METHOD FOR OPTICALLY CLARIFYING A TISSUE SAMPLE WITH AN EMBEDDING MEDIUM

Description

[0001] The invention relates to a method according to the preamble of claim 1, a kit for preparing biological tissue samples according to claim 10 and a use according to claim 12.

[0002] Transparent biological tissue samples are required to enable three-dimensional imaging of tissue samples, for example, using light-sheet microscopy. To achieve transparency in biological preparations, heme groups of the blood pigment hemoglobin and lipids must be removed. In dehydration procedures, the tissue is treated with various mixtures of a water-miscible organic solvent and water. The treatment is carried out with an increasing proportion of the organic solvent to completely remove the water from the tissue. Several options exist, such as tetrahydrofuran, methanol, isopropanol, tert-butanol, and ethanol. Ethanol is currently the most commonly used dehydration medium for pathological tissue clearing. The end result of all dehydration procedures is an anhydrous sample.

[0003] The final step in the various clearing procedures is adjusting the refractive index to that of the tissue being examined under the microscope. According to Spalteholz ("On Making Human and Animal Specimens Transparent," S. Hirzel Publishing House, 1911, German Imperial Patent No. 229044), the refractive index of dehydrated tissue is n = 1.547 for bone. The refractive index of other tissues can be estimated at approximately n = 1.551 based on the optimal mixtures he empirically determined. Such a high refractive index necessitates the use of aromatic compounds, which are generally immiscible with water. For his investigations, Spalteholz used mixtures of wintergreen oil (methyl salicylate) and benzyl benzoate or isosafrole in a ratio adapted to the different tissues.

[0004] A variant of Spalteholz's method is known from Dodt, Leischner, Schierloh, Jährling, Mauch, Deininger, Deussing, Eder, Zieglgänsberger, Becker "Ultramicroscopy: three-dimensional visualization of neuronal networks in the whole mouse brain". Nat Methods. 2007;4(4):331-6.), in which "Murray's clear" (a 2:1 mixture of benzyl benzoate and benzyl alcohol with a refractive index of n = 1.559) is used as the embedding medium. Another variant is known from Becker, Jährling, Saghafi, Weiler, Dodt H "Chemical Clearing and Dehydration of GFP Expressing Mouse Brains" (2012) PLoS ONE 7(3): e33916. https: / / doi.org / 10.1371 / journal.pone.0033916. Embedding media for spalted wood, as well as several other aromatic compounds, were tested. Dibenzyl ether (DBE, refractive index n = 1.562) proved to be the most suitable compound and has become the de facto gold standard in tissue clearing.The advantage of DBE also lies in its ease of use, because the solution does not need to be mixed and then checked for the refractive index; it can be used directly as a pure substance.

[0005] WO 2017 / 093323 A1 discloses the use of ethyl cinnamate (ECi) and related cinnamyl esters as non-toxic alternatives to the previously common embedding media. It exhibits lower acute and chronic toxicity than other aromatic compounds used for tissue treatment, such as dibenzyl ether, benzyl alcohol, or benzyl benzoate. Since it achieves the desired refractive index as a pure substance, a mixing step is eliminated, thus eliminating a work step and a potential source of error.

[0006] The selection of pure liquid substances suitable as embedding media for dehydrated samples is currently limited to two compounds: dibenzyl ether and ethyl cinnamate. Both substances have disadvantages: dibenzyl ether is toxic. While ethyl cinnamate has low toxicity, its melting point is 6–9°C, meaning that samples clarified with ethyl cinnamate cannot be stored in a refrigerator, as the embedding medium would crystallize. Ethyl cinnamate also has a comparatively high vapor pressure.

[0007] The invention is based on the objective of providing a method for producing a transparent tissue sample of a biological tissue for light microscopic examination, which overcomes the aforementioned disadvantages and is in particular less costly than producing mixtures as an embedding medium, avoids embedding media of increased toxicity and allows for the cooled storage of the dehydrated tissue samples.

[0008] The problem is solved according to the invention by a method according to claim 1. The problem is further solved by a kit according to claim 10 and the use according to claim 12.

[0009] Further embodiments are the subject of the dependent claims or are described below.

