Analytical method, analytical system and analytical surface

Abstract

The purpose of the present invention is to provide a method capable of associating the position information of a cell with the constituent components of the cell in single-cell decomposition. The present technology provides an analysis method, comprising: an imaging step, imaging a specimen in a state where the specimen overlaps with a surface, and a molecule having a linking group, a barcode sequence, and a target capture portion that can be cleaved by stimulation is fixed to the surface via the linking group; an association step, associating the position of the cell with the barcode sequence of the molecule at the position by using the specimen image obtained by imaging; a cleavage step, by selectively applying stimulation to the position of the cell to cleave the linking group of the molecule at the position; and a binding step, binding the molecule released from the surface by cleavage to the constituent components of the cell via the target capture portion of the molecule.

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 acquiring the type and amount of constituent components contained in a cell in association with positional information of the cell. Background Art

[0002] In order to analyze biological tissues, the type and amount of mRNA contained in cells forming the biological tissues are measured. In the analysis of biological tissues, not only the type and amount of mRNA contained in the cells but also the positional information of the cells is important. In view of this, some methods for acquiring the positional information of cells contained in the biological tissues and the information on the mRNA contained in the cells in association with each other have been proposed.

[0003] For example, the non-patent document 1 cited below discloses a method called spatial transcriptomics. The probe used in this method contains a cleavage site, T7 amplification and sequencing processing, a spatial barcode, a UMI, and an mRNA capture region (non-patent document 1). Figure 2 ). In this method, a tissue section is placed on a slide fixed with a probe, and 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 an analysis step, such as sequencing.

[0004] Reference List

[0005] Non-patent literature

[0006] Non-patent literature 1: Patrik L. Stahl et al., Visualization and analysis of gene expression in tissue sections by spatial transcriptomics, Science, July 1, 2016, Vol. 353, No. 6294, pp. 78-82 Summary of the invention

[0007] Problems to be solved by the present invention

[0008] It is believed that if the positional information of cells and information about the constituent components contained in the cells can be associated with each other for each cell forming the biological tissue, the biological tissue can be analyzed in more detail. In view of this, the purpose of the present technology is to provide a method capable of associating the positional information of cells with unit constituent components at single-cell resolution.

[0009] Solution to the problem

[0010] The inventors of the present technology have discovered that the above-mentioned problems can be solved by a specific analysis method.

[0011] That is, the present technology provides an analysis method, comprising:

[0012] an imaging step of imaging the specimen in a state where the specimen and the surface overlap, wherein the molecule having a linker group that can be cleaved by stimulation, a barcode sequence, and a target capture portion is fixed to the surface via the linker group;

[0013] a correlating step of correlating the location of the cell with the barcode sequence of the molecule at the location by using the specimen image obtained by imaging;

[0014] a cleavage step of selectively stimulating a location of the cell to cleave a linker group of the molecule at the location; and

[0015] The binding step is to bind the molecule released from the surface by cleavage to the constituent components of the cell through the target capture portion of the molecule.

[0016] Specimens may include tissue samples.

[0017] In the cleavage step, a location of the cell may be selectively stimulated so as not to cleave the attachment group of the molecule at locations other than said location of the cell.

[0018] The stimulus may be light stimulus.

[0019] The binding step may include a moving step in which the molecules are moved toward the cells by applying an electric field, a magnetic field, or a centrifugal force.

[0020] The binding step may include a movement step whereby the cell moves towards the molecule by natural diffusion.

[0021] The binding step may include an incubation step to allow the molecule to bind to constituent components of the cell.

[0022] The analysis method of the present technology may further include an analysis step of analyzing the conjugate of the molecule and the constituent components of the cell after the binding step.

[0023] The conjugate can be subjected to a sequencing process in an analysis step.

[0024] The analyzing step may include a two-dimensional mapping step of performing two-dimensional mapping based on a result of the correlation in the correlating step by using the analysis result and the specimen image.

[0025] In addition, the present technology also provides an analysis system, comprising:

[0026] an analytical substrate having a surface, a molecule having a linking group cleavable by stimulation, a barcode sequence, and a target capture portion immobilized to the surface via the linking group;

[0027] an imaging device for imaging and analyzing the specimen overlapped with the substrate;

[0028] an associating unit that associates the position of the cell selected in the specimen image obtained by the imaging with the barcode sequence of the molecule at the position; and

[0029] A stimulation supply device, the stimulation supply device selectively stimulates the location of the cell, wherein,

[0030] A conjugate in which the molecule released from the surface due to the stimulus is bound to a constituent component of the cell via the target capturing portion of the molecule is used as an analysis target.

[0031] Specimens may include tissue samples.

[0032] The stimulus supply means may be formed to selectively stimulate the location of the cell without cleaving the linking group of the molecule at a location other than the location of the cell.

[0033] The stimulus supply device may be a light irradiation device.

[0034] The analysis system may include an electric field applying device, a magnetic field applying device, or a centrifugal force applying device for applying an electric field, a magnetic field, or a centrifugal force, wherein the electric field, the magnetic field, or the centrifugal force is used to move the molecules released from the surface due to the stimulation of the stimulation supply device toward the cells.

[0035] The analysis system may further include an incubation device that causes the molecules released from the surface due to stimulation by the stimulation supply means to bind to constituent components of the cells.

[0036] The analysis system may further include an analysis device that analyzes the conjugate of the molecules and the constituent components of the cells released from the surface due to the stimulation by the stimulation supply device.

[0037] The analyzing device may be a sequencer.

[0038] The analysis system may further include a two-dimensional mapping unit that performs two-dimensional mapping based on the correlation result of the correlation unit by using the analysis result of the analysis device and the specimen image obtained by the imaging.

