A gentle method for preparing transparent biological samples for examination using an optical microscope.

Abstract

The present invention relates to a method for preparing a transparent tissue sample of biological tissue for observation with an optical microscope, comprising the steps of: a) incubating the tissue in a strongly basic solution; and b) electrophoresis of the dehydrated tissue sample in a neutral, acidic, or weakly basic aqueous electrophoresis solution, which is 1 to 100 mol / m³. 3 The process includes the steps of immersing the sample in an electrophoretic solution containing a buffering agent of a certain concentration and at least one nonionic detergent in a concentration of 0.1 to 10% by weight, and then clearing it by electrophoresis by exposing it to an electric field in the electrophoretic solution. Electrophoresis for clearing and, if necessary, staining is performed at a weakly acidic or neutral pH value.

Description

Technical Field

[0001] The present invention relates to the method described in the preamble of claim 1.

Background Art

[0002] A transparent biological tissue sample is necessary, for example, to image a tissue sample in three dimensions using a light sheet microscope. To make a biological sample transparent, in particular, the heme group of hemoglobin, which is a blood pigment, and lipids must be removed from the biological sample. For example, by strong basic pretreatment, tissue components that limit transparency, such as heme compounds and lipids, are released. The tissue components thus released are then actively removed from the tissue by electrophoresis. In the next stage of the "electrical removal" process, the tissue is usually dehydrated with an alcohol series and then placed in a suitable solvent, such as an organic solvent, to adjust the refractive index.

[0003] Document US2005 / 0130317A1 describes a parallel analyzer for biological molecules having a reaction space extending between two electrodes. A voltage is applied to the electrodes to move biological molecules introduced between the electrodes.

[0004] In the tissue clearing method described in document DE102016123458B3, after performing strong basic pretreatment using an alkali metal hydroxide solution with pH > 14, fixed tissue is usually electrophoresed in a similar basic solution with 8 < pH < 10. Some types of tissues, particularly the brain and lungs, swell during basic electrophoresis.

Summary of the Invention

[0005] As is known in histological practice, shrinkage of fixed tissue is generally observed during dehydration. Therefore, while tissue shrinkage is acceptable for further analysis, swelling poses greater structural stress to the tissue material. The swelling-shrinkage cycle presents the problem of sensitive tissue samples being subjected to mechanical damage. Cracks can occur if the tissue does not allow sufficient elastic deformation, or, for example, if it is encapsulated in an inelastic capsule. Often, mechanical structural defects only occur or become noticeable when the tissue shrinks again after previously swelling (expanding). The hardening of the material and the forces generated during deformation at this time have a particularly significant impact on the tissue. Samples with little extent in any dimension of three-dimensional space, i.e., flat shapes, appear particularly vulnerable in this respect. Tissue samples are at their smallest dimensions after dehydration. In all processing steps leading up to dehydration, including dehydration, the tissue should be allowed to shrink in the most controlled manner possible, and swelling of the tissue should be prevented in particular, especially for the reasons mentioned above. Therefore, in sensitive tissue samples, it is desirable that electrophoresis be performed without swelling at least.

[0006] The present invention aims to overcome the above-mentioned drawbacks and, in particular, to provide a method for preparing transparent tissue samples of biological tissue for examination by optical microscope, which can reliably clear tissue samples without damaging or degrading them, and which minimizes the effort required for the clearing process and sample preparation.

[0007] According to the present invention, this objective is achieved by the method described in claim 1.

[0008] Other embodiments are the means of the dependent claims or are described below.

[0009] The present invention provides a method for preparing a transparent tissue sample of biological tissue for optical microscopy examination, a) The step of incubating the tissue in a strongly basic solution, preferably an alkali metal hydroxide solution, b) The tissue is immersed in an aqueous electrophoresis solution and exposed to an electric field in this electrophoresis solution, thereby clearing the dehydrated tissue sample by electrophoresis. Includes.

[0010] Electrophoresis solutions should be 1 to 100 mol / m³. 3 The solution contains a buffering agent at a concentration of 0.1 to 10% by weight of at least one nonionic detergent. A concentration of 1 to 5% by weight is also possible. The pH of the electrophoresis solution is 4.5 or higher. The buffering agent is an organic compound having at least one carboxylic acid group or at least one sulfonic acid group and at least one amine group. The pH of the electrophoresis solution is obtained by adding this substance to an aqueous solution, that is, the pH is obtained without adding a strong acid or strong base.

[0011] In step a), the tissue incubation is preferably carried out in an alkali metal hydroxide solution containing ethanol and at least one nonionic detergent in addition to the alkali metal hydroxide. In this incubation, it is desirable that the following parameters be at least partially satisfied. Incubation is carried out for 30 to 120 minutes, preferably 45 to 90 minutes. Incubation is performed at a temperature of 20°C to 50°C, preferably 35°C to 40°C, i.e., approximately 37.5°C. The concentration of the alkaline aqueous solution is 50 to 2,000 mol / m³. 3 Preferably 100 to 1,000 mol / m³ 3 The alkaline aqueous solution contains NaOH to provide an alkaline concentration. The alkaline aqueous solution contains C1-5 alcohol at a concentration of 10 to 70% by volume, or preferably 40 to 60% by volume (v / v). The alkaline aqueous solution contains ethanol and detergent at a concentration of 0.1 to 10% by weight, preferably 0.5 to 2.5% by weight. This solution is strongly basic and has a pH of 13 or higher, preferably 14.

