Preparation method of biological tissue patch

Abstract

The invention discloses a preparation method of a biological tissue patch. The preparation method comprises the following steps: providing a biological tissue embedding block; carrying out cutting and trimming on the embedding block; fitting the cross section of the trimmed embedding block on a preheated dielectric plate, and then freezing the embedding block adhered with the dielectric plate at low temperature; and separating the dielectric plate and the embedding block, naturally drying, and preserving at low temperature to obtain the biological tissue patch. The preparation method can be used for preparing a semi-thin tissue patch sample with a single-layer fluorescence signal, can be used for subsequent fixation and immunolabeling, particularly can obtain effective 3D imaging data in super-resolution imaging, is simple to operate and good in repeatability, has a good sample preparation effect, and can effectively solve the sample preparation problem of super-resolution tissue fluorescence imaging.

Description

Technical Field

[0001] The invention belongs to the technical field of biological sample preparation, and in particular relates to a method for preparing a biological tissue patch. Background Art

[0002] In the biological field, super-resolution imaging technology is widely used. It can break through the diffraction limit of traditional optical microscopes and achieve nanometer-level resolution, so as to observe the subtle structure and dynamic changes inside cells. This makes super-resolution imaging technology play an important role in organelle imaging, rare disease diagnosis, cancer research and diagnosis, gene editing and cell dynamics research, live cell imaging and biomolecule tracking, and drug screening. Although super-resolution imaging technology can realize the imaging of thinner samples such as cells, it is still difficult to achieve histological research that is closer to physiology and pathology, especially MINFLUX super-resolution technology. At present, MINFLUX super-resolution microscopy imaging technology can achieve a spatial resolution of 1-3 nanometers, but this technology has certain limitations and requirements on the labeling and thickness of the sample: (1) Imaging depth: Although MINFLUX technology has a high resolution, its imaging depth is still subject to certain limitations; for thicker samples, the light propagation path inside the sample becomes longer, the light scattering and absorption effects are aggravated, and the fluorescence background increases, resulting in a decrease in imaging quality; therefore, MINFLUX technology is more suitable for thin layer samples or samples that have been specially treated (such as DNA origami, cell samples, and ultra-thin slice samples). (2) Signal attenuation: As the sample thickness increases, the fluorescence signal will gradually attenuate during the propagation process; this may result in weaker fluorescence signals detected in the deep area of ​​the sample, thereby affecting the sensitivity and resolution of the imaging. (3) Imaging time: For thicker samples, the imaging time of MINFLUX technology may increase significantly, increasing the complexity and time cost of imaging.

[0003] At present, conventional tissue sectioning techniques mainly include frozen sections, paraffin sections, and ultrathin sections. Frozen sections are a common method for tissue immunofluorescence staining. The tissue is frozen at a certain hardness at low temperature and then frozen at a constant temperature. This method can preserve the cell membrane surface and the immune activity of multiple enzymes in the cell and the antigen, which is beneficial to subsequent immunofluorescence staining and fluorescence microscopy. However, the thickness of the sample section is usually greater than 5μm. Especially for tissues with poor hardness, such as brain tissue, it is very difficult to slice below 5μm. Paraffin sections are mainly embedded in paraffin, which can maintain the high hardness of the sample and perform continuous thin sections. However, during the sample preparation process, a series of organic solvent treatments will lose a lot of antigen activity, which will have a great impact on subsequent immunofluorescence labeling and cannot be applied to MINFLUX super-resolution microscopy. Semi-thin (0.5~3μm) or ultra-thin (50~300nm) sections can reach a thickness of 50nm and are mainly used for electron microscopy observation. Their operation is complicated, the cost is high, and they cannot be combined with immunofluorescence labeling.

[0004] Due to the above reasons, super-resolution imaging technology, especially MINFLUX resolution technology, cannot be applied in biological tissue samples. Summary of the invention

[0005] In view of this, the primary purpose of the present invention is to provide a method for preparing a biological tissue patch, which is a heat-assisted semi-thin tissue patch that can effectively retain tissue samples, and the sample thickness is between 0.5 and 2 μm, which can be used for subsequent fixation and immunolabeling, and can be applied to optical imaging, especially to obtain effective 3D imaging data in super-resolution imaging, thereby solving the problem of sample preparation of biological tissue samples in super-resolution imaging.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions: One aspect of the present invention discloses a method for preparing a biological tissue patch, comprising the following steps: Provide embedded blocks of biological tissues; cutting and trimming the embedded block; The trimmed embedded block cross section is attached to a preheated medium plate, and then the embedded block attached to the medium plate is cryogenically frozen; The medium plate and the embedding block are separated, naturally dried and then stored at low temperature to obtain a biological tissue patch.