[0039] Furthermore, the present technology also provides an analysis surface, wherein,

[0040] A molecule having a cleavable linker, a barcode sequence, and a target capture portion is immobilized to the analytical surface via the linker; and

[0041] The barcode sequence is used to provide information about the position where the molecule having the barcode sequence is immobilized. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 An example of a flow chart showing an analysis method according to the present technology.

[0043] Figure 2 is a schematic diagram for describing the operation in each step included in the analysis method according to the present technology.

[0044] Figure 3 is a schematic diagram showing an analysis surface used in an analysis method according to the present technology and molecules immobilized to the surface.

[0045] Figure 4 is a schematic diagram for describing associated steps included in a method according to the present technology.

[0046] Figure 5 is a block diagram of an example of an analysis system according to the present technology. DETAILED DESCRIPTION

[0047] Hereinafter, a preferred mode for carrying out the present technology will be described. It should be noted that the embodiments described below are typical examples of the present technology, and the scope of the present technology is not limited to these embodiments. It should be noted that the present technology will be described in the following order.

[0048] 1. First embodiment (analysis method)

[0049] (1) Description of the First Embodiment

[0050] (2) Example of the first embodiment

[0051] (2-1) Imaging target preparation steps

[0052] (2-2) Imaging steps

[0053] (2-3) Association steps

[0054] (2-4) Cracking step

[0055] (2-5) Combination steps

[0056] (2-6) Analysis steps

[0057] 2. Second Embodiment (Analysis System)

[0058] 3. Third Embodiment (Analysis Surface)

[0059] 1. First embodiment (analysis method)

[0060] (1) Description of the First Embodiment

[0061] In the analysis method of the present technology, the specimen is imaged in a state where the specimen overlaps with the surface, and a molecule having a linking group that can be cleaved by stimulation, a barcode sequence, and a target capture portion is fixed to the surface via the linking group. Then, the position of the cell contained in the specimen is associated with the barcode sequence of the molecule at the position by using the specimen image obtained by imaging. After the association is performed, the position of the cell to be analyzed is stimulated, and the linking group of the molecule present at the position is cleaved. Due to the cleavage, the molecule is released from the surface, and the molecule is bound to the constituent components of the cell to be analyzed via the target capture portion.

[0062] Thus, the molecule bound to the constituent components of the cell has a barcode sequence, and the barcode sequence is associated with the position of the cell. Therefore, when analyzing the molecule bound to the constituent components of the cell (e.g., identification of the constituent components of the cell, identification of the barcode sequence, etc.), information about the constituent components of the cell associated with the position information of the cell can be obtained. In addition, in the analysis method, the specimen image is also acquired as described above. Therefore, for example, data about the constituent components of the cell can be mapped on the specimen image.

[0063] In the present technology, the specimen can be a fixed specimen, for example, a frozen section or a FFPE section. In many cases, the analysis method of the related art is only applicable to frozen sections or FFPE sections. However, the analysis method of the present technology is applicable to these two sections. The specimen can be, for example, a tissue sample, and can particularly be a biological tissue sample.

[0064] In the analysis method of the present technology, the position of the cell is selectively stimulated, and the molecules present at the position are released from the surface and then combined with the constituents of the cell. More preferably, in the lysis step, the position of the cell is selectively stimulated so as not to cleave the connecting groups of the molecules at the position other than the position of the cell. This makes it possible to analyze the constituents (e.g., mRNA or protein) of the cell at single cell resolution. That is, the analysis method of the present technology can be an analysis method for analyzing the constituents of the cell contained in the specimen at single cell resolution.

[0065] In addition, the constituent components of various cells contained in the specimen can be comprehensively analyzed by using barcode sequences associated with the position information of the cells. For example, in the analysis of mRNA in a tissue sample according to the related art, a specific area can be specified based on the morphological information or immunostaining information of the cells in the tissue sample, the specific area can be cut out with a laser, etc., and then the specific area can be analyzed for mRNA. In the present technology, the expression information of mRNA in the tissue sample can be comprehensively performed for each cell contained in the tissue sample without, for example, cutting out the tissue sample. That is, the analysis method of the present technology can be an analysis method for analyzing the constituent components of multiple cells contained in a specimen at a single-cell resolution.

[0066] (2) Example of the first embodiment

[0067] Figure 1 An example of a flow chart showing an analysis method according to the present technology is shown. Figure 1 As shown, the analysis method of the present technology may include an imaging target preparation step S101, an imaging step S102, an association step S103, a lysis step S104, a binding step S105, and an analysis step S106. Each step will be described below.

[0068] (2-1) Imaging target preparation steps

[0069] In the imaging target preparation step S101, an imaging target in the imaging step S102 described later is prepared. The imaging target may be a laminate obtained by overlapping a specimen with a surface having a linker cleavable by a stimulus, a barcode sequence, and a molecule of a target capture part fixed thereto via the linker.

[0070] In the imaging target preparation step S101, for example, Figure 2 As shown in (A), an analysis substrate (e.g., a slide glass, etc.) 102 having a surface 101 on which a plurality of molecules 100 are fixed overlapped with a substrate (e.g., a slide glass, etc.) 104 on which a specimen 103 is placed. The overlap may be performed so that the surface 101 and the specimen 103 face each other, and, for example, the surface 101 and the specimen 103 may be in contact with each other via a buffer solution, etc. In addition, after the surface 101 and the specimen 103 are overlapped, the laminate of the analysis substrate 102 and the substrate 104 may be immersed in a buffer solution such as PBS.

[0071] The positional relationship between the substrates 102 and 104 may be fixed so that the positional relationship between the surface 101 and the specimen 103 overlapped thereby is not changed in steps described later (specifically, the imaging step S102 to the bonding step S105 ).

