Space omics device and method based on laser ejection sorting-sequencing

Abstract

The invention provides a spatial omics device and method based on laser ejection sorting-sequencing, and the spatial omics device comprises a laser control module, a beam shaping module, a sorting receiving motion control module, a fluorescence microscopic imaging module and a program control unit. Target cell subgroups or single cells with different sizes are sorted from animal and plant tissues with various thicknesses and different types, and high-throughput and low-damage sorting is realized; according to the present invention, the separation of small to single-cell regions of interest (ROI) from cells, the high-throughput selective separation of ROI for stained tissues, and barcode labeling are achieved, full-length gene data of single base resolution are obtained, sequencing is associated with spatial and staining information, and high-resolution spatial information on the cell and tissue levels is provided.

Description

Technical Field

[0001] The present invention belongs to the technical field of the intersection of biology, optics, biomedical engineering and gene sequencing, in particular to a spatial omics device and method based on laser ejection sorting-sequencing. Background Art

[0002] Spatial omics is a cutting-edge technology used to study the spatial distribution and interrelationships of different molecules in biological tissues. It combines different omics data such as genomics, transcriptomics, proteomics and metabolomics to provide high-resolution spatial information at the cellular and tissue levels. Spatial omics technology aims to quantitatively measure the gene expression of cells and provide specific location information of cells in tissue space. Compared with traditional technologies, spatial omics technology can obtain the true gene expression characteristics of cells in the physiological environment of tissues, as well as the relationship with the microenvironment, and further promote the understanding of cell characteristics under normal and pathological conditions. Spatial transcriptomics is a method for studying gene expression in cells and tissues. By analyzing the differences in gene expression at different locations in tissues, the spatial gene regulatory network is revealed. Unlike traditional whole-tissue or whole-cell RNA sequencing methods, spatial transcriptomics allows scientists to detect gene expression levels in specific regions or cell types in tissues.

[0003] To accurately understand cancer and improve diagnostic performance, it is important to link spatial and phenotypic histopathological information with objective and massively parallel measurements of the cellular genomic alteration status in a high-throughput manner; however, large amounts of sequencing data cannot be linked to spatial and phenotypic histopathological information because cells comprising the tissue must be pooled or dissociated in solution prior to sequencing; this loss of information linking subclonal genomic heterogeneity to histopathological context hinders deeper analysis of tumor masses.

[0004] The following is a summary of the main existing spatial omics technologies:

[0005] 1. Spatial Transcriptomics: This is a high-resolution method that combines RNA sequencing with the spatial information of tissues to simultaneously measure the expression levels of hundreds to thousands of genes in tissue sections. This technology usually uses chips or sequencing platforms with high spatial resolution.

[0006] 2. FISSEQ (Fluorescent In Situ Sequencing): FISSEQ technology allows in situ RNA sequencing in tissue sections by sequencing RNA molecules one by one in the sections. This method is very useful for revealing the distribution of genes in tissues and the spatial relationship of cell types.

[0007] 3. MERFISH (Multiplexed Error-Robust Fluorescence In SituHybridization): MERFISH is a method based on fluorescence in situ hybridization that allows the spatial localization of hundreds to thousands of genes. By using multiple DNA probes labeled with different fluorescent colors, the expression of multiple genes can be detected simultaneously.

[0008] 4. ISS (In Situ Sequencing): ISS technology uses DNA primers fixed on the slice to guide the sequencing primers to the location of specific genes, thereby achieving in situ sequencing in tissues.

[0009] 5. Slide-seq (Spatially Resolved Transcriptomics Sequencing): Slide-seq achieves RNA sequencing at different locations in the tissue by arranging tiny RNA sequencing reaction areas on the slice. This method allows high-resolution spatial localization of gene expression.

[0010] 6.Cytometry in situ Sequencing (CITE-seq): CITE-seq combines protein labeling and RNA sequencing to provide joint analysis of cell types and gene expression with spatial resolution by simultaneously measuring cell surface proteins and gene expression.

[0011] 7. Laser microdissection-sequencing technology (LCM-seq): This technology can quickly and accurately capture target cells from slices visually while retaining spatial location information. Currently, spatial omics technology based on laser microdissection has been applied to very small numbers of cells.

[0012] Currently, spatial omics technology based on laser microdissection has played an active role in the research fields of tumor biology, developmental biology and various disease biology. The specific spatial position of cells is obtained through laser microdissection, and different sequencing libraries are constructed for the sorted cells according to the research purpose. Through high-throughput sequencing, a large amount of expression information of known and unknown genes can be obtained.

[0013] However, the existing technology still has insurmountable technical defects, such as:

[0014] ① The throughput of the laser capture microdissection process is low and cannot be applied to process a large number of cells at one time;

[0015] ②The ultraviolet (UV) beam used in laser capture microdissection not only burns the periphery and hinders flux, but also causes damage to cells;

[0016] ③ Even in infrared (IR) microdissection using the direct contact method, cell debris or unselected cells may cross-contaminate target cells.

[0017] ④The resolution is low and it is impossible to achieve precise single-cell level.

[0018] These issues limit the use of microdissection to spatially map heterogeneous genomes in a high-throughput and high-resolution manner; therefore, there is an urgent need to develop devices and methods that can effectively detect tumor subclones and variants with low-level allele fractions with high sensitivity and accuracy while preserving the spatial location and histopathological phenotype of gene expression. Summary of the invention

[0019] In order to solve the above technical problems, the present invention provides a spatial omics device and method based on laser catapult sorting-sequencing. By constructing a laser catapult sorting device, beam shaping is combined with laser-induced forward transfer to achieve sorting of target cell subpopulations or single cells of different sizes from various thicknesses and different types of animal and plant tissues, thereby achieving high-throughput (100 cells / minute) and low-damage sorting; combined with barcode-based high-throughput sequencing, single-cell whole genome, transcriptome or proteome sequencing is performed; regions of interest (ROI) as small as single cells are separated from cells, and ROIs are selectively separated at high throughput for immunofluorescence (IF)-stained tissues, and barcode labeling is performed to obtain full-length gene data with single-base resolution, and sequencing is associated with spatial and staining information. As the cost of sequencing continues to decrease, this method is of great significance for studying spatial omics.

[0020] Spatial omics device and method based on laser ejection sorting-sequencing, wherein:

[0021] The spatial omics device based on laser ejection sorting-sequencing includes:

[0022] Laser control module, beam shaping module, sorting and receiving motion control module, fluorescence microscopy imaging module and program control unit.

[0023] The laser control module is used to emit one or more lasers for ejecting cells, and can adjust the energy and spot of the laser to control the ejection range;

[0024] As an example, the laser control module includes: a laser, an energy adjustment module and a beam expansion module;

[0025] The laser is used to emit one or more lasers to eject cells.

[0026] The energy regulating module is used to regulate the laser energy, and adopts a half-wave plate and a polarizing plate to realize continuous adjustment of the laser energy or power.

[0027] The beam expansion module can expand the diameter of the collimated light beam and reduce the energy density of the emitted laser, including a first lens and a second lens combined structure.

[0028] As a preferred example, the laser is: a single pulse laser or a continuous pulse laser.

[0029] The beam shaping module is used to modulate the laser into a vortex beam or a light spot consistent with the cell contour;

[0030] As an example, the beam shaping module includes: a first reflector, a second reflector, a beam shaping element and a sorting objective lens; the beam shaping element is conjugate with the rear focal plane of the sorting objective lens, and the laser is modulated into different mode beams on the focal plane after passing through the sorting objective lens; after being shaped by the beam shaping module, the laser is focused on the laser ejection chip;

[0031] As a preferred example, the beam shaping element is: a vortex glass or a spatial light modulator (SLM) or a DMD reflector; vortex beams of different sizes are generated by beam shaping elements such as a vortex glass, and under the action of a laser, transfer effects of different materials are presented.

[0032] The sorting and receiving motion control module is used to eject and receive the target cells under the action of laser energy;

[0033] As an example, the sorting and receiving motion control module includes: a laser ejection chip and a cell receiver;

[0034] The laser ejection chip comprises: a glass bottom plate and a covering film, wherein the covering film is located on the glass bottom plate and the sample is attached to the covering film;

[0035] The cell receiver is located below the laser ejection chip. When the laser ejection chip receives laser energy, the cells in the sample are ejected into the cell receiver to achieve ejection sorting of the cells.

[0036] As an example, the cell receiver is a 96-well plate, an 8-well PCR tube strip or a single PCR tube.

[0037] The fluorescence microscopy module is used to perform bright field and fluorescence imaging on the tissue on the laser ejection chip and obtain the position and outline of each cell in the tissue.

[0038] As an example, the fluorescence microscopy imaging module includes: an optical microscopy imaging device and an image recognition and positioning system;

[0039] The optical microscopic imaging device is used to perform optical imaging on the sample and generate an image;

[0040] The image recognition and positioning system is used to obtain the position and outline of each cell in the image by image recognition.

[0041] As an example, the specific structural design of the fluorescence microscopy imaging module includes: a camera, a first condensing lens, a second condensing lens, a switchable fluorescence filter set, a fluorescence light source, a third reflector and an imaging objective lens.

[0042] The program control unit is used to control the laser control module, the beam shaping module, the sorting and receiving motion control module and the fluorescence microscopy imaging module to modulate different mode beams;

[0043] As an example, the program control unit is used to image tissue samples, select target single cells, and navigate tissue images through a graphical user interface; by automatically controlling the LEM-seq instrument, it can adjust the laser energy, control the movement of the laser ejection chip and the cell receiver; and automatically separate the tissue samples, with a high level of automated control.

[0044] As an example, the program control unit can also recognize the contour of the tissue sample and adaptively shape the light beam into light beams of different modes through an algorithm; and record the sorting position each time a single cell is sorted.

[0045] The spatial omics method based on laser ejection sorting-sequencing includes:

[0046] Step 1: Collect samples, embed them in OCT or paraffin, slice and stain them to form tissue samples, and place them on a laser ejection chip;

[0047] Step 2: Perform bright field and fluorescence imaging of the tissue sample on the laser ejection chip through a fluorescence microscopy imaging module, and obtain the position and outline of each target cell in the tissue sample;

[0048] As an example, each target cell is: a single cell or a plurality of cells.

[0049] Step 3: Using a beam shaping module, the laser is modulated into a vortex light or a light spot consistent with the contour of the target cell;

[0050] Step 4: Adjust the laser control module to emit lasers of different energy levels to adapt to target cells of different sizes and types;

[0051] As an example, the laser is a pulsed laser.

[0052] Step 5: Control the movement of the laser ejection chip in the sorting and receiving motion control module so that different areas of the tissue sample can be correspondingly sorted for single cells, and control the cell receiver to receive the ejected target cells into different holes of the cell receiver;

[0053] Step 6: Taking the spatial transcriptome as an example, the received target cells are lysed and RNA is extracted, and then single-cell PCR amplification is performed; the amplified cDNA is quality tested and purified, and a sequencing library is constructed, followed by sequencing;

[0054] Step 7: Downstream of sequencing, the bioinformatics pipeline reconstructs the visualization of sequencing data in 3D or 2D graphs and is used to identify coding genes to map cell populations or single cells to specific locations in tissue samples.

[0055] Beneficial effects of the present invention:

[0056] The present invention is based on the spatial omics of laser ejection sorting and sequencing, which can effectively detect tumor subclones and variants with low-level allele fractions with high sensitivity and accuracy, while retaining the 3D spatial organization and histopathological phenotype of information cells; making full use of spatial and phenotypic information to group cancer cells in tumor tissue sections into potential subclones by computation or pathology;

[0057] The processes (including cell observation, selection and separation) are highly automated through program software to achieve high-throughput sorting and analysis; by establishing this new method, different subclones and their somatic variations were identified in cancer tissues; the heterogeneous genomic landscape of subclones can be directly mapped to the spatial and phenotypic organization of tumor masses; a genomic map is constructed in the spatial context of two-dimensional or three-dimensional space of cancer tissue, and the dimensions of tissue pathology and genomics are expanded to understand the evolutionary history of cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] Figure 1 The figure is a schematic diagram of the spatial transcriptomics analysis process of the spatial omics method based on laser ejection sorting-sequencing of the present invention.