[0012] In a preferred embodiment of the method according to the present invention, the pH value of the electrophoresis solution is in the range of 4.5 to 8.5, preferably 5.0 to 7.9, and more preferably the pH value is in the range of 5.5 to 7.9. Preferably, the pH value of the electrophoresis solution is spontaneously established in water. That is, no strong acid or strong base is added additionally to adjust the pH value so as to deviate from the spontaneous pH value. A strong acid is understood to refer to a subgroup of acids with an acid dissociation constant (pKa) of 4.5 or less in this context. A strong base is understood to refer to a subgroup of bases with a base dissociation constant (pKb) of 4.5 or less in this context, which corresponds to an acid dissociation constant (pKa) of 9.5 or more. Strong acids and strong bases exist in a significantly ionized state in an aqueous solution, unnecessarily increasing the conductivity of the electrophoresis solution. Therefore, the electrophoresis solution does not contain a pH adjuster that unnecessarily increases the conductivity. In principle, the lower the conductivity, the more advantageous it is.

[0013] An aqueous neutral, acidic or weakly basic electrophoresis solution is preferably weakly basic (pH 8.5 or 7.9) rather than neutral to weakly acidic (up to pH 4.5). In chemistry, generally, a solution with a pH value in the range of 4 to 7 is called weakly acidic, and a solution with a pH value in the range of 7 to 11 is called weakly basic. A solution with a pH value of 7 is called neutral.

[0014] The concentration of the buffer substance in the electrophoresis solution is from 5 to 50 mol / m 3 and more preferably from 15 to 25 mol / m 3 is desirable.

[0015] The concentration of the non-ionic detergent in the electrophoresis solution is preferably from 0.5 to 2.5% by weight.

[0016] (Buffer substance) The buffer substance is preferably 2-dimensions cm 2has a molar conductivity of less than / mol. For this purpose, in a dilution series of low concentrations (e.g., 5, 10, 20, 40 mM), the specific conductivity is measured using a conductivity meter. The measured values in μS / cm are plotted as a function of the concentration in mM units. In this dilution range, the specific conductivity should show a linear dependence on the concentration. Therefore, this dependence is determined using linear regression. The slope of the resulting straight line represents the molar conductivity in Siemens cm 2 / mol.

[0017] Preferably, a buffer substance showing a low self-conductivity as a pure substance solution is selected from weakly acidic to neutral buffer substances. These buffer substances preferably have an upper limit of molar conductivity of 1.5 S cm 2 / mol, and particularly preferably 1 S cm 2 / mol.

[0018] The buffer substance is preferably an organic compound containing only one ionizable acid group capable of generating one proton per molecule or an organic compound containing only one ionizable base group capable of generating one hydroxyl ion per molecule, and at the pH value spontaneously formed by dissolution in water, contains at most one positive charge and at most one negative charge per molecule and has no other charged groups. Particularly preferably, the buffer substance is an organic compound having only one carboxylic acid group or only one sulfonic acid group and having at least one amine group.

[0019] The buffer substance is an organic compound having at least one carboxylic acid group or at least one sulfonic acid group and having at least one amine group. Preferably, the buffer substance is selected from the group consisting of aminocarboxylic acids, aminosulfonic acids, aminocarboxylate salts, or mixtures thereof. More preferably, the buffer substance is aminoalkanesulfonic acid, hydroxyalkylaminosulfonic acid, piperidinealkanoic acid, amino acids, N-hydroxyalkylaminocarboxylic acids, or aminocarboxylate salts, aminosulfonate salts, or mixtures thereof, and when measured by the above dilution method, has a molar conductivity of 2 Siemens cm2 It is less than / mol. In this invention, for linguistic simplification, a mixture of multiple buffering substances is also referred to as a buffering substance, just like a pure substance.

[0020] (aminosulfonic acid) The buffering agent may be selected from the aminoalkanesulfonic acid group according to general formula I.

[0021] [ka]

[0022] Here, n is 1-4, R 1 is a hydrogen atom or a methyl group, R 2 These include cyclohexyl residues, alkyl residues having 1 to 4 carbon atoms, or hydroxylalkyl residues having 1 to 4 carbon atoms and at least one hydroxyl group. That is the case.

[0023] Suitable buffering agents in the form of aminoalkanesulfonic acid include, for example, those with the general formula (molecular formula) C6H 11 -NH-[CH2] n -SO2OH This is a 2-cyclohexylaminoalkanesulfonic acid, where n=1-4, and chemical formula II is as shown in chemical formula 2. Examples include 2-(cyclohexylamino)ethanesulfonic acid (CHES) (CAS number 103-47-9), 3-(cyclohexylamino)-1-propanesulfonic acid (CAPS) (CAS number 1135-40-6), and 4-(cyclohexylamino)-1-butanesulfonic acid (CABS) (CAS number 161308-34-5).

[0024] [ka]

[0025] Other suitable aminoalkanesulfonic acids include hydroxyalkylaminosulfonic acids, for example, those with the molecular formula C[COH]3-NH-[CH2] n -SO2OH Tris(hydroxymethyl)methylaminoalkanesulfonic acid for n=1-4 for example, It is N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid (TAPS) (CAS number 29915-38-6), and its chemical formula III is as shown in Chemical Formula 3.

[0026] [ka]

[0027] (aminocarboxylic acid) A buffering substance even more suitable for the method according to the present invention is an aminocarboxylic acid.

[0028] Suitable buffering substances in the form of aminocarboxylic acids include, for example, non-proteinogenic amino acids, for example, those with a molecular formula. C6H9NH-[CH2]n-COOH These are piperidinecarboxylic acids (piperidinealkanoic acids) with n=0-3, and their chemical formula IV is as shown in Chemical Formula 4.

[0029] [ka]

[0030] Here, n is 0-3, These include piperidine-2-carboxylic acid, also known as pipecolic acid (CAS number 535-75-1), n=0; piperidine-3-carboxylic acid, also known as nipecotinic acid (CAS number 498-95-3), n=0; piperidineacetic acid, such as piperidine-2-acetic acid and piperidine-4-acetic acid, n=1; piperidinepropionic acid, n=2; and piperidinebutyric acid, n=3.

[0031] Other aminocarboxylic acids suitable as buffering substances have the general formula, The amino acids of NH2-CHR-COOH and the dipeptide NH2-CHR-CONH-CHR-COOH, such as aminoacetic acid, also known as glycine (CAS number 56-40-6), are represented by the following chemical formula:

[0032] [ka]

[0033] Glycylglycine (CAS number 556-50-3) is represented by the following chemical formula V.

[0034] [ka]

[0035] Pyrrolidine-2-carboxylic acid, also known as proline (CAS number 609-36-9), is represented by the following chemical formula VI.

[0036] [ka]

[0037] N-hydroxyalkylaminocarboxylic acids are also suitable as buffering agents. The general molecular formula for this group of amino acids is: C[COH]3-NH-[CH2] n -COOH, Tris(hydroxymethyl)methylaminocarboxylic acids with n=1-3 are suitable buffering agents. One suitable tris(hydroxymethyl)methylaminocarboxylic acid is, for example, N-[tris(hydroxymethyl)methyl]aminoacetic acid, also known as tricine, or N-[tris(hydroxymethyl)methyl]glycine (CAS number 5704-04-1), which is represented by the following chemical formula VII.

[0038] [ka]

[0039] Another suitable N-hydroxylalkyl amino acid is bis(hydroxyethyl)aminoacetic acid, also known as N,N-bis(2-hydroxyethyl)glycine (CAS number 150-25-4), which is represented by the following chemical formula VIII.

[0040] [ka]

[0041] (salts) Further suitable buffering materials for the method according to the present invention are aminocarboxylate salts and aminosulfonates.

[0042] A suitable aminocarboxylate salt is, for example, one with the molecular formula, N + (CH3)3-[CH2] n -COO - This group includes trialkylammonium alkanoates, such as trimethylammonium alkanoates with n=1-3. Suitable trimethylammonium alkanoates are represented by the following chemical formula IX, for example, trimethylammonium acetate, also known as trimethylglycine, betaine (CAS number 107-43-7),

[0043] [ka]

[0044] These are amino acid salts with longer alkanoic acid chains, such as trimethylammonium propionate (n=2) or trimethylammonium bnitrate (4C). These compounds are salts because they have a nitrogen atom with a permanent charge.

[0045] In addition to N-methyl-substituted ammoniacalkanates, ethyl-substituted or higher N-alkyl-substituted salts are also suitable.

[0046] In addition to trialkylammonium alkyl sulfonates, structurally similar trialkylammonium alkyl sulfonates, i.e., aminosulfonates, can also be used. One suitable alkylammonium alkyl sulfonate is, for example, 3-[dimethyl-(2-hydroxyethyl)ammonium]-1-propanesulfonate (NDSB211), CAS number 38880-58-9, represented by the following chemical formula X.

[0047] [ka]

[0048] Other suitable alkylammonioalkylsulfonic acids are, in general formula N-(1,1-dimethyl-2-hydroxyethyl)-3-amino-2-hydroxypropanesulfonic acid (AMPSO), CAS number 68399-79-1, is represented by the following chemical formula XI.

[0049] [ka]

[0050] Surprisingly, buffering substances may be used not only as pure substances but also mixed with other buffering substances or buffering substrates for clearing by electrophoresis. For example, two buffering substances suitable for the present invention can be mixed. In-house tests have shown that, for example, when N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid (TAPS) and 3-(cyclohexylamino)-1-propanesulfonic acid (CAPS) are mixed in a 1:1 ratio, the conductivity of the mixture is identical to that of a pure CAPS solution, even though TAPS itself has a higher conductivity. Such a mixture of multiple buffering substances is referred to as a buffering substance in the present invention.

[0051] The buffering substance can be mixed with the buffering base, and the mixture preferably contains more than 50% by weight, particularly more than 75% by weight, of the weakly acidic to neutral buffering substance, that is, the buffering substance is the main component compared to the buffering base.

[0052] The buffering agent is an aminoalkanesulfonic acid following chemical formula I (general formula I), with the molecular formula C[COH]3-NH-[CH2] n -SO2OH, n=1-4 tris(hydroxymethyl)methylaminoalkanesulfonic acid, molecular formula C6H9NH-[CH2] n -COOH, n=0-3 piperidine carboxylic acid, molecular formula NH2-CHR-COOH and dipeptide NH2-CHR-CONH-CHR-COOH, N-hydroxyalkylaminocarboxylic acid, molecular formula C[COH]3-NH-[CH2] n -COOH, n=1-3 tris(hydroxymethyl)methylaminocarboxylic acid, N-hydroxylalkyl amino acid, trialkylammonioalkanoate, for example, N + (CH3)3-[CH2] n -COO - Preferably, the molecular formula is selected from the group consisting of trimethylammonioalkanoates and trialkylammonioalkylsulfonates having n=1-3.