[0010] The inventive method for producing transparent tissue samples of a biological tissue for light microscopic examination comprises the following steps: a) Dehydrating the tissue sample with a dehydrating solvent and b) Clarifying the dehydrated tissue sample by immersing it in a liquid embedding medium with a refractive index suitable for the tissue.

[0011] The embedding medium contains a benzaldehyde anisole ether, preferably selected from 3-methoxybenzaldehyde, 4-methoxybenzaldehyde, 2-hydroxy-5-methoxybenzaldehyde or 4-ethoxybenzaldehyde.

[0012] The method according to the invention serves to achieve optical transparency of a biological or human tissue sample for light microscopy. The biological tissue is, for example, human or animal tissue.

[0013] The clarification step of the clearing procedure is to adjust the refractive index to that of the tissue being examined under the microscope. For this purpose, the tissue sample is transferred to a refractive index-adjusting solution, the embedding medium. The embedding medium must be miscible with the solvent used in the dehydration step.

[0014] In one embodiment, the embedding medium contains 10 to 100 vol% of the benzaldehyde ether and 0 to 90 vol% of an optically suitable, inert organic solvent with a refractive index of about 1.3, preferably about 1.5. For example, the embedding medium contains 90 to 100 vol% of the benzaldehyde ether.

[0015] In another embodiment, the embedding medium contains 10 to 100 vol% of the benzaldehyde ether and 0 to 90 vol% of an optically suitable, inert organic solvent with a refractive index of about 2.0, preferably about 1.65. For example, the embedding medium contains 90 to 100 vol% of the benzaldehyde ether.

[0016] All percentages according to the invention are given in volume percent (Vol.-%).

[0017] In the preferred embodiment, the embedding medium consists of one of the benzaldehyde anisole ethers according to the invention, i.e., the benzaldehyde anisole ether is used as a pure substance. "Pure substance" here refers to the benzaldehyde anisole ether in its technically available purity.

[0018] Benzaldehyde ether is a benzaldehyde anisole ether. Preferably, the benzaldehyde anisole ether is selected from 3-methoxybenzaldehyde (meta-anisaldehyde; CAS No. 591-31-1), 4-methoxybenzaldehyde (para-anisaldehyde; CAS No. 123-11-5), 2-hydroxy-5-methoxybenzaldehyde (6-hydroxy-m-anisaldehyde; CAS No. 672-13-9), or 4-ethoxybenzaldehyde (homo-anisaldehyde; CAS No. 10031-82-0). 3-Methoxybenzaldehyde, 4-methoxybenzaldehyde, and 2-hydroxy-5-methoxybenzaldehyde have a purity of approximately 98.0% as a technical product. 4-Ethoxybenzaldehyde, as a technical product, has a purity of approximately 99%.

[0019] Dehydration step a) serves to obtain an anhydrous tissue sample. In one embodiment, the tissue sample is preferably treated in several passes with different dehydration compositions with a descending water series, i.e., mixtures with an ascending proportion of a water-miscible organic solvent, to remove the water from the tissue. Alcohols, ketones, or ethers are used as the dehydrating medium. Suitable dehydrating solvents include, for example, ethanol, methanol, isopropanol, tert-butanol (IUPAC: 2-methylpropan-2-ol), tetrahydrofuran, or acetone.

[0020] The dehydration medium has the following properties: 1. it mixes completely with water so that water and fixative can be gradually removed from the tissue via an ascending concentration series, and 2. it mixes completely with the embedding medium, which adjusts the refractive index to that of the dehydrated tissue.

[0021] In an alternative embodiment, the dehydration step a) is carried out with 2,2-dimethoxypropane (DMP), which removes the water contained in the tissue by a chemical reaction.

[0022] In the inventive method, the dehydration step a) in one embodiment to obtain an anhydrous tissue sample comprises the application of dehydrating compositions. from aqueous ethanol with increasing concentrations of ethanol, wherein the ethanol concentrations of the dehydration compositions range from 30% to 100 vol%, or from aqueous tetrahydrofuran with increasing concentrations of tetrahydrofuran, wherein the tetrahydrofuran concentration of the dehydration compositions ranges from 30% to 100 vol%, or from aqueous methanol with increasing concentrations of methanol, wherein the methanol concentrations of the dehydration compositions range from 30% to 100 vol%, or from an aqueous mixture of another alcohol, ketone, or ether, wherein the solvent concentration of the dehydration compositions ranges from 30% to 100 vol%.