[0059] Figure 2 It is a schematic diagram of the structure of the spatial omics device based on laser ejection sorting-sequencing of the present invention.

[0060] Figure 3 Schematic diagram of the beam shaping and effect of the spatial omics method based on laser ejection sorting-sequencing in the present invention. (Beam shaping generates an annular beam with a diameter of 1 to 45 μm and its effect on the transfer of materials produced by coating)

[0061] Figure 4This is a diagram of the sorting effect of various tissue samples by bright field and fluorescence imaging based on the spatial omics method of laser ejection sorting-sequencing in the present invention. (The sorting of frozen unstained sections, paraffin HE stained sections, frozen HE stained sections and frozen immunofluorescence stained sections was achieved respectively)

[0062] Figure 5 Schematic diagram of Barcode-based high-throughput sequencing based on the spatial omics method of laser ejection sorting-sequencing in the present invention. (bMDA-seq allows multiple analysis of a large number of single cells at single nucleotide resolution, thereby expanding the understanding of heterogeneous cell populations; after bMDA-seq, each single cell data shows a genome coverage depth sufficient to perform spatial genomics)

[0063] Figure 6 This is a spatial omics analysis diagram of the present invention based on the spatial omics method of laser ejection sorting-sequencing. (Each transcriptome and epigenome data point is mapped to the tissue according to the barcode)

[0064] Figure 7 Schematic diagram of the process design of the spatial omics method based on laser ejection sorting-sequencing of the present invention. DETAILED DESCRIPTION

[0065] Below, reference Figures 1 to 7 As shown, a spatial omics device and method based on laser ejection sorting-sequencing, wherein:

[0066] The spatial omics device based on laser ejection sorting-sequencing includes:

[0067] Laser control module 101, beam shaping module 102, sorting and receiving motion control module 103, fluorescence microscopy imaging module 104 and program control unit.

[0068] The laser control module 101 is used to emit one or more lasers for ejecting cells, and can adjust the energy and spot of the laser to control the ejection range;

[0069] As an example, the laser control module 101 includes: a laser 201, an energy adjustment module and a beam expansion module;

[0070] The laser 201 is used to emit one or more lasers to eject cells.

[0071] The energy regulating module is used to regulate the laser energy, and adopts the half-wave plate 202 and the polarizing plate 203 to realize the continuous adjustment of the laser energy or power.

[0072] The beam expansion module can expand the diameter of the collimated light beam and reduce the energy density of the emitted laser, including a first lens 204 and a second lens 205 combined in an upper and lower structure.

[0073] As a preferred example, the laser 201 is: a single pulse laser or a continuous pulse laser.

[0074] The beam shaping module 102 is used to modulate the laser into a vortex beam or a light spot consistent with the cell contour;

[0075] As an example, the beam shaping module 102 includes: a first reflector 301, a second reflector 303, a beam shaping element 302 and a sorting objective lens 304; the beam shaping element 302 is conjugate with the rear focal plane of the sorting objective lens 304, and the laser is modulated into different mode beams on the focal plane after passing through the sorting objective lens 304; after being shaped by the beam shaping module, the laser is focused on the laser ejection chip 401;

[0076] As a preferred example, the beam shaping element 302 is: a vortex glass or a spatial light modulator (SLM) or a DMD reflector; vortex beams of different sizes are generated by beam shaping elements such as a vortex glass, and under the action of a laser, transfer effects of different materials are presented.

[0077] The sorting and receiving motion control module 103 is used to eject and receive the target cells under the action of laser energy;

[0078] As an example, the sorting receiving motion control module 103 includes: a laser ejection chip 401 and a cell receiver 402;

[0079] The laser ejection chip 401 comprises: a glass bottom plate and a covering film, wherein the covering film is located on the glass bottom plate and the sample is attached to the covering film;

[0080] The cell receiver 402 is located below the laser ejection chip 401. When the laser ejection chip 401 receives laser energy, the cells in the sample are ejected into the cell receiver 402, thereby realizing ejection sorting of the cells.