[0072] Each molecule 100 has a stimulus-cleavable linker, a barcode sequence, and a target capture moiety.

[0073] In the present specification, a molecule (i.e., a molecule having a linking group, a barcode sequence, and target capture) is used to capture a target and may also be referred to as a target capture molecule. A target capture molecule is a name indicating a molecule used for the present technology, and may be used in the present specification, for example, to refer to a molecule that has captured a target, and also to a molecule in which a linking group has been cleaved in a cleavage step described later. A target capture molecule may be, for example, a single molecule or a complex molecule. A single molecule may mean, for example, a type of molecule having multiple functions, and may be, for example, a nucleic acid portion configured as a linking group, a nucleic acid portion configured as a barcode sequence, and a nucleic acid portion configured as a target capture portion (e.g., DNA or RNA). A composite molecule may be, for example, a molecular assembly containing two or more types of molecules (e.g., a binding substance of two or more types of molecules), and may be, for example, a conjugate of a nucleic acid portion configured as a linking group and a nucleic acid portion configured as a barcode sequence and a polypeptide configured as a target capture portion (e.g., a protein or a portion thereof, an oligopeptide, etc.).

[0074] Reference Figure 3 An example of the structure of molecule 100 is described. Figure 3 The molecule 100 in FIG. 1 has a linker 1, a collection sequence portion 2, an amplification sequence portion 3, a barcode sequence portion 4, a unique molecular identifier (UMI) portion 5, and a target capture portion 6. In addition, the molecule 100 is immobilized to a surface 101 via the linker 1.

[0075] The collection sequence part 2, the amplification sequence part 3, the barcode sequence part 4, and the UMI part 5 may also be constituted as a continuous nucleic acid (particularly DNA). In the case where the target capture part 6 is a nucleic acid, not only the collection sequence part 2, the amplification sequence part 3, the barcode sequence part 4, and the UMI part 5, but also the target capture part 6 may be constituted as a continuous nucleic acid (particularly DNA). In those cases, for example, the end close to the part where the molecule 100 is fixed to the surface 101 may be the 5'-end, and the other end may be the 3'-end.

[0076] The constituent components of molecule 100 will now be described.

[0077] The linking group 1 may be cleavable by stimulation, for example by light stimulation or temperature stimulation, and is preferably cleavable by light stimulation. Light stimulation is particularly suitable for selectively stimulating a specific location in the cleavage step described below.

[0078] The linking group 1 may contain, for example, any one selected from arylcarbonylmethyl, nitroaryl, coumarin-4-ylmethyl, arylmethyl, metal-containing groups and other groups as a linking group that can be cleaved by light stimulation. These groups may be described in, for example, Photoremovable Protecting Groups in Chemistry and Biology: Reaction Mechanisms and Efficacy, Chem. Rev. 2013, 113, 119-191.

[0079] For example, arylcarbonylmethyl can be phenacyl, o-alkylbenzoyl or p-hydroxybenzoyl. Nitroaryl can be, for example, o-nitrobenzyl, o-nitro-2-phenylethoxycarbonyl or o-nitroaniline. Arylmethyl can be, for example, an aryl group into which a hydroxyl group is introduced or an aryl group into which a hydroxyl group is not introduced.

[0080] In the case where the linking group 1 can be cleaved by light stimulation, the linking group can preferably be cleaved by light having a wavelength of 360 nm or more. The linking group can preferably be cleaved at 0.5 μJ / μm 2 or less. (Light-sheet fluorescence microscopy for quantitative biology, Nat Methods. 2015 Jan; 12 (1): 23-6. doi: 10.1038 / nmeth.3219.). By adopting a linking group that is cut by light having the above wavelength or at the above energy, cell damage (specifically, the cutting of DNA or RNA, etc.) that may occur when light stimulation is applied can be reduced.

[0081] In the case where the linker 1 can be cleaved by temperature stimulation, the linker 1 can include, for example, a temperature-responsive polymer. The temperature-responsive polymer can change from hydrophilic to hydrophobic or from hydrophobic to hydrophilic in response to, for example, temperature changes. By such changes, the target capture molecule can be released from the surface 1.

[0082] Particularly preferably, the linking group can be cleaved by light in the short wavelength region, specifically, light in the wavelength region of 360nm to 410nm, or the linking group can be cleaved by light in the near infrared region or infrared region, specifically, light in the wavelength region of more than 800nm. In the case where the linking group is effectively cut off by light with a wavelength in the visible light region, it may be difficult to process the analysis surface. Therefore, the linking group is preferably cleaved by light in the short wavelength region or light in the near infrared region or infrared region.

[0083] The collection sequence part 2 comprises nucleic acids for collecting molecules 100 released from the surface 101 in the analysis step described later. The nucleic acid can be DNA or RNA, and is particularly DNA. For example, Figure 3 As shown in (D), the sequence of the nucleic acid contained in the collection sequence section 2 is complementary to the sequence of the nucleic acid 8 fixed to the bead 9. The molecule 100 having the collection sequence section 2 can be efficiently collected by the bead 9 to which a plurality of nucleic acids 8 are fixed. The base sequence of the nucleic acid contained in the collection sequence section 2 can be appropriately set by a person skilled in the art.

[0084] The amplification sequence part 3 may include, for example, a nucleic acid having a primer sequence for amplifying nucleic acid or a promoter sequence for transcribing nucleic acid in an analysis step described later. The nucleic acid may be DNA or RNA, and in particular DNA. The amplification sequence part 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.

