Method for optical clearing of tissue samples using embedding media

Abstract

The invention relates to a method for preparing a transparent tissue sample of a biological or human tissue for optical microscopic examination, having the following steps: a) dehydrating the tissue sample using a dehydrating solvent, and b) transparentizing the dehydrated tissue sample by transferring it into an embedding medium containing benzaldehyde anisole. In comparison with the benzyl benzoate / benzyl alcohol mixtures and the methyl salicylate-benzyl benzoate mixtures which have hitherto generally been used as embedding medium, benzaldehyde anisole has the advantage that it can be used as a pure substance in the same way as diphenylmethylether: this is because the pure substance already has the desired refractive index, so that it is not necessary to set the refractive index by mixing the embedding medium. Furthermore, the benzaldehyde anisole according to the invention penetrates the dehydrated tissue more quickly than the previously known embedding media and also makes the tissue transparent more quickly.

Description

[0001] The present invention relates to the method according to the preamble of claim 1, the kit for preparing biological tissue samples as described in claim 10, and the application as described in claim 13.

[0002] To enable three-dimensional imaging of tissue samples, such as through light microscopy, transparent biological tissue samples are required. Achieving transparency in biological agents requires the removal of heme groups and lipids, particularly from hemoglobin. In the "dehydration" process, the tissue is treated with various mixtures of water and water using water-miscible organic solvents. To completely remove water from the tissue, treatment is performed with increasing proportions of organic solvents. Many options are available, such as tetrahydrofuran, methanol, isopropanol, tert-butanol, and ethanol. Ethanol is currently the most commonly used dehydrating medium for achieving tissue transparency in pathology. The final result of all "dehydration" processes is an anhydrous sample.

[0003] The final step in various clarification methods is to adjust the refractive index to match that of the tissue being microscopically examined. According to Spalteholz (“ das Durchsichtigmachen von menschlichen und tierischen "[On the transparency of human and animal preparations], published by S. Hirzel, 1911 (German Patent No. 229044), states that the refractive index of dehydrated tissue for bone is n = 1.547. Based on his empirically determined optimal mixture, the refractive index of other tissues can be estimated to be approximately n = 1.551. This high refractive index necessitates the use of aromatic compounds that are generally immiscible with water. For his research, Spalteholz used a mixture of wintergreen oil (methyl salicylate) and benzyl benzoate or isoflavone, the mixing ratio customized according to various tissues."

[0004] Available from Dodt, Leischner, Schierloh, Mauch, Deininger, Deussing, Eder, Learn about a variant of the Spalteholz method in Becker's "Ultramicroscopy: three-dimensional visualization of neural networks in the whole mouse brain," which uses "Murray clearing agent" (a 2:1 mixture of benzyl benzoate and benzyl alcohol, with a refractive index n = 1.559) as the embedding medium. From Becker, Another variant can be found in Saghafi, Weiler, and Dodt, “Chemical Clearing and Dehydration of GFP Expressing Mouse Brains” (2012) PLoS ONE 7(3):e33916. https: / / doi.org / 10.1371 / journal.pone.003391. Spalteholz embedding media and many other aromatic compounds were tested. Diphenylmethyl ether (DBE, refractive index n = 1.562) emerged as the most suitable compound and has become the de facto gold standard in tissue clearing. The advantage of DBE is also its ease of use, as there is no need to mix the solution and then check its refractive index; it can be used directly in pure substance form.

[0005] Ethyl cinnamate (ECi) and related cinnamates are known from WO 2017 / 093323A1 as non-toxic alternatives to commonly used embedding media at the time. It exhibits lower acute and chronic toxicity than other aromatic compounds used in tissue treatment, such as diphenylmethyl ether, benzyl alcohol, or benzyl benzoate. Because it achieves the desired refractive index as a pure substance, the mixing step is eliminated, thereby eliminating working steps and potential sources of error.

