CN110006862B - Expansion cutting microscopic imaging method and super-absorbent hydrogel suitable for method - Google Patents
Expansion cutting microscopic imaging method and super-absorbent hydrogel suitable for method Download PDFInfo
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- 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
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- 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
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- 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]
Abstract
The invention discloses an expansion cutting microscopic imaging method and a super water-absorbing hydrogel suitable for the method, wherein the method comprises the steps of applying an expansion substance to a biological tissue to be imaged, expanding the biological tissue into an expanded tissue with the hardness of not less than 15KPa under the action of the expansion substance, and then cutting and imaging the expanded tissue by combining mechanical cutting, so that the three-dimensional expansion cutting microscopic imaging of the biological tissue is realized, and the three-dimensional super resolution imaging is realized. The invention can effectively solve the problems of limitation of super-resolution imaging on the tissue thickness, soft tissue sample, difficult cutting, easy sliding to cause image drift and the like by controlling the action of key swelling substances, particularly the hardness value requirement and the like of biological tissues under the action of the swelling substances and matching with the subsequent repeated cutting-imaging process, and can realize the three-dimensional super-resolution imaging of thicker biological tissues which is difficult to achieve by the prior art.
Description
Technical Field
The invention belongs to the field of fluorescence microscopic imaging, and particularly relates to an expansion cutting microscopic imaging method and a super water-absorbing hydrogel suitable for the method.
Background
The expansion microscope (ExM) is a super-resolution technology developed in recent years, which is to polymerize a biological sample in a swellable polyelectrolyte hydrogel to form a dense cross-linked network, physically and uniformly amplify the sample after absorbing water, and after such physical amplification, the molecules in the diffraction limited region are separated to a greater distance in space, so that the resolution can be performed even by using a conventional diffraction limited microscope. Unlike other super-resolution techniques that rely on specialized instruments (e.g., super-resolution microscopes), the ExM technique is compatible with conventional microscopes (e.g., wide-field microscopes, confocal microscopes, etc.) to allow three-dimensional nanoscale resolution imaging of specimens from cells and tissues under normal, fast, diffraction-limited microscopes.
ExM has been developed for various expansion factors (a multiple of uniform expansion in three dimensions). The imaging resolution is also developed from 70nm to 15nm, from the initial 4X to 10X, and the latest technology even reaches-4.5X 4.5 ≈ 20X. Researchers have neglected the stiffness of superabsorbent hydrogels in pursuing the attainment of a high expansion factor (i.e., a multiple of expansion, with a higher expansion factor and a corresponding higher imaging resolution). The super water-absorbing polymers used for the expansion microscope at present all use sodium acrylate as a water-absorbing reagent, and a small amount of acrylamide and bisacrylamide are crosslinked with the sodium acrylate to form a polymer network, so that the gel texture of the hydrogel after water absorption and expansion is very soft, the hydrogel cannot be directly supported by hands, and the actual operation is difficult. When the hydrogel is placed on a glass slide in the imaging process, the hydrogel is easy to slide, the image can drift, the focusing is fuzzy, and the subsequent image processing is troublesome. Above all, this kind of hydrogel with soft quality can not be used for mechanical cutting imaging, even though the microscope is illuminated by the light sheet with large volume imaging, and is limited by the working distance of the objective lens (8mm), at most, the data of the tissue with the thickness of 2mm (in the case of 4 times swelling) can be obtained, which is not beneficial for the data acquisition of the sample with larger volume. In addition, all current super-resolution microscopes cannot image large volumes of biological tissue.
Based on the problems and the current requirement for the large-volume biological tissue three-dimensional network super-resolution imaging, a new super-resolution imaging method is urgently needed to be established, the expansion microscope is used for realizing the super-resolution imaging of the biological tissue, meanwhile, the expanded sample is ensured to have enough hardness, the expansion microscope is combined with the mechanical cutting fluorescence microscope, the limitation of the current super-resolution imaging on the thickness of the sample is broken through by using the cutting principle, and the three-dimensional super-resolution imaging of the complete tissue block is realized.
Disclosure of Invention
In view of the above defects or needs for improvement in the prior art, the present invention provides an expansion cutting micro-imaging method and a super-absorbent hydrogel suitable for the method, wherein the method is matched with a subsequent repeated cutting-imaging process (such as a sequential cutting-imaging process) by controlling the action of a key expansion substance, especially the hardness value requirement of a biological tissue under the action of the expansion substance, and can effectively solve the problems of soft tissue sample, difficult cutting, easy sliding, image drift, difficult realization of three-dimensional super-resolution imaging, and the like, and can realize the three-dimensional super-resolution imaging of thick or large-volume biological tissue, which is difficult to achieve in the prior art. The super-absorbent hydrogel with specific composition and proportion is particularly suitable for the biological tissue expansion cutting imaging method, the hardness of the expanded tissue after expansion can reach more than 15KPa, the three-dimensional direction magnification of the sample tissue can be more than or equal to 4 times (the volume magnification is more than or equal to 100 times), and the super-resolution effect (the resolution ratio of more than or equal to 250nm/4 to 62.5 nm) is realized under the condition of common optical microscope imaging; meanwhile, the method is combined with a mechanical cutting strategy, so that the limitation of the conventional super-resolution imaging on the thickness of the sample is broken, and the three-dimensional super-resolution imaging of the large-volume biological sample is realized.
In order to achieve the above object, according to one aspect of the present invention, there is provided an expansive cutting micro-imaging method, characterized in that a expansive substance is applied to an original biological tissue to be imaged, the original biological tissue is expanded into an expanded tissue with a hardness of not less than 15KPa by the action of the expansive substance, and then the expanded tissue is cut and micro-imaged in combination with mechanical cutting, thereby realizing three-dimensional expansive cutting micro-imaging of the biological tissue, acquiring three-dimensional super-resolution data, and realizing three-dimensional super-resolution imaging.
