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

Abstract

The application relates to a method and system for simultaneously obtaining complete morphology and omics molecular information of a single neuron, which comprises the following steps: obtaining a sample in which the cell body, dendrite and axon of a neuron are labeled by fluorescence; performing a cycle operation of scanning imaging and cutting on the section of the sample until the scanning imaging of the whole sample is completed, and collecting the cut section containing the fluorescence-labeled neuron cell body; based on the three-dimensional image of the sample section obtained by the scanning imaging operation, reconstructing the complete morphology of the neuron, and extracting the fluorescence-labeled neuron cell body from the collected section to obtain the omics molecular information of the neuron by omics analysis. The application can effectively integrate the fine complete morphology and omics molecular information of the same neuron, and successfully solves the problem that the two key information cannot be simultaneously obtained at the single neuron precision level.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a method and system for simultaneously acquiring the complete morphology and omics molecular information of a single neuron. Background Technology

[0002] In the field of neuroscience research, the diversity of neurons is a core issue. Neurons exhibit rich diversity in molecular composition, functional properties, morphological structure, and interconnections. To better understand and classify these complex neuronal types, scientists typically classify them based on multiple criteria, including detailed morphology, molecular information, and electrophysiological characteristics. The complete morphology of a neuron not only provides detailed morphological features and precise anatomical location but also constitutes the true structural basis of neural circuits, serving as a key basis for defining neuronal types. Cell typing methods based on molecular information obtain and classify rich intrinsic molecular information within cells through omics analysis techniques such as RNA transcriptomics, epigenomics, and proteomics. Combining the complete morphological information of neurons with molecular information can provide multi-dimensional fusion information for the accurate classification of neurons, which is of great significance in neuroscience research.

[0003] The rapid development of various omics analysis and imaging technologies based on molecular and morphological information in recent years has greatly facilitated the study of neuronal typing. The development of single-cell transcriptome sequencing technology has enabled the tracking of cell lineages in multiple brain regions and even throughout the entire brain, and has established cell type databases. The application of spatial transcriptomics technology allows researchers to obtain spatially distributed molecular maps of the omics from mouse brain slices. However, these omics analyses all target manually dissected nuclei / brain regions, extracting single cells or groups of cells, and then using sequencing technology to obtain omics analysis information from these cells. This process only utilizes the neuronal cell body and cannot obtain the complex morphological information of the neuron's dendrites and axons, lacking the acquisition of detailed and complete morphological information of the neuron.

[0004] The advent of automated microscopic optical imaging technology, by combining optical tomography and precision cutting techniques, has enabled sub-micron resolution three-dimensional fine imaging of large samples of biological tissue at the centimeter level, revealing significant differences in morphology and projection characteristics among neurons of different types and anatomical locations. However, existing imaging techniques only acquire morphological information of neurons and cannot simultaneously obtain the rich molecular information of neurons.

[0005] In summary, existing technical solutions have limitations in practical applications, only able to acquire detailed and complete morphological or omics molecular information of neurons separately. This limitation prevents the correlation between complete morphological information and molecular information at the precision level of a single neuron, thus failing to provide multi-dimensional fusion information for accurate neuronal typing. Summary of the Invention

[0006] The purpose of this invention is to provide a method and system for simultaneously acquiring the complete morphology and molecular omics information of a single neuron. This method acquires the fine morphology of neurons through whole-brain imaging, preserves the slices, extracts the cell bodies of the same neurons from them, and then acquires molecular information through single-cell omics analysis, thereby integrating the complete morphology and molecular omics information of the same neuron.

[0007] To achieve this objective, the present invention provides the following technical solution:

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

[0009] Obtain fluorescently labeled samples of neuronal cell bodies, dendrites, and axons;

[0010] The cross-section of the sample is subjected to a cyclical operation of scanning imaging and cutting until the scanning imaging of the entire sample is completed, and the slices containing fluorescently labeled neuronal cell bodies obtained from the cutting are collected.

[0011] Based on the three-dimensional images of sample cross-sections obtained from scanning imaging, the complete morphology of neurons is reconstructed, and fluorescently labeled neuronal cell bodies are extracted from the collected slices. Omics analysis is then used to obtain the molecular omics information of the neurons.

[0012] According to an embodiment of the present invention, the cyclical operation of scanning and imaging the cross-section of the sample and cutting it until the scanning and imaging of the entire sample is completed, and the processing of collecting the slices containing fluorescently labeled neuronal cell bodies obtained from the cutting, includes:

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

[0014] S4. Determine whether the slice to be cut contains fluorescently labeled neuronal cell bodies based on the three-dimensional image of the sample cross-section. If yes, proceed to step S6; otherwise, proceed to step S5.

[0015] S5, the tool cuts the shallow part of the sample that has been imaged, discards the cut slices, and then proceeds to step S7;

[0016] S6, the tool cuts the shallow part of the sample that has been imaged and collects the cut slices, and then proceeds to step S7;

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

[0018] According to an embodiment of the present invention, the process of reconstructing the complete morphology of a neuron from a three-dimensional image of a sample cross-section obtained based on a scanning imaging operation, and extracting fluorescently labeled neuronal cell bodies from collected slices and obtaining omics molecular information of the neuron through omics analysis, includes:

[0019] S8, acquire three-dimensional images of all sample cross-sections and slices containing fluorescently labeled neuronal cell bodies;

[0020] S9. Select the three-dimensional cross-sectional images of the sample containing fluorescently labeled neurons from all the sample cross-sectional three-dimensional images, and reconstruct the fine and complete morphology of the neurons based on the sample cross-sectional three-dimensional images of the sample containing fluorescently labeled neurons.

[0021] S10, extract the cell bodies of the corresponding fluorescently labeled neurons from the slices containing fluorescently labeled neuronal cell bodies, and obtain 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 neuronal cell bodies, dendrites, and axons have been fluorescently labeled includes:

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

[0024] S2, using resin embedding technology, was used to embed fluorescently labeled mouse brains to prepare samples with micron-level cutting hardness.

[0025] In a further refined scheme, the above method also includes the step of performing a joint analysis of individual neurons based on complete morphological and omics molecular information.

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

[0027] The imaging unit is used to scan and image cross-sections of fluorescently labeled samples of neuronal cell bodies, dendrites, and axons to obtain three-dimensional images of the sample cross-sections.

[0028] A slicing unit is used to cut the sample to obtain slices;

[0029] A mobile platform is used to move the sample back and forth between the imaging unit and the slicing unit;

[0030] A collection unit for collecting slices containing fluorescently labeled neuronal cell bodies obtained from cutting;

[0031] The analysis unit is used to determine whether the slice contains fluorescently labeled neuronal cell bodies, to reconstruct the complete morphology of the neuron based on the three-dimensional image of the sample cross-section, and to extract fluorescently labeled neuronal cell bodies from the collected slices, and to obtain the omics molecular information of the neuron 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 cutting tool. The tank is used to hold a processing fluid, the sample is immersed in the processing fluid, and the cutting tool 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 pick up slices. One end of the sorting pipe is connected to the water pump and is used to sort slices containing fluorescently labeled neuronal cell bodies collected in the delivery pipe into the collector, and to sort slices without fluorescently labeled neuronal cell bodies collected in the delivery pipe into the receiving container.

[0035] In the above scheme, the sorting tube is connected to the water pump, and the slices in the delivery tube can enter the sorting tube. Then, the slices containing fluorescently labeled neuronal inclusions go directly into the collector, and the slices without fluorescently labeled neuronal inclusions go directly into the receiving container. This can realize automatic sorting of slices. Moreover, the pipeline structure of the whole system is simple and the control method is simple. It is only necessary to control the slices in the delivery tube to enter the receiving container or collector. In addition, the slices flow with the processing liquid in the pipeline in a unidirectional flow, which 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 collection 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 scheme, the solenoid valve and the branch pipe work together to achieve more accurate slice sorting, preventing slices without fluorescently labeled neuronal bodies from entering the collector, and also preventing slices with fluorescently labeled neuronal bodies from entering the receiving container, thus ensuring the accuracy and reliability of the two types of slice sorting.