[0006] Therefore, the selection of pure liquid substances suitable as embedding media for dehydrated samples is currently limited to two compounds: diphenylmethyl ether and ethyl cinnamate. However, both substances have drawbacks: diphenylmethyl ether is toxic. Although ethyl cinnamate has low toxicity, its melting point is 6°C to 9°C, so samples cleared with ethyl cinnamate cannot be stored in a refrigerator, as the embedding medium will crystallize therein. Ethyl cinnamate also has a relatively high vapor pressure.

[0007] The object of this invention is to provide a method for preparing transparent tissue samples of biological tissues for optical microscopy examination. This method overcomes the above-mentioned disadvantages and, in particular, involves a more labor-saving approach compared to the need to generate a mixture as an embedding medium, avoids highly toxic embedding media, and allows for the cold storage of dehydrated tissue samples.

[0008] According to the present invention, this objective is achieved by the method as described in claim 1. This objective is also achieved by the kit as described in claim 9 and the application as described in claim 11.

[0009] Other embodiments are the subject of the dependent claims or the description below.

[0010] The method according to the present invention for preparing transparent tissue samples of biological tissue for optical microscopy includes the following steps:

[0011] a) Dehydrate the tissue sample using a dehydrating solvent, and

[0012] b) Transparent the dehydrated tissue sample by placing it in an embedding medium with a refractive index that matches the group.

[0013] The embedding medium contains benzaldehyde anisole, preferably selected from 3-methoxybenzaldehyde, 4-methoxybenzaldehyde, 2-hydroxy-5-methoxybenzaldehyde and 4-ethoxybenzaldehyde.

[0014] The purpose of the method according to the invention is to achieve optical transparency in biological or human tissue samples used for optical microscopy. Biological tissues include, for example, human or animal tissues.

[0015] The clearing step in the clearing method involves adjusting the refractive index to match that of the tissue to be microscopically examined. For this, the tissue sample is transferred to a solution used to adjust the refractive index: an embedding medium. This embedding medium must be miscible with the solvent used in the dehydration step.

[0016] In one embodiment, the embedding medium contains 10 vol% to 100 vol% benzaldehyde ether and 0 vol% to 90 vol% 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 vol% to 100 vol% benzaldehyde ether.

[0017] In another embodiment, the embedding medium contains 10 vol% to 100 vol% benzaldehyde ether and 0 vol% to 90 vol% 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 vol% to 100 vol% benzaldehyde ether.

[0018] All percentages mentioned in this invention are volume percentages (volume %).

[0019] In a preferred embodiment, the encapsulation medium consists of benzaldehyde anisole of the present invention, i.e., benzaldehyde anisole is used as a pure substance. Pure substance is understood herein to mean benzaldehyde anisole having an industrially available purity.

[0020] Benzaldehyde ether is benzaldehyde anisole. Benzaldehyde anisole is preferably selected from 3-methoxybenzaldehyde (m-anisaldehyde; CAS No. 591-31-1), 4-methoxybenzaldehyde (p-anisaldehyde; CAS No. 123-11-5), 2-hydroxy-5-methoxybenzaldehyde (6-hydroxy-m-anisaldehyde; CAS No. 672-13-9), and 4-ethoxybenzaldehyde (homoanisaldehyde; CAS No. 10031-82-0). 3-methoxybenzaldehyde, as an industrial product, has a purity of approximately 97.0%. 4-methoxybenzaldehyde, as an industrial product, has a purity of approximately 97.0%. 2-hydroxy-5-methoxybenzaldehyde, as an industrial product, has a purity of approximately 98.0%. 4-ethoxybenzaldehyde, as an industrial product, has a purity of approximately 99%.

[0021] The purpose of dehydration step a) is to obtain an anhydrous tissue sample. In one embodiment, the tissue sample is preferably treated multiple times with various dehydration compositions in a decreasing series of water (i.e., mixtures containing increasing proportions of water-miscible organic solvents) to remove water from the tissue. Alcohols, ketones, or ethers are used as the dehydration medium. Suitable dehydration solvents are, for example, ethanol, methanol, isopropanol, tert-butanol (IUPAC: 2-methylprop-2-ol), tetrahydrofuran, or acetone.

