Method for improving h&e-stained image quality of spatio-temporal chip, reagent combination, kit, and device

Abstract

The present invention relates to the technical field of biology, and in particular to a method for improving the H&E-stained image quality of a spatio-temporal chip, a reagent combination, a kit, and a device. The present invention provides a new mounting medium, which uses a simple reagent and corresponding process design, achieving the same effects of mRNA in-situ capture and comprehensive enhancement of mRNA diffusion as a glycerol mounting medium, and effectively solving the problems of fading and color diffusion after tissue staining. Registration of an H&E image and a spatio-temporal expression matrix of a BGI Stereo-seq chip provides possibility for high-precision subcellular-level omics analysis.

Description

Method, reagent combination, kit and device for improving the quality of H&E staining images on spatiotemporal chips Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a method, a reagent combination, a kit and a device for improving the quality of H&E staining images of spatiotemporal chips. Background Art

[0002] Spatial transcriptomics is a technology dedicated to quantifying transcriptional information corresponding to spatial locations. It provides detailed spatial information beyond traditional transcriptome sequencing, helping researchers identify the location of transcripts within tissues at cellular levels or even higher resolution. This will facilitate the understanding and interpretation of both individual cells and cell populations within entire tissues.

[0003] H&E (hematoxylin-eosin staining) is a relatively mature experimental technique with a wide range of applications. It can clarify the structure, properties, distribution and quantity of cells and tissues, and plays an important role in tumor pathological diagnosis, pathogenesis research and treatment plan formulation.

[0004] ssDNA staining or DAPI staining are currently the conventional staining protocols for spatiotemporal transcriptomics. By capturing fluorescent images of stained tissue cell nuclei and combining them with the spatiotemporal transcriptomics expression matrix, single-cell omics analysis can be achieved. Compared to spatiotemporal transcriptomics combined with conventional staining protocols, H&E staining on spatiotemporal microarrays can help us observe tissue morphology more clearly, assisting in more accurate determination of tissue typing and obtaining expression information of specific tissue regions in pathological research and applications, allowing for downstream differential analysis and enrichment analysis of selected regions.

[0005] The conventional H&E staining process can be simplified into three steps: hematoxylin staining, blueing, and eosin staining. However, H&E staining of tissues on spatiotemporal transcriptome chips requires consideration of both H&E image quality and spatiotemporal transcriptome capture.

[0006] The current H&E staining process for spatiotemporal transcriptomics involves washing with 5× SSC after hematoxylin and blue staining to remove excess dye, followed by gentle, slow drying. The tissue mounting medium used is 100% glycerol. However, even after washing with 5× SSC and drying, some salt residue remains, which can affect the visualization of microarray track lines under the microscope. While using glycerol as a mounting medium effectively addresses issues such as unclear ssDNA imaging, uneven capture, and RNA diffusion within tissues, it provides poor mounting results after H&E staining. Because the eosin used in H&E staining is readily soluble in water, while glycerol is highly hygroscopic, the eosin quickly dissolves due to contact with the aqueous phase after mounting, making it difficult to preserve images with good quality for extended periods. Furthermore, glycerol-mounted spatiotemporal microarrays also struggle to capture clear track lines under the microscope. Images without track lines cannot be registered with the spatiotemporal expression matrix, and manual registration accuracy is limited for subsequent single-cell delineation and cell bin analysis.

[0007] Using the current method, H&E images that meet the requirements of pathological diagnosis can be obtained, but there are also certain problems:

[0008] (1) The color of the stained tissue is dark, and the contrast between the nucleus and cytoplasm is not clear enough;

[0009] (2) Due to the strong water absorption of glycerol, eosin easily fades after sealing, making it difficult to maintain H&E images for a long time. This places high demands on experimental operation and photography time.

[0010] (3) It is impossible to stably capture the track lines on the chip and complete automatic image registration and subsequent analysis.

[0011] Summary of the Invention

[0012] In view of this, the present invention provides a method, a reagent combination, a kit and an apparatus for improving the quality of spatiotemporal chip H&E staining images.

[0013] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0014] The present invention provides the use of polyethylene glycol or a polyethylene glycol composition as a sealing agent and / or a low-salt solution as a cleaning agent in any of the following items:

[0015] (I), mounting slides after tissue staining; and / or

[0016] (II) Avoiding discoloration or blurred staining of cells or tissues after staining; and / or

[0017] (III) improving the image quality of the spatiotemporal chip; and / or

[0018] (IV) Improve the image clarity of the space-time chip track line; and / or

[0019] (V), increasing the nuclear-cytoplasmic ratio of stained cells; and / or

[0020] (VI), prolonging the preservation time of the stained image; and / or

[0021] (VII), cell typing or tissue typing; and / or

[0022] (VIII), cellbin expression analysis; and / or

[0023] (IX) Improving the registration accuracy of the stained image and the spatiotemporal expression matrix; and / or

[0024] (X) does not affect or improve spatiotemporal capture rate and / or coverage; and / or

[0025] (XI) improving the accuracy of obtaining mRNA and / or protein expression information;

[0026] The low salt solution includes but is not limited to 0.1×SSC.

[0027] In some specific embodiments of the present invention, the staining includes but is not limited to a combination of one or more of spatial omics H&E staining, spatial omics ssDNA staining, and DAPI staining.

[0028] In some specific embodiments of the present invention, the polyethylene glycol composition includes but is not limited to any of the following:

[0029] (I), a composition of polyethylene glycols of different molecular weights; and / or

[0030] (II), a combination of water and / or additives and polyethylene glycol; and / or

[0031] The additives include, but are not limited to, RNase inhibitors or glycerol.

[0032] In some specific embodiments of the present invention, the polyethylene glycol includes but is not limited to a combination of one or more of polyethylene glycol 400, polyethylene glycol 6000 or polyethylene glycol 8000.

[0033] The present invention also provides a sealing agent, comprising polyethylene glycol or a composition of polyethylene glycol;

[0034] The polyethylene glycol or polyethylene glycol composition is used as a sealing agent for any of the following items;

[0035] (I), mounting slides after tissue staining; and / or

[0036] (II) Avoiding discoloration or blurred staining of cells or tissues after staining; and / or

[0037] (III) improving the image quality of the spatiotemporal chip; and / or

[0038] (IV) Improve the image clarity of the space-time chip track line; and / or

[0039] (V), increasing the nuclear-cytoplasmic ratio of stained cells; and / or

[0040] (VI), prolonging the preservation time of the stained image; and / or

[0041] (VII), cell typing or tissue typing; and / or

[0042] (VIII), cellbin expression analysis; and / or

[0043] (IX) Improving the registration accuracy of the stained image and the spatiotemporal expression matrix; and / or

[0044] (X) does not affect or improve spatiotemporal capture rate and / or coverage; and / or

[0045] (XI) Improve the accuracy of obtaining mRNA and / or protein expression information.

[0046] In some specific embodiments of the present invention, the polyethylene glycol composition includes but is not limited to any of the following:

[0047] (I), a composition of polyethylene glycols of different molecular weights; and / or

[0048] (II), a combination of water and / or additives and polyethylene glycol; and / or

[0049] The additives include, but are not limited to, RNase inhibitors or glycerol.

[0050] In some specific embodiments of the present invention, the polyethylene glycol includes but is not limited to a combination of one or more of polyethylene glycol 400, polyethylene glycol 6000 aqueous solution or polyethylene glycol 8000 aqueous solution.

[0051] In some specific embodiments of the present invention, in g / mL, the mass volume ratio of polyethylene glycol 6000 to water in the polyethylene glycol 6000 aqueous solution includes but is not limited to 1:2; the mass volume ratio of polyethylene glycol 8000 to water includes but is not limited to 1:2.

[0052] In some specific embodiments of the present invention, the volume ratio of the polyethylene glycol 400, the polyethylene glycol 6000 aqueous solution and the polyethylene glycol 8000 aqueous solution includes but is not limited to 1:1:1.

[0053] The present invention also provides cleaning agents, including but not limited to low-salt solutions;

[0054] The low salt solution includes but is not limited to 0.1×SSC;

[0055] The low salt solution is used as a cleaning agent for any of the following items:

[0056] (I), mounting slides after tissue staining; and / or

[0057] (II) Avoiding discoloration or blurred staining of cells or tissues after staining; and / or

[0058] (III) improving the image quality of the spatiotemporal chip; and / or

[0059] (IV) Improve the image clarity of the space-time chip track line; and / or

[0060] (V), increasing the nuclear-cytoplasmic ratio of stained cells; and / or

[0061] (VI), prolonging the preservation time of the stained image; and / or

[0062] (VII), cell typing or tissue typing; and / or

[0063] (VIII), cellbin expression analysis; and / or

[0064] (IX) Improving the registration accuracy of the stained image and the spatiotemporal expression matrix; and / or

[0065] (X) does not affect or improve spatiotemporal capture rate and / or coverage; and / or

[0066] (XI) Improve the accuracy of obtaining mRNA and / or protein expression information.

[0067] In some specific embodiments of the present invention, the cleaning agent is used for any of the following:

[0068] (i) cleaning excess hematoxylin and / or bluing dye from the stained tissue; and / or

[0069] (ii) Using polyethylene glycol or a polyethylene glycol composition as a sealing medium to seal the slides, taking pictures, and then removing the sealing medium with the cleaning agent.

[0070] The present invention also provides a combination of reagents, including but not limited to:

[0071] the mounting medium; and

[0072] The cleaning agent.

[0073] The present invention also provides a kit, including but not limited to:

[0074] (I), the mounting medium; and / or

[0075] (II), the cleaning agent; and / or

[0076] (III), the reagent combination.

[0077] The present invention also provides the use of the reagent combination or the kit in any of the following items:

[0078] (I), mounting slides after tissue staining; and / or

[0079] (II) Avoiding discoloration or blurred staining of cells or tissues after staining; and / or

[0080] (III) improving the image quality of the spatiotemporal chip; and / or

[0081] (IV) Improve the image clarity of the space-time chip track line; and / or

[0082] (V), increasing the nuclear-cytoplasmic ratio of stained cells; and / or

[0083] (VI), prolonging the preservation time of the stained image; and / or

[0084] (VII), cell typing or tissue typing; and / or

[0085] (VIII), cellbin expression analysis; and / or

[0086] (IX) Improving the registration accuracy of the stained image and the spatiotemporal expression matrix; and / or

[0087] (X) does not affect or improve spatiotemporal capture rate and / or coverage; and / or

[0088] (XI) Improve the accuracy of obtaining mRNA and / or protein expression information.

[0089] The present invention also provides a method for processing tissue sections, which comprises contacting the stained tissue sections with the cleaning agent, the cleaning agent in the reagent combination, or the cleaning agent in the kit to clean excess hematoxylin and / or bluing dye on the stained tissue, drying the sections, and then sealing the sections with the sealing medium, the sealing medium in the reagent combination, or the sealing medium in the kit.

[0090] In some specific embodiments of the present invention, the cleaning agent is used for cleaning at least once, preferably once or three times.

[0091] In some specific embodiments of the present invention, the chip surface needs to be washed three times with a 0.1×SSC solution after hematoxylin staining, and needs to be washed once with a 0.1×SSC solution after blue staining.

[0092] In some specific embodiments of the present invention, the drying rate includes but is not limited to: 5 m / s to 10 m / s.

