Ionic liquid preparations, tissue processing methods and their applications

Abstract

The present invention relates to a hydrophilic amorphous ionic liquid formulation, a method for preparing the same, a method for treating biological tissue using the ionic liquid formulation, and the application of the ionic liquid formulation in biological tissue treatment. The ionic liquid formulation contains: (a) an ionic liquid containing specific cations and specific anions; (b) an auxiliary agent that adjusts the properties of component (a); and (c) water.

Description

[Technical Field]

[0001] The present invention relates to ionic liquids and their applications. Specifically, the present invention relates to a hydrophilic, high refractive index amorphous ionic liquid preparation, a biological tissue treatment method using the ionic liquid preparation, and the application of the ionic liquid in these biological tissue treatment methods. [Background technology]

[0002] Ionic liquids are molten salt systems composed of organic cations and inorganic (or organic) anions that are liquid at room temperature. Ionic liquids are tasteless, non-flammable, have extremely low vapor pressure, are virtually non-volatile, and have high thermal and chemical stability. Functional ionic liquids can be designed to meet a variety of requirements by modifying functional groups. Based on these properties of ionic liquids, it is possible to explore ionic liquid formulations suitable for biological tissue treatment.

[0003] Biological tissue processing methods include, but are not limited to, non-deforming tissue clearing methods, ultra-low temperature vitrification non-destructive tissue preservation methods, low temperature fluorescence enhancement methods, ice crystal-free frozen sections, expanded tissue frozen sections, and super-resolution imaging methods.

[0004] 1. Organizational transparency Tissue clearing involves removing materials and pigments with non-uniform refractive indexes through degreasing and bleaching processes, and then adjusting the refractive index of the tissue to a high matching level using a refractive index matching liquid, thereby reducing light scattering, refraction, and reflection, resulting in a clearing effect on the treated tissue.Currently, conventional refractive index matching reagents are divided into organic solvents and hydrophilic reagents. Organic solvent clearing methods, including the BABB and DISCO series, use alcohols or ethers (e.g., tetrahydrofuran) to dehydrate and degrease the tissue, removing the low-refractive-index water component, followed by organic solvent clearing. While organic solvents have a high refractive index and are effective for clearing tissue, they have poor fluorescence preservation properties and can generate significant amounts of autofluorescence. High concentrations of organic solvents can cause serious tissue damage. Furthermore, organic solvents contain highly toxic reagents, such as tetrahydrofuran, which pose a health risk to humans. Furthermore, the dehydration process can cause tissue shrinkage. Aqueous clearing methods cause tissue swelling. Many hydrophilic reagents, including urea, tend to denaturate proteins and cause tissue swelling due to their hyperhydration. Many clearing methods also have slow clearing rates, and refractive index matching agents, such as antipyrine, tend to crystallize upon saturation, damaging the tissue. Furthermore, hydrophilic reagents have a high water content, making it difficult to achieve a high refractive index. Due to their high water content, their transparency is poor. To prevent crystallization of high-concentration refractive index matching agents, they can only be stored at room temperature, which is unfavorable for fluorescence. Furthermore, tissues treated with aqueous clearing have reduced rigidity, making them prone to deformation and breakage, making them unfavorable for sample storage.

[0005] 2. Ultra-low temperature vitrification preservation Cryo-vitrification uses high concentrations of cryoprotectants and liquid nitrogen to lower the temperature and vitrify dehydrated samples without ice crystal formation, enabling long-term cryopreservation of cells, tissues, or organs while maintaining their basic structure and vital functions. In the vitrified state, water molecules do not rearrange, and structural and volume changes do not occur, preventing mechanical or solution-induced tissue damage. However, the cryoprotectants used in conventional cryoprotection techniques are very high in concentration, and some components, such as dimethyl sulfoxide (DMSO), are highly toxic to materials. Therefore, the dehydration process and the permeability of the cryoprotectant must be strictly controlled. Furthermore, while cryoprotectants can reduce ice crystal formation in practice, they cannot completely suppress it, resulting in some degree of tissue damage. Furthermore, while conventional cryovitrification methods prevent ice crystal formation by rapid freezing with liquid nitrogen, recrystallization of cryoprotected samples is likely to occur during the rewarming process, resulting in sample damage. Therefore, the procedure is difficult and risky.

[0006] 3. Low-temperature fluorescence enhancement Fluorescence intensity responds highly sensitively to temperature. When the temperature rises, molecules can be excited to receive additional thermal energy, converting the excited energy to ground-state vibrational energy, which then rapidly oscillates and relaxes, losing vibrational energy. Furthermore, in solution systems, decreasing the temperature increases the viscosity of the medium, reducing collisions between the fluorescent substance and solvent molecules and decreasing the probability of inactivation. Therefore, the fluorescence intensity of fluorescent substances at low temperatures is significantly stronger than that at room temperature. In recent years, the low-temperature properties of fluorescence have attracted attention. In fluorescence imaging, photobleaching of fluorescent proteins is reduced at low temperatures. However, aqueous systems are prone to ice crystal formation at low temperatures, causing tissue damage, making sample preparation a major challenge for cryofluorescence imaging. Furthermore, there are currently no imaging systems dedicated to low-temperature fluorescence enhancement, and most samples for cryoimaging are used for photoelectron correlation imaging in cryo-electron microscopy. Cryo-electron microscopy samples also face the problem of recrystallization, and the risk of the fluorescence excitation laser inducing freeze-thaw cycles in the sample limits the widespread use of this method.

[0007] 4, Frozen section Frozen sectioning is a method in which tissue is allowed to harden to an appropriate degree in a short period of time in a low-temperature environment, after which it is sliced. One of the important factors in section preparation is the freezing rate. If the freezing rate is too slow, large ice crystal particles are generated, compressing cells or widening intercellular spaces, or the ice crystal edges disrupt fine tissues, leaving numerous vacuoles and hollow nuclei when the ice crystals melt, leading to phenomena such as widened intercellular spaces and structural changes in tissue morphology. Conventional methods primarily use cryoprotectants and improved sectioning techniques (e.g., rapid freezing with liquid nitrogen) to reduce ice crystal damage to cellular tissues in frozen sections. However, these methods only reduce ice crystal formation to a certain extent and cannot completely suppress it. Furthermore, conventional frozen sectioning methods involve dehydration using substances such as sucrose or cryoprotection using cell membrane-permeable cryoprotectants. These methods can only suppress ice crystal formation at low temperatures and have very strict requirements for temperature control; otherwise, sample damage due to crystallization or recrystallization will occur. Furthermore, ice crystals formed during freezing can cause sample damage during the sectioning process and alter the ultrastructure.

[0008] 5. Expansion Microscopic Imaging Expansion microscopy imaging is a novel super-resolution imaging technique that utilizes the expansion of a water gel to uniformly magnify biological samples, enabling super-resolution imaging under standard optical imaging conditions. Expansion imaging is applicable to a variety of samples, including cells and tissue sections, and super-resolution imaging of biological macromolecules such as proteins, nucleic acids, and lipids can all be achieved using expansion imaging. Expansion imaging can also be used in multi-scale combinations with confocal microscopes, light sheet microscopes, and super-resolution microscopes. In super-resolution microscopes, the combination of expansion and sectioning can further improve resolution to the nanometer level. However, the water content of expanded samples is extremely high, making them fragile and prone to fracture, making subsequent tissue processing (e.g., transfer, cryosectioning, etc.) difficult. Furthermore, the dilution of fluorescence intensity due to expansion and the destruction of fluorescent proteins during enzymatic digestion weaken the fluorescence intensity and affect imaging quality. Conventional tissue expansion methods have several drawbacks: they make it difficult to preserve tissue, make cryosectioning impossible, and require long-working-distance, low-magnification objectives for imaging.

[0009] Therefore, it is necessary to provide a biological tissue processing method that can solve the above problems. Summary of the Invention

[0010] To solve the above-mentioned problems in the prior art, the present invention provides a hydrophilic ionic liquid formulation, which can be applied to various tissue processing techniques in histology, including, but not limited to, tissue clearing methods, ultra-low temperature vitrification tissue preservation methods, low temperature fluorescence enhancement methods, ice crystal-free frozen sections, expanded tissue frozen sections, and super-resolution imaging methods.

[0011] In a first aspect of the present invention there is provided a hydrophilic amorphous ionic liquid formulation, a) an ionic liquid containing a cation containing a nitrogen-containing heterocyclic compound or an imine-based compound substituted with an amino group and an anion containing a compound containing a carboxyl group or a sulfo group; Optionally, b) an adjuvant for modifying the properties of component a), including one or more of antipyrine, nicotinamide, nicotinic acid, 1,4-diazabicyclo[2.2.2]octane, and a reducing agent (e.g., pyrosulfite, pyrosulfite salts, metaxylylenediamine, etc.); and Selectable c) water, including.

[0012] The reducing agent in component b) is used to protect other components from the effects of oxygen in the air, and is preferably one that can be stably present in the ionic liquid formulation, and preferably includes potassium pyrosulfite.

[0013] Preferably, the molar ratio of the cations to the anions in component a) is close to or equal to the reciprocal of their charge ratio, so that the cations and the anions are sufficiently dissociated and have similar or nearly identical charges. Preferably, the cation comprises 1-(3-aminopropyl)-imidazole. Preferably, the anion comprises orthophthalic acid.

[0014] The charge of a cation and anion as described herein is "approximate or nearly the same" if the difference in the total charge between the two is 5%, 2%, or 1% or less of the total charge of the cation. Similarly, the molar ratio of a cation to anion as described herein is "close" to the reciprocal of their charge ratio if the charge of the cation and anion is "approximate or nearly the same."

[0015] In one embodiment, the nitrogen-containing heterocyclic compound substituted with the amino group as a cation may be a nitrogen-containing heterocycle substituted with one or more aminoalkyl groups. In one embodiment, the one or more aminoalkyl groups are each independently an amino C 1-8 Alkyl group, amino C 1-6 Alkyl group, amino C 1-3 Alkyl group, amino C 3-6It may be a cycloalkyl group, etc. In one embodiment, the nitrogen-containing heterocycle may be a single ring or a fused ring, for example, a 5- or 6-membered nitrogen-containing heterocycle such as imidazole, imidazoline, imidazolidine, pyrrole, pyrroline, pyrrolidine, pyrazole, pyrazoline, pyrazolidine, triazole, tetrazole, pyridine, piperidine, piperazine, pyridazine, pyrimidine, pyrazine, triazine, etc. It may also be an 8- to 14-membered nitrogen-containing fused heterocycle, for example, dihydropyrrolopyrrole, tetrahydrocyclopentadienopyrrole, indole, isoindole, indoline, indole, benzimidazole, azaindole, quinoline, isoquinoline, or a hydrogenated product thereof. In one embodiment, the nitrogen-containing heterocycle may optionally further contain another heteroatom such as O or S. In one embodiment, the substitution position of the aminoalkyl group in the nitrogen-containing heterocycle is not limited, and is preferably a nitrogen atom. In one embodiment, the nitrogen-containing heterocyclic compound substituted with the amino group is N-(amino C 1-6 alkyl)-5- or 6-membered nitrogen-containing heterocyclic monocyclic ring, preferably N-(amino C 1-6 alkyl)-imidazoles, for example 1-(3-aminopropyl)-imidazole.

[0016] In one embodiment, the compound containing a carboxyl group or a sulfo group as an anion may be an aliphatic or aromatic monocarboxylic acid, dicarboxylic acid, tricarboxylic acid, monosulfonic acid, disulfonic acid, etc. In one embodiment, the compound containing a carboxyl group or a sulfo group may be carbonic acid, acetic acid, succinic acid, citric acid, benzoic acid, orthophthalic acid, terephthalic acid, isophthalic acid, sulfonic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, benzenedisulfonic acid, etc. In one preferred embodiment, the compound containing a carboxyl group or a sulfo group is a benzenedicarboxylic acid such as benzoic acid or orthophthalic acid.

[0017] Preferably, the auxiliary agent of component b) is capable of being stably present in the ionic liquid system, has a protective effect on fluorescence and tissue structure, and / or is capable of improving the refractive index, and / or is capable of reducing viscosity, and / or is capable of performing an antioxidant function, and / or is capable of adjusting the pH.

[0018] Preferably, the water of component c) is ultrapure water, preferably deoxygenated ultrapure water such as boiling deoxygenated ultrapure water.

[0019] Preferably, the mass concentration of component a) is 30 w / w% to 100 w / w%, the concentration of component b) is 0 w / w% to 70 w / w%, and the concentration of component c) is 0 w / w% to 60 w / w%, based on the total weight of the ionic liquid formulation. More preferably, the mass concentration of component a) is 50 w / w% to 70 w / w%, the concentration of component b) is 10 w / w% to 40 w / w%, and the concentration of component c) is 5 w / w% to 60 w / w%, based on the total weight of the ionic liquid formulation. More preferably, the ionic liquid formulation contains, based on the total weight of the ionic liquid formulation, water at a concentration of 10 w / w % to 40 w / w %, 1-(3-aminopropyl)-imidazole at a concentration of 20 w / w % to 30 w / w %, orthophthalic acid at a concentration of 20 w / w % to 30 w / w %, nicotinamide at a concentration of 5 w / w % to 20 w / w %, antipyrine at a concentration of 5 w / w % to 20 w / w %, metaxylylenediamine at a concentration of 2 w / w % to 5 w / w %, and potassium pyrosulfite at a concentration of 0.2 w / w % to 1 w / w %.

[0020] Preferably, the mass volume concentration (w / v%) of component a) is 35% to 95%, the mass volume concentration of component b) is 4% to 50% (preferably, the mass volume concentration of the reducing agent is 0.1% to 2.0%), and the mass volume concentration of component c) is 5% to 45%, based on the total volume of the ionic liquid formulation. More preferably, the mass volume concentration of component a) is 75% to 90%, preferably 40% to 70%, the mass volume concentration of component b) is 6% to 23%, preferably 25% to 40% (preferably, the mass volume concentration of the reducing agent is 0.1% to 1.0%), and the mass volume concentration of component c) is 10% to 40%, preferably 10% to 50%, based on the total volume of the ionic liquid formulation.

[0021] In a more preferred specific embodiment of the present invention, the ionic liquid formulation contains water at a mass / volume concentration (w / v%) of 10% to 40%, 1-(3-aminopropyl)-imidazole at a mass / volume concentration of 40% to 60%, potassium pyrosulfite at a mass / volume concentration of 0.2% to 0.8%, orthophthalic acid at a mass / volume concentration of 35% to 65%, antipyrine at a mass / volume concentration of 8% or less (e.g., 1% to 8%, 2% to 6%), and nicotinamide at a mass / volume concentration of 5% to 15%, wherein the concentrations of 1-(3-aminopropyl)-imidazole as the cation and orthophthalic acid as the anion satisfy the molar ratio of cation to anion specified in component a) above.

[0022] Most preferably, in an exemplary embodiment of the present invention, the ionic liquid formulation comprises water at a weight to volume concentration (w / v %) of 26%, 1-(3-aminopropyl)-imidazole at a weight to volume concentration of 56%, potassium pyrosulfite at a weight to volume concentration of 0.5%, orthophthalic acid at a weight to volume concentration of 40%, antipyrine at a weight to volume concentration of 5%, and nicotinamide at a weight to volume concentration of 10%.

[0023] The ionic liquid formulation is in an amorphous state and preferably remains in a liquid state at room temperature.

[0024] Preferably, the refractive index of the ionic liquid formulation is 1.50 or higher, preferably 1.50 to 1.55, and more preferably 1.51 to 1.54. The refractive index of the ionic liquid formulation is achieved by adjusting the ratios of components a), b), and c).

[0025] In a second aspect of the present invention, there is provided a method for preparing the above-described ionic liquid formulation, the method comprising: preparing the formulation from components a) to c) in a container; injecting an inert gas such as nitrogen, argon, or helium into the container to inertize it; and sealing the container.

[0026] In the above formulations and methods, the agents and reagents used are directly purchased or prepared analytical reagents or more. The preparation method of the ionic liquid can be a method known to those skilled in the art, for example, but not limited to, the use of physical means such as ultrasound and microwaves to enhance the ionic liquid.

[0027] Optionally, in addition to determining the proportion in advance during blending, the formulation may be freeze-dried, vacuum-dried, or added with a desiccant to reduce the water content and increase the refractive index or meet other requirements.

[0028] A third aspect of the present invention provides a method for treating biological tissue, specifically a method for making biological tissue transparent, which comprises treating defatted biological tissue or a biological tissue slice with the above-mentioned ionic liquid formulation to perform refractive index matching, preferably by perfusion, immersion, or infiltration. A method for preserving low-temperature vitrified tissue comprises infiltrating biological tissue with the above-mentioned ionic liquid formulation and storing the infiltrated biological tissue at a temperature below room temperature, for example, below 0°C, −20°C, −40°C, or −80°C. A method for low-temperature fluorescence optically enhanced imaging comprises treating fluorescent biological tissue with the above-mentioned ionic liquid formulation and imaging the treated biological tissue at a temperature below room temperature, for example, below 0°C, −20°C, −40°C, or −80°C. A method for ice-crystal-free frozen sectioning comprises treating biological tissue with the above-mentioned ionic liquid formulation and freezing and slicing the treated biological tissue. The method for preserving expanded tissue and performing frozen sectioning includes expanding biological tissue after fixation with paraformaldehyde to perform secondary fixation, immersing the expanded biological tissue sample after fixation in the ionic liquid preparation, freezing it after its shape has stabilized, and then performing preservation or frozen sectioning.