[0023] In an alternative embodiment of the method according to the invention, the dehydration step a) to obtain a tissue sample with a residual water content >2% is carried out using dehydrating compositions. from aqueous ethanol with increasing concentrations of ethanol, wherein the ethanol concentrations of the dehydration compositions range from 50% to 98 vol%, preferably 70% to 98%, particularly preferably 75% to 98 vol%, or from an aqueous mixture of another alcohol, ketone or ether, wherein the solvent concentration of the dehydration compositions ranges from 50% to 98 vol%, preferably 70% to 98%, particularly preferably 75% to 98 vol%.

[0024] The dehydration compositions used preferably contain 2-30% water by volume, even in the final stage of the dehydration process. In this embodiment, for example, denatured ethanol with a residual water content of 4-6% by volume can also be used.

[0025] In a further alternative embodiment of the method according to the invention, the dehydration step a) is carried out in a gradient mixer. For this purpose, a tissue sample is placed in a mixing vessel (gradient mixer). A dehydrating solvent is introduced via an inlet until a residual water content of 5 to 20 vol% is reached. The dehydrating solvent can have a solvent concentration of 100 vol% or a residual water content between 2 and 50 vol%. In one variant, the tissue sample is initially placed directly into a dehydrating solvent with a solvent concentration of 50 vol% in the gradient mixer, and the gradient is then increased in small steps.

[0026] Preferably, the tissue sample is transferred directly from the gradient mixer into the embedding medium without prior incubation in high-purity solvent. An incubation step in high-purity solvent is preferably omitted in steps a) and b).

[0027] It is surprising that tissue samples can be clarified in this way. According to the state of the art, the procedure in a gradient mixer proceeds as follows: A tissue sample is placed in a mixing vessel (gradient mixer). This vessel has, for example, an inlet at the top and an outlet on the side. If the sample is placed in this mixing vessel in 50% ethanol and a high concentration (> 95 vol%) of solvent is introduced via the inlet using a metering pump, a gradient builds up that asymptotically attempts to reach 100 vol%. In this way, the sample is provided with a beneficial, steadily increasing ethanol concentration. Since 100% can never be reached during mixing, the sample is usually subsequently incubated in high-purity ethanol. According to the state of the art, this incubation step is repeated several times.Therefore, in practice, the dosing pump can be filled with technical ethanol for cost reasons.

[0028] According to the invention, 1. the last (possibly repeated) incubation step with absolute ethanol has become unnecessary and 2. the endpoint reached with the gradient mixer, as long as it is above approximately 80% ethanol, is sufficient for the further clarification of the tissue sample in step b).

[0029] In another alternative embodiment, dehydration step a) is performed in a single pass. Instead of a multi-pass dehydration series, dehydration according to step a) is carried out in a single pass by immersing the tissue sample in a dehydrating solvent with a solvent concentration of at least 70% by volume. This embodiment is particularly suitable for tissue samples with compact tissue that absorb little water. In the subsequent step b), the tissue sample is immediately placed in the embedding medium, again without incubation in a high-purity solvent for complete dehydration. This results in a significant time saving and also a considerable reduction in the amount of reagents used. The reduction in reagents results, in part, from the smaller quantity of solvent required.Secondly, it is no longer necessary to use a highly purified anhydrous solvent; a technical-grade solvent can be used. For example, with ethanol, it is not necessary to use expensive 100% high-purity ethanol, but rather the significantly more cost-effective denatured ethanol. According to the prior art, the incubation with highly purified anhydrous solvent is repeated to completely remove any remaining water; in many protocols, the incubation in the embedding medium is also repeated for the same reason. Since, according to the invention, the final incubations for the reliable removal of residual water from the tissue are eliminated in both embodiments, reagents are saved according to the inventive method.

[0030] In one embodiment of the method according to the invention, the tissue sample is dehydrated in step a) and optically clarified in step b), before it is dehydrated in step a), fixed and / or fixed with formaldehyde and / or washed and / or washed with water and / or incubated in an aqueous alkaline solution with non-ionic detergent, and / or delipidated with a detergent solution (delipidation), and / or delipidated with an organic solvent (delipidation), and / or bleached with oxidizing reagents, and / or decolorized with amino alcohols.