[0093] The present invention also provides tissue sections prepared by the processing method.

[0094] The present invention also provides a method for improving the image quality of the space-time chip and / or improving the image clarity of the track line of the space-time chip.

[0095] A tissue section is fixed on a probe chip, dried, fixed, stained, and the dye is discarded to obtain a stained tissue chip;

[0096] The cleaning agent, the cleaning agent in the reagent combination, or the cleaning agent in the kit is brought into contact with the stained tissue chip to clean excess hematoxylin and / or bluing dye on the stained tissue, which is then blown dry and then sealed with the sealing medium, the sealing medium in the reagent combination, or the sealing medium in the kit.

[0097] In some specific embodiments of the present invention, the cleaning agent is used for cleaning at least once, preferably once or three times.

[0098] In some specific embodiments of the present invention, the chip surface needs to be washed three times with a 0.1×SSC solution after hematoxylin staining, and needs to be washed once with a 0.1×SSC solution after blue staining.

[0099] In some specific embodiments of the present invention, the drying rate includes but is not limited to: 5 m / s to 10 m / s.

[0100] The present invention also provides a spatiotemporal chip prepared by the method.

[0101] The present invention also provides a method for improving the accuracy of obtaining mRNA and / or protein expression information, comprising the following steps:

[0102] Step 1: Take a tissue section and fix it on a chip with a probe, dry it, fix it, stain it, and discard the dye to obtain a stained tissue chip;

[0103] Step 2: contacting the stained tissue chip with the cleaning agent, the cleaning agent in the reagent combination, or the cleaning agent in the kit to wash away residual dye and air-dry;

[0104] Step 3: sealing the sample with the sealing medium, the sealing medium in the reagent combination, or the sealing medium in the kit, and taking a photo;

[0105] Step 4: removing the sealing medium using the cleaning agent, the cleaning agent in the reagent combination, or the cleaning agent in the kit, adjusting the pH value, permeabilizing, and reverse transcribing the mRNA to obtain cDNA;

[0106] Step 5: After further enzyme digestion, cDNA with positional probe information is obtained, library is constructed, and sequencing is performed to obtain mRNA and / or protein expression information.

[0107] In some specific embodiments of the present invention, the cleaning agent is used for cleaning at least once, preferably once or three times.

[0108] In some specific embodiments of the present invention, the chip surface needs to be washed three times with a 0.1×SSC solution after hematoxylin staining, and needs to be washed once with a 0.1×SSC solution after blue staining.

[0109] In some specific embodiments of the present invention, the drying rate includes but is not limited to: 5 m / s to 10 m / s.

[0110] In some specific embodiments of the present invention, the step 4 of removing the sealing medium is performed by soaking in 0.1×SSC for 3 to 5 seconds;

[0111] The pH value is adjusted in step 4 using a hydrochloric acid solution with a pH of 2.

[0112] The present invention also provides a method for capturing spatiotemporal transcriptomes, comprising the following steps:

[0113] Step 1: Take a tissue section and fix it on a chip with a probe, dry it, fix it, stain it, and discard the dye to obtain a stained tissue chip;

[0114] Step 2: contacting the stained tissue chip with the cleaning agent, the cleaning agent in the reagent combination, or the cleaning agent in the kit to wash away residual dye and air-dry;

[0115] Step 3: sealing the sample with the sealing medium, the sealing medium in the reagent combination, or the sealing medium in the kit, and taking a photo;

[0116] Step 4: removing the sealing medium using the cleaning agent, the cleaning agent in the reagent combination, or the cleaning agent in the kit, adjusting the pH value, permeabilizing, and reverse transcribing the mRNA to obtain cDNA;

[0117] Step 5: After further enzyme digestion, cDNA with position probe information is obtained, library is constructed, and sequencing is performed to obtain capture information.

[0118] In some specific embodiments of the present invention, the cleaning agent is used for cleaning at least once, preferably once or three times.

[0119] In some specific embodiments of the present invention, the chip surface needs to be washed three times with a 0.1×SSC solution after hematoxylin staining, and needs to be washed once with a 0.1×SSC solution after blue staining.

[0120] In some specific embodiments of the present invention, the drying rate includes but is not limited to: 5 m / s to 10 m / s.

[0121] In some specific embodiments of the present invention, the step 4 of removing the sealing medium is performed by soaking in 0.1×SSC for 3 to 5 seconds;

[0122] The pH value of step 4 is adjusted using a hydrochloric acid solution with a pH of 2.

[0123] The present invention also provides a device comprising the tissue slice or the spatiotemporal chip.

[0124] The present invention also provides a storage medium storing computer-readable instructions. When the computer-readable instructions are executed by one or more processors, the one or more processors execute the steps of the method for capturing spatiotemporal transcriptomes.

[0125] The present invention also provides a computer device comprising: one or more processors, and a memory; the memory stores computer-readable instructions, and when the computer-readable instructions are executed by the one or more processors, the steps of the method for capturing spatiotemporal transcriptomes are performed.

[0126] The present invention includes but is not limited to the following beneficial effects:

[0127] The sample processing method provided by the present invention is a sample processing method that can maintain the H&E staining state of tissue sections on the chip for a longer period of time in spatiotemporal omics and obtain relatively clear track line images under bright field illumination. Using polyethylene glycol (PEG400, PEG6000, PEG8000), a commonly used and low-cost industrial raw material, and through a simple process design, it systematically solves the problems of blurred H&E staining of cells and tissues, short maintenance time, and inability to achieve automatic alignment, thereby improving the registration accuracy of H&E images and spatiotemporal expression matrices. At the same time, using polyethylene glycol instead of glycerol does not affect subsequent spatiotemporal capture, and even improves the capture effect on some samples.

[0128] This method is low-cost, easy to operate, and has significant effects. It is applicable to various types of fresh and PFA samples and has broad application prospects. Considering the market demand for animal individual development and pathological diagnosis, it is expected to bring higher economic benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0129] Figure 1 shows the H&E+ transcriptome workflow;

[0130] Figure 2 shows a comparison of microarray and slide staining images using different mounting media; 1 - H&E staining of mouse stomach sections on glass slides; 2 - Photograph of microarray stained with polyethylene glycol 400; 3 - Photograph of microarray stained with glycerol mounting media;

[0131] Figure 3 shows a comparison of details of tissue regions and blank regions using different mounting media; 1 - H&E chip, photographic results of a tissue region mounted with polyethylene glycol 400; 2 - H&E chip, photographic results of a tissue region mounted with glycerol; 3 - H&E chip, photographic results of a blank region mounted with polyethylene glycol 400; 4 - H&E chip, photographic results of a blank region mounted with glycerol;

[0132] Figure 4 shows a comparison of chip staining images using different mounting media; 1 - a photograph of a mouse testis section stained with H&E on a chip and then sealed with polyethylene glycol 400; 2 - a photograph of a mouse testis section stained with H&E on a chip and then sealed with polyethylene glycol 8000; 3 - a photograph of a mouse testis section stained with H&E on a chip and then sealed with glycerol;

[0133] Figure 5 shows a comparison of details of the tissue edge and intra-tissue cavity regions using different mounting media; 1 - H&E chip, photographic results of the tissue edge using polyethylene glycol 400 mounting; 2 - H&E chip, photographic results of the tissue edge using glycerol mounting; 3 - H&E chip, photographic results of the intra-tissue cavity using polyethylene glycol 400 mounting; 4 - H&E chip, photographic results of the intra-tissue cavity using glycerol mounting;

[0134] Figure 6 shows a comparison of microarray staining images using different mounting media; 1 - Photograph of a coronal section of a mouse hemibrain stained on a microarray using H&E staining followed by mounting with polyethylene glycol 6000; 2 - Photograph of a coronal section of a mouse hemibrain stained on a microarray using glycerol mounting;

[0135] Figure 7 shows the comparison of multiple photographs after polyethylene glycol sealing;

[0136] Figure 8 shows the comparison of photographs taken on the first and third days of sealing with different sealing agents;

[0137] Figure 9 shows a comparison of spatiotemporal capture results for H&E staining processes using different mounting media; 1 - glycerol mounting, original cleaning process, bin 50; 2 - polyethylene glycol (PEG400, 100%) mounting, original cleaning process, bin 50; 3 - polyethylene glycol (PEG400, 100%) mounting, optimized cleaning process, bin 50;

[0138] Figure 10 shows a comparison of spatiotemporal capture results of the H&E staining process using different mounting media; among them, 1- glycerol mounting is visualized, Bin 50; 2- PEG400 mounting (polyethylene glycol 400) is visualized, Bin 50; 3- PEG8000 mounting (polyethylene glycol 8000) is visualized, Bin 50;

[0139] Figure 11 shows a comparison of chip staining images using different mounting media; 1 - H&E chip, H&E staining results using glycerol mounting media; 2 - H&E chip, H&E staining results using polyethylene glycol 400 mounting media; 3 - H&E chip, H&E staining results using polyethylene glycol 8000 mounting media;

[0140] Figure 12 shows a comparison of spatiotemporal capture results of the H&E staining process using different mounting media; 1-no mounting, Bin 50; 2-glycerol mounting for visualization, Bin 50; 3-PEG400 mounting (polyethylene glycol 400) for visualization, Bin 50; 4-PEG6000 mounting (polyethylene glycol 8000) for visualization, Bin 50;

[0141] Figure 13 shows a comparison of chip staining images using different mounting media; 1 - H&E staining image of a chip mounted with glycerol; 2 - H&E staining image of a chip mounted with PEG400; 3 - H&E staining image of a chip mounted with PEG6000;

[0142] FIG14 shows a comparison of H&E staining images of different mounting media; among them, H&E staining images of 1-glycerol mounting media; H&E staining images of 2-polyethylene glycol (mixed) mounting media;

[0143] Figure 15 shows a comparison of spatiotemporal capture results of H&E staining processes using different mounting media; among them, the spatiotemporal omics analysis results of 1-glycerol mounting; 2-polyethylene glycol (mixed) mounting, spatiotemporal omics analysis results of bin50;

[0144] FIG16 shows a comparison of the spatiotemporal capture results of the DAPI staining process using different mounting media; 1-glycerol mounting, bin 50; 2-polyethylene glycol (PEG400, 100%) mounting, bin 50;

[0145] FIG17 shows a comparison of spatiotemporal capture results of ssDNA staining processes using different mounting media; 1-glycerol mounting, bin 50; 2-polyethylene glycol (PEG400, 100%) mounting, bin 50;

[0146] Figure 18 shows a comparison of the clarity of the track lines; 1 - glycerol mounting, the effect of the original cleaning process; 2 - H&E mounting medium mounting, the effect of the optimized cleaning process; 3 - H&E image QC pass rate statistics (46 cases before optimization, 50 cases after optimization). DETAILED DESCRIPTION

[0147] The present invention discloses methods, reagent combinations, kits, and devices for improving the quality of spatiotemporal chip H&E staining images. Those skilled in the art can refer to the contents of this article and appropriately improve the process parameters to achieve the desired effect. It should be noted in particular that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of the present invention to implement and apply the technology of the present invention.