[0081] The fluorescence microscopy imaging module 104 is used to perform bright field and fluorescence imaging on the tissue on the laser ejection chip 401 and obtain the position and outline of each cell in the tissue.

[0082] As an example, the fluorescence microscopic imaging module 104 includes: an optical microscopic imaging device and an image recognition and positioning system;

[0083] The optical microscopic imaging device is used to perform optical imaging on the sample and generate an image;

[0084] The image recognition and positioning system is used to obtain the position and outline of each cell in the image by image recognition.

[0085] As an example, the specific structural design of the fluorescence microscopy imaging module 104 includes: a camera 507, a first condensing lens 506, a second condensing lens 504, a switchable fluorescence filter set 503, a fluorescence light source 505, a third reflector 502 and an imaging objective lens 501.

[0086] The program control unit is used to control the laser control module 101, the beam shaping module 102, the sorting and receiving motion control module 103 and the fluorescence microscopy imaging module 104 to modulate different mode beams;

[0087] As an example, the program control unit is used to image tissue samples, select target single cells, and navigate tissue images through a graphical user interface; by automatically controlling the LEM-seq instrument, it can adjust the laser energy, control the movement of the laser ejection chip 401 and the cell receiver 402; and automatically separate the tissue samples, with a high level of automated control.

[0088] As an example, the program control unit can also recognize the contour of the tissue sample and adaptively shape the light beam into light beams of different modes through an algorithm; and record the sorting position each time a single cell is sorted.

[0089] The spatial omics method based on laser ejection sorting-sequencing includes:

[0090] Step 1: Collect samples, embed them with OCT or paraffin, slice and stain them, form tissue samples, and place them on the laser ejection chip 401;

[0091] As an example, the tissue section refers to: the tissue sample is frozen and stored in an environment of minus 80°C, cut into 10 μm thick slices using a cryostat; and the slices are thawed onto the laser ejection chip.

[0092] As an example, the laser ejection chip 401 is coated with a special coating, which is a vaporized coating that facilitates laser cutting and ejection. The laser ejection chip is stored in a -20°C environment before the analysis operation.

[0093] As an example, the staining refers to drying the sections at room temperature for 15 minutes and then performing HE and immunofluorescence staining.

[0094] Step 2: Perform bright field and fluorescence imaging of the tissue sample on the laser ejection chip 401 through the fluorescence microscopy imaging module 104, and obtain the position and outline of each target cell in the tissue sample;

[0095] As an example, each of the target cells is a single cell.

[0096] As an example, the bright field and fluorescence imaging refers to: imaging the whole tissue slice of the tissue sample to generate a whole slice scan image.

[0097] As an example, the whole slice scan image refers to: for the whole slice tissue imaging, the slice is taken out of the refrigerator and placed at room temperature for 2 minutes, and then scanned using the imaging objective lens 501, and then the image is stitched.

[0098] Step 3: using the beam shaping module 102 to modulate the laser into a vortex light or a light spot consistent with the contour of the target cell;

[0099] Specific operations include:

[0100] First, target cells to be isolated were selected based on the whole-slice scan image;

[0101] Secondly, the beam profile that needs to be modulated is determined based on the morphology and contour of the target cells;

[0102] Finally, a phase map is generated according to the target cell contour through a beam shaping algorithm. After the light beam passes through the phase map, it is focused by the sorting objective lens 304 onto the surface of the laser ejection chip 401 to generate a light spot consistent with the contour of the target cell.

[0103] As an example, the beam shaping algorithm includes:

[0104] ① Input intensity is S, set initial phase to fai_s0;

[0105] ② Perform Fourier transform on S*exp(i*fai_s0) to obtain the amplitude distribution and phase distribution fai_t0 on the Fourier plane;

[0106] ③Replace the amplitude distribution of the Fourier plane with the target intensity distribution T;

[0107] ④ Perform inverse Fourier transform on T*exp(ifai_t0) to obtain the amplitude distribution and phase distribution fai_s1 of the SLM plane, replace the amplitude distribution with S, and keep the phase distribution unchanged;

[0108] ⑤Perform Fourier transform to obtain the amplitude and phase distribution in the Fourier plane, and then repeat the above steps for iteration.

[0109] Step 4: Adjust the laser control module 101 to emit lasers of different energy levels to adapt to target cells of different sizes and types;

[0110] As an example, the laser is a pulsed laser.

[0111] As an example, after the target cells are identified, a single green laser (532 nm) is irradiated to each target area of ​​the tissue sample; when the green laser irradiates the vaporized coating, the target cells on the target cell area are transferred downward under the action of pressure.

[0112] Compared to laser microdissection using UV cutting and ejection, the present invention uses a green laser and a one-time separation step, does not cause damage to nucleic acids, and can be performed quickly, thereby enabling the recovery of high-quality nucleic acids; extracts DNA from single cells or a small number of cells with high throughput; on the other hand, standard laser microdissection uses a green laser to heat a thermoplastic film, which expands and captures the target cells; although this method is not subject to UV-induced damage, the thermoplastic film-based method requires physical contact between the film and the sample and tearing off the cells by removing the cell capture cap; this physical contact method leads to inherent problems such as limited throughput and scalability, debris or non-selective cell adhesion, and limited applicability depending on sample conditions;

[0113] Step 5: Control the laser ejection chip 401 in the sorting and receiving motion control module 103 to move, so that different areas of the tissue sample can be correspondingly single-cell sorted, and control the cell receiver 402 to receive the ejected target cells into different holes of the cell receiver 402;

[0114] As an example, the special coating of the laser catapulting chip 401 is applied on glass by sputtering deposition; a green laser is irradiated onto the target area, evaporating the special coating and subjecting the target cells in the area to force; a laser catapulting chip with a 50nm thick coating is used; single cells can be separated from tissue sections 4 to 16μm thick; using a green laser in combination with the coating, cell separation can be completed without damaging single cells.

[0115] Step 6: Taking the spatial transcriptome as an example, the received target cells are lysed and RNA is reverse transcribed, and then cDNA is amplified; the amplified cDNA is quality tested and purified, and a sequencing library is constructed, followed by sequencing;

[0116] Specific operations include:

[0117] ① After centrifugation through the PCR tube, use cell lysis buffer to lyse the target cells;

[0118] ② Use RNA reverse transcription kit to reverse transcribe RNA;

[0119] ③ Amplify the reverse transcribed cDNA and purify all amplified products using a kit immediately after the amplification reaction;

[0120] ④ In order to verify the quality of sample RNA extraction, the product was quality checked using a nucleic acid fragment analyzer;

[0121] ⑤ When most of the amplified products are 1500 bp in size, they are judged to be qualified in quality inspection, and the library is constructed using the NGS library preparation kit and sequenced using the Illimina sequencing system.

[0122] As an example, to prevent carryover contamination, pipette tips, PCR tubes, and caps were stored in a clean bench equipped with UV light, and real-time amplification of non-template controls was monitored to ensure that no contaminants were carried over.

[0123] Furthermore, barcode multiple displacement amplification (bMDA) can prepare single-cell genome sequencing libraries in multiple ways, achieving economical, efficient, and higher-throughput single-nucleotide resolution single-cell genome analysis; single-cell RNA barcode sequencing (SCRB-seq) is a high-throughput transcriptome method based on a higher-throughput cell receiver, adopts a 3' end enrichment strategy, uses barcode sequences to mark cells, and integrates specific molecular barcodes UMIs to distinguish the source of the amplified molecules and more accurately quantify transcripts.

[0124] Step 7: Downstream of sequencing, the sequencing data is reconstructed and visualized in 3D or 2D plots through a bioinformatics pipeline and used to identify coding genes to map cell populations or single cells to specific locations in tissue samples to obtain an image of CNA heterogeneity.

[0125] As an illustration, by analyzing serial sections of tissue, we discovered how heterogeneous tumor subclones exist in the three-dimensional space of tissue samples; we demonstrated that laser ejection sorting-sequencing is a tool that connects genomics and histopathology; before isolation, cells with various phenotypes were identified by histopathological evaluation with HE and immunofluorescence; based on the phenotypic information, several cells were selected to discover the genetic heterogeneity of cell populations with various phenotypes; after isolating the target cell clusters, we performed sequencing to obtain an image of CNA heterogeneity.