[0031] In one embodiment of the method according to the invention, the tissue sample is fixed before it is dehydrated in step a) and optically clarified in step b), and the fixing agent is selected from Crosslinking fixatives such as formaldehyde, glutaraldehyde, acrolein, carbodiimides, diethyl pyrocarbonate, bisimodo esters or glyoxal or mixtures thereof, and / or coagulating fixatives such as alcohols and other organic solvents, acids, potassium dichromate, lead nitrate, copper sulfate and mercuric chloride and mixtures thereof.

[0032] The process according to the invention is preferably carried out after tissue preparation steps and after electrophoresis steps. The tissue sample treated in the process according to the invention is thus already pretreated. The pretreatment by electrophoresis is preferably carried out according to the method for electrophoretic clarification described in DE 10 2016 123 458 B3. For the execution of the electrophoretic clarification, reference is made to patent specification DE 10 2016 123 458 B3, the contents of which are hereby incorporated into this application.

[0033] In the inventive method, the optically clarified tissue sample is preferably examined in a further step under a microscope in order to obtain an image of the internal structure of the sample, wherein the microscope is a light microscope, preferably a light sheet microscope, a confocal microscope, a two-photon microscope or an optical projection tomography microscope (OPT).

[0034] The previously used embedding medium, dibenzyl ether (DBE), has a refractive index of n = 1.562. The second pure substance, ethyl cinnamate, has a refractive index of n = 1.559. Of the benzaldehyde anisole ethers according to the 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. When using the benzaldehyde anisole ethers according to the invention as an embedding medium for tissue clearing of dehydrated tissue samples, at least identical, and in some cases improved, transparency was achieved compared to tissue samples treated with methyl salicylate / benzyl benzoate and with ethyl cinnamate.

[0035] Spalteholz's work already suggests that the refractive indices of different tissues vary slightly. In his original 1911 publication, he lists the following series of human tissues with ascending refractive indices, empirically determined using the optimal mixing ratio of methyl salicylate and BB: young embryos (5:1–3:1 depending on weight) < adult decalcified bones (5:3) n = 1,547 < adult musculature approximately like older embryos (2:1) < brain and spinal cord (1:1). This variability has direct consequences for the transparency of the different tissues and their microscopic examination, and especially for quantitative spectroscopic determinations that can be carried out with a microscope.The method according to the invention offers the advantage that various pure substances can be used as an embedding medium, which can be selected depending on the tissue in order to approximate the refractive index of the tissue.

[0036] Compared to the previously commonly used embedding media, benzyl benzoate / benzyl alcohol mixtures (BABB) and methyl salicylate / benzyl benzoate mixtures, benzaldehyde anisole ethers have the advantage that, like dibenzyl ethers, they can be used as a pure substance: Since the pure substance already achieves the desired refractive index, the refractive index does not need to be adjusted by mixing the embedding medium. This eliminates a mixing step, thus removing a redundant step and a potential source of error.

[0037] Surprisingly, the benzaldehyde anisole ethers according to the invention penetrate the dehydrated tissue significantly faster than previously known embedding media and quickly render the tissue transparent. This saves time during sample preparation, allowing the sample to be examined under a light microscope more quickly. Consequently, the results of the light microscopic examinations are also available more rapidly. The density of the dehydrated tissue is determined by the density of the dehydrating solvent still present in the tissue, e.g., ethanol. Since the embedding medium typically has a higher density, its complete penetration into the tissue sample can be readily monitored by observing the settling of the tissue sample within the embedding medium. For example, with 2-hydroxy-5-methoxybenzaldehyde, complete penetration of the embedding medium into the tissue sample is achieved after only 30-45 minutes.The tissue samples lie on the bottom of the container. In contrast, with conventional embedding media such as dibenzyl ether or methyl salicylate-benzyl benzoate mixtures, this process step typically takes several hours or even overnight. The inventive method thus offers the advantage that the embedding medium diffuses more quickly into the tissue of the tissue sample, resulting in a significant time saving during sample preparation. The tissue samples can therefore be examined more quickly using light microscopy.

