Method and system for simultaneously acquiring complete form and omics molecular information of single neuron

Abstract

The invention relates to a method and a system for simultaneously acquiring complete form and omics molecular information of a single neuron. The method comprises the following steps: acquiring fluorescently-labeled samples of cell bodies, dendrites and axons of the neuron; performing cyclic operation of scanning imaging and cutting on the section of the sample until the scanning imaging of the whole sample is completed, and collecting a section containing a fluorescence-labeled neuron cell body obtained by cutting; the method comprises the following steps: reconstructing a sample section three-dimensional image obtained based on scanning imaging operation to obtain a complete form of neurons, extracting fluorescently-labeled neuronal cell bodies from collected slices, and obtaining omics molecular information of the neurons through omics analysis. According to the method, the fine and complete form and omics molecular information of the same neuron can be effectively integrated, and the problem that the two kinds of key information cannot be obtained at the same time at the single neuron precision level is successfully solved.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and particularly to a method and system for simultaneously obtaining the complete morphology and omics molecular information of single neurons. Background Art

[0002] In the field of neuroscience research, the diversity of neurons is a core issue. Neurons exhibit rich diversity in terms of molecular composition, functional characteristics, morphological structure, and mutual connection. To better understand and classify these complex neuron types, scientists usually classify them according to various criteria such as fine morphology, molecular information, or electrophysiological characteristics. The complete morphology of neurons not only provides detailed morphological features and accurate anatomical localization, but also constitutes the real structural basis of neural circuits and is the key basis for defining neuron types. The cell typing method based on molecular information is to obtain the rich internal molecular information of cells through omics analysis techniques such as RNA transcriptome, epigenome, and proteome and then classify them. Combining the complete morphological information and molecular information of neurons can provide multi-dimensional fusion information for the accurate classification of neurons, which is of great significance in neuroscience research.

[0003] In recent years, various omics analysis or imaging techniques based on molecular and morphological information have developed rapidly, greatly promoting the research on neuron typing. The development of single-cell transcriptome sequencing technology has enabled cell lineage tracing in multiple brain regions and even the whole brain, and a cell type database has been established. The application of spatial transcriptomics technology allows people to obtain omics molecular maps with spatial distribution information from mouse brain slice samples. However, these omics analyses are all for manually dissected nuclei / brain regions, from which single cells or a group of cells are extracted, and then sequencing technology is used to obtain the omics analysis information of these cells. In this process, only the cell bodies of neurons are utilized, and the complex morphological information of dendrites and axons of neurons cannot be obtained, lacking the acquisition of fine and complete morphological information of neurons.

[0004] The emergence of automated microscopic optical imaging technology, by combining optical tomography technology and precision cutting technology, has achieved sub-micron resolution three-dimensional fine imaging of centimeter-scale large biological tissue samples, revealing the huge differences in the morphological and projection characteristics of different types and anatomically located neurons. However, the existing imaging technology only obtains the morphological information of neurons and cannot simultaneously obtain the rich molecular information of neurons.

[0005] In summary, the existing technical solutions have limitations in practical applications and can only obtain the fine and complete morphology or omics molecular information of neurons separately. This limitation results in the inability to correlate the complete morphological information with the molecular information at the precision level of a single neuron, thus unable to provide multi-dimensional fusion information for the accurate typing of neurons. Summary of the Invention

[0006] The object of the present invention is to provide a method and system for simultaneously obtaining the complete morphology and omics molecular information of a single neuron. This method, while obtaining the fine morphology of neurons through whole-brain imaging, preserves the sections and extracts the cell bodies of the same neurons from them, and then obtains molecular information through single-cell omics analysis, and further integrates to obtain the complete morphology and omics molecular information of the same neuron.

[0007] To achieve this object, the present invention provides the following technical solutions:

[0008] On the one hand, the present invention provides a method for simultaneously obtaining the complete morphology and omics molecular information of a single neuron, including the following steps:

[0009] Obtain a sample in which the cell body, dendrites and axons of neurons are fluorescently labeled;

[0010] Perform a cyclic operation of scanning imaging and cutting on the cross-section of the sample until the entire sample is scanned and imaged, and collect the sections containing the fluorescently labeled neuron cell bodies obtained by cutting;

[0011] Based on the three-dimensional image of the sample cross-section obtained by the scanning imaging operation, reconstruct the complete morphology of the neuron, and extract the fluorescently labeled neuron cell bodies from the collected sections, and obtain the omics molecular information of the neuron through omics analysis.

[0012] According to an embodiment of the present invention, the process of performing a cyclic operation of scanning imaging and cutting on the cross-section of the sample until the entire sample is scanned and imaged, and collecting the sections containing the fluorescently labeled neuron cell bodies obtained by cutting includes:

[0013] S3, scan and image the cross-section of the sample to obtain a three-dimensional image of the sample cross-section, and move the sample to the position where the tool is located;

[0014] S4, judge whether the section to be cut next contains a fluorescently labeled neuron cell body according to the three-dimensional image of the sample cross-section. If so, enter step S6; otherwise, enter step S5;

[0015] S5, the tool cuts the shallow part of the imaged sample and discards the section generated by cutting, and then enters step S7;

[0016] S6, the tool cuts the shallow part of the imaged sample and collects the section generated by cutting, and then enters step S7;

[0017] S7, judge whether the imaging of the entire sample is completed. If not, jump to step S3 to scan and image the newly exposed cross-section of the sample.

[0018] According to an embodiment of the present invention, the complete morphology of neurons is reconstructed from the three-dimensional image of the sample cross-section obtained by the scanning imaging operation; and the processing of extracting the cell bodies of fluorescently labeled neurons from the collected sections and obtaining the omics molecular information of the neurons through omics analysis includes:

[0019] S8, obtaining all the three-dimensional images of the sample cross-sections and the sections containing the cell bodies of the fluorescently labeled neurons;

[0020] S9, screening out the three-dimensional images of the sample cross-sections containing the fluorescently labeled neurons from all the three-dimensional images of the sample cross-sections, and reconstructing the fine and complete morphology of the neurons based on the three-dimensional images of the sample cross-sections containing the fluorescently labeled neurons;

[0021] S10, extracting the corresponding cell bodies of the fluorescently labeled neurons from the sections containing the cell bodies of the fluorescently labeled neurons, and obtaining the omics molecular information of the neurons through omics analysis.

[0022] According to an embodiment of the present invention, the processing of obtaining a sample in which the cell bodies, dendrites and axons of neurons have been fluorescently labeled includes:

[0023] S1, using the fluorescent labeling method to label the cell bodies, dendrites and axons of mouse brain neurons;

[0024] S2, using the resin embedding technique to embed the fluorescently labeled mouse brain, and preparing a sample with a micro-scale cutting hardness.

[0025] In a further improved solution, the above method further includes the step of performing a joint analysis on a single neuron based on the complete morphology and omics molecular information.

[0026] On the other hand, the present invention also provides a system for simultaneously obtaining the complete morphology and omics molecular information of a single neuron, including:

[0027] An imaging unit for scanning and imaging the cross-section of a sample in which the cell bodies, dendrites and axons of neurons have been fluorescently labeled to obtain a three-dimensional image of the sample cross-section;

[0028] A sectioning unit for performing a cutting operation on the sample to obtain sections;

[0029] A moving platform for driving the sample to move back and forth between the imaging unit and the sectioning unit;

[0030] A collection unit for collecting the sections containing the cell bodies of the fluorescently labeled neurons obtained by cutting;

[0031] An analysis unit for determining whether a slice contains a fluorescently labeled neuron cell body, reconstructing the complete morphology of neurons based on the three-dimensional image of the sample cross-section, extracting the fluorescently labeled neuron cell bodies from the collected slices, and obtaining the omics molecular information of the neurons through omics analysis.

[0032] The imaging unit includes a camera and a microscopic optical imaging module.

[0033] The slicing unit includes a tank and a cutter. The tank is used to hold the processing fluid, the sample is immersed in the processing fluid, and the cutter is used to cut the sample to form slices.

[0034] The collection unit includes a delivery pipe, a water pump, a sorting pipe, a receiving container, and a collector. One end of the delivery pipe is connected to the water pump, and the other end faces the cutter to suck the slices. One end of the sorting pipe is connected to the water pump, and is used to sort the slices containing fluorescently labeled neuron cell bodies collected in the delivery pipe into the collector, and sort the slices containing no fluorescently labeled neuron cell bodies collected in the delivery pipe into the receiving container.

[0035] In the above solution, the sorting pipe is connected to the water pump, and the slices in the delivery pipe can enter the sorting pipe. Then, the slices containing fluorescently labeled neuron cell bodies directly enter the collector, and the slices containing no fluorescently labeled neuron cell bodies directly enter the receiving container. It can not only achieve automatic sorting of slices, but also the pipeline structure of the whole system is simple, and the control method is simple. It only needs to control the slices in the delivery pipe to enter the receiving container or the collector. Moreover, the flow mode of the slices with the processing fluid in the pipeline is downstream unidirectional flow, and it is not easy to block the pipeline.

[0036] The collection unit further includes a first branch pipe, a second branch pipe, a first solenoid valve, and a second solenoid valve. One end of the first branch pipe is connected to the receiving container, and the other end is connected to the sorting pipe. The first solenoid valve is installed on the first branch pipe; one end of the second branch pipe is connected to the sorting pipe, and the other end faces the collector. The second solenoid valve is installed on the second branch pipe.

[0037] In the above solution, the solenoid valve cooperates with the branch pipe, which can more accurately achieve slice sorting, prevent the slices containing no fluorescently labeled neuron cell bodies from entering the collector, and also prevent the slices containing fluorescently labeled neuron cell bodies from entering the receiving container, ensuring the accurate and reliable sorting of the two types of slices.

[0038] Compared with the prior art, the present invention has the following technical advantages:

[0039] A novel idea is proposed to ingeniously combine microscopic optical imaging and omics sequencing analysis at the single-cell precision level: a method of combining scanning imaging with sectioning is used to obtain high-resolution fine and complete morphological data of fluorescently labeled neurons. Meanwhile, sections containing the cell bodies of fluorescently labeled neurons are collected, and the cell bodies are extracted from them for omics analysis to obtain molecular information. This solution can effectively integrate the fine and complete morphology and omics molecular information of the same neuron, successfully solving the long-standing problem that has plagued the accurate classification of neurons - the inability to obtain these two key pieces of information at the single-neuron precision level.

[0040] Other advantages of the present invention can be found in the relevant descriptions in the embodiment section. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following will briefly introduce the drawings required for use in the embodiments or the description of the prior art. Obviously, the following drawings are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0042] Figure 1 It is a flowchart of the method for simultaneously obtaining the complete morphology and omics molecular information of a single neuron in the present invention.

[0043] Figure 2 It is a schematic diagram of injecting virus to label the fine and complete morphology of neurons and preparing samples by resin embedding in the test example of the present invention.

[0044] Figure 3 It is a schematic diagram of scanning imaging of the sample cross-section and collecting sections containing the cell bodies of fluorescently labeled neurons in the test example of the present invention.

[0045] Figure 4 It is a schematic diagram of extracting neuron cell bodies from sections and obtaining molecular information through omics analysis in the test example of the present invention.

[0046] Figure 5 It is a schematic diagram of jointly analyzing a single neuron based on morphological and omics molecular information in the test example of the present invention.

[0047] Figure 6 It is a schematic diagram of the system structure for simultaneously obtaining the complete morphology and omics molecular information of a single neuron in the present invention.

[0048] The names represented by the reference numerals in the drawings are as follows:

[0049] 1. Mouse brain; 2. Inject virus; 3. Sample; 4. Fluorescently labeled neurons with fine and complete morphology; 5. Camera; 6. Microscopic optical imaging module; 7. Tank; 8. Aqueous solution; 9. Tool; 10. Delivery tube; 11. Section; 12. Three-dimensional image of the sample cross-section; 13. Section containing fluorescently labeled neuron cell bodies; 14. Cell bodies of fluorescently labeled neurons; 15. Single-cell omics analysis device; 16. Single-cell omics analysis data; 17. Fine and complete morphology of neurons; 18. Water pump; 19. Receiver; 20. First branch pipe; 21. First solenoid valve; 22. Second solenoid valve; 23. Second branch pipe; 24. Collection plate; 25. Liquid collection tank; 26. On the X-Y translation stage; 27. Computer; 28. Three-dimensional image data of the sample cross-section; 29. Control signal; 30. Flow direction of the section containing fluorescently labeled neuron cell bodies; 31. Flow direction of the section not containing fluorescently labeled neuron cell bodies; 32. Sorting tube. Detailed implementation manner

[0050] In order to make the objectives, technical solutions and advantages of the present invention clearer and more understandable, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0051] Please refer to Figure 1 , the method for simultaneously obtaining the complete morphology and omics molecular information of single neurons provided in this embodiment includes the following steps:

[0052] S1. Use the fluorescent labeling method to label the fine morphology of mouse brain neurons, including cell bodies, dendrites and axons.