[0053] Particularly preferred, the buffering agent is selected from the group consisting of 2-(cyclohexylamino)ethanesulfonic acid, 3-(cyclohexylamino)-1-propanesulfonic acid, 4-(cyclohexylamino)-1-butanesulfonic acid, N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid, piperidine-2-carboxylic acid, piperidine-3-carboxylic acid, piperidineacetic acid, piperidinepropionic acid, aminoacetic acid, glycylglycine, pyrrolidine-2-carboxylic acid, N-[tris(hydroxymethyl)methyl]aminoacetic acid, bis(hydroxyethyl)aminoacetic acid, trimethylammonium acetate, trimethylammonium propionate, trimethylammonium btyrate, 3-[dimethyl(2-hydroxyethyl)ammonium]-1-propanesulfonate, N-(1,1-dimethyl-2-hydroxyethyl)-3-amino-2-hydroxypropanesulfonic acid, and mixtures thereof.

[0054] (detergent) In the method according to the present invention, a nonionic detergent is used. A nonionic detergent is a surfactant that has a head group that is polar, hydrophilic, and water-soluble, and has no charge in the neutral pH range. It should be understood that the term "nonionic detergent" does not refer only to detergents with zero ionicity. Rather, this term refers to all detergents that do not exhibit significant ionicity, i.e., at least essentially ionicity, and in particular detergents that have no net charge per molecule in the pH range of pH > 4.5. Furthermore, it is preferable that the nonionic detergent is selected to have the lowest possible affinity for proteins. Suitable nonionic detergents include, for example, alkylphenyl ethoxylates such as octylphenyl ethoxylate, fatty acid ethoxylates, aliphatic alcohol polyglycol ethers, alkyl polyglucosides, secondary alcohol ethoxylates, betaines and alkyl sulfobetaines, also known as sultaines, and mixtures of these detergents.

[0055] Practical tests of the method according to the present invention have shown that at least the following detergents are suitable as nonionic detergents in the method according to the present invention. - Polyoxyethylene (20) sorbitan monolaurate (CAS number 9005-64-5), trade name Tween20, - Polyoxyethylene sorbitan monooleate (CAS number 9005-65-6), trade name Tween80, - Polyethylene glycol 4-tert-octylphenyl ether (molecular formula t-Oct-C6H4-(OCH2CH2) x OH, x=~5, CAS number 9002-93-1), product name TritonX45, - Octylphenoxypolyethoxyethanol (molecular formula t-Oct-C6H4-(OCH2CH2)) x OH, x=9-10, CAS number 9036-19-5), product name TritonX100 - Polyethylene glycol-tert-octylphenyl ether (CAS number 9036-19-5), trade name TritonX102, - Polyethylene glycol monoalkyl ether (CAS number 9043-30-5), trade name Genapol, - n-octyl-β-D-glucopyranoside (CAS number 29836-26-8) - Decaethylene glycol monododecyl ether (CAS number 9002-92-0), trade name Brij, - Octylphenoxypolyethyleneoxyethanol (molecular formula (C2H4O)) n C 14 H 22 O, CAS number 9002-93-1), product name IGEPAL CA-630, - Secondary alcohol ethoxylate (CAS number 84133-50-6), trade name Tergitol, and, - 3-[(3-Colamidopropyl)dimethylammonio]-1-propanesulfonate hydrate (CAS number 75621-03-3), trade name CHAPS, - Lauryl sulfobetaine (CAS number 14933-08-5).

[0056] The properties particularly favorable for use in the present invention are demonstrated by nonionic detergents, specifically polyoxyethylene sorbitan monooleate (Tween 80), octylphenoxypolyethoxyethanol (Triton X 100), polyethylene glycol 4-tert-octylphenyl ether (Triton X 45), polyethylene glycol-tert-octylphenyl ether (Triton X 102), polyethylene glycol monoalkyl ether (Genapol), n-octyl β-D-glucopyranoside, decaethylene glycol monododecyl ether (Brij), octylphenoxypolyethyleneoxyethanol (IGEPAL CA-630), and secondary alcohol ethoxylate (Tergitol). The most outstanding properties are demonstrated by octylphenoxypolyethoxyethanol (Triton X100), decaethylene glycol monododecyl ether (Brij), secondary alcohol ethoxylate (Tergitol), polyethylene glycol monoalkyl ether (Genapol), and n-octyl-β-D-glucopyranoside.

[0057] The nonionic detergent used for electrophoresis may be a combination of several of the detergents mentioned above. The selection and / or mixture of detergents can be intentionally adjusted to suit the various components of each biological tissue to be removed during clearing by electrophoresis.

[0058] The use of a nonionic detergent in the method according to the present invention means that no current in the form of detergent ions is generated by the applied electric field, and this current is merely parasitic (unwanted) with respect to the desired clearing of the biological tissue. Such parasitic ionic currents result in heating of the biological tissue, but this heating is unrelated to the clearing of the biological tissue. Parasitic currents prevent the formation of an electric field with a greater electric field strength throughout the biological tissue because the large current generated by the parasitic currents overheats the biological tissue. However, an electric field with a greater electric field strength is advantageous for electrically removing larger, and consequently slower-moving, micelles from the biological tissue.

[0059] The method according to the present invention is preferably, 0) The step of fixing the tissue sample with formaldehyde, glutaraldehyde, or other cross-linking fixative, a) A step of pretreatment with an alkali metal hydroxide solution, b) A step of performing electrophoresis in a solution containing a low concentration of buffering material and without further addition of a strong alkali or strong base, with a pH value of 4.5 to 8.5, preferably 5.0 to 7.9, more preferably 5.5 to 7.9, in the presence of a nonionic detergent, c) A step of making the material transparent for a period of time determined by the course of change in electrical values, particularly ohmic resistance and / or conductivity, until the rate of change of ohmic resistance and / or conductivity decreases to a predetermined extent, d) Dehydration step, followed by, e) The step of embedding the tissue in a solvent or solvent mixture having a refractive index > 1.5, so that it becomes transparent.

