WO2020199328A1 - 膨胀切削显微成像方法及适用于该方法的超吸水水凝胶 - Google Patents

膨胀切削显微成像方法及适用于该方法的超吸水水凝胶 Download PDF

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WO2020199328A1
WO2020199328A1 PCT/CN2019/088308 CN2019088308W WO2020199328A1 WO 2020199328 A1 WO2020199328 A1 WO 2020199328A1 CN 2019088308 W CN2019088308 W CN 2019088308W WO 2020199328 A1 WO2020199328 A1 WO 2020199328A1
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imaging
cutting
tissue
expanded
expansion
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French (fr)
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骆清铭
曾绍群
陈瑞希
刘秀丽
杨雄
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华中科技大学
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    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
    • C08F220/00—Copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and only one being terminated by only one carboxyl radical or a salt, anhydride ester, amide, imide or nitrile thereof
    • C08F220/02—Monocarboxylic acids having less than ten carbon atoms; Derivatives thereof
    • C08F220/52—Amides or imides
    • C08F220/54—Amides, e.g. N,N-dimethylacrylamide or N-isopropylacrylamide
    • C08F220/56—Acrylamide; Methacrylamide
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
    • C08F220/00—Copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and only one being terminated by only one carboxyl radical or a salt, anhydride ester, amide, imide or nitrile thereof
    • C08F220/02—Monocarboxylic acids having less than ten carbon atoms; Derivatives thereof
    • C08F220/52—Amides or imides
    • C08F220/54—Amides, e.g. N,N-dimethylacrylamide or N-isopropylacrylamide
    • C08F220/58—Amides, e.g. N,N-dimethylacrylamide or N-isopropylacrylamide containing oxygen in addition to the carbonamido oxygen, e.g. N-methylolacrylamide, N-(meth)acryloylmorpholine
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J3/00—Processes of treating or compounding macromolecular substances
    • C08J3/02—Making solutions, dispersions, lattices or gels by other methods than by solution, emulsion or suspension polymerisation techniques
    • C08J3/03—Making solutions, dispersions, lattices or gels by other methods than by solution, emulsion or suspension polymerisation techniques in aqueous media
    • C08J3/075—Macromolecular gels
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/645—Specially adapted constructive features of fluorimeters
    • G01N21/6456—Spatial resolved fluorescence measurements; Imaging
    • G01N21/6458—Fluorescence microscopy
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
    • C08F220/00—Copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and only one being terminated by only one carboxyl radical or a salt, anhydride ester, amide, imide or nitrile thereof
    • C08F220/02—Monocarboxylic acids having less than ten carbon atoms; Derivatives thereof
    • C08F220/52—Amides or imides
    • C08F220/54—Amides, e.g. N,N-dimethylacrylamide or N-isopropylacrylamide
    • C08F220/58—Amides, e.g. N,N-dimethylacrylamide or N-isopropylacrylamide containing oxygen in addition to the carbonamido oxygen, e.g. N-methylolacrylamide, N-(meth)acryloylmorpholine
    • C08F220/585—Amides, e.g. N,N-dimethylacrylamide or N-isopropylacrylamide containing oxygen in addition to the carbonamido oxygen, e.g. N-methylolacrylamide, N-(meth)acryloylmorpholine and containing other heteroatoms, e.g. 2-acrylamido-2-methylpropane sulfonic acid [AMPS]

Definitions

  • the present invention belongs to the field of fluorescence microscopy imaging, and more specifically, relates to a swelling cutting microscopic imaging method and a superabsorbent hydrogel suitable for the method.
  • the swelling cutting microscopy imaging method can particularly realize large-volume biological tissue samples. Three-dimensional super-resolution imaging.
  • Expansion microscopy is a super-resolution technology developed in recent years. It polymerizes biological samples in a swellable polyelectrolyte hydrogel to form a dense cross-linked network. After absorbing water, the sample is physically uniformly enlarged. After such physical magnification, the molecules in the diffraction limited area are separated to a greater distance in space, so they can be resolved even with a traditional diffraction limited microscope. Unlike other super-resolution technologies that require special instruments (such as super-resolution microscopes), ExM technology is compatible with traditional microscopes (such as wide-field microscopes, confocal microscopes, etc.), allowing cells and tissues to be ordinary, fast, and diffracting. The three-dimensional nano-resolution imaging of the specimen can be realized under the restricted microscope.
  • ExM with various expansion factors has been developed. From the initial 4 ⁇ to 10 ⁇ , the latest technology even reached ⁇ 4.5 ⁇ 4.5 ⁇ 20 ⁇ , and the imaging resolution was also developed from 70nm to 15nm.
  • researchers are pursuing the ultimate in swelling factor (that is, the swelling factor, the higher the swelling factor, the higher the corresponding imaging resolution), but they ignore the hardness of the superabsorbent hydrogel.
  • the super-absorbent polymers currently used for expansion microscopes all use sodium acrylate as a water-absorbing agent, and a small amount of acrylamide and bisacrylamide are cross-linked to form a polymer network.
  • the colloidal texture of the hydrogel is very soft after swelling with water.
  • the purpose of the present invention is to provide a swelling cutting microscopic imaging method and a superabsorbent hydrogel suitable for the method, in which it acts on key swelling substances, especially for biological tissues.
  • the hardness value requirements are controlled, and the subsequent repeated cutting-imaging process (such as sequential cutting-imaging process) can effectively solve the problem that tissue samples are soft, difficult to cut, and easy to slide compared with the existing technology.
  • the superabsorbent hydrogel with a specific composition and ratio in the present invention is particularly suitable for the expansion and cutting imaging method of biological tissues.
  • an expansive cutting microscopic imaging method is provided, which is characterized in that the method is to apply an expansive substance to the initial biological tissue to be imaged, and make the The initial biological tissue is expanded into an expanded tissue with a hardness of not less than 15KPa, and then the expanded tissue is cut and microscopically imaged in combination with mechanical cutting, so as to realize the three-dimensional expanded cutting microscopic imaging of the biological tissue and obtain the three-dimensional super-resolution data.
  • the swelling material is a superabsorbent hydrogel
  • the initial biological tissue and the superabsorbent hydrogel are cross-linked and polymerized after absorbing water and swelling to form a gel block with a hardness of not less than 15KPa.
  • the glue block is the swelling tissue; the swelling multiple of the superabsorbent hydrogel after absorbing water is ⁇ 2 times, preferably ⁇ 4 times.
  • the applying an expanding substance to the initial biological tissue to be imaged, and expanding the initial biological tissue into an expanded tissue with a hardness of not less than 15KPa under the action of the expanding substance specifically includes the following steps:
  • Tissue anchoring Use fluorescently labeled biological tissues to anchor proteins in biological tissues with methacrylic acid N-hydroxysuccinimide (MA-NHS) or 6-propenylaminoacetic acid succinate (AcX) ;
  • Dialysis and swelling in deionized water Place the digested biological tissue in step (4) for dialysis in deionized water, change the deionized water every 1 hour, and repeat 3 to 5 times until the sample volume is maximized.
  • the cutting and microscopic imaging of the expanded tissue in combination with mechanical cutting specifically includes the following steps:
  • step (b) Use the surface layer of the expanded tissue obtained in step (a) as the second imaging layer, excite imaging under a fluorescence microscope to obtain the second imaging layer, and then cut off the second imaging layer to obtain Expanded tissue after secondary cutting;
  • the cutting and microscopic imaging of the expanded tissue in combination with mechanical cutting is specifically an automatic cutting fluorescence microscope.
  • the present invention provides a superabsorbent hydrogel suitable for swelling cutting microscopic imaging methods, characterized in that the superabsorbent hydrogel is mainly composed of superabsorbent compounds, monomers, and cross-linking It is composed of super-absorbent compound, monomer, cross-linking agent, initiator and accelerator.
  • the mass ratio of the five components meets (10-15): (10-20): (0.5-2 ): (0.5-1): (0.5-1).
  • every 100 parts of superabsorbent hydrogel includes 10-15 parts of superabsorbent compound, 10-20 parts of monomer, 0.5-2 parts of crosslinking agent, 0.5-1 part of initiator and 0.5-1 part of accelerator, and the remaining components are deionized water.
  • the super absorbent compound is a hydrophilic compound, preferably acrylic acid, methacrylic acid, alginic acid, itaconic acid, crotonic acid, maleic acid, Italian acid, 2-acrylamido-2- One or more of methyl-1-propanesulfonic acid (AMPS), vinyl alcohol, vinyl acetate, and hydroxyethyl methacrylate; more preferably AMPS, itaconic acid, maleic acid, Italian acid, vinyl alcohol , One or more of vinyl acetate;
  • AMPS methyl-1-propanesulfonic acid
  • the monomer is acrylamide or N,N-dimethylacrylamide, preferably acrylamide;
  • the crosslinking agent is bisacrylamide
  • the initiator is ammonium persulfate (APS) or potassium persulfate (KPS), preferably APS;
  • the accelerator is tetramethylethylenediamine (TEMED).
  • the present invention provides the application of the above-mentioned superabsorbent hydrogel as a swelling substance in cutting microscopic imaging of biological tissues to realize three-dimensional super-resolution imaging of biological tissues.
  • the biological tissue and the swelling material are expanded, and then the expanded biological sample is sliced and imaged layer by layer, and repeated cycles are used to obtain three-dimensional ultrasound. Resolve the data to get the microscopic imaging method of expansion cutting.
  • the present invention uses expanding substances to expand biological tissues into tissues with a hardness of not less than 15KPa, which is convenient for mechanical cutting; if the hardness is too low, the tissue sample is very soft and cannot be directly supported by hand, and the cutting operation will be very difficult.