[0126] In order to better illustrate the design principle of the present invention, a specific experiment is now used to illustrate the effect as follows:

[0127] Embodiment 1:

[0128] Guided by the population information of the cells, 2 to 3 cells were selected to cover all populations and effectively represent the spatial organization of the tumor;

[0129] By using laser pulses to eject each cell on the laser ejection chip 401, each cell is separated into different wells of the cell receiver 402;

[0130] RNA extracted from different wells of the cell recipients was amplified and sequenced; tissue samples were immunofluorescently stained and full-length transcriptome data were obtained with single-base resolution, associated with spatial and staining information; specifically, selective isolation of each ROI allowed barcoding of entire sections of the transcriptome, allowing for in-depth multimodal analysis of specific cellular niches.

[0131] The present invention is based on the spatial omics of laser ejection sorting and sequencing, which can effectively detect tumor subclones and variants with low-level allele fractions with high sensitivity and accuracy, while retaining the 3D spatial organization and histopathological phenotype of information cells; making full use of spatial and phenotypic information to group cancer cells in tumor tissue sections into potential subclones by computation or pathology;

[0132] The processes (including cell observation, selection and separation) are highly automated through program software to achieve high-throughput sorting and analysis; by establishing this new method, different subclones and their somatic variations were identified in cancer tissues; the heterogeneous genomic landscape of subclones can be directly mapped to the spatial and phenotypic organization of tumor masses; a genomic map is constructed in the spatial context of two-dimensional or three-dimensional space of cancer tissue, and the dimensions of tissue pathology and genomics are expanded to understand the evolutionary history of cancer.

[0133] The above are only preferred embodiments of the present invention. It should be understood that the description of the above embodiments is only used to help understand the method and core ideas of the present invention, and is not used to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, etc. made within the ideas and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A spatial omics device based on laser ejection sorting-sequencing, characterized in that: include: Laser control module, beam shaping module, sorting and receiving motion control module, fluorescence microscopy imaging module and program control unit; The laser control module is used to emit one or more lasers for ejecting cells, and can adjust the energy and spot of the laser to control the ejection range; The beam shaping module is used to modulate the laser into a vortex beam or a light spot consistent with the cell contour; The sorting and receiving motion control module is used to eject and receive the target cells under the action of laser energy; The fluorescence microscopy module is used to perform bright field and fluorescence imaging of the tissue on the laser ejection chip and obtain the position and outline of each cell in the tissue; The program control unit is used to control the laser control module, the beam shaping module, the sorting and receiving motion control module and the fluorescence microscopy imaging module to modulate light beams of different modes.

2. The spatial omics device based on laser ejection sorting-sequencing according to claim 1, characterized in that: The laser control module includes: a laser, an energy adjustment module and a beam expansion module; The laser is used to emit one or more lasers for catapulting cells; The energy adjustment module is used to adjust the laser energy, and adopts a half-wave plate and a polarizing plate to achieve continuous adjustment of the laser energy or power; The beam expansion module can expand the diameter of the collimated light beam and reduce the energy density of the emitted laser, including a first lens and a second lens combined structure.

3. The spatial omics device based on laser ejection sorting-sequencing according to claim 1, characterized in that: The laser is a single pulse laser or a continuous pulse laser.

4. The spatial omics device based on laser ejection sorting-sequencing according to claim 1, characterized in that: The beam shaping module includes: a first reflector, a second reflector, a beam shaping element and a sorting objective lens; the beam shaping element is conjugate with the rear focal plane of the sorting objective lens, and the laser is modulated into different mode beams on the focal plane after passing through the sorting objective lens; after being shaped by the beam shaping module, the laser is focused on the laser ejection chip.

5. The spatial omics device based on laser ejection sorting-sequencing according to claim 4, characterized in that: The beam shaping element is a vortex glass slide or a spatial light modulator or a DMD reflector. Vortex beams of different sizes are generated by the beam shaping element, and under the action of laser, transfer effects of different materials are presented.