[0029] A fourth aspect of the present invention provides an application of the above-mentioned ionic liquid formulation in biological tissue processing, the biological tissue processing including one or more of tissue clearing, cryovitrification tissue preservation, cryofluorescence enhancement, ice-free cryosectioning, expanded tissue cryosectioning, and super-resolution imaging.

[0030] To more clearly explain the details and exemplary embodiments of the present invention, the following description of the embodiments of the present invention will be given with reference to the drawings, in which: [Brief explanation of the drawings]

[0031] [Figure 1] 1 is a rheometer analysis of an ionic liquid according to an exemplary embodiment of the present invention, illustrating the mechanical properties of the ionic liquid. [Figure 2]FIG. 1 shows an ionic liquid according to an exemplary embodiment of the present invention stored at −80° C. and then removed and crushed, demonstrating the vitrification properties of the ionic liquid. [Figure 3] 1A-1C are photographs of various biological tissue samples after ionic liquid clearing according to exemplary embodiments of the present invention. [Figure 4] FIG. 10 is a light sheet imaging diagram of a disembodied brain after ionic liquid clearing according to an exemplary embodiment of the present invention. [Figure 5] FIG. 10 shows the section transparency after clarifying an ionic liquid according to an exemplary embodiment of the present invention. [Figure 6] 1A-1C are spinning disk confocal images of tissue sections after ionic liquid clearing according to an exemplary embodiment of the present invention, showing reduced light scattering in the XY axis and no light attenuation in the Z axis after clearing. [Figure 7] FIG. 1 shows the state of tissue infiltrated with an ionic liquid at −80° C. according to an exemplary embodiment of the present invention. [Figure 8] 1 shows fluorescence intensity data of GFPuv at different temperatures in ionic liquids according to an exemplary embodiment of the present invention. [Figure 9] FIG. 1 is a statistical plot of low temperature fluorescence decay of GFPuv in ionic liquids according to an exemplary embodiment of the present invention. [Figure 10] 1 is a flow chart showing the tissue treatment and sectioning process using an ionic liquid according to Example 1 of the present application. [Figure 11] FIG. 1 shows the results of patch tissue treatment using an ionic liquid according to Example 2 of the present application. [Figure 12] FIG. 10 shows the results of expanded and transparent tissue treatment using an ionic liquid according to Example 3 of the present application. DETAILED DESCRIPTION OF THE INVENTION

[0032] Specific embodiments of the present invention will be described below.

[0033] In one embodiment of the present invention, a hydrophilic amorphous ionic liquid formulation is provided, a) an ionic liquid containing a cation containing a nitrogen-containing heterocyclic compound or an imine-based compound substituted with an amino group and an anion containing a compound containing a carboxyl group or a sulfo group; Optionally, b) an adjuvant for modifying the properties of component a), including one or more of antipyrine, nicotinamide, nicotinic acid, 1,4-diazabicyclo[2.2.2]octane, and a reducing agent (e.g., pyrosulfite, pyrosulfite salts, metaxylylenediamine, etc.); and Selectable c) water, including.

[0034] The reducing agent in component b) is used to protect other components from the effects of oxygen in the air, and is preferably one that can be stably present in the ionic liquid formulation, and preferably includes potassium pyrosulfite.

[0035] The hydrophilic ionic liquid preparation of the present invention can be adjusted to have a refractive index close to that of the tissue itself, has an amorphous state, has a high elastic modulus, does not form solid crystals, and can vitrify tissue at ultralow temperatures.The hydrophilic ionic liquid preparation of the present invention can improve the quantum yield of fluorescent molecules at low temperatures.After immersing an expanded tissue sample in the hydrophilic ionic liquid preparation of the present invention, it can maintain the expanded state while replacing the water content.

[0036] In an exemplary embodiment of the invention, the ionic liquid formulation comprises water at a weight to volume concentration (w / v %) of 26%, 1-(3-aminopropyl)-imidazole at a weight to volume concentration of 56%, potassium pyrosulfite at a weight to volume concentration of 0.5%, orthophthalic acid at a weight to volume concentration of 40%, antipyrine at a weight to volume concentration of 5%, and nicotinamide at a weight to volume concentration of 10%.

[0037] FIG. 1 is a rheometer analysis diagram of an ionic liquid formulation according to the above exemplary embodiment of the present invention, showing the changes in the storage modulus and loss modulus of the ionic liquid with temperature. It demonstrates that the ionic liquid formulation of the present invention has a high modulus at low temperatures. The ratio of the loss modulus to the storage modulus represents the viscosity of the ionic liquid. In a viscous flow state at high temperatures, increasing the temperature reduces the viscosity of the ionic liquid, facilitating perfusion. At low temperatures, the storage modulus increases rapidly, and the material is close to a solid state, but its loss modulus is also high, making it close to the semi-solid, highly elastic gel state of biological tissue. It does not form ice crystals, making it suitable for cryosectioning.

[0038] FIG. 2 shows the ionic liquid according to the exemplary embodiment of the present invention stored at −80° C. for 1 hour, then removed and crushed, demonstrating the vitrification properties of the ionic liquid.

[0039] Based on the above properties, the hydrophilic ionic liquid formulation of the present invention can be used in a variety of tissue treatment methods and exhibits excellent properties.

[0040] I. Tissue transparency The hydrophilic ionic liquid formulation of the present invention can be used as a refractive index matching agent in non-deforming tissue clearing methods. Specifically, the hydrophilic ionic liquid formulation of the present invention has a refractive index close to that of the tissue itself, allowing for rapid and effective tissue clearing without the tissue distortion problems associated with conventional aqueous and organic solvent clearing methods.