[0085] The barcode sequence unit 4 contains a nucleic acid having a barcode sequence. The nucleic acid may be specifically DNA or RNA, and more specifically DNA. The barcode sequence is used, for example, to identify the position of a target capture molecule on the surface 101. The barcode sequence can be used as a label for distinguishing a target capture molecule having a certain barcode sequence from a target capture molecule having other barcode sequences. The barcode sequence may be associated with information about the position at which the target capture molecule having the barcode sequence is fixed (hereinafter, also referred to as "position information"). The position information may be used to identify the position on the surface 101, and is, for example, information about 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 steps in the imaging step and in steps after the imaging step. The ID number may correspond one-to-one to the barcode sequence, and may be used as data corresponding to the barcode sequence in the imaging step and in steps thereafter.

[0086] A plurality of target capture molecules fixed in a specific area of ​​the surface 101 may have the same barcode sequence. Therefore, the specific area and the barcode sequence are associated with each other. By setting the size of the specific area to be smaller than the size of the cell, the target capture molecule with the barcode sequence can be associated with the location where a cell exists. As described above, the surface used for the analysis method of the present technology may have a plurality of areas, and a plurality of target capture molecules with the same barcode sequence are fixed to each of the plurality of areas. The barcode sequence may be different in each area. The size of each area is preferably smaller than the size of the cell and may be, for example, 50 μm or less, preferably, 10 μm or less, and more preferably, 5 μm or less.

[0087] In one embodiment of the present technology, a target capture molecule having a barcode sequence whose sequence is known can be fixed in a predetermined area. For example, the surface 101 has a plurality of areas, and a plurality of target capture molecules fixed to each of the plurality of areas can have the same barcode sequence. The plurality of areas can be set to be smaller than the size of the cells to be analyzed. With respect to the surface 101 formed as described above, each of the plurality of areas can be associated with a barcode sequence of a plurality of target capture molecules fixed to each area.

[0088] In this specification, the area where the target capture molecules having the same barcode sequence are immobilized (as described above) will also be referred to as a spot. The size of the spot may be, for example, 50 μm or less, preferably 10 μm or less, and more preferably 5 μm or less.

[0089] In the surface 101 formed as described above, the barcode sequences of certain target capture molecules and the positions where certain target capture molecules exist can be associated with each other when the target capture molecules are fixed to the surface 101. For fixation, for example, biotin is bound to the linker 1 of the target capture molecule, streptavidin is bound to the surface 101 to which the target capture molecule is to be fixed, and biotin is bound to streptavidin, thereby fixing the target capture molecule to the surface 101.

[0090] In another embodiment of the present technology, the target capture molecules with barcode sequences can be randomly arranged on the surface 101. In this case, after the target capture molecules with barcode sequences are fixed to the surface 101, the barcode sequences of the fixed target capture molecules are read, whereby the barcode sequences of certain target capture molecules are associated with the locations where certain target capture molecules exist. The reading can be performed by a method, such as sequencing by synthesis, sequencing by connection, or sequencing by hybridization.

[0091] In this embodiment, for example, beads (e.g., gel beads) bound to multiple target capture molecules having the same barcode sequence can be used, and the beads (e.g., gel beads) can be fixed to surface 101. The size of the beads (e.g., gel beads) can be, for example, 50 μm or less, preferably 10 μm or less, and more preferably 5 μm or less. In order to bind the target capture molecules to the beads (e.g., gel beads), for example, a combination of biotin and streptavidin can be used. For example, biotin is bound to the linker 1 of the target capture molecules, streptavidin is bound to the beads, and the biotin is bound to the streptavidin, whereby the target capture molecules are fixed to the beads.

[0092] The surface 101 may have a plurality of recesses. One spot or one bead in the present embodiment may be placed in each of the plurality of recesses. Due to the plurality of recesses, the spot or bead may be placed on the surface 101 more easily. For example, the recess preferably has a size in which one bead may be placed. The recess may have a shape such as a circle, an ellipse, a hexagon, or a quadrilateral, but is not limited thereto.

[0093] In addition, in surface 101, the surface state of the surface portion on which a spot or a bead is placed can be different from the surface state of other surface portions. For example, the surface portion on which a spot or a bead is placed can be hydrophilic, and other surface portions can be hydrophobic, or other surface portions can be hydrophobic and have projections. The example of the method for imparting hydrophilicity to the surface includes reactive ion etching in the presence of oxygen and irradiation with deep ultraviolet light in the presence of ozone. In these methods, a mask can be used, and its part to be imparted with hydrophilicity is penetrated. In addition, the example of the method for imparting hydrophobicity to the surface includes silicone spray (spraying on silicone), for example, Techspray 2101-12S etc. can be used. In addition, in the case of imparting hydrophobicity, for example, a mask to be imparted with hydrophobicity can be used.

[0094] For example, target capture molecules can also be synthesized on a substrate by using DNA microarray preparation technology, etc. For example, target capture molecules can be synthesized at specific positions using technologies such as digital micromirror devices (DMDs), liquid crystal shutters, or spatial light phase modulators for photolithography. The synthesis method is described in, for example, Basic Concepts of Microarrays and Potential Applications in Clinical Microbiology, CLINICAL MICROBIOLOGY REVIEWS, Oct. 2009, pp. 611-633. Note that in the case of synthesizing target capture molecules on a substrate by synthesis, information about the position of the synthetic target capture molecules is obtained when synthesizing the target capture molecules, and the barcode sequence is associated with the position information. At this time, an ID number can be given.

[0095] In one embodiment of the present technology, all target capture molecules fixed to the surface can have a common oligonucleotide sequence. By using a fluorescently labeled nucleic acid with a sequence complementary to the oligonucleotide sequence, the position where the target capture molecule is fixed (particularly, the position of the spot or the position of the bead) can be confirmed, and the position in the dark field can be confirmed in particular. In addition, in the absence of the above-mentioned recesses or protrusions on the surface, it may be difficult to grasp the position where the target capture molecule is fixed. In this case, the fluorescent label makes it easier to grasp the position where the target capture molecule is fixed.

[0096] UMI part 5 may contain nucleic acid, particularly DNA or RNA, more particularly DNA. UMI part 5 may have a sequence of, for example, 5 bases to 30 bases, particularly 6 bases to 20 bases, more particularly 7 bases to 15 bases.

[0097] The UMI part 5 may be configured so that the target capture molecules immobilized on the surface 101 have different sequences. For example, in the case where the UMI part has a nucleic acid sequence of 10 bases, the type of the UMI sequence is four to ten, that is, one million or more.

[0098] The UMI section 5 can be used to quantify the target molecule. For example, a UMI sequence is added to cDNA obtained by reverse transcription of 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 another mRNA molecule having the same sequence as the mRNA have a different UMI sequence. Therefore, the copy number of mRNA can be determined by counting the number of types of UMI sequences having the same cDNA sequence.

[0099] For example, the UMI section 5 can be configured so that multiple target capture molecules with the same barcode sequence fixed to a spot or bead have different sequences. That is, multiple target capture molecules fixed to a spot or bead can have the same barcode sequence and have different UMIs.

[0100] The target capture part 6 has a constituent component for capturing molecules contained in the cell. The constituent component may be, for example, a nucleic acid or a protein. In the case where the constituent component is a nucleic acid, the nucleic acid may be, for example, a poly-T sequence, so as to comprehensively capture the mRNA contained in the cell. Alternatively, the nucleic acid may have a sequence complementary to the target sequence. In the case where the constituent component is a protein, the protein may be, for example, an antibody. The constituent component may be an aptamer or a molecular imprinting polymer.

[0101] The target capture part 6 may include two or more types of constituents for capturing molecules contained in cells. The target capture part 6 may include both proteins and nucleic acids, and may include, for example, both antibodies and poly-T sequences. Therefore, both proteins and mRNAs may be detected simultaneously.

[0102] The surface 101 may preferably be the surface of a transparent substrate. The entire substrate may be transparent, or only a portion to which the target capture molecules are to be fixed may be transparent. The surface of the substrate is preferably planar so as to advantageously bring the surface into contact with the specimen. The transparent substrate may be, for example, a glass substrate or a resin substrate. The substrate may be, for example, a glass slide. Because the substrate is transparent, the specimen can be imaged in the imaging step described later. In addition, because the substrate is transparent, association can be easily performed in the association step described later.

[0103] The specimen 4 can be, for example, a specimen containing cells, and more specifically, a fixed specimen containing cells. The specimen can be a biological tissue sample, in particular a frozen tissue sample or a formalin-fixed paraffin-embedded (FFPE) specimen, more particularly a frozen tissue section or a FFPE section. The specimen 4 can be loaded on, for example, a substrate, and can be loaded on the surface of a transparent substrate in particular. The entire substrate can be transparent, or only the portion on which the specimen is to be loaded can be transparent. The transparent substrate can be, for example, a glass substrate or a resin substrate. The substrate can be, for example, a glass slide. The surface of the substrate is preferably planar so as to advantageously bring the surface into contact with the surface on which the target capture molecules are fixed.

[0104] Specimen 4 can be stained so that it is easier to perform the segmentation of cells described later. Staining can be, for example, cell membrane staining, hematoxylin and eosin (HE) staining, DAPI staining, or a combination of two or more thereof. For cell membrane staining, a cell membrane staining reagent that is incorporated into a lipid bilayer membrane to emit fluorescence can be used, and those skilled in the art can appropriately select the reagent. HE staining can be used for bright field observation, for example, to identify cell morphology. DAPI staining can be used for nuclear staining.

[0105] (2-2) Imaging steps

[0106] In the imaging step S102, for example, Figure 2 As shown in (B), the specimen 103 is imaged in a state where the surface 101 and the specimen 103 overlap. The imaging can be performed at a resolution capable of identifying individual cells included in the specimen 103. The imaging element 110 can be, for example, a CCD or a CMOS.

[0107] For example, imaging can be performed by the imaging element 110 via the objective lens 111. That is, the captured image can be a microscopic image. The magnification of the objective lens 111 can be appropriately selected according to the size of the cell.

[0108] The imaging may be bright field imaging 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, the imaging may be performed once or multiple times for a partial area selected by a user or a control unit, or may be performed once or multiple times to comprehensively cover the entire or partial specimen 103.

[0109] Imaging of the imaging element 110 can be controlled by a control unit (not shown) connected to the imaging element 110. The control unit may include, for example, a hard disk, a CPU, and a memory, and the function of the control unit may be realized by, for example, a general-purpose computer or an information processing device.

[0110] In addition, the control unit may be provided in the imaging element 110. For example, the imaging element including the control unit may be formed as a monolithic semiconductor device having a laminated structure in which a plurality of crystal grains (e.g., two or three crystal grains) are laminated. One of the crystal grains includes a plurality of pixels arranged side by side in two dimensions. Components (e.g., CPU, memory, etc.) for implementing the functions of the control unit may be mounted on the remaining crystal grains. An example of an imaging element including a control unit is an imaging element disclosed, for example, in WO 2018 / 051809. By using an imaging element including a control unit as an imaging element, various processes can be performed without outputting specimen image data to the outside of the imaging element. This results in an increase in the speed of information processing.

[0111] The imaging element 110 may 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.

[0112] Furthermore, the specimen image data received by the control unit may be stored in, for example, a storage unit connected to the control unit. The storage unit may be a general-purpose storage device. In the case of performing subsequent steps, the control unit may acquire the specimen image data from the storage unit.

[0113] (2-3) Association steps

[0114] In the associating step S103, by 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 the position are associated with each other. The association can be performed in advance via the position information associated with the barcode sequence. In the case where an ID number is assigned to each barcode sequence, the position of the cell and the ID number can be associated. Therefore, the position of the cell and the barcode sequence can be associated via the ID number.

[0115] The location of a cell may mean the area occupied by the cell in the specimen image. In order to identify the area, image processing may be performed on the specimen image, for example, cell segmentation may be performed thereon. Cell segmentation makes it easier to identify the target capture molecule present at the location of the cell.

[0116] For example, in the association step S103, the position information of the cell to be analyzed in the cell in the specimen image is obtained. The position information of the cell can be obtained by using the image data obtained by segmentation. Because the specimen image is obtained by imaging the surface 101 and the specimen 103 overlapped thereby, 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, in the imaging target preparation step S101, the position information of the target capture molecule is associated with the barcode sequence of the target capture molecule. Therefore, by acquiring the position information of the cell in the specimen image, the position of the cell can be associated with the barcode sequence of the target capture molecule at that position.

[0117] This association can preferably be performed so that one barcode sequence is not associated with the location of two or more cells. For example, in some cases, two or more cells are present in one spot as described above. In this case, the barcode sequence of the target capture molecule fixed to the one spot is preferably not associated with any location of the two or more cells.

[0118] Will refer to Figure 4 An example is described of how to distinguish between spots on a surface that are to be correlated and spots that are not to be correlated.

[0119] When zoomed in Figure 4 When obtaining a portion of a specimen image of a tissue in the left portion of the image or an enlarged image of a portion thereof, such as in Figure 4 As shown in the central part of , cells in a tissue sample can be visually identified. The specimen is imaged while overlapping with the analysis surface, and is therefore imaged in a state where the specimen overlaps with a large number of circular spots, such as Figure 4 Each circular spot has multiple target capture molecules with the same barcode sequence.

[0120] exist Figure 4 In the right part of FIG. 1 , a large number of circular spots are shown for better understanding. However, these spots may not be confirmed or may be confirmed in the image acquired by the imaging element.

[0121] Furthermore, 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.

[0122] exist Figure 4In the specimen image of the center portion of , it can be confirmed that two types of cells exist. One type of cells is colored in dark gray, and the other type of cells is colored in light gray. For example, for those two types of cells, the position of each cell is associated with the barcode sequence of the target capture molecule present at that position, and the spots indicated by the solid circle are associated, as shown in FIG. Figure 4 As shown in the right part of FIG. Each solid circle exists in the region of the image of one cell. As described above, a spot existing in the region of the image of one cell can be a target to be associated.

[0123] Meanwhile, for example, each dotted circle overlaps two or more cells, extends over a cell and an area outside the cell, or exists outside the area of ​​the image of the cell. These dotted circles may not be the target to be associated.

[0124] For example, the associating step S103 may be performed by the control unit. For example, the control unit may associate the positions of all or some types of cells among the cells present in the specimen image with the barcode sequence of the molecule present at the position of the cell. In the case of associating some types of cells, the control unit may identify the cells to be associated based on, for example, the characteristics of the cells (shape, size, color, pattern, or a combination thereof). For example, only cells of a specific type may be associated. For example, in Figure 4 In the center portion of the image, the position of the cells with dark gray is associated with the barcode sequence of the target capture molecule present at that position, while the cells with light gray are not associated.

[0125] In addition, when an imaging element including a control unit is used as the imaging element, the association step S103 can be performed without outputting the specimen image data to the outside of the imaging element. Therefore, the association step S103 can be performed at a higher speed.

[0126] The associating step S103 may be performed in response to a user operation. For example, the user selects a cell to be associated from among cells present in the specimen image by, for example, clicking a mouse. The control unit may then associate the position of the cell selected by the user with the barcode sequence of the target capture molecule at that position.

[0127] (2-4) Cracking step

[0128] In the cleavage step S104, the location of the cell is selectively stimulated to cleave the linking group of the molecule at the location. Figure 2 As shown in (C), the location of the selected cells can be irradiated with light by the stimulus supply device 130.

[0129] The location where stimulation is provided may be the location of some cells associated in the association step S103 or may be the location of all cells associated therein. Preferably, the location of the cell is selectively stimulated so as not to cleave the linking group of the target capture molecule at a location other than the location of the cell. The stimulation supply device 130 is preferably configured to selectively provide stimulation, as described above. That is, in the cleavage step S104, the linking group of the target capture molecule having a barcode sequence that has not yet been associated may not be cleaved.

[0130] For example, in Figure 4 In the right image, all associated spots represented by solid circles can be stimulated. As described above, all positions of the associated cells can be stimulated.

[0131] Alternatively, in Figure 4 In the right image of , the position represented by the solid circular spot in the dark gray cell or the position represented by the solid circular spot in the light gray cell can be stimulated. As described above, the position of some associated cells (specifically, cells of a specific type) can be stimulated.

[0132] Alternatively, in Figure 4 In the right image of , the position represented by the solid circular spot in one or more cells selected from the related cells can be stimulated. As described above, the position of some associated cells (specifically, the selected cells) can be stimulated.

[0133] By cleaving the linking group in step S104, the target capture molecule is released from the surface 101. For example, Figure 3 As shown in (B), the linking group 1 of the molecule 100 is cleaved and the molecule 100 is released from the surface 101.

[0134] The stimulus may be, for example, a light stimulus or a temperature stimulus (also called thermal stimulus) and may preferably be a light stimulus. Light stimulation is particularly suitable for selectively stimulating a specific narrow range.

[0135] For example, the lysis step S104 may be performed by the control unit. More specifically, the control unit may drive the stimulus supply device to selectively stimulate the location of the cell. An example of an employable stimulus supply device will be described below.

[0136] In order to selectively apply light stimulation to the position of the cell, a light irradiation device can be used as the stimulation supply device 130. For example, the light irradiation device can be a digital micromirror device (DMD) or a liquid crystal display device. The micromirrors included in the DMD can irradiate the selected position on the surface 101 with light. For example, the liquid crystal display device can be a reflective liquid crystal display, and a specific example thereof includes 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.

[0137] In addition, liquid crystal shutters or spatial light modulators can be used to selectively apply light stimulation to the location of the cell. Likewise, through these units, light stimulation can be applied to selected locations.

[0138] Those skilled in the art can appropriately select the wavelength of the irradiation light according to the type of linking group contained in the target capture molecule.

[0139] For example, a combination of infrared light and infrared light absorbing material can be used to selectively apply temperature stimulation to the location of the cell. In this case, the stimulation supply device can be, for example, an infrared laser generating device. For example, the substrate 102 having the surface 101 is made of an infrared light absorbing material, and then the location of the cell is selectively irradiated with infrared light by the infrared laser generating device, thereby selectively applying temperature stimulation to the location.

[0140] (2-5) Combination steps

[0141] In the binding step S105 , the target capturing molecules released from the surface 101 by the cleavage in step S104 bind to the constituent components of the cell via the target capturing portion of the target capturing molecules.

[0142] For example, if Figure 2 As shown in the schematic enlarged view surrounded by a single-point chain line in (C), only the linking group of the target capture molecule at the position irradiated with light is cleaved and taken into the cell immediately below it. Then, for example, Figure 3 As shown in (C), the cell constituent component 7 can be bound to the target capturing portion 6 of the target capturing molecule 100 in the cell.

[0143] The combination 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, because 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 placing a laminate of substrate 102 and substrate 104 between opposing electrodes. In addition, a metal film or a transparent electrode (such as an indium tin oxide (ITO) electrode) having a thickness that does not hinder bright field observation or dark field observation (specifically, fluorescence observation) can be placed on substrate 102 and substrate 104. An electric field applying device for applying an electric field, a magnetic field applying device for applying a magnetic field, or a centrifugal force applying device for applying a centrifugal force can be a device known in the art. Examples of centrifugal force applying devices include swing rotor type centrifugal force applying devices. For example, the application of an electric field, a magnetic field or a centrifugal force by these devices can be controlled by a control unit.

[0144] The binding step S105 may include a moving step of moving the target capturing molecules toward the cells by natural diffusion.

[0145] In the moving step, an electric field, a magnetic field or a combination of centrifugal force and natural diffusion can be used. The moving step preferably includes applying an electric field. This makes it possible to increase the probability that the released target capture molecules are taken up into cells.

[0146] The binding step S105 may include an incubation step for binding the target capture molecule to the constituent components of the cell. The time and temperature of the incubation step can be selected according to the target capture part and the cell constituent components captured by the target capture part. The incubation step can be performed in a state where the space between the surface 101 and the specimen is filled with a buffer solution. For example, in the case where both the target capture part and the cell constituent components are nucleic acids, for example, the incubation can be performed in a constant temperature chamber at 30°C to 40°C, specifically 30°C to 37°C, for example, for 5 hours to 30 hours, specifically 16 hours to 24 hours. For example, in the case where the target capture part is an antibody and the cell constituent component is a constituent captured by the antibody, the incubation can be performed at, for example, 0°C to 30°C, specifically 4°C to room temperature (e.g., 25°C), for example, for 5 hours to 30 hours, specifically 16 hours to 24 hours.

[0147] In the binding step S105, the target capture molecule can be brought into the cell or can be bound to the cell surface. Then, the target capture molecule is bound to the constituent components of the cell via the target capture portion 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. The washing method can be, for example, immersing the specimen 103 in a buffer.

[0148] (2-6) Analysis steps

[0149] In the analysis step S106, the conjugate produced by binding the target capture molecule to the cell constituent component in the binding step S105 can be analyzed. Figure 2 As shown in (D), analysis can be performed using an analysis device 200. For example, in analysis step S106, the conjugate can be subjected to a sequencing process, and the analysis device 200 can be a sequencer. The sequencing process can be performed in the case where, for example, the cell constituents are nucleic acids, particularly DNA or RNA, more particularly mRNA. The sequencing process can be performed by a sequencer, or can be performed by a next generation sequencer or by a sequencer of the Sanger method. In order to comprehensively analyze the cells forming the tissue at a higher speed, a sequencing process can be performed by a next generation sequencer.

[0150] In order to perform a sequencing process in the analysis step, the analysis step may further include a step of preparing a nucleic acid (e.g., cDNA, etc.) to be subjected to a sequencing process and a step of purifying the nucleic acid. Through the preparation step and the purification step, for example, a library for a next generation sequencing process can be prepared.

[0151] In the preparation of the library, the collection sequence part 2 can be used, for example Figure 3 The molecules 100 bound to the cell constituent component 7 can be collected by using beads 9 to which nucleic acids having sequences complementary to the nucleic acid sequences contained in the collection sequence portion 2 are fixed.

[0152] In the preparation step, the laminate of substrates 102 and 104 bonded in the bonding step S105 may be washed with a buffer such as PBS to remove unbound target capture molecules. Then, next, substrate 102 is removed from substrate 104, and, for example, a cDNA synthesis step of synthesizing cDNA from mRNA and an amplification step of amplifying the synthesized cDNA may be performed on cells from which target capture molecules have been obtained. Prior to the cDNA synthesis step and the amplification step, a lysis step of lysing the cells may be performed. When the conjugate of the target capture molecule and the target is collected after the lysis step, the cDNA synthesis step and the amplification step may be performed more efficiently.

[0153] After the preparation step, a purification step for purifying the nucleic acid obtained in the preparation step can be performed. The purification step can include, for example, a process of decomposing the constituent components other than the nucleic acid by using an enzyme (such as proteinase K). In addition, a nucleic acid collection process can be performed in the purification step. In the nucleic acid collection process, for example, commercially available nucleic acid purification reagents can be used, and examples thereof include magnetic beads such as AMPure XP. Note that dsDNA in cells can also be collected in the purification step, but dsDNA can be prevented from being subjected to sequencing during the sequencing process. For example, when the target capture molecule has an aptamer sequence for a sequencing process (specifically, for a next-generation sequencing process), only nucleic acids with the aptamer sequence can be subjected to sequencing.

[0154] In the analysis step S106, the cell constituents can be analyzed for each cell based on the results of the sequencing process. For example, in the analysis step S106, the sequence of the mRNA contained in the cell and / or the copy number of each mRNA can be determined for each cell. In addition, in the analysis step S106, the type and / or quantity of the antigen or the type and / or quantity of the transcription factor can be determined for each cell. Such analysis of the cell constituents of each cell can be performed based on the barcode sequence of the sequence determined by the sequencing process. For example, a sequence with the same barcode sequence is selected from a large number of sequences determined by the sequencing process. The sequence with the same barcode sequence is based on the target capture molecule taken into a cell. Therefore, analyzing the cell constituents for each barcode sequence refers to analyzing the cell constituents for each cell.

[0155] The analyzing step S106 may include a two-dimensional mapping step of performing two-dimensional mapping based on the result of the association in the associating step S103 by using the analysis result and the specimen image. For example, in the analyzing step S106, the analysis result of the cell constituents of each cell may be mapped onto the specimen image obtained in the imaging step based on the position information associated with the barcode sequence. By mapping, for example, Figure 2 As shown in (E), the specimen image ( Figure 2 (E) in the upper part) and a mapping image showing the distribution of the types of cell constituent components at various positions in the specimen image ( Figure 2 (E) in the lower part). Based on the image obtained by mapping, it is possible to grasp which cell at which position contains which molecule in what amount. That is, the position information obtained by imaging can be combined with the quantitative information obtained by sequence analysis, and thus the molecules in the tissue sample can be comprehensively analyzed while maintaining spatial information at single-cell resolution.

[0156] 2. Second Embodiment (Analysis System)

[0157] Figure 5is a block diagram of an example of an analysis system according to the present technology. Figure 5 As shown, the analysis system 10 of the present technology includes an analysis substrate 102 , an imaging element 110 , a control unit 120 , a storage unit 125 , and a stimulus supply device 130 .

[0158] The analysis substrate 102 , the imaging element 110 , and the stimulus supply device 130 may be those described in Section 1 above, and the description thereof also applies to the present embodiment.

[0159] The control unit 120 may be the control unit described in the above section 1, and its description also applies to the present 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 units will be described in more detail below.

[0160] The image processing unit 121 processes the specimen image acquired by the imaging element 110. For example, the image processing unit 121 can perform the cell segmentation described in the above section 1. In addition, the image processing unit 121 can identify the cells to be associated in the association step from among the cells present in the specimen image. For example, the identification can be performed based on the characteristics of the cells (shape, size, color, pattern, or a combination thereof). The identification can also be performed based on the color data in the image. For example, cells emitting specific fluorescence can be identified as cells to be associated in the association step.

[0161] 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 can be performed by using the image obtained by segmentation.

[0162] For example, the associating unit 122 may acquire position data of a cell based on a specimen image, identify a barcode sequence to which position data corresponding to the position data is assigned, and associate the barcode sequence with the position of the cell.

[0163] The association unit 122 may perform association of the position of the cell with the barcode sequence of the target capture molecule for all cells in the specimen image or some cells in the specimen image. For example, the association unit 122 may associate only some types of cells or only cells present in some regions of all cells present in the specimen image.

[0164] The stimulation control unit 123 causes the stimulation supply device 130 to selectively stimulate the position of the cell associated by the association unit 122. The stimulation control unit 123 can drive the stimulation supply device 130 to stimulate all or some of the cells associated by the association unit 122. The stimulation supply device 130 is preferably configured to selectively stimulate the position of the cell without cleaving the linking group of the target capture molecule at a position other than the position of the cell. The stimulation supply device 130 is preferably the light irradiation device described in the above section 1.

[0165] The control unit 120 may include, for example, a hard disk, a CPU, and a memory, and the functions of the control unit may be implemented by, for example, a general-purpose computer or an information processing device. The functions of the image processing unit 121, the association unit 122, and the stimulation control unit 123 may also be implemented by a general-purpose computer or an information processing device.

[0166] As described in the above section (2-2), the control unit 120 may be provided in the imaging element 110. By using an imaging element including the control unit as an imaging element, various types of processing can be performed without outputting the specimen image data to the outside of the imaging element. This results in an increase in the speed of information processing. For example, the processing of the above-mentioned image processing unit 121, the association unit 122, and the stimulation control unit 123 can be performed at a higher speed.

[0167] In order to perform the binding step S105 described in the above section 1, the analysis system 10 may include, for example, an electric field or magnetic field applying device that applies an electric field or a magnetic field, or a centrifugal force applying device that applies a centrifugal force, for moