Analysis method, analysis system, and surface for analysis

Abstract

To provide a method capable of associating position information of a cell with a cell component with single cell resolution.SOLUTION: This technology provides an analysis method including an imaging step for imaging a sample in a state in which a molecule containing a linker cleavable by stimulation, a barcode sequence and a target capture portion is overlapped on a surface fixed through the linker, an association step for associating the position of a cell with the barcode sequence of the molecule existing the position by using a sample image acquired by the imaging, a cleavage step for selectively giving stimulation to the position of the cell and cleaving the linker of the molecule existing at the position, and a binding step for binding the molecule released from the surface by the cleavage to the component of the cell through the target capture portion of the cell.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present technology relates to an analysis method, an analysis system, and an analysis surface. More specifically, the present technology relates to an analysis method, an analysis system, and an analysis surface for obtaining the types and amounts of components contained in cells in association with the position information of the cells.

Background Art

[0002] In order to analyze biological tissues, the types and amounts of mRNA contained in the cells that make up the biological tissues are measured. In the analysis of biological tissues, in addition to the types and amounts of mRNA contained in the cells, the position information of the cells is also important. Therefore, several methods have been proposed so far for obtaining the position information of the cells contained in a biological tissue in association with the information of the mRNA contained in the cells.

[0003] For example, Non-Patent Document 1 below discloses a method called Spatial transcriptomics. The probe used in this method includes a cleavage site, a T7 amplification and sequencing handle, a Spatial barcode, a UMI, and an mRNA capture region (Figure 2 of the same document). In this method, a tissue section is placed on a slide glass on which the probe is fixed, the mRNA in the tissue section is captured by the molecule, and then reverse transcribed to synthesize cDNA. The synthesized cDNA is cleaved at the cleavage site, collected in a tube, and subjected to analysis steps such as sequencing.

Prior Art Documents

Non-Patent Documents

[0004]

Non-Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0005] If the association between the positional information of cells and the information of the components contained in the cells can be carried out for each cell constituting a biological tissue, it is considered that a more detailed analysis of the biological tissue becomes possible. Therefore, the present technology aims to provide a method that enables the association of cell positional information and cell components at single-cell resolution.

Means for Solving the Problems

[0006] The present inventors have found that the above problems can be solved by a specific analysis method. That is, the present technology includes an imaging step of imaging a specimen in a state where a molecule containing a linker cleavable by stimulation, a barcode sequence, and a target capture part is overlaid on a surface to which the molecule is fixed via the linker, an association step of associating the position of a cell and the barcode sequence of the molecule at that position using the specimen image obtained by the imaging, a cleavage step of selectively stimulating the position of the cell to cleave the linker of the molecule at that position, a binding step of binding the molecule released from the surface by the cleavage to the components of the cell via the target capture part of the molecule, and provides an analysis method including the above steps. The specimen may include a tissue sample. In the cleavage step, selective stimulation may be applied to the position of the cell so that the linker of the molecule at a position other than the position of the cell does not cleave. The stimulation may be light stimulation. The binding step may include a moving step of moving the molecule toward the cell by applying an electric field, a magnetic field, or a centrifugal force. The binding step may include a moving step of moving the molecule toward the cell by natural diffusion. The binding step may include an incubating step for binding the molecule and a constituent component of the cell. The analysis method of the present technology may further include an analysis step of analyzing the conjugate of the molecule and the constituent component of the cell after the binding step. In the analysis step, sequencing processing may be performed on the conjugate. The analysis step may include a two-dimensional mapping step of performing two-dimensional mapping based on the association result in the association step using the analysis result and the specimen image.

[0007] Further, the present technology includes an analysis substrate having a surface on which a molecule including a linker cleavable by a stimulus, a barcode sequence, and a target capture portion is fixed via the linker, An imaging device that images a specimen overlaid on the analysis substrate, An associating unit that associates the position of a selected cell in the specimen image obtained by the imaging and the barcode sequence of the molecule at the position, A stimulation device that selectively applies a stimulus to the position of the cell, and using, as an analysis target, a conjugate in which the molecule released from the surface by the stimulus and the constituent component of the cell are bound via the target capture portion of the molecule. An analysis system is also provided. The specimen may include a tissue sample. The stimulation device may be configured to selectively apply a stimulus to the position of the cell without cleaving the linker included in the molecule at a position other than the position of the cell. The stimulation device may be a light irradiation device. The analysis system may include an electric field application device, a magnetic field application device, or a centrifugal force application device that applies an electric field, a magnetic field, or a centrifugal force to move the molecules released from the surface toward the cells by the application of a stimulus by the stimulus application device. The analysis system may further include an incubation device for promoting the binding of the molecules released from the surface by the application of a stimulus by the stimulus application device to the components of the cells. The analysis system may further include an analysis device for analyzing the conjugate of the molecules released from the surface by the application of a stimulus by the stimulus application device and the components of the cells. The analysis device may be a sequencer. The analysis system may further include a two-dimensional mapping unit that performs two-dimensional mapping based on the association result by the association unit using the analysis result by the analysis device and the specimen image obtained by the imaging.

[0008] In addition, in the present technology, a molecule including a cleavable linker, a barcode sequence, and a target capture unit is fixed via the linker, and the barcode sequence is used to provide information regarding the position where the molecule including the barcode sequence is fixed. An analysis surface is also provided.

Brief Description of Drawings

[0009]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

[0010] Hereinafter, preferred embodiments for carrying out the present technology will be described. Note that the embodiments described below show typical embodiments of the present technology, and the scope of the present technology is not limited to only these embodiments. The description of the present technology will be made in the following order. 1. First Embodiment (Analysis Method) (1) Description of the First Embodiment (2) Example of the First Embodiment (2-1) Preparation Step of Imaging Target (2-2) Imaging Step (2-3) Association Step (2-4) Cleavage Step (2-5) Binding Step (2-6) Analysis Step 2. Second Embodiment (Analysis System) 3. Third Embodiment (Analysis Surface)

[0011] 1. First Embodiment (Analysis Method)

[0012] (1) Description of the First Embodiment

[0013] In the analysis method of the present technology, a specimen is imaged in a state where molecules including a linker cleavable by stimulation, a barcode sequence, and a target capture part are fixed via the linker on a surface. Then, using the specimen image obtained by the imaging, the position of cells contained in the specimen and the barcode sequence of the molecules at that position are associated. After the association is made, stimulation is applied to the position of the cells to be analyzed, and the linker of the molecules present at that position is cleaved. By the cleavage, the molecules are released from the surface, and the molecules bind to the constituent components of the cells to be analyzed via the target capture part.

[0014] A molecule bound to a cell component in this way has a barcode sequence, and the barcode sequence is associated with the position of the cell. Therefore, by analyzing the molecule bound to the cell component (such as identifying the cell component and the barcode sequence), information about the cell component can be obtained in association with the cell position information. Furthermore, in this analysis method, a specimen image is also obtained as described above. Therefore, for example, data on cell components can be mapped onto the specimen image.