[0053] In this step, inject virus into the target brain region of a specific transgenic mouse type, so that the virus infects neurons and expresses fluorescent proteins, thereby realizing the fluorescent labeling of the fine morphology of specific types of neurons in this brain region. A specific transgenic mouse type refers to a mouse strain in which foreign genes are introduced into the mouse body through genetic engineering technology and can stably inherit and express these foreign genes. Fluorescently labeling the fine morphology of mouse brain neurons is a commonly used technique in current neuroscience research, and the specific processing details are not elaborated here.

[0054] S2. Embed in resin to prepare a sample.

[0055] In this step, use the resin embedding technique to embed the mouse brain and prepare a sample with a micron-level cutting hardness.

[0056] Such as Figure 2As shown, an operation of injecting virus 2 is performed on the target brain region of the mouse brain 1 of a specific transgenic type. The virus infects a small number of neurons of a specific type in this brain region and expresses a fluorescent protein in the neurons, thereby generating neurons 4 with finely complete and morphologically fluorescently labeled in the mouse brain 1. Then, a sample 3 with a micro-level cutting hardness is prepared by resin embedding to meet the precise cutting requirements in subsequent imaging processes.

[0057] In this embodiment, GMA resin is used to embed the sample. First, the perfused mouse brain sample is fixed with a mixed solution of 4% paraformaldehyde and 0.1% glutaraldehyde at 4°C for 12 - 24 hours to shape the tissue; then gradient dehydration is carried out, and the sample is successively placed in 50%, 75%, 95%, and 100% ethanol solutions to remove the moisture in the sample; then a GMA resin solution is prepared and mixed with an initiator in a certain proportion, and the dehydrated sample is subjected to gradient infiltration and successively placed in 50%, 75%, and 100% GMA resin solutions; then it infiltrates in 100% GMA resin for 48 - 72 hours and the resin is changed 2 times to ensure that the resin fully infiltrates the sample; finally, the sample is placed in a gelatin capsule and polymerized at a set temperature to cure the resin and form a resin-embedded sample block, which is convenient for subsequent microtomy operations and at the same time maintains the ultrastructure of the sample and the integrity of the fluorescent signal.

[0058] It should be noted that if there are ready-to-use samples, steps S1 and S2 above do not need to be performed.

[0059] S3, Scanning and imaging the cross-section of the sample to obtain a three-dimensional image of the sample cross-section.

[0060] As Figure 3 shown, the sample 3 is fixed in a tank 7 filled with an aqueous solution 8, and a camera 5 and a microscopic optical imaging module 6 are used to scan and image the cross-section of the sample to obtain a three-dimensional image of the sample cross-section.

[0061] It should be noted that Figure 3 does not fix two samples 3 in the tank 7, but means that after the sample is scanned and imaged, it is moved to the position of the tool 9 for slicing, that is, Figure 3 the sample 3 shown on the right in

[0062] is actually the state after the left sample is moved. The tank 7 is fixed on a moving platform. After the sample 3 completes scanning and imaging, it is driven by the moving platform to move to the position of the tool 9. After the tool 9 slices the sample 3, the moving platform drives the tank 7 to move below the microscopic optical imaging module 6 for scanning and imaging of the sample 3.

[0063] Based on the tomographic imaging results of the sample cross-section obtained in step S3, the dynamic threshold segmentation method and morphological operations are used to quickly identify and determine whether the cell bodies of the fluorescently labeled intact and fine neurons 4 are included therein.

[0064] In cell body recognition, the dynamic threshold segmentation method can effectively handle the situation of uneven background or large changes in cell body gray level. By assigning different thresholds to different regions of the image, it can better adapt to the local gray level changes in the cell body recognition task. Morphological operations refer to changing the shape of a binary image to enhance or remove certain features when processing the binary image, thereby optimizing the cell body recognition result. By combining these two methods, the accuracy and robustness of cell body recognition can be significantly improved. The dynamic threshold segmentation method and morphological operations are common techniques for cell body recognition, and the specific processing details will not be elaborated here.

[0065] S5. The tool cuts the shallow part of the sample that has been imaged, discards the slices generated by the cutting, and then proceeds to step S7.