[0041] In tissue clearing methods, refractive index matching can be achieved by adjusting formulation components based on the refractive index of the tissue's main components after delipidation and decolorization. Furthermore, the auxiliary components in the ionic liquid contribute to achieving the goal of non-deforming clearing of ionic liquids. This refractive index matching method can be applied to perfusion clearing, large isolated tissue clearing, or section clearing, and maintains the macroscopic size and microscopic morphology of the tissue unchanged.

[0042] "Perfusion clearing" refers to the process of injecting undiluted or diluted ionic liquid by perfusion, using a circulation system to sufficiently diffuse the refractive index matching agent throughout the tissue, thereby homogenizing the refractive index of the tissue.

[0043] "Large isolated tissue clearing" refers to the use of undiluted or diluted ionic liquids to homogenize the refractive index of the target tissue by immersion / infiltration.

[0044] Optionally, for tissues infiltrated with diluted ionic liquids, methods such as freeze-drying, vacuum drying, and desiccant addition may be used to reduce the water content in the tissue to increase the refractive index of the tissue or achieve other requirements.

[0045] "Section clearing" refers to refractive index matching after dewaxing of tissue sections following cryosectioning, paraffin sectioning, or vibration sectioning. Dewaxing can be performed by methods well known to those skilled in the art.

[0046] Preferably, based on the non-deformable properties, the tissue section may be collected on a glass slide and then subjected to the above-mentioned delipidation and refractive index matching, which is called patch clearing.

[0047] Patch transparency can be achieved by the following steps. Step 1: Prepare tissue sections by methods such as frozen sections, paraffin sections, or vibration sections. Step 2: The tissue section is attached to an adhesive slide. Step 3: Immerse the slide with the tissue section attached in the degreasing reagent, and the aqueous degreasing reagent may be one well known to those skilled in the art. Step 4: After the tissue in the tissue section is completely degreased, the slide and tissue are immersed in phosphate buffer to wash off the degreasing reagent. This process can be performed in three rounds, each lasting 20 minutes, until no bubbles remain. Step 5: Remove the slide and immerse it in an ionic liquid formulation diluted with water to wash off the phosphate buffer solution. Step 6: Remove the slide, absorb excess water with dust-free paper, and air-dry the tissue section until it is nearly transparent. Step 7: The ionic liquid formulation is dropped onto a glass slide, and the tissue section is thoroughly infiltrated to achieve a uniform refractive index. Step 8: The tissue slice is sealed with a cover glass, and resin is applied to the edge of the cover glass to seal the inside. If the tissue slice is thick, it may be supported with a support.

[0048] Alternatively, tissue sections can be collected in culture dishes or well plates and then refractive index matched by the addition of an ionic liquid before being mounted on glass slides, a process known as float clearing.

[0049] The float process can be realized by the following steps. Step 1: Prepare tissue sections by methods such as frozen sections, paraffin sections, or vibration sections. Step 2: Place tissue sections in phosphate buffered saline for storage. Step 3: Transfer tissue sections to a delipidation reagent for delipidation. Step 4, transfer the tissue sections to phosphate buffer and wash. Step 5, the washed tissue sections are transferred to the diluted ionic liquid formulation and washed. Step 6, refractive index matching, may be performed in one of two ways: step 6.1 or step 6.2. In step 6.1, the tissue section washed with diluted ionic liquid is transferred to undiluted ionic liquid and immersed in it until its refractive index becomes uniform. Then, the tissue section is transferred to an adhesive slide glass, and ionic liquid is dropped onto the slide glass to perform the sealing operation. Resin is applied to the edge of the cover glass to seal the inside. In step 6.2, the tissue section washed with diluted ionic liquid is transferred to an adhesive slide, excess water is absorbed with dust-free paper, and the tissue section is air-dried until it becomes nearly transparent. After the refractive index of the tissue section becomes uniform, a drop of ionic liquid is added to the slide, and then the section is mounted by applying resin to the edge of the cover glass to seal the inside.

[0050] Figure 3 shows photographs of various biological tissue samples after clearing with ionic liquid according to the above exemplary embodiment of the present invention. Animal tissues fixed with 4% paraformaldehyde or untreated plant tissues were immersed in ionic liquid by direct immersion. As can be seen, the heart, liver, spleen, lung, kidney, brain, and plant leaf tissues immersed in the ionic liquid of the present invention were cleared.

[0051] 4 is a light sheet imaging image of an isolated brain after clearing with ionic liquid according to the above exemplary embodiment of the present invention obtained in Example 1. As can be seen, the clearing based on the ionic liquid method is satisfactory for large-scale tissue light sheet imaging and has excellent effects.

[0052] Figure 5 shows the transparency of a sample section after ionic liquid clearing according to the above exemplary embodiment of the present invention. Brightfield and widefield imaging was performed using a Zeiss Axio Scan 2 slide scanner with a 561 nm emission filter to obtain 16-bit monochrome intensity data. "IL RI matching" refers to the imaging results after refractive index matching using an ionic liquid. As can be seen, the ionic liquid clearing effect is very good.

[0053] Figure 6 shows spinning disk confocal images of tissue sections after clearing with an ionic liquid according to the above exemplary embodiment of the present invention. The images show reduced light scattering in the XY axes (left image) and no light attenuation in the Z axis (right image). The tissue was a 200 μm tissue section prepared by vibrating sectioning from a mouse brain perfused with 4% paraformaldehyde. The confocal microscope used was an Andor spinning disk confocal microscope, and images were taken with a 20x objective. As can be seen, the Z axis deformation of the cleared tissue treated with the ionic liquid of the present invention was suppressed, and the high level of clearing significantly reduced the attenuation of both excitation and emission light, resulting in excellent imaging results.

[0054] Therefore, the ionic liquid of the present invention can achieve non-deforming tissue clearing, and its beneficial effects compared to conventional techniques include avoiding tissue destruction and resulting deformation caused by conventional high-concentration organic solvents, and avoiding protein denaturation and tissue swelling caused by conventional hydrophilic reagents.The novel ionic liquid of the present invention has a high refractive index of up to 1.53 and does not cause sample deformation.