[0038] If a tissue sample contains residual water and is transferred to an organic solvent as an embedding medium in which this water is no longer soluble, the water becomes trapped in the tissue. Due to its incomplete miscibility, this leads to turbidity that impairs transparency. Therefore, tissue samples are currently typically incubated multiple times with high-purity ethanol and often subsequently again with the embedding medium to prevent this impairment of the sample's transparency. According to the state of the art, as already explained, the incubation with high-purity anhydrous solvent is repeated to completely remove any remaining water. Many protocols also repeat the incubation in the embedding medium for the same reason.

[0039] According to the invention, the tissue is clarified with a benzaldehyde anisole ether. Surprisingly, it has been found that good transparency can be achieved even with tissue samples containing residual water. At low residual water contents of 2-5 vol%, no impairment of transparency is observed, and even at high water contents of up to 20% or up to 30 vol%, depending on the type of tissue treated, only very low turbidity is observed, which still allows for light microscopic examination of the sample.

[0040] The method according to the invention therefore offers a particularly fast and simple method for dewatering a tissue sample, since, unlike all previously known embedding media, a small residual water content of up to 10 vol% does not impair the light microscopic results and even larger residual water quantities still lead to an acceptable transparency of the tissue sample.

[0041] The method according to the invention also allows for a simplified process compared to clarification methods using conventional embedding media. According to the prior art, the tissue sample is transferred from vessel to vessel during dehydration with an increasing concentration of the dehydrating solvent, e.g., an increasing ethanol concentration. This requires a relatively large number of individual steps, and these steps are discrete. Furthermore, in the last step(s), a highly purified solvent, e.g., highly purified ethanol, must always be used.

[0042] In the process according to the invention, a dehydrating solvent with a residual water content can also be used. For example, denatured 95-97% technical ethanol can be used instead of high-purity ethanol. The process according to the invention therefore also leads to cost savings in the solvents used.

[0043] The inventive method also allows for a simplified process when using a gradient mixer, because here too, no subsequent complete dehydration with a high-purity solvent is necessary.

[0044] Benzaldehyde anisole ethers also exhibit low toxicity. This is advantageous when used as an embedding medium, as the tissue sample is located directly below the user's airways in the microscope, and the user can inhale fumes rising from the sample. According to the European Chemicals Agency (ECHA), the toxicity of para-anisaldehyde is considered safe, unlike that of dibenzyl ethers or benzyl benzoate / benzyl alcohol mixtures.

[0045] The kit according to the invention for the preparation of biological tissue samples for light microscopy contains: a dehydrating solvent for dehydrating the tissue sample and an embedding medium for clarifying the dehydrated tissue sample by immersion in the embedding medium, where The embedding medium is a benzaldehyde anisole ether, as described above, selected from 3-methoxybenzaldehyde, 4-methoxybenzaldehyde, 2-hydroxy-5-methoxybenzaldehyde, and 4-ethoxybenzaldehyde. The benzaldehyde anisole ether is preferably used in the concentrations and purities described above.

[0046] The dehydrating solvent in the kit is an ether, ketone or alcohol; preferably the solvent is selected from ethanol, methanol, isopropanol, tert-butanol, 2,2'-thiodiethanol, trichloroethanol, tetrahydrofuran and acetone.

[0047] According to the invention, a benzaldehyde anisole ether is used. selected from 3-methoxybenzaldehyde, 4-methoxybenzaldehyde, 2-hydroxy-5-methoxybenzaldehyde or 4-ethoxy-benzaldehyde as an embedding medium for the preparation of a biological, especially human, tissue sample for light microscopic examination.

[0048] The benzaldehyde anisole ether used according to the invention is preferably used in the concentrations and purities described above. Examples Example 1

[0049] Several tissue samples (pig lung) with a volume of approximately 0.25 ml (0.5 x 0.5 x 1 cm³) were dehydrated in absolute ethanol and photographed. The ethanol-dehydrated tissue samples are in Figure 1 , shown on the left side.