  • these soft samples When placed on a glass slide during the imaging stage, it will be very easy to slide, the image will drift, resulting in blurred focus, and three-dimensional super-resolution imaging will be very difficult.
  • the hardness of the expanded sample is controlled to be no less than 15KPa, followed by repeated fluorescence excitation imaging-cutting (ie fluorescence excitation imaging first, and subsequent cutting) process, repeated tomographic imaging is used to obtain a two-dimensional image of the expanded sample, and then The two-dimensional image is superimposed to realize the three-dimensional super-resolution imaging of the entire sample; through the superimposing process, three-dimensional stereo imaging can be realized.
  • the present invention can realize super-resolution imaging of thick and large-volume tissues, which is a great breakthrough in the limitation of thickness imaging of various super-resolution technologies at present.
  • the method can be applied in life science research, for example, the method can be used to obtain continuous biological sample fine structure information.
  • the expansion cutting microscopic imaging method of the present invention uses expansion materials to expand biological tissues and form expanded tissues with a hardness of not less than 15KPa.
  • it can be combined with an automatic cutting microscopic imaging system to simultaneously cut and image the expanded tissues, thereby breaking the super Resolution imaging limits the thickness of tissues, especially three-dimensional super-resolution imaging of thick tissues.
  • the thickness of the tissue that can be imaged by traditional super-resolution microscope imaging is often only 10-20 ⁇ m.
  • the thickness of the expanded tissue is also required to not exceed 8 mm (in the case of expansion by 4 times, The initial tissue thickness is not more than 2mm), and using the present invention, on the basis of ensuring that the hardness of the expanded tissue is not less than 15KPa, imaging of the expanded tissue with a thickness of centimeters can be achieved (the thickness of the initial biological tissue before expansion can also reach Cm), breaking the limitation of super-resolution imaging on tissue thickness in the prior art, and can realize three-dimensional super-resolution imaging of thick tissue.
  • the super absorbent polymer of the present invention is preferably used as a swelling material. On the one hand, it can have a certain hardness after absorbing water and swell, and it is compatible with automatic slice tomography microscopes, and realizes super-resolution imaging of tissues. On the other hand, it can break the current Super-resolution imaging limits the thickness of the sample and is used to obtain three-dimensional super-resolution data of large-volume tissues.
  • the superabsorbent hydrogel of specific composition and ratio which is further preferred in the present invention, still has a certain hardness after absorbing water and swells and can be used for mechanical cutting, especially as a swelling substance applied to the above-mentioned machinable optical imaging method; for example,
  • the biological tissue and superabsorbent hydrogel are cross-linked and polymerized. After absorbing water and swelling, they can still maintain sufficient hardness for automatic cutting and imaging by mechanical cutting fluorescence microscope. Through repeated cutting and imaging, the image data of the entire sample is obtained, and then through three-dimensional After processing such as registration, complete three-dimensional image information can be obtained.
  • the present invention controls the proportion of crosslinking agent in the superabsorbent hydrogel, which not only ensures the mechanical strength of the hydrogel, but also ensures the expansion ratio of the hydrogel, and effectively solves the problem that the tissue sample is soft and difficult to cut. , And easy to slide, resulting in image drift and other problems, especially can achieve three-dimensional super-resolution imaging of thicker biological tissue that is difficult to achieve in the prior art.
  • the superabsorbent hydrogel can not only ensure isotropic expansion of the tissue without causing disorder of the cell structure, but also has a good retention rate for signals such as fluorescent proteins or fluorescent dyes labeled in biological tissues, which is convenient for imaging.
  • the expansion cutting microscopy method provided by the present invention breaks the limitation of traditional expansion microscopes and super-resolution microscopes (for example, stimulated emission loss microscope STED, light activated positioning microscope PALM and random optical reconstruction microscope STORM) on tissue thickness, It is suitable for super-resolution imaging of biological tissues of any thickness and volume.
  • traditional expansion microscopes and super-resolution microscopes for example, stimulated emission loss microscope STED, light activated positioning microscope PALM and random optical reconstruction microscope STORM
  • the expanded ablation microscopic imaging method provided by the present invention can be applied to any fluorescently labeled biological tissue samples, including genetically modified and immunohistochemically labeled biological samples, and has a good retention rate for sample signals and is convenient for imaging.
  • the superabsorbent hydrogel with specific components and proportions is used as the swelling material to obtain the corresponding swelling cutting microscopy method.
  • the superabsorbent hydrogel is dialyzed in an aqueous solution to reach equilibrium , It can be expanded ⁇ 4 times in all directions and has a certain degree of hardness. It is compatible with various soft-embedded automatic cutting fluorescence microscopes, and is used to obtain three-dimensional nano-level super-resolution data of thick and large-volume biological tissues.
  • Fig. 1 is a flowchart of the imaging method of the machinable expansion microscope of the present invention.
  • Figure 2 is a schematic diagram of 3D printing of a superabsorbent hydrogel polymerization tank of thick biological tissue.
  • Figure 3 is a photograph of the brain slice of Example 1 after water absorption and swelling by cross-linking and polymerizing the hydrogel.
  • the left image is a photo placed on a graph paper, and the right image is a photo supported by a hand.
  • Figure 4 shows the comparison of results before and after expansion of transgenic fluorescent protein GFP brain slices in Example 2 (Figure A and Figure B in Figure 4), expansion factor calculation (Figure C in Figure 4) and examples of image registration results before and after expansion ( Panel D in Figure 4).
  • Figure 5 shows the comparison of the results before and after expansion of the immunohistochemically labeled Alexa546 brain slice in Example 3 ( Figure A and Figure B in Figure 5), the calculation of the expansion factor (Figure C in Figure 5) and the image registration before and after expansion Example of results (Panel D in Figure 5).
  • Figure 6 is an example of the three-dimensional imaging results of the transgenic fluorescent protein GFP brain mass of Example 4 after expansion, in the figure X is 5mm, Y is 4mm, and Z is 3mm.
  • Fig. 7 is an example of the three-dimensional imaging result of the immunohistochemical labeled fluorescent dye Alexa546 after expansion in Example 5.
  • X is 5mm
  • Y is 5.5mm
  • Z is 11.3mm.
  • the three-dimensional imaging method of machinable expansion microscopic imaging mainly relies on the sample being able to maintain sufficient hardness (hardness not less than 15KPa) after absorbing water to a certain volume.
  • the swelling material exemplified in the present invention is a superabsorbent hydrogel.
  • the superabsorbent hydrogel mainly includes five components: superabsorbent compounds, monomers, crosslinkers, initiators, and accelerators. The hydrogel components are calculated in parts by weight.
  • the superabsorbent compound is 10-15 parts, the monomer is 10-20 parts, the crosslinking agent is 0.5-2 parts, the initiator and The accelerator is 0.5-1 part each, and the other components are deionized water.
  • the macromolecular chain of superabsorbent hydrogel contains hydrophilic groups such as -SO 3 H, -OH, -COOH, -CONH 2 that can form a large number of hydrogen bonds with water molecules, so the molecular network can continue to expand From the physical structure point of view, due to the existence of the three-dimensional network structure of polymer cross-linking, osmotic pressure can be generated inside and outside the water-absorbent resin network. After water molecules enter, due to the constraints of the grid, they restrict their diffusion and movement, which has a very Good water retention performance. Therefore, the higher the content of hydrophilic groups, the stronger the hydrophilicity, which will increase the water absorption capacity of the superabsorbent polymer.
  • hydrophilic groups such as -SO 3 H, -OH, -COOH, -CONH 2 that can form a large number of hydrogen bonds with water molecules, so the molecular network can continue to expand From the physical structure point of view, due to the existence of the three-dimensional network structure of
  • AMPS contains a sulfonic acid group, which has stronger hydrophilicity compared with the carboxyl group in sodium acrylate; itaconic acid, maleic acid and Italian acid all have two carboxyl groups, which is better than sodium acrylate with only one carboxyl group They have stronger hydrophilicity.
  • the superabsorbent compounds in the formulation include but are not limited to AMPS, itaconic acid, maleic acid, Italian acid, vinyl alcohol and/or vinyl acetate.
  • the crosslinking agent mainly supports the hydrogel network.
  • the crosslinking agent in the present invention includes but is not limited to bisacrylamide.
  • reaction mechanism of superabsorbent hydrogel polymerization is as follows:
  • X 1 C(O)NH 2 , C(O)NHCH 2 NHC(O), C(O)NHC(CH 3 ) 2 CH 2 SO 3 H
  • Tissue anchoring Use N-hydroxysuccinimide methacrylate (MA-NHS) or 6-propenylaminoacetic acid succinate (AcX) to anchor proteins in biological tissues to fluorescently labeled tissues.
  • the anchoring reagent solvent can be an aqueous solution of 2-(N-morpholine)ethanesulfonic acid (MES), including 100mM MES, 150mM NaCl, the concentration of the anchoring reagent is 0.1mg/ml, the reaction temperature is 4°C, and the anchoring The time is 12h.
  • the purpose of anchoring is to introduce alkenyl groups into the proteins of biological tissues for cross-linking reactions with the components of the hydrogel, and to fix the proteins in the biological tissues to the hydrogel network.
  • Hydrogel penetration the anchored biological tissue is placed in the prepared superabsorbent hydrogel solution for penetration, the penetration time is preferably 2 to 4 hours, and the penetration temperature is preferably 4°C. Penetration is the filling of tissues with the various components of the hydrogel.