As a further preferred aspect of the present invention, the swelling substance is a super absorbent hydrogel, and the initial biological tissue and the super absorbent hydrogel undergo cross-linking polymerization and then swell with water to form a gel mass having a hardness of not less than 15KPa, which is a swollen tissue; the expansion multiple of the super water-absorbing hydrogel after water absorption is more than or equal to 2 times, and preferably more than or equal to 4 times.
As a further preference of the present invention, the applying of a swelling substance to the original biological tissue to be imaged, under the effect of which the original biological tissue is swelled to a swelled tissue with a hardness of not less than 15KPa, specifically comprises the following steps:
(1) tissue anchoring: anchoring proteins in biological tissue using N-hydroxysuccinimide-methacrylate (MA-NHS) or 6-acrylamidoacetic acid succinate (AcX);
(2) hydrogel permeation: placing the anchored biological tissue obtained in the step (1) in a prepared super-absorbent hydrogel solution for infiltration;
(3) hydrogel polymerization: placing the biological sample obtained in the step (2) after permeation into a polymerization tank, adding a new super-absorbent hydrogel solution, sealing the surface of the polymerization tank, placing the polymerization tank into a wet box containing water, and placing the wet box into an oven for polymerization;
(4) and (3) protease K digestion: placing the polymerized biological sample obtained in the step (3) into proteinase K digestion solution for digestion;
(5) dialysis expansion in deionized water: and (5) putting the digested biological tissue obtained in the step (4) into deionized water for dialysis, changing the deionized water every 1h, and repeating for 3-5 times until the volume of the sample is expanded to the maximum.
As a further preferred embodiment of the present invention, the cutting and microscopic imaging of the dilated tissue in combination with mechanical cutting specifically comprises the following steps:
(a) taking the surface layer of the expanded tissue as a first imaging layer, carrying out excitation imaging under a fluorescence microscope to obtain an image of the first layer, and then cutting off the first imaging layer to obtain the once-cut expanded tissue;
(b) taking the surface layer of the primarily cut expanded tissue obtained in the step (a) as a second imaging layer, carrying out excitation imaging under a fluorescence microscope to obtain an image of the second layer, and then cutting off the second imaging layer to obtain a secondarily cut expanded tissue;
(c) and repeating the processes of fluorescence excitation imaging and cutting, sequentially obtaining the imaging of a third layer, a fourth layer and more layers, thereby obtaining a series of two-dimensional images of the expansive tissue, and then superposing the two-dimensional images to realize the three-dimensional super-resolution imaging of the whole expansive tissue.
As a further preference of the present invention, said cutting and microscopic imaging of the dilated tissue in combination with mechanical cutting is in particular by means of an automatic cutting fluorescence microscope.
According to another aspect of the present invention, there is provided a superabsorbent hydrogel suitable for use in a swelling cut microscopic imaging method, characterized in that the superabsorbent hydrogel mainly comprises a superabsorbent compound, a monomer, a crosslinking agent, an initiator, and an accelerator, wherein the mass ratio of the superabsorbent compound, the monomer, the crosslinking agent, the initiator, and the accelerator satisfies (10-15): (10-20): (0.5-2): (0.5-1): (0.5-1).
As a further preference of the invention, every 100 parts of the super absorbent hydrogel comprises 10 to 15 parts of super absorbent compound, 10 to 20 parts of monomer, 0.5 to 2 parts of cross-linking agent, 0.5 to 1 part of initiator and 0.5 to 1 part of accelerator by weight, and the rest is deionized water.
As a further preference of the present invention, the superabsorbent compound is a hydrophilic compound, preferably one or more of acrylic acid, methacrylic acid, alginic acid, itaconic acid, crotonic acid, maleic acid, italic acid, 2-acrylamido-2-methyl-1-propanesulfonic Acid (AMPS), vinyl alcohol, vinyl acetate, hydroxyethyl methacrylate; more preferably one or more of AMPS, itaconic acid, maleic acid, Italian acid, vinyl alcohol and vinyl acetate;
the monomer is acrylamide or N, N-dimethylacrylamide, and acrylamide is preferred;
the cross-linking agent is bisacrylamide;
the initiator is Ammonium Persulfate (APS) or potassium persulfate (KPS), preferably APS;
the accelerator is Tetramethylethylenediamine (TEMED).
According to another aspect of the present invention, the present invention provides the use of the above-mentioned superabsorbent hydrogel as a swelling substance in cutting microscopic imaging of biological tissues to achieve three-dimensional super-resolution imaging of biological tissues.
Compared with the prior art, the technical scheme provided by the invention has the advantages that the expansion cutting microscopic imaging method is obtained by performing expansion treatment on the biological tissue and the expansion substance, then performing layer-by-layer cutting imaging on the expanded biological sample, and repeating the steps. The invention utilizes the expansion substance to expand the biological tissue into the tissue with the hardness not lower than 15KPa, thereby facilitating the mechanical cutting; if the hardness is too low, the tissue samples are very soft and cannot be directly supported by hands, the cutting operation is very difficult, and the soft samples are very easy to slide when placed on a glass slide in the imaging stage, the image can have a drift phenomenon, so that the focusing is fuzzy, and the three-dimensional super-resolution imaging is very difficult. According to the invention, the hardness of the sample after expansion is controlled to be not less than 15KPa, a repeated fluorescence excitation imaging-cutting (namely, fluorescence excitation imaging and cutting-back) process is adopted subsequently, a two-dimensional image of the expanded sample is obtained by utilizing repeated tomography, and then the two-dimensional image is subjected to superposition processing, so that the three-dimensional super-resolution imaging of the whole sample is realized; by the superimposition processing, three-dimensional stereoscopic imaging can be realized. The invention can realize the super-resolution imaging of thick and large-volume tissues, and is a great breakthrough in the limitation of various super-resolution technologies to the thickness imaging. The method can be applied to life science research, and for example, continuous fine structure information of biological samples can be obtained by the method.