[0050] The dehydrated tissue samples were then immersed in 5 ml of the respective embedding medium for 3 hours. According to the invention, 2-hydroxy-5-methoxybenzaldehyde and, as a comparative example, a mixture of methyl salicylate with benzyl benzoate, having a refractive index adjusted to that of 2-hydroxy-5-methoxybenzaldehyde, were used as embedding media. The result of the clarification step was also photographed. The results are presented in Figure 1 The right side is shown. It is evident that clarification with methyl salicylate / benzyl benzoate already results in good transparency of the tissue sample. However, even better transparency was achieved with the 2-hydroxy-5-methoxybenzaldehyde according to the invention. Example 2

[0051] Several tissue samples (pig lung) with a volume of approximately 0.25 ml (0.5x0.5x1 cm 3< ) were prepared as described in Example 1, using 2-hydroxy-5-methoxybenzaldehyde, ethyl cinnamate and dibenzyl ether as embedding media.

[0052] The tissue samples were then stored overnight in absolute ethanol, allowing the embedding medium to be washed out. The tissue samples were transferred to ethanol with defined water contents to achieve a specific residual water content in the tissue, incubated for 2 hours, and then returned to their respective embedding medium for photography. The tissue samples were placed in ethanol with 95%, 90%, and 80% vol. ethanol concentrations. The results were photographed and are shown in Figure 2The first row shows the tissue samples, 100% dehydrated, in the embedding media. The second row shows the tissue samples incubated in ethanol with 5 vol% water content in the embedding medium. The third row shows the tissue samples incubated in ethanol with 10 vol% water content in the embedding medium, and the fourth row shows the tissue samples incubated in ethanol with 20% water content in the embedding medium. The bottom row shows the same tissue samples in pure ethanol, i.e., before they are placed in the respective embedding media. When completely dehydrated in ethanol, all samples are equally opaque.

[0053] It has been shown that the embedding media known from the prior art, dibenzyl ether and ethyl cinnamate, only achieve the desired transparency with no residual water or with only a very small amount of residual water. With dibenzyl ether, a residual water content of just 5 vol% is sufficient for the sample to become opaque and therefore unsuitable for microscopic examination. Ethyl cinnamate also shows significant turbidity at a residual water content of 95%, and the tissue sample is no longer completely transparent. In contrast, tissue samples clarified with 2-hydroxy-5-methoxybenzaldehyde remain completely transparent even with a residual water content of 10 vol% (90 vol% ethanol) and only show slight turbidity at 20% residual water (80% ethanol content). Example 3

[0054] Tissue samples with a volume of approximately 0.25 ml (0.5 x 0.5 x 1 cm³) were dehydrated in absolute ethanol. They were then transferred to an embedding medium in a sample container. According to the invention, 2-hydroxy-5-methoxybenzaldehyde was used as an embedding medium, and, for comparison, dibenzyl ether and mixtures of methyl salicylate with benzyl benzoate were used. It was observed that with 2-hydroxy-5-methoxybenzaldehyde, the tissue sample was clearly below the surface after only 15 minutes and, depending on the type of tissue, lay at the bottom of the container after 30–45 minutes. In dibenzyl ether, the tissue sample only sank to the bottom after several hours, comparable to the time required for mixtures of methyl salicylate / benzyl benzoate and dibenzyl ether.

[0055] In a further experiment, the rate at which the dehydrating solvent ethanol was replaced by the embedding media in a tissue sample was determined. The embedding media used were 2-hydroxy-5-methoxybenzaldehyde according to the invention, and dibenzyl ether and ethyl cinnamate were used as comparative examples. The tissue samples were placed in the embedding medium for 15 min, 30 min, and 70 min and then photographed. The results are presented in Figure 3The images are shown. In the top row, the tissue samples were photographed after 15 minutes of clarification. In the middle row, the tissue samples were photographed after 30 minutes of clarification. In the bottom row, the tissue samples were photographed after 70 minutes of clarification. It can be seen that the tissue sample treated with 2-hydroxy-5-methoxybenzaldehyde is already transparent after 70 minutes, while the sample treated with ECi shows only slight transparency, and the tissue sample treated with DBE is not yet transparent at all. The clarification method according to the invention thus achieves a significantly faster clarification of the tissue sample.

[0056] The invention is not limited to one of the embodiments described above, but can be modified in many ways.

[0057] All features and advantages arising from the claims, the description and the drawing, including process steps, can be essential to the invention both individually and in various combinations.

