IMPROVED METHOD FOR OPTICAL CLARIFICATION OF A TISSUE SAMPLE FOR LIGHT MICROSCOPIC EXAMINATION

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 image the tissue samples three-dimensionally, for example, using light-sheet microscopy. To achieve transparency in biological specimens, heme groups of the blood pigment hemoglobin and lipids, in particular, must be removed from the biological specimens. In "dehydrating" 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 dehydrating medium for pathological tissue clearing. The end result of all "dehydrating" procedures is an anhydrous sample.

[0003] The final step in the various clearing procedures is to adjust the refractive index to the refractive index of the tissue to be examined under the microscope. According to Spalteholz ("Über das Durchsichtigmachen von Menschenrechte und Tierischen Präparats", published by S. Hirzel, 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 requires the use of aromatic compounds that are generally immiscible or only slightly miscible with water. For his investigations, Spalteholz used mixtures of wintergreen oil (methyl salicylate) and benzyl benzoate or isosafrole in a mixing ratio adapted to the different tissues.

[0004] A variant of the Spalteholz 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 made from split wood and several other aromatic compounds were tested. Dibenzyl ether (DBE, refractive index n = 1.562) emerged as the most suitable compound and has become the de facto gold standard for tissue clearing.The advantage of DBE is also that it is easy to use because the solution does not need to be mixed and then checked for 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 nontoxic alternatives to the previously commonly used embedding media. It exhibits lower toxicity than other aromatic compounds used for tissue treatment, such as dibenzyl ether, benzyl alcohol, or benzyl benzoate. Because it achieves the desired refractive index as a pure substance, a mixing step is eliminated, thus eliminating a processing step and a potential source of error.

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

[0007] The invention is based on the object of providing a method for producing transparent tissue samples of a biological tissue for light microscopic examination, which overcomes the above disadvantages and which, in particular, is less complex than producing mixtures as embedding media, avoids embedding media of increased toxicity and allows the refrigerated storage of the dehydrated tissue samples.

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

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

[0010] The method according to the invention for producing transparent tissue samples of a biological tissue for light microscopic examination comprises the steps: a) Dehydrating the tissue sample with a dehydrating solvent and b) clarifying the dehydrated tissue sample by placing it in a liquid embedding medium with a refractive index matching the tissue.

[0011] The embedding medium contains an aromatic ester according to formula I or formula IV: where R 1< = -H, -CH 3 or -CH 2 -CH 3, R 2< is a radical of formula II or formula III R 3< = -H, -CH 3 , -CH 2 -CH 3 or -OCH 3 R 4< = -H or -CH 3 R 5< = -H or -CH 3 and n = 1 or 2; or

[0012] The ester according to the invention is preferably an aromatic ester according to formula I.

[0013] 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.

[0014] The clarification step of the clearing process involves matching the refractive index to the refractive index of the tissue to be examined. For this purpose, the tissue sample is transferred into a refractive index-matching solution, the embedding medium. The embedding medium must be miscible with the solvent used in the dehydration step.

[0015] In one embodiment, the embedding medium contains 10 to 100 vol.% of the aromatic ester 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 aromatic ester according to formula I.

[0016] In another embodiment, the embedding medium contains 10 to 100 vol.% of the aromatic ester 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 aromatic ester according to formula I.

[0017] All percentages according to the invention are given in volume percent (vol%).

[0018] In the preferred embodiment, the embedding medium consists of an aromatic ester according to formula I or formula IV, preferably formula I, i.e., the aromatic ester is used as a pure substance. The pure substance is understood to mean the ester in the technically available purity.

[0019] The aromatic ester is preferably a phenylacetic acid ester with aromatic alcohols. The aromatic ester is particularly preferably selected from benzyl 2-phenyl acetate, 4-methoxybenzyl phenyl acetate, phenethyl benzoate, or 2-furylmethyl phenyl acetate, particularly preferably benzyl 2-phenyl acetate. Benzyl 2-phenyl acetate (CAS No. 102-16-9) has a purity of approximately 98.0% as a technical product. 4-methoxybenzyl phenyl acetate (CAS No. 102-17-0) has a purity of approximately 99.0% as a technical product. 2-phenethyl phenyl acetate (CAS No. 102-20-5) has a purity of approximately 99.0% as a technical product. 2-Furylmethyl phenylacetate (CAS No. 36707-28-5) has a technical purity of approximately 96.0%. Phenethyl benzoate (CAS No. 94-47-3) has a technical purity of approximately 98%.

[0020] The dehydration step a) serves to obtain a water-free tissue sample. In a preferred embodiment, the tissue sample is treated in several passes, preferably with different dehydration compositions with a descending water series, i.e. mixtures with an ascending proportion of a water-miscible organic solvent, in order to completely remove the water from the tissue. Alcohols, ethers, or ketones are used as the dehydrating medium. Suitable dehydrating solvents are, for example, ethanol, methanol, isopropanol, tert-butanol (IUPAC: 2-methylpropan-2-ol), tetrahydrofuran, or acetone. In the process according to the invention, the dehydration step a) comprises, for example, the application of dehydration compositions from aqueous ethanol with increasing concentrations of ethanol, wherein the ethanol concentrations of the dehydration compositions range from 30% to 100% by volume, or from aqueous tetrahydrofuran with increasing concentrations of tetrahydrofuran, wherein the tetrahydrofuran concentration of the dehydration compositions range from 30% to 100% by volume, or from aqueous methanol with increasing concentrations of methanol, wherein the methanol concentrations of the dehydration compositions range from 30% to 100% by volume, or from an aqueous mixture of another alcohol, ether, or ketone, wherein the solvent concentration of the dehydration compositions ranges from 30% to 100% by volume.

[0021] The dehydration medium has the following properties: 1. it mixes completely with water so that the water and the 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.

[0022] 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 with the water contained in the tissue.

[0023] In one embodiment of the method according to the invention, the tissue sample is dehydrated in step a) and optically clarified in step b), 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.

[0024] In one embodiment of the method according to the invention, the tissue sample is fixed before being dehydrated in step a) and optically clarified in step b) and the fixative is selected from cross-linking 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.

[0025] The method according to the invention is preferably carried out after tissue preparation steps and after electrophoresis steps. The tissue sample treated in the method according to the invention is thus preferably 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 implementation of electrophoretic clarification, reference is made to patent specification DE 10 2016 123 458 B3, the content of which is hereby incorporated into this application.

[0026] In the method according to the invention, 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).

[0027] 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.

[0028] Of the aromatic esters according to formula I, for example, phenylmethylbenzyl acetate has a refractive index of n = 1.555-1.558, 4-methoxybenzylphenyl acetate a refractive index of n = 1.559, 2-phenethylphenyl acetate a refractive index of n = 1.551, and 2-furylmethylphenyl acetate a refractive index of n = 1.548 (calculated). When using the aromatic esters according to formula I as embedding media for tissue clearing of dehydrated tissue samples, identical transparency to tissue samples treated with methyl salicylate / benzyl benzoate and ethyl cinnamate could be achieved. In addition to alkoxy substitutions, short alkyl substitutions of the phenylacetic acid esters also yield refractive indices in this range, which are similar to the refractive indices of the tissues determined by Spalteholz.

[0029] Spalteholz's work already suggests that the refractive indices of different tissues differ slightly from one another. His original publication from 1911 lists the following series of human tissues, empirically determined based on the optimal mixing ratio of methyl salicylate and BB, in ascending order of refractive index: young embryos (5:1-3:1 depending on weight) < adult decalcified bones (5:3) n = 1,547 < adult muscle, approximately the same as older embryos (2:1) < brain and spinal cord (1:1). This variability has direct consequences for the transparency of the various tissues and their microscopic examination, and especially for quantitative spectroscopic determinations, which can be performed using a microscope.The method according to the invention offers the advantage that different pure substances are used as embedding medium, which can be selected depending on the tissue in order to approximate the refractive index of the tissue.

[0030] Compared to the previously commonly used embedding media, benzyl benzoate / benzyl alcohol mixtures (BABB) and methyl salicylate / benzyl benzoate mixtures, the aromatic esters according to Formula I have the advantage that, just like dibenzyl ether, they can be used as pure substances: 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 eliminating a redundant process and a potential source of error.

