Ionic liquid preparation and tissue processing method and application thereof

Abstract

The present invention provides a hydrophilic amorphous ionic liquid formulation, a biological tissue treatment method using the ionic liquid formulation, and the use of the ionic liquid formulation in biological tissue treatment. The ionic liquid formulation comprises: a) an ionic liquid containing specific cations and specific anions; b) an auxiliary agent for adjusting the properties of component a); c) water; and d) a reducing agent capable of being stably present in the ionic liquid formulation.

Description

Technical Field

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

[0002] Ionic liquids are molten salt systems composed of organic cations and inorganic (or organic) anions that are liquid at room temperature. They are odorless, non-flammable, have extremely low vapor pressures, are virtually non-volatile, and possess high thermal and chemical stability. By modifying functional groups, ionic liquids can be tailored to meet diverse requirements. These properties of ionic liquids have enabled the development of ionic liquid formulations suitable for biological tissue treatment.

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

[0004] 1. Organizational transparency

[0005] Tissue clearing utilizes a defatting and decolorization process to remove materials and pigments with uneven refractive indices. A refractive index matching solution is then used to match the tissue's refractive index to a higher, more uniform level, thereby reducing light scattering, refraction, and reflection, resulting in a transparent effect on the treated tissue. Currently, conventional refractive index matching reagents are divided into organic solvents and hydrophilic reagents.

[0006] Organic solvent clearing methods include the BABB and DISCO series, which use substances such as alcohols or ethers (such as tetrahydrofuran) for dehydration and degreasing, thereby removing the water component with a lower refractive index, and then using organic solvents for clearing. Although organic solvents have a high refractive index and good clearing effect, they have poor fluorescence preservation effects and may also produce a large amount of autofluorescence. High concentrations of organic solvents can cause great damage to tissues. In addition, organic solvent formulas contain highly toxic reagents such as tetrahydrofuran, which may be harmful to human health. Moreover, the dehydration process will cause tissue shrinkage.

[0007] The aqueous clearing method will cause the tissue to swell. Most hydrophilic reagents are based on the superhydration property of urea, which will lead to the denaturation of proteins and cause tissue swelling. In addition, many methods have slow clearing speeds, and refractive index matching substances such as antipyrine are prone to crystallization and damage to the tissue when they reach saturation. In addition, the hydrophilic reagent has a high water content, and its refractive index is difficult to reach a high level. Due to the high water content, its transparency will be low, and in order to prevent the crystallization of high-concentration refractive index matching agents, it can only be stored at room temperature, which is not friendly to fluorescence. At the same time, the rigidity of tissues treated with aqueous clearing decreases, and it is easy to deform and break, so it is also not friendly to sample preservation.

[0008] 2. Ultra-low temperature vitrification preservation

[0009] Ultra-low temperature vitrification uses high-concentration cryoprotectants and liquid nitrogen to cool the sample, vitrifying it without producing ice crystals, thereby achieving long-term cryopreservation of cells, tissues or organs and maintaining their basic structure or viability.

[0010] In the glassy state, water molecules do not rearrange, and no changes in structure and volume occur, so no tissue damage will be caused by mechanical damage or solution effects. However, the concentration of cryoprotectants used in existing ultra-low temperature vitrification preservation technologies is very high, and some components, such as dimethyl sulfoxide (DMSO), are extremely toxic to the material, requiring strict control of the dehydration process and the permeability of the cryoprotectant. In addition, in actual operation, cryoprotectants can only reduce the formation of ice crystals, but cannot completely inhibit them, so they still cause tissue damage to a certain extent. In addition, existing ultra-low temperature vitrification methods mainly use liquid nitrogen quick freezing to prevent ice crystal formation, but ultra-low temperature vitrification samples are prone to form recrystallized ice crystals that damage the sample during the rewarming process, making this method difficult to operate and very risky.

[0011] 3. Low-temperature fluorescence enhancement

[0012] Fluorescence intensity is sensitive to temperature. When the temperature rises, the excited molecules receive additional thermal energy, which may convert the excitation energy into ground-state vibrational energy, which is then rapidly lost through vibrational relaxation. Furthermore, in a solution system, a decrease in temperature increases the viscosity of the medium, reducing collisions between the fluorescent substance and solvent molecules and the probability of deactivation. Consequently, the fluorescence intensity of fluorescent substances at low temperatures is significantly enhanced compared to room temperature.

[0013] In recent years, the low-temperature properties of fluorescence have received increasing attention. In fluorescence imaging, the photobleaching of fluorescent proteins is reduced at lower temperatures. However, aqueous systems are prone to forming ice crystals at low temperatures, causing damage to tissues. Therefore, sample preparation has become a major challenge for low-temperature fluorescence imaging. In addition, there is currently no imaging system in the field that specifically enhances fluorescence at low temperatures. Most samples for cryo-imaging are used for combined photoelectric imaging of cryo-electron microscopy. For cryo-electron microscopy samples, there is also the problem of recrystallization, and the laser that excites fluorescence may also cause freeze-thaw of the sample, which has prevented much promotion of this method.

[0014] 4. Cryosectioning

[0015] Cryosectioning involves using a low-temperature environment to achieve the desired hardness within a short period of time, followed by sectioning. A key factor in sectioning is the freezing rate. A slow freezing rate can easily produce large ice crystals, which can squeeze cells, widen intercellular spaces, or sever microstructures through the edges of ice crystals. When the ice crystals melt, they leave behind numerous vacuoles and empty nuclei, which can also cause tissue morphology to exhibit increased gaps and structural changes.