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

[0039] This study proposes a novel approach that ingeniously combines microscopic optical imaging with omics sequencing analysis at the single-cell precision level. It employs scanning imaging combined with slicing to acquire high-resolution, detailed, and complete morphological data of fluorescently labeled neurons. Simultaneously, slices containing the cell bodies of fluorescently labeled neurons are collected, and the cell bodies are extracted for omics analysis to obtain molecular information. This approach effectively integrates the detailed and complete morphology of the same neuron with omics molecular information, successfully solving the long-standing problem of accurate neuronal typing—the inability to simultaneously acquire these two key pieces of information at the single-neuron precision level.

[0040] Other advantages of this invention are described in the embodiments section. Attached Figure Description

[0041] To more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0042] Figure 1 This is a flowchart of the method for simultaneously acquiring the complete morphology and omics molecular information of a single neuron according to the present invention.

[0043] Figure 2 This is a schematic diagram of the fine and complete morphology of virus-labeled neurons and the preparation of samples by resin embedding in the experimental examples of this invention.

[0044] Figure 3 This is a schematic diagram of cross-sectional scanning imaging of samples and collection of slices containing fluorescently labeled neuronal cell bodies in an experimental example of the present invention.

[0045] Figure 4 This is a schematic diagram illustrating the extraction of neuronal cell bodies from slices and the acquisition of molecular information through omics analysis in an experimental example of the present invention.

[0046] Figure 5 This is a schematic diagram illustrating the joint analysis of a single neuron based on morphological and omics molecular information in the experimental examples of this invention.

[0047] Figure 6 This is a schematic diagram of the system structure for simultaneously acquiring the complete morphology of a single neuron and omics molecular information according to the present invention.

[0048] The names represented by the labels in the attached diagram are as follows:

[0049] 1. Mouse brain; 2. Injected 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. Cutting tool; 10. Delivery tube; 11. Slice; 12. Three-dimensional image of sample cross-section; 13. Slice containing fluorescently labeled neuronal cell bodies; 14. Fluorescently labeled neuronal cell bodies; 15. Single-cell omics analysis equipment; 16. Single-cell omics analysis data; 17. Fine and complete morphology of neurons; 18. Water pump; 19. Collection container; 20. First branch tube; 21. First solenoid valve; 22. Second solenoid valve; 23. Second branch tube; 24. Collection plate; 25. Collection tank; 26. On XY translation stage; 27. Computer; 28. Three-dimensional image data of sample cross-section; 29. ​​Control signal; 30. Flow direction of slice containing fluorescently labeled neuronal cell bodies; 31. Flow direction of slice without fluorescently labeled neuronal cell bodies; 32. Sorting tube. Detailed Implementation

[0050] To make the objectives, technical solutions, and advantages of this invention clearer, the 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 merely illustrative and not intended to limit the invention.

[0051] Please see Figure 1 The method for simultaneously acquiring the complete morphology and omics molecular information of a single neuron provided in this embodiment includes the following steps:

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

[0053] In this step, a virus is injected into the target brain region of a specific transgenic mouse strain, causing the virus to infect neurons and express fluorescent proteins, thereby achieving fluorescent labeling of the fine morphology of specific neuronal types in that brain region. A specific transgenic mouse strain refers to a mouse breed in which foreign genes have been introduced into the mouse body through genetic engineering techniques, enabling it to stably inherit and express these foreign genes. Fluorescent labeling of the fine morphology of mouse brain neurons is a commonly used technique in neuroscience research; specific processing details are not elaborated here.

[0054] S2, resin embedding is used to prepare the sample.

[0055] In this step, the mouse brain is embedded using resin embedding technology to prepare a sample with micron-level cutting hardness.

[0056] like Figure 2As shown, virus 2 was injected into the target brain region of a specific transgenic mouse brain 1. The virus infected a small number of neurons of a specific type in this brain region and expressed fluorescent proteins in the neurons, thereby producing fluorescently labeled neurons 4 with fine and intact morphology in mouse brain 1. Then, a sample 3 with micron-level cutting hardness was prepared by resin embedding to meet the precision cutting requirements in the subsequent imaging process.

[0057] In this embodiment, GMA resin was used to embed the sample. First, the perfused mouse brain sample was fixed at 4°C for 12-24 hours using a mixture of 4% paraformaldehyde and 0.1% glutaraldehyde to allow the tissue to solidify. Next, a gradient dehydration process was performed, in which the sample was successively immersed in 50%, 75%, 95%, and 100% ethanol solutions to remove water. Then, a GMA resin solution was prepared and mixed with an initiator in a certain proportion. The dehydrated sample was then subjected to a gradient permeation process, in which it was successively immersed in 50%, 75%, and 100% GMA resin solutions. Finally, the sample was infiltrated in 100% GMA resin for 48-72 hours, with the resin being replaced twice to ensure that the resin fully wetted the sample. Finally, the sample was placed in a gelatin capsule and polymerized at a set temperature to solidify the resin, forming a resin-embedded sample block. This facilitates subsequent micron-sectioning operations while maintaining the integrity of the sample's ultrastructure and fluorescence signal.

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

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

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

[0061] It should be noted that, Figure 3 The middle part does not mean that the two samples 3 are fixed in the groove 7, but rather that after the samples are scanned and imaged, they are moved to the right to the position of the cutter 9 to facilitate slicing. Figure 3 The sample 3 shown on the right is actually the state of the sample on the left after it has been moved. The tank 7 is fixed on a moving platform. After the sample 3 is scanned and imaged, it is moved to the position of the cutter 9 by the moving platform. After the cutter 9 slices the sample 3, the moving platform moves the tank 7 to below the microscopic optical imaging module 6 to facilitate scanning and imaging of the sample 3.

[0062] S4. Based on the three-dimensional image of the sample cross-section, determine whether the slice to be cut contains fluorescently labeled neuronal cell bodies, that is, whether it contains target cells. If it does, proceed to step S6; otherwise, proceed to step S5.

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

[0064] In cell body recognition, dynamic thresholding effectively handles situations with uneven backgrounds or significant variations in cell grayscale. By assigning different thresholds to different regions of the image, it can better adapt to local grayscale changes in cell body recognition tasks. Morphological operations, when processing binary images, involve altering the image shape to enhance or remove certain features, thereby optimizing cell body recognition results. Combining these two methods can significantly improve the accuracy and robustness of cell body recognition. Dynamic thresholding and morphological operations are commonly used techniques in cell body recognition; their specific processing details will not be elaborated upon here.

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

[0066] See also Figure 3 The shallow portion of sample 3 that has already been imaged is cut away by tool 9, and the slices that do not contain fluorescently labeled neuronal cell bodies are discarded. 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 image in step S3.

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

[0068] See also Figure 3 and Figure 6 The shallow portion of sample 3 that has been imaged is cut off by the blade 9, and a slice 13 containing fluorescently labeled neuronal cell bodies is collected. A fluid-based slice collection method is used: the port of the delivery tube 10 is placed near the blade, and the aqueous solution 8 continuously flowing into the delivery tube 10 can drive the slice 11 produced by cutting into the delivery tube 10, thereby achieving slice collection.

[0069] S7. Determine whether the imaging of the entire sample is complete. If not, proceed to step S3 to scan and image the newly exposed sample cross-section. If complete, proceed to step S8.

[0070] To determine whether the imaging of the entire sample is complete, we directly judge whether there are still samples based on the current cross-sectional imaging results. If there are, it means that the imaging of the regular sample is not complete; otherwise, the imaging is complete.

[0071] S8, acquire a three-dimensional image of the sample cross-section and a slice containing fluorescently labeled neuronal cell bodies.

[0072] By continuously performing cross-sectional scanning imaging and cutting of the sample until the entire sample 3 is imaged, a complete three-dimensional image 12 of the sample cross-section is obtained, as well as a slice 13 containing fluorescently labeled neuronal cell bodies, such as... Figure 3 As shown.

[0073] S9, based on the reconstruction of three-dimensional images of sample cross-sections, obtains the fine and complete morphology of neurons.

[0074] like Figure 5 As shown, using all the obtained sample cross-sectional three-dimensional images 12, sample cross-sectional three-dimensional images containing fluorescently labeled neurons are selected, and the fine and complete morphology 17 of the fluorescently labeled neurons is reconstructed. After obtaining the sample cross-sectional three-dimensional images, the neuronal cell bodies, dendrites, and axonal fibers in the images are identified and tracked through image preprocessing and automatic / semi-automatic methods, thereby obtaining the complete neuronal morphology.