[0007] The preparation method of the present invention cuts and trims the tissue embedding block, and then uses heat assistance to fit the cross section of the embedding block to the preheated medium plate, so that the sample is quickly dehydrated and the affinity between the sample and the medium plate is changed, thereby effectively retaining the biological tissue sample on the medium plate and the sample thickness is 0.5-2μm, and effective 3D imaging data can be obtained in optical imaging, especially super-resolution imaging.

[0008] Another aspect of the present invention provides the use of the biological tissue patch prepared by the preparation method as described above in optical imaging.

[0009] The biological tissue patch prepared in the present invention is a semi-thin tissue patch (thickness is 0.5-2 μm) and can be used for subsequent fixation and immunolabeling, so it can be applied to optical imaging, especially super-resolution imaging, to solve the problem of sample preparation of biological tissue samples in super-resolution imaging.

[0010] Beneficial effects of the present invention: The preparation method of the biological tissue patch provided by the present invention is a heat-assisted semi-thin tissue patch method, which can be obtained by using a conventional freezing slicer and a constant temperature heating device, is simple to operate, has a good sample preparation effect, and has good repeatability. Specifically, in the present invention, the embedded block is cut and trimmed and then bonded to the preheated medium plate, the sample is quickly dehydrated by heating, and the affinity between the sample and the cover glass is changed, so that the tissue sample is effectively retained on the medium plate, and the prepared tissue patch has a thickness of 0.5~2μm, and can be used for subsequent fixation and immunolabeling, and can be applied to optical imaging, especially in super-resolution imaging such as STORM and MINFLUX to obtain effective 3D imaging data.

[0011] In general, this simple and easy-to-operate heat-assisted semi-thin tissue patch sample preparation method can effectively solve the sample preparation problem of super-resolution tissue fluorescence imaging. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 The figure is a schematic diagram of the preparation process of a mouse brain semi-thin tissue patch in a preferred embodiment of the present invention.

[0013] Figure 2 This is the scanning electron microscopy staining of the mouse brain semi-thin tissue patch sample in the embodiment ( Figure 2 a) and the local magnification results of imaging ( Figure 2 b).

[0014] Figure 3 This is a graph showing the synaptic protein PSD-95 and RIM confocal XZ plane imaging results of the mouse brain semi-thin tissue patch sample in the example.

[0015] Figure 4 The STORM imaging results of the semi-thin tissue patch sample (HATS) in the embodiment and the slice sample (Slice) in the comparative example; wherein, Figure 4 a is the STORM imaging image of the sample; Figure 4 b is the gray value distribution of STORM imaging; Figure 4 c is the statistical diagram of the gray value variance of the sample imaging frame.

[0016] Figure 5 The distribution results of the postsynaptic protein PSD-95 in the STORM imaging of the semi-thin tissue patch sample in the embodiment and the slice sample in the comparative example; wherein, Figure 5 a is a schematic diagram of the alignment of presynaptic protein RIM and postsynaptic protein PSD-95 nanoclusters to form transsynaptic nanocolumns; Figure 5 b is an example of the nanoscale distribution of synaptic protein PSD-95 imaged by STORM imaging of a semi-thin tissue patch sample in the embodiment, in which the red area is a local high-density area; Figure 5 c is the transsynaptic alignment of RIM and PSD-95 protein in the semi-thin tissue patch sample in the embodiment and the slice sample in the comparative example; Figure 5 d is the average volume of PSD-95 synapses in the semi-thin tissue patch sample in the embodiment and the slice sample in the comparative example; Figure 5 e is the statistical result of the number of PSD-95 nanoclusters in the semi-thin tissue patch sample in the embodiment and the slice sample in the comparative example; Figure 5 f is the statistical result of the PSD-95 nanocluster volume of the semi-thin tissue patch sample in the embodiment and the slice sample in the comparative example.

[0017] Figure 6 The characterization results of synaptic protein PSD-95 in the semi-thin tissue patch sample in the embodiment, wherein: Figure 6 a is the synaptic protein PSD-95 Confocal, Figure 6 b is the MINFLUX imaging result, Figure 6 c is the maximum projection image of single-molecule localization reconstruction in MINFLUX imaging. Figure 6 d is the minimum projection image of single-molecule localization reconstruction in MINFLUX imaging. DETAILED DESCRIPTION

[0018] Embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0019] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art of the present invention. The terms used herein in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. In addition, unless otherwise specified, methods that do not specifically record conditions or steps are conventional methods, and the reagents and materials used can be obtained from commercial sources.