According to the expansion cutting microimaging method, the expansion substance is utilized to expand the biological tissue and form the expanded tissue with the hardness not lower than 15KPa, and particularly, the automatic cutting microimaging system can be combined to simultaneously cut and image the expanded tissue, so that the limitation of the super-resolution imaging on the thickness of the tissue is broken, and particularly, the three-dimensional super-resolution imaging of the thick tissue can be realized. In the prior art, the thickness of the tissue which can be imaged by the traditional super-resolution microscope imaging is usually only 10-20 μm, and the thickness of the expanded tissue is required to be not more than 8mm (under the condition of 4 times of expansion, the thickness of the initial tissue is not more than 2mm) when the expansion microscope is used for imaging by using the objective lens with the longest working distance.
The super water-absorbing polymer preferably used as an expanding substance has certain hardness after water absorption and expansion, is compatible with a tomography microscope which is automatically sliced, realizes tissue super-resolution imaging, and breaks the limitation of the current super-resolution imaging on the thickness of a sample to obtain three-dimensional super-resolution data of a large-volume tissue.
The super water-absorbing hydrogel with the further optimized specific composition and the proportion still has certain hardness after water absorption and expansion, can be used for mechanical cutting, and particularly can be used as an expansion substance to be applied to the optical imaging method for cutting; for example, biological tissues and super water-absorbing hydrogel can be subjected to cross-linking polymerization, the water-absorbing hydrogel can still keep enough hardness after water swelling for automatic cutting imaging of a mechanical cutting fluorescence microscope, and three-dimensional complete image information can be obtained by repeated cutting imaging until image data of the whole sample is obtained and post-processing such as three-dimensional registration. The invention particularly controls the proportion of the cross-linking agent in the super-absorbent hydrogel, thereby not only ensuring the mechanical strength of the hydrogel, but also ensuring the expansion multiple of the hydrogel, effectively solving the problems of image drift and the like caused by softer tissue samples, difficult cutting and easy sliding, and particularly realizing the three-dimensional super-resolution imaging of thicker biological tissues which is difficult to achieve in the prior art. In addition, the super water-absorbing hydrogel can ensure that the tissue expands isotropically, does not cause disorder of a cell structure, has good retention rate on signals such as fluorescent protein or fluorescent dye marked in the biological tissue, and is convenient for imaging.
In general, compared with the prior art, the above technical solution contemplated by the present invention can achieve the following beneficial effects:
(1) the dilation cutting microscopy method provided by the invention breaks the limitation of the traditional dilation microscope and super-resolution microscope (such as stimulated emission depletion microscope (STED), light-activated positioning microscope (PALM) and random optical reconstruction microscope (STORM)) on the tissue thickness, and can be suitable for super-resolution imaging of biological tissues with any thickness and any volume.
(2) The expansion cutting microscopic imaging method provided by the invention can be suitable for any fluorescence-labeled biological tissue sample, including transgenic-labeled and immunohistochemical-labeled biological samples, has good retention rate on sample signals, and is convenient for imaging.
(3) In the invention, the expansion cutting microscopic method correspondingly obtains the super water-absorbing hydrogel with specific components and proportions as the expansion substance, and in addition to the characteristics, after the super water-absorbing hydrogel is dialyzed in aqueous solution to reach balance, the super water-absorbing hydrogel can expand by more than or equal to 4 times in all three-dimensional directions and has certain hardness, can be compatible with various soft-embedded automatic cutting fluorescence microscopes, and is used for obtaining three-dimensional nanometer super resolution data of thick large-volume biological tissues.
Drawings
FIG. 1 is a flow chart of a method of imaging a machinable expansion microscope of the present invention.
Fig. 2 is a schematic 3D printing view of a superabsorbent hydrogel polymerization tank for thick biological tissue.
FIG. 3 is a photograph of the brain piece and hydrogel of example 1 after swelling due to water absorption by crosslinking polymerization, the left photograph being a photograph placed on a paper grid, and the right photograph being a photograph supported by a hand.
Fig. 4 is a comparison graph of the results before and after brain expansion of the transgenic fluorescent protein GFP in example 2 (panels a and B in fig. 4), calculation of the expansion factor (panel C in fig. 4), and an example of the image registration results before and after expansion (panel D in fig. 4).
Fig. 5 is a comparison graph of the results before and after brain slice expansion (panels a and B in fig. 5), expansion factor calculation (panel C in fig. 5), and an example of the image registration results before and after expansion (panel D in fig. 5) of the immunohistochemically labeled fluorescent dye Alexa546 in example 3.
FIG. 6 is an example of the three-dimensional imaging results of the transgenic fluorescent protein GFP brain block of example 4 after swelling, wherein X is 5mm, Y is 4mm, and Z is 3 mm.
FIG. 7 is an example of the results of three-dimensional imaging of the immunohistochemically labeled fluorescent dye Alexa546 after swelling, where X is 5mm, Y is 5.5mm, and Z is 11.3mm in example 5.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention. In addition, the technical features involved in the embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.
The invention provides a three-dimensional imaging method for machinable expansion microscopic imaging, which mainly depends on that a sample can still keep enough hardness (the hardness is not lower than 15KPa) after absorbing water to a certain volume. To illustrate specifically, the swelling substance of the present invention is a superabsorbent hydrogel, which mainly comprises five components: the hydrogel comprises, by weight, 10-15 parts of super absorbent compound, 10-20 parts of monomer, 0.5-2 parts of cross-linking agent, 0.5-1 part of initiator and accelerator respectively, and deionized water as other components per 100 parts of super absorbent hydrogel.
The macromolecular chain of the super water-absorbing hydrogel contains-SO capable of forming a large number of chemical bonds such as hydrogen bonds and the like with water molecules3H、-OH、-COOH、-CONH2Hydrophilic groups are equal, so that the molecular network can be continuously expanded; from the physical structure, the existence of the polymer cross-linked three-dimensional network structure can generate osmotic pressure inside and outside the water-absorbent resin network, and water molecules are restricted from diffusing outwards due to the constraint of the grid after entering, so that the water-absorbent resin has good water retention performance. Therefore, the greater the content of hydrophilic groups, the greater the hydrophilic ability, which results in an increase in the water-absorbing ability of the super absorbent polymer. In addition, the water absorption capacity and strength of the super water-absorbent hydrogel are in a mutually restricted relationship, and the water absorption capacity is strong under the condition of the same expansion ratioThe hydrogel of (2) will retain a higher mechanical strength. AMPS contains sulfonic group, and has stronger hydrophilic capability compared with carboxyl in sodium acrylate; itaconic acid, maleic acid and Italian acid all have two carboxyl groups, have stronger hydrophilic ability than sodium acrylate with only one carboxyl, and compared with sodium acrylate as a water-absorbing compound, the mechanical strength of the hydrogel can be improved after swelling under the condition of the same swelling multiple; in addition, when various water-absorbing substances (for example, AMPS and acrylamide or vinyl alcohol or vinyl acetate) participate in the polymerization at the same time, the mechanical strength of the hydrogel is also improved due to the formation of a mutual coordination function of various groups. Superabsorbent compounds in the formulation include, but are not limited to, AMPS, itaconic, maleic, italic, vinyl alcohol, and/or vinyl acetate. The crosslinking agent mainly acts to support the hydrogel network, and the higher the content of the crosslinking agent, the higher the mechanical strength of the hydrogel, but at the same time, the higher the content of the crosslinking agent, the higher the expansion factor of the hydrogel is limited. The crosslinking agent in the present invention includes, but is not limited to, bisacrylamide.
The reaction mechanism of the polymerization of superabsorbent hydrogels is as follows:
(1) free radical initiation
HSO3O-+H2O→H2SO4+HO·
(2) Propagation of free radicals
R＝NH2，NHC(CH3)2CH2SO3H，OH，COOH
In·＝TEMED·，HSO3O·，HO·
(3) Polymeric chain propagation and termination
X1＝C(O)NH2，C(O)NHCH2NHC(O)，C(O)NHC(CH3)2CH2SO3H
X2＝X1Or a growing chain
The treatment and imaging of biological tissue using the above-described expanded polymer, as shown in figure 1, comprises the following steps:
(1) tissue anchoring: fluorescently labeled tissue is anchored to proteins in biological tissue using N-hydroxysuccinimide methacrylate (MA-NHS) or 6-acrylamidoacetic acid succinate (AcX). The anchoring reagent solvent may be an aqueous solution of 2- (N-morpholine) ethanesulfonic acid (MES) including 100mM MES,150mM NaCl, the concentration of the anchoring reagent being 0.1mg/ml, the temperature of the reaction being 4 ℃ and the anchoring time being 12 h. The purpose of anchoring is to introduce alkenyl groups into proteins in the biological tissue for cross-linking reactions with the components of the hydrogel, fixing the proteins in the biological tissue to the hydrogel network.
(2) Hydrogel permeation: and (3) placing the anchored biological tissue in the prepared super-absorbent hydrogel solution for infiltration, wherein the infiltration time is preferably 2-4 hours, and the infiltration temperature is preferably 4 ℃. Infiltration is the filling of the tissue with the individual components of the hydrogel.
(3) Hydrogel polymerization: placing the permeated biological sample in a polymerization tank (the polymerization tank can adopt a structure shown in figure 2), adding a new super-absorbent hydrogel solution, sealing the surface of the polymerization tank, placing the polymerization tank in a wet box containing water, and placing the wet box in an oven for polymerization at the polymerization temperature of 37 ℃ for 2 hours.
(4) And (3) protease K digestion: the polymerized biological sample is digested in proteinase K digestion solution, wherein the digestion buffer solution is preferably a mixed solution of 50mM Tris (pH 8),1mM EDTA, 0.5% Triton X-100,1M NaCl, the concentration of the proteinase K is preferably 8units/mL, the digestion time is preferably 12h, and the digestion temperature is preferably 37 ℃. The purpose of proteinase K digestion is to release protein interactions in biological tissues, enabling isotropic swelling of the tissues.
(5) Dialysis expansion in deionized water: and (3) dialyzing the digested biological tissue in deionized water, changing the deionized water every 1h, and repeating for 3-5 times until the biological tissue expands to the maximum sample volume.
(6) Three-dimensional cutting imaging: imaging the expanded biological sample in a fluorescence microscope with an automatic cutting function, imaging the surface layer, mechanically cutting off the imaged surface layer to expose a new surface layer, and exciting to perform fluorescence imaging;
(7) and (4) repeating the process of the step (6), and repeating tomography in such a way until a three-dimensional image of the whole sample is obtained.
According to the cutting swelling imaging method, after the super water-absorbing hydrogel is crosslinked with the biological tissue and polymerized to absorb water to swell, the fluorescence signals of the biological sample marked by the fluorescent protein and the fluorescent dye can still be kept, the biological tissue can also be guaranteed to swell isotropically, and the disturbance rate of the marked signals is below 4% compared with that before swelling after swelling. More importantly, the super water-absorbent polymer provided by the invention still has enough hardness to be used for cutting imaging on the premise of ensuring that the expansion coefficient is more than or equal to 4, and is particularly suitable for acquiring super resolution data of a large-volume biological sample.
The imaging method can be used for selecting any imaging system for three-dimensional imaging, and the sample is in a completely transparent state after expansion treatment, and is preferably an automatic cutting imaging system based on light sheet illumination; the hardness of the sample after expansion is similar to that of agarose embedded soft embedding systems, and is preferably a soft embedded automatic cutting imaging system.