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

[0076] like Figure 4 As shown, fluorescently labeled neuronal cell bodies 14 were extracted from the collected slice 13 containing fluorescently labeled neuronal cell bodies using laser microdissection or capillary microneedle sampling methods. The single-cell omics analysis data 16 of these neurons were then obtained through analysis using a single-cell omics analysis device 15. The single-cell omics analysis device 15 employs single-cell transcriptome sequencing technology, extracting RNA from individual cells and performing high-throughput sequencing to obtain the transcriptome information of each cell.

[0077] S11 performs a combined analysis of the complete morphology and omics molecular information of a single neuron.

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

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

[0080] S111 extracts features of the fine and complete morphology of neurons, such as the number of branches, protrusion length, branch angle, dendrite complexity, and cell volume, and constructs a morphological feature matrix.

[0081] S112 performs quality control, alignment, and counting of RNA transcriptome data to construct a gene expression matrix.

[0082] S113. Dimensionality reduction is performed on the morphological feature matrix and gene expression matrix respectively. Principal component analysis (PCA), t-SNE, or UMAP can be used to extract the main feature information and obtain the dimensionality-reduced morphological features and gene expression features respectively.

[0083] S114 integrates the dimensionality-reduced morphological and gene expression features using methods such as feature splicing or weighted fusion to construct a comprehensive feature matrix. Then, a clustering algorithm is used to perform cluster analysis on the comprehensive feature matrix, grouping neurons with similar morphology and gene expression patterns into one class. Clustering algorithms can include, for example, hierarchical clustering, k-means clustering, and graph-based clustering methods.

[0084] S115 uses visualization methods such as clustering dendrograms, heatmaps, and scatter plots to display clustering results.

[0085] Differential and functional enrichment analyses of gene expression among different clusters can be performed to understand the role of these genes in biological processes. At the same time, the clustering results can be combined with the morphological characteristics of neurons to analyze the morphological differences of neurons in different clusters, such as branch complexity, process length, and cell volume, and to explore the correlation between morphological characteristics and gene expression.

[0086] This invention employs a slice-based whole-brain imaging method, performing an imaging-slicing cycle on samples to acquire whole-brain images, from which the detailed and complete morphology of individual neurons can be obtained. During the imaging process, target cell bodies are identified based on real-time imaging results, and slices containing these cell bodies are collected. Cell bodies are then extracted from the slices for omics analysis. This invention efficiently couples whole-brain imaging for acquiring neuronal morphological information with single-cell tissue analysis at the single-cell precision level, achieving the acquisition of both omics molecular information and complete morphological information of the same neuron.

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

[0088] For details, please refer to Figure 6The 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 cross-sections of samples with fluorescently labeled neuronal cell bodies, dendrites, and axons to obtain three-dimensional images of the sample cross-sections. The slicing unit is mainly used to cut the samples to obtain slices. The collection unit is mainly used to collect the slices containing fluorescently labeled neuronal cell bodies obtained from the cutting process. The analysis unit is mainly used to determine whether the slices contain fluorescently labeled neuronal cell bodies, and to reconstruct the complete morphology of neurons based on the three-dimensional images of the sample cross-sections obtained from the scanning imaging operation. It also extracts the cell bodies of fluorescently labeled neurons from the collected slices and obtains the omics molecular information of the neurons through omics analysis. The analysis unit can be implemented by a computer 27.

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

[0090] For more details, please 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 a computer 27 to determine whether the slice to be cut contains fluorescently labeled neuronal cell bodies.

[0091] Please see 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 is a diamond cutting tool, which is installed above the sample 3 and is used to cut the sample 3 to form a slice 11.

[0092] See also Figure 6 The collection unit may include a delivery pipe 10, a water pump 18, a sorting pipe 32, a collection container 19, and a collector. The delivery pipe 10 is fixed above the cutting edge of the cutter 9, with one end immersed in the processing fluid and facing the cutter 9. This end is beveled to facilitate the aspiration of the slices. The other end is connected to the water pump 18 to aspirate the processing fluid containing the slices. In this embodiment, the water pump 18 is a peristaltic pump. This type of pump uses a rotor to deform a rubber hose, thereby propelling the water flow forward. For small slices (such as slices of resin-embedded samples a few millimeters wide), they can pass smoothly through the peristaltic pump under the influence of the water flow without breaking or being damaged.