[0015] In this technology, the specimen may be a fixed specimen, for example, a frozen section or an FFPE section. Many of the analysis methods of the prior art are applicable only to either a frozen section or an FFPE section, but the analysis method of this technology can be applied to both of these sections. The specimen may be, for example, a tissue sample, particularly a biological tissue sample.

[0016] In the analysis method of this technology, selective stimulation is applied to the position of the cell, so that the molecule present at that position is released from the surface and binds to the cell component. More preferably, in the cleavage step, selective stimulation is applied to the position of the cell so that the linker of the molecule at a position other than the position of the cell does not cleave. Thereby, analysis of cell components (such as mRNA or protein) with single-cell resolution becomes possible. That is, the analysis method of this technology may be an analysis method for analyzing cell components contained in a specimen with single-cell resolution. Furthermore, by using barcode sequences associated with the positional information of cells, the components of each cell can be comprehensively analyzed for various cells included in the specimen. For example, in the analysis of mRNA in a tissue sample according to the prior art, a specific region can be designated based on the morphological information or immunostaining information of the cells in the tissue sample, cut out with a laser or the like, and then subjected to mRNA analysis. In this technology, without performing operations such as cutting out the tissue sample, the expression information of mRNA in the tissue sample can be comprehensively obtained for each cell included in the tissue sample. That is, the analysis method of this technology may be an analysis method for analyzing the components of a plurality of cells included in a specimen with single-cell resolution.

[0017] (2) Example of the first embodiment

[0018] An example of the flowchart of the analysis method of this technology is shown in FIG. 1. As shown in FIG. 1, the analysis method of this technology may include an imaging target preparation step S101, an imaging step S102, an association step S103, a cleavage step S104, a binding step S105, and an analysis step S106. These steps will be described below.

[0019] (2-1) Imaging target preparation step

[0020] In the imaging target preparation step S101, the imaging target in the subsequent imaging step S102 is prepared. The imaging target may be a laminate obtained by overlaying a specimen on a surface where molecules including a linker cleavable by stimulation, a barcode sequence, and a target capture part are fixed via the linker.

[0021] In the imaging target preparation step S101, for example, as shown in FIG. 2(A), an analysis substrate 102 (such as a slide glass) having a surface 101 to which a plurality of molecules 100 are fixed is superposed on a substrate 104 (such as a slide glass) on which a specimen 103 is placed. The superposition may be performed such that the surface 101 and the specimen 103 face each other. For example, the surface 101 and the specimen 103 may be brought into contact with each other via a buffer or the like. Further, after the surface 101 and the specimen 103 are superposed, the laminate of the analysis substrate 102 and the substrate 104 may be immersed in a buffer such as PBS. The positional relationship between the substrates 102 and 104 can be fixed so that the positional relationship between the superposed surface 101 and the specimen 103 is not changed in the subsequent steps (particularly the imaging step S102 to the binding step S105).

[0022] The molecule 100 includes a linker cleavable by stimulation, a barcode sequence, and a target capture unit. In this specification, a molecule (i.e., a molecule including the linker, the barcode sequence, and the target capture) is a molecule used for capturing a target and may also be referred to as a target capture molecule. The target capture molecule is a name for meaning the molecule used in this technology, and in this specification, it is used, for example, to refer to the molecule after capturing the target, and may also be used to refer to the molecule after the linker is cleaved in the cleavage step described later. The target capture molecule may be, for example, either a single molecule or a complex molecule. A single molecule may, for example, mean one type of molecule having a plurality of functions, and may be, for example, one nucleic acid (e.g., DNA or RNA) including a nucleic acid portion constituting the linker, a nucleic acid portion constituting the barcode sequence, and a nucleic acid portion constituting the target capture portion. A complex molecule may be, for example, a molecular aggregate composed of two or more types of molecules (e.g., a conjugate of two or more types of molecules), and may be, for example, a conjugate of a nucleic acid including a nucleic acid portion constituting the linker and a nucleic acid portion constituting the barcode sequence, and a polypeptide (e.g., a protein or a part thereof, or an oligopeptide, etc.) constituting the target capture portion. An example of the structure of molecule 100 will be described with reference to FIG. 3. The molecule 100 shown in FIG. 3 includes a linker 1, a recovery sequence portion 2, an amplification sequence portion 3, a barcode sequence portion 4, a UMI (Unique molecular identifier) portion 5, and a target capture portion 6. Further, the molecule 100 is fixed to the surface 101 via the linker 1. The recovery sequence portion 2, the amplification sequence portion 3, the barcode sequence portion 4, and the UMI portion 5 may be configured as a continuous nucleic acid (particularly DNA). When the target capture portion 6 is a nucleic acid, in addition to the recovery sequence portion 2, the amplification sequence portion 3, the barcode sequence portion 4, and the UMI portion 5, the target capture portion 6 may also be configured as a continuous nucleic acid (particularly DNA). In these cases, for example, the end close to the fixing portion between the surface 101 and the molecule 100 may be the 5' end, and the other end may be the 3' end. The components of molecule 100 will be described below.

[0023] Linker 1 may be a linker cleavable by stimulation, for example, a linker cleavable by light stimulation or temperature stimulation, preferably a linker cleavable by light stimulation. Light stimulation is particularly suitable for selectively stimulating a specific position in the cleavage step described below.

[0024] As a linker cleavable by, for example, light stimulation, Linker 1 may contain any one selected from an arylcarbonylmethyl group, a nitroaryl group, a coumarin-4-ylmethyl group, an arylmethyl group, a metal-containing group, and other groups. As these groups, for example, those described in Photoremovable Protecting Groups in Chemistry and Biology: Reaction Mechanisms and Efficacy, Chem. Rev. 2013, 113, 119-191 may be used. For example, the arylcarbonylmethyl group may be a phenacyl group, an o-alkylphenacyl group, or a p-hydroxyphenacyl group. The nitroaryl group may be, for example, an o-nitrobenzyl group, an o-nitro-2-phenethyloxycarbonyl group, or an o-nitroanilide. The arylmethyl group may be, for example, one introduced with a hydroxy group or may not be introduced.

[0025] When Linker 1 is a linker cleavable by light stimulation, the linker may preferably be cleaved by light having a wavelength of 360 nm or more. The linker is preferably 0.5 μJ / μm 2It may be a linker cleaved by the following energy. (Light-sheet fluorescence microscopy for quantitative biology, Nat Methods. 2015 Jan;12(1):23-6. doi: 10.1038 / nmeth.3219.) By employing a linker cleaved by light of the above wavelength or the above energy, cell damage (such as DNA or RNA cleavage) that may occur when applying a light stimulus can be reduced.

[0026] When Linker 1 is a linker cleavable by a temperature stimulus, Linker 1 may contain, for example, a temperature-responsive polymer. The temperature-responsive polymer can change, for example, from hydrophilic to hydrophobic or from hydrophobic to hydrophilic in response to a temperature change. Due to such a change, the target-capturing molecule can be released from Surface 1.