[0066] For reference Figure 3 , the shallow part of the sample 3 that has been imaged is cut off by the tool 9, and the slices that do not contain the cell bodies of the fluorescently labeled neurons are discarded. The tool 9 is a diamond tool, and the slice thickness is less than or equal to the depth range of the sample cross-section scanning imaging in step S3.

[0067] S6. The tool cuts the shallow part of the sample that has been imaged, collects the slices generated by the cutting, and then proceeds to step S7.

[0068] For reference Figure 3 and Figure 6 , the shallow part of the sample 3 that has been imaged is cut off by the tool 9, and the slices 13 that contain the cell bodies of the fluorescently labeled neurons are collected. The fluid slice collection method is adopted: the port of the delivery pipe 10 is placed near the cutting edge, and by using the aqueous solution 8 continuously flowing into the delivery pipe 10, the slices 11 generated by the cutting can be driven into the delivery pipe 10 to achieve slice collection.

[0069] S7. Determine whether the imaging of the entire sample is completed. If not, jump to step S3 to perform tomographic imaging on the newly exposed sample cross-section; if it is completed, execute step S8.

[0070] To determine whether the imaging of the entire sample is completed, it is directly judged based on the current cross-section imaging results whether there is still a sample. If there is, it means that the imaging of the regular sample is not completed; otherwise, the imaging is completed.

[0071] S8. Obtain the three-dimensional image of the sample cross-section and the slices that contain the cell bodies of the fluorescently labeled neurons.

[0072] By continuously and cyclically performing sample cross-section scanning imaging and cutting until the imaging of the entire sample 3 is completed, a complete three-dimensional image 12 of the sample cross-section and a section 13 containing fluorescently labeled neuron cell bodies are obtained, as Figure 3 shown.

[0073] S9, Reconstruct the fine and complete morphology of neurons based on the three-dimensional image of the sample cross-section.

[0074] As Figure 5 shown, using all the obtained three-dimensional images 12 of the sample cross-section, select the three-dimensional images of the sample cross-section containing fluorescently labeled neurons, and reconstruct the fine and complete morphology 17 of the fluorescently labeled neurons. After obtaining the three-dimensional image of the sample cross-section, identify and track the neuron cell bodies, dendrites, and axon fibers in the image through image preprocessing and automatic / semi-automatic methods, so as to obtain the complete neuron morphology.

[0075] S10, Extract the corresponding cell bodies of the fluorescently labeled neurons from the sections containing fluorescently labeled neuron cell bodies, and obtain the omics molecular information of the neurons through omics analysis.

[0076] As Figure 4 shown, adopt laser microdissection or capillary micro-needle sampling methods to extract the cell bodies 14 of the fluorescently labeled neurons from the collected sections 13 containing fluorescently labeled neuron cell bodies, and analyze to obtain the single-cell omics analysis data 16 of the neurons through the single-cell omics analysis device 15. The single-cell omics analysis device 15 adopted uses single-cell transcriptome sequencing technology for analysis. By extracting the RNA of a single cell and performing high-throughput sequencing, the transcriptome information of a single cell is obtained.

[0077] S11, Perform a joint analysis of the complete morphology and omics molecular information of a single neuron.

[0078] As Figure 5 shown, use the fine and complete morphology 17 of the neurons and the single-cell omics analysis data 16 obtained in steps S9 and S10 respectively to perform a joint analysis based on the fine and complete morphology of the neurons and omics molecular information.

[0079] In this embodiment, by way of example, the joint analysis based on the fine and complete morphology of the neurons and omics molecular information may include the following steps:

[0080] S111, Extract features from the fine and complete morphology of the neurons, such as the number of branches, the length of the protrusions, the branching angle, the dendritic complexity, the cell volume, etc., and construct a morphological feature matrix.

[0081] S112, Perform quality control, alignment, and counting on the RNA transcriptome data, and construct a gene expression matrix.

[0082] S113. Perform dimensionality reduction on the morphological feature matrix and the gene expression matrix respectively. Methods such as principal component analysis (PCA), t-SNE, or UMAP can be used to extract the main feature information, and the dimensionality-reduced morphological features and gene expression features are obtained respectively.

[0083] S114. Integrate the dimensionality-reduced morphological features and gene expression features using methods such as feature splicing or weighted fusion to construct a comprehensive feature matrix, and then use a clustering algorithm to perform clustering analysis on the comprehensive feature matrix to group neurons with similar morphological and gene expression patterns into one category. The clustering algorithm can be, for example, hierarchical clustering, k-means clustering, graph-based clustering methods, etc.