[0022] The dehydration medium here has the following properties: (1) it is completely miscible with water so that the fixative can be gradually removed from the tissue by an increasing concentration series, and (2) it is completely miscible with the embedding medium used to adjust the refractive index to that of the dehydrated tissue.

[0023] In an alternative embodiment, the dehydration step a) is carried out with 2,2-dimethoxypropane (DMP), which removes the water from the tissue by chemical reaction with the water present in the tissue.

[0024] In the method according to the invention, in one embodiment, the dehydration step a) of obtaining an anhydrous tissue sample involves using a dehydration composition as follows:

[0025] - It consists of aqueous ethanol with increasing ethanol concentrations, wherein the ethanol concentration of the dehydrated composition is 30% to 100% by volume, or

[0026] - It consists of an aqueous tetrahydrofuran having an increasing tetrahydrofuran concentration, wherein the tetrahydrofuran concentration of the dehydrated composition is 30% to 100% by volume, or

[0027] - It consists of aqueous methanol with increasing methanol concentrations, wherein the methanol concentration of the dehydrated composition is 30% to 100% by volume, or

[0028] - It consists of an aqueous mixture of another alcohol, ketone or ether, wherein the solvent concentration of the dehydrating composition is 30% to 100% by volume.

[0029] In an alternative embodiment of the method according to the invention, the dehydration step a) is performed using the following dehydration composition to obtain a tissue sample having a residual water content of >2%:

[0030] - It consists of a composition of aqueous ethanol having an increasing concentration of ethanol, wherein the ethanol concentration of the dehydration composition is 50 vol% to 98 vol%, preferably 70 vol% to 98 vol%, more preferably 75 vol% to 98 vol%, or

[0031] - It consists of an aqueous mixture of another alcohol, ketone or ether, wherein the solvent concentration of the dehydrating composition is 50 vol% to 98 vol%, preferably 70 vol% to 98 vol%, more preferably 75 vol% to 98 vol%.

[0032] 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, denatured ethanol with a residual water content of, for example, 4% to 6% by volume can also be used.

[0033] In another alternative embodiment of the method according to the invention, the dehydration step a) is performed in a gradient mixer. For this purpose, the tissue sample is placed in a mixing container (gradient mixer). A dehydrating solvent is introduced through the inlet until a residual water content of 5% to 20% by volume is achieved. The dehydrating solvent may have a solvent concentration of 100% by volume or a residual water content of 2% to 50% by volume. In a variation, the tissue sample in the gradient mixer is initially introduced directly into a dehydrating solvent with a solvent concentration of 50% by volume, and then the gradient is increased in small increments.

[0034] Tissue samples are preferably transferred directly from the gradient mixer to the embedding medium without prior incubation in a high-purity solvent. Preferably, the incubation step in a high-purity solvent is omitted in step a) or step b).

[0035] Surprisingly, tissue sample clearing in this manner is possible. According to existing technology, the method in a gradient mixer is as follows: The tissue sample is placed in a mixing container (gradient mixer). The container has, for example, an inlet at the top and a side outlet. If a sample in 50% ethanol is placed in this mixing container, and a high concentration (>95 vol% solvent) is introduced through the inlet using a metering pump, a gradient is established attempting to asymptotically reach a value of 100 vol%. This results in the sample being supplied with a favorable, steadily increasing ethanol content. Since it is impossible to reach 100% with the mixture, the sample is typically subsequently incubated in high-purity ethanol. According to existing technology, this incubation step is repeated multiple times. Therefore, for cost reasons, the metering pump can routinely be filled with industrial-grade ethanol.

[0036] In the method of the present invention, (1) the final (optionally repeated) incubation step with anhydrous ethanol becomes redundant, and (2) the endpoint reached using a gradient mixer is sufficient for further clearing of the tissue sample in step b), provided that the ethanol content is higher than about 80%.

[0037] In another alternative embodiment, dehydration step a) is performed in a single round. Instead of using a dehydration series comprising two or more rounds, dehydration according to step a) is performed in a single round by placing 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 dense tissues that are virtually non-absorbent. In the subsequent step b), the tissue sample is immediately placed in the embedding medium without incubation in a high-purity solvent to achieve complete dehydration. This results in significant time savings, as well as significant savings in reagents used. The savings in reagents are primarily due to the smaller amount of solvent required. Secondly, the use of high-purity anhydrous solvents is no longer necessary; industrial-grade solvents can be used instead. In the case of ethanol, the use of, for example, expensive 100% high-purity ethanol is unnecessary; cheaper denatured ethanol can be used instead. According to the prior art, incubation with high-purity anhydrous solvent is repeated to completely remove the last trace amounts of water, and for the same reason, many protocols also repeat incubation in the embedding medium. Since the final incubation for reliably removing residual water from the tissue is omitted in both embodiments of the invention, the method of the present invention saves reagents.

[0038] In one embodiment of the method according to the invention, the tissue sample is dehydrated in step a) and optically transparentized in step b).

[0039] • Fixed and / or

[0040] Fixation with formaldehyde and / or

[0041] • After washing and / or

[0042] • Wash with water and / or

[0043] • Incubate in an alkaline aqueous solution containing a nonionic detergent and / or

[0044] • Degreasing with detergent solution and / or

[0045] • Delipidation with organic solvents and / or

[0046] • Bleaching with oxidizing agents and / or

[0047] • Decolorize with amino alcohols.

[0048] In one embodiment of the method according to the invention, prior to dehydration in step a) and optical clearing in step b), the tissue sample is fixed, and the fixative is selected from...

[0049] - Crosslinking fixatives, such as formaldehyde, glutaraldehyde, acrolein, carbodiimide, diethyl pyrocarbonate, diimino ester, or glyoxal or mixtures thereof, and / or

[0050] - Coagulants and fixatives, such as alcohols and other organic solvents, acids, potassium dichromate, lead nitrate, copper sulfate and mercuric chloride and mixtures thereof.

[0051] The method according to the invention is preferably performed after the tissue preparation step and the electrophoresis step. Therefore, the tissue sample processed in the method according to the invention has been pretreated. The electrophoretic pretreatment is preferably performed according to the electrophoretic clearing method described in DE 102016123458B3. For the performance of the electrophoretic clearing, refer to patent specification DE102016123458B3, the contents of which are incorporated herein by reference.

[0052] In the method according to the invention, the optically transparent tissue sample is preferably examined under a microscope in another step to obtain an image of the internal structure of the sample, wherein the microscope is an optical microscope, preferably a light sheet microscope, a confocal microscope, a two-photon microscope, or an optical projection tomography (OPT) microscope.

[0053] The refractive index of the embedding medium diphenylmethyl ether (DBE) used to date is n = 1.562. The refractive index of the second pure substance, ethyl cinnamate, is n = 1.559. In the benzaldehyde anisole of the present invention, the refractive index of 3-methoxybenzaldehyde is, for example, n = 1.552, and the refractive index of 2-hydroxy-5-methoxybenzaldehyde is n = 1.580. The application of the benzaldehyde anisole of the present invention as an embedding medium for tissue clearing of dehydrated tissue samples achieves at least the same transparency as, and in some cases even better transparency, than, tissue samples treated with methyl salicylate / benzyl benzoate and ethyl cinnamate.