[0148] Compared to glycerol mounting, the polyethylene glycol (PEG400, PEG6000, PEG8000) mounting method used in this invention exhibits better performance and longer retention on H&E-stained fresh frozen-embedded tissue sections. Polyethylene glycol mounting effectively alleviates eosin bleed-out on tissues, enhancing the color vividness and nuclear-cytoplasmic contrast of H&E-stained images. For BGI spatiotemporal microarrays, polyethylene glycol mounting effectively improves the clarity of track lines under epi- or reflected-field illumination.

[0149] This study uses a BGI-produced imaging reagent, which includes polyethylene glycol, to effectively prevent eosin blurring after H&E staining, while maintaining spatial and temporal transcriptome capture. This reagent provides clearer tracking lines. Based on precisely registered H&E images, we can perform single-cell delineation and tissue region segmentation, enabling subsequent cell bin expression analysis.

[0150] The present invention relates to a novel method for processing protein chips with biological tissues attached, which uses a gas cylinder to quickly blow away the low-salt solution on the surface of the chip and the biological tissue slice to reduce the occlusion of the unmodified blank area on the chip by salt stains, improve the imaging quality of the track line on the protein chip, and realize the automatic alignment of the incident bright field image and the spatiotemporal expression matrix. At the same time, a type of polyethylene glycol with a certain water absorption effect is used to seal the stained tissue, effectively alleviating the blurring of the dye on the tissue due to contact with the aqueous phase, thereby obtaining an H&E image with a clear nuclear-cytoplasmic contrast that meets the needs of pathological diagnosis. Using this process, the quality inspection pass rate of the track line can be improved, so that the H&E image can be automatically aligned with the spatiotemporal expression matrix; compared with manual alignment, the alignment accuracy is improved, and mRNA clustering analysis can be well performed based on the H&E image to achieve accurate cell typing, assist in pathological research and application to more accurately judge tissue typing and obtain expression information of specific tissue areas, and perform downstream difference analysis and enrichment analysis on selected areas.

[0151] The characteristics of this process are that after a tissue section of a certain thickness is attached to a chip with a single-stranded DNA probe, the tissue is baked dry, fixed with an organic solvent, and stained with H&E. After discarding the hematoxylin and bluing reagent, the chip is cleaned with a low-salt solution, and the low-salt solution on the surface of the tissue and chip is quickly blown dry to minimize salt residue on the chip. Then, polyethylene glycol is dripped on the chip and a coverslip is applied. After the polyethylene glycol completely infiltrates the tissue, the chip can be photographed. (Procedure: Drop PEG onto the center of the tissue on the chip, then seal the chip using a standard coverslip. Liquid volume: 3.5 μL for a 1 cm x 1 cm chip; adjust the volume accordingly for chips of different sizes. PEG dilution ratios for different molecular weights: PEG 8000 (Rigaku brand) at 50% (w / v), and PEG 6000 powder diluted with NF·H2O at a 50% concentration.) The track line is clearly visible when photographing, and the chip can be left for extended periods, during which the dye on the tissue remains stable and does not fade. The PEG is then washed away with a low-salt solution. The chip is pre-washed with pH 2 hydrochloric acid to adjust its pH. Permeabilization reagent is then added to permeabilize the tissue and subsequently reverse transcribe the mRNA. After obtaining cDNA, the chip is digested with enzymes to obtain cDNA containing the positional probe information. This cDNA is then used for subsequent library construction, sequencing, and other procedures. Obtaining sequence information of mRNA with position information, and then analyzing the data to obtain in situ expression information of mRNA or protein, where:

[0152] 1) Use low-salt solution (0.1×SSC) to wash away excess hematoxylin and blue dye on the tissue. Use a gas cylinder to quickly blow away the low-salt solution on the chip surface to minimize salt residue on the chip.

[0153] 2) Dry the tissue on the chip as much as possible to avoid eosin dispersal when sealing the stained tissue;

[0154] 3) When photographing the stained tissues and cells on the chip, use polyethylene glycol to seal the slides and take photos;

[0155] 4) Using polyethylene glycol to mount the slides can help obtain H&E stained images that meet pathological judgment requirements and clearer tracking lines that meet registration requirements, helping with automatic registration and cell cutout.

[0156] 5) After taking the photo, briefly soak the sample in low-salt solution (0.1×SSC), pre-wash with 0.01N hydrochloric acid, and then permeabilize the sample.

[0157] 6) Through the design of the whole process, it helps to realize the automatic registration of H&E images and provide a data basis for subsequent cell bin analysis.

[0158] The specific process is shown in Figure 1.

[0159] The technical problems solved by the present invention are as follows:

[0160] 1) Solve the problem of eosin staining on tissues. The present invention uses polyethylene glycol to mount the slides and take photos to obtain H&E staining images. Under the protection of polyethylene glycol, the eosin staining on the tissue is less likely to spread.

[0161] 2) Compared with glycerol mounting, polyethylene glycol mounting can maintain tissue staining for a longer time, allowing the experimenter to take pictures multiple times, which increases the error tolerance of H&E image taking.

[0162] 3) Address the issue of unclear nuclear-cytoplasmic contrast after staining. Compared to glycerol mounting, H&E images obtained using polyethylene glycol 400 have clearer nuclear-cytoplasmic contrast and more vivid colors.

[0163] 4) Solve the problem of unclear track line imaging on the chip after sealing. A lower concentration (0.1×SSC) of salt residue can effectively improve the imaging clarity of the track line of the spatiotemporal chip; compared with using glycerol sealing, using polyethylene glycol 400 can obtain clearer track line imaging;

[0164] 5) Combining all the above improvements, the H&E image quality and track line quality inspection pass rate are greatly improved, and automatic registration of H&E images with spatiotemporal expression matrices is achieved, providing the possibility for subsequent tissue cell bin-level mRNA analysis.

[0165] The raw materials and reagents used in the method, reagent combination, kit, and device for improving the quality of H&E staining images of spatiotemporal chips provided by the present invention can all be purchased on the market.

[0166] Explanation of terms

[0167] Analyte can be broadly divided into one of two groups: nucleic acid analytes and non-nucleic acid analytes. Examples of non-nucleic acid analytes include, but are not limited to, lipids, carbohydrates, peptides, proteins, glycoproteins (N-connected or O-connected), lipoproteins, phosphoproteins, specific phosphorylation or acetylation variants of proteins, amidation variants of proteins, hydroxylation variants of proteins, methylation variants of proteins, ubiquitination variants of proteins, sulfated variants of proteins, viral proteins (e.g., viral capsids, viral envelopes, viral coats, viral auxiliary proteins, viral glycoproteins, viral spikes, etc.), extracellular and intracellular proteins, antibodies, and antigen binding fragments. In addition, analyte can be located in a subcellular position, including, for example, organelles, such as mitochondria, Golgi apparatus, endoplasmic reticulum, chloroplasts, endocytic vesicles, secretory vesicles, vacuoles, lysosomes, etc. Analyte can also be a peptide or protein, including but not limited to antibodies and enzymes. Analyte can also be detected indirectly, such as by detecting an intermediate, such as a connection product or an analyte capture agent (e.g., an oligonucleotide-conjugated antibody).

[0168] "Biological sample" is typically obtained from a subject to be analyzed using any of a variety of techniques, including but not limited to biopsy, surgery, and laser capture microscopy (LCM), and typically includes cells and / or other biological materials from the subject. In some embodiments, the biological sample can be a tissue section. In some embodiments, the biological sample can be a fixed and / or stained biological sample (e.g., a fixed and / or stained tissue section). Non-limiting examples of staining agents include histological stains (e.g., hematoxylin and / or eosin) and immunological stains (e.g., fluorescent stains). In some embodiments, the biological sample (e.g., a fixed and / or stained biological sample) can be imaged.

[0169] The biological sample can be permeabilized with one or more permeabilization agents. For example, permeabilization of the biological sample can facilitate analyte capture.

[0170] Array-based spatial analysis methods involve transferring one or more analytes from a biological sample to an array of features on a substrate, wherein each feature is associated with a unique spatial position on the array. Subsequent analysis of the transferred analyte includes determining the identity of the analyte and the spatial position of the analyte within the biological sample. The spatial position of the analyte within the biological sample is determined based on the features of the analyte combined (e.g., directly or indirectly) on the array and the relative spatial position of the feature within the array.

[0171] "Capture probe" refers to any molecule capable of capturing (directly or indirectly) and / or labeling an analyte (e.g., an analyte of interest) in a biological sample. In some embodiments, the capture probe is a nucleic acid or a polypeptide. The capture probe may include a cleavage domain and / or a functional domain (e.g., a primer binding site, such as for next generation sequencing (NGS)). The generation of the capture probe can be achieved by any suitable method, and the present invention is not limited thereto.

[0172] In some embodiments, detection of one or more analytes (eg, protein analytes) can be performed using one or more analyte-capture agents.

[0173] Spatial information can provide information of biological and / or medical importance. For example, the methods and compositions described herein can allow: identification of one or more biomarkers for a disease or condition (e.g., diagnostic biomarkers, prognostic biomarkers, and / or biomarkers for determining therapeutic efficacy); identification of candidate drug targets for treating a disease or condition; identification (e.g., diagnosis) of a subject as having a disease or condition; identification of the stage and / or prognosis of a subject's disease or condition; identification of a subject as having an increased likelihood of developing a disease or condition; monitoring the progression of a subject's disease or condition; determination of the efficacy of a treatment for a subject's disease or condition; identification of a subpopulation of patients for whom a treatment is effective for a disease or condition; improvement of treatment for a subject having a disease or condition; selection of subjects for participation in a clinical trial; and / or selection of a treatment for a subject having a disease or condition.

[0174] Spatial information can provide information of biological importance. For example, the methods and compositions described herein can allow: identification of transcriptome and / or proteome expression profiles (e.g., in healthy and / or diseased tissues); identification of multiple analyte types in close proximity (e.g., nearest neighbor analysis); determination of upregulated and / or downregulated genes and / or proteins in diseased tissues; characterization of the tumor microenvironment; characterization of tumor immune responses; characterization of cell types and their co-localization in tissues; and identification of genetic variants within tissues (e.g., based on gene and / or protein expression profiles associated with specific disease or condition biomarkers).

[0175] Typically, for methods based on spatial arrays, the substrate acts as a support for the direct or indirect attachment of capture probes to the features of the array. A "feature" is an entity that serves as a support or reservoir for the various molecular entities used in spatial analysis. In some embodiments, some or all of the features in the array are functionalized for analyte capture.

[0176] Typically, analytes and / or intermediaries (or portions thereof) can be captured when a biological sample is brought into contact with a substrate comprising capture probes (e.g., a substrate into which the capture probes are embedded, dotted, printed, fabricated, or having features (e.g., beads, wells) comprising capture probes). As used herein, "contact" or "contacted" or "contacting" a biological sample with a substrate refers to any contact (e.g., direct or indirect) that allows the capture probes to interact (e.g., covalently bind or non-covalently bind (e.g., hybridize)) with an analyte from the biological sample. Capture can be achieved actively (e.g., using electrophoresis) or passively (e.g., using diffusion).

[0177] During the analysis of spatial information, sequence information associated with the analyte is obtained, and this sequence information can be used to provide information about the spatial distribution of the analyte in the biological sample. Various methods can be used to obtain spatial information. In some embodiments, specific capture probes and the analytes they capture are associated with specific locations in an array of features on a substrate.