[0084] S115. Display the clustering results through visualization methods such as drawing a clustering dendrogram, heat map, scatter plot, etc.

[0085] Differential analysis and functional enrichment analysis of gene expression between different clustering groups can also be performed to understand the roles of these genes in biological processes. At the same time, the clustering results are combined with the morphological features of neurons to analyze the morphological differences between neurons in different clustering groups, such as branch complexity, process length, cell volume, etc., and explore the correlation between morphological features and gene expression.

[0086] Adopt a slice-based whole-brain imaging method to perform imaging-slice cycles on the sample to obtain whole-brain imaging pictures, and then fine and complete morphologies of individual neurons can be obtained therefrom; during the imaging process, identify the target soma according to the real-time imaging results, collect the slices containing the soma, and extract the soma from the slices for omics analysis. The present invention efficiently couples and correlates the two technologies of obtaining neuron morphological information by whole-brain imaging and single-cell tissue analysis at the precision level of a single cell, and realizes the acquisition of omics molecular information and complete morphological information of the same neuron.

[0087] This embodiment also provides a system for simultaneously obtaining the complete morphology and omics molecular information of a single neuron. Figure 1 The method shown is implemented based on this system.

[0088] Specifically, please refer to Figure 6, the system mainly includes an imaging unit, a slicing unit, a collection unit, and an analysis unit. The imaging unit is mainly used to scan and image the cross-section of a sample in which neuron cell bodies, dendrites, and axons have been fluorescently labeled to obtain a three-dimensional image of the sample cross-section; the slicing unit is mainly used to perform cutting operations on the sample to obtain slices; the collection unit is mainly used to collect the slices containing fluorescently labeled neuron cell bodies obtained by cutting; the analysis unit is mainly used to determine whether the slices contain fluorescently labeled neuron cell bodies, and based on the three-dimensional image of the sample cross-section obtained by the scanning imaging operation, reconstruct the complete morphology of the neurons, and extract the cell bodies of the fluorescently labeled neurons from the collected slices, and obtain the omics molecular information of the neurons through omics analysis. The analysis unit can be implemented by a computer 27.

[0089] In the implementation process, the cross-section of the sample is first scanned and imaged, and then cut, and these two operations are performed cyclically. The scanning imaging and cutting operations are carried out at different workstations. Therefore, in order to facilitate the movement of the sample from the imaging position to the cutting position, the above system may further include a moving platform for driving the sample to move back and forth between the imaging unit and the slicing unit.

[0090] More specifically, refer to Figure 3 and Figure 6 , the imaging unit includes a camera 5 and a microscopic optical imaging module 6. The three-dimensional image data 28 of the sample cross-section obtained by scanning imaging is transmitted to the computer 27 to determine whether the slices to be obtained by cutting contain fluorescently labeled neuron cell bodies.

[0091] Please refer to Figure 3 and Figure 6 , the slicing unit includes a tank 7 and a cutting tool 9. The tank 7 is used to hold the processing fluid. In the test example, the processing fluid is an aqueous solution 8. The sample 3 is immersed in the aqueous solution 8. The cutting tool 9 uses a diamond cutting tool and is installed above the sample 3, and is used to cut the sample 3 to form a slice 11.

[0092] Refer to Figure 6 , the collection unit may include a delivery pipe 10, a water pump 18, a sorting pipe 32, a receiver 19, and a collector. The delivery pipe 10 is fixed above the cutting edge of the cutting tool 9, one end is immersed in the processing fluid and faces the cutting tool 9, and this end is beveled to more conveniently suck the slices, and the other end is connected to the water pump 18 for sucking the processing fluid containing the slices. In this embodiment, the water pump 18 uses a peristaltic pump. This pump deforms the rubber hose by the rotor to push the water flow in the hose forward. For slices with a small size (such as slices of resin-embedded samples with a width of several millimeters), they can pass through the peristaltic pump smoothly under the drive of the water flow without breaking or being damaged.

[0093] One end of the sorting tube 32 is connected to the water pump 18, which is used to sort the slices 13 containing fluorescently labeled neuron cell bodies collected in the delivery tube 10 into the collector, and to sort the slices collected in the delivery tube 10 that do not contain fluorescently labeled neuron cell bodies into the receiving container 19. The receiving container 19 is a leak-proof container that can collect the discarded slices and the processing fluid.