[0031] The aromatic esters according to Formula I also exhibit low toxicity. This is advantageous when used as an embedding medium, as the tissue sample in the microscope is located directly below the user's respiratory tract, and the user can inhale vapors rising from the sample. According to the European Chemicals Agency (ECHA), phenylmethylbenzyl acetate is toxicologically safe, unlike dibenzyl ether or benzyl benzoate / benzyl alcohol mixtures.

[0032] The phenylacetic acid esters according to Formula I are also low-volatility, as they have a significantly lower vapor tension than the known embedding media, so fewer vapors rise from the tissue sample. This further reduces the already low toxicity of the aromatic esters according to Formula I.

[0033] One disadvantage of the aromatic compounds previously used as embedding media is their corrosive effect. It is known that aromatic compounds attack the objective lens and the microscope, and that the vapors of these volatile compounds alone are sufficient to corrode the surfaces of the microscope facing the liquid over time.

[0034] Due to the lower volatility of the aromatic esters according to Formula I, the embedding media according to the invention offer significantly higher material compatibility, as significantly fewer vapors rise that could lead to corrosion. In summary, the low volatility of the aromatic esters according to Formula I reduces odor formation and respiratory irritation and toxicity, as well as the corrosive effect of the vapors on the microscope.

[0035] Phenylmethylbenzyl acetate also has a pleasant odor, with a sweet floral scent, hints of jasmine and chocolate, which increases the acceptance of the embedding medium among laboratory personnel. Thus, during microscopy, the user is not bothered by unpleasant odors rising from the stage, as is the case with conventional embedding media: dibenzyl ether has an unpleasant plastic smell, and benzyl alcohol has a phenolic odor.

[0036] The phenylacetic acid esters according to Formula I and Formula IV also allow clarified samples to be stored in the refrigerator. The melting point of phenylmethylbenzyl acetate, for example, is between -13.54 °C and -9 °C, meaning the compound is refrigerator-safe and even allows storage or transport on ice or in cold packs.

[0037] 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 placing it in the embedding medium, where the embedding medium is an aromatic ester of formula I or formula IV, as described above. The aromatic ester in the kit according to the invention is preferably benzyl 2-phenyl acetate, 4-methoxybenzyl phenyl acetate, phenethyl benzoate, or 2-furylmethyl phenyl acetate, preferably benzyl 2-phenyl acetate. The aromatic ester is preferably used in the concentrations and purities described above.

[0038] According to the invention, the dehydrating solvent in the kit is selected from ethanol, methanol, isopropanol, tert-butanol, 2,2'thiodiethanol, trichloroethanol, tetrahydrofuran, or acetone.

[0039] According to the invention, an aromatic ester according to formula I or formula IV is used with where R 1< = -H, -CH 3 or -CH 2 -CH 3, R 2< is a radical of formula II or formula III R 3< = -H, -CH 3 , -CH 2 -CH 3 or OCH 3 R 4< = -H or -CH 3 R 5< = -H or -CH 3 and n = 1 or 2; or as an embedding medium for the preparation of a biological, particularly human, tissue sample for light microscopic examination.

[0040] The aromatic ester is preferably benzyl 2-phenyl acetate, 4-methoxybenzyl phenyl acetate, phenethyl benzoate, or 2-furylmethyl phenyl acetate, particularly preferably benzyl 2-phenyl acetate. The aromatic ester is preferably used in the concentrations and purities described above.

[0041] The vapor pressures of various embedding media are known in the literature.

[0042] Fig. 1shows the vapor pressures of various embedding media. The phenylmethylbenzyl acetate (benzylphenyl acetate (BPA)) according to the invention has a significantly lower vapor pressure than the embedding media currently used. Of the compared embedding media, BPA is the least volatile and has the lowest vapor pressure. The vapor pressure of BPA (at 25 °C) is approximately 275 times lower than that of ethyl cinnamate. The two embedding media BABB and BBMS also have significantly (approximately 180 times) higher vapor pressures.