[0178] When sequence information about capture probes and / or analytes is obtained during the analysis of spatial information, the position of the capture probes and / or analytes can be determined by reference to the stored information that uniquely associates each space with an array feature position. In this way, a specific capture probe and the captured analyte are associated with a specific position in the array of features. Each array feature position represents a position relative to a coordinate reference point (e.g., array position, fiducial marker) of the array. Therefore, each feature position has an "address" or position in the coordinate space of the array.

[0179] In some embodiments, specialized hardware and / or software may be used to perform spatial analysis.

[0180] Suitable systems for carrying out spatial analysis may include components such as chambers (for example, flow cells or sealable, fluid-tight chambers) for accommodating biological samples. Biological samples may, for example, be installed in biological sample holders. One or more fluid chambers may be connected to chambers and / or sample holders via fluid conduits, and fluid may be delivered to chambers and / or sample holders via fluid pumps, vacuum sources or other devices coupled to fluid conduits, which generate pressure gradients to drive fluid streams. One or more valves may also be connected to fluid conduits to regulate the flow of reagent from a reservoir to chambers and / or sample holders.

[0181] The system may optionally include a control unit (which includes one or more electronic processors), an input interface, an output interface (such as a display) and a storage unit (e.g., a solid-state storage medium, such as but not limited to magnetic, optical or other solid-state, persistent, writable and / or rewritable storage medium). The control unit may optionally be connected to one or more remote devices via a network. The control unit (and its components) can generally perform any of the steps and functions described herein. In the case where the system is connected to a remote device, the remote device (or multiple remote devices) can perform any of the steps or features described herein. The system may optionally include one or more detectors (e.g., CCD, CMOS) for capturing images. The system may also optionally include one or more light sources (e.g., LED-based, diode-based, laser) for irradiating the sample, a substrate with features, an analyte from the biological sample captured on the substrate, and various control and calibration media.

[0182] The system may optionally include software instructions encoded in and / or implemented in one or more tangible storage media and hardware components such as application specific integrated circuits. When executed by a control unit (particularly an electronic processor) or an integrated circuit, the software instructions may cause the control unit, the integrated circuit, or other component executing the software instructions to perform any of the method steps or functions described herein.

[0183] In some cases, the systems described herein can detect (eg, register images of) biological samples on an array.

[0184] Prior to transferring the analyte from the biological sample to the array of features on the substrate, the biological sample can be aligned with the array. Alignment of the biological sample with the array of features comprising capture probes can facilitate spatial analysis that can be used to detect differences in the presence and / or levels of analytes at different locations in the biological sample.

[0185] In some cases, one or more fiducial markers can be used to align a map of the presence and / or levels of an analyte with an image of a biological sample, and the fiducial markers can be used as reference points or measurement scales for alignment (e.g., aligning a sample with an array, aligning two substrates, determining the position of a sample or array on a substrate relative to the fiducial marker) and / or for quantitative measurements of size and / or distance.

[0186] The present invention will be further described below in conjunction with the embodiments:

[0187] Example 1 Preparation of polyethylene glycol (PEG400 (100%)) mounted tissue

[0188] The experimental materials were prepared into tissue sections.

[0189] Tissue fixation: 10 μm thick tissue sections were attached to a chip with a single-stranded DNA probe (the chip was obtained from the Spatiotemporal Kit, specifically the Stereo-seq Transcriptome Reagent Set (Vector Edition), Specification: 4RXN, Catalog Number: 201ST114). The tissue was then oven-dried and fixed with an organic solvent.

[0190] H&E staining: Pre-cool 1 tube of 30 mL of anhydrous methanol (Sigma, 34860-1L-R) and 1 tube of 30 mL of alcohol-soluble eosin (Eosin Y, Sangon No.A600190-0025, 0.013 g dissolved in 25 mL of anhydrous methanol, mix and set aside), place the chip with the tissue attached in the first tube of methanol for 27 minutes, then place it in alcohol-soluble eosin for staining for 3 minutes, then put it back in the first tube for rinsing for 1 minute, take it out and place it in the fume hood, wait for the tissue and chip to dry naturally. Then add hematoxylin (Sigma, No.51275) dropwise on the chip surface (the amount of liquid added depends on the chip area, per 1 cm 2 100 μL was added dropwise), and after staining for 7 minutes, the surface liquid was aspirated with a pipette, and then washed once with 0.1×SSC solution (prepared with 20×SSC buffer solution and nuclease-free water) (cleaning method: add dropwise to the surface and then aspirate with a pipette. The amount of washing liquid added depends on the chip area, and the amount per 1 cm is 0.1×SSC solution). 2 After washing, prepare Bluing Buffer (Dako, No. CS70230-2) and add Bluing Buffer to the chip surface (the amount of solution added depends on the chip area, per 1 cm 2 Add 100μL), then wash once with 0.1XSSC (same method as above) and blow dry with a gas cylinder at a wind speed of 5m / s~10m / s. After drying, add a sealing medium and prepare for staining and washing: after hematoxylin staining, use 0.1×SSC solution (prepared by 20×SSC buffer solution and nuclease-free water) to wash the chip surface 3 times. After blue staining, use 0.1×SSC solution to wash the chip surface once. After pouring off the low-salt solution on the tissue for the last time, quickly (wind speed 5m / s~10m / s) use a gas cylinder to blow dry the chip and the low-salt solution on the chip surface to minimize the residual low-salt solution on the chip. This step is the key to determining the quality of the track line imaging on the chip.

[0191] Mounting: Use a pipette to slowly draw up an appropriate amount of polyethylene glycol (PEG400 (100%)) and drop it onto the center of the chip. Then, seal the chip with a coverslip. Once the polyethylene glycol completely covers the chip surface, you can prepare to photograph. (Procedure: Drop PEG onto the center of the tissue on the chip, then follow the standard coverslip mounting procedure. Liquid volume: For a 1cm*1cm chip, add 3.5μL. Adjust the amount accordingly for chips of different sizes.) The track line should be clearly visible when photographing, and the slide can be left for a long time, during which time the dye on the tissue remains stable and does not fade.

[0192] The specific process is shown in Figure 1.

[0193] Example 2 Preparation of polyethylene glycol (PEG6000 (50%)) mounted tissue

[0194] The experimental materials were prepared into tissue sections.

[0195] Tissue fixation: 10 μm thick tissue sections were attached to a chip with a single-stranded DNA probe (the chip was obtained from the Spatiotemporal Kit, specifically the Stereo-seq Transcriptome Reagent Set (Vector Edition), Specification: 4RXN, Catalog Number: 201ST114). The tissue was then oven-dried and fixed with an organic solvent.

[0196] H&E staining and mounting were performed in the same manner as described in Example 1, except that polyethylene glycol (PEG6000 (50%), prepared by diluting PEG6000 powder with NF·H2O to a concentration of 50%) was used as the mounting medium.

[0197] The specific process is shown in Figure 1.

[0198] Example 3 Preparation of polyethylene glycol (PEG8000 (50%)) mounted tissue

[0199] The experimental materials were prepared into tissue sections.

[0200] Tissue fixation: 10 μm thick tissue sections were attached to a chip with a single-stranded DNA probe (the chip was obtained from the Spatiotemporal Kit, specifically the Stereo-seq Transcriptome Reagent Set (Vector Edition), Specification: 4RXN, Catalog Number: 201ST114). The tissue was then oven-dried and fixed with an organic solvent.

[0201] H&E staining and mounting were performed in the same manner as described in Example 1, except that polyethylene glycol (PEG8000 (50%), purchased from RIGAKU, was prepared by diluting PEG8000 powder with ASTM Type I water to a concentration of 50%) was used as the mounting medium.

[0202] The specific process is shown in Figure 1.

[0203] Example 4 Preparation of polyethylene glycol (mixed) mounted tissue

[0204] The experimental materials were prepared into tissue sections.

[0205] Tissue fixation: 10 μm thick tissue sections were attached to a chip with a single-stranded DNA probe (the chip was obtained from the Spatiotemporal Kit, specifically the Stereo-seq Transcriptome Reagent Set (Vector Edition), Specification: 4RXN, Catalog Number: 201ST114). The tissue was then oven-dried and fixed with an organic solvent.

[0206] H&E staining and mounting were performed in the same manner as described in Example 1, except that the mounting medium was a polyethylene glycol mixture prepared by mixing PEG400 (100%), PEG6000 (50%), and PEG8000 (50%) in a volume ratio of 1:1:1.

[0207] The brand of PEG8000 is RIGAKU, 50% (w / v), and its preparation method is as follows: PEG8000 powder is diluted with ASTM Type I water to a concentration of 50%; PEG6000 powder is diluted with NF·H2O to a concentration of 50%.

[0208] The specific process is shown in Figure 1.

[0209] Example 5 Spatiotemporal capture of tissues using polyethylene glycol sealing

[0210] Prepare tissue coverslips using the experimental method described in Example 1 and photograph them. Discard the coverslips and soak in a low-salt solution (0.1×SSC buffer) for 3–5 seconds to remove the polyethylene glycol. Excessive wash concentration or excessive washes will affect the capture of the spatiotemporal transcriptome to varying degrees. Prewash the chip with pH 2 hydrochloric acid (0.01N hydrochloric acid) to adjust the pH of the chip. Add the permeabilization reagent (derived from the spatiotemporal kit). Refer to the kit instructions for the following steps (Stereo-seq Transcriptome Reagent Set (Vector Edition), Specification: 4RXN, Catalog Number: 201ST114, Kit Version V1.2).

[0211] After permeabilization of the tissue and subsequent mRNA reverse transcription reaction, cDNA is obtained. The chip is then digested with enzymes to obtain cDNA with positional probe information. This cDNA is then subjected to a series of subsequent operations such as library construction and sequencing. Sequence information of the mRNA with positional information is obtained, and the data is then analyzed to obtain in situ expression information of the mRNA or protein.

[0212] Chip preparation and sequencing were performed according to the standard procedures of MGI DNB make and sequencing reagent preparation, and data analysis was performed using the BGI spatiotemporal omics visualization system.

[0213] The specific process is shown in Figure 1.

[0214] Example 6 Spatiotemporal capture of tissues using polyethylene glycol sealing

[0215] Prepare tissue coverslips using the experimental method described in Example 2 and photograph them. Discard the coverslips and soak in a low-salt solution (0.1×SSC buffer) for 3–5 seconds to remove the polyethylene glycol. Excessive wash concentrations or excessive washes can affect the capture of the spatiotemporal transcriptome to varying degrees. Prewash the chip with pH 2 hydrochloric acid (0.01N hydrochloric acid) to adjust the pH of the chip. Add the permeabilization reagent (derived from the spatiotemporal kit). Follow the steps in the kit instructions (Stereo-seq Transcriptome Reagent Set (Vector Edition), Specification: 4RXN, Catalog Number: 201ST114, Kit Version V1.2). Specifically:

[0216] After permeabilization of the tissue and subsequent mRNA reverse transcription reaction, cDNA is obtained. The chip is then digested with enzymes to obtain cDNA with positional probe information. This cDNA is then subjected to a series of subsequent operations such as library construction and sequencing. Sequence information of the mRNA with positional information is obtained, and the data is then analyzed to obtain in situ expression information of the mRNA or protein.

[0217] Chip preparation and sequencing were performed according to the standard procedures of MGI DNB make and sequencing reagent preparation, and data analysis was performed using the BGI spatiotemporal omics visualization system.