[0027] Particularly preferably, the linker may be a linker cleaved by light in the short-wavelength region, specifically light in the wavelength region of 360 nm to 410 nm, or may be a linker cleaved by light in the near-infrared region or infrared region, specifically light in the wavelength region of 800 nm or more. When the linker is a linker efficiently cleaved by light having a wavelength in the visible light region, it may be difficult to handle the analysis surface. Therefore, the linker is preferably a linker cleaved by the light in the short-wavelength region or the near-infrared region or infrared region.

[0028] The recovery sequence portion 2 contains a nucleic acid used to recover the molecule 100 released from the surface 101 in the analysis step described later. The nucleic acid may be DNA or RNA, particularly DNA. For example, as shown in (D) of FIG. 3, the sequence of the nucleic acid contained in the recovery sequence portion 2 is complementary to the sequence of the nucleic acid 8 immobilized on the bead 9. The molecule 100 having the recovery sequence portion 2 can be efficiently recovered by the beads 9 on which a plurality of nucleic acids 8 are immobilized. The base sequence of the nucleic acid contained in the recovery sequence portion 2 may be appropriately set by those skilled in the art.

[0029] The amplification array unit 3 may contain a nucleic acid having a primer sequence used for nucleic acid amplification or a promoter sequence used for nucleic acid transcription in, for example, the analysis process described later. The nucleic acid may be DNA or RNA, particularly DNA. The amplification array unit 3 may have both a primer sequence and a promoter sequence. The primer sequence may be, for example, a PCR handle. The promoter sequence may be, for example, a T7 promoter sequence.

[0030] The barcode sequence unit 4 contains a nucleic acid having a barcode sequence. The nucleic acid may particularly be DNA or RNA, more particularly DNA. The barcode sequence is used, for example, to identify the presence position of the target capture molecule on the surface 101. The barcode sequence can be used as an identifier to distinguish a target capture molecule containing a certain barcode sequence from a target capture molecule containing another barcode sequence. The barcode sequence may be associated with information regarding the position where the target capture molecule containing the barcode sequence is fixed (hereinafter also referred to as "position information"). The position information may be for specifying a position on the surface 101, and may be, for example, information regarding XY coordinates, but is not limited thereto. An ID number may be assigned to the barcode sequence associated with the position information. The ID number may be used in the processes after the imaging process. The ID number may correspond one-to-one with the barcode sequence and may be used as data corresponding to the barcode sequence in the processes after the imaging process.

[0031] A plurality of target-capturing molecules fixed within a certain region on the surface 101 may have the same barcode sequence. Thereby, the certain region and the barcode sequence are associated with each other. By setting the size of the certain region to be smaller than the size of a cell, the target-capturing molecules containing the barcode sequence can be associated with the position where one cell exists. Thus, the surface used in the analysis method of the present technology may have a plurality of regions where a plurality of target-capturing molecules having the same barcode sequence are fixed. The barcode sequence may be different for each region. The size of each region may preferably be smaller than the size of a cell, for example, 50 μm or less, preferably 10 μm or less, more preferably 5 μm or less.

[0032] In one embodiment of the present technology, target-capturing molecules containing a barcode sequence with a known sequence may be fixed in a predetermined region. For example, the surface 101 has a plurality of regions, and a plurality of target-capturing molecules fixed to each of the plurality of regions may contain the same barcode sequence. The plurality of regions may be set to be smaller than the size of the cell to be analyzed. Regarding the surface 101 configured as described above, each of the plurality of regions and the barcode sequence included in the plurality of target-capturing molecules fixed to each region can be associated with each other. The region where target-capturing molecules containing the same barcode sequence are fixed as described above is also referred to as a spot in this specification. The size of the spot may be, for example, 50 μm or less, preferably 10 μm or less, more preferably 5 μm or less. When the target-capturing molecules are immobilized on the surface 101 configured as described above, the barcode sequence included in a certain target-capturing molecule and the position where the certain target-capturing molecule exists can be associated with each other. For this immobilization, for example, biotin is bound to the linker 1 of the target-capturing molecule and streptavidin is bound to the surface 101 to which the target-capturing molecule is to be fixed, and the target-capturing molecule is immobilized on the surface 101 by the binding of the biotin and the streptavidin.

[0033] In other embodiments of the present technology, target capture molecules containing barcode arrays may be randomly arranged on the surface 101. In this case, after the target capture molecules containing barcode arrays are fixed to the surface 101, by reading the barcode arrays contained in the fixed target capture molecules, the barcode array contained in a certain target capture molecule and the position where the certain target capture molecule exists are associated. The reading can be performed by techniques such as Sequencing By Synthesis, sequencing by ligation, or sequencing by hybridization, for example. In this embodiment, for example, beads (such as gel beads) to which a plurality of target capture molecules containing the same barcode array are bound may be used, and the beads (such as gel beads) can be fixed to the surface 101. The size of the beads (such as gel beads) can be, for example, 50 μm or less, preferably 10 μm or less, more preferably 5 μm or less. To bind the target capture molecules to the beads (such as gel beads), for example, a combination of biotin and streptavidin may be used. For example, biotin is bound to the linker 1 of the target capture molecule and streptavidin is bound to the beads, and by the binding of the biotin and the streptavidin, the target capture molecule is immobilized on the beads.

[0034] The surface 101 may be provided with a plurality of recesses. One spot or one bead in the above embodiment may be arranged in each of the plurality of recesses. In the plurality of recesses, the spot or the bead can be more easily arranged on the surface 101. The size of the recess is preferably, for example, a size into which one bead can fit. The shape of the recess may be circular, elliptical, hexagonal, or square, but is not limited thereto.

[0035] In addition, among the surface 101, the surface state of the surface portion where the spot or the beads are disposed may be different from that of other surface portions. For example, the surface portion where the spot or the beads are disposed may be hydrophilic, and other surface portions may be hydrophobic, or other surface portions may have hydrophobicity and convex portions. As a method for imparting hydrophilicity to the surface, for example, reactive ion etching in the presence of oxygen and irradiation with deep ultraviolet light in the presence of ozone can be mentioned. In these methods, a mask through which the portion for imparting hydrophilicity is penetrated can be used. Further, as a method for imparting hydrophobicity to the surface, spray-on-silicone can be mentioned, and for example, Techspray 2101-12S may be used. Also in the case of imparting hydrophobicity, for example, a mask through which the portion for imparting hydrophobicity is penetrated can be used.

[0036] For example, the target capture molecules can also be synthesized on the substrate using techniques such as DNA microarray fabrication technology. For example, the target capture molecules can be synthesized at specific positions using techniques such as DMD (Digital Mircomirror Device), liquid crystal shutter, or spatial light phase modulator used in photolithography. The method for such synthesis is described, for example, in Basic Concepts of Microarrays and Potential Applications in Clinical Microbiology, CLINICAL MICROBIOLOGY REVIEWS, Oct. 2009, p. 611-633. When the target capture molecules are synthesized on the substrate by such synthesis, information on the position where the target capture molecules are synthesized is obtained when the target capture molecules are synthesized, and the barcode sequence and the position information are associated. At that time, an ID number may be assigned.