[0054] Spalteholz's research had previously shown that the refractive indices of different tissues differ slightly from one another. His original 1911 publication listed a series of human tissues in ascending order of refractive index, determined empirically based on the optimal mixing ratio of methyl salicylate and BB: early embryos (5:1–3:1, depending on body weight) < adult decalcified bone (5:3) n = 1.547 < adult muscle, approximately the same as later embryos (2:1) < brain and spinal cord (1:1). This variation has a direct impact on the transparency of different tissues and on microscopic examination of said tissues, and particularly on quantitative spectroscopic measurements. The method according to the invention offers the advantage of using various pure substances as embedding media, which can be selected according to tissue type to approximate the tissue's refractive index.

[0055] Compared to benzyl benzoate / benzyl alcohol mixtures (BABB) and methyl salicylate / benzyl benzoate mixtures, which have been commonly used as embedding media to date, benzaldehyde anisole has the following advantages: Like diphenylmethyl ethers, they can be used in pure substance form; since the desired refractive index is already achieved in pure substance form, there is no need to set the refractive index by mixing embedding media. This eliminates the mixing step, thereby eliminating redundant working steps and potential sources of error.

[0056] Surprisingly, the benzaldehyde anisole of the present invention penetrates dehydrated tissue more quickly than any known embedding medium to date, and rapidly makes the tissue transparent. This saves time in sample preparation, allowing samples to be provided more rapidly for examination under an optical microscope. Therefore, the results of optical microscopy examination are also made more readily available. The density of dehydrated tissue is determined by the density of the dehydrating solvent, such as ethanol, still present in the tissue. Since embedding media typically have a higher density, complete penetration of the embedding medium into the tissue sample can be easily monitored by the settling of the tissue sample in the embedding medium. For example, using 2-hydroxy-5-methoxybenzaldehyde, the embedding medium is completely penetrated into the tissue sample in just 30 to 45 minutes, i.e., the tissue sample is at the bottom of the container. On the other hand, using conventional embedding media such as diphenylmethyl ether or methyl salicylate / benzyl benzoate mixtures, this process typically takes several hours to overnight. The method according to the invention therefore offers the advantage of more rapid diffusion of the embedding medium into the tissue sample, resulting in significant time savings in sample preparation. Tissue samples can therefore be examined more rapidly under an optical microscope.

[0057] If a tissue sample contains residual water and is transferred to an organic solvent used as an embedding medium in which the water can no longer be dissolved, the water is trapped in the tissue, resulting in turbidity that adversely affects transparency due to its incomplete miscibility. Therefore, it has been customary to incubate tissue samples multiple times, for example, with high-purity ethanol, followed by repeated incubation with the embedding medium, to avoid adversely affecting the sample's transparency. According to existing techniques, as explained, repeated incubation with high-purity anhydrous solvents is to completely remove any remaining trace amounts of water; for the same reason, many procedures also repeat incubation in the embedding medium.

[0058] According to the present invention, tissue clearing is performed using benzaldehyde anethole. Surprisingly, it was found that tissue samples containing residual water also achieved good transparency. At low residual water contents of 2% to 5% vol%, no adverse effect on transparency was observed, and even at high water contents of up to 20% or 30% vol, depending on the type of tissue treated, only extremely low turbidity was observed, which still allowed for sample examination by optical microscopy.

[0059] Therefore, the method according to the invention provides a particularly rapid and simple method for dehydrating tissue samples because, unlike all known embedding media to date, a low residual water content of up to 10% by volume has no adverse effect on the results of optical microscopy examination, and acceptable transparency in tissue samples is achieved even in the case of larger amounts of residual water.

[0060] Compared to clearing methods using conventional embedding media, the method according to the invention also allows for a simplified procedure. According to the prior art, during dehydration, tissue samples are transferred between containers with increasing concentrations of dehydrating solvent (e.g., ethanol with increasing concentrations). This requires several separate steps. Furthermore, a high-purity solvent, such as high-purity ethanol, is always required in one or more final steps.

[0061] In the method according to the invention, a dehydrating solvent with residual water content can also be used. For example, denatured 95% to 97% industrial-grade ethanol can be used instead of high-purity ethanol. Therefore, the method according to the invention also saves on the cost of the solvent used.