[0218] The specific process is shown in Figure 1.

[0219] Example 7 Spatiotemporal capture of tissues using polyethylene glycol sealing

[0220] Prepare tissue coverslips using the experimental method described in Example 3 and photograph them. Discard the coverslips and soak in a low-salt solution (0.1×SSC buffer) for 3–5 seconds to remove the polyethylene glycol. Excessive wash concentration or excessive washes will affect the capture of the spatiotemporal transcriptome to varying degrees. Prewash the chip with pH 2 hydrochloric acid (0.01N hydrochloric acid) to adjust the pH of the chip. Add the permeabilization reagent (derived from the spatiotemporal kit). Follow the steps in the kit instructions (Stereo-seq Transcriptome Reagent Set (Vector Edition), Specification: 4RXN, Catalog Number: 201ST114, Kit Version V1.2). Specifically:

[0221] After permeabilization of the tissue and subsequent mRNA reverse transcription reaction, cDNA is obtained. The chip is then digested with enzymes to obtain cDNA with positional probe information. This cDNA is then subjected to a series of subsequent operations such as library construction and sequencing. Sequence information of the mRNA with positional information is obtained, and the data is then analyzed to obtain in situ expression information of the mRNA or protein.

[0222] Chip preparation and sequencing were performed according to the standard procedures of MGI DNB make and sequencing reagent preparation, and data analysis was performed using the BGI spatiotemporal omics visualization system.

[0223] The specific process is shown in Figure 1.

[0224] Example 8 Spatiotemporal capture of tissues using polyethylene glycol sealing

[0225] Prepare tissue coverslips using the experimental method described in Example 4 and photograph them. Discard the coverslips and soak in a low-salt solution (0.1×SSC buffer) for 3–5 seconds to remove the polyethylene glycol. Excessive wash concentration or excessive washes will affect the capture of the spatiotemporal transcriptome to varying degrees. Prewash the chip with pH 2 hydrochloric acid (0.01N hydrochloric acid) to adjust the pH of the chip. Add the permeabilization reagent (derived from the spatiotemporal kit). Follow the steps in the kit instructions (Stereo-seq Transcriptome Reagent Set (Vector Edition), Specification: 4RXN, Catalog Number: 201ST114, Kit Version V1.2). Specifically:

[0226] After permeabilization of the tissue and subsequent mRNA reverse transcription reaction, cDNA is obtained. The chip is then digested with enzymes to obtain cDNA with positional probe information. This cDNA is then subjected to a series of subsequent operations such as library construction and sequencing. Sequence information of the mRNA with positional information is obtained, and the data is then analyzed to obtain in situ expression information of the mRNA or protein.

[0227] Chip preparation and sequencing were performed according to the standard procedures of MGI DNB make and sequencing reagent preparation, and data analysis was performed using the BGI spatiotemporal omics visualization system.

[0228] The specific process is shown in Figure 1.

[0229] Example 9 Spatiotemporal capture of polyethylene glycol-sealed tissue (DAPI staining spatiotemporal capture)

[0230] The experimental materials were prepared into tissue sections.

[0231] Tissue fixation: 10 μm thick tissue sections were attached to a chip with a single-stranded DNA probe (the chip was obtained from the Spatiotemporal Kit, specifically the Stereo-seq Transcriptome Reagent Set (Vector Edition), Specification: 4RXN, Catalog Number: 201ST114). The tissue was then oven-dried and fixed with an organic solvent.

[0232] DAPI staining: DAPI dye (Thermo 62248) was added dropwise to the chip surface (1 cm per chip). 2 Add 100 μL of HCl dropwise and incubate in the dark for 5 minutes.

[0233] Staining and cleaning: After hematoxylin staining, use 0.1×SSC solution (prepared from 20×SSC buffer solution and nuclease-free water) to clean the chip surface three times. After blue staining, use 0.1×SSC solution to clean the chip surface once. After pouring off the low-salt solution on the tissue for the last time, quickly blow dry the chip and the low-salt solution on the chip surface with a gas cylinder (wind speed 5m / s-10m / s) to minimize the residual low-salt solution on the chip. This step is key to determining the quality of the track line imaging on the chip.

[0234] Mounting: Use a pipette to slowly draw up an appropriate amount of polyethylene glycol (PEG400 (100%)) and drop it onto the center of the chip. Then, seal the chip with a coverslip. Once the polyethylene glycol completely covers the chip, you can prepare to photograph. (Procedure: Drop PEG onto the center of the chip tissue and then follow standard coverslip mounting procedures. Liquid volume: For a 1cm*1cm chip, add 3.5μL. Adjust the amount for different chip sizes accordingly. Dilution ratios for PEG of different molecular weights: PEG8000 (Rigaku brand) 50% (w / v), PEG6000 powder diluted with NF·H2O to a concentration of 50%).

[0235] After taking the photo, discard the coverslip and soak in a low-salt solution (0.1× SSC buffer) for 3–5 seconds to remove the polyethylene glycol. Excessive wash concentrations or excessive washes can affect spatiotemporal transcriptome capture to varying degrees. Prewash the chip with pH 2 hydrochloric acid (0.01N) to adjust the chip's pH. Add a permeabilization reagent to permeabilize the tissue and perform the subsequent mRNA reverse transcription reaction. After obtaining cDNA, perform enzyme digestion on the chip to obtain cDNA with positional probe information. This cDNA is then subjected to a series of subsequent operations, including library construction and sequencing. Sequence information of the mRNA with positional information is obtained, and the data is then analyzed to determine in situ expression information of the mRNA or protein.

[0236] Chip preparation and sequencing were performed according to the standard procedures of MGI DNB make and sequencing reagent preparation, and data analysis was performed using the BGI spatiotemporal omics visualization system.

[0237] Example 10 Spatiotemporal capture of polyethylene glycol-sealed tissue (ssDNA staining spatiotemporal capture)

[0238] The experimental materials were prepared into tissue sections.

[0239] Tissue fixation: 10 μm thick tissue sections were attached to a chip with a single-stranded DNA probe (the chip was obtained from the Spatiotemporal Kit, specifically the Stereo-seq Transcriptome Reagent Set (Vector Edition), Specification: 4RXN, Catalog Number: 201ST114). The tissue was then oven-dried and fixed with an organic solvent.

[0240] ssDNA staining: dilute qubit ssDNA staining solution (Qubit, catalog number Q10212) with 0.1XSSC. Dilution method: 0.5μL ssDNA staining solution + 99.5μL 0.1XSSC solution. After preparation, drop it on the chip surface. The amount of drop (per 1cm 2 100 μL was added dropwise) and incubated in the dark for 5 min.

[0241] Staining and cleaning: After hematoxylin staining, use 0.1×SSC solution (prepared from 20×SSC buffer solution and nuclease-free water) to clean the chip surface three times. After blue staining, use 0.1×SSC solution to clean the chip surface once. After pouring off the low-salt solution on the tissue for the last time, quickly blow dry the chip and the low-salt solution on the chip surface with a gas cylinder (wind speed 5m / s-10m / s) to minimize the residual low-salt solution on the chip. This step is key to determining the quality of the track line imaging on the chip.

[0242] Mounting: Use a pipette to slowly draw up an appropriate amount of polyethylene glycol (PEG400 (100%)) and drop it onto the center of the chip. Then, seal the chip with a coverslip. Once the polyethylene glycol completely covers the chip, you can prepare to photograph. (Procedure: Drop PEG onto the center of the chip tissue and then follow standard coverslip mounting procedures. Liquid volume: For a 1cm*1cm chip, add 3.5μL. Adjust the amount for different chip sizes accordingly. Dilution ratios for PEG of different molecular weights: PEG8000 (Rigaku brand) 50% (w / v), PEG6000 powder diluted with NF·H2O to a concentration of 50%).

[0243] After taking the photo, discard the coverslip and soak in a low-salt solution (0.1× SSC buffer) for 3–5 seconds to remove the polyethylene glycol. Excessive wash concentrations or excessive washes can affect spatiotemporal transcriptome capture to varying degrees. Prewash the chip with pH 2 hydrochloric acid (0.01N) to adjust the chip's pH. Add a permeabilization reagent to permeabilize the tissue and perform the subsequent mRNA reverse transcription reaction. After obtaining cDNA, perform enzyme digestion on the chip to obtain cDNA with positional probe information. This cDNA is then subjected to a series of subsequent operations, including library construction and sequencing. Sequence information of the mRNA with positional information is obtained, and the data is then analyzed to determine in situ expression information of the mRNA or protein.

[0244] Chip preparation and sequencing were performed according to the standard procedures of MGI DNB make and sequencing reagent preparation, and data analysis was performed using the BGI spatiotemporal omics visualization system.

[0245] Example 11 Comparison of photographic results of polyethylene glycol (PEG400, PEG6000, PEG8000) and glycerol sealants

[0246] Experimental materials: mouse stomach, mouse testis, mouse hemibrain, 100% methanol (Sigma-Aldrich), polyethylene glycol 400 (Sangon Biotech), alcohol-soluble eosin (Sangon Biotech), hematoxylin stain (Sigma), blue stain (Agilent), 0.1×SSC, coverslip (Shitai), 100% glycerol (Diomand).

[0247] Experimental equipment: microtome (Leica), compressed air tank (CINE EDC GEAR), microscope (Motic PA53).

[0248] Experimental groups:

[0249] H&E slide group: Mouse stomach was used as the experimental material, and 10 μm thick tissue sections were attached to slides; H&E staining and mounting were performed as described in Example 1;

[0250] Control group 1: Mouse stomach was used as the experimental material, and the operation was carried out according to the instructions of BGI-Stereo-seq Transcriptome Reagent Set (compatible with H&E) (A0), kit 201SP118 (8RXNs), and glycerol sealing was performed;

[0251] Control group 2: Mouse testes were used as experimental materials and glycerol-sealed according to the instructions of the BGI-Stereo-seq Transcriptome Reagent Kit (compatible with H&E) (A0), kit 201SP118 (8RXNs);

[0252] Control group 3: Mouse hemibrain was used as experimental material, and the operation was carried out according to the instructions of BGI-Stereo-seq Transcriptome Reagent Set (compatible with H&E) (A0), kit 201SP118 (8RXNs), and glycerol sealing was performed;

[0253] Test Group 1: Mouse stomach was used as the experimental material. Tissue mounts were prepared using polyethylene glycol 400 as the mounting medium according to the experimental method described in Example 1 and photographed.

[0254] Test Group 2: Mouse testicles were used as experimental materials. Tissue mounts were prepared using polyethylene glycol 400 as a mounting medium according to the experimental method described in Example 1 and photographed.

[0255] Test Group 3: Mouse testicles were used as experimental materials. Tissue mounts were prepared and photographed using polyethylene glycol 8000 as the mounting medium according to the experimental method described in Example 3.

[0256] Test Group 4: Mouse hemibrain was used as the experimental material. Tissue mounts were prepared using polyethylene glycol 6000 as the mount according to the experimental method described in Example 2 and photographed.