The following are examples:
example 1:
an expansive cutting microscopic imaging method, wherein the key is to apply an expansive substance to an initial biological tissue to be imaged, and the initial biological tissue is expanded into an expanded tissue with the hardness of not less than 15KPa under the action of the expansive substance. Fig. 1 is a flow chart of an imaging method of a cuttable expansion microscope, and example 1 is an example of a sample after crosslinking and expansion with hydrogel are completed, and cutting imaging is not performed (table 1 below also shows the test results of expansion factors and hardness performed after the completion of the expansion of the tissue), and specifically includes the following steps:
(1) protein anchoring: anchoring proteins in biological tissues by using the mouse brain slice through MA-NHS, and introducing double bonds into the biological tissues for cross-linking with hydrogel. The anchoring solvent is 2- (N-morpholine) ethanesulfonic acid (MES) aqueous solution, including 100mM MES,150mM NaCl, the anchoring compound concentration is preferably 0.1mg/ml, at 4 ℃ in a horizontal shaker, anchoring for 12 hours;
(2) hydrogel permeation: placing the anchored biological tissue in a super water-absorbing hydrogel solution for permeation, and placing the biological tissue in a horizontal shaking table at the temperature of 4 ℃ for permeation for 1 h;
(3) hydrogel polymerization: the infiltrated biological sample is placed in the polymerization tank shown in figure 2 again, new super-absorbent hydrogel solution is added, the top of the polymerization tank is covered with a cover glass to prevent air bubbles from entering, then the covered polymerization tank is placed in a wet box containing water, and the wet box is placed in an oven and polymerized for 2 hours at 37 ℃.
(4) And (3) protease K digestion: the polymerized biological sample is placed in proteinase K digestion solution for digestion, wherein the digestion solution buffer solution is a mixed solution of 50mM Tris (pH 8),1mM EDTA, 0.5% Triton X-100 and 1M NaCl, the concentration of the proteinase K is 8units/mL, and the digestion solution is placed in a 37 ℃ air bath oscillator for digestion for 12 hours.
(5) Dialysis expansion in deionized water: and (3) placing the digested biological tissue into deionized water for dialysis expansion, changing the deionized water every 1 hour, and repeating for 3-5 times until the biological tissue is expanded to the maximum volume.
Example 2:
results before and after expansion of GFP fluorescent protein transgene-labeled mouse brain slices are exemplified:
(1) imaging before expansion: imaging a brain slice marked by fluorescent protein GFP with the thickness of 100 mu m under a confocal microscope, and marking the imaged position;
(2) protein anchoring: the fluorescently labeled tissue was used to anchor proteins in biological tissue using MA-NHS, introducing double bonds into biological tissue for cross-linking with hydrogels. The anchoring solvent is 2- (N-morpholine) ethanesulfonic acid (MES) in water, comprising 100mM MES and 150mM NaCl, the anchoring compound concentration is 0.1mg/ml, and the anchoring solvent is placed in a horizontal shaker at 4 ℃ for 12 h;
(3) hydrogel permeation: placing the anchored biological tissue in a super water-absorbing hydrogel solution for permeation, and placing the biological tissue in a horizontal shaking table at the temperature of 4 ℃ for permeation for 1 h;
(4) hydrogel polymerization: the infiltrated biological sample is placed in the polymerization tank shown in figure 2 again, new super-absorbent hydrogel solution is added, the top of the polymerization tank is covered with a cover glass to prevent air bubbles from entering, then the covered polymerization tank is placed in a wet box containing water, and the wet box is placed in an oven and polymerized for 2 hours at 37 ℃.
(5) And (3) protease K digestion: the polymerized biological sample is placed in proteinase K digestion solution for digestion, wherein the digestion solution buffer solution is a mixed solution of 50mM Tris (pH 8),1mM EDTA, 0.5% Triton X-100 and 1M NaCl, the concentration of the proteinase K is 8units/mL, and the digestion solution is placed in a 37 ℃ air bath oscillator for digestion for 12 hours.
(6) Dialysis expansion in deionized water: placing the digested biological tissue in deionized water for dialysis expansion, changing the deionized water every 1 hour, and repeating for 3-5 times until the biological tissue is expanded to the maximum volume;
(7) cutting by a vibrating slicer: slicing the sample obtained in the step (6) after expansion (the thickness of the sample after expansion is approximately equal to 500 mu m) on a vibrating microtome, and cutting the sample into slices with the thickness of 50 mu m;
(8) imaging after swelling: the swollen 50 μm-thick sheet obtained in (7) was mounted on a slide glass, and the same position as the imaging result in step (1) was imaged.
Example 3:
results before and after expansion of immunohistochemically labeled mouse brain sheets are exemplified:
(1) brain slice immunohistochemical labeling: rinsing 100 μ M brain slice in PBS for 5min for 3 times, and placing the rinsed brain slice in PBS/0.2% Triton X-100/20% DMSO/0.3M glycine solution for 2 hr; then transferred into PBS/0.2% Triton X-100/10% DMSO/6% sheep serum for 2h, after which the samples were placed in PBS/0.2% Tween-20 containing 10mg/ml heparin sodium (PTwH) solution for 30 min; the samples were then transferred to a solution of PTwH/5% DMSO/3% sheep serum in which primary antibody was dissolved (primary anti-rabbit anti-GFP dilution ratio 1:600), and incubated on a shaker at 37 ℃ for 8 hours; rinsing the sample with PTWH solution on a shaker at 37 deg.C for 8 times, 5min each time, and standing overnight after the last rinsing and changing; the next day the samples were transferred to PTwH/3% sheep serum solution with secondary antibody dissolved and incubated for 6h on a shaker at 37 ℃ (secondary antibody rabbit Alexa546 dilution ratio 1: 600); finally, the sample was rinsed 4 times with PTWH solution 5min each time on a horizontal shaker at room temperature.