[0037] In one embodiment of the present technology, any of the target-capturing molecules immobilized on the surface may contain a common oligo sequence. By using a nucleic acid having a sequence complementary to the oligo sequence and being fluorescently labeled, the position where the target-capturing molecule is immobilized (particularly the position of the spot or the position of the bead) can be confirmed, and particularly can be confirmed in a dark field. Also, when there are no recesses or protrusions on the surface as described above, it may become difficult to grasp the position where the target-capturing molecule is immobilized. In this case, the fluorescent label makes it easier to grasp the position where the target-capturing molecule is immobilized.

[0038] The UMI part 5 may contain a nucleic acid, particularly may contain DNA or RNA, and more particularly contains DNA. The UMI part 5 may have a sequence of, for example, 5 bases to 30 bases, particularly 6 bases to 20 bases, and more particularly 7 bases to 15 bases. The UMI part 5 may be configured to have different sequences among the target-capturing molecules immobilized on the surface 101. For example, when the UMI part has a nucleic acid sequence of 10 bases, the number of types of UMI sequences is 4 to the 10th power, that is, 1 million or more. The UMI part 5 can be used to quantify target molecules. For example, a UMI sequence is added to cDNA obtained by reverse-transcribing mRNA. A large number of cDNAs obtained by amplifying cDNA transcribed from one mRNA molecule have the same UMI sequence, but a large number of cDNAs obtained by amplifying cDNA transcribed from other mRNA molecules having the same sequence as the mRNA have different UMI sequences. Therefore, by counting the number of types of UMI sequences having the same cDNA sequence, the copy number of mRNA can be determined.

[0039] For example, the UMI part 5 may be configured to have different sequences among a plurality of target-capturing molecules containing the same barcode sequence immobilized on the spot or the bead. That is, a plurality of target-capturing molecules immobilized on the spot or the bead have the same barcode sequence while having different UMIs from each other.

[0040] The target capture unit 6 includes components for capturing molecules contained in cells. The components can be, for example, nucleic acids or proteins. When the component is a nucleic acid, the nucleic acid may be, for example, a poly-T sequence in order to comprehensively capture mRNA contained in cells. Alternatively, the nucleic acid may have a sequence complementary to the target sequence. When the component is a protein, the protein may be, for example, an antibody. The component may be an aptamer or a molecular imprinted polymer. The target capture unit 6 may include two or more types of components for capturing molecules contained in cells. The target capture unit 6 may include both proteins and nucleic acids, and may include, for example, both an antibody and a poly-T sequence. Thereby, both proteins and mRNA can be detected simultaneously.

[0041] The surface 101 can preferably be the surface of a transparent substrate. The entire substrate may be transparent, or only the portion to which the target capture molecule is fixed may be transparent. The surface of the substrate is preferably flat in order to make good contact with the specimen. The transparent substrate can be, for example, a glass substrate or a resin substrate. The substrate may be, for example, a slide glass. By being transparent, the specimen can be imaged in the imaging process described later. Also, by being transparent, the association is easily performed in the association process described later.

[0042] Specimen 4 may be a specimen containing cells, for example, and more particularly may be a fixed specimen containing cells. The specimen may be a biological tissue specimen, particularly may be a frozen tissue specimen or an FFPE (formalin-fixed paraffin-embedded) specimen, and more particularly may be a frozen tissue section or an FFPE section. Specimen 4 may be placed on a substrate, for example, and particularly may be on the surface of a transparent substrate. The substrate may be entirely transparent or only the portion where the specimen is placed may be transparent. The transparent substrate may be, for example, a glass substrate or a resin substrate. The substrate may be a slide glass, for example. The surface of the substrate is preferably flat in order to make good contact with the surface on which the target capture molecules are immobilized.

[0043] Specimen 4 may be stained to more easily perform the cell segmentation described later. The staining may be, for example, cell membrane staining, HE (Hematoxylin Eosin) staining, or DAPI staining, or a combination of two or more of these. For the cell membrane staining, a cell membrane staining reagent that is incorporated into the lipid bilayer and emits fluorescence may be used, and the reagent may be appropriately selected by those skilled in the art. The HE staining is used for bright-field observation and may be used, for example, to identify cell morphology. The DAPI staining may be used for nuclear staining.

[0044] (2-2) Imaging step

[0045] In the imaging step S102, for example, as shown in (B) of FIG. 2, the specimen 103 is imaged in a state where the surface 101 and the specimen 103 are overlapped. The imaging can be performed with a resolution that allows individual cells contained in the specimen 103 to be recognized. The imaging element 110 may be, for example, a CCD or a CMOS. The imaging may be performed, for example, by the imaging element 110 through the objective lens 111. That is, the image to be imaged may be a microscopic image. The magnification of the objective lens 111 may be appropriately selected according to the size of the cells.

[0046] The imaging may be bright-field or dark-field imaging, or both bright-field imaging and dark-field imaging may be performed. The imaging may be performed once or multiple times. For example, it may be performed once or multiple times for some regions selected by the user or the control unit, or it may be performed once or multiple times so as to cover the entire area or a part of the specimen 103.

[0047] The imaging by the imaging element 110 can be controlled by a control unit (not shown) connected to the imaging element 110. The control unit may be composed of, for example, a hard disk, a CPU, and a memory, and the function of the control unit can be realized by, for example, a general-purpose computer or an information processing device. In addition, the control unit may be provided inside the imaging element 110. The imaging element provided with the control unit may be configured as, for example, a one-chip semiconductor device having a stacked structure in which a plurality of dies (for example, two or three dies) are stacked. One of the dies includes a plurality of pixels arranged two-dimensionally. Components (such as a CPU and a memory) for realizing the function of the control unit can be mounted on the remaining dies. As an example of the imaging element including the control unit, for example, the imaging element disclosed in International Publication No. 2018 / 051809 can be cited. By using the imaging element provided with the control unit as the imaging element, various processes can be performed without outputting the specimen image data to the outside of the imaging element, which leads to speeding up of information processing.

[0048] The imaging element 110 can transmit the specimen image data obtained by imaging to the control unit. The control unit receives the specimen image data and uses the specimen image data in subsequent steps. In addition, the specimen image data received by the control unit may be stored, for example, in a storage unit connected to the control unit. The storage unit may be a general-purpose storage device. When the control unit performs subsequent processes, it can acquire the specimen image data from the storage unit.

[0049] (2-3) Association step

[0050] In the association step S103, using the specimen image obtained by imaging in the imaging step S102, the position of the cell and the barcode sequence of the target capture molecule at that position are associated. The association may be performed via position information pre-associated with the barcode sequence. When an ID number is assigned to each barcode sequence, the position of the cell and the ID number may be associated. Thereby, the position of the cell and the barcode sequence can be associated via the ID number.