[0060] Instead of step d), the sample may be immersed in a suitable aqueous medium, such as a concentrated sugar solution, an aromatic compound, or an iodine-containing radiographic contrast agent, without dehydration.

[0061] The maximum temperature of the electrophoresis solution during clearing by electrophoresis may be maintained in the range of 20 to 90°C. In any case, the temperature must be kept below the boiling point of the electrophoresis solution. Typically, during clearing by electrophoresis, it is preferable to maintain the maximum temperature of the electrophoresis solution at 40 to 60°C, i.e., about 50°C. However, if the temperature exceeds this, the binding sites of the antibody to the protein can be exposed by heat, and as a result, these proteins can be labeled with the antibody.

[0062] The method according to the present invention involves an increase in the temperature of the biological tissue due to the introduction of electrical energy converted into heat. This is essentially unavoidable in electrophoresis. However, in the method according to the present invention, a particularly large degree of clearing of the biological tissue is achieved in relation to the introduced electrical energy. This makes it particularly easy to limit the temperature of the biological tissue to the highest temperature within the above range without the need to actively cool the electrophoretic solution.

[0063] During clearing by electrophoresis, fresh buffer and / or fresh detergent may be added to the electrophoresis solution. Alternatively, the electrophoresis solution may be continuously replaced.

[0064] When implementing the method according to the present invention, the pH value of the electrophoretic solution may be maintained in the range of 4.5 to 8.5, preferably 5.0 to 7.9, during clearing by electrophoresis. The current flowing as a result of the applied electric field usually lowers the pH value of the electrophoretic solution in the anodic buffer region. On the other hand, in the cathode buffer region, the pH value of the electrophoretic solution usually rises. Therefore, in order to maintain a high efficiency in clearing biological tissue, it is useful to maintain the pH value of the electrophoretic solution within the above range. For this purpose, fresh buffer material may be added during clearing by electrophoresis. In addition, detergent consumed by the removal of micelles under the influence of the electric field may be replenished by adding fresh detergent to the electrophoretic solution during clearing by electrophoresis. The maximum efficiency of the method according to the present invention is achieved by continuously replacing the electrophoretic solution with unused electrophoretic solution.

[0065] In electrophoresis, the power supplied to the electrophoretic solution and the tissue immersed therein may be adjusted during electrophoretic clearing. The power may be adjusted to a fixed value for at least a portion of the electrophoretic clearing period.

[0066] During electrophoretic clearing, the electrical conductivity of the electrophoretic solution and the biological tissue immersed therein may be recorded. If the electrical conductivity or its change over time exceeds and / or falls below predetermined limit values, the individual conditions of the electrophoretic clearing may be changed and / or the electrophoretic clearing may be terminated.

[0067] In the method according to the present invention, the power supplied to the electrophoretic solution and the tissue immersed therein may be controlled during electrophoretic clearing. This control may be performed according to the temperature of the biological tissue or the electrophoretic solution, and may be controlled to a constant value to ensure that a particular maximum temperature is maintained. At least for a portion of the electrophoretic clearing period, the power may be controlled to such a fixed value. If the clearing of the biological tissue has progressed considerably, and / or if the electrophoretic solution has already been largely consumed, it is often not meaningful to further set the power to a fixed value.

[0068] The progress of tissue clearing may be observed by recording the electrical conductivity of the electrophoretic solution and the tissue immersed therein. First, as micelles to be removed are formed within the tissue, the electrical resistance decreases, i.e., the electrical conductivity increases. Due to the consumption of the electrophoretic solution and / or the already completed extraction of the heme groups and major lipid components to be removed, the electrical conductivity decreases again. Therefore, in the method according to the present invention, the conditions for electrophoretic clearing may be changed and / or the electrophoretic clearing may be terminated when the electrical resistance or its change over time exceeds and / or falls below a predetermined limit value.

[0069] Prior to clearing the biological tissue by electrophoresis according to the method of the present invention, the biological tissue may undergo other treatments. This includes, for example, fixation of the biological tissue with formaldehyde, but unlike the hydrogel used for fixation, formaldehyde does not restrict the mobility of micelles. Steps that can be performed before actual clearing by electrophoresis include, for example, washing the biological tissue with water or an electrophoretic solution that will be used later during clearing by electrophoresis. Furthermore, the biological tissue may be incubated in an alkaline aqueous solution before clearing by electrophoresis.

[0070] After biological tissue has been cleared by electrophoresis, it can be further prepared for observation under a light microscope. To do this, at least one of the following steps can be performed:

[0071] The biological tissue is incubated with at least one type of antibody. The biological tissue is exposed to an electric field while in a solution of antibodies. The biological tissue is washed with an organic solvent. The biological tissue is washed with xylene or dichloromethane. The biological tissue is introduced into a solution with a refractive index ranging from n=1.4 to n=1.6. A refractive index range of n=1.4 to n=1.6, i.e., n=approximately 1.5, means that this solution has the same refractive index as the cleared biological tissue. As a result, when observing the biological tissue with an optical microscope, no scattering occurs at its interface.

[0072] Specifically, the biological tissue cleared by electrophoresis may be introduced into an aqueous solution and / or sugar solution, polyol solution, aromatic solution, or iodine-containing contrast agent solution with a refractive index of n=approximately 1.5 within the above range. Alternatively, the biological tissue cleared by electrophoresis may be dehydrated in a series of alcohol solutions with gradually increasing alcohol concentrations and / or introduced into an organic solvent, methyl salicylate, or a methyl salicylate solution with a refractive index in the range of n=approximately 1.5.