[0020] The first aspect of the present invention provides a method for preparing a biological tissue patch, the main steps of which are as follows: S1. Provide embedding blocks of biological tissues The biological tissue described in the present invention is not limited to commonly used experimental animals such as mice, rabbits, sheep, monkeys or cattle. The specific biological tissue can be brain tissue, liver tissue, lung tissue, kidney tissue, heart tissue or nerve tissue, but it is not limited to this. Any biological tissue with optical imaging requirements in the art can be used. Preferably, the biological tissue is brain tissue, which comes from vertebrates or some invertebrates with clear brain structures. In some specific embodiments of the present invention, the biological tissue is mouse brain tissue.

[0021] It is understandable that obtaining biological tissue is a conventional means or method in the art and will not be elaborated in detail here.

[0022] In the present invention, the preparation of the biological tissue embedding block can be carried out in a manner known in the art, such as freezing the biological tissue at low temperature and then embedding it into a block using an OCT embedding agent. The specific operation can be, for example, placing the biological tissue block in an embedding box containing a sufficient amount of OCT embedding agent so that it completely covers the biological tissue, and then placing it in the freezing table of a freezing microtome to completely freeze the tissue into a block. However, it is not limited to this, and any method for preparing the embedding block in the art can be used, and those skilled in the art can select a specific method as needed. There is no particular limitation on the size of the specific embedding block, and the conventional size in the art can be used.

[0023] S2, cutting and trimming the embedded block In the present invention, the cutting and trimming is to fix the embedded block on a device (more commonly used is a frozen section sample holder) and then cut and trim it to obtain a suitable tissue section. The specific cross-sectional size is not particularly limited and can be adjusted according to actual imaging needs and the size of the medium plate. In the present invention, the preferred cross-sectional size is (1~2)×(1~2) cm. Preferably, the cut and trimmed cross-sectional size is a square, for example, it can be any size of 1cm×1cm, 1.5cm×1.5cm, 2cm×2cm, but it is not limited thereto.

[0024] S3, attach the trimmed embedded block to the preheated dielectric plate, and place the embedded block with the dielectric plate on the lower Warm Freezing In the present invention, the heat-treated medium plate is attached to the cross section of the embedding block, the sample is heated to rapidly dehydrate, and the affinity between the sample and the medium plate is changed by rapid heating and freezing, thereby effectively retaining the tissue sample on the medium plate.

[0025] In the present invention, the dielectric plate refers to a flat, transparent cover plate with a certain degree of adhesion to the tissue, preferably made of glass, such as a cover glass that is more common in the art, but not limited to this. Before bonding the embedding block, the dielectric plate is preheated in advance to achieve the purpose of bonding and heating at the same time. The preheating temperature should not be too high, which will damage the tissue; it should not be too low, which will not achieve the purpose of heating and dehydration. At the same time, the thermal conductivity of the dielectric plate needs to be considered. Therefore, it can be adjusted and optimized by known experimental methods. In some specific embodiments of the present invention, for mouse brain tissue, the dielectric plate is a cover glass, and its preheating temperature is 45°C~55°C, for example, it can be any temperature among 45°C, 48°C, 50°C, 52°C, and 55°C.

[0026] Furthermore, the bonding time between the embedding block and the dielectric plate should not be too long or too short, and can be selected according to needs. In some embodiments of the present invention, the bonding time is 3 to 5 seconds.

[0027] After lamination, the embedding block with the medium plate is frozen at low temperature. In some specific embodiments of the present invention, the low temperature freezing is performed at -23°C to -17°C for 5 to 10 seconds.

[0028] It is understandable that for different tissues of the same organism, or the same tissues of different organisms, the specific processing temperature and time will be different based on the difference in tissue properties. The effect of adjusting the thickness and integrity of the tissue can be achieved by designing the fitting time between the embedding block cross section and the preheating medium plate and the low-temperature freezing time. This can be determined by experiments for those skilled in the art, so there is no special limitation.

[0029] S4. Separate the medium plate and embedding block, dry naturally and store at low temperature to obtain a biological tissue patch In the present invention, the separation of the medium plate and the embedding block must ensure that the tissue integrity is maintained as much as possible during the entire separation process. Therefore, preferably, after warming up at room temperature (23-30° C.) for 20-40 seconds, the medium plate is vertically peeled off to leave a biological tissue slice on the medium plate.

[0030] Finally, the biological tissue is naturally dried (such as air-dried) and then stored at a low temperature (depending on the biological tissue, generally -80°C or -20°C), to obtain a biological tissue patch with a thickness of 0.5μm~2μm.

[0031] In addition, it can be understood that the preparation of the biological tissue patch in the present invention also includes the steps of patch fixation and labeling.