  • the polymerized biological sample is digested in a proteinase K digestion solution, where the digestion buffer is preferably a mixed solution of 50mM Tris (pH 8), 1mM EDTA, 0.5% Triton X-100, 1M NaCl, The concentration of proteinase K is preferably 8 units/mL, the digestion time is preferably 12 h, and the digestion temperature is preferably 37°C.
  • the purpose of proteinase K digestion is to relieve the interaction of proteins in biological tissues, so that the tissues can expand isotropically.
  • Dialysis and swelling in deionized water The digested biological tissue is placed in deionized water for dialysis, and the deionized water is changed every 1 hour, and repeated 3 to 5 times until the sample volume is expanded to the maximum.
  • Stereoscopic cutting imaging the expanded biological sample is placed in a fluorescent microscope with automatic cutting function for imaging. The surface layer is imaged first, and then the imaged surface layer is mechanically cut off to expose the new surface layer, and then excited to make it Fluorescence imaging
  • step (6) Repeat the process of step (6), and repeat the tomographic imaging until a three-dimensional image of the entire sample is obtained.
  • the superabsorbent hydrogel after the superabsorbent hydrogel is cross-linked and polymerized with biological tissue to absorb water and swell, it can still maintain the fluorescent signal of the biological sample labeled with fluorescent protein and fluorescent dye, and can also ensure that the biological tissue is isotropic After expansion, compared with before expansion, the disturbance rate of the marked signal is below 4%. More importantly, the superabsorbent polymer of the present invention has sufficient hardness to be used for cutting imaging under the premise that the expansion coefficient reaches ⁇ 4, and is especially suitable for the acquisition of super-resolution data of large-volume biological samples.
  • the imaging method of the present invention can choose any imaging system for three-dimensional imaging. Since the sample is in a completely transparent state after the expansion process, it is preferably an automatic cutting imaging system based on light sheet illumination; the hardness of the sample after the expansion is similar to agarose embedding
  • the soft embedding system is preferably an automatic cutting imaging system for soft embedding.
  • FIG. 1 is the flow chart of the imaging method of the machinable expansion microscope.
  • Example 1 is an example after the cross-linking and expansion of the sample and the hydrogel is completed, and the cutting imaging has not been carried out yet (the following table 1 also shows the operation after the expansion of the tissue is completed. Expansion factor and hardness test results), specifically including the following steps:
  • the anchoring solvent is an aqueous solution of 2-(N-morpholine)ethanesulfonic acid (MES), including 100mM MES, 150mM NaCl, the concentration of the anchoring compound is preferably 0.1mg/ml, placed on a horizontal shaker at 4°C, Anchor for 12 hours;
  • MES 2-(N-morpholine)ethanesulfonic acid
  • Proteinase K digestion Place the polymerized biological sample in a proteinase K digestion solution for digestion, where the digestion solution buffer is a mixture of 50mM Tris (pH 8), 1mM EDTA, 0.5% Triton X-100, 1M NaCl The solution, the concentration of proteinase K is 8units/mL, and it is digested in a gas bath shaker at 37°C for 12h.
  • the digestion solution buffer is a mixture of 50mM Tris (pH 8), 1mM EDTA, 0.5% Triton X-100, 1M NaCl
  • the solution, the concentration of proteinase K is 8units/mL, and it is digested in a gas bath shaker at 37°C for 12h.
  • Dialysis swelling in deionized water The digested biological tissues are placed in deionized water for dialysis swelling. The deionized water is changed every 1 hour and repeated 3-5 times until the volume is expanded to the maximum.
  • Imaging before swelling First, image a 100 ⁇ m thick fluorescent protein GFP-labeled brain slice under a confocal microscope, and mark the imaging position;
  • Protein anchoring Use MA-NHS to anchor the protein in the biological tissue to the fluorescently labeled tissue, and introduce double bonds into the biological tissue for cross-linking with the hydrogel.
  • the anchoring solvent is an aqueous solution of 2-(N-morpholine)ethanesulfonic acid (MES), including 100mM MES, 150mM NaCl, and the concentration of the anchoring compound is 0.1mg/ml. Place it on a horizontal shaker at 4°C. Set 12h;
  • Proteinase K digestion Place the polymerized biological sample in a proteinase K digestion solution for digestion, where the digestion solution buffer is a mixture of 50mM Tris (pH 8), 1mM EDTA, 0.5% Triton X-100, 1M NaCl The solution, the concentration of proteinase K is 8units/mL, and it is digested in a gas bath shaker at 37°C for 12h.
  • the digestion solution buffer is a mixture of 50mM Tris (pH 8), 1mM EDTA, 0.5% Triton X-100, 1M NaCl
  • the solution, the concentration of proteinase K is 8units/mL, and it is digested in a gas bath shaker at 37°C for 12h.
  • Dialysis swelling in deionized water The digested biological tissue is placed in deionized water for dialysis swelling, and the deionized water is changed every 1 hour, and repeated 3 to 5 times until the volume is expanded to the maximum;
  • the expanded sample (the thickness of the expanded sample ⁇ 500 ⁇ m) obtained in step (6) is sliced on a vibrating microtome, and cut into 50 ⁇ m thick slices;
  • Pre-expansion imaging firstly image the immunohistochemically labeled brain slice under a confocal microscope, and mark the imaging position;
  • Protein anchoring Use MA-NHS to anchor the protein in the biological tissue to the fluorescently labeled tissue, and introduce double bonds into the biological tissue for cross-linking with the hydrogel.
  • the anchoring solvent is an aqueous solution of 2-(N-morpholine)ethanesulfonic acid (MES), including 100mM MES, 150mM NaCl, and the concentration of the anchoring compound is 0.1mg/ml. Place it on a horizontal shaker at 4°C. Set 12 hours;
  • MES 2-(N-morpholine)ethanesulfonic acid
  • Hydrogel polymerization The infiltrated biological sample is placed in the polymerization tank shown in Figure 2, and a new superabsorbent hydrogel solution is added. The top of the polymerization tank is sealed with a cover glass to prevent bubbles from entering Then put the sealed polymerization tank in a wet box containing water, put it in an oven, and polymerize at 37°C for 2h.
  • Proteinase K digestion Place the polymerized biological sample in a proteinase K digestion solution for digestion, where the digestion solution buffer is a mixture of 50mM Tris (pH 8), 1mM EDTA, 0.5% Triton X-100, 1M NaCl The solution, the concentration of proteinase K is 8units/mL, and it is digested in a gas bath shaker at 37°C for 12h.
  • the digestion solution buffer is a mixture of 50mM Tris (pH 8), 1mM EDTA, 0.5% Triton X-100, 1M NaCl
  • the solution, the concentration of proteinase K is 8units/mL, and it is digested in a gas bath shaker at 37°C for 12h.
  • Dialysis and swelling in deionized water The digested biological tissue is placed in deionized water for dialysis and swelling. The deionized water is changed every 1 hour and repeated 3-5 times until the swelling reaches the maximum volume;
  • Vibrating microtome cutting the biological sample (after expansion ⁇ 500 ⁇ m) obtained in step (7) is sliced on a vibrating microtome, and cut into 50 ⁇ m thick slices;
  • Protein anchoring A fluorescently labeled mouse brain block (volume 1250 ⁇ 1000 ⁇ 750 ⁇ m 3 ) is used to anchor proteins in biological tissues using MA-NHS, and double bonds are introduced into the biological tissues for use with water Gel cross-linking.
  • the anchoring solvent is an aqueous solution of 2-(N-morpholine)ethanesulfonic acid (MES), including 100mM MES, 150mM NaCl, and the concentration of the anchoring compound is 0.2mg/ml. Place it on a horizontal shaker at 4°C. Set 12 hours;
  • MES 2-(N-morpholine)ethanesulfonic acid
  • Proteinase K digestion Place the polymerized biological sample in a proteinase K digestion solution for digestion, where the digestion solution buffer is a mixture of 50mM Tris (pH 8), 1mM EDTA, 0.5% Triton X-100, 1M NaCl The solution, the concentration of proteinase K is 8 units/mL, and it is digested in an air bath shaker at 37°C for 24 hours.
  • the digestion solution buffer is a mixture of 50mM Tris (pH 8), 1mM EDTA, 0.5% Triton X-100, 1M NaCl
  • the solution, the concentration of proteinase K is 8 units/mL, and it is digested in an air bath shaker at 37°C for 24 hours.
  • Dialysis swelling in deionized water The digested biological tissue is placed in deionized water for dialysis swelling, and the deionized water is changed every 1 hour, and repeated 3 to 5 times until the volume is maximized;
  • Stereoscopic cutting imaging place the expanded biological sample obtained in step (5) in a fluorescent microscope with automatic cutting function for imaging, first image the surface layer, and then mechanically cut off the imaged surface layer to expose the new The surface layer is then excited to make its fluorescence imaging;
  • step (6) Repeat the process of step (6), and repeat the tomographic imaging until a three-dimensional image of the entire sample is obtained.
  • Immunostaining Put the pretreated sample in PBS/0.2% Triton X-100/20% DMSO/0.3M Glycine solution for 1 d, and then transfer the sample into PBS/0.2% Triton X-100/10% DMSO/6% In the goat serum solution for 1 d, the sample was placed in PBS/0.2% Tween-20/10 mg/ml heparin (PTwH) solution overnight. On the second day, the sample was transferred to a solution of PTwH/5%DMSO/3% goat serum with the primary antibody (diluted ratio of primary antibody to rabbit anti-GFP 1:300), and incubated with the primary antibody on a shaker at 37°C for 2 days.