[0055] II. Cryogenic vitrification tissue preservation The hydrophilic ionic liquid formulation of the present invention can be used for cryogenic vitrification tissue preservation. Due to its amorphous nature, the hydrophilic ionic liquid formulation of the present invention does not form solid crystals regardless of temperature fluctuations. At cryogenic temperatures below -40°C, tissue immersed in the ionic liquid forms a vitrified state, without ice crystal formation and without damage to the microstructure of the preserved tissue. Therefore, after sufficient immersion in the ionic liquid, tissues requiring preservation can be placed in a cryogenic medical freezer for long-term storage, eliminating the problem of recrystallization that occurs when cryogenically vitrifying tissues using conventional liquid nitrogen.

[0056] 7 shows the state of tissues infiltrated with an ionic liquid at -80°C according to the above exemplary embodiment of the present invention. The tissues are mouse heart, liver, spleen, lung, and kidney tissues fixed by perfusion with 4% paraformaldehyde, and are placed together on the slide shown. After infiltration, the tissues do not form any crystals at -80°C and remain transparent and glassy. As can be seen, no ice crystal damage occurs in the tissues cleared at -80°C.

[0057] Therefore, the ionic liquid of the present invention can be applied to vitrification for long-term cryopreservation of tissues, and its beneficial effect is that, compared to conventional ultra-low temperature vitrification methods (e.g., rapid freezing using liquid nitrogen), the ionic liquid of the present invention has an extremely low vapor pressure, does not evaporate, does not crystallize, and forms a glassy state rather than a crystal when cooled, so that recrystallized ice crystals are not generated, which is advantageous for long-term cryopreservation of samples.

[0058] III. Low-temperature fluorescence enhancement The hydrophilic ionic liquid formulations of the present invention can be applied to low-temperature fluorescence enhancement of tissue samples. Because the hydrophilic ionic liquid formulations of the present invention have the amorphous properties described above, they can maintain fluorescent tissues intact at low temperatures without causing damage to the microstructure of the sample due to ice crystal damage at ultralow temperatures. Due to the hydrophilic polarity of the hydrophilic ionic liquid formulations and their photochemical properties that increase the quantum yield of fluorescent molecules at low temperatures, fluorescent tissues treated or infiltrated with the ionic liquid formulations can achieve fluorescence enhancement at ultralow temperatures. This can be used to improve the quality of optical imaging in photoelectron correlation cryo-electron microscopy, cryo-optical imaging with ultralow-temperature fluorescence enhancement, and detection of weak fluorescent signals.

[0059] Alternatively, the polarity of the ionic liquid preparation at room temperature has the property of enhancing fluorescence, so that it can be used as a fluorescence enhancer even at room temperature.

[0060] Figure 8 shows the fluorescence intensity data of GFPuv in an ionic liquid according to the above exemplary embodiment of the present invention at different temperatures, with PBS as the control. The samples to be measured were GFPuv protein mixed with different reagents, incubated overnight at the indicated temperatures (room temperature, -20°C, and -80°C), and then transferred to a PerkinElmer EnVision microplate reader at the same temperatures for fluorescence intensity measurement. The excitation wavelength of the microplate reader was 395 nm, and the emission wavelength was 506 nm. As can be seen, the fluorescence intensity of the sample at -80°C (IL-80) was significantly enhanced compared to that at room temperature (IL-RT) and -20°C (IL-20).

[0061] Figure 9 is a statistical diagram of the fluorescence decay of GFPuv at low temperatures in the ionic liquid according to the above exemplary embodiment of the present invention. The method used in Figure 9 is the same as that in Figure 8. As can be seen, on the third day, the fluorescence in the ionic liquid stored at -80°C (IL-80) barely decayed, while the fluorescence in the ionic liquids stored at room temperature (IL-RT) and -20°C (IL-20) significantly decayed, indicating that the ionic liquid of the present invention has excellent preservation ability for fluorescent tissues at low temperatures.

[0062] Therefore, the ionic liquid of the present invention can be applied to enhance fluorescence properties at low temperatures. Currently, there are no imaging systems dedicated to fluorescence enhancement using low temperatures, and most frozen imaging samples are used for photoelectron correlation imaging in cryo-electron microscopes. Cryo-electron microscope samples also face the problem of recrystallization, and the fluorescence excitation laser may induce freezing and thawing of the sample, limiting the widespread use of this method. The ionic liquid used in the present invention does not attenuate the intensity of fluorescent proteins; instead, it enhances their fluorescence intensity at low temperatures. Furthermore, it does not cause evaporation or crystallization, resulting in minimal damage to the sample.

[0063] IV, ice crystal-free cryosections The hydrophilic ionic liquid formulation of the present invention can be used to prepare frozen sections of tissue samples without ice crystal damage. The hydrophilic ionic liquid formulation of the present invention is amorphous and has high shear modulus properties (see Figure 1), which prevents the destruction of the microstructure of frozen sections due to ice crystal formation during freezing. Therefore, tissues treated or infiltrated with the ionic liquid formulation can be frozen sections without ice crystal damage within a specific temperature range.

[0064] In contrast, conventional frozen sectioning methods involve dehydration using substances such as sucrose or cryoprotection using cell membrane-permeable cryoprotectants, which can only suppress ice crystal formation at low temperatures and have extremely strict temperature control requirements; otherwise, sample damage due to crystallization or recrystallization occurs. Furthermore, ice crystals still form during freezing, causing sample damage during the sectioning process and altering the microstructure. The ionic liquid of the present invention forms a glassy state rather than a crystalline state upon cooling (see Figure 2), making it applicable to frozen sections without ice crystal damage.