[0094] To achieve the automatic sorting of slices containing and not containing fluorescently labeled neuron cell bodies, the collection unit may further include a first branch pipe 20, a second branch pipe 23, a first solenoid valve 21, and a second solenoid valve 22. One end of the first branch pipe 20 is connected to the receiving container 19, and the other end is connected to the sorting tube 32. The first solenoid valve 21 is installed on the first branch pipe 20 to control the entry of slices that do not contain fluorescently labeled neuron cell bodies into the receiving container 19. One end of the second branch pipe 23 is connected to the sorting tube 32, and the other end faces the collector. The second solenoid valve 22 is installed on the second branch pipe 23 to control the entry of slices 13 containing fluorescently labeled neuron cell bodies into the collector. The computer 27 sends control signals 29 to the first solenoid valve 21 and the second solenoid valve 22, and the corresponding valves can be automatically opened and closed.

[0095] The delivery tube 10, the first branch pipe 20, and the second branch pipe 23 are all made of transparent polytetrafluoroethylene hard tubes, with smooth tube walls and low friction coefficients, and excellent corrosion resistance.

[0096] As Figure 6 shown, the collector may include a collection plate 24 and a liquid collection tank 25. The collection plate 24 is a perforated plate, that is, a number of holes are provided on the collection plate 24, and a filter screen is arranged at each hole. The liquid collection tank 25 is arranged below the collection plate 24. The processing fluid with slices flows into the holes of the collection plate 24, the slices remain on the filter screen, and the processing fluid flows through the filter screen into the lower liquid collection tank 25. The processing fluid can be effectively collected through the liquid collection tank 25 to ensure the cleanliness and hygiene of the environment, and it is more conducive to the collection of slices on the filter screen.

[0097] One hole in the collection plate 24 can only collect one slice. Therefore, during the slice collection process, the collection plate 24 needs to be continuously moved so that the idle hole is aligned with the nozzle of the second branch pipe 23. In order to achieve fully automated collection and sorting, the collection plate 24 can be fixed on the X-Y translation stage 26 (the X-Y translation stage 26 refers to a mechanism that can move along the X-axis and the Y-axis. The two double-headed arrows in the figure respectively represent the movement directions along the X-axis and the Y-axis). The X-Y translation stage 26 drives the collection plate 24 to move so as to align different holes of the collection plate 24 with the second branch pipe 23 for sequentially packaging different slices in different holes. In this embodiment, in order to facilitate the installation of the X-Y translation stage 26, the collection plate 24 is fixed on the liquid collection tank 25, the liquid collection tank 25 is fixed on the X-Y translation stage 26, and the X-Y translation stage 26 drives the liquid collection tank 25 to move, and the collection plate 24 can follow the liquid collection tank 25 to move synchronously.

[0098] The above-described embodiments are only specific implementation manners of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art within the technical scope disclosed by the present invention can easily think of various equivalent modifications, substitutions, and improvements, etc. These modifications, substitutions, and improvements should all be covered within the protection scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.

Claims

1. A method for simultaneously acquiring complete morphological and omics molecular information of a single neuron, characterized in that: The following steps are involved: Obtain samples in which the cell bodies, dendrites, and axons of neurons have been fluorescently labeled; Performing a cyclic operation of scanning imaging and cutting on the cross section of the sample until the scanning imaging of the entire sample is completed, and collecting the slices obtained by cutting that contain fluorescently labeled neuronal cell bodies; Based on the three-dimensional images of the sample cross-section obtained by scanning imaging operations, the complete morphology of the neuron is reconstructed, and the fluorescently labeled neuronal cell bodies are extracted from the collected slices, and the omics molecular information of the neuron is obtained through omics analysis.

2. A method for simultaneously acquiring complete morphology and omics molecular information of a single neuron according to claim 1, characterized in that: The cyclic operation of scanning and imaging the cross section of the sample and cutting until the scanning and imaging of the entire sample is completed, and the processing of the slices containing fluorescently labeled neuronal cell bodies obtained by cutting is collected, including: S3, scanning and imaging the sample cross section to obtain a three-dimensional image of the sample cross section, and moving the sample to the position of the tool; S4, judging whether the slice to be cut contains fluorescently labeled neuronal cell bodies according to the three-dimensional image of the sample cross section, if yes, proceeding to step S6, otherwise proceeding to step S5; S5, the tool cuts the imaged shallow part of the sample, and discards the slices produced by the cutting, and then enters step S7; S6, the tool cuts the imaged shallow part of the sample, collects the slices produced by the cutting, and then enters step S7; S7, judging whether the imaging of the whole sample is completed, if not, jumping to step S3, scanning and imaging the newly exposed sample section.