  • Protein anchoring Use MA-NHS to anchor the protein in biological tissues to the fluorescently labeled mouse brain block, and introduce double bonds into the biological tissues for cross-linking with the hydrogel.
  • the anchoring solvent is an aqueous solution of 2-(N-morpholine)ethanesulfonic acid (MES), including 100mM MES, 150mM NaCl, and the concentration of the anchoring compound is 0.2mg/ml. Place it on a horizontal shaker at 4°C. Set 12 hours;
  • MES 2-(N-morpholine)ethanesulfonic acid
  • Proteinase K digestion Place the polymerized biological sample in a proteinase K digestion solution for digestion, where the digestion solution buffer is a mixture of 50mM Tris (pH 8), 1mM EDTA, 0.5% Triton X-100, 1M NaCl The solution, the concentration of proteinase K is 8 units/mL, and it is digested in an air bath shaker at 37°C for 24 hours.
  • the digestion solution buffer is a mixture of 50mM Tris (pH 8), 1mM EDTA, 0.5% Triton X-100, 1M NaCl
  • the solution, the concentration of proteinase K is 8 units/mL, and it is digested in an air bath shaker at 37°C for 24 hours.
  • Dialysis swelling in deionized water The digested biological tissue is placed in deionized water for dialysis swelling, and the deionized water is changed every 1 hour, and repeated 3 to 5 times until the volume is expanded to the maximum;
  • Stereoscopic cutting imaging place the expanded biological sample obtained in step (5) in a fluorescent microscope with automatic cutting function for imaging, first image the surface layer, and then mechanically cut off the imaged surface layer to expose the new The surface layer is then excited to make its fluorescence imaging;
  • step (6) Repeat the process of step (6), and repeat the tomographic imaging until a three-dimensional image of the entire sample is obtained.
  • Table 1 is the swelling factor and hardness test of sample numbers 1-6.
  • the swelling factor is to trim the gel sample before swelling into small pieces with a cross-section of 1 cm 2 and accurately measure the side length of the cross-section with a vernier caliper. After the expansion is completed in deionized water, use a vernier caliper to accurately measure the side length.
  • the ratio of the side length after expansion to the length before expansion is the expansion factor; the gel strength after expansion is measured by using an electronic dynamic and static fatigue testing machine to test.
  • the resulting elastic modulus represents the hardness.
  • Fig. 2 is a schematic diagram of 3D printing of a superabsorbent hydrogel polymerization tank for thick biological tissues.
  • the polymerization tank designed in the present invention can be adapted to the polymerization of any size and volume of sample tissue by changing the setting of the XYZ direction.
  • Figure 3 is a representative photo of the tissues of Example 1 and the hydrogel cross-linked polymer after swelling.
  • the swollen tissue is completely transparent, and the black grid on the background paper (left picture) can be clearly seen.
  • the hydrogel is cross-linked and swelled, it still maintains sufficient hardness and can be supported by hands without deformation (right picture).
  • Figure 4 is a comparison of Example 2 transgenic fluorescent protein GFP mouse brain slices before and after expansion.
  • Figure A and Figure B are the comparison results before and after expansion.
  • Figure B is the enlargement of the corresponding white frame in Figure A.
  • Figure C in Figure 4 is the expansion factor calculation. The actual distance of the same position before and after the expansion shown in Figure A in Figure 4 (indicated by short lines c and d in Figure C), the distance after expansion (56.27 ⁇ m) and the expansion The ratio of the front distance (12.45 ⁇ m), the expansion factor is 4.52.
  • Figure D in Figure 4 shows the image registration before and after the expansion. After registration, it can be seen that the images before and after the expansion can basically be completely overlapped, indicating that the image information after the expansion is less turbulent and the fidelity is higher.
  • Fig. 5 is the comparison of the mouse brain slices labeled with the fluorescent dye Alexa546 in Example 3 before and after expansion.
  • the representation method is the same as that in Figure 4.
  • Figure A and Figure B in Figure 5 are the comparison results before and after expansion.