[0257] Experimental results:

[0258] (1) The results of sealing and photographing the mouse stomach are shown in Figures 2 and 3:

[0259] Figure 2 shows images of mouse stomach sections stained with H&E on glass slides, microarray stained with polyethylene glycol 400-sealed microarray (test group 1), and microarray stained with glycerol-sealed microarray (control group 1). The overall staining effect of the two microarray-stained images is similar to that of the glass slides, but the H&E microarray and polyethylene glycol 400-sealed microarrays show clearer tissue boundaries and more vivid image colors.

[0260] Comparing the tissue and blank areas of the polyethylene glycol 400 and glycerol mounts, we can find that the eosin staining in the tissue area of ​​the polyethylene glycol 400 mount is very uniform, with no eosin bleed, while the tissue area in the glycerol mount image has obvious eosin bleed. Compared with the glycerol mount, the polyethylene glycol 400 mount can capture clearer track lines in the blank area of ​​the chip (as shown in Figure 3).

[0261] (2) The results of mounting and photographing the mouse testicles are shown in Figures 4 and 5:

[0262] Figure 4 shows photographs of mouse testis sections 1, 2, and 3 after H&E staining on a chip using polyethylene glycol 400 sealing (test group 2), polyethylene glycol 8000 sealing (test group 3), and glycerol sealing (control group 2). Compared to glycerol sealing, the two H&E staining images using polyethylene glycol sealing showed more vivid colors and no eosin bleed.

[0263] Comparing the tissue edge areas and tissue cavity areas of polyethylene glycol-sealed films and glycerol-sealed films, we can find that the tissue boundaries of the tissue area edges and tissue cavity areas of polyethylene glycol-sealed films are very clear, the eosin staining is very uniform, and there is no eosin bleaching. However, in the images of glycerol-sealed films, obvious eosin bleaching appears in both the tissue edge and the tissue cavity areas (as shown in Figure 5).

[0264] (3) The results of the mounted photographs of the mouse hemibrain are shown in Figure 6:

[0265] Figures 1 and 2 in Figure 6 are photographs of coronal sections of a mouse hemibrain stained on a chip using polyethylene glycol 6000 (test group 4) and glycerol (control group 3). For H&E chip staining, the polyethylene glycol 6000 chip has clearer tissue boundaries and more vivid image colors; the glycerol chip has obvious eosin haloing at the tissue edge, and obvious eosin haloing can also be observed in the tissue cavity.

[0266] In summary, we can see that compared to glycerol mounting, polyethylene glycol (PEG400, PEG6000, PEG8000) mountings all perform better on H&E-stained fresh frozen-embedded tissue sections. Polyethylene glycol mounting can effectively alleviate the problem of eosin bleed on tissues and improve the color vividness and nuclear-cytoplasmic contrast of H&E-stained images. For BGI spatiotemporal microarrays, polyethylene glycol mounting can effectively improve the clarity of track lines under epi- and reflected-field illumination.

[0267] Example 12 Retention time after polyethylene glycol (PEG400) sealing

[0268] Experimental materials: mouse small intestine, tumor samples, 100% methanol (Sigma-Aldrich), polyethylene glycol 400 (Sangon Biotech), alcohol-soluble eosin (Sangon Biotech), hematoxylin stain (Sigma), blue stain (Agilent), 0.1× SSC, coverslips (Shitai), 100% glycerol (Diomand).

[0269] Experimental equipment: microtome (Leica), compressed air tank (CINE EDC GEAR), microscope (Motic PA53).

[0270] Experimental groups:

[0271] Control group: Mouse small intestine was used as experimental material, and the operation was carried out according to the instructions of BGI-Stereo-seq Transcriptome Reagent Set (compatible with H&E) (A0), kit 201SP118 (8RXNs), and glycerol sealing was performed;

[0272] Test Group 1: Tumor samples were used as experimental materials. Tissue mounts were prepared using PEG400 as a mountant according to the experimental method described in Example 1 and photographed.

[0273] Test Group 2: Mouse small intestine was used as the experimental material. Tissue mounting slides were prepared using PEG400 as the mounting medium according to the experimental method described in Example 1 and photographed.

[0274] Experimental results:

[0275] (1) Tumor samples:

[0276] After the tumor sample was mounted with polyethylene glycol 400 (test group 1), the H&E staining image could be preserved for a longer period of time, with clear tissue edges and uniform staining. There was no eosin bleaching 31 minutes after mounting (as shown in Figure 7).

[0277] (2) Mouse small intestine:

[0278] As shown in Figure 8, compared with the glycerol mounting (control group), the H&E staining image of the mouse small intestine after polyethylene glycol 400 mounting (test group 2) can be retained for a longer time. If the subsequent capture amount is not considered, the image quality is still good after three days.

[0279] In summary, we can see that compared to glycerol mounting, polyethylene glycol (PEG400) mounting performs better on H&E-stained fresh frozen-embedded tissue sections and has a longer retention time. Polyethylene glycol mounting can effectively alleviate the problem of eosin bleed on tissues and improve the color vividness and nuclear-cytoplasmic contrast of H&E-stained images. For BGI spatiotemporal microarrays, polyethylene glycol mounting can effectively improve the clarity of track lines under epi- and reflected-field illumination.

[0280] Example 13: Comparison of spatiotemporal capture results using polyethylene glycol sealing and different cleaning processes

[0281] Experimental materials: mouse heart, 100% methanol (Sigma-Aldrich), Qubit™ ssDNA Reagent (Invitrogen), 5× SSC, 0.1× SSC, coverslips (Shitai), 100% PEG400 (Sigma), 100% glycerol (Diomand), STOmics Gene Expression Kit-S1, STOmics Library Preparation Kit-S1 (BGI), Seq2000 Sequencing Kit PE100 (MGI);

[0282] Experimental equipment: microtome (Leica), compressed air tank (CINE EDC GEAR), microscope (Mitoc PA53), Seq2000 sequencer (MGI)

[0283] Experimental methods: The three chips were operated according to the instructions of the BGI Stereo-seq transcriptome reagent set (compatible with H&E);

[0284] Experimental groups:

[0285] The control group was obtained by the experimenter using the same tissue for H&E staining and following the standard protocol in the manual, followed by glycerol sealing and mRNA capture results.

[0286] Test Group 1: Mouse heart slices were used as experimental materials and sealed with polyethylene glycol 400. The spatiotemporal capture results were obtained by following the standard cleaning process in the instruction manual (original cleaning process).

[0287] Test Group 2: Mouse heart slices were used as experimental materials and sealed with polyethylene glycol 400. Spatiotemporal capture results were obtained by following the experimental method described in Example 5 (with optimized cleaning process);

[0288] Experimental results:

[0289] (1) The results of spatiotemporal omics analysis are shown in Figure 9;

[0290] Figure 9 shows glycerol mounting, original cleaning process, bin 50 (control group);

[0291] Figure 9 2 shows polyethylene glycol (PEG400, 100%) mounting, original cleaning process (test group 1), bin 50;

[0292] Figure 9 3 Polyethylene glycol (PEG400, 100%) sealing, optimized cleaning process (test group 2), bin 50;

[0293] (2) The mRNA capture results are shown in Table 1:

[0294] Table 1 mRNA capture results of different sealing agents and cleaning processes in mouse heart

[0295] Judging from the results, the optimized cleaning process can significantly improve spatiotemporal capture. When Dup is lower, the capture amount (Bin200 Median Gene Type and Median MID) is improved to varying degrees compared with the original cleaning schemes of glycerol sealing and polyethylene glycol sealing.

[0296] Example 14 Comparison of spatiotemporal capture results between polyethylene glycol-sealed tissue and glycerol-sealed tissue

[0297] Experimental materials: mouse testis, 100% methanol (Sigma-Aldrich), Qubit™ ssDNA Reagent (Invitrogen), 5×SSC, 0.1×SSC, coverslips (Shitai), 100% PEG400 (Sangon Biotech), 50% PEG8000 (Sigma), 100% glycerol (Diomand), STOmics Gene Expression Kit-S1, STOmics Library Preparation Kit-S1 (BGI), Seq2000 Sequencing Kit PE100 (MGI).

[0298] Experimental equipment: microtome (Leica), compressed air tank (CINE EDC GEAR), microscope (Mitoc PA53), Seq2000 sequencer (MGI).

[0299] Experimental groups:

[0300] The control group (H&E staining, glycerol mounting) was operated according to the instructions of the BGI-Stereo-seq transcriptome reagent set (compatible with H&E). The experimenters used the same tissue for H&E staining and obtained the mRNA capture results according to the standard process of the BGI spatiotemporal transcriptome kit. The glycerol mounting was photographed and the mRNA capture results were obtained;

[0301] Test Group 1 (H&E staining, PEG400 mounting): Mouse testicles were used as experimental materials, and spatiotemporal capture results were obtained according to the experimental method described in Example 5;

[0302] Test group 2 (H&E staining, PEG8000 mounting): Mouse testicles were used as experimental materials, and spatiotemporal capture results were obtained according to the experimental method described in Example 7;

[0303] Spatiotemporal omics analysis results:

[0304] (1) The visualization image (Bin50) is shown in Figure 10;

[0305] (2) The mRNA capture results are shown in Table 2:

[0306] Table 2 mRNA capture results of different mounting media in mouse testis

[0307] Note: The brackets are the comparison of the amount of Genetype and MID data when the dup value is adjusted to a unified value.

[0308] (3) H&E staining images are shown in Figure 11:

[0309] From the results, compared with glycerol mounting (control group), polyethylene glycol (PEG400, PEG8000) (test group 1, test group 2) mounting effectively improved the image quality (as shown in Figure 11), while the coverage (MID_UT / Unique Reads) remained unchanged. Under the same Dup, the capture amount (Bin200 Gene Type and MID) was also unaffected.

[0310] Example 15 Comparison of spatiotemporal capture results between polyethylene glycol-sealed tissue and glycerol-sealed tissue

[0311] Experimental materials: mouse thymus, 100% methanol (Sigma-Aldrich), Qubit™ ssDNA Reagent (Invitrogen), 5× SSC, 0.1× SSC, coverslips (Shitai), 100% PEG400 (Sigma), 50% PEG6000 (Sigma), 100% glycerol (Diomand), STOmics Gene Expression Kit-S1, STOmics Library Preparation Kit-S1 (BGI), Seq2000 Sequencing Kit PE100 (MGI);

[0312] Experimental equipment: microtome (Leica), compressed air tank (CINE EDC GEAR), microscope (Mitoc PA53), Seq2000 sequencer (MGI);

[0313] Experimental groups:

[0314] The control group followed the instructions for use of the BGI-Stereo-seq transcriptome reagent set (compatible with H&E). The experimenters used the same tissue for H&E staining and obtained the mRNA capture results according to the standard procedures in the instructions. Among them, control group 1 did not perform sealing operation, and control group 2 used glycerol for sealing.

[0315] Test group 1 (H&E staining, PEG400 mounting): using mouse thymus as the experimental material, the spatiotemporal capture results were obtained according to the experimental method described in Example 5.

[0316] Test group 2 (H&E staining, PEG6000 mounting): using mouse thymus as the experimental material, the spatiotemporal capture results were obtained according to the experimental method described in Example 6.