Claims

1. Method for producing a transparent tissue sample of a biological tissue for light-microscopic examination, comprising the steps of: a) dehydrating the tissue sample with a dehydrating solvent and b) clearing the dehydrated tissue sample by placing it into an embedding medium containing a benzaldehyde anisole ether selected from 3-methoxybenzaldehyde, 4-methoxybenzaldehyde, 2-hydroxy-5-methoxybenzaldehyde or 4-ethoxybenzaldehyde.

2. Method according to Claim 1, characterized in that the embedding medium contains 10% to 100% by volume of the benzaldehyde anisole ether and contains 0% to 90% by volume of an optically suitable, inert organic solvent having a refractive index of about 1.3, preferably 1.5.

3. Method according to Claim 1, characterized in that the embedding medium contains 10% to 100% by volume of the benzaldehyde anisole ether and contains 0% to 90% by volume of an optically suitable, inert organic solvent having a refractive index of about 2.0, preferably 1.65.

4. Method according to any of the preceding claims, characterized in that the embedding medium consists of a benzaldehyde anisole ether selected from 3-methoxybenzaldehyde, 4-methoxybenzaldehyde, 2-hydroxy-5-methoxybenzaldehyde or 4-ethoxybenzaldehyde.

5. Method according to any of the preceding claims, characterized in that the dehydration step a) comprises the use of dehydrating compositions composed of aqueous alcohol, ketone or ether and the dehydrating compositions are an aqueous mixture of an alcohol, ketone or ether having a solvent concentration of the dehydrating compositions from 50% by volume to 98% by volume, preferably 70% by volume to 98% by volume, particularly preferably 75% to 98% by volume, preferably an aqueous mixture of aqueous ethanol having increasing concentrations of ethanol, where the ethanol concentrations of the dehydrating compositions range from 50% by volume to 98% by volume.

6. Method according to any of the preceding claims, characterized in that the dehydration step a) is carried out in a gradient mixer and the tissue sample is introduced preferably at the start of the dehydration step directly into a dehydrating solvent having a solvent concentration of 50% and the gradient is then increased in small increments.

7. Method according to any of the preceding claims, characterized in that the tissue sample, before it is dehydrated in step a) and optically cleared in step b), • is fixed and / or • is fixed with formaldehyde and / or • is washed and / or • is washed with water and / or • is incubated in an aqueous alkaline solution and / or • is delipidated with a detergent solution and / or • is delipidated with an organic solvent and / or • is bleached with oxidizing reagents and / or • is decolorized with amino alcohols.

8. Method according to any of the preceding claims, characterized in that the tissue sample, before it is dehydrated in step a) and optically cleared in step b), is fixed and the fixing agent is selected from - crosslinking fixatives such as formaldehyde, glutaraldehyde, acrolein, carbodiimides, diethyl pyrocarbonate, bisimido esters or glyoxal or mixtures thereof, and / or - coagulating fixatives such as alcohols and other organic solvents, acids, potassium dichromate, lead nitrate, copper sulfate and mercury chloride and mixtures thereof.

9. Method according to any of the preceding claims, characterized in that in a further step the optically cleared tissue sample is examined under a microscope in order to obtain an image of the internal structure of the sample, where the microscope is a light microscope.

10. Kit for preparing biological tissue samples for light microscopy, containing: - a dehydrating solvent for dehydrating the tissue sample and - an embedding medium for clearing the dehydrated tissue sample by placing the sample into the embedding medium, where the dehydrating solvent is selected from ethanol, isopropanol or tert-butanol and is in the form of dehydrating compositions composed of aqueous solvent with increasing concentrations of solvent, and where the embedding medium is a benzaldehyde anisole ether selected from 3-methoxybenzaldehyde, 4-methoxybenzaldehyde, 2-hydroxy-5-methoxybenzaldehyde or 4-ethoxybenzaldehyde.

11. Kit according to Claim 10, characterized in that the benzaldehyde anisole ether is selected from 3-methoxybenzaldehyde, 4-methoxybenzaldehyde, 2-hydroxy-5-methoxybenzaldehyde and 4-ethoxybenzaldehyde.

12. Use of a benzaldehyde anisole ether selected from 3-methoxybenzaldehyde, 4-methoxybenzaldehyde, 2-hydroxy-5-methoxybenzaldehyde or 4-ethoxybenzaldehyde as an embedding medium for preparing a biological tissue sample for light-microscopic examination.