[0062] When using a gradient mixer, the method according to the invention also allows for a simplified procedure, since subsequent complete dehydration with a high-purity solvent is not required.

[0063] Furthermore, benzaldehyde anisaldehyde has low toxicity. This is advantageous when used as an embedding medium because the tissue sample in the microscope is located directly below the user's airway, and the user can inhale vapors rising from the sample. According to the European Chemicals Agency (ECHA), unlike diphenylmethyl ether or benzyl benzoate / benzyl alcohol mixtures, anisaldehyde has no toxicological effects.

[0064] The kit for preparing biological tissue samples for optical microscopy according to the present invention comprises:

[0065] - The dehydrating solvent used to dehydrate the tissue sample, and

[0066] - An embedding medium used to make a dehydrated tissue sample transparent by placing it in the embedding medium.

[0067] in,

[0068] The encapsulation medium is benzaldehyde anisole as described above, selected from 3-methoxybenzaldehyde, 4-methoxybenzaldehyde, 2-hydroxy-5-methoxybenzaldehyde, and 4-ethoxybenzaldehyde. Benzaldehyde anisole is preferably used at the concentrations and purities described above.

[0069] The dehydration 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.

[0070] According to the present invention, benzaldehyde anethole:

[0071] Selected from 3-methoxybenzaldehyde, 4-methoxybenzaldehyde, 2-hydroxy-5-methoxybenzaldehyde, and 4-ethoxybenzaldehyde.

[0072] Used as an embedding medium for preparing biological tissue samples, particularly human tissue samples, for optical microscopy examination.

[0073] The benzaldehyde anisole used according to the present invention is preferably used at the concentration and purity described above. Example

[0074] Example 1

[0075] A volume of approximately 0.25 ml (0.5 × 0.5 × 1 cm) 3 Several tissue samples (pig lungs) were dehydrated in anhydrous ethanol and photographed. The tissue samples dehydrated with ethanol are shown below. Figure 1 On the left side.

[0076] The dehydrated tissue sample was then placed in 5 ml of the appropriate embedding medium for 3 hours. The embedding medium used was a mixture of 2-hydroxy-5-methoxybenzaldehyde according to the present invention and, as a comparative example, methyl salicylate and benzyl benzoate, the refractive index of which was adjusted to the refractive index of 2-hydroxy-5-methoxybenzaldehyde. The results of the clearing step were also photographed. The results are shown in... Figure 1 On the right side. It was found that clearing with methyl salicylate / benzyl benzoate had resulted in good transparency of the tissue samples. However, the transparency obtained with the 2-hydroxy-5-methoxybenzaldehyde of this invention was even better.

[0077] Example 2

[0078] Using 2-hydroxy-5-methoxybenzaldehyde, ethyl cinnamate, and diphenylmethyl ether as the embedding medium, the volume prepared as described in Example 1 was approximately 0.25 ml (0.5 × 0.5 × 1 cm). 3 Several tissue samples (pig lungs).

[0079] The tissue samples were then stored overnight in anhydrous ethanol, and the embedding medium was washed out of the tissue samples again. The tissue samples were then transferred to ethanol with the specified water content to obtain the specified residual water content in the tissue, incubated for 2 hours, and then returned to the corresponding embedding medium for imaging. Tissue samples were placed in ethanol with contents of 95%, 90%, and 80% vol. The results were captured and shown below. Figure 2 The first row shows 100% dehydrated tissue samples in the embedding medium; the second row shows tissue samples in the embedding medium incubated in ethanol with a water content of 5% by volume; the third row shows tissue samples in the embedding medium incubated in ethanol with a water content of 10% by volume; and the fourth row shows tissue samples in the embedding medium incubated in ethanol with a water content of 20%. The bottom row shows the same tissue samples in anhydrous ethanol, i.e., before they were placed in the corresponding embedding medium. All samples have equal opacity when completely dehydrated in ethanol.