[0051] The position of the cell may mean the region occupied by the cell in the specimen image. To specify the region, image processing on the specimen image may be performed, for example, cell segmentation may be performed. Cell segmentation makes it easier to identify the target capture molecule present at the position of the cell.

[0052] For example, in the association step S103, the position information of the cell to be analyzed among the cells in the specimen image is acquired. The position information of the cell may be acquired using the image data obtained by the above segmentation. Since the specimen image is captured with the surface 101 and the specimen 103 superimposed, the position information of the cell corresponds to the position information of the target capture molecule at the position of the cell. Here, as described above, the position information of the target capture molecule is associated with the barcode sequence included in the target capture molecule in the imaging target preparation step S101. Therefore, by acquiring the position information of the cell in the specimen image, the position of the cell and the barcode sequence included in the target capture molecule at that position can be associated.

[0053] Preferably, the association may be performed so that one barcode sequence is not associated with the positions of two or more cells. For example, there may be a case where two or more cells are present in one spot described above. In this case, preferably, the barcode sequence of the target capture molecule fixed to the one spot is not associated with the position of any of the two or more cells.

[0054] An example of how to distinguish between spots that are associated and spots that are not associated among the spots on the surface will be described with reference to FIG. 4.

[0055] By magnifying a part of the tissue specimen image shown on the left of FIG. 4 or obtaining an enlarged image of the part, cells in the tissue sample can be visualized as shown in the center of FIG. 4. The specimen is imaged with the analysis surface superimposed, and for example, as shown on the right of FIG. 4, it is imaged with a large number of circular spots overlapping. Each circular spot contains a plurality of target capture molecules having the same barcode sequence. On the right of FIG. 4, a large number of circular spots are shown for better understanding, but these spots do not have to be visible or may be visible in the image obtained by the imaging device. In addition, when the position of the spot cannot be confirmed in the specimen image, the position of the spot may be displayed in the specimen image by image processing.

[0056] It can be confirmed that two types of cells exist in the specimen image in the center of FIG. 4. One type of cell is shown in dark gray, and the other type of cell is shown in light gray. When associating the position of the cell and the barcode sequence of the target capture molecule present at that position for these two types of cells, for example, as shown on the right of FIG. 4, the spots indicated by the solid circles are associated. The solid circles are within the region of the image of one cell. Thus, the spots within the region of the image of one cell can be the target of association. On the other hand, the dotted circles, for example, overlap two or more cells, span the region outside the cells, or exist outside the region of the image of the cell. These dotted circles do not have to be the target of association.

[0057] The association step S103 can be performed, for example, by the control unit. For example, the control unit can associate the position of a cell with the barcode sequence of the molecule at the position of the cell for all or some types of cells among the cells present in the specimen image. When the association is performed for some types of cells, the control unit can identify the cells for which the association is performed based on, for example, cell characteristics (shape, size, color, or pattern, or a combination thereof). For example, among the images shown in the center of FIG. 4, while associating the position of the cell indicated by dark gray with the barcode sequence of the target capture molecule at that position, the association is not performed for the cells indicated by light gray, etc., and the association may be performed only for specific types of cells. Also, when using an image pickup device equipped with a control unit as the image pickup device, the association step S103 can be performed without outputting the specimen image data outside the image pickup device. Thereby, the association step S103 can be processed more quickly.

[0058] The association step S103 may be performed based on a user operation. For example, the user selects, for example, by clicking a mouse, the cells for which the user wishes to perform the association from among the cells present in the specimen image. Then, for the cells selected by the user, the control unit can associate the position of the cell with the barcode sequence of the target capture molecule at that position.

[0059] (2-4) Cleavage step

[0060] In the cleavage step S104, selective stimulation is applied to the position of the cell to cleave the linker of the molecule at that position. For example, as shown in (C) of FIG. 2, light can be irradiated to the position of the selected cell by the stimulation application device 130.

[0061] The position where the stimulus is applied may be the position of some of the cells associated in the association step S103, or may be the positions of all of the cells associated. Preferably, the stimulus is selectively applied to the position of the cells so that the linker of the target capture molecule located at a position other than the position of the cells does not cleave. The stimulus applying device 130 is preferably configured to selectively apply the stimulus in this way. That is, in the cleavage step S104, the linker of the target capture molecule containing the barcode sequence that has not been associated may not be cleaved.

[0062] For example, in the image shown on the right in FIG. 4, the stimulus may be applied to all of the spots indicated by the solid circles where the association has been made. In this way, the stimulus may be applied to all of the positions of the associated cells. Alternatively, in the image shown on the right in FIG. 4, the stimulus may be applied to the position indicated by the solid circle spot in the cells indicated by the dark gray, or to the position indicated by the solid circle spot in the cells indicated by the light gray. In this way, the stimulus may be applied to the positions of some of the cells (particularly certain types of cells) among the associated cells. Alternatively, in the image shown on the right in FIG. 4, the stimulus may be applied to the position indicated by the solid circle spot in one or more cells selected from among the associated cells. In this way, the stimulus may be applied to the positions of some of the cells (particularly the selected cells) among the associated cells.

[0063] By cleavage of the linker in step S104, the target capture molecule is released from the surface 101. For example, as shown in FIG. 3(B), when the linker 1 of the molecule 100 is cleaved, the molecule 100 is released from the surface 101.

[0064] The stimulus may be, for example, a light stimulus or a temperature stimulus (also referred to as a heat stimulus), and may preferably be a light stimulus. The light stimulus is particularly suitable for selectively applying the stimulus to a specific narrow range.

[0065] The cleavage step S104 may be performed, for example, by the control unit. More specifically, the control unit can drive the stimulation application device to selectively apply stimulation to the position of the cell. Examples of the stimulation application device that can be adopted will be described below.

[0066] To selectively apply light stimulation to the position of the cell, a light irradiation device can be used as the stimulation application device 130. The light irradiation device may be, for example, a DMD (Digital Micromirror Device) or a liquid crystal display device. The selected position on the surface 101 can be irradiated with light by the micromirrors constituting the DMD. The liquid crystal display device may be, for example, a reflective liquid crystal display, and a specific example can be SXRD (Sony Corporation). By controlling the liquid crystal of the liquid crystal display device, the selected position on the surface 101 can be irradiated with light. Also, a liquid crystal shutter or a spatial light modulator may be used to selectively apply light stimulation to the position of the cell. Also by these, light stimulation can be applied to the selected position. The wavelength of the irradiated light may be appropriately selected by those skilled in the art according to the type of the linker contained in the target capture molecule.

[0067] To selectively apply temperature stimulation to the position of the cell, for example, a combination of infrared light and an infrared light absorbing material can be used. In this case, for example, an infrared laser light generating device can be adopted as the stimulation application device. For example, by forming the substrate 102 having the surface 101 from an infrared light absorbing material and selectively irradiating the position of the cell with infrared light by the infrared laser light generating device, temperature stimulation can be selectively applied to the position.