[0016] Existing methods mainly reduce the damage of ice crystals to cell tissues in frozen sections by using cryoprotectants and improving slicing techniques (such as liquid nitrogen sudden cooling), but they can only reduce ice crystal formation to a certain extent, and cannot completely inhibit ice crystal formation. In addition, existing frozen sectioning methods use substances such as sucrose for dehydration, or use cryoprotectants that can penetrate the cell membrane for cryoprotection. These methods can only inhibit the formation of ice crystals at low temperatures, and have extremely high requirements for temperature control, otherwise they will face damage to the sample due to crystallization or recrystallization. In addition, the ice crystals formed during freezing will cause damage to the sample during the slicing process, resulting in changes in the microstructure.

[0017] 5. Expansion Microscopy

[0018] Expansion microscopy is a novel super-resolution imaging technique that utilizes hydrogel expansion to uniformly magnify biological samples, enabling super-resolution imaging under conventional optical imaging conditions. Expansion imaging is applicable to a variety of sample types, including cells and tissue sections. Biomacromolecules such as proteins, nucleic acids, and lipids can all be imaged super-resolution using expansion imaging. Furthermore, expansion imaging can be combined with confocal microscopy, light-sheet microscopy, and super-resolution microscopy at multiple scales. Under super-resolution microscopy, expansion combined with sectioning can further improve resolution to the nanometer level.

[0019] However, after expansion, the sample becomes extremely watery and fragile, making it difficult to perform further tissue processing (such as transfer and cryosectioning). Furthermore, the dilution of fluorescence intensity caused by expansion and the destruction of fluorescent proteins during enzymatic digestion both weaken the fluorescence intensity, thus affecting imaging quality. Furthermore, existing tissue expansion methods face challenges in preserving tissue, making cryosectioning impossible and requiring imaging only through low-magnification objectives with long working distances.

[0020] Therefore, there is an urgent need to provide a biological tissue processing method that can solve the above problems. Summary of the Invention

[0021] To solve the above-mentioned problems in the prior art, the present invention provides a hydrophilic ionic liquid preparation, which can be used in 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 section methods, expanded tissue frozen section methods and super-resolution imaging methods.

[0022] The first aspect of the present invention provides a hydrophilic amorphous ionic liquid formulation comprising the following components:

[0023] a) an ionic liquid containing a cation and an anion, wherein the cation comprises an amino-substituted nitrogen-containing heterocyclic compound or an imine compound, and the anion comprises a carboxyl- or sulfonyl-containing compound;

[0024] b) an adjuvant for adjusting the properties of component a), which comprises one or more of antipyrine, nicotinamide, nicotinic acid and 1,4-diazabicyclo[2.2.2]octane;

[0025] c) water; and

[0026] d) a reducing agent capable of being stably present in the ionic liquid formulation.

[0027] Preferably, the molar ratio of the cation to the anion in component a) is close to or equal to the inverse of their charge ratio, so that the cation and the anion are fully ionized and have substantially the same charge. The cation preferably comprises 1-(3-aminopropyl)-imidazole. The anion preferably comprises phthalic acid.

[0028] In one embodiment, the amino-substituted nitrogen-containing heterocyclic compound may be a nitrogen-containing heterocyclic ring substituted with one or more aminoalkyl groups. In one embodiment, the one or more aminoalkyl groups may each independently be an amino C 1-8 Alkyl, amino C 1-6 Alkyl, amino C 1-3 Alkyl, amino C 3-6Cycloalkyl, etc. In one embodiment, the nitrogen-containing heterocycle can be a monocyclic or condensed ring, for example, it can be a five-membered or six-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 can also be an eight- to fourteen-membered nitrogen-containing heterocycle condensed ring, such as dihydropyrrolopyrrole, tetrahydrocyclopentadiene pyrrole, indole, isoindole, indoline, indazole, benzimidazole, azaindole, quinoline, isoquinoline and its hydrogenation product, etc. In one embodiment, the nitrogen-containing heterocycle can also optionally contain other heteroatoms such as O and S. In one embodiment, the substitution position of the aminoalkyl group on the nitrogen-containing heterocycle is not limited, but is preferably a nitrogen atom. In a preferred embodiment, the amino-substituted nitrogen-containing heterocyclic compound is N-(amino C 1-6 Alkyl)-five-membered or six-membered nitrogen-containing heterocyclic monocyclic ring, preferably N-(amino C 1-6 alkyl)-imidazole, for example 1-(3-aminopropyl)-imidazole.

[0029] In one embodiment, the carboxyl or sulfonyl group-containing compound can be an aliphatic or aromatic monocarboxylic acid, dicarboxylic acid, tricarboxylic acid, monosulfonic acid, disulfonic acid, etc. In one embodiment, the carboxyl or sulfonyl group-containing compound can be carbonic acid, acetic acid, succinic acid, citric acid, benzoic acid, phthalic acid, terephthalic acid, isophthalic acid, sulfonic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, benzene disulfonic acid, etc. In a preferred embodiment, the carboxyl or sulfonyl group-containing compound is benzoic acid or phthalic acid, such as phthalic acid.

[0030] Preferably, the auxiliary agent of component b) can be stably present in the ionic liquid system, and has a protective effect on fluorescence and tissue structure, and / or can increase the refractive index, and / or can reduce viscosity.

[0031] Preferably, the water in component c) is ultrapure water, preferably deoxygenated ultrapure water, such as ultrapure water that has been subjected to boiling and deoxygenation treatment.

[0032] Preferably, the reducing agent of component d) is used to protect the other components from oxygen in the air. The reducing agent of component d) preferably comprises potassium metabisulfite.

[0033] Preferably, based on the total volume of the ionic liquid formulation, the mass volume ratio concentration (w / v%) of component a) is 35% to 95%, the mass volume ratio concentration of component b) is 4% to 50%, the mass volume ratio concentration of component c) is 5% to 45%, and the mass volume ratio concentration of component d) is 0.1% to 2.0%; more preferably, based on the total volume of the ionic liquid formulation, the mass volume ratio concentration of component a) is 75% to 90%, the mass volume ratio concentration of component b) is 6% to 23%, the mass volume ratio concentration of component c) is 10% to 40%, and the mass volume ratio concentration of component d) is 0.1% to 1.0%.

[0034] In a more preferred embodiment of the present invention, the ionic liquid formulation comprises: 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 metabisulfite at a mass volume concentration of 0.2% to 0.8%, phthalic 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 a cation and phthalic acid as an anion satisfy the molar ratio of cation to anion defined in component a) above.

[0035] Most preferably, in an exemplary embodiment of the present invention, the ionic liquid formulation comprises: water at a mass volume ratio concentration (w / v%) of 26%, 1-(3-aminopropyl)-imidazole at a mass volume ratio concentration of 56%, potassium metabisulfite at a mass volume ratio concentration of 0.5%, phthalic acid at a mass volume ratio concentration of 40%, antipyrine at a mass volume ratio concentration of 5%, and nicotinamide at a mass volume ratio concentration of 10%.

[0036] The ionic liquid formulation is amorphous and preferably remains liquid at room temperature.

[0037] Preferably, the refractive index of the ionic liquid preparation is greater than 1.50, preferably 1.50 to 1.55, more preferably 1.51 to 1.54. The refractive index of the ionic liquid preparation can be achieved by adjusting the ratio of components a), b), and c).

[0038] A second aspect of the present invention provides a method for preparing the above-mentioned ionic liquid formulation, comprising: preparing the components a)-d) into a formulation in a container, filling the container with an inert gas such as nitrogen, argon, helium, etc. for inert protection, and sealing the container.

[0039] In the above-mentioned preparations and methods, the drugs and reagents used are purchased or prepared directly and are of analytical grade or higher. The ionic liquids are prepared using methods known to those skilled in the art, such as, but not limited to, methods using physical acceleration methods such as ultrasound and microwaves.

[0040] Alternatively, in addition to determining the ratio in advance during preparation, the preparation can also be freeze-dried, vacuum-dried, or added with a desiccant to reduce the water content and increase the refractive index or meet other requirements.

[0041] The third aspect of the present invention provides a biological tissue processing method, specifically, a biological tissue transparent method, comprising: using the above-mentioned ionic liquid preparation to treat defatted biological tissue or biological tissue slices to perform refractive index matching, wherein the treatment preferably includes perfusion, immersion or infiltration; a low-temperature vitrified tissue preservation method, comprising: using the above-mentioned ionic liquid preparation to infiltrate biological tissue, and preserving the infiltrated biological tissue at a temperature below room temperature, for example, below 0°C, -20°C, below -40°C or below -80°C; a low-temperature fluorescence optical enhancement imaging .... The invention relates to a method for preserving and freezing expanded tissue, comprising: expanding and re-fixing biological tissue fixed with paraformaldehyde, immersing the fixed expanded biological tissue sample in the ionic liquid preparation, freezing it after its shape stabilizes, and preserving or freezing-slicing it.

[0042] A fourth aspect of the present invention provides the use of the above-mentioned ionic liquid formulation in biological tissue processing, wherein the biological tissue processing includes one or more of tissue clearing, low-temperature vitrified tissue preservation, low-temperature fluorescence enhancement, ice-crystal-free frozen sections, expanded tissue frozen sections and super-resolution imaging. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] In order to more clearly illustrate the details and exemplary embodiments of the present invention, the embodiments of the present invention will be described in detail below with reference to the accompanying drawings, but the present invention is not limited thereto. In the accompanying drawings:

[0044] Figure 1 FIG1 is a rheometer analysis graph of the ionic liquid according to an exemplary embodiment of the present invention, which shows the mechanical properties of the ionic liquid.

[0045] Figure 2The ionic liquid according to the exemplary embodiment of the present invention is shown as being taken out and broken after being stored at -80°C, confirming the vitrification property of the ionic liquid.

[0046] Figure 3 are photographs of various biological tissue samples after being transparentized using the ionic liquid according to an exemplary embodiment of the present invention.

[0047] Figure 4 This is an in vitro brain light sheet image after being transparentized using the ionic liquid according to an exemplary embodiment of the present invention.

[0048] Figure 5 Graph showing the transparency of a slice after being transparentized using the ionic liquid according to an exemplary embodiment of the present invention.

[0049] Figure 6 This is a confocal image of a rotating disk of tissue sections after clearing with an ionic liquid according to an exemplary embodiment of the present invention, showing reduced scattering in the XY axis and no light attenuation in the Z axis direction after clearing.

[0050] Figure 7 This is a state of the tissue infiltrated with the ionic liquid according to an exemplary embodiment of the present invention at -80°C.

[0051] Figure 8 Figure 2 shows the fluorescence intensity data of GFPuv in the ionic liquid at different temperatures according to an exemplary embodiment of the present invention.

[0052] Figure 9 Statistical graph of fluorescence decay of GFPuv in ionic liquid at low temperature according to an exemplary embodiment of the present invention.

[0053] Figure 10 A flow chart of the tissue processing and slicing process using ionic liquids in Example 1 of the present application is shown.

[0054] Figure 11 The figure shows the result of patch tissue treatment using ionic liquid in Example 2 of the present application.

[0055] Figure 12 The diagram shows the results of using ionic liquid combined with swelling and transparent tissue treatment in Example 3 of the present application. DETAILED DESCRIPTION

[0056] The specific embodiments of the present invention are described in detail below.

[0057] One embodiment of the present invention provides a hydrophilic amorphous ionic liquid formulation comprising the following components:

[0058] a) an ionic liquid containing a cation and an anion, wherein the cation comprises an amino-substituted nitrogen-containing heterocyclic compound or an imine compound, and the anion comprises a carboxyl- or sulfonyl-containing compound;

[0059] b) an adjuvant for adjusting the properties of component a), which comprises one or more of antipyrine, nicotinamide, nicotinic acid and 1,4-diazabicyclo[2.2.2]octane;

[0060] c) water; and

[0061] d) a reducing agent capable of being stably present in the ionic liquid formulation.

[0062] The hydrophilic ionic liquid preparation of the present invention can be adjusted to have a refractive index close to that of the tissue itself, and has an amorphous state with a high elastic modulus, does not form solid crystals, and can make the tissue enter a vitrified state at ultra-low temperatures. The hydrophilic ionic liquid preparation of the present invention can increase the quantum yield of fluorescent molecules at low temperatures. After soaking in a swollen tissue sample, the hydrophilic ionic liquid preparation of the present invention can replace the water in the tissue sample while still maintaining its swollen state.

[0063] In an exemplary embodiment of the present invention, the ionic liquid formulation includes: water at a mass volume ratio concentration (w / v%) of 26%, 1-(3-aminopropyl)-imidazole at a mass volume ratio concentration of 56%, potassium metabisulfite at a mass volume ratio concentration of 0.5%, phthalic acid at a mass volume ratio concentration of 40%, antipyrine at a mass volume ratio concentration of 5%, and nicotinamide at a mass volume ratio concentration of 10%.

[0064] Figure 1 : This is a rheometer analysis diagram of the ionic liquid formulation of the above exemplary embodiment of the present invention, which shows the change of the storage modulus and loss modulus of the ionic liquid with temperature. It is confirmed that the ionic liquid formulation of the present invention has a high elastic modulus at low temperatures. The ratio of the loss modulus to the storage modulus characterizes the viscosity of the ionic liquid. In the viscous flow state with a higher temperature, heating causes the viscosity of the ionic liquid to decrease, making it easy to perfuse. In the state with a lower temperature, the storage modulus rises rapidly and the material approaches a solid state. However, since its loss modulus is also high, it is close to the semi-solid highly elastic gel state of biological tissue, and no ice crystals are formed, which can be used for frozen sections.

[0065] Figure 2 The ionic liquid according to the exemplary embodiment of the present invention is shown as being broken after being taken out after being stored at -80°C for 1 hour, confirming the vitrification property of the ionic liquid.

[0066] Based on the above characteristics, the hydrophilic ionic liquid preparation of the present invention can be used in a variety of tissue treatment methods and exhibits excellent properties.

[0067] I. Organizational transparency

[0068] The hydrophilic ionic liquid formulation of the present invention can be used as a refractive index matching agent in a deformation-free tissue clearing method. Specifically, the hydrophilic ionic liquid formulation of the present invention has a refractive index close to that of the tissue itself, enabling rapid and efficient tissue clearing while avoiding the tissue deformation associated with traditional aqueous and organic solvent clearing methods.

[0069] In tissue clearing methods, for biological tissues that have undergone defatting and decolorization, the refractive index of the tissue's primary components can be matched by adjusting the formulation composition. Furthermore, the additives in the ionic liquid aid in achieving the desired clearing effect without deformation. This refractive index matching method can be applied to perfusion clearing, clearing large ex vivo tissues, or clearing sections, while preserving the tissue's macroscopic size and microscopic morphology.

[0070] "Perfusion clearing" refers to the perfusion of undiluted or diluted ionic liquids, using the circulatory system to fully diffuse the refractive index matching agent throughout the tissue, thereby homogenizing the tissue refractive index.

[0071] "Large-scale ex vivo tissue clearing" refers to the use of undiluted or diluted ionic liquids to homogenize the refractive index of the target tissue by immersion / infiltration.

[0072] Alternatively, for tissue infiltrated with diluted ionic liquid, the water content in the tissue can be reduced by freeze drying, vacuum drying, or adding a desiccant to increase the tissue refractive index or meet other requirements.

[0073] "Section clearing" refers to refractive index matching after defatting frozen, paraffin, or vibratome tissue sections. Defatting can be performed using methods known to those skilled in the art.

[0074] Preferably, based on its non-deformable nature, the tissue sections can be collected onto glass slides and then subjected to the above-mentioned defatting and refractive index matching. This operation is called slide clearing.

[0075] The transparency of the patch can be achieved by the following steps:

[0076] Step 1: Prepare tissue sections by methods such as frozen sectioning, paraffin sectioning, or vibratome sectioning.

[0077] Step 2: Attach the tissue sections to adhesive slides.

[0078] Step 3: Soak the glass slide with the tissue section in the degreasing agent, wherein the aqueous degreasing agent can be well known to those skilled in the art.

[0079] Step 4: After the tissue in the tissue section is completely defatted, the slide and tissue are soaked in phosphate buffer to wash away the defatting agent; this step can be performed for 3 rounds, each lasting 20 minutes, until no foam is generated.

[0080] Step 5: Take out the glass slide and soak it in the ionic liquid preparation diluted with water to wash away the phosphate buffer.

[0081] Step 6. Remove the slide, absorb excess moisture with dust-free paper, and let the tissue sections dry in the air until they become slightly transparent.

[0082] Step 7: Add the ionic liquid preparation dropwise onto the glass slide to fully soak the tissue section and make its refractive index uniform.

[0083] Step 8. Seal the slice with a coverslip and apply resin to the edge of the coverslip to seal the inside. If the tissue slice is thick, you can use a support.

[0084] Alternatively, tissue sections can be collected in a culture dish or well plate and then treated with ionic liquid for refractive index matching before proceeding to the next step of mounting. This operation is called clearing.

[0085] The above-mentioned transparent film can be achieved by the following steps:

[0086] Step 1: Prepare tissue sections by methods such as frozen sectioning, paraffin sectioning, or vibratome sectioning.

[0087] Step 2: Store the tissue sections in phosphate buffered saline.

[0088] Step 3: Transfer the tissue sections to a degreasing reagent for degreasing.

[0089] Step 4: Transfer the tissue sections to phosphate buffered saline for rinsing.

[0090] Step 5: Transfer the rinsed tissue sections to the diluted ionic liquid preparation for rinsing.

[0091] Step 6: Refractive index matching can be performed in one of two ways: step 6.1 or step 6.2.

[0092] Step 6.1: Transfer the tissue sections rinsed in the diluted ionic liquid to undiluted ionic liquid for immersion. After the refractive index is uniform, transfer them to an adhesive slide, add ionic liquid for sealing, and apply resin to the edge of the coverslip to seal the inside.

[0093] Step 6.2: Transfer the tissue sections rinsed in the diluted ionic liquid to adhesive slides. Remove excess moisture with lint-free paper and air-dry the sections until slightly transparent. Add the ionic liquid dropwise to the slides. Once the refractive index of the sections is uniform, seal the slides and apply resin around the edges of the coverslip to seal the interior.

[0094] Figure 3 Photos of various biological tissue samples after being transparentized using the ionic liquid according to the exemplary embodiment of the present invention are shown. Animal tissues fixed with 4% paraformaldehyde or untreated plant tissues were directly immersed in the ionic liquid. It can be seen that the heart, liver, spleen, lung, kidney, brain, and plant leaf tissues were transparentized after being immersed in the ionic liquid according to the present invention.

[0095] Figure 4 This is an in vitro brain light sheet image obtained after clearing using the ionic liquid according to the exemplary embodiment of the present invention, as obtained in Example 1. It can be seen that the clearing method based on the ionic liquid method can meet the requirements of large-scale tissue light sheet imaging with excellent results.

[0096] Figure 5 This image demonstrates the transparency of a sample slice after being cleared using the ionic liquid according to the exemplary embodiment of the present invention. Brightfield and widefield imaging was performed using a Zeiss Axio Scan 2 slide scanner with a 561nm emission filter, acquiring 16-bit monochromatic brightness data. "IL RI matching" refers to the imaging results after refractive index matching using the ionic liquid. It can be seen that the ionic liquid clearing effect is very excellent.

[0097] Figure 6 This is a confocal image of a rotating disk of a tissue slice after being transparentized with the ionic liquid of the above exemplary embodiment of the present invention, which shows the reduction of scattering in the XY axis (left picture) and the absence of light attenuation in the Z axis (right picture) after transparentization. The tissue is a mouse brain fixed by perfusion with 4% paraformaldehyde, and 200μm tissue slices were prepared by vibrating sectioning. The confocal microscope is a rotating disk confocal microscope produced by Andor, and the images were taken using a 20x lens. It can be seen that the deformation of the transparent tissue treated with the ionic liquid of the present invention on the Z axis is suppressed, and the high degree of transparency greatly reduces the attenuation of both the excitation light and the emission light, presenting an excellent imaging effect.

[0098] Therefore, the ionic liquids of the present invention can achieve deformation-free tissue transparency. Compared to existing technologies, they avoid the tissue damage and deformation caused by conventional high-concentration organic solvents, and avoid the protein denaturation and tissue swelling caused by conventional hydrophilic reagents. The novel ionic liquids of the present invention have a refractive index as high as 1.53 and do not cause sample deformation.

[0099] II. Ultra-low temperature vitrification tissue preservation

[0100] The hydrophilic ionic liquid formulation of the present invention can be used for ultra-low temperature vitrified tissue preservation. Due to its amorphous nature, the hydrophilic ionic liquid formulation of the present invention does not form solid crystals regardless of temperature changes. At ultra-low temperatures below -40°C, tissues infiltrated with the ionic liquid enter a vitrified state without ice crystal formation, thus preventing damage to the microstructure of the preserved tissue. Therefore, after being fully infiltrated with the ionic liquid, the tissue to be preserved can be placed in an ultra-low temperature medical refrigerator for long-term storage, thus eliminating the problem of recrystallization caused by rewarming of traditional liquid nitrogen ultra-low temperature vitrified tissue.

[0101] Figure 7 This image shows the state of tissues infiltrated with an ionic liquid according to the exemplary embodiment of the present invention at -80°C. The tissues are mouse heart, liver, spleen, lung, and kidney fixed by perfusion with 4% paraformaldehyde and placed on the glass slide shown in the figure. After infiltration, the tissues exhibit no crystallization at -80°C, remaining transparent and glassy. As can be seen, the transparentized tissues exhibit no ice crystal damage at -80°C.

[0102] Therefore, the ionic liquid of the present invention can be applied to vitrification for long-term frozen tissue storage. Its beneficial effect is that, compared with existing ultra-low temperature vitrification methods (such as liquid nitrogen quick freezing), 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 crystals when cooled. Therefore, recrystallized ice crystals will not be formed, thereby facilitating the long-term frozen storage of samples.

[0103] III. Low-temperature fluorescence enhancement

[0104] The hydrophilic ionic liquid preparation of the present invention can be applied to low-temperature fluorescence enhancement of tissue samples. Due to the above-mentioned amorphous characteristics, the hydrophilic ionic liquid preparation of the present invention will not be damaged by ice crystals at ultra-low temperatures, resulting in microscopic tissue damage, thereby making it possible to place fluorescent tissues intact at low temperatures. Based on the hydrophilic polarity of the ionic liquid preparation and the photochemical properties that increase the quantum yield of fluorescent molecules at low temperatures, fluorescent tissues treated or infiltrated with the ionic liquid preparation can achieve fluorescence enhancement at ultra-low temperatures, and can be used to improve the optical imaging quality of cryo-electron microscopy photoelectric combination, for cryo-optical imaging of ultra-low temperature fluorescence enhancement, for detecting weak fluorescence signals, etc.