[0073] The method according to the present invention makes it possible to achieve clearing of biological tissue within a few hours for the preparation of biological preparations for optical microscopy.

[0074] Optionally, tissue samples may be stained with dyes or antibodies before or during electrophoresis. Biological tissues may be exposed to an electric field in a dye solution, preferably during electrophoresis. Since staining is performed during clearing by electrophoresis, no separate step for staining is required.

[0075] A preferred modification is to perform staining during electrophoresis. The electric field increases the rate of the staining process because the dye or stained biomolecules move through the tissue by their charge rather than by diffusion.

[0076] In the method according to the present invention, the clearing step by electrophoresis can be performed within a pH range from basic (pH > 7.0) to neutral (pH = 7.0) and acidic (pH = 4.5), allowing for the selection of a pH value at which the dye / antibody becomes charged, and enabling simultaneous clearing and activation staining by utilizing the effect of an electric field. The pH value during electrophoresis may be adjusted to a pH value at which the dye / antibody has the optimal charge for activation staining. The pH range extending to acidic conditions expands the options for combining clearing and staining by electrophoresis. A further advantage of staining under the influence of an electric field is that the electric field efficiently removes unbound dye / antibody from the tissue. Therefore, this method replaces the time-consuming washing and fractionation steps in regression staining methods, saving time and materials. Furthermore, by limiting both processes to a single electrophoresis step, the Joule heating of the tissue generated during each electrophoresis can be further minimized.

[0077] To specifically implement the method according to the present invention, tissue can be placed in a reaction chamber having a constricted portion and a reaction space that can be filled with an electrophoretic solution, which is rotationally symmetrical around the vertical axis, for clearing by electrophoresis. Below the constricted portion, there is a downward-opening annular channel for the reaction space, which is connected to an upward-extending gas discharge channel, and a first annular electrode is located in the reaction space within the annular channel and / or below the annular channel, and a second annular electrode is located in the reaction space above the constricted portion. The first and second electrodes are connected to two outputs of a DC voltage source to expose the biological tissue to an electric field. The tissue is placed within the reduced free cross-sectional area of ​​the reaction space, and a substantially uniform electric field is concentrated between the electrodes. Bubbles formed at the lower electrode and rising therefrom are discharged through the gas discharge channel, so that bubbles do not accumulate in the reaction space and obstruct the flow of current. Furthermore, if the gas bubbles contain hydrogen produced by hydrolysis, this hydrogen may mix with oxygen produced by hydrolysis at the upper electrode, thus preventing the formation of oxyhydrogen. The reaction chamber may have other features, such as those described in reference US2005 / 0130317A1.

[0078] The method for preparing transparent tissue samples according to the present invention has many advantages over conventional methods, particularly basic electrophoresis for clearing tissue samples.

[0079] Surprisingly, following a strongly basic pretreatment, tissue samples can be cleared by electrophoresis at pH values ​​ranging from neutral to weakly acidic (pH ≥ 4.5) and even weakly basic (pH values ​​of 8.5 or 7.9). Below a pH of 4.5, charge carriers are no longer released from the tissue, and as a result, the tissue no longer becomes clear.

[0080] Furthermore, it is surprising that tissue samples do not swell during electrophoresis at pH values ​​ranging from weakly basic to neutral and weakly acidic, and in some cases, the volume even decreases. Since tissues shrink during dehydration compared to their initial size in formaldehyde, the change in tissue size throughout the clearing process is favorably maintained by the fact that the tissue volume remains constant or decreases during electrophoresis.

[0081] Compared to processes where tissue volume increases during basic electrophoresis and then decreases during dehydration, this new continuous process is preferable, resulting in smaller maximum and overall changes. This eliminates the problem of mechanical damage to sensitive tissue samples caused by the swelling and shrinking cycle.

[0082] The fact that tissue clearing is achieved by electrophoresis at weakly basic, neutral, or weakly acidic pH values ​​in the method according to the present invention opens up new application possibilities, such as simultaneously clearing and staining tissue samples in a single electrophoresis step. By adjusting the pH value of the electrophoresis solution, the direction and speed of electrically driven transport of dyes and biomolecules with ionizable groups can be controlled through the change in charge caused by the change in pH value. This allows the desired contact time with the tissue to be adjusted to match the clearing time. In the case of purely passive staining before electrophoresis (with or without subsequent electrically driven decolorization), the tensile force during clearing by electrophoresis can be limited to prevent the loss of previous binding during this step.

[0083] When staining with charged dyes / antibodies before electrophoresis, there is a risk that the charged dyes / antibodies may be removed from the tissue again if the electrical "attraction force" exceeds the acceptable binding strength of the tissue component being stained. Therefore, the most important application is staining during electrophoresis. Due to the influence of the electric field, the dye (or stained biomolecule) is attracted into the tissue by its charge rather than penetrating through diffusion, thus increasing the rate of the staining process. However, the "attraction force" should not be so high that there is insufficient time for interaction for binding. The optimal conditions for active staining with dyes / antibodies usually do not coincide with the optimal conditions for the clearing step by electrophoresis at basic pH values. Therefore, the method according to the present invention solves the problem of the currently lacking but very useful pH range of 4.5 to 7.9 in electrophoresis, and complements the possibility of effective staining before or during electrophoresis, i.e., during the clearing process.

[0084] Therefore, in the method according to the present invention, both the clearing and staining electrophoresis steps may be carried out at a weakly acidic, neutral, or weakly basic pH value. This offers the following advantages: • The volume change of the tissue occurs in only one direction, the volume decreases, the volume change becomes smaller, and both electrophoresis steps for clearing and staining can be performed simultaneously in a single step. • Electrophoretic staining can be performed at the optimal pH value for the stained molecules and the staining process. This shortens the clearing process compared to basic electrophoresis.