[0068] (2-5) Binding step

[0069] In the binding step S105, the target capture molecule released from the surface 101 by the cleavage in step S104 is bound to the constituent components of the cell via the target capture portion of the target capture molecule.

[0070] As shown in the schematic enlarged view surrounded by the dashed line in (C) of FIG. 2, for example, only the linker of the target capture molecule at the irradiated position is cleaved and taken into the cell directly below. Then, as shown in (C) of FIG. 3, for example, in the cell, the cell component 7 can bind to the target capture portion 6 of the target capture molecule 100.

[0071] The binding step S105 may include a moving step of moving the target capture molecule toward the cell by applying an electric field, a magnetic field, or a centrifugal force. For example, since the nucleic acid contained in the target capture molecule has a negative charge, the target capture molecule can be moved toward the cell by a positive charge. For example, an electric field can be applied by arranging a laminate of the substrate 102 and the substrate 104 between opposing electrodes. Further, a metal thin film or a transparent electrode such as ITO (Indium Tin Oxide) having a thickness that does not interfere with bright-field observation or dark-field observation (particularly fluorescence observation) may be arranged on the substrate 102 and the substrate 104. As the electric field applying device for applying an electric field, the magnetic field applying device for applying a magnetic field, or the centrifugal force applying device for applying a centrifugal force, a device known in the art may be employed. As the centrifugal force applying device, for example, a swinging rotor type centrifugal force applying device can be mentioned. The application of the electric field, the magnetic field, or the centrifugal force by these devices may be controlled by, for example, a control unit.

[0072] The binding step S105 may include a moving step of moving the target capture molecule toward the cell by natural diffusion. In the moving step, a combination of applying an electric field, a magnetic field, or a centrifugal force and natural diffusion may be used. The moving step preferably includes applying an electric field, thereby increasing the probability that the released target capture molecule is taken into the cell.

[0073] The binding step S105 may include an incubation step for binding the target capture molecule to the components of the cell. The time and temperature of the incubation step may be selected according to the target capture part and the cell components captured by the target capture part. The incubation step may be performed, for example, with the space between the surface 101 and the specimen filled with a buffer. For example, when both the target capture part and the cell components are nucleic acids, incubation can be carried out in a thermostat at, for example, 30°C to 40°C, particularly 30°C to 37°C, for, example, 5 hours to 30 hours, particularly 16 hours to 24 hours. For example, when the target capture part is an antibody and the cell components are the components captured by the antibody, incubation can be carried out at, for example, 0°C to 30°C, particularly 4°C to room temperature (e.g., 25°C), for, example, 5 hours to 30 hours, particularly 16 hours to 24 hours.

[0074] In the binding step S105, the target capture molecule may be taken into the cell or bound to the cell surface. Then, the target capture molecule binds to the components of the cell through the target capture part of the target capture molecule. Thereafter, the surface 101 and the specimen 103 are separated, and the unbound target capture molecules are removed by washing. As a washing method, the specimen 103 may be immersed in a buffer or the like.

[0075] (2-6) Analysis step

[0076] In the analysis step S106, an analysis can be performed on the conjugate generated by the binding of the molecule for target capture and the cell components in the binding step S105. As shown in (D) of FIG. 2, the analysis can be performed using the analyzer 200. For example, in the analysis step S106, a sequencing process may be performed on the conjugate, and the analyzer 200 may be a sequencer. The sequencing process can be performed, for example, when the cell component is a nucleic acid, particularly DNA or RNA, more particularly mRNA. The sequencing process may be performed by a sequencer, and may be performed by a next-generation sequencer or a sequencer using the Sanger method. In order to perform a more comprehensive analysis of the cells constituting the tissue at a higher speed, the sequencing process can be performed by a next-generation sequencer.

[0077] In order to perform a sequencing process in the analysis step, the analysis step may further include a preparation step of a nucleic acid (such as cDNA) to be a target of the sequencing process and a purification step of the nucleic acid. By these preparation step and purification step, for example, a library for performing a next-generation sequencing process may be prepared. In the preparation of the library, for example, as shown in (D) of FIG. 3, the recovery sequence portion 2 can be used. Using beads 9 to which a nucleic acid having a sequence complementary to the nucleic acid sequence contained in the recovery sequence portion 2 is immobilized, the molecule 100 bound to the cell component 7 can be recovered.

[0078] In the preparation step, the laminate of the substrates 102 and 104 after the binding in the binding step S105 is washed using a buffer such as PBS, for example, so that unbound target capture molecules can be removed. Then, the substrate 102 is removed from the substrate 104, and for the cells incorporating the target capture molecules, for example, a cDNA synthesis step of synthesizing cDNA from mRNA and an amplification step of amplifying the synthesized cDNA can be performed. Before the cDNA synthesis step and the amplification step, a lysis step of lysing the cells may be performed. By recovering the conjugate of the target capture molecule and the target after the lysis step, the cDNA synthesis step and the amplification step can be performed more efficiently.

[0079] After the preparation step, a purification step of purifying the nucleic acid obtained in the preparation step may be performed. The purification step may include a decomposition treatment of components other than the nucleic acid using an enzyme such as proteinase K. Further, in the purification step, a nucleic acid recovery treatment may be performed. In the nucleic acid recovery treatment, for example, commercially available nucleic acid purification reagents may be used, and examples thereof include magnetic beads such as AMPure XP. In addition, although intracellular dsDNA can also be recovered in the purification step, it is possible to prevent dsDNA from being sequenced in the sequencing treatment. For example, by including an adapter sequence for sequencing treatment (particularly for next-generation sequencing treatment) in the target capture molecule, only the nucleic acid containing the adapter sequence can be sequenced.

[0080] In the analysis step S106, based on the sequencing treatment result, the cell components can be analyzed for each cell. For example, in the analysis step S106, for each cell, the sequence and / or the copy number of each mRNA contained in the cell can be determined. Further, in the analysis step S106, for each cell, the type and / or number of antigens, or the type and / or number of transcription factors can be determined. Such analysis of cell components for each cell can be performed based on the barcode sequence in the sequence determined by the sequencing treatment. For example, among a large number of sequences determined by the sequencing treatment, sequences containing the same barcode sequence are selected. The sequences containing the same barcode sequence are based on the target capture molecules incorporated into one cell. Therefore, analyzing cell components for each barcode sequence means analyzing cell components for each cell.

[0081] The analysis step S106 may include a two-dimensional mapping step of performing two-dimensional mapping based on the association result in the association step S103 using the result of the analysis and the specimen image. For example, in the analysis step S106, the analysis result of the cell components for each cell can be mapped to the specimen image obtained in the imaging step based on the position information associated with the barcode array. By this mapping, for example, as shown in (E) of FIG. 2, a specimen image (above (E) of FIG. 2) and a mapping image (below (E) of FIG. 2) showing the distribution of the types of cell components at each position in the specimen image can be obtained. From the image obtained by this mapping, it is possible to grasp which cells at which positions contain which molecules in what amounts. That is, the position information obtained by imaging and the quantitative information obtained by sequence analysis can be combined, and comprehensive analysis of the molecules in the tissue sample becomes possible while retaining the spatial information at single cell resolution.