[0317] Spatiotemporal omics analysis results:

[0318] (1) The visualization image (Bin50) is shown in Figure 12;

[0319] In Figure 12, 1 shows no sealing, Bin 50

[0320] Figure 12, 2 shows the visualization of glycerol sealant, Bin50

[0321] Figure 12 3 shows the visualization of PEG400 sealing (polyethylene glycol 400), Bin50

[0322] Figure 12 shows PEG6000 (polyethylene glycol 6000) sealant visualization, Bin 50

[0323] (2) The mRNA capture results are shown in Table 3:

[0324] Table 3 mRNA capture results of different sealing agents for mouse thymus

[0325] Note: The brackets are the comparison of the amount of Genetype and MID data when the dup value is adjusted to a unified value.

[0326] (3) H&E staining images: (no sealing treatment and no photographing), the photographic results are shown in Figure 13:

[0327] From the results, compared with no sealing, the three sealing agents can effectively improve the uniformity of tissue capture. The visualization results are basically consistent with the H&E staining results (as shown in Figure 13), while the non-sealing method can clearly show uneven capture. The three sealing agents can effectively improve the coverage of mRNA capture (MID_UT / Unique Reads, the higher the coverage, the better the in situ capture), and there is no significant difference in capture amount compared with no sealing.

[0328] Example 16 Comparison of Spatiotemporal Capture Results of Polyethylene Glycol (Mixed) Seals

[0329] Experimental materials: mouse liver, 100% methanol (Sigma-Aldrich), Qubit™ ssDNA Reagent (Invitrogen), 5×SSC, 0.1×SSC, coverslips (Sita), 100% PEG400 (Sigma), 50% PEG6000 (Sigma), 50% PEG8000 (Sigma), 100% glycerol (Diomand), STOmics Gene Expression Kit-S1, STOmics Library Preparation Kit-S1 (BGI), Seq2000 Sequencing Kit PE100 (MGI).

[0330] Experimental equipment: microtome (Leica), compressed air tank (CINE EDC GEAR), microscope (Mitoc PA53), Seq2000 sequencer (MGI).

[0331] Experimental method: Two sets of chips were operated according to the instruction manual of BGI-Stereo-seq transcriptome reagent set (compatible with H&E).

[0332] Experimental groups:

[0333] The control group was obtained by using the same tissue for H&E staining and glycerol mounting, and following the standard protocol in the manufacturer's instructions.

[0334] The test group followed the experimental method described in Example 8. 3.5 μL of a mixture of PEG400 (100%), PEG6000 (50%), and PEG8000 (50%) in a 1:1:1 ratio was used to seal the slides and obtain mRNA capture results. PEG8000 was prepared by diluting PEG8000 powder with ASTM Type I water to a concentration of 50%, and diluting PEG6000 powder with NF·H2O to a concentration of 50%.

[0335] Experimental results:

[0336] (1) H&E staining images are shown in Figure 14;

[0337] (2) The results of spatiotemporal omics analysis are shown in Figure 15 ;

[0338] In FIG15 , 1 shows a glycerol mount, bin 50;

[0339] In FIG15 , 2 shows polyethylene glycol (mixed) sealing, bin 50;

[0340] (3) The mRNA capture results are shown in Table 4:

[0341] Table 4 mRNA capture results of different mounting media in mouse liver

[0342] Note: The brackets are the comparison of the amount of Genetype and MID data when the dup value is adjusted to a unified value.

[0343] The results show that the mixed polyethylene glycol as a mounting medium can also effectively improve image quality and alleviate the problem of eosin blurring. When the Dup is lower, the capture amount (Bin200Median Gene Type and Median MID) is improved compared with glycerol mounting. With the same Dup of 52%, the capture is increased by 134%.

[0344] Example 17 Comparison of spatiotemporal capture results between polyethylene glycol-sealed tissue and glycerol-sealed tissue

[0345] Experimental materials: mouse hemibrain, mouse lymph node, 100% methanol (Sigma-Aldrich), Qubit™ ssDNA Reagent (Invitrogen), 5× SSC, 0.1× SSC, coverslips (Shitai), 100% PEG400 (Sigma), 50% PEG6000 (Sigma), 50% PEG8000 (Sigma), 100% glycerol (Diomand), STOmics Gene Expression Kit-S1, STOmics Library Preparation Kit-S1 (BGI), Seq2000 Sequencing Kit PE100 (MGI);

[0346] Experimental equipment: microtome (Leica), compressed air tank (CINE EDC GEAR), microscope (Mitoc PA53), Seq2000 sequencer (MGI)

[0347] Experimental groups:

[0348] 1. Using mouse testicles as experimental materials:

[0349] Control group 1 (H&E staining, glycerol mounting) was performed according to the instructions of the BGI-Stereo-seq transcriptome reagent set (compatible with H&E). The experimenters used the same tissue for H&E staining and obtained the mRNA capture results according to the standard process of the BGI spatiotemporal transcriptome kit. The glycerol mounting was photographed and the mRNA capture results were obtained;

[0350] Test Group 1 (H&E staining, PEG400 mounting): Mouse testicles were used as experimental materials, and spatiotemporal capture results were obtained according to the experimental method described in Example 5;

[0351] Test group 2 (H&E staining, PEG8000 mounting): Mouse testicles were used as experimental materials, and spatiotemporal capture results were obtained according to the experimental method described in Example 7;

[0352] 2. Using mouse thymus as experimental material:

[0353] Control group 2 followed the instructions for use of the BGI-Stereo-seq transcriptome reagent set (compatible with H&E). The experimenters used the same tissue for H&E staining and obtained the mRNA capture results according to the standard procedures in the instructions. Among them, control group 1 did not perform sealing operation, while control group 2 used glycerol for sealing.

[0354] Test group 3 (H&E staining, PEG400 mounting): using mouse thymus as the experimental material, the spatiotemporal capture results were obtained according to the experimental method described in Example 5.

[0355] Test group 4 (H&E staining, PEG6000 mounting): using mouse thymus as the experimental material, the spatiotemporal capture results were obtained according to the experimental method described in Example 6.

[0356] 3. Mouse liver was used as experimental material:

[0357] Control group 3 was the mRNA capture result obtained by the experimenter using the same tissue for H&E staining and glycerol mounting according to the standard protocol in the manufacturer's instructions;

[0358] Test Group 5 followed the experimental method described in Example 8. 3.5 μL of a mixture of PEG 400 (100%), PEG 6000 (50%), and PEG 8000 (50%) in a 1:1:1 ratio was used to seal the slides and obtain mRNA capture results. PEG 8000 was prepared from RIGAKU brand, and 50% (w / v) was prepared by diluting PEG 8000 powder with ASTM Type I water to a concentration of 50%, and diluting PEG 6000 powder with NF·H2O to a concentration of 50%.

[0359] 4. Using mouse hemibrain as experimental material:

[0360] Control group 4 (using Glycerol as mounting medium): Mouse hemibrain was used as experimental material. Following the instructions of the BGI-Stereo-seq Transcriptome Reagent Kit (compatible with H&E), the experimenters used the same tissue for H&E staining and obtained mRNA capture results according to the standard procedures in the instructions.

[0361] Test Group 6 (PEG400 as mounting medium): Mouse hemibrain was used as experimental material, and the results of mRNA capture were obtained according to the experimental method described in Example 5;

[0362] Test Group 7 (PEG8000 as mounting medium): Mouse hemibrain was used as experimental material, and the results of mRNA capture were obtained according to the experimental method described in Example 6;

[0363] Test Group 8 (PEG6000 as mounting medium): Mouse hemibrain was used as experimental material, and the results of mRNA capture were obtained according to the experimental method described in Example 7;

[0364] 5. Using mouse lymph nodes as experimental materials:

[0365] Control group 5 (using Glycerol as mounting medium): Mouse lymph nodes were used as experimental materials. Following the instructions of the BGI-Stereo-seq transcriptome reagent kit (compatible with H&E), the experimenters used the same tissue for H&E staining and obtained mRNA capture results according to the standard procedures in the instructions.

[0366] Test group 9 (PEG400 was used as the sealing agent): mouse lymph nodes were used as experimental materials, and the results of mRNA capture were obtained according to the experimental method described in Example 5.

[0367] The experimental results are shown in Table 5:

[0368] Table 5 Comparison of capture differences using different mounting media for the same sample (same dup)

[0369] In summary, it can be seen that compared with glycerol sealing, polyethylene glycol (PEG400, PEG6000, PEG8000) sealing performs better on H&E stained fresh frozen embedded tissue sections. These three polyethylene glycol reagents as sealing agents can play a similar role as glycerol in improving in situ capture while not affecting the temporal and spatial capture of the processed samples.

[0370] Example 18 Polyethylene glycol (PEG400) sealing and DAPI staining for temporal and spatial capture results comparison

[0371] Experimental materials: mouse stomach, 100% methanol (Sigma-Aldrich), DAPI staining solution (Thermo, 62248), 5×SSC, 0.1×SSC, coverslip (Shitai), 100% PEG400 (Sigma), 100% glycerol (Diomand), STOmics Gene Expression Kit-S1, STOmics Library Preparation Kit-S1 (BGI), Seq2000 Sequencing Kit PE100 (MGI).

[0372] Experimental equipment: microtome (Leica), compressed air tank (CINE EDC GEAR), microscope (Mitoc PA53), Seq2000 sequencer (MGI).

[0373] Experimental methods: Two sets of chips were operated according to the instructions of the BGI-Stereo-seq transcriptome reagent set;

[0374] Experimental groups:

[0375] (1) The control group (DAPI staining, glycerol mounting) is the mRNA capture result of the experimenter using the same tissue and performing the standard transcriptome process according to the instructions;

[0376] (2) The test group (DAPI staining, PEG400 mounting) was prepared according to the experimental method described in Example 9 to obtain spatiotemporal capture results;

[0377] Experimental results:

[0378] (1) The results of spatiotemporal omics analysis are shown in Figure 16:

[0379] In FIG16 , 1 shows a glycerol mount, bin 50;

[0380] Figure 16 2 shows polyethylene glycol (PEG400, 100%) sealing, bin 50;

[0381] (2) The mRNA capture results are shown in Table 6:

[0382] Table 6 mRNA capture results of mouse stomach DAPI staining with different mounting media

[0383] From the results, for DAPI staining, PEG sealing increased the capture by 20% compared with glycerol sealing, and the coverage rate was higher than that of glycerol sealing.

[0384] Example 19 Polyethylene glycol (PEG400) sealing, ssDNA staining and temporal and spatial capture results comparison

[0385] Experimental materials: mouse stomach, 100% methanol (Sigma-Aldrich), Qubit™ ssDNA Reagent (Invitrogen), 5×SSC, 0.1×SSC, coverslips (Sita), 100% PEG400 (Sigma), 100% glycerol (Diomand), STOmics Gene Expression Kit-S1, STOmics Library Preparation Kit-S1 (BGI), Seq2000 Sequencing Kit PE100 (MGI).

[0386] Experimental equipment: microtome (Leica), compressed air tank (CINE EDC GEAR), microscope (Mitoc PA53), Seq2000 sequencer (MGI).