[0085] According to the method of the present invention, a favorable size profile can be obtained during the clearing process. That is, the volume / size of the tissue sample is reduced in a stable and controlled manner, and size variability is minimized.

[0086] Surprisingly, and contrary to expectations, electrophoresis in the pH range from weakly acidic (pH ≥ 4.5) to weakly basic yields high-quality clarity of tissue samples. Particularly for gentle electroclearing processes, electrophoresis in the pH range from weakly acidic (pH ≥ 4.5) to neutral, and weakly basic, is advantageous because it prevents sample swelling during electrophoresis and supports or gently initiates shrinkage to match size after dehydration. [Brief explanation of the drawing]

[0087] [Figure 1] This figure shows the optical size changes during electrical clearing in the human brain. [Figure 2] This figure shows the optical size change during electroclearing of pig lungs. [Figure 3] This figure shows the size change due to weight during the electrotransparency treatment. [Modes for carrying out the invention]

[0088] The mild effects of acidic / neutral electrophoresis were tested using lung and brain tissues, which are particularly susceptible to size changes.

[0089] Human brain and pig lung tissue samples were electrophoresed at a weakly acidic pH using 20 mMCAPS buffer (pH 6) and 20 mMCHES buffer (pH 5), and also at a basic pH (pH 10) using 20 mMTris buffer (comparative example). The buffers were used in their pure form without mixing with other buffers, bases, or acids to adjust the pH. This minimized the charge carriers added to the liquid and, consequently, the conductivity of the solution. All solutions contained 1% w / v of a nonionic detergent, in this case Triton X-100, which is octylphenol ethoxylate. All samples became clear after clearing treatment.

[0090] The tissues were fixed with 4% formaldehyde and stored in the fixation solution for an extended period. Human brain and pig lungs stored for two years were evaluated. Because tissues fixed for extended periods become mechanically sensitive to handling and clearing, brain tissue stored in formaldehyde solution for seven years was also cleared for comparison. The size of all tissues at the start of treatment was 10x10x2mm. 3 That was the case.

[0091] As an indicator of volume change, tissue samples were weighed after each processing step and photographed for sizing. Weights were normalized relative to the initial weight of the fixed sample. The aqueous solutions used for fixation, basic pretreatment, and electrophoresis have similar densities. Since the density of ethanol is approximately 80% of water, the volume change during the dehydration step is slightly overestimated; however, accurate measurement is impossible with this method because the dry content of the sample is unknown. Photographs of the samples after each step also record the size change (see Figures 1 and 2).

[0092] Both tissue types showed a decrease in volume during the transition from fixed tissue ("Fix") to dehydrated sample ("EtOH") (see Figure 3). Brain tissue showed little change during strong basic pretreatment. In contrast, lung tissue showed shrinkage at this step. Both tissues showed a clearly undesirable increase in size during basic electrophoresis with Tris (comparative example, basic electrophoresis). This increase could be completely prevented in the tests of the present invention by using CAPS, particularly CHES. In the case of brain tissue after 7 years, a decrease in volume was observed, and this decrease continued even during dehydration. A decrease in volume was also observed when the buffering substances CAPS and CHES according to the present invention were used on pig lungs. In this case, the volume had already almost reached the post-dehydration volume during electrophoresis. Therefore, by using a weakly acidic (CHES) or (CAPS) buffering substance during electrophoresis, tissue electrophoresis can be performed without the size increase that occurs when using a basic buffering substance (Tris). The mechanical stress caused by swelling and subsequent contraction could be clearly demonstrated using examples of human brain tissue samples that had been fixed for seven years.

[0093] This beneficial effect was tested and observed with several different buffering materials, in addition to CHES and CAPS, ranging from pH 4.5 to 5.5 (AMPSO, CABS, commonly known as N,N-bis(2-hydroxyethyl)glycine), pH 5.5 to 7 (Trisine, TAPS, CAPS glycylglycine), and to neutral pH (glycine). CHES and CAPS exhibit high electrical resistance (low conductivity), i.e., they allow for a high potential difference between electrodes, making them even more advantageous for the clearing process by electrophoresis. For comparison, tissue swelling is generally observed with basic buffering materials. Electrophoresis using Tris solution (comparative) showed relatively large cracks in the samples. Human brain samples that had been fixed for 7 years decomposed into multiple parts during dehydration. Similar experiments have shown that such sensitive materials are often damaged even after electrophoresis.

[0094] Figure 1 shows the optical size change during electroclearization in the human brain. 10x10x2mm 3 The two brain samples were made clear as described above. The samples had previously been preserved in formalin for two years (top row) or seven years (bottom row). Each row shows the size progression after electrophoresis using the specified buffer. In the order specified in the images, the samples are shown after pretreatment with formalin ("Fix"), 1M NaOH, 50% ethanol, and 1% TritonX-100, electrophoresis with 20mM of the specified buffer (Tris, CHES, CAPS), complete dehydration with ethanol ("EtOH"), and embedding in a mixture of benzyl benzoate and wintergreen oil ("BBWGO").

[0095] Figure 2 shows the change in optical size during electroclearing of pig lungs. The pig lungs had been preserved in formalin for two years prior to the clearing treatment. The treatment and conditions were the same as in Figure 1.