[0032] Among them, patch fixation is a conventional biological tissue fixation method in the art, such as common chemical fixation, that is, fixation using a fixative, such as paraformaldehyde, etc. In some specific embodiments of the present invention, PFA with a concentration of 4% is used for fixation.

[0033] The labeling process is performed according to different specific optical imaging techniques. For example, in some specific embodiments of the present invention, the labeling process specifically refers to immune labeling. Since it is a conventional method in the art, it will not be described in detail here.

[0034] The second aspect of the present invention provides the use of the biological tissue patch prepared by the preparation method described in the first aspect of the present invention in optical imaging.

[0035] The biological tissue patch prepared by the method of the present invention can effectively retain tissue samples, and the prepared tissue patch has a thickness of 0.5-2 μm, which can be used for subsequent fixation and labeling, and can meet most optical imaging requirements in this field, especially optical imaging based on fluorescent labeling. The typical application field is super-resolution imaging, and specific examples include but are not limited to STED super-resolution imaging, STORM super-resolution imaging or MINFLUX super-resolution imaging. It can effectively obtain 3D imaging data and solve the sample preparation problem of super-resolution tissue fluorescence imaging.

[0036] The following uses the brain tissue of mice as an example to further specifically illustrate the technical solution of the present invention.

[0037] Example This embodiment provides a method for preparing a semi-thin tissue patch sample of mouse brain tissue for super-resolution fluorescence imaging. The schematic diagram of the process is shown in Figure 1 , the specific steps are as follows: (1) Take fresh mouse brain tissue and freeze it quickly at low temperature; (2) Embed the quick-frozen brain tissue into blocks using OCT at low temperature; (3) Fixing the embedded block obtained in step (2) on a conventional frozen section specimen holder and trimming the block, and trimming the cross section of the embedded block to a size of 1.5×1.5 cm; (4) Preheat the coverslip at 50°C and place the trimmed tissue block with the cross section facing downward on the coverslip for 3–5 seconds. (5) Then place the entire embedded block with the coverslip at -20°C for 5 to 10 seconds; (6) After returning to room temperature for 20 to 40 seconds, remove the coverslip vertically, let it dry and store it at low temperature. The semi-thin brain tissue sample patch is completed.

[0038] The prepared semi-thin brain tissue sample patches were fixed with 4% PFA and processed by conventional super-resolution fluorescence microscopy sample labeling, and were used for STED, STORM, and MINFLUX super-resolution imaging, respectively.

[0039] Comparative Example This comparative example provides a method for preparing a conventional brain tissue frozen section, which is different from the example in that the embedding block is not subjected to heat-assisted treatment.

[0040] The preparation method of conventional brain tissue frozen sections in this comparative example comprises the following specific steps: (1) Take fresh rat brain tissue and freeze it quickly at low temperature; (2) Quickly frozen brain tissue was embedded in OCT at low temperature into blocks; (3) After the embedded block is fixed on a regular frozen section specimen holder, it is trimmed and sliced ​​at a thickness of 5 μm. The slice is attached to a cover slip, dried, and stored at low temperature to complete the slicing.

[0041] The obtained brain tissue cryosections were fixed with 4% PFA and labeled with conventional super-resolution fluorescence microscopy samples for STORM or MINFLUX super-resolution imaging, respectively.

[0042] Results and Analysis Figure 2 The scanning electron microscopy results of the semi-thin tissue patch of mouse brain prepared in the example show that some typical structures in the tissue cells can be clearly seen, including mitochondria, synaptic vesicles, nerve myelin sheaths and postsynaptic density, etc., indicating that the preparation method of the present invention can preserve the structure of the tissue sample more completely.

[0043] Figure 3 The confocal XZ plane imaging result diagram of the synaptic protein PSD-95 and RIM of the mouse brain semi-thin tissue patch in the embodiment shows that the brain tissue patch obtained by the method in the embodiment was subsequently successfully subjected to immunofluorescence staining of synaptic protein PSD-95 and RIM, and the effective thickness of the tissue patch in the Z axis was measured in the confocal imaging, which was about 0.5~2μm, and was a semi-thin sample with a single layer of fluorescence signal.

[0044] pass Figure 4 It can be seen from the results that after the brain tissue patch was obtained by the method of this embodiment and the conventional 5 μm frozen section in the comparative example, the synaptic protein PSD-95 was labeled and STORM imaging was performed. It can be seen that the immunofluorescence background obtained by the method of the present invention is smaller than the result of the conventional 5 μm frozen section in the comparative example, indicating that the patch sample prepared by the preparation method of the present invention has a higher signal-to-background ratio (SBR) in STORM super-resolution imaging.