[0065] V. Cryosectioning and super-resolution imaging of post-expansion tissue The hydrophilic ionic liquid formulation of the present invention can be used for frozen sectioning and super-resolution imaging of expanded tissues. After undergoing secondary gel fixation, expanded biological tissues can maintain their expanded state even when immersed in the ionic liquid formulation of the present invention to replace the water content. By utilizing the state transition of ionic liquids at low temperatures, biological tissues undergo a phase transition to an amorphous, highly elastic state, enabling ultrathin frozen sections. This enables super-resolution imaging using conventional optical microscopes, and by combining this with technologies such as confocal microscopes, light sheet microscopes, and super-resolution microscopes, it is possible to improve resolution to the nanometer level. At the same time, low temperatures increase fluorescence intensity, mitigating the problem of dilution of fluorescence brightness due to sample expansion.

[0066] This invention overcomes the challenges posed by conventional methods, such as the difficulty of preserving expanded tissues, the excessive fragility of the expanded specimens, the difficulty of subsequent tissue processing (e.g., transfer and sectioning), and the dilution of fluorescence intensity due to expansion. As a result, expanded tissue specimens can be sectioned and fitted into high-magnification objective lenses with short working distances, enabling super-resolution imaging beyond the limits of optical microscopes.

[0067] Example The following examples provide some embodiments of the present invention, but the embodiments of the present invention are not limited to the following examples.

[0068] Example 1 The ionic liquid according to the exemplary embodiment of the present invention was used to perform perfusion clearing of mouse brains, followed by freeze-drying and frozen sectioning to perform super-resolution imaging of the cleared tissue sections. The basic flow is shown in Figure 10, and the specific procedures are as follows: 1. Transcardial perfusion of the mouse was performed using pre-chilled heparin-saline solution until sufficient blood was drained. 2. Transcardial perfusion of mice was performed for 40 minutes using 4% paraformaldehyde solution containing 0.01 M phosphate buffer. 3. The mouse is subjected to whole-body delipidation perfusion using a delipidant until it is completely delipidated. 4. Transcardial perfusion of mice was performed overnight using a 4-fold diluted aqueous solution of ionic liquid. 5. The mouse brain was removed and placed in 1.5-fold diluted ionic liquid. 6. The liquid and mouse brain are frozen in a freezer at -80°C, and then placed in a vacuum freeze dryer for freeze-drying until the mouse brain becomes transparent. 7. A cleared mouse brain was imaged using a light sheet microscope, and the imaging liquid was the ionic liquid itself. 8. The cleared mouse brain is embedded in a freezing embedding medium and then completely frozen at -80°C. 9. After freezing is complete, transfer the mouse brain to the cryosection machine using dry ice. 10. Cut the mouse brain into 50 μm thin sections at -40 °C, and attach the sections to gelatin-coated adhesive glass slides. 11. The sections are subjected to super-resolution imaging using an Airyscan microscope and data is acquired.

[0069] The imaging results are shown in Figure 4, which shows that the ionic liquid-based clearing method is satisfactory for large-scale tissue light-sheet imaging and has excellent effectiveness.

[0070] In addition, the heart, liver, spleen, lung, kidney, brain, and plant leaves were treated in a similar manner, and the imaging results are shown in Figure 3. As can be seen from the results, all of the tissues immersed in the ionic liquid of the present invention became transparent.

[0071] Example 2 The ionic liquid of the above exemplary embodiment of the present invention is used to perform a non-deforming transparency treatment on the attached tissue section, and the specific operation is as follows. 1. Immerse the prepared tissue section slide in the degreasing reagent for 30 minutes. 2. Immerse the tissue section in 4-fold diluted ionic liquid three times, immersing for 20 minutes each time, to wash off the degreasing reagent. 3. Wipe the slide and air dry the brain slice until it becomes slightly transparent. 4. Ionic liquid is dropped onto the tissue slice and refractive index matching is performed, which takes about 1 minute. 5. After the tissue sections have become transparent, they are mounted. 6. After mounting, photographs were taken using a 60x zoom lens on an Andor Dragonfly spinning disk confocal microscope.

[0072] The imaging results of this example are shown in Figure 11. The tissue is not deformed after treatment with the ionic liquid.

[0073] Example 3 The ionic liquid of the above exemplary embodiment of the present invention is used to perform tissue cryosectioning after expansion, and the specific procedures are as follows: 1. Mice were perfused transcardially with paraformaldehyde, and the brains were removed. After fixation for one day, the brains were transferred to a glycine solution and then subjected to vibrotomy. The brain slices were then stored at 4°C. 2. Suspend AcX in anhydrous DMSO at a concentration of 10 mg / mL. Dilute AcX with PBS to 0.1 mg / mL before use. Place the sample in the diluted AcX and incubate it in a shaking incubator at room temperature for at least 6 hours. 3. Concentrated ammonium persulfate initiator and tetramethylethylenediamine were added to the monomer solution at a maximum concentration of 0.2% (w / w), and 4-hydroxy-TEMPO was added as an inhibitor. The tissue sections and the monomer solution + APS / TEMED were incubated at 4°C for 30 minutes, and then transferred to a humidified 37°C incubator for 2 hours for gelation. 4. Immerse the gel completely in the protease solution and incubate at room temperature overnight or at 37°C for 4 hours. 5. Place the digested gel in excess double deionized water for 0.25-2 hours to allow it to swell. Repeat this process 3-5 times until the size of the swollen sample stabilizes. 6. After swelling, the gel was placed in 2 mL of PEG-DA solution overnight at room temperature in the dark. After that, the gel was removed from the PEG-DA solution, and excess PEG-DA solution was removed from the surface. The gel was then placed under 4 mW / cm 2 Irradiate with ultraviolet light for 10 minutes. 7. The gel is immersed in ionic liquid until the shape is stable, then sectioned and imaged with a 10x zoom lens.

[0074] The imaging results of this example are shown in FIG.