[0093] One end of the sorting tube 32 is connected to the water pump 18, and is used to sort the slices 13 containing fluorescently labeled neuronal cell bodies collected in the delivery tube 10 into the collector, and to sort the slices without fluorescently labeled neuronal cell bodies collected in the delivery tube 10 into the receiving container 19. The receiving container 19 is a watertight container that can collect discarded slices and processing fluid.

[0094] To achieve automatic sorting of slices containing and without fluorescently labeled neuronal 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 pipe 32. The first solenoid valve 21 is installed on the first branch pipe 20 to control the entry of slices without fluorescently labeled neuronal cell bodies into the receiving container 19. One end of the second branch pipe 23 is connected to the sorting pipe 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 neuronal cell bodies into the collector. The computer 27 sends control signals 29 to the first solenoid valve 21 and the second solenoid valve 22 to automatically open and close the corresponding valves.

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

[0096] like Figure 6 As shown, the collector may include a collection plate 24 and a collection tank 25. The collection plate 24 is a perforated plate, meaning it has several holes, each with a filter screen. The collection tank 25 is located below the collection plate 24. Processing fluid containing slices flows into the holes of the collection plate 24, while the slices remain on the filter screen. The processing fluid then flows through the filter screen into the collection tank 25 below. The collection tank 25 effectively collects the processing fluid, ensuring a clean and hygienic environment, and also facilitates the collection of slices from the filter screen.

[0097] Each hole in the collecting plate 24 can only collect one slice. Therefore, during the slice collection process, the collecting plate 24 needs to be continuously moved so that the empty hole is aligned with the opening of the second branch pipe 23. To achieve fully automated collection and sorting, the collecting plate 24 can be fixed on the XY translation stage 26 (the XY translation stage 26 refers to a mechanism that can move along the X-axis and Y-axis; the two bidirectional arrows in the figure represent the movement directions along the X-axis and Y-axis, respectively). The XY translation stage 26 drives the collecting plate 24 to move, aligning different holes of the collecting plate 24 with the second branch pipe 23, so that different slices can be sequentially packed into different holes. In this embodiment, to facilitate the installation of the XY translation stage 26, the collecting plate 24 is fixed on the liquid collection tank 25, and the liquid collection tank 25 is fixed on the XY translation stage 26. The XY translation stage 26 drives the liquid collection tank 25 to move, and the collecting plate 24 can move synchronously with the liquid collection tank 25.

[0098] The embodiments described above are merely specific implementations of the present invention, but the scope of protection of the present invention is not limited thereto. Any person skilled in the art can easily conceive of various equivalent modifications, substitutions, and improvements within the technical scope disclosed in the present invention, and these modifications, substitutions, and improvements should all be covered within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A method for simultaneously acquiring single neuron complete morphology and omics molecular information, characterized in that, The method comprises the following steps: Obtaining a sample in which the cell body, dendrite and axon of a neuron are fluorescently labeled, comprising: S1, labeling the cell body, dendrite and axon of a neuron in a mouse brain by using a fluorescent labeling method; S2, embedding the mouse brain that has been fluorescently labeled by using a resin embedding technique to prepare a sample with micron-level cutting hardness; Performing a cycle of scanning imaging and cutting on the section of the sample until the scanning imaging of the entire sample is completed, and collecting the slices containing the fluorescently labeled neuron cell bodies obtained by cutting; the scanning imaging is performed by using a microscopic optical imaging module; and the cutting is performed by using a diamond tool; Based on the three-dimensional images of the sample sections obtained by the scanning imaging operation, the complete morphology of the neuron is reconstructed, and the fluorescently labeled neuron cell bodies are extracted from the collected slices, and the omics molecular information of the neuron is obtained by omics analysis; the omics analysis is performed by using a single-cell transcriptome sequencing technology, RNA of a single cell is extracted, high-throughput sequencing is performed, and then the transcriptome information of the single cell is obtained.