  • Figure B is the magnification of the corresponding white box in Figure A.
  • Figure C in Figure 5 is the expansion factor calculation. The actual distance of the same position before and after the expansion shown in Figure A in Figure 5 is measured (indicated by short lines c and d in Figure C), the distance after expansion (70.11 ⁇ m) and the expansion The ratio of the front distance (14.94 ⁇ m), the expansion factor is 4.69.
  • Picture D in Fig. 5 is the image registration before and after expansion at the position framed by the white frame in Fig. A. After registration, it can be seen that the images before and after expansion can basically be completely overlapped, indicating that the image information after expansion is less turbulent and fidelity Higher.
  • Fig. 6 and Fig. 7 are the three-dimensional imaging results of the brain masses of transgenic fluorescent protein GFP mice and immunohistochemical mice after swelling. The results are all imaged by automatic cutting fluorescence microscope, indicating that the superabsorbent polymer of the present invention has suitable mechanical strength , It can be applied to cutting imaging.
  • the present invention can realize super-resolution imaging of thick and large-volume tissues, which is a great breakthrough for the current limitations of various super-resolution technologies on thickness imaging.
  • the present invention can directly use the automatic cutting imaging system based on light sheet illumination in the prior art (such as the oblique light sheet mechanical cutting system), or directly use the soft embedded automatic cutting imaging system in the prior art (such as the sequential two-photon imaging system, Correspondingly, the cutting-imaging process repeated multiple times is the sequential cutting-imaging process).
  • the biological tissue swelling and cutting microscopic imaging method of the present invention can also use other swelling substances, as long as the hardness of the biological tissues after expansion is not less than 15KPa, It is sufficient if stable cutting can be achieved. It is believed that with the deepening of follow-up research, more and more expansive materials suitable for the expansive cutting microscopic imaging method of the present invention will appear in the future.

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Abstract

一种膨胀切削显微成像方法及适用于该方法的超吸水水凝胶，其中方法是将向待成像的生物组织施加膨胀物质，在该膨胀物质的作用下使生物组织膨胀成硬度不低于15kPa的膨胀组织，然后结合机械切削对该膨胀组织进行切削和成像，从而实现生物组织的三维膨胀切削显微成像，实现三维超分辨成像。该方法通过对生物组织在这些膨胀物质作用下硬度值要求等进行控制，与后续重复切削-成像过程相配合，能够有效解决超分辨成像对组织厚度的限制以及组织样本较软、难以切削、且易滑动导致图像漂移等问题，可以实现现有技术难以达到的对于较厚的生物组织三维超分辨成像。

Description

膨胀切削显微成像方法及适用于该方法的超吸水水凝胶 【技术领域】
本发明属于荧光显微成像领域，更具体地，涉及一种膨胀切削显微成像方法及适用于该方法的超吸水水凝胶，利用膨胀切削显微成像方法尤其可实现大体积生物组织样本的三维超分辨成像。
【背景技术】
膨胀显微镜(ExM)是近年来发展起来一种超分辨的技术，它是将生物样本在可膨胀的聚电解质水凝胶中聚合形成致密的交联网络，吸水后将样本在物理上均匀放大，经过这样的物理放大后，衍射受限区域的分子在空间中被分离到更大的距离，因此即使使用传统的衍射受限显微镜也可以分辨。不像其他的超分辨技术需要依赖于特殊的仪器(如超分辨显微镜)，ExM技术与传统显微镜(例如，宽场显微镜、共聚焦显微镜等)相兼容，使细胞和组织在普通、快速、衍射受限的显微镜下实现对标本进行三维纳米级分辨率成像。
目前各种膨胀因子(三维方向均匀膨胀的倍数)的ExM已经被发展起来。由最初的4×发展到了10×，最新的技术甚至达到了～4.5×4.5≈20×，成像分辨率也由70nm发展到了15nm。研究者们在追求膨胀因子(即膨胀倍数，膨胀因子越高，相应的成像分辨率越高)达到极致的情况下，却忽略了超吸水水凝胶的硬度。目前用于膨胀显微镜的超吸水聚合物均是以丙烯酸钠作为吸水试剂，另外以少量的丙烯酰胺和双丙烯酰胺与之交联形成聚合物网络，吸水膨胀后水凝胶的胶体质地非常软，无法直接用手支撑，实际操作困难。在成像过程中将水凝胶置于玻片上时极易滑动，图像会出现漂移现象，导致聚焦模糊，后期图像处理麻烦。最重要的是，这种质地较软的水凝胶无法用于机械切削成像，即使利用大体积成像的光片照明显微 镜，受限于其物镜工作距离(8mm)，最多只能也获取厚度为2mm的组织(膨胀4倍的情况下)的数据，不利于更大体积的样本的数据获取。另外，目前所有的超分辨显微镜都无法实现大体积生物组织的成像。
基于以上提出的问题以及当前对大体积生物组织三维网络超分辨成像的需求，急需建立一种新的超分辨成像方法，利用膨胀显微镜实现生物组织的超分辨成像，同时保证膨胀的样本具有足够的硬度，与机械切削荧光显微镜相结合，利用切削原理打破目前超分辨成像对样本厚度的限制，实现完整组织块的三维超分辨成像。
【发明内容】
针对现有技术的以上缺陷或改进需求，本发明的目的在于提供膨胀切削显微成像方法及适用于该方法的超吸水水凝胶，其中通过对关键的膨胀物质作用，尤其是对生物组织在这些膨胀物质作用下硬度值要求等进行控制，与后续重复切削-成像过程(如序列切削-成像过程)相配合，与现有技术相比能够有效解决组织样本较软、难以切削、且易滑动导致图像漂移、难以实现三维超分辨成像等问题，可以实现现有技术难以达到的对于厚的、或大体积生物组织三维超分辨成像。并且，本发明中特定组成及配比的超吸水水凝胶，尤其适用于该生物组织膨胀切削成像方法，可以使膨胀后的膨胀组织其硬度达到15KPa以上，并且能够使样本组织三维各个方向放大≥4倍(体积放大≥100倍)，在普通光学显微镜成像的条件下实现超分辨(≥250nm/4＝62.5nm的分辨率)的效果；同时与机械切削策略结合，打破目前超分辨成像对样本厚度的限制，实现大体积生物样本的三维超分辨成像。
为实现上述目的，按照本发明的一个方面，提供了一种膨胀切削显微成像方法，其特征在于，该方法是将向待成像的初始生物组织施加膨胀物质，在该膨胀物质的作用下使所述初始生物组织膨胀成硬度不低于15KPa的膨胀组织，然后结合机械切削对该膨胀组织进行切削和显微成像，从而 实现生物组织的三维膨胀切削显微成像，获取三维超分辨数据，实现三维超分辨成像。
作为本发明的进一步优选，所述膨胀物质为超吸水水凝胶，所述初始生物组织与所述超吸水水凝胶交联聚合后吸水膨胀形成硬度不低于15KPa的凝胶块，该凝胶块即为膨胀组织；所述超吸水水凝胶的吸水后膨胀倍数≥2倍，优选为≥4倍。
作为本发明的进一步优选，所述将向待成像的初始生物组织施加膨胀物质，在该膨胀物质的作用下使所述初始生物组织膨胀成硬度不低于15KPa的膨胀组织，具体包括以下步骤：
(1)组织锚定：将荧光标记的生物组织使用甲基丙烯酸N-羟琥珀酸亚胺酯(MA-NHS)或者6-丙烯氨基乙酸琥珀酸酯(AcX)对生物组织中蛋白质进行锚定；
(2)水凝胶渗透：将步骤(1)中获得的锚定后的生物组织置于配制完成的超吸水水凝胶溶液中进行渗透；