[0105] Optionally, the polarity of the ionic liquid preparation at room temperature also has the property of enhancing fluorescence, and thus can also be used as a fluorescence enhancer at room temperature.

[0106] Figure 8 The fluorescence intensity data of GFPuv at different temperatures in the ionic liquid of the above exemplary embodiment of the present invention are shown, and PBS is used as a control. The sample to be tested is a GFPuv protein mixed with different reagents, and is kept overnight at the predetermined temperatures (room temperature, -20°C and -80°C) shown in the figure, and transferred to a PerkinElmer EnVision microplate reader at the same temperature for fluorescence intensity measurement. The excitation light wavelength of the microplate reader is 395nm, and the emission light wavelength is 506nm. It can be seen that the fluorescence brightness of the sample at minus 80°C (IL--80) is greatly enhanced compared to that at room temperature (IL-RT) and minus 20°C (IL--20).

[0107] Figure 9 Statistical graph of the fluorescence decay of GFPuv at low temperature in the ionic liquid according to the exemplary embodiment of the present invention. Figure 9 The method used is Figure 8 It can be seen that by the third day, the fluorescence in the ionic liquid stored at -80°C (IL-80) had almost no attenuation, while the fluorescence in the ionic liquids stored at room temperature (IL-RT) and -20°C (IL-20) showed obvious attenuation, which proves that the ionic liquid of the present invention has excellent preservation ability for fluorescent tissue at low temperatures.

[0108] Therefore, the ionic liquid of the present invention can be applied to enhance fluorescence properties at low temperatures. Currently, there is no imaging system that specifically enhances fluorescence at low temperatures in the field. Most samples for cryo-imaging are used for photoelectric combined imaging of cryo-electron microscopes. For cryo-electron microscope samples, the same problem of recrystallization is faced, and the laser that excites fluorescence may also cause freeze-thaw of the sample, making this method not widely promoted. However, the ionic liquid used in the present invention does not weaken the intensity of fluorescent protein. Instead, it can enhance the fluorescence intensity at low temperatures, and does not evaporate or crystallize, causing minimal damage to the sample.

[0109] IV. Ice-free frozen sections

[0110] The hydrophilic ionic liquid preparation of the present invention can be used to prepare frozen sections of tissue samples without ice crystal damage. The hydrophilic ionic liquid preparation of the present invention has the characteristics of being amorphous and having a high shear elastic modulus state (see Figure 1 ), which can avoid microstructural damage caused by ice crystal formation during sample freezing in frozen sections. Therefore, tissues treated or infiltrated with the ionic liquid preparation can achieve frozen sections without ice crystal damage in a specific temperature range.

[0111] In contrast, the existing frozen sectioning method uses substances such as sucrose for dehydration, or uses cryoprotectants that can penetrate the cell membrane for cryoprotection. This can only inhibit the formation of ice crystals at low temperatures and has extremely high requirements for temperature control. Otherwise, crystallization or recrystallization will damage the sample. Ice crystals will still form during freezing, causing damage to the sample and changes in the microstructure during the sectioning process. The ionic liquid of the present invention will form a glassy state rather than a crystal when cooled (see Figure 2 ), thus enabling the use of frozen sections without ice crystal damage.

[0112] V. Cryosectioning and Super-resolution Imaging of Expanded Tissue

[0113] The hydrophilic ionic liquid formulation of the present invention can be used for cryosectioning and super-resolution imaging of expanded tissue. After undergoing secondary gel fixation, expanded biological tissue can be immersed in the ionic liquid formulation of the present invention to displace moisture while maintaining its expanded state. By utilizing the state transformation of the ionic liquid at low temperatures, biological tissue is converted into an amorphous, highly elastic state, enabling cryosectioning and ultrathin sectioning, enabling super-resolution imaging using conventional optical microscopes. Combined with confocal microscopy, light-sheet microscopy, and ultra-high-resolution microscopy, resolution can be further increased to the nanometer level. Furthermore, low temperatures increase fluorescence intensity, alleviating the problem of fluorescence brightness dilution caused by sample expansion.

[0114] The present invention solves the problems of traditional expanded tissue preservation difficulties, excessive fragility of expanded samples, difficulty in further tissue processing (such as transfer, slicing, etc.), and dilution of fluorescence intensity caused by expansion. As a result, expanded tissue samples can also be sliced ​​and adapted to high-power objectives with short working distances, achieving super-resolution imaging that breaks the limits of optical microscopy.

[0115] Example

[0116] The following examples provide some embodiments of the present invention, but the embodiments of the present invention are not limited to the following examples.

[0117] Example 1

[0118] The ionic liquid of the exemplary embodiment of the present invention was used to perform perfusion clearing of rat brain, and super-resolution imaging of cleared tissue sections was performed in combination with freeze drying and frozen sectioning. The basic process is as follows Figure 10 The specific operations are as follows.

[0119] 1. Perfuse the mouse transcardially with ice-cold heparin-saline solution until the blood is fully drained.

[0120] 2. Mice were transcardially perfused with 4% paraformaldehyde solution in 0.01 M phosphate buffer for 40 minutes.

[0121] 3. Use degreasing reagent to perform whole body degreasing perfusion on the mouse until it is completely degreased.

[0122] 4. Mice were transcardially perfused overnight with a 4-fold diluted ionic liquid aqueous solution.

[0123] 5. Remove the brain and place the mouse brain in 1.5 times diluted ionic liquid.