2. The method of claim 1, wherein, The cycle of scanning imaging and cutting on the section of the sample until the scanning imaging of the entire sample is completed, and the collection of the slices containing the fluorescently labeled neuron cell bodies obtained by cutting, comprises: S3, scanning imaging of the sample section to obtain a three-dimensional image of the sample section, and moving the sample to the position of the tool; S4, judging whether the slice to be cut contains the fluorescently labeled neuron cell body according to the three-dimensional image of the sample section, and if yes, proceeding to step S6, otherwise, proceeding to step S5; S5, cutting the imaged shallow part of the sample by the tool, and discarding the slice generated by the cutting, and then proceeding to step S7; S6, cutting the imaged shallow part of the sample by the tool, and collecting the slice generated by the cutting, and then proceeding to step S7; S7, judging whether the imaging of the entire sample is completed, and if not, jumping to step S3 to perform scanning imaging on the newly exposed sample section.

3. The method of claim 2, wherein, The three-dimensional images of the sample sections obtained by the scanning imaging operation are used to reconstruct the complete morphology of the neuron; and the fluorescently labeled neuron cell bodies are extracted from the collected slices, and the molecular information of the neuron is obtained by omics analysis, the processing of the omics molecular information of the neuron, comprises: S8, obtaining all the three-dimensional images of the sample sections and the slices containing the fluorescently labeled neuron cell bodies; S9, screening the three-dimensional images of the sample sections containing the fluorescently labeled neuron from all the three-dimensional images of the sample sections, and reconstructing the fine complete morphology of the neuron based on the three-dimensional images of the sample sections containing the fluorescently labeled neuron; S10, extracting the cell bodies of the corresponding fluorescently labeled neuron from the slices containing the fluorescently labeled neuron cell bodies, and obtaining the omics molecular information of the neuron by omics analysis.

4. The method of claim 1, wherein, Further comprising: performing joint analysis of a single neuron based on the complete morphology and the omics molecular information.

5. A system for simultaneously acquiring single neuron complete morphology and omics molecular information, characterized in that, Comprises: An imaging unit is configured to scan and image a section of a sample in which cell bodies, dendrites and axons of neurons have been fluorescently labeled, to obtain a three-dimensional image of the section of the sample; the imaging unit comprises a camera and a microscopic optical imaging module; the sample is obtained by: S1, labeling cell bodies, dendrites and axons of neurons in a mouse brain using a fluorescent labeling method; S2, embedding the mouse brain that has been fluorescently labeled using a resin embedding technique to prepare a sample with micron-level cutting hardness; A slicing unit is configured to perform a cutting operation on the sample to obtain a slice; the slicing unit comprises a tank and a cutter, the tank is configured to hold a processing liquid, the sample is immersed in the processing liquid, and the cutter is configured to cut the sample to form a slice, the cutter being a diamond cutter; A moving platform is configured to move the sample back and forth between the imaging unit and the slicing unit; A collecting unit is configured to collect the slice containing the fluorescently labeled neuron cell bodies obtained by cutting; An analysis unit is configured to determine whether the slice contains fluorescently labeled neuron cell bodies, to reconstruct a complete morphology of the neurons based on the three-dimensional image of the section of the sample, and to extract fluorescently labeled neuron cell bodies from the collected slice, to obtain omics molecular information of the neurons through omics analysis; the omics analysis is performed using single-cell transcriptome sequencing technology, by extracting RNA from a single cell and performing high-throughput sequencing to obtain transcriptome information of the single cell.

6. The system for simultaneously acquiring single neuron complete morphology and omics molecular information according to claim 5, characterized in that, The collecting unit comprises a delivery pipe, a water pump, a sorting pipe, a storage 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 slice, one end of the sorting pipe is connected to the water pump, and the sorting pipe is configured to sort the slice containing fluorescently labeled neuron cell bodies collected in the delivery pipe into the collector, and to sort the slice not containing fluorescently labeled neuron cell bodies collected in the delivery pipe into the storage container.

7. The system for simultaneously acquiring single neuron complete morphology and omics molecular information according to claim 6, characterized in that, The collecting unit further comprises a first branch pipe, a second branch pipe, a first electromagnetic valve and a second electromagnetic valve, one end of the first branch pipe is connected to the storage container, and the other end is connected to the sorting pipe, the first electromagnetic valve is installed in 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 electromagnetic valve is installed in the second branch pipe.

Images