(2) Imaging before expansion: imaging the immunohistochemically marked brain slice under a confocal microscope, and marking the imaging position;
(3) protein anchoring: the fluorescently labeled tissue was used to anchor proteins in biological tissue using MA-NHS, introducing double bonds into biological tissue for cross-linking with hydrogels. The anchoring solvent was 2- (N-morpholine) ethanesulfonic acid (MES) in water, containing 100mM MES and 150mM NaCl, and the anchoring compound concentration was 0.1mg/ml, and the anchoring was carried out in a horizontal shaker at 4 ℃ for 12 hours;
(4) hydrogel permeation: placing the anchored biological tissue in a super water-absorbing hydrogel solution for permeation, and placing the biological tissue in a horizontal shaking table at the temperature of 4 ℃ for permeation for 1 h;
(5) hydrogel polymerization: the infiltrated biological sample is placed in the polymerization tank shown in figure 2 again, new super-absorbent hydrogel solution is added, the top of the polymerization tank is covered with a cover glass to prevent air bubbles from entering, then the covered polymerization tank is placed in a wet box containing water, and the wet box is placed in an oven and polymerized for 2 hours at 37 ℃.
(6) And (3) protease K digestion: the polymerized biological sample is placed in proteinase K digestion solution for digestion, wherein the digestion solution buffer solution is a mixed solution of 50mM Tris (pH 8),1mM EDTA, 0.5% Triton X-100 and 1M NaCl, the concentration of the proteinase K is 8units/mL, and the digestion solution is placed in a 37 ℃ air bath oscillator for digestion for 12 hours.
(7) Dialysis expansion in deionized water: dialyzing and expanding the digested biological tissue in deionized water, changing the deionized water every 1 hour, and repeating for 3-5 times until the biological tissue is expanded to the maximum volume;
(8) cutting by a vibrating slicer: slicing the biological sample (after swelling, the thickness is approximately equal to 500 mu m) obtained in the step (7) on a vibration microtome, and cutting the biological sample into slices with the thickness of 50 mu m;
(9) imaging after swelling: the swollen 50 μm-thick sheet obtained in (8) was mounted on a slide glass, and the same position as the imaging result in step (2) was imaged.
Example 4:
results before and after expansion of GFP fluorescent protein transgene-labeled mouse brain blocks are exemplified:
(1) protein anchoring-fluorescence-labeled mouse brain blocks (volume 1250 × 1000 × 750 μm)3) The proteins in the biological tissue were anchored using MA-NHS, introducing double bonds into the biological tissue for cross-linking with the hydrogel. The anchoring solvent was 2- (N-morpholine) ethanesulfonic acid (MES) in water, containing 100mM MES and 150mM NaCl, and the anchoring compound concentration was 0.2mg/ml, and the anchoring was carried out in a horizontal shaker at 4 ℃ for 12 hours;
(2) hydrogel permeation: placing the anchored biological tissue in a super water-absorbing hydrogel solution for infiltration, placing the biological tissue in a horizontal shaking table at 4 ℃ for infiltration for 2 hours, replacing fresh penetrating fluid, and continuing the infiltration for 2 hours;
(3) hydrogel polymerization: the infiltrated biological sample is placed in the polymerization tank shown in figure 2 again, new super-absorbent hydrogel solution is added, the top of the polymerization tank is covered with a cover glass to prevent air bubbles from entering, then the covered polymerization tank is placed in a wet box containing water, and the wet box is placed in an oven and polymerized for 2 hours at 37 ℃.
(4) And (3) protease K digestion: the polymerized biological sample is placed in proteinase K digestion solution for digestion, wherein the digestion solution buffer solution is a mixed solution of 50mM Tris (pH 8),1mM EDTA, 0.5% Triton X-100 and 1M NaCl, the concentration of the proteinase K is 8units/mL, and the digestion solution is placed in a 37 ℃ air bath oscillator for digestion for 24 hours.
(5) Dialysis expansion in deionized water: placing the digested biological tissue in deionized water for dialysis expansion, changing the deionized water every 1 hour, and repeating for 3-5 times until the biological tissue is expanded to the maximum volume;
(6) three-dimensional cutting imaging: imaging the expanded biological sample obtained in the step (5) in a fluorescence microscope with an automatic cutting function, imaging the surface layer, mechanically cutting off the imaged surface layer to expose a new surface layer, and exciting to perform fluorescence imaging;
(7) and (4) repeating the process of the step (6), and repeating tomography in such a way until a three-dimensional image of the whole sample is obtained.
Example 5:
immunohistochemically labeled mouse brain blocks (volume 1250 × 1375 × 2825 μm)3) Results before and after inflation of (c):
(1) immunohistochemical labeling of mouse brain blocks: the tissue was first rinsed 3 times with 0.01M PBS buffer for 10min each, and then dehydrated with 20%, 40%, 60%, 80%, 100% methanol solutions, respectively, at 4 ℃ for 20min each. The sample is placed in a mixed solution of 5% H2O 2/20% DMSO/CH3OH and bleached for 12H at 4 ℃. The samples were then reconstituted using 100%, 80%, 60%, 40%, 20% methanol solution and 0.01M PBS buffer in sequence, 20min each time. Immunostaining: the pre-treated samples were placed in PBS/0.2% Triton X-100/20% DMSO/0.3M glycine solution for 1d, then the samples were transferred into PBS/0.2% Triton X-100/10% DMSO/6% sheep serum for 1d, after which the samples were placed in PBS/0.2% Tween-20/10mg/ml heparin (PTwH) solution overnight. The next day, the samples were transferred to a solution of primary antibody in PTwH/5% DMSO/3% goat serum (primary antibody rabbit anti-GFP dilution ratio 1:300) and incubated on a shaker at 37 ℃ for 2 days. After the primary antibody incubation was complete, the samples were rinsed 5 times with PTWH solution on a 37 ℃ shaker for 1h each time overnight after completion of the last rinsing step. The next day the samples were transferred to a PTw H/3% sheep serum solution containing secondary antibodies and incubated on a shaker at 37 ℃ (secondary antibody to rabbit Alexa546 at a dilution ratio of 1:300) for 2 d. After the incubation with the secondary antibody, the samples were rinsed 5 times with PTWH solution for 1h and finally 3 times in PBS solution for 30min on a constant temperature shaker at 37 ℃.