[0387] Experimental methods: Two sets of chips were operated according to the instructions of the BGI-Stereo-seq transcriptome reagent set;

[0388] Experimental groups:

[0389] (1) The control group was the mRNA capture results of the experimenter using the same tissue according to the standard transcriptome process (using ssDNA staining and glycerol mounting);

[0390] (2) Test group (ssDNA staining, PEG400 mounting): spatiotemporal capture results were obtained according to the experimental method described in Example 10;

[0391] Experimental results:

[0392] (1) The results of spatiotemporal omics analysis are shown in Figure 17:

[0393] In FIG17 , 1 shows a glycerol mount, bin 50;

[0394] 2 in FIG17 shows polyethylene glycol (PEG400, 100%) sealing, bin 50;

[0395] (2) The mRNA capture results are shown in Table 7:

[0396] Table 7 mRNA capture results of mouse stomach staining with different mounting media

[0397] Judging from the results, for ssDNA staining, PEG sealing has no significant effect on capture and coverage compared to glycerol sealing, and can be used as a substitute for glycerol.

[0398] Example 20: Track line clarity comparison

[0399] Experimental materials: The samples were commercially available OCT-embedded fresh-frozen samples; using a Leica microtome, slices were selected at the target location, with each slice being 10 μm thick.

[0400] Experimental methods:

[0401] Control group: Follow the instructions of BGI-Stereo-seq Transcriptome Reagent Set (compatible with H&E) (A0), kit 201SP118 (8RXNs), perform glycerol sealing, and select non-tissue areas for photography.

[0402] Test group: Tissue mounts were prepared using PEG400 as a mountant according to the experimental method described in Example 1, and non-tissue areas were selected for photography.

[0403] Test results:

[0404] Figure 18 shows glycerol sealing and the original cleaning process;

[0405] Figure 18, 2 shows the new process of sealing with PEG400 sealing medium;

[0406] FIG18(3) shows the statistical H&E image QC pass rate (46 cases before optimization, 50 cases after optimization).

[0407] Compared with glycerol sealing, the H&E sealing process can effectively improve the clarity of the track line and greatly improve the QC pass rate of the automatic registration process. After optimization, the H&E automatic process registration pass rate increased from 15% to 64%. After the process is stabilized, the QC pass rate reaches 90%.

[0408] The above is a detailed introduction to the method, reagent combination, kit and device provided by the present invention for improving the quality of H&E staining images of spatiotemporal chips. This article uses specific examples to illustrate the principles and implementation methods of the present invention. The description of the above embodiments is only used to help understand the method of the present invention and its core ideas. It should be pointed out that for those skilled in the art, without departing from the principles of the present invention, several improvements and modifications can be made to the present invention, and these improvements and modifications also fall within the scope of protection of the claims of the present invention.

Claims

1. Use of polyethylene glycol or a composition of polyethylene glycol as a mounting medium and a low-salt solution as a cleaning agent in any of the following; (I), Mounting after tissue staining; and / or (II), Avoiding discoloration or staining halo of cells or tissues after staining; and / or (III), Improving the image quality of the spatial-temporal chip; and / or (IV), Improving the imaging clarity of the track line of the spatial-temporal chip; and / or (V), Improving the nuclear-cytoplasmic ratio of cells after staining; and / or (VI), Prolonging the storage time of stained images; and / or (VII), Cell typing or tissue typing; and / or (VIII), Cellbin expression analysis; and / or (IX), Improving the registration accuracy between the stained image and the spatial-temporal expression matrix; and / or (X), Not affecting or improving the spatial-temporal capture rate and / or coverage rate; and / or (XI), Improving the accuracy of obtaining mRNA and / or protein expression information; The low-salt solution includes but is not limited to 0.1×SSC.

2. The application according to claim 1, wherein The staining includes but is not limited to a combination of one or more of H&E staining in spatial omics, ssDNA staining in spatial omics, and DAPI staining; or The composition of polyethylene glycol includes but is not limited to any of the following: (I), Compositions of polyethylene glycol with different molecular weights; and / or (II), Compositions of water and / or additives with polyethylene glycol; and / or The additives include but are not limited to RNase inhibitors or glycerol.

3. The application according to any one of claims 1 or 2, characterized in that, The polyethylene glycol includes but is not limited to a combination of one or more of polyethylene glycol 400, polyethylene glycol 6000, or polyethylene glycol 8000.

4. Mounting medium, characterized in that, Comprising polyethylene glycol or a composition of polyethylene glycol; The polyethylene glycol or a composition of polyethylene glycol is used as a mounting medium for any of the following; (I), Mounting after tissue staining; and / or (II), Avoiding discoloration or staining halo of cells or tissues after staining; and / or (III), Improving the image quality of the spatial-temporal chip; and / or (IV), Improving the imaging clarity of the track line of the spatial-temporal chip; and / or (V), Improving the nuclear-cytoplasmic ratio of cells after staining; and / or (VI), Prolonging the storage time of stained images; and / or (VII), Cell typing or tissue typing; and / or (VIII), Cellbin expression analysis; and / or (IX), Improving the registration accuracy between the stained image and the spatial-temporal expression matrix; and / or (X), Not affecting or improving the spatial-temporal capture rate and / or coverage rate; and / or (XI), Improving the accuracy of obtaining mRNA and / or protein expression information; The composition of polyethylene glycol includes but is not limited to any of the following: (I), Compositions of polyethylene glycol with different molecular weights; and / or (II), Compositions of water and / or additives with polyethylene glycol; and / or The additives include but are not limited to RNase inhibitors or glycerol.

5. The mounting medium according to claim 4, wherein The polyethylene glycol includes but is not limited to a combination of one or more of polyethylene glycol 400, aqueous solution of polyethylene glycol 6000, or aqueous solution of polyethylene glycol 8000.

6. The mounting medium according to claim 5, wherein, In g / mL, the mass-to-volume ratio of polyethylene glycol 6000 to water in the aqueous solution of polyethylene glycol 6000 includes but is not limited to 1:2; the mass-to-volume ratio of polyethylene glycol 8000 to water includes but is not limited to 1:2; or the volume ratio of polyethylene glycol 400, the aqueous solution of polyethylene glycol 6000, and the aqueous solution of polyethylene glycol 8000 includes but is not limited to 1:1:

1.

7. Reagent combination, characterized in that, including but not limited to the mounting medium and cleaning agent described in any one of claims 4 to 6; the cleaning agent includes but is not limited to 0.1×SSC.

8. Kit, characterized in that, including but not limited to: (I), the mounting medium described in any one of claims 4 to 6; and / or (II), the reagent combination described in claim 7.

9. Use of the reagent combination described in claim 7 or the kit described in claim 8 in any of the following; (I), mounting after tissue staining; and / or (II), avoiding color fading or staining halo dispersion of cells or tissues after staining; and / or (III), improving the image quality of the spatial-temporal chip; and / or (IV), improving the imaging clarity of the track line of the spatial-temporal chip; and / or (V), increasing the nuclear-cytoplasmic ratio of cells after staining; and / or (VI), extending the storage time of stained images; and / or (VII), cell typing or tissue typing; and / or (VIII), cellbin expression analysis; and / or (IX), improving the registration accuracy between the stained image and the spatial-temporal expression matrix; and / or (X), not affecting or improving the spatial-temporal capture rate and / or coverage rate; and / or (XI), improving the accuracy of obtaining mRNA and / or protein expression information.

10. A method for processing tissue sections, characterized in that, Contact the stained tissue section with the cleaning agent in the reagent combination described in claim 7 or the cleaning agent in the kit described in claim 8 to wash away the excess hematoxylin and / or blueing dye on the stained tissue, air dry, and then mount with the mounting medium described in any one of claims 4 to 6, the mounting medium in the reagent combination described in claim 7, or the mounting medium in the kit described in claim 8.

11. The processing method according to claim 10, wherein The number of times of washing with the cleaning agent is not less than 1 time, preferably 1 time or 3 times; or The air drying rate includes but is not limited to: 5 m / s to 10 m / s.

12. A tissue section prepared by the treatment method described in any one of claims 10 or 11.

13. A method for improving the image quality of a spatial-temporal chip and / or improving the imaging clarity of the track line of a spatial-temporal chip, characterized in that a tissue section is fixed on a chip with probes, dried by baking, fixed, stained, and the dye is discarded to obtain a stained tissue chip; Contact the stained tissue chip with the cleaning agent in the reagent combination described in claim 7 or the cleaning agent in the kit described in claim 8 to wash away the excess hematoxylin and / or blueing dye on the stained tissue, air dry, and then mount with the mounting medium described in any one of claims 4 to 6, the mounting medium in the reagent combination described in claim 7, or the mounting medium in the kit described in claim 8.

14. The method according to claim 13, wherein The number of times of washing with the cleaning agent is not less than 1 time, preferably 1 time or 3 times; or The drying rate includes, but is not limited to: 5 m / s to 10 m / s.

15. A spatio-temporal chip obtained by the method according to any one of claims 13 to 14.

16. A method for improving the accuracy of obtaining mRNA and / or protein expression information, characterized in that, Comprising the following steps: Step 1: Fix a tissue section on a chip with probes, dry by baking, fix, stain, discard the dye, and obtain a stained tissue chip; Step 2: Contact the stained tissue chip with the cleaning agent in the reagent combination according to claim 7 or the cleaning agent in the kit according to claim 8 to wash away the residual dye, and dry; Step 3: Then mount the section with the mounting medium according to any one of claims 4 to 6, the mounting medium in the reagent combination according to claim 7, or the mounting medium in the kit according to claim 8, and take a picture; Step 4: Use the cleaning agent in the reagent combination according to claim 7 or the cleaning agent in the kit according to claim 8 to remove the mounting medium, adjust the pH value, permeabilize, and reverse transcribe mRNA to obtain cDNA; Step 5: Then perform enzymatic digestion to obtain cDNA with position probe information, construct a library and sequence to obtain mRNA and / or protein expression information.

17. The method according to claim 16, wherein The number of cleaning times of the cleaning agent is not less than 1 time, preferably 1 time or 3 times; or The drying rate includes, but is not limited to: 5 m / s to 10 m / s; or In step 4, removing the mounting medium is carried out by soaking in 0.1×SSC for 3 to 5 s; In step 4, adjusting the pH value is carried out using a hydrochloric acid solution with a pH of 2.

18. A method for spatiotemporal transcriptome capture, characterized in that, Comprising the following steps: Step 1: Fix a tissue section on a chip with probes, dry by baking, fix, stain, discard the dye, and obtain a stained tissue chip; Step 2: Contact the stained tissue chip with the cleaning agent in the reagent combination according to claim 7 or the cleaning agent in the kit according to claim 8 to wash away the residual dye, and dry; Step 3: Then mount the section with the mounting medium according to any one of claims 4 to 6, the mounting medium in the reagent combination according to claim 7, or the mounting medium in the kit according to claim 8, and take a picture; Step 4: Use the cleaning agent in the reagent combination according to claim 7 or the cleaning agent in the kit according to claim 8 to remove the mounting medium, adjust the pH value, permeabilize, and reverse transcribe mRNA to obtain cDNA; Step 5: Then perform enzymatic digestion to obtain cDNA with position probe information, construct a library and sequence to obtain capture information.

19. The method according to claim 18, wherein The number of cleaning times of the cleaning agent is not less than 1 time, preferably 1 time or 3 times; or The drying rate includes, but is not limited to: 5 m / s to 10 m / s; or In step 4, removing the mounting medium is carried out by soaking in 0.1×SSC for 3 to 5 s; In step 4, adjusting the pH value is carried out using a hydrochloric acid solution with a pH of 2.

20. Device, characterized in that, Comprising the tissue section according to claim 12 or the spatio-temporal chip according to claim 15.

Images