[0096] Figure 3 shows the size change due to weight during the electrotransparency treatment. The samples in Figures 1 and 2 were weighed after each step. The change in weight is used here as a measure of the change in size. The weights were normalized relative to the initial weight of the sample in formalin. Therefore, the vertical axis corresponds to the relative change in weight. Differences are observed between the two tissues. In the case of brain tissue samples, pretreatment has little effect on weight, but in the case of basic electrophoresis with Tris (comparative example), the weight approximately doubles. On the other hand, the use of the other two buffering agents according to the present invention avoids the increase in weight. In the case of lungs, the tissue shrinks during pretreatment and swells to almost its original size during electrophoresis with Tris (comparative example). Electrophoresis using the other two buffering agents according to the present invention also prevents swelling here, and instead causes contraction to near the final weight after dehydration.

[0097] The present invention is not limited to any of the embodiments described above and can be modified in various ways.

[0098] All features and advantages evident from the claims, detailed description and drawings, including structural details, spatial arrangement and steps of method, can be essential elements of the present invention, either individually or in various combinations.

Claims

1. A method for preparing transparent tissue samples of biological tissue for observation with an optical microscope, a) A step of incubating the tissue in a strongly alkaline solution, b) The dehydrated tissue sample is subjected to electrophoresis in a neutral, acidic, or weakly basic aqueous solution containing 1 to 100 mol / m³ of solution. ３ The process includes immersing the sample in an electrophoresis solution containing a buffering agent of a certain concentration and at least one nonionic detergent in a concentration of 0.1 to 10% by weight, and then exposing the sample to an electric field in the electrophoresis solution to make it transparent by electrophoresis. The electrophoretic solution has a pH value of 4.5 or higher. The buffering substance is an organic compound having at least one carboxylic acid group or at least one sulfonic acid group and at least one amine group, A method in which the pH value of the electrophoretic solution is determined by introducing the buffering substance into an aqueous solution.

2. The method according to claim 1, wherein the electrophoretic solution has a pH value of 4.5 to 8.5, preferably 5.5 to 7.

9.

3. The method according to claim 2, wherein the pH value of the electrophoretic solution is spontaneously adjusted in water.

4. The buffer substance is 20 mol / m ３ At this concentration, 2 siemens·cm ２ The method according to any one of claims 1 to 3, having a molar electrical conductivity of less than / mol.

5. The method according to any one of claims 1 to 4, wherein the buffering substance is an organic compound containing only one ionizable acid group capable of generating one proton per molecule, or only one ionizable base group capable of generating one hydroxyl ion per molecule, and is an organic compound containing at most one positive charge and at most one negative charge per molecule at a pH value spontaneously formed by dissolution in water, and does not contain any other charged groups, and the electrophoretic solution contains at least one buffering substance or a mixture of buffering substances containing a buffer base.

6. The method according to claim 4 or 5, wherein the buffering substance is selected from the group consisting of aminocarboxylic acids, aminosulfonic acids, aminocarboxylic acid salts, aminosulfonate salts, or mixtures thereof.

7. The method according to claim 6, wherein the buffering substance is an aminoalkanesulfonic acid, a hydroxyalkylaminosulfonic acid, a piperidinealkanoic acid, an amino acid, an N-hydroxyalkylaminocarboxylic acid, or an aminocarboxylate or aminosulfonate, or a mixture thereof.

8. The method according to any one of claims 1 to 7, wherein the mixture of a buffering substance and a buffering base contains, based on the total weight of the buffering substance and the buffering base, preferably more than 50% by weight and preferably more than 75% by weight of the buffering substance in the mixture.

9. Nonionic detergents include polyoxyethylene (20) sorbitan monolaurate, polyoxyethylene sorbitan monooleate, polyethylene glycol 4-tert-octylphenyl ether, octylphenoxypolyethoxyethanol, polyethylene glycol-tert-octylphenyl ether, n-octyl-β-D-glucopyranoside, octylphenol ethoxylate, decaethylene glycol monododecyl ether, octylphenoxypolyethoxyethanol, and secondary alcohols. The method according to any one of claims 1 to 8, wherein the nonionic detergent is selected from the group consisting of toxylates, betaines, alkyl sulfobetaines, or mixtures thereof, and preferably, the nonionic detergent is selected from octylphenoxypolyethoxyethanol, decaethylene glycol monododecyl ether, secondary alcohol ethoxylate, n-octyl-β-D-glucopyranoside, 3-[(3-collamidopropyl)dimethylammonio]-1-propanesulfonate hydrate, and lauryl sulfobetaine.

10. 0) The step of fixing the tissue sample with formaldehyde, glutaraldehyde, or other cross-linking fixative, a) A step of pretreatment with a solution containing alkali metal hydroxide, b) A step of performing electrophoresis in a solution containing a low concentration of buffering material with a pH of 4.5 to 8.5, without adding a strong alkali or strong base with an acid constant (pKa) of 9.5 or higher, in the presence of a nonionic detergent. c) A step of performing transparency over a period determined by the changes in ohmic resistance and / or conductivity until the rate of change of these values ​​decreases to a predetermined extent, Optionally, d. The dehydration step, and then, e) The step of embedding the tissue in a solvent or solvent mixture having a refractive index that makes it transparent, The method according to any one of claims 1 to 9, including the method described in any one of claims 1 to 9.

11. The method according to any one of claims 1 to 10, wherein the biological tissue is stained with at least one dye or antibody.

12. The method according to claim 10, wherein the staining is performed during electrophoresis.

13. The method according to any one of claims 1 to 12, wherein the maximum temperature of the electrophoretic solution during clearing by electrophoresis is maintained in a range of 20°C to 90°C, which is lower than the boiling point of the electrophoretic solution.

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