(2) Protein anchoring: the fluorescently labeled mouse brain block was used to anchor proteins in biological tissues using MA-NHS, introducing double bonds into biological tissues for cross-linking with hydrogels. The anchoring solvent was 2- (N-morpholine) ethanesulfonic acid (MES) in water, containing 100mM MES and 150mM NaCl, and the anchoring compound concentration was 0.2mg/ml, and the anchoring was carried out in a horizontal shaker at 4 ℃ for 12 hours;
(3) hydrogel permeation: placing the anchored biological tissue in a super water-absorbing hydrogel solution for infiltration, placing the biological tissue in a horizontal shaking table at 4 ℃ for infiltration for 2 hours, replacing fresh penetrating fluid, and continuing the infiltration for 2 hours;
(4) hydrogel polymerization: the infiltrated biological sample is placed in the polymerization tank shown in figure 2 again, new super-absorbent hydrogel solution is added, the top of the polymerization tank is covered with a cover glass to prevent air bubbles from entering, then the covered polymerization tank is placed in a wet box containing water, and the wet box is placed in an oven and polymerized for 2 hours at 37 ℃.
(5) And (3) protease K digestion: the polymerized biological sample is placed in proteinase K digestion solution for digestion, wherein the digestion solution buffer solution is a mixed solution of 50mM Tris (pH 8),1mM EDTA, 0.5% Triton X-100 and 1M NaCl, the concentration of the proteinase K is 8units/mL, and the digestion solution is placed in a 37 ℃ air bath oscillator for digestion for 24 hours.
(6) Dialysis expansion in deionized water: placing the digested biological tissue in deionized water for dialysis expansion, changing the deionized water every 1 hour, and repeating for 3-5 times until the biological tissue is expanded to the maximum volume;
(7) three-dimensional cutting imaging: imaging the expanded biological sample obtained in the step (5) in a fluorescence microscope with an automatic cutting function, imaging the surface layer, mechanically cutting off the imaged surface layer to expose a new surface layer, and exciting to perform fluorescence imaging;
(8) and (4) repeating the process of the step (6), and repeating tomography in such a way until a three-dimensional image of the whole sample is obtained.
The samples obtained in examples 1 to 5 above were tested as follows:
in example 1, Table 1 shows the swelling factor and hardness test for sample No. 1 to 6, wherein the swelling factor is obtained by trimming a gel specimen before swelling to a cross section of 1cm2The section is accurately measured by a vernier caliperAfter the side length of the pipe is placed in deionized water to be expanded, the side length of the pipe is accurately measured by using a vernier caliper, and the ratio of the expanded side length to the side length before expansion is an expansion factor; the gel strength after swelling was measured by using an electronic dynamic and static fatigue tester, and the hardness was represented by the obtained elastic modulus.
Table 1 summary of test results of superabsorbent materials superabsorbent polymers, exemplified by AMPS, on swelling factor and hardness parameters at different compounding ratios
It can be seen from Table 1 that when the mass ratio of the superabsorbent compound, the monomer, the crosslinking agent, the initiator, and the accelerator satisfies (10-15): (10-20): (0.5-2): (0.5-1): (0.5-1), the expansion factors are all more than or equal to 4, and the hardness is not less than 15 KPa.
Examples 2 and 3 after the sample expansion was completed, the sample was sliced using a vibrating microtome, cut into 50 μm thick slices, mounted with a glass slide and a cover slip, and finally subjected to image acquisition under an inverted fluorescence confocal microscope (ZISS, 710).
Examples 4 and 5 three-dimensional data were acquired by performing ablation imaging using an auto-ablation fluorescence microscope after sample expansion was completed.
FIG. 2 is a schematic 3D printing diagram of a super-absorbent hydrogel polymerization tank for thick biological tissues, and the polymerization tank designed by the invention can be suitable for polymerization of sample tissues with any size and any volume by changing the arrangement of XYZ directions.
FIG. 3 is a photograph showing the swollen tissue and hydrogel crosslinked polymer of example 1, wherein the swollen tissue is completely transparent, and the black grid on the background paper is clearly seen (left panel), and the swollen tissue and hydrogel crosslinked polymer still maintains enough rigidity to be supported by hand and not deformed (right panel).
FIG. 4 is a comparison of the transgenic fluorescent protein GFP mouse brain slice before and after expansion in example 2. In fig. 4, both the graph a and the graph B are the comparison results before and after expansion, and the graph B is the enlargement of the out position of the corresponding white frame in the graph a, so that the resolution ratio after expansion is obviously improved compared with that before expansion, and the detail of the dendritic structure after expansion is clearly distinguished through the comparison of the detail. Graph C in fig. 4 is the calculation of the expansion factor, and the actual distance (indicated by short lines C and d in graph C) at the same position before and after the expansion shown in graph a in fig. 4 is measured, and the ratio of the distance after the expansion (56.27 μm) to the distance before the expansion (12.45 μm) is obtained, that is, the expansion factor is 4.52. Fig. 4 is a diagram D of image registration before and after dilation, and it can be seen after registration that the images before and after dilation can be basically completely overlapped, which shows that the image information after dilation has less disturbance and higher fidelity.