[0080] It was found that the known embedding media, diphenylmethyl ether and ethyl cinnamate, only achieved the desired transparency when there was no residual water or only a very small amount of residual water. In the case of DBE, a residual water content of 5% by volume was sufficient to make the sample no longer transparent and therefore unexamined under a microscope. Significant turbidity was also observed at a residual water content of 95% ECi, and the tissue sample was no longer completely transparent. On the other hand, tissue samples cleared with 2-hydroxy-5-methoxybenzaldehyde remained completely transparent even at a residual water content of 10% by volume (90% by volume ethanol), and showed only slight turbidity at 20% residual water (80% ethanol content).

[0081] Example 3

[0082] Tissue samples with a volume of approximately 0.25 ml (0.5 × 0.5 × 1 cm³) were dehydrated in anhydrous ethanol. They were then transferred to an embedding medium in a sample container. The embedding medium used was 2-hydroxy-5-methoxybenzaldehyde according to the invention, and, as a comparative example, a mixture of diphenylmethyl ether and methyl salicylate with benzyl benzoate. It was observed that in the case of 2-hydroxy-5-methoxybenzaldehyde, the tissue samples had clearly sunk below the surface after 15 minutes, and, depending on the tissue type, reached the bottom of the container after 30 to 45 minutes. In diphenylmethyl ether, the tissue samples took several hours to sink to the bottom, which is comparable to the time required for the methyl salicylate / benzyl benzoate mixture and DBE.

[0083] In further experiments, the rate at which the dehydrating solvent ethanol was replaced by the embedding medium in the tissue samples was determined. The embedding medium used was 2-hydroxy-5-methoxybenzaldehyde according to the present invention and, as comparative examples, diphenylmethyl ether and ethyl cinnamate. The tissue samples were placed in the embedding medium for 15 minutes, 30 minutes, and 70 minutes, and then photographed. The results are as follows: Figure 3 As shown. In the top row, tissue samples were photographed 15 minutes after clearing. In the middle row, tissue samples were photographed 30 minutes after clearing. In the bottom row, tissue samples were photographed 70 minutes after clearing. It can be seen that tissue samples treated with 2-hydroxy-5-methoxybenzaldehyde were already clear after 70 minutes, while samples treated with ECi showed only slight transparency, and tissue samples treated with DBE were completely opaque. Therefore, the clearing method according to the present invention achieves faster clearing of tissue samples.

[0084] The present invention is not limited to one of the above embodiments, but can be modified in various ways.

[0085] All features and advantages apparent from the claims, description and drawings, including method steps, are essential to the invention, individually or in various combinations.

Claims

1. A method for preparing a transparent tissue sample of biological tissue for optical microscopy examination, the method comprising the following steps: a) Dehydrate the tissue sample using a dehydrating solvent, and b) Transparentening a dehydrated tissue sample by placing it in an embedding medium containing benzaldehyde anisole selected from 3-methoxybenzaldehyde, 4-methoxybenzaldehyde, 2-hydroxy-5-methoxybenzaldehyde and 4-ethoxybenzaldehyde.

2. The method as described in claim 1, characterized in that, The embedding medium contains 10% to 100% by volume of the benzaldehyde anisole and 0% to 90% by volume of an optically suitable inert organic solvent with a refractive index of about 1.

3.

3. The method as described in claim 1, characterized in that, The embedding medium contains 10% to 100% by volume of the benzaldehyde anisole and 0% to 90% by volume of an optically suitable inert organic solvent with a refractive index of about 1.

5.

4. The method as described in claim 1, characterized in that, The embedding medium contains 10% to 100% by volume of the benzaldehyde anisole and 0% to 90% by volume of an optically suitable inert organic solvent with a refractive index of about 2.

0.

5. The method as described in claim 1, characterized in that, The embedding medium contains 10% to 100% by volume of the benzaldehyde anisole and 0% to 90% by volume of an optically suitable inert organic solvent with a refractive index of about 1.

65.