(3)水凝胶聚合：将步骤(2)中获得渗透后的生物样本置于聚合槽中，加入新的超吸水水凝胶溶液中，聚合槽表面进行封片，然后将聚合槽置于含有水的湿盒中，放入烘箱进行聚合；
(4)蛋白酶K消化：将步骤(3)中获得聚合后的生物样本置于蛋白酶K消化液中消化；
(5)去离子水中透析膨胀：将步骤(4)获得消化后的生物组织置于去离子水中透析，每隔1h换一次去离子水，重复3～5次，直至膨胀至样本体积最大。
作为本发明的进一步优选，所述结合机械切削对膨胀组织进行切削和显微成像，具体包括如下步骤：
(a)以所述膨胀组织的表层作为第一层成像层，在荧光显微镜下激发成像，获得第一层的成像，然后切削掉该第一层成像层，得到一次切削后 的膨胀组织；
(b)将步骤(a)得到的一次切削后的膨胀组织其表层作为第二层成像层，在荧光显微镜下激发成像，获得第二层的成像，然后切削掉该第二层成像层，得到二次切削后的膨胀组织；
(c)重复先荧光激发成像、后切削的过程，依次得到第三层、第四层、……的成像，从而得到所述膨胀组织的系列二维图像，然后对这些二维图像进行叠加处理，即可实现整个膨胀组织的三维超分辨成像。
作为本发明的进一步优选，所述结合机械切削对膨胀组织进行切削和显微成像具体是利用自动切削荧光显微镜。
按照本发明的另一方面，本发明提供了一种适用于膨胀切削显微成像方法的超吸水水凝胶，其特征在于，该超吸水水凝胶主要由超吸水化合物、单体、交联剂、引发剂和加速剂组成，其中，超吸水化合物、单体、交联剂、引发剂和加速剂五者的质量之比满足(10-15)：(10-20)：(0.5-2)：(0.5-1)：(0.5-1)。
作为本发明的进一步优选，按重量份计，每100份的超吸水水凝胶中，包括10-15份的超吸水化合物，10-20份的单体，0.5-2份的交联剂，0.5-1份的引发剂，以及0.5-1份的加速剂，其余组分为去离子水。
作为本发明的进一步优选，所述超吸水化合物为亲水化合物，优选为丙烯酸、甲基丙烯酸、海藻酸、衣康酸、巴豆酸、马来酸、意大利酸、2-丙烯酰氨基-2-甲基-1-丙磺酸(AMPS)、乙烯醇、醋酸乙烯、甲基丙烯酸羟乙酯中的一种或多种；更优选为AMPS、衣康酸、马来酸、意大利酸、乙烯醇、醋酸乙烯中的一种或多种；
所述单体为丙烯酰胺或N,N-二甲基丙烯酰胺，优选为丙烯酰胺；
所述交联剂为双丙烯酰胺；
所述引发剂为过硫酸铵(APS)或者过硫酸钾(KPS)，优选为APS；
所述加速剂为四甲基乙二胺(TEMED)。
按照本发明的又一方面，本发明提供了上述超吸水水凝胶作为膨胀物质在生物组织的切削显微成像实现生物组织三维超分辨成像中的应用。
通过本发明所构思的以上技术方案，与现有技术相比，通过向生物组织与膨胀物质进行膨胀处理，然后对膨胀后的生物样本进行逐层的切削成像，反复循环，由此获取三维超分辨数据，从而得到膨胀切削显微成像方法。本发明利用膨胀物质使生物组织膨胀为硬度不低于15KPa的组织，便于机械切削；硬度过低的话，组织样本非常软，无法直接用手支撑，切削操作将会非常困难，并且，这些软样本在成像阶段置于玻片上时将极易滑动，图像会出现漂移现象，导致聚焦模糊，三维超分辨成像将非常困难。本发明通过将膨胀后样本的硬度控制为不低于15KPa，后续采用重复荧光激发成像－切削(即先荧光激发成像、后切削)过程，利用反复断层成像获取膨胀样品的二维图像，然后对二维图像进行叠加处理，实现整个样本的三维超分辨成像；通过叠加处理，可实现三维立体成像。本发明可实现厚的大体积组织实现超分辨成像，对目前各种超分辨技术对厚度成像存在限制是极大的突破。该方法可应用于生命科学研究中，例如可利用该方法获取连续的生物样品精细结构信息。
本发明膨胀切削显微成像方法，利用膨胀物质，使生物组织膨胀，并形成硬度不低于15KPa的膨胀组织，尤其可以结合自动切削显微成像系统对该膨胀组织同时切削和成像，从而打破超分辨成像对组织厚度的限制，尤其可以实现厚组织的三维超分辨成像。现有技术中，传统的超分辨显微镜成像能够成像的组织厚度往往只有10～20μm，膨胀显微镜利用最长工作距离的物镜成像时也要求膨胀组织的厚度不超过8mm(膨胀4倍的情况下，初始组织厚度不超过2mm)，而利用本发明，在确保膨胀组织的硬度不低于15KPa的基础上，可实现厘米量级厚度的膨胀组织的成像(膨胀前的初始生物组织的厚度也可达厘米量级)，打破了现有技术中超分辨成像对组织厚度的限制，可以实现厚组织的三维超分辨成像。
本发明优选作为膨胀物质的超吸水聚合物，一方面能在吸水膨胀后具有一定的硬度，与自动切片的断层切削显微镜相兼容，实现组织超分辨成像的同时，另一方面，又能打破目前超分辨成像对样本厚度的限制，用于获取大体积组织的三维超分辨数据。
本发明中进一步优选的特定组成及配比的超吸水水凝胶，吸水膨胀后仍然具有一定的硬度能够用于机械切削，尤其可作为膨胀物质应用于上述可切削的光学成像方法；例如，可以将生物组织与超吸水水凝胶进行交联聚合，吸水膨胀后仍然能够保持足够的硬度用于机械切削荧光显微镜进行自动切削成像，通过反复切削成像，直至获取整个样本的图像数据，再通过三维配准等后处理即可得到三维完整图像信息。本发明尤其通过对超吸水水凝胶中交联剂的占比进行控制，既保证了水凝胶的机械强度，又确保了水凝胶的膨胀倍数，有效解决了组织样本较软、难以切削、且易滑动导致图像漂移等问题，尤其可以实现现有技术难以达到的对于较厚的生物组织三维超分辨成像。并且，该超吸水水凝胶既可保证组织各向同性膨胀，不会引起细胞结构的紊乱，又能够对生物组织中标记的荧光蛋白或者荧光染料等信号具有良好的保持率，便于成像。
总体而言，通过本发明所构思的以上技术方案与现有技术相比，能够取得下列有益效果：
(1)本发明提供的膨胀切削显微方法打破了传统的膨胀显微镜以及超分辨显微镜(例如，受激发射损耗显微镜STED，光激活定位显微镜PALM和随机光学重建显微镜STORM)对组织厚度的限制，可适用于任何厚度、任何体积的生物组织超分辨成像。
(2)本发明所提供的膨胀切削显微成像方法可适用于任何荧光标记的生物组织样本，包括转基因标记和免疫组化标记的生物样本，对样本信号具有良好的保持率，便于成像。
(3)本发明中将特定组分及配比的超吸水水凝胶作为膨胀物质由此对 应得到的膨胀切削显微方法，除了上述特点外，超吸水水凝胶在水溶液中透析达到平衡后，三维各个方向可膨胀≥4倍且具有一定的硬度，能够与各种软包埋的自动切削荧光显微镜相兼容，用于获取厚的大体积生物组织的三维纳米级超分辨数据。
【附图说明】
图1为本发明的可切削膨胀显微镜的成像方法的流程图。
图2为厚生物组织的超吸水水凝胶聚合槽3D打印示意图。
图3为实施例1脑片与水凝胶交联聚合吸水膨胀后的照片，左图为放在方格纸上的照片，右图为手支撑起的照片。
图4为实施例2转基因荧光蛋白GFP脑片膨胀前后结果对比图(图4中的图A、图B)、膨胀因子计算(图4中的图C)和膨胀前后的图像配准结果示例(图4中的图D)。
图5为实施例3免疫组化标记荧光染料Alexa546脑片膨胀前后结果对比图(图5中的图A、图B)、膨胀因子计算(图5中的图C)和膨胀前后的图像配准结果示例(图5中的图D)。
图6为实施例4转基因荧光蛋白GFP脑块膨胀后三维成像结果示例，图中X为5mm，Y为4mm，Z为3mm。
图7为实施例5免疫组化标记荧光染料Alexa546膨胀后三维成像结果示例，图中X为5mm，Y为5.5mm，Z为11.3mm。
【具体实施方式】
为了使本发明的目的、技术方案及优点更加清楚明白，以下结合附图及实施例，对本发明进行进一步详细说明。应当理解，此处所描述的具体实施例仅仅用以解释本发明，并不用于限定本发明。此外，下面所描述的本发明各个实施方式中所涉及到的技术特征只要彼此之间未构成冲突就可以相互组合。
本发明提供的一种可切削的膨胀显微成像三维成像方法，主要是依赖 于样本在吸水后到一定的体积后依然能够保持足够的硬度(硬度不低于15KPa)。为具体说明，本发明例举的膨胀物质为超吸水水凝胶，超吸水水凝胶主要共包括五种成分：超吸水化合物、单体、交联剂、引发剂和加速剂，所述的水凝胶组分按重量份计，每100份的超吸水水凝胶中，超吸水化合物为10-15份，单体为10-20份，交联剂为0.5-2份，引发剂和加速剂各0.5-1份，其他成分为去离子水。
超吸水水凝胶的大分子链上含有能与水分子之间形成大量氢键等化学键的-SO 3H、-OH、-COOH、-CONH 2等亲水基团，因而分子网络能够不断扩张；从物理结构来看，由于聚合物交联的立体网络结构的存在，能够使吸水树脂网络内外产生渗透压，水分子进入后，由于网格的约束，限制其往外扩散运动，因而又具有很好的保水性能。因此，亲水基团的含量越多，亲水能力越强，会使超吸水聚合物的吸水能力增强。另外，超吸水水凝胶的吸水倍率和强度是相互制约的关系，在同样膨胀倍数的情况下，吸水能力强的水凝胶则会保持更高的机械强度。AMPS中含有磺酸基基团，与丙烯酸钠中的羧基相比具有更强的亲水能力；衣康酸、马来酸和意大利酸均具有两个羧基基团，比只有一个羧基的丙烯酸钠具有更强的亲水能力，它们与丙烯酸钠作为吸水化合物相比，在同等膨胀倍数的情况下，膨胀后可以提高水凝胶机械强度；另外当多种吸水物质(例如，AMPS与丙烯酰胺或乙烯醇或醋酸乙烯)同时参与聚合时，由于各种基团形成相互协调作用，也会提高水凝胶的机械强度。配方中的超吸水化合物包括但不限于AMPS、衣康酸、马来酸、意大利酸、乙烯醇和/或醋酸乙烯。交联剂主要是支撑水凝胶网络的作用，交联的含量越多，水凝胶的机械强度就会越高，但是同时交联剂含量高时又会限制水凝胶的膨胀倍数。本发明中的交联剂包括并不限于双丙烯酰胺。
超吸水水凝胶聚合的反应机理如下：
(1)自由基引发
Figure PCTCN2019088308-appb-000001
(2)自由基传播
Figure PCTCN2019088308-appb-000002
R＝NH 2，NHC(CH 3) 2CH 2SO 3H，OH，COOH
Figure PCTCN2019088308-appb-000003
In *＝TEMED *，HSO 3O *，HO *
(3)聚合链增长及终止
Figure PCTCN2019088308-appb-000004
X 1＝C(O)NH 2，C(O)NHCH 2NHC(O)，C(O)NHC(CH 3) 2CH 2SO 3H
X 2＝X 1或者增长的链
使用上述膨胀聚合物对生物组织进行处理并成像，如图1所示，包括以下步骤：