6. The spatial omics device based on laser ejection sorting-sequencing according to claim 1, characterized in that: The sorting and receiving motion control module includes: a laser ejection chip and a cell receiver; The laser ejection chip comprises: a glass bottom plate and a covering film, wherein the covering film is located on the glass bottom plate and the sample is attached to the covering film; The cell receiver is located below the laser ejection chip. When the laser ejection chip receives laser energy, the cells in the sample are ejected into the cell receiver to achieve ejection sorting of the cells.

7. The spatial omics device based on laser ejection sorting-sequencing according to claim 1, characterized in that: The fluorescence microscopy imaging module comprises: a camera, a first condensing lens, a second condensing lens, a switchable fluorescence filter set, a fluorescence light source, a third reflector and an imaging objective lens.

8. A method of spatial omics based on laser ejection sorting-sequencing, characterized in that: include: Step 1: Collect samples, embed them in OCT or paraffin, slice and stain them to form tissue samples, and place them on a laser ejection chip; Step 2: Perform bright field and fluorescence imaging of the tissue sample on the laser ejection chip through a fluorescence microscopy imaging module, and obtain the position and outline of each target cell in the tissue sample; Step 3: using a beam shaping module to modulate the laser into a vortex light or a light spot consistent with the contour of the target cell; including: First, target cells to be isolated were selected based on the whole-slice scan image; Secondly, the beam profile that needs to be modulated is determined based on the morphology and contour of the target cells; Finally, a phase map is generated according to the target cell contour through a beam shaping algorithm. After the light beam passes through the phase map, it is focused on the surface of the laser ejection chip by the sorting objective lens to generate a light spot consistent with the contour of the target cell. Step 4: Adjust the laser control module to emit lasers of different energy levels to adapt to target cells of different sizes and types; after determining the target cells, irradiate each target area of ​​the tissue sample with a single green laser; when the green laser irradiates the vaporized coating, the target cells on the target cell area move downward under the action of pressure; Step 5: Control the movement of the laser ejection chip in the sorting and receiving motion control module so that different areas of the tissue sample can be correspondingly sorted for single cells, and control the cell receiver to receive the ejected target cells into different holes of the cell receiver; Step 6: Lyse the received target cells and extract RNA, then perform single-cell PCR amplification; perform quality inspection and purification on the amplified cDNA, construct a sequencing library, and then perform sequencing; the specific operations include: ① After centrifugation through the PCR tube, use protease to lyse the target cells; ② For whole genome amplification by multiple displacement amplification, use an amplification kit to amplify and monitor the amplification in real time; ③ Use the kit to purify all amplified products immediately after the amplification reaction; ④ To verify whether the sample has been thoroughly amplified, real-time whole genome amplification monitoring and PCR verification and internal regional primer sets are used; ⑤ When the yield of most amplified products exceeds 1 μg, they are used for Illumina library construction; Step 7: Downstream of sequencing, the sequencing data is reconstructed and visualized in 3D or 2D plots through a bioinformatics pipeline and used to identify coding genes to map cell populations or single cells to specific locations in tissue samples to obtain an image of CNA heterogeneity.

9. The method of spatial omics based on laser ejection sorting-sequencing according to claim 8, characterized in that: The tissue section refers to: the tissue sample is frozen and stored in an environment of minus 80°C, and is cut into 10 μm thick slices using a cryostat; and the slices are thawed onto a laser ejection chip; The laser ejection chip is coated with a special coating, which is a vaporized coating that facilitates laser cutting and ejection. The laser ejection chip is stored in a -20°C environment before the analysis operation; The staining refers to: drying the sections at room temperature for 15 minutes, and then performing HE and immunofluorescence staining.

10. The method of spatial omics based on laser ejection sorting-sequencing according to claim 8, characterized in that: The bright field and fluorescence imaging refers to: performing whole tissue slice imaging on the tissue sample to generate a whole slice scan image; The whole slice scanning image refers to: for the whole slice tissue imaging, the slice is taken out of the refrigerator and placed at room temperature for 2 minutes, and then scanned with an imaging objective lens and image stitching is performed.

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