[0082] 2. Second Embodiment (Analysis System)

[0083] An example of the block diagram of the analysis system of the present technology is shown in FIG. 5. As shown in FIG. 5, the analysis system 10 of the present technology includes an analysis substrate 102, an imaging device 110, a control unit 120, a storage unit 125, and a stimulation device 130.

[0084] The analysis substrate 102, the imaging device 110, and the stimulation device 130 may be as described in 1. above, and the description also applies to this embodiment.

[0085] The control unit 120 may be the control unit described in 1. above, and the description also applies to this embodiment. The control unit 120 may include, for example, an image processing unit 121, an association unit 122, and a stimulation control unit 123. These will be described in more detail below.

[0086] The image processing unit 121 processes the specimen image acquired by the imaging device 110. The image processing unit 121 can perform, for example, the cell segmentation described in the above 1. Further, the image processing unit 121 can identify the cells to which the association is made in the association step among the cells existing in the specimen image. The identification can be performed, for example, based on the characteristics (shape, size, color, or pattern, or a combination thereof) of the cells. The identification may be performed based on the color data in the image. For example, a cell that emits a certain fluorescence can be identified as a cell to which the association is made in the association step.

[0087] The association unit 122 associates the position of the cell with the barcode sequence of the target capture molecule at the position of the cell. The association may be performed using the image obtained by the above segmentation. For example, the association unit 122 acquires the position data of the cell based on the specimen image, identifies the barcode sequence assigned to the position data corresponding to the position data, and can associate the barcode sequence with the position of the cell.

[0088] The association unit 122 may perform the association between the position of the cell and the barcode sequence of the target capture molecule at the position of the cell for all the cells in the specimen image, or may perform the association for some of the cells in the specimen image. For example, the association unit 122 can perform the association only for some types of cells among all the cells existing in the specimen image, or can perform the association only for the cells existing in some regions.

[0089] The stimulation control unit 123 causes the stimulation application device 130 to selectively apply stimulation to the positions of the cells associated by the association unit 122. The stimulation control unit 123 can drive the stimulation application device 130 to apply stimulation to all or part of the cells associated by the association unit 122. The stimulation application device 130 is preferably configured to selectively apply stimulation to the positions of the cells without cleaving the linker contained in the target capture molecule located at a position other than the position of the cells. The stimulation application device 130 is preferably the light irradiation device described in 1. above.

[0090] The control unit 120 may be composed of, for example, a hard disk, a CPU, and a memory, and the functions of the control unit may be realized in a general-purpose computer or information processing device. The functions of the image processing unit 121, the association unit 122, and the stimulation control unit 123 can also be realized by a general-purpose computer or information processing device. As described above in (2-2), the control unit 120 may be provided in the image pickup device 110. By using an image pickup device provided with a control unit as the image pickup device, various processes can be performed without outputting the specimen image data to the outside of the image pickup device, which leads to speeding up of information processing. For example, the processes by the image processing unit 121, the association unit 122, and the stimulation control unit 123 described above can be performed at higher speed.

[0091] In order to perform the binding step S105 described in 1. above, the analysis system 10 may include, for example, an electric field or magnetic field application device that applies an electric field or magnetic field for moving the target capture molecule released from the surface of the analysis substrate 102 toward the cells by applying stimulation by the stimulation application device 130, or a centrifugal force application device that applies a centrifugal force. By means of this device, the target capture molecule is more efficiently taken up by the cells.

[0092] In order to perform the binding step S105 described in 1. above, the analysis system 10 may further include an incubation device for promoting the binding of the target capture molecules released from the surface of the analysis substrate 102 to the cell components by applying a stimulus with the stimulus application device 130. The incubation device may include, for example, a thermostatic bath.

[0093] In order to perform the analysis step S106 described in 1. above, the analysis system 10 may further include an analyzer for analyzing the conjugate of the target capture molecule released from the surface of the analysis substrate 102 and the cell components by applying a stimulus with the stimulus application device 130. The analyzer may be, for example, a sequencer. The sequencer may be, for example, a next-generation sequencer or a sequencer that performs sequencing by the Sanger method.

[0094] The analysis system 10 may further include a two-dimensional mapping unit that performs two-dimensional mapping based on the association result by the association unit using the analysis result by the analyzer and the specimen image. The two-dimensional mapping unit may be configured as an element of the control unit 120 and may perform, for example, the two-dimensional mapping step described in 1. above. By the two-dimensional mapping unit, for example, information regarding the components of the cell can be mapped to the specimen image. The information regarding the components of the cell may be, for example, the type or amount of the components of the cell. From the image obtained by the mapping, it is possible to grasp which cells at which positions contain which molecules in what amounts. That is, the position information obtained by imaging and the quantitative information obtained by sequence analysis can be combined, and comprehensive analysis of the molecules in the tissue sample can be performed while retaining the spatial information at single cell resolution.

[0095] The analysis system 10 may further include an output unit. The output unit may include, for example, a display device and / or a printing device. The control unit 120 can cause the output unit to output an image obtained by performing an analysis method according to the present technology, such as a specimen image acquired by the imaging device 110 and a mapping image generated by the two-dimensional mapping unit. Further, the control unit 120 can cause the output unit to output an analysis result (e.g., a sequencing processing result, etc.) by the analysis device.

[0096] 3. Third Embodiment (Analysis Surface)

[0097] The present technology also provides an analysis surface on which a target capture molecule including a cleavable linker, a barcode array, and a target capture unit is fixed via the linker, and the barcode array is used to provide information regarding the position where the molecule including the barcode array is fixed. An example of the analysis surface is the surface 101 of the analysis substrate 102 described in 1. above, and the description of the surface also applies to this embodiment.