3. A method for simultaneously acquiring complete morphology and omics molecular information of a single neuron according to claim 2, characterized in that: The three-dimensional image of the sample cross section obtained based on the scanning imaging operation is used to reconstruct the complete morphology of the neuron; And extract the cell bodies of fluorescently labeled neurons from the collected slices, and obtain the molecular information of the cells through omics analysis. The processing of the omics molecular information of the neurons includes: S8, obtain three-dimensional images of all sample sections and slices containing fluorescently labeled neuronal cell bodies; S9, selecting the sample cross-sectional three-dimensional images containing fluorescently labeled neurons from all the sample cross-sectional three-dimensional images, and reconstructing the detailed and complete morphology of the neurons based on the sample cross-sectional three-dimensional images containing fluorescently labeled neurons; S10, extracting the cell bodies of corresponding fluorescently labeled neurons from the slices containing fluorescently labeled neuronal cell bodies, and obtaining the omics molecular information of the neurons through omics analysis.

4. A method for simultaneously acquiring complete morphology and omics molecular information of a single neuron according to claim 1, characterized in that: The processing of obtaining the sample whose neuronal cell bodies, dendrites and axons have been fluorescently labeled comprises: S1, the cell bodies, dendrites, and axons of mouse brain neurons were labeled using fluorescent labeling; S2, the fluorescently labeled mouse brain was embedded using resin embedding technology to prepare a sample with micron-level cutting hardness.

5. The method for simultaneously acquiring complete morphology and omics molecular information of a single neuron according to claim 1, characterized in that: It also includes the step of conducting a joint analysis of single neurons based on complete morphological and omics molecular information.

6. A system for simultaneously acquiring complete morphological and omics molecular information of a single neuron, characterized in that: include: An imaging unit is used to scan and image the cross section of a sample whose cell bodies, dendrites and axons have been fluorescently labeled, to obtain a three-dimensional image of the sample cross section; A slicing unit, used for performing a cutting operation on the sample to obtain slices; A moving platform, used for driving the sample to move back and forth between the imaging unit and the slicing unit; A collecting unit, used for collecting the slices obtained by cutting and containing the fluorescently labeled neuronal cell bodies; The analysis unit is used to determine whether the slice contains fluorescently labeled neuronal cell bodies, and to reconstruct the complete morphology of neurons based on the three-dimensional image of the sample section, and to extract fluorescently labeled neuronal cell bodies from the collected slices, and to obtain the omics molecular information of the neurons through omics analysis.

7. A system for simultaneously acquiring complete morphology and omics molecular information of a single neuron according to claim 6, characterized in that: The imaging unit includes a camera and a microscopic optical imaging module.

8. A system for simultaneously acquiring complete morphology and omics molecular information of a single neuron according to claim 6, characterized in that: The slicing unit comprises a trough body and a cutter, wherein the trough body is used to contain a processing liquid, the sample is immersed in the processing liquid, and the cutter is used to cut the sample to form a slice.

9. A system for simultaneously acquiring complete morphology and omics molecular information of a single neuron according to claim 8, characterized in that: The collecting unit comprises a conveying tube, a water pump, a sorting tube, a collecting container and a collector. One end of the conveying tube is connected to the water pump, and the other end faces the tool to absorb the slices. One end of the sorting tube is connected to the water pump, and is used to sort the slices containing fluorescently labeled neuronal cell bodies collected in the conveying tube into the collector, and to sort the slices not containing fluorescently labeled neuronal cell bodies collected in the conveying tube into the collecting container.

10. A system for simultaneously acquiring complete morphology and omics molecular information of a single neuron according to claim 9, characterized in that: The collection unit also includes a first branch pipe, a second branch pipe, a first solenoid valve, and a second solenoid valve. One end of the first branch pipe is connected to the collecting container, and the other end is connected to the sorting tube. The first solenoid valve is installed on the first branch pipe; one end of the second branch pipe is connected to the sorting tube, and the other end faces the collector. The second solenoid valve is installed on the second branch pipe.

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