6. The method according to any one of claims 1 to 5, characterized in that, The embedding medium is composed of benzaldehyde anisole selected from 3-methoxybenzaldehyde, 4-methoxybenzaldehyde, 2-hydroxy-5-methoxybenzaldehyde and 4-ethoxybenzaldehyde.

7. The method according to any one of claims 1 to 5, characterized in that, Dehydration step a) includes using a dehydration composition consisting of an aqueous alcohol, ketone or ether, wherein the dehydration composition is an aqueous mixture of alcohol, ketone or ether, and wherein the solvent concentration of the dehydration composition is 50% to 98% by volume.

8. The method as described in claim 7, characterized in that, The solvent concentration of the dehydration composition is from 70% to 98% by volume.

9. The method as described in claim 7, characterized in that, The solvent concentration of the dehydration composition is from 75% to 98% by volume.

10. The method as described in claim 7, characterized in that, The dehydration composition is an aqueous mixture of aqueous ethanol with an increasing ethanol concentration, wherein the ethanol concentration of the dehydration composition is from 50% to 98% by volume.

11. The method according to any one of claims 1 to 5, characterized in that, The dehydration step a) is carried out in a gradient mixer, and the tissue sample is introduced directly into a dehydration solvent with a solvent concentration of 50% by volume at the beginning of the dehydration step, and then the gradient is increased in small increments.

12. The method according to any one of claims 1 to 5, characterized in that, The tissue sample prior to dehydration in step a) and optical transparency in step b) • Fixed and / or • After washing and / or • Incubate in an alkaline aqueous solution and / or • Degreasing with detergent solution and / or • Degreasing with organic solvents and / or • Bleaching with oxidizing agents and / or • Decolorize with amino alcohols.

13. The method of claim 12, wherein, The tissue sample is fixed with formaldehyde before dehydration in step a) and optical clearing in step b).

14. The method of claim 12, wherein, The tissue sample is washed with water before dehydration in step a) and optical transparency in step b).

15. The method according to any one of claims 1 to 5, characterized in that, The tissue sample was fixed before dehydration in step a) and optical clearing in step b), and the fixative was selected from... - Crosslinking fixative, and / or - Coagulant and fixative.

16. The method as described in claim 15, characterized in that, The crosslinking fixative is selected from formaldehyde, glutaraldehyde, acrolein, carbodiimide, diethyl pyrocarbonate, diimino ester, glyoxal, and mixtures thereof.

17. The method as described in claim 15, characterized in that, The coagulant / fixing agent is selected from alcohols and other organic solvents, acids, potassium dichromate, lead nitrate, copper sulfate, mercuric chloride, and mixtures thereof.

18. The method according to any one of claims 1 to 5, characterized in that, The optically transparent tissue sample is then examined under a microscope in another step to obtain an image of the internal structure of the sample; the microscope is an optical microscope.

19. A kit for preparing biological tissue samples for optical microscopy, the kit comprising: - Dehydrating solvents used to dehydrate tissue samples, and - An embedding medium for making a dehydrated tissue sample transparent by placing the sample in the embedding medium. Wherein, the dehydration solvent is an alcohol, ketone, or ether, and The embedding medium is benzaldehyde anisole selected from 3-methoxybenzaldehyde, 4-methoxybenzaldehyde, 2-hydroxy-5-methoxybenzaldehyde and 4-ethoxybenzaldehyde.

20. The kit according to claim 19, characterized in that, The dehydration solvent is selected from ethanol, methanol, tetrahydrofuran, isopropanol, tert-butanol, 2,2'-thiodiethanol, trichloroethanol, and acetone.

21. The application of benzaldehyde anisole as an embedding medium for preparing biological tissue samples for optical microscopy examination, wherein the benzaldehyde anisole is selected from 3-methoxybenzaldehyde, 4-methoxybenzaldehyde, 2-hydroxy-5-methoxybenzaldehyde and 4-ethoxybenzaldehyde.

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