[0124] 6. Freeze the liquid and mouse brain in a -80°C refrigerator, then place them in a vacuum freeze dryer for freeze drying until the mouse brain becomes transparent.

[0125] 7. The transparent mouse brain was imaged using a light sheet microscope, where the imaging liquid was the ionic liquid itself.

[0126] 8. After embedding the transparent mouse brain with a freezing embedding medium, place it in a freezer at -80°C.

[0127] 9. After freezing, transfer the mouse brain to the cryostat using dry ice.

[0128] 10. Cut the mouse brain into 50 μm thin sections at -40°C and mount the sections on gelatin-coated adhesive slides.

[0129] 11. Use Airyscan microscope to perform super-resolution imaging on the slices to obtain data.

[0130] Imaging results such as Figure 4 As shown, it can be seen that the transparentization based on the ionic liquid method can meet the needs of large-scale tissue light sheet imaging with excellent results.

[0131] In addition, similar methods were used to process the heart, liver, spleen, lung, kidney, and plant leaves, and the imaging results are as follows: Figure 3 It can be seen that all tissues soaked in the ionic liquid of the present invention are in a transparent state.

[0132] Example 2

[0133] The ionic liquid of the exemplary embodiment of the present invention is used to perform a transparent treatment on the attached tissue slice without any deformation. The specific operation is as follows:

[0134] 1. Soak the prepared tissue slides in the degreasing reagent for 30 minutes.

[0135] 2. Soak the tissue sections in 4-fold diluted ionic liquid three times for 20 minutes each time to wash away the degreasing agent.

[0136] 3. Dry the slides and air dry the brain slices until they become slightly transparent.

[0137] 4. Add the ionic liquid dropwise onto the tissue section for refractive index matching, which takes about 1 minute.

[0138] 5. After the tissue sections become transparent, proceed with the sealing operation.

[0139] 6. After sealing, use an Andor Dragonfly spinning disk confocal microscope with a 60x lens to photograph.

[0140] The imaging results of this embodiment are as follows Figure 11 The tissue showed no deformation after being treated with ionic liquid.

[0141] Example 3

[0142] The ionic liquid according to the exemplary embodiment of the present invention is used to perform frozen sectioning of expanded tissue, and the specific operation is as follows:

[0143] 1. Perfuse mice transcardially with paraformaldehyde, remove the brain, post-fix for one day, transfer to glycine solution, and vibrate to obtain brain slices stored at 4°C.

[0144] 2. AcX was suspended in anhydrous DMSO at a concentration of 10 mg / mL. Before use, AcX was diluted to 0.1 mg / mL with PBS. The sample was incubated in the diluted AcX on a shaker at room temperature for at least 6 h.

[0145] 3. Concentrated ammonium persulfate initiator and tetramethylethylenediamine were added to the monomer solution, up to 0.2% (w / w) each; the inhibitor 4-hydroxy-TEMPO was added, and the tissue sections were incubated with the monomer solution plus APS / TEMED at 4°C for 30 minutes, and then transferred to a humidified 37°C incubator for 2 hours for gelation.

[0146] 4. Completely immerse the gel in the protease solution at room temperature overnight / 37°C for 4 hours.

[0147] 5. Place the digested gel in excess double-deionized water for 0.25-2 hours to swell, repeat 3-5 times until the size of the swollen sample reaches a steady state.

[0148] 6. Place the swollen gel in 2 mL of PEG-DA solution at room temperature overnight in the dark. Then remove the gel from the PEG-DA solution, remove the excess PEG-DA solution from the surface, and use 4 mW / cm 2 UV irradiation for 10 min.

[0149] 7. Immerse the gel in ionic liquid until its shape is stable, slice it, and image it using a 10x microscope.

[0150] The imaging results of this embodiment are as follows Figure 12 shown.

[0151] The technical concept and specific embodiments of the present invention are described above, but it should be understood that the above specific embodiments do not limit the scope of the present invention in any way. Those skilled in the art will understand that, without departing from the essence of the present invention, various modifications and / or changes can be made to the invention shown in the specific embodiments, and the modified and / or changed embodiments are also included in the scope of the present invention. Therefore, the embodiments of the present invention are merely illustrative and non-restrictive.

Claims

1. A hydrophilic amorphous ionic liquid formulation for biological tissue treatment, comprising the following components: a) an ionic liquid containing a cation and an anion, wherein the cation comprises a nitrogen-containing heterocycle substituted with one or more aminoalkyl groups, and the nitrogen-containing heterocycle is a five-membered or six-membered nitrogen-containing heterocyclic monocycle, and the anion comprises a carboxyl-containing compound; b) an adjuvant for adjusting the properties of component a), which comprises one or more of antipyrine and nicotinamide; c) water; and d) a reducing agent capable of being stably present in the ionic liquid formulation; in, Based on the total volume of the ionic liquid formulation, the mass volume ratio concentration of component a) is 35 w / v% to 95 w / v%, the mass volume ratio concentration of component b) is 4 w / v% to 50 w / v%, the mass volume ratio concentration of component c) is 5 w / v% to 45 w / v%, and the mass volume ratio concentration of component d) is 0.1 w / v% to 2.0 w / v%.

2. The ionic liquid formulation according to claim 1, wherein The molar ratio of the cations to the anions in component a) is close to or equal to the inverse of their charge ratio, so that the cations and the anions are fully ionized and have substantially the same charge.

3. The ionic liquid formulation according to claim 1, wherein The one or more aminoalkyl groups are each independently amino C 1-8 Alkyl or amino C 3-6 Cycloalkyl.

4. The ionic liquid formulation according to claim 1, wherein The one or more aminoalkyl groups are each independently amino C 1-6 alkyl.

