according to any one of claims 1 to 3, wherein the embedding medium comprises a benzaldehyde anithole ether selected from 3-methoxybenzaldehyde, 4-methoxybenzaldehyde, 2-hydroxy-5-methoxybenzaldehyde, and 4-ethoxybenzaldehyde.

5. The method according to any one of claims 1 to 4, wherein the dehydration step a) is carried out by a gradient mixer, the tissue sample is placed in the gradient mixer, the dehydrating solvent is introduced from the inlet until the residual moisture content reaches 5 to 20 volume%, and the method comprises using a dehydrating composition comprising an aqueous alcohol, ketone or ether, wherein the dehydrating composition is an aqueous mixture of alcohol, ketone or ether having a solvent concentration of 50 to 98 volume%, preferably 70 to 98 volume%, more preferably 75 to 98 volume%, preferably an aqueous mixture of aqueous ethanol having an increasing ethanol concentration, and the ethanol concentration of the dehydrating composition is in the range of 50 to 98 volume%,.

6. The method according to any one of claims 1 to 4, wherein the dehydration step a) is performed in a gradient mixer, the tissue sample is directly introduced into a dehydrating solvent having a solvent concentration of 50% at the start of the dehydration step, and thereafter the gradient is increased in small increments.

7. Before the tissue sample is dehydrated in step a) and optically cleared in step b), - Fixed and / or, - Fixed using formaldehyde, and / or • Washed and / or, • Washed with water and / or, • Incubated in an alkaline aqueous solution and / or, - Degreased using a detergent solution and / or, - Degreased using an organic solvent, and / or - Bleached using an oxidizing agent, and / or - Decolorized using amino alcohol, The method according to any one of claims 1 to 6.

8. The tissue sample is fixed before it is dehydrated in step a) and optically cleared in step b), The fixative is - Crosslinking fixatives such as formaldehyde, glutaraldehyde, acrolein, carbodiimide, diethyl pyrocarbonate, bisimide esters or glyoxal or mixtures thereof, and / or - Selected from coagulation fixatives such as alcohols and other organic solvents, acids, potassium dichromate, lead nitrate, copper sulfate, mercury chloride, and mixtures thereof, The method according to any one of claims 1 to 6.

9. In a further step, the optically cleared tissue sample is examined under a microscope to obtain an image of the internal structure of the sample. The aforementioned microscope is an optical microscope. The method according to any one of claims 1 to 8.

10. A kit for preparing biological tissue samples for optical microscopy, - A dehydrating solvent for dehydrating the tissue sample, - An embedding medium for making the dehydrated tissue sample transparent by placing it within the embedding medium, Equipped with, The dehydrating solvent exists in the form of a dehydrating composition in a descending water series, which is a series in which the water content decreases, i.e., a mixture in which the proportion of a water-miscible organic solvent selected from ethanol, isopropanol, or tert-butanol gradually increases. The kit wherein the embedding medium is a benzaldehyde anithole ether selected from 3-methoxybenzaldehyde, 4-methoxybenzaldehyde, 2-hydroxy-5-methoxybenzaldehyde, and 4-ethoxybenzaldehyde.

11. The kit according to claim 10, wherein the benzaldehyde anithol ether is selected from 3-methoxybenzaldehyde, 4-methoxybenzaldehyde, 2-hydroxy-5-methoxybenzaldehyde, and 4-ethoxybenzaldehyde.

12. The kit according to claim 10 or claim 11, wherein the dehydrating solvent is selected from ethanol, methanol, tetrahydrofuran, isopropanol, tert-butanol, 2,2'-thiodiethanol, trichloroethanol, and acetone.

13. Use of benzaldehyde anithole ethers selected from 3-methoxybenzaldehyde, 4-methoxybenzaldehyde, 2-hydroxy-5-methoxybenzaldehyde, and 4-ethoxybenzaldehyde as embedding media for preparing biological tissue samples for examination by optical microscope.

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