[0098] Note that the present technology can also have the following configuration. 〔1〕An imaging step of imaging a specimen in a state where the specimen is overlaid on a surface on which a molecule including a linker cleavable by stimulation, a barcode array, and a target capture unit is fixed via the linker; An association step of associating the position of a cell with the barcode array of the molecule at the position using the specimen image obtained by the imaging; A cleavage step of selectively applying stimulation to the position of the cell to cleave the linker of the molecule at the position; A binding step of binding the molecule released from the surface by the cleavage to a constituent component of the cell via the target capture unit of the molecule; An analysis method including 〔2〕The analysis method according to 〔1〕, wherein the specimen includes a tissue sample. 〔3〕The analysis method according to 〔1〕 or 〔2〕, wherein in the cleavage step, selective stimulation is applied to the position of the cell so that the linker of the molecule at a position other than the position of the cell is not cleaved. 〔4〕The analysis method according to any one of 〔1〕 to 〔3〕, wherein the stimulation is light stimulation. 〔5〕The analysis method according to any one of 〔1〕 to 〔4〕, wherein the binding step includes a moving step of moving the molecule toward the cell by applying an electric field, a magnetic field, or a centrifugal force. 〔6〕The analysis method according to any one of 〔1〕 to 〔4〕, wherein the binding step includes a moving step of moving the molecule toward the cell by natural diffusion. 〔7〕The analysis method according to any one of 〔1〕 to 〔6〕, wherein the binding step includes an incubating step for binding the molecule and a constituent component of the cell. 〔8〕The analysis method according to any one of 〔1〕 to 〔7〕, further including an analysis step of analyzing the conjugate of the molecule and the constituent component of the cell after the binding step. 〔9〕The analysis method according to 〔8〕, wherein in the analysis step, a sequencing process is performed on the conjugate. 〔10〕The analysis method according to 〔8〕 or 〔9〕, wherein the analysis step includes a two-dimensional mapping step of performing two-dimensional mapping based on the association result in the association step using the analysis result and the specimen image. 〔11〕An analysis substrate having a surface on which a molecule including a linker cleavable by stimulation, a barcode sequence, and a target capture portion is fixed via the linker, An imaging device for imaging a specimen stacked on the analysis substrate, An association unit for associating the position of a selected cell in the specimen image obtained by the imaging with the barcode sequence of the molecule at the position, A stimulation application device for selectively applying stimulation to the position of the cell, comprising using, as an analysis target, a conjugate in which a molecule released from the surface by the stimulation and a constituent component of the cell are bound via the target capture portion of the molecule. Analysis system. 〔12〕The analysis system according to 〔11〕, wherein the specimen includes a tissue sample. 〔13〕The analysis system according to 〔11〕 or 〔12〕, wherein the stimulation device is configured to selectively apply stimulation to the position of the cell without cleaving a linker contained in a molecule at a position other than the position of the cell. 〔14〕The analysis system according to any one of 〔11〕 to 〔13〕, wherein the stimulation device is a light irradiation device. 〔15〕The analysis system according to any one of 〔11〕 to 〔14〕, further including an electric field application device, a magnetic field application device, or a centrifugal force application device that applies an electric field, a magnetic field, or a centrifugal force for moving the molecule released from the surface toward the cell by the application of stimulation by the stimulation device. 〔16〕The analysis system according to any one of 〔11〕 to 〔15〕, further including an incubation device for promoting the binding of the molecule released from the surface by the application of stimulation by the stimulation device to a constituent component of the cell. 〔17〕The analysis system according to any one of 〔11〕 to 〔16〕, further including an analysis device for analyzing a conjugate of the molecule released from the surface by the application of stimulation by the stimulation device and a constituent component of the cell. 〔18〕The analysis system according to 〔17〕, wherein the analysis device is a sequencer. 〔19〕The analysis system according to 〔17〕 or 〔18〕, further including a two-dimensional mapping unit that performs two-dimensional mapping based on the association result by the association unit using the analysis result by the analysis device and the specimen image obtained by the imaging. 〔20〕A molecule including a cleavable linker, a barcode sequence, and a target capture unit is fixed via the linker, and the barcode sequence is used to provide information regarding the position where the molecule including the barcode sequence is fixed. Analysis surface.

Explanation of symbols

[0099] 100 molecules 101 analysis surface 102 substrate 103 specimen 104 substrate 110 imaging device 120 control unit 130 stimulation device

Claims

1. an imaging step of imaging the specimen in a state where the specimen is superimposed on a surface on which a molecule including a linker that can be cleaved by a stimulus, a barcode sequence, and a target capture portion is immobilized via the linker; an association step of associating the position of a cell with the barcode sequence of a molecule at that position using the specimen image obtained by the imaging; a cleavage step of selectively applying a stimulus to a location of the cell to cleave the linker of the molecule at that location; a binding step of binding the molecule released from the surface by the cleavage to a component of the cell via the target capture portion of the molecule; Analytical methods including:

2. The method of claim 1 , wherein the specimen comprises a tissue sample.

3. The analytical method according to claim 1 , wherein in the cleavage step, a stimulus is selectively applied to the location of the cell so that a linker of a molecule at a location other than the location of the cell is not cleaved.

4. The method of claim 1 , wherein the stimulus is a light stimulus.

5. The method according to claim 1 , wherein the binding step includes a migration step of migrating the molecule toward the cell by application of an electric field, a magnetic field, or centrifugal force.

6. The analytical method according to claim 1 , wherein the binding step includes a transfer step of transferring the molecule toward the cell by natural diffusion.

7. The analytical method according to claim 1 , wherein the binding step includes an incubation step for binding the molecule to a component of the cell.

8. The method according to claim 1 , further comprising an analysis step of performing an analysis on the conjugate between the molecule and the cell component after the binding step.

9. The analytical method according to claim 8 , wherein the binding entity is subjected to a sequencing process in the analyzing step.

10. The analysis method according to claim 8 , wherein the analyzing step includes a two-dimensional mapping step of performing two-dimensional mapping based on the association result in the associating step, using the analysis result and the specimen image.

11. an analytical substrate having a surface on which a molecule including a linker that can be cleaved by a stimulus, a barcode sequence, and a target capture portion is immobilized via the linker; an imaging device for imaging the sample placed on the analytical substrate; an associating unit that associates the position of a selected cell in the specimen image obtained by the imaging with a barcode sequence of a molecule at that position; A stimulation device that selectively stimulates the location of the cell; Including, a conjugate in which the molecule released from the surface by the stimulation and a component of the cell are bound to each other via the target capture portion of the molecule is used as an analysis subject; Analysis system.

12. The analytical system of claim 11 , wherein the specimen comprises a tissue sample.

13. The analysis system of claim 11, wherein the stimulus application device is configured to selectively apply a stimulus to the location of the cell without cleaving a linker contained in a molecule at a location other than the location of the cell.

14. The analysis system according to claim 11 , wherein the stimulation device is a light irradiation device.

15. The analysis system according to claim 11, further comprising an electric field application device, a magnetic field application device, or a centrifugal force application device that applies an electric field, a magnetic field, or a centrifugal force to move the molecules released from the surface by the application of a stimulus by the stimulus application device toward the cells.

16. The analytical system according to claim 11 , further comprising an incubation device for encouraging the molecules released from the surface by the application of a stimulus by the stimulus application device to bind to components of the cells.

17. The analytical system according to claim 11 , further comprising an analytical device that analyzes a bond between the molecule and a component of the cell that has been released from the surface by the application of a stimulus by the stimulus application device.

18. The analytical system according to claim 17 , wherein the analytical device is a sequencer.

19. The analysis system according to claim 17 , further comprising a two-dimensional mapping unit that performs two-dimensional mapping based on the association result by the association unit using the analysis result by the analysis device and the specimen image obtained by the imaging.

20. A molecule comprising a cleavable linker, a barcode sequence and a target capture portion is immobilized via the linker; and The barcode sequence is used to provide information regarding the location at which a molecule containing the barcode sequence is fixed. Analytical surface.

Images