5. The ionic liquid preparation according to claim 1 or 2, wherein The cation contains N-(amino C 1-6 alkyl)-five-membered or six-membered nitrogen-containing heterocyclic monocyclic ring.

6. The ionic liquid preparation according to claim 1 or 2, wherein The cation comprises 1-(3-aminopropyl)-imidazole.

7. The ionic liquid preparation according to claim 1 or 2, wherein The anion comprises an aliphatic or aromatic monocarboxylic acid, dicarboxylic acid or tricarboxylic acid.

8. The ionic liquid preparation according to claim 1 or 2, wherein The anion comprises benzoic acid or phthalic acid.

9. The ionic liquid preparation according to claim 1 or 2, wherein The anion comprises phthalic acid.

10. The ionic liquid preparation according to claim 1 or 2, wherein Component c) is deoxygenated ultrapure water.

11. The ionic liquid preparation according to claim 1 or 2, wherein The reducing agent of component d) comprises potassium metabisulfite.

12. The ionic liquid formulation according to claim 1 or 2, wherein Based on the total volume of the ionic liquid formulation, the mass volume ratio concentration of component a) is 75 w / v% to 90 w / v%, the mass volume ratio concentration of component b) is 6 w / v% to 23 w / v%, the mass volume ratio concentration of component c) is 10 w / v% to 40 w / v%, and the mass volume ratio concentration of component d) is 0.1 w / v% to 1.0 w / v%.

13. The ionic liquid formulation according to claim 1 or 2, wherein The cation comprises 1-(3-aminopropyl)-imidazole, the anion comprises phthalic acid, component b) comprises antipyrine and nicotinamide, the reducing agent of component d) comprises potassium metabisulfite, and The ionic liquid formulation comprises: water at a mass volume ratio concentration of 10 w / v% to 40 w / v%, 1-(3-aminopropyl)-imidazole at a mass volume ratio concentration of 40 w / v% to 60 w / v%, potassium metabisulfite at a mass volume ratio concentration of 0.2 w / v% to 0.8 w / v%, phthalic acid at a mass volume ratio concentration of 35 w / v% to 65 w / v%, antipyrine at a mass volume ratio concentration of 8 w / v% or less, and nicotinamide at a mass volume ratio concentration of 5 w / v% to 15 w / v%.

14. The ionic liquid formulation according to claim 13, comprising: water at a mass volume ratio concentration of 26 w / v%, 1-(3-aminopropyl)-imidazole at a mass volume ratio concentration of 56 w / v%, potassium metabisulfite at a mass volume ratio concentration of 0.5 w / v%, phthalic acid at a mass volume ratio concentration of 40 w / v%, antipyrine at a mass volume ratio concentration of 5 w / v%, and nicotinamide at a mass volume ratio concentration of 10 w / v%.

15. The ionic liquid formulation according to claim 1 or 2, wherein The refractive index of the ionic liquid preparation is greater than 1.

50.

16. The ionic liquid formulation according to claim 15, wherein The refractive index of the ionic liquid formulation is 1.50 to 1.

55.

17. The ionic liquid formulation according to claim 15, wherein The refractive index of the ionic liquid formulation is 1.51 to 1.

54.

18. The ionic liquid formulation according to claim 1 or 2, wherein The auxiliary agent b) further comprises one or more of nicotinic acid and 1,4-diazabicyclo[2.2.2]octane.

19. A method for preparing the ionic liquid formulation according to any one of claims 1 to 18, the method comprising: The components a) to d) are formulated in a container, and The container is filled with inert gas for inert protection, and the container is sealed.

20. The method of claim 19, wherein: The inert gas is selected from nitrogen, argon and helium.

21. A method for clearing biological tissue, comprising: The ionic liquid formulation according to any one of claims 1 to 18 is used to treat defatted biological tissue or biological tissue sections to perform refractive index matching.

22. The method of claim 21, wherein: The treatment includes perfusion, soaking or infiltration.

23. A method for low-temperature vitrification tissue preservation, comprising: Infiltrating biological tissue with the ionic liquid formulation according to any one of claims 1 to 18, and preserving the infiltrated biological tissue at a temperature below room temperature.

24. The method of claim 23, wherein: The temperature is below 0°C.

25. The method of claim 23, wherein: The temperature is below -20°C.

26. The method of claim 23, wherein: The temperature is below -80°C.

27. A low-temperature fluorescence optical enhancement imaging method, comprising: The ionic liquid formulation according to any one of claims 1 to 18 is used to treat fluorescent biological tissue, and the treated biological tissue is imaged at a temperature below room temperature.

28. The method of claim 27, wherein: The temperature is below 0°C.

29. The method of claim 27, wherein: The temperature is below -20°C.

30. The method of claim 27, wherein: The temperature is below -80°C.

31. A method for freezing sections without ice crystals, the method comprising: The biological tissue is treated with the ionic liquid formulation according to any one of claims 1 to 18, and the treated biological tissue is frozen and sliced.

32. A method for preserving and freezing sections of expanded tissue, the method comprising: The biological tissue after the primary fixation is expanded and fixed for a second time, and the expanded biological tissue sample after the second fixation is immersed in the ionic liquid preparation according to any one of claims 1 to 18, and is frozen after its shape is stabilized, and is stored or frozen sectioned.

33. The method of claim 32, wherein: The primary fixation was performed with paraformaldehyde, and the secondary fixation was performed with gel.

34. Use of the ionic liquid formulation of any one of claims 1 to 18 in biological tissue processing, wherein the biological tissue processing comprises one or more of tissue clearing, low-temperature vitrification tissue preservation, low-temperature fluorescence enhancement, ice-crystal-free frozen sections, expanded tissue frozen sections, and super-resolution imaging.

Images