[0045] Figure 5 For the STORM super-resolution imaging in the embodiment, the sample preparation method in the embodiment is compared with the synaptic protein 3D structure results prepared by the conventional 5 μm frozen section method. It is known to those skilled in the art that the presynaptic protein RIM and the postsynaptic protein PSD-95 nanoclusters are aligned to form transsynaptic nanopillars ( Figure 5 a), by observing the transsynaptic nanopillars and the nanoscale distribution of the synaptic protein PSD-95, it can be determined that the sample preparation method of the present invention has no significant difference in the alignment degree, number, volume and synaptic volume of the pre- and post-synaptic protein nanoclusters in the sample compared with the 5 μm frozen section in the comparative example ( Figure 5 b- Figure 5 f). This indicates that the patch sample prepared by the preparation method of the present invention retains the original nano-level fine distribution.

[0046] Figure 6 The MINFLUX super-resolution imaging results of the brain tissue PSD-95 synaptic protein obtained in the example show that the sample prepared by the method of the present invention has a lower fluorescence background and can be applied to MINFLUX super-resolution imaging, which can perform MINFLUX imaging of tissue samples that was previously difficult to carry out.

[0047] It should be noted that, for the preparation of brain tissue samples, when the experimental process meets the following conditions, the purpose of the present invention can be achieved: The cross-sectional size of the embedding block may be, for example, any of 1 cm×1 cm, 1.5 cm×1.5 cm, and 2 cm×2 cm.

[0048] The preheating temperature of the cover glass is between 45°C and 55°C, for example, it can be any temperature among 45°C, 48°C, 50°C, 52°C, and 55°C.

[0049] The low temperature freezing treatment parameters for the embedded block with the medium plate can be -23°C to -17°C for 5 to 10 seconds, for example, it can be -23°C, -20°C, -18°C, or -17°C.

[0050] The room temperature return time may be 20 to 40 seconds, for example, 20 seconds, 25 seconds, 30 seconds, 32 seconds, 35 seconds, 40 seconds, etc.

[0051] Those skilled in the art can make appropriate selections of the above process parameters according to actual needs, and all of them can achieve the purpose of the present invention.

[0052] In addition, other tissues can also be prepared by the biological tissue patch preparation method of the present invention, so as to achieve the purpose of super-resolution imaging. In addition, the biological tissue patch prepared by the preparation method of the present invention is not limited to super-resolution imaging, but can be generally applied to most optical imaging based on fluorescent labeling. The specific experimental process conditions can be determined by known methods or experiments, and will not be described one by one here.

[0053] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0054] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A method for preparing a biological tissue patch, characterized in that: The following steps are involved: Provide embedding blocks of biological tissues; cutting and trimming the embedded block; The trimmed embedded block cross section is attached to a preheated medium plate, and then the embedded block attached to the medium plate is cryogenically frozen; The medium plate and the embedding block are separated, and then naturally dried and stored at low temperature to obtain a biological tissue patch; Preferably, the biological tissue patch has a thickness of 0.5 μm to 2 μm.

2. The preparation method according to claim 1, characterized in that The embedding block is formed by freezing the biological tissue at low temperature and then embedding it in an OCT embedding agent.

3. The preparation method according to claim 1, characterized in that: The biological tissue is brain tissue.

4. The preparation method according to claim 1, characterized in that: The cross-sectional size of the embedded block after cutting and trimming is (1-2)×(1-2) cm; Preferably, the cutting and trimming is achieved by fixing the embedding block on a frozen section sample tray.

5. The preparation method according to claim 1, characterized in that: The medium plate is a cover glass, and the preheating temperature is 45° C. to 55° C.

6. The preparation method according to claim 1, characterized in that: The low temperature freezing is carried out at -23°C to -17°C for 5 to 10 seconds.

7. The preparation method according to claim 1, characterized in that: The separation of the medium plate and the embedding block comprises the following steps: After the cryogenically frozen embedded block with the medium plate attached is warmed up to 23-30°C for 20-40 seconds, the medium plate is peeled off vertically.

8. The preparation method according to claim 1, characterized in that: The preparation method also includes the steps of patch fixing and marking treatment; Preferably, the patch is fixed with PFA having a mass fraction of 4%; Preferably, the marker is an immune marker.

9. Application of the biological tissue patch prepared by the preparation method according to any one of claims 1 to 8 in optical imaging.

10. The use according to claim 9, characterized in that The optical imaging is super-resolution imaging; Preferably, the super-resolution imaging is STED super-resolution imaging, STORM super-resolution imaging or MINFLUX super-resolution imaging.

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