[0043] In an enlarged section in Figure 1 It can be seen that BPA is even less volatile (about 4 times) than DBE, which also contains two aromatic ring groups, and which is also less volatile than ECi due to its significantly lower vapor tension, with lower strength than methyl salicylate (also in the mixture with BB) and ECi. Examples

[0044] A tissue sample (pig lung) with a volume of approximately 0.25 ml (0.5 x 0.5 x 1 cm 3 ) was dehydrated with ethanol in several passes. In the final pass, ultrapure ethanol was used, and the dehydrated tissue sample was photographed. The results are shown in Figure 2 , left side. The tissue sample was then placed in 5 ml of the respective embedding media for 3 hours. A mixture of methyl salicylate with benzyl benzoate and the benzyl phenyl acetate according to the invention were used as embedding media. The result of the clarification step was also photographed and Figure 2 right side. It can be seen that while methyl salicylate / benzyl benzoate already leads to good transparency of the tissue sample, even better transparency was achieved with the benzyl phenyl benzoate according to the invention.

[0045] The invention is not limited to one of the embodiments described above, but can be modified in many ways.

Claims

1. Method for preparing transparent tissue samples of a biological tissue for examination by light microscopy, comprising the steps of: a) dehydrating the tissue sample with a dehydrating solvent and b) clearing the dehydrated tissue sample by placing it in an embedding medium comprising an aromatic ester corresponding to formula I or formula IV, where where R1 is -H, -CH3 or -CH2-CH3, R2 is a radical of the formula II or formula III R3 is -H, -CH3, -CH2-CH3 or -OCH3, R4 is -H or -CH3, R5 is -H or -CH3 and n is 1 or 2; or 2. Method according to Claim 1, characterized in that the embedding medium contains 10% to 100% by volume of the aromatic ester and 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 aromatic ester and 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 an aromatic ester corresponding to formula I or formula IV.

5. Method according to any of the preceding claims, characterized in that the aromatic ester is benzyl 2-phenylacetate, 4-methoxybenzyl phenylacetate, phenethyl benzoate or 2-furylmethyl phenylacetate, preferably benzyl 2-phenylacetate.

6. Method according to any of the preceding claims, characterized in that the dehydration step a) involves the use of dehydrating compositions composed of aqueous alcohol, ketone or ether.

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 decolourized 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 that the fixative is preferably selected from crosslinking fixatives such as formaldehyde, glutaraldehyde, acrolein, carbodiimides, diethyl pyrocarbonate, bisimidoesters or glyoxal or mixtures thereof, and / or coagulant fixatives such as alcohols and other organic solvents, acids, potassium dichromate, lead nitrate, copper sulfate and mercuric chloride, and mixtures thereof.

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

10. Kit for preparing biological tissue samples for light microscopy, comprising: - a dehydrating solvent for dehydrating the tissue sample and - an embedding medium for clearing the dehydrated tissue sample by placing the sample in the embedding medium, wherein the dehydrating solvent is selected from ethanol, tetrahydrofuran, isopropanol, tert-butanol, 2,2'-thiodiethanol, trichloroethanol or acetone and is present in the form of dehydrating compositions of aqueous solvent with increasing concentrations of solvent and the embedding medium is an aromatic ester of the formula I or formula IV, where where R1 is -H, -CH3 or -CH2-CH3, R2 is a radical of the formula II or formula III R3 is -H, -CH3, -CH2-CH3 or OCH3 , R4 is -H or -CH3, R5 is -H or -CH3 and n is 1 or 2; or 11. Kit according to Claim 10, characterized in that the aromatic ester is benzyl 2-phenylacetate, 4-methoxybenzyl phenylacetate, phenethyl benzoate or 2-furylmethyl phenylacetate, preferably benzyl 2-phenylacetate.

12. Use of an aromatic ester corresponding to formula I or corresponding to formula IV, where where R1 is -H, -CH3 or -CH2-CH3, R2 is a radical of the formula II or formula III R3 is -H, -CH3, -CH2-CH3 or OCH3 , R4 is -H or -CH3, R5 is -H or -CH3 and n is 1 or 2; or as an embedding medium for preparing a biological tissue sample for examination by light microscopy.