(1)组织锚定：将荧光标记的组织使用甲基丙烯酸N-羟琥珀酸亚胺酯 (MA-NHS)或者6-丙烯氨基乙酸琥珀酸酯(AcX)对生物组织中蛋白质进行锚定。锚定试剂溶剂可以为2-(N-吗啉)乙磺酸(MES)的水溶液，包括100mM MES，150mM NaCl，锚定试剂的浓度为0.1mg/ml，反应的温度为4℃，锚定时间为12h。锚定的目的是向生物组织的蛋白质中引入烯基，用于和水凝胶各组分进行交联反应，将生物组织中的蛋白固定到水凝胶网络上。
(2)水凝胶渗透：锚定后的生物组织置于配制完成的超吸水水凝胶溶液中进行渗透，渗透时间优选为2～4小时，渗透温度优选为4℃。渗透是将组织中充满水凝胶的各个组分。
(3)水凝胶聚合：渗透后的生物样本置于聚合槽中(聚合槽可以采用如图2所示的结构)，加入新的超吸水水凝胶溶液中，聚合槽表面进行封片，然后将聚合槽置于含有水的湿盒中，放入烘箱进行聚合，聚合温度为为37℃，聚合时间为2h。
(4)蛋白酶K消化：聚合后的生物样本置于蛋白酶K消化液中消化，其中消化缓冲液优选为50mM Tris(pH 8),1mM EDTA,0.5％Triton X-100,1M NaCl的混合溶液，蛋白酶K的浓度优选为8units/mL，消化时间优选为12h，消化温度优选为37℃。蛋白酶K消化的目的是解除生物组织中蛋白的相互作用，使组织能够各向同性膨胀。
(5)去离子水中透析膨胀：消化后的生物组织置于去离子水中透析，每隔1h换一次去离子水，重复3～5次，直至膨胀至样本体积最大。
(6)立体切削成像：膨胀后的生物样本置于自动切削功能的荧光显微镜中成像，先对表层进行成像，然后机械切削掉所述的已成像表层，露出新表层，然后再进行激发使其荧光成像；
(7)重复所述步骤(6)的过程，如此反复断层成像直至获取整个样品的三维图像。
本发明的切削膨胀成像方法，超吸水水凝胶在与生物组织交联聚合吸 水膨胀后，依然能够保持对荧光蛋白和荧光染料标记的生物样本的荧光信号，也能够保证生物组织是各向同性的膨胀，膨胀后与膨胀前相比，所标记信号的紊乱率在4％以下。更重要的本发明的超吸水聚合物在保证了膨胀系数达到≥4的前提下，仍然具有足够的硬度能够用于切削成像，特别适用于大体积生物样本的超分辨数据的获取。
本发明的成像方法可选择用于三维成像的任意成像系统，由于样本膨胀处理后处于完全透明状态，优选为基于光片照明的自动切削成像系统；样本膨胀完成后硬度是类似的琼脂糖包埋的软包埋体系，又优选为软包埋的自动切削成像系统。
以下为实施例：
实施例1：
一种膨胀切削显微成像方法，其中的关键是将向待成像的初始生物组织施加膨胀物质，在该膨胀物质的作用下使所述初始生物组织膨胀成硬度不低于15KPa的膨胀组织。图1是可切削膨胀显微镜的成像方法的流程，实施例1是样本与水凝胶交联膨胀完成后的示例，还未进行切削成像(后文表1还给出了膨胀组织完成后进行的膨胀因子和硬度的测试结果)，具体包括以下步骤：
(1)蛋白质锚定：将小鼠脑片用MA-NHS对生物组织中蛋白质进行锚定，向生物组织中引入双键，用于与水凝胶交联。锚定溶剂为2-(N-吗啉)乙磺酸(MES)的水溶液,包括100mM MES,150mM NaCl，锚定化合物的浓度优选为0.1mg/ml,在4℃下置于水平摇床，锚定12小时；
(2)水凝胶渗透：将锚定后的生物组织置于超吸水水凝胶溶液中渗透，在4℃下置于水平摇床渗透1h；
(3)水凝胶聚合：渗透后的生物样本重新置于图2所示的聚合槽中，加入新的超吸水水凝胶溶液中，聚合槽顶部用盖玻片封片，防止有气泡进入，然后将封片后的聚合槽置于含有水的湿盒中，放入烘箱，37℃聚合2h。
(4)蛋白酶K消化：将聚合后的生物样本置于蛋白酶K消化液中进行消化，其中消化液缓冲液为50mM Tris(pH 8),1mM EDTA,0.5％Triton X-100,1M NaCl的混合溶液，蛋白酶K的浓度为8units/mL，置于37℃气浴振荡器中消化12h。
(5)去离子水中透析膨胀：消化后的生物组织置于去离子水中透析膨胀，每隔1小时换一次去离子水，重复3-5次，直到膨胀至体积最大。
实施例2：
GFP荧光蛋白转基因标记的小鼠脑片的膨胀前后的结果示例：
(1)膨胀前成像：先对100μm厚的荧光蛋白GFP标记的脑片在共聚焦显微镜下成像，标记好成像的位置；
(2)蛋白质锚定：将荧光标记的组织使用MA-NHS对生物组织中蛋白质进行锚定，向生物组织中引入双键，用于与水凝胶交联。锚定溶剂为2-(N-吗啉)乙磺酸(MES)的水溶液,包括100mM MES,150mM NaCl，锚定化合物的浓度为0.1mg/ml,在4℃下置于水平摇床，锚定12h；
(3)水凝胶渗透：将锚定后的生物组织置于超吸水水凝胶溶液中渗透，在4℃下置于水平摇床渗透1h；
(4)水凝胶聚合：渗透后的生物样本重新置于图2所示的聚合槽中，加入新的超吸水水凝胶溶液中，聚合槽顶部用盖玻片封片，防止有气泡进入，然后将封片后的聚合槽置于含有水的湿盒中，放入烘箱，37℃聚合2h。
(5)蛋白酶K消化：将聚合后的生物样本置于蛋白酶K消化液中进行消化，其中消化液缓冲液为50mM Tris(pH 8),1mM EDTA,0.5％Triton X-100,1M NaCl的混合溶液，蛋白酶K的浓度为8units/mL，置于37℃气浴振荡器中消化12h。
(6)去离子水中透析膨胀：消化后的生物组织置于去离子水中透析膨胀，每隔1小时换一次去离子水，重复3～5次，直到膨胀至体积最大；
(7)振动切片机切削：将步骤(6)所获得的膨胀后样本(膨胀后样 本的厚度≈500μm)在振动切片机上进行切片，切成50μm厚的薄片；
(8)膨胀后成像：将(7)中所获得的膨胀后的50μm厚的薄片在玻片上进行封片，对步骤(1)成像结果的相同位置进行成像。
实施例3：
免疫组化标记的小鼠脑片的膨胀前后的结果示例：
(1)脑片免疫组化标记：将100μm厚的脑片置于PBS溶液中漂洗3次，每次5min，漂洗后的脑片置于PBS/0.2％Triton X-100/20％DMSO/0.3M甘氨酸溶液中2h；然后移入PBS/0.2％Triton X-100/10％DMSO/6％羊血清的溶液中2h，之后将样本置于PBS/0.2％Tween-20含有10mg/ml肝素钠(PTwH)溶液中30min；然后将样本移入溶解有一抗的PTwH/5％DMSO/3％羊血清溶液中(一抗兔抗GFP稀释比例为1:600)，在37℃摇床上一抗孵育8小时；用PTWH溶液在37℃摇床上对样本漂洗8次，每次5min，最后一次漂洗换液后过夜；次日将样本移入溶解有二抗的PTwH/3％羊血清溶液中，在37℃摇床上二抗孵育6h(二抗羊抗兔Alexa546稀释比例为1:600)；最后在常温下，水平摇床上，采用PTWH溶液对样本漂洗4次，每次5min。
(2)膨胀前成像：先对免疫组化标记的脑片在共聚焦显微镜下成像，标记好成像的位置；
(3)蛋白质锚定：将荧光标记的组织使用MA-NHS对生物组织中蛋白质进行锚定，向生物组织中引入双键，用于与水凝胶交联。锚定溶剂为2-(N-吗啉)乙磺酸(MES)的水溶液,包括100mM MES,150mM NaCl，锚定化合物的浓度为0.1mg/ml,在4℃下置于水平摇床，锚定12小时；
(4)水凝胶渗透：将锚定后的生物组织置于超吸水水凝胶溶液中渗透，在4℃下置于水平摇床渗透1h；
(5)水凝胶聚合：渗透后的生物样本重新置于图2所示的聚合槽中，加入新的超吸水水凝胶溶液中，聚合槽顶部用盖玻片封片，防止有气泡进 入，然后将封片后的聚合槽置于含有水的湿盒中，放入烘箱，37℃聚合2h。
(6)蛋白酶K消化：将聚合后的生物样本置于蛋白酶K消化液中进行消化，其中消化液缓冲液为50mM Tris(pH 8),1mM EDTA,0.5％Triton X-100,1M NaCl的混合溶液，蛋白酶K的浓度为8units/mL，置于37℃气浴振荡器中消化12h。
(7)去离子水中透析膨胀：消化后的生物组织置于去离子水中透析膨胀，每隔1小时换一次去离子水，重复3-5次，直到膨胀至体积最大；
(8)振动切片机切削：将步骤(7)所获得的生物样本(膨胀后≈500μm)在振动切片机上进行切片，切成50μm厚的薄片；
(9)膨胀后成像：将(8)中所获得的膨胀后的50μm厚的薄片在玻片上进行封片，对步骤(2)成像结果的相同位置进行成像。
实施例4：
GFP荧光蛋白转基因标记的小鼠脑块的膨胀前后的结果示例：
(1)蛋白质锚定：将荧光标记的小鼠脑块(体积为1250×1000×750μm 3)使用MA-NHS对生物组织中蛋白质进行锚定，向生物组织中引入双键，用于与水凝胶交联。锚定溶剂为2-(N-吗啉)乙磺酸(MES)的水溶液,包括100mM MES,150mM NaCl，锚定化合物的浓度为0.2mg/ml,在4℃下置于水平摇床，锚定12小时；
(2)水凝胶渗透：将锚定后的生物组织置于超吸水水凝胶溶液中渗透，在4℃下置于水平摇床渗透2h，换新鲜的渗透液，继续渗透2h；
(3)水凝胶聚合：渗透后的生物样本重新置于图2所示的聚合槽中，加入新的超吸水水凝胶溶液中，聚合槽顶部用盖玻片封片，防止有气泡进入，然后将封片后的聚合槽置于含有水的湿盒中，放入烘箱，37℃聚合2h。
(4)蛋白酶K消化：将聚合后的生物样本置于蛋白酶K消化液中进行消化，其中消化液缓冲液为50mM Tris(pH 8),1mM EDTA,0.5％Triton X-100,1M NaCl的混合溶液，蛋白酶K的浓度为8units/mL，置于37℃气 浴振荡器中消化24h。
(5)去离子水中透析膨胀：消化后的生物组织置于去离子水中透析膨胀，每隔1小时换一次去离子水，重复3～5次，直到膨胀至体积最大；
(6)立体切削成像：将步骤(5)中所得的膨胀后的生物样本置于自动切削功能的荧光显微镜中成像，先对表层进行成像，然后机械切削掉所述的已成像表层，露出新表层，然后再进行激发使其荧光成像；
(7)重复所述步骤(6)的过程，如此反复断层成像直至获取整个样品的三维图像。
实施例5：
免疫组化标记的小鼠脑块(体积为1250×1375×2825μm 3)的膨胀前后的结果示例：
(1)小鼠脑块进行免疫组化标记：首先用0.01M PBS缓冲液对组织进行漂洗3次，每次10min，然后分别用20％、40％、60％、80％、100％、100％的甲醇溶液，在4℃环境下进行脱水，每20min。将样本置于5％H2O2/20％DMSO/CH3OH混合溶液中，在4℃环境下进行漂白12h。之后依次使用100％、80％、60％、40％、20％的甲醇溶液和0.01M PBS缓冲液，对样本进行复水，每次20min。免疫染色：将经过预处理的样本置于PBS/0.2％Triton X-100/20％DMSO/0.3M甘氨酸溶液中1d，然后将样本移入PBS/0.2％Triton X-100/10％DMSO/6％羊血清的溶液中1d，之后将样本置于PBS/0.2％Tween-20/10mg/ml肝素(PTwH)溶液中过夜。第二天将样本移入溶解有一抗的PTwH/5％DMSO/3％羊血清溶液中(一抗兔抗GFP稀释比例1:300)，在37℃环境下，摇床上一抗孵育2d。一抗孵育结束后，用PTWH溶液在37℃摇床上漂洗样本5次，每次1h，完成最后一次漂洗步骤后过夜。第二天将样本移入含有二抗的PTw H/3％羊血清溶液中在37℃环境下(二抗羊抗兔Alexa 546稀释比例1:300)，摇床上孵育2d。二抗孵育结束后，在37℃恒温摇床上，用PTWH溶液漂洗样本5次， 每次1h，最后置于PBS溶液中漂洗3次，每次30min。