FIG. 5 is a comparison of the immunohistochemical fluorescent dye Alexa 546-labeled mouse brain slice before and after expansion in example 3. In the same way as in the representation method of fig. 4, both the graphs a and B in fig. 5 are the comparison results before and after expansion, and the graph B is the enlargement of the corresponding white frame out position in the graph a. Graph C in fig. 5 is the calculation of the expansion factor, and the actual distance (indicated by short lines C and d in graph C) at the same position before and after the expansion shown in graph a in fig. 5 is measured, and the ratio of the distance after the expansion (70.11 μm) to the distance before the expansion (14.94 μm) is obtained, that is, the expansion factor is 4.69. Fig. 5, D, is the registration of the images before and after expansion at the out-of-frame position in the white frame in fig. a, and it can be seen after the registration that the images before and after expansion can be basically completely overlapped, which indicates that the image information after expansion has less disturbance and higher fidelity.
FIGS. 6 and 7 are the results of three-dimensional imaging of brain masses of transgenic GFP mice and immunohistochemical mice, respectively, using an autoreactive fluorescence microscope to image, which shows that the superabsorbent polymer of the present invention has suitable mechanical strength and is fully suitable for ablation imaging. The invention can realize the super-resolution imaging of thick and large-volume tissues, and is a great breakthrough in the limitation of various super-resolution technologies to the thickness imaging.
The invention can directly adopt an automatic cutting imaging system (such as an oblique optical sheet mechanical cutting system) based on optical sheet illumination in the prior art, and can also directly adopt a soft-embedded automatic cutting imaging system (such as a sequence two-photon imaging system, and a corresponding cutting-imaging process which is a sequence cutting-imaging process is repeated for a plurality of times) in the prior art.
In addition to the super-absorbent hydrogel with specific components used in the above-mentioned examples, the tissue expansion cutting microscopic imaging method of the present invention may also use other expansive materials as long as the hardness of the expanded biological tissue is not less than 15KPa, and stable cutting can be achieved. It is believed that with the advancement of subsequent research, more and more intumescent materials will be available in the future for use in the present invention for use in the method of expansive ablation microscopy imaging.
It will be understood by those skilled in the art that the foregoing is only a preferred embodiment of the present invention, and is not intended to limit the invention, and that any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the scope of the present invention.
Claims (6)
1. A super-absorbent hydrogel suitable for an expansion cutting microscopic imaging method is characterized by comprising a super-absorbent compound, a monomer, a cross-linking agent, an initiator and an accelerator, wherein the mass ratio of the super-absorbent compound to the monomer to the cross-linking agent to the initiator to the accelerator satisfies (10-15): (10-20): (0.5-2): (0.5-1): (0.5-1);
in addition, every 100 parts of the super-absorbent hydrogel comprises 10-15 parts of super-absorbent compound, 10-20 parts of monomer, 0.5-2 parts of cross-linking agent, 0.5-1 part of initiator and 0.5-1 part of accelerator by weight, and the rest is deionized water;
the super water-absorbing compound is one or more of 2-acrylamido-2-methyl-1-propanesulfonic Acid (AMPS), itaconic acid, maleic acid, italian acid, vinyl alcohol and vinyl acetate;
the monomer is acrylamide or N, N-dimethylacrylamide;
the cross-linking agent is bisacrylamide;
the initiator is Ammonium Persulfate (APS) or potassium persulfate (KPS);
the accelerator is Tetramethylethylenediamine (TEMED).
2. The use of the superabsorbent hydrogel of claim 1 as a swelling agent in cutting microscopy of biological tissue for three-dimensional super-resolution imaging of biological tissue.
3. The application of claim 2, wherein the application is to apply a swelling substance to an initial biological tissue to be imaged, swell the initial biological tissue into a swollen tissue with hardness of not less than 15KPa under the action of the swelling substance, and then cut and microimage the swollen tissue in combination with mechanical cutting, thereby realizing three-dimensional swelling cutting microimaging of the biological tissue, acquiring three-dimensional super-resolution data and realizing three-dimensional super-resolution imaging.
4. Use according to claim 3, wherein said application of a swelling substance to the initial biological tissue to be imaged, under the effect of which the initial biological tissue is swelled into a swelled tissue having a hardness not lower than 15KPa, comprises the following steps:
(1) tissue anchoring: anchoring proteins in biological tissue using N-hydroxysuccinimide-methacrylate (MA-NHS) or 6-acrylamidoacetic acid succinate (AcX);
(2) hydrogel permeation: placing the anchored biological tissue obtained in the step (1) in a prepared super-absorbent hydrogel solution for infiltration;
(3) hydrogel polymerization: placing the biological sample obtained in the step (2) after permeation into a polymerization tank, adding a new super-absorbent hydrogel solution, sealing the surface of the polymerization tank, placing the polymerization tank into a wet box containing water, and placing the wet box into an oven for polymerization;
(4) and (3) protease K digestion: placing the polymerized biological sample obtained in the step (3) into proteinase K digestion solution for digestion;
(5) dialysis expansion in deionized water: and (5) putting the digested biological tissue obtained in the step (4) into deionized water for dialysis, changing the deionized water every 1h, and repeating for 3-5 times until the volume of the sample is expanded to the maximum.
5. Use according to claim 3, wherein the ablation and microscopic imaging of the dilated tissue in combination with mechanical ablation comprises the steps of:
(a) taking the surface layer of the expanded tissue as a first imaging layer, carrying out excitation imaging under a fluorescence microscope to obtain an image of the first layer, and then cutting off the first imaging layer to obtain the once-cut expanded tissue;
(b) taking the surface layer of the primarily cut expanded tissue obtained in the step (a) as a second imaging layer, carrying out excitation imaging under a fluorescence microscope to obtain an image of the second layer, and then cutting off the second imaging layer to obtain a secondarily cut expanded tissue;
(c) and repeating the processes of fluorescence excitation imaging and cutting, sequentially obtaining the imaging of a third layer, a fourth layer and more layers, thereby obtaining a series of two-dimensional images of the expansive tissue, and then superposing the two-dimensional images to realize the three-dimensional super-resolution imaging of the whole expansive tissue.
6. Use according to claim 3, wherein the cutting and microscopic imaging of the dilated tissue in combination with mechanical cutting is carried out in particular by means of an auto-cutting fluorescence microscope.
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