[0075] Although the technical concept and specific embodiments of the present invention have been described above, it should be understood that the above specific embodiments do not limit the scope of the present invention in any way. As will be understood by those skilled in the art, multiple modifications and / or changes can be made to the invention shown in the specific embodiments without departing from the essence of the present invention, and the modified and / or changed embodiments are also included within the scope of the present invention. Therefore, the embodiments of the present invention are merely illustrative and not limiting.

Claims

1. 1. A hydrophilic amorphous ionic liquid formulation comprising: a) an ionic liquid containing a cation containing a nitrogen-containing heterocyclic compound or an imine-based compound substituted with an amino group and an anion containing a compound containing a carboxyl group or a sulfo group; Optionally, b) an adjuvant that modifies the properties of component a), comprising one or more of antipyrine, nicotinamide, nicotinic acid, 1,4-diazabicyclo[2.2.2]octane, pyrosulfite, pyrosulfite salts, and metaxylylenediamine; and Optionally, c) water.

2. 2. The ionic liquid formulation of claim 1, wherein the molar ratio of the cations to the anions in component a) is close to or equal to the reciprocal of their charge ratio, such that the cations and the anions are sufficiently dissociated and have similar or nearly identical charges.

3. The cation comprises a nitrogen-containing heterocycle substituted with one or more aminoalkyl groups, preferably each of the one or more aminoalkyl groups independently being an amino C 1-8 Alkyl group, amino C 1-6 Alkyl group, amino C 1-3 Alkyl group or amino group C 3-6 Preferably, the nitrogen-containing heterocycle is a 5- or 6-membered nitrogen-containing heterocyclic monocycle or an 8- to 14-membered nitrogen-containing heterocyclic condensed ring, and preferably, the cation is an N-(amino C) group such as 1-(3-aminopropyl)-imidazole. 1-6 The ionic liquid formulation according to claim 1 or 2, comprising a (alkyl)-5- or 6-membered nitrogen-containing heterocyclic monocycle.

4. 3. The ionic liquid formulation of claim 1 or 2, wherein the anion comprises an aliphatic or aromatic monocarboxylic, dicarboxylic, tricarboxylic, monosulfonic or disulfonic acid, preferably a benzenedicarboxylic acid such as benzoic acid or orthophthalic acid.

5. 3. The ionic liquid formulation according to claim 1 or 2, wherein component c) is ultrapure water, preferably deoxygenated ultrapure water such as ultrapure water that has been subjected to a boiling deoxygenation treatment.

6. The ionic liquid formulation according to claim 1 or 2, wherein the pyrosulfite is potassium pyrosulfite.

7. the concentration of component a) is 30 w / w% to 100 w / w%, the concentration of component b) is 0 w / w% to 70 w / w%, and the concentration of component c) is 0 w / w% to 60 w / w%, based on the total weight of the ionic liquid formulation; The ionic liquid formulation according to claim 1 or 2, wherein the concentration of component a) is preferably 50 w / w % to 70 w / w %, the concentration of component b) is 10 w / w % to 40 w / w %, and the concentration of component c) is 5 w / w % to 60 w / w %, based on the total weight of the ionic liquid formulation.

8. 3. The ionic liquid formulation according to claim 1, comprising, based on the total weight of the ionic liquid formulation, water at a concentration of 10 w / w% to 40 w / w%, 1-(3-aminopropyl)-imidazole at a concentration of 20 w / w% to 30 w / w%, orthophthalic acid at a concentration of 20 w / w% to 30 w / w%, nicotinamide at a concentration of 5 w / w% to 20 w / w%, antipyrine at a concentration of 5 w / w% to 20 w / w%, metaxylylenediamine at a concentration of 2 w / w% to 5 w / w%, and potassium pyrosulfite at a concentration of 0.2 w / w% to 1 w / w%.

9. 3. The ionic liquid formulation according to claim 1, wherein the refractive index of the ionic liquid formulation is 1.50 or more, preferably 1.50 to 1.55, and more preferably 1.51 to 1.

54.

10. A method for preparing the ionic liquid formulation of any one of claims 1 to 9, comprising: preparing a formulation of components a) to c) in a container; and inertizing the container by injecting an inert gas such as nitrogen, argon, or helium into the container and sealing the container.

11. A method for making biological tissue transparent, comprising: The method comprises treating a delipidated biological tissue or biological tissue slice with the ionic liquid formulation of any one of claims 1 to 9 to perform refractive index matching, wherein the treatment preferably comprises perfusion, immersion, or infiltration.

12. 1. A method for cryovitrifying tissue for preservation, comprising: The method comprises infiltrating a biological tissue with the ionic liquid preparation according to any one of claims 1 to 9, and storing the infiltrated biological tissue at a temperature below room temperature, for example, at a temperature below 0°C, -20°C, -40°C, or -80°C.

13. 1. A method for low-temperature fluorescence optically enhanced imaging, comprising: The method comprises treating fluorescent biological tissue with the ionic liquid formulation according to any one of claims 1 to 9, and imaging the treated biological tissue at a temperature below room temperature, for example, at a temperature below 0°C, -20°C, -40°C, or -80°C.

14. 1. A method for ice crystal-free cryosectioning, comprising: The method comprises treating biological tissue with the ionic liquid formulation of any one of claims 1 to 9, and freezing and slicing the treated biological tissue.

15. 1. A method for preserving and cryosectioning tissue after expansion, comprising: The method comprises expanding a biological tissue after primary fixation to perform secondary fixation, immersing the expanded biological tissue sample after secondary fixation in the ionic liquid preparation according to any one of claims 1 to 9, freezing the tissue after its shape has stabilized, and then storing or cryosectioning the tissue. Preferably, the primary fixation is performed with paraformaldehyde, and preferably, the secondary fixation is performed with a gel.

16. 10. An application of the ionic liquid formulation according to any one of claims 1 to 9 in biological tissue processing, wherein the biological tissue processing comprises one or more of tissue clearing, cryovitrification tissue preservation, cryofluorescence enhancement, ice-free cryosectioning, expanded tissue cryosectioning, and super-resolution imaging.

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