(2)蛋白质锚定：将荧光标记的小鼠脑块使用MA-NHS对生物组织中蛋白质进行锚定，向生物组织中引入双键，用于与水凝胶交联。锚定溶剂为2-(N-吗啉)乙磺酸(MES)的水溶液,包括100mM MES,150mM NaCl，锚定化合物的浓度为0.2mg/ml,在4℃下置于水平摇床，锚定12小时；
(3)水凝胶渗透：将锚定后的生物组织置于超吸水水凝胶溶液中渗透，在4℃下置于水平摇床渗透2h，换新鲜的渗透液，继续渗透2h；
(4)水凝胶聚合：渗透后的生物样本重新置于图2所示的聚合槽中，加入新的超吸水水凝胶溶液中，聚合槽顶部用盖玻片封片，防止有气泡进入，然后将封片后的聚合槽置于含有水的湿盒中，放入烘箱，37℃聚合2h。
(5)蛋白酶K消化：将聚合后的生物样本置于蛋白酶K消化液中进行消化，其中消化液缓冲液为50mM Tris(pH 8),1mM EDTA,0.5％Triton X-100,1M NaCl的混合溶液，蛋白酶K的浓度为8units/mL，置于37℃气浴振荡器中消化24h。
(6)去离子水中透析膨胀：消化后的生物组织置于去离子水中透析膨胀，每隔1小时换一次去离子水，重复3～5次，直到膨胀至体积最大；
(7)立体切削成像：将步骤(5)中所得的膨胀后的生物样本置于自动切削功能的荧光显微镜中成像，先对表层进行成像，然后机械切削掉所述的已成像表层，露出新表层，然后再进行激发使其荧光成像；
(8)重复所述步骤(6)的过程，如此反复断层成像直至获取整个样品的三维图像。
按以下方法将上述实施例1-5所得的样品进行测试：
实施例1中，表1是样品编号1-6的膨胀因子和硬度测试，其中膨胀因子是将膨胀前凝胶样本修成截面为1cm 2的小块，用游标卡尺精确量取截面的边长，置于去离子水中膨胀完成后，再用游标卡尺精确量取其边长，膨 胀后与膨胀前边长的比值即为膨胀因子；膨胀后凝胶强度的测定是使用电子动静态疲劳试验机进行测试，以所得的弹性模量来代表硬度。
表1超吸水物质以AMPS为例的超吸水聚合物在不同的配比条件下膨胀因子和硬度参量的测试结果一览表
Figure PCTCN2019088308-appb-000005
从表1中可以看出当超吸水化合物、单体、交联剂、引发剂以及加速剂五者的质量之比满足(10-15)：(10-20)：(0.5-2)：(0.5-1)：(0.5-1)时，膨胀因子均≥4且硬度均不低于15KPa。
实施例2和3完成样本膨胀后，先使用振动切片机进行切片，切成50μm厚的薄片后，用载玻片和盖玻片进行封片，最后在倒置荧光共聚焦显微镜(ZISS,710)下进行图像获取。
实施例4和5完成样本膨胀后，采用自动切削功能的荧光显微镜进行切削成像来获取三维数据。
图2是厚生物组织的超吸水水凝胶聚合槽3D打印示意图，本发明设计的聚合槽可以通过改变对XYZ方向的设置适用于任意大小，任意体积样本 组织的聚合。
图3是实施例1组织与水凝胶交联聚合物膨胀后的代表照片，另外膨胀后的组织是完全透明的，可以清晰的看出背景纸上的黑色网格(左图)，组织与水凝胶交联膨胀后，依然保持了足够的硬度，可以用手支撑且不变形(右图)。
图4是实施例2转基因荧光蛋白GFP小鼠脑片膨胀前后的对比。图4中，图A、图B均为膨胀前后的对比结果，图B为图A中对应白框框出位置的放大，通过细节的对比可以清晰的看出膨胀后比膨胀前的分辨率明显提高，膨胀后树突结构细节清晰可辨。图4中图C为膨胀因子计算，分别测出图4中图A所示膨胀前后相同位置的实际距离(在图C中分别用短线c和d表示)，膨胀后距离(56.27μm)与膨胀前距离(12.45μm)的比值，即得到膨胀因子为4.52。图4中图D为膨胀前后的图像配准，配准后可以看出，膨胀前后的图像基本可以完全重合，说明膨胀后图像信息紊乱度较小，保真度较高。
图5是实施例3免疫组化荧光染料Alexa546标记的小鼠脑片膨胀前后的对比。与图4的表示方法相同，图5中图A、图B均为膨胀前后的对比结果，图B为图A中对应白框框出位置的放大，同样的，对比可看出膨胀后比膨胀前的分辨率显著提高，膨胀后细节信息清晰可辨。图5中图C为膨胀因子计算，分别测出图5中图A所示膨胀前后相同位置的实际距离(在图C中分别用短线c和d表示)，膨胀后距离(70.11μm)与膨胀前距离(14.94μm)的比值，即得到膨胀因子为4.69。图5中图D为图A中白框框出位置的膨胀前后的图像配准，配准后可以看出，膨胀前后的图像基本可以完全重合，说明膨胀后图像信息紊乱度较小，保真度较高。
图6和图7是分别是转基因荧光蛋白GFP小鼠和免疫组化小鼠脑块膨胀后三维成像结果，该结果均是采用自动切削荧光显微镜成像，说明本发明的超吸水聚合物机械强度合适，完全可以适用于切削成像。本发明可实 现厚的大体积组织实现超分辨成像，对目前各种超分辨技术对厚度成像存在限制是极大的突破。
本发明可直接采用现有技术中基于光片照明的自动切削成像系统(如斜光片机械切削系统)，也可直接采用现有技术中软包埋的自动切削成像系统(如序列双光子成像系统，相应的，重复多次的切削-成像过程即序列切削-成像过程)。
除了上述实施例中所采用的特定组分的超吸水水凝胶外，本发明中的生物组织膨胀切削显微成像方法也可以采用其他膨胀物质，只要生物组织膨胀后的硬度不低于15KPa、能够实现稳定的切削即可。相信随着后续研究的深入，未来也会有越来越多的适用于本发明膨胀切削显微成像方法的膨胀物质出现。
本领域的技术人员容易理解，以上所述仅为本发明的较佳实施例而已，并不用以限制本发明，凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等，均应包含在本发明的保护范围之内。

Claims (9)

  1. 一种膨胀切削显微成像方法，其特征在于，该方法是将向待成像的初始生物组织施加膨胀物质，在该膨胀物质的作用下使所述初始生物组织膨胀成硬度不低于15KPa的膨胀组织，然后结合机械切削对该膨胀组织进行切削和显微成像，从而实现生物组织的三维膨胀切削显微成像，获取三维超分辨数据，实现三维超分辨成像。
  2. 如权利要求1所述膨胀切削显微成像方法，其特征在于，所述膨胀物质为超吸水水凝胶，所述初始生物组织与所述超吸水水凝胶交联聚合后吸水膨胀形成硬度不低于15KPa的凝胶块，该凝胶块即为膨胀组织；所述超吸水水凝胶的吸水后膨胀倍数≥2倍，优选为≥4倍。
  3. 如权利要求2所述膨胀切削显微成像方法，其特征在于，所述将向待成像的初始生物组织施加膨胀物质，在该膨胀物质的作用下使所述初始生物组织膨胀成硬度不低于15KPa的膨胀组织，具体包括以下步骤：
    (1)组织锚定：将荧光标记的生物组织使用甲基丙烯酸N-羟琥珀酸亚胺酯(MA-NHS)或者6-丙烯氨基乙酸琥珀酸酯(AcX)对生物组织中蛋白质进行锚定；
    (2)水凝胶渗透：将步骤(1)中获得的锚定后的生物组织置于配制完成的超吸水水凝胶溶液中进行渗透；
    (3)水凝胶聚合：将步骤(2)中获得渗透后的生物样本置于聚合槽中，加入新的超吸水水凝胶溶液中，聚合槽表面进行封片，然后将聚合槽置于含有水的湿盒中，放入烘箱进行聚合；
    (4)蛋白酶K消化：将步骤(3)中获得聚合后的生物样本置于蛋白酶K消化液中消化；
    (5)去离子水中透析膨胀：将步骤(4)获得消化后的生物组织置于去离子水中透析，每隔1h换一次去离子水，重复3～5次，直至膨胀至样 本体积最大。
  4. 如权利要求1所述膨胀切削显微成像方法，其特征在于，所述结合机械切削对膨胀组织进行切削和显微成像，具体包括如下步骤：
    (a)以所述膨胀组织的表层作为第一层成像层，在荧光显微镜下激发成像，获得第一层的成像，然后切削掉该第一层成像层，得到一次切削后的膨胀组织；
    (b)将步骤(a)得到的一次切削后的膨胀组织其表层作为第二层成像层，在荧光显微镜下激发成像，获得第二层的成像，然后切削掉该第二层成像层，得到二次切削后的膨胀组织；
    (c)重复先荧光激发成像、后切削的过程，依次得到第三层、第四层、……的成像，从而得到所述膨胀组织的系列二维图像，然后对这些二维图像进行叠加处理，即可实现整个膨胀组织的三维超分辨成像。
  5. 如权利要求1所述膨胀切削显微成像方法，其特征在于，所述结合机械切削对膨胀组织进行切削和显微成像具体是利用自动切削荧光显微镜。
  6. 一种适用于膨胀切削显微成像方法的超吸水水凝胶，其特征在于，该超吸水水凝胶主要由超吸水化合物、单体、交联剂、引发剂和加速剂组成，其中，超吸水化合物、单体、交联剂、引发剂和加速剂五者的质量之比满足(10-15)：(10-20)：(0.5-2)：(0.5-1)：(0.5-1)。
  7. 如权利要求6所述适用于膨胀切削显微成像方法的超吸水水凝胶，其特征在于，按重量份计，每100份的超吸水水凝胶中，包括10-15份的超吸水化合物，10-20份的单体，0.5-2份的交联剂，0.5-1份的引发剂，以及0.5-1份的加速剂，其余组分为去离子水。
  8. 如权利要求6所述适用于膨胀切削显微成像方法的超吸水水凝胶，其特征在于，所述超吸水化合物为亲水化合物，优选为丙烯酸、甲基丙烯酸、海藻酸、衣康酸、巴豆酸、马来酸、意大利酸、2-丙烯酰氨基-2-甲基-1- 丙磺酸(AMPS)、乙烯醇、醋酸乙烯、甲基丙烯酸羟乙酯中的一种或多种；更优选为AMPS、衣康酸、马来酸、意大利酸、乙烯醇、醋酸乙烯中的一种或多种；
    所述单体为丙烯酰胺或N,N-二甲基丙烯酰胺，优选为丙烯酰胺；
    所述交联剂为双丙烯酰胺；
    所述引发剂为过硫酸铵(APS)或者过硫酸钾(KPS)，优选为APS；
    所述加速剂为四甲基乙二胺(TEMED)。
  9. 如权利要求6－8任意一项所述超吸水水凝胶作为膨胀物质在生物组织的切削显微成像实现生物组织三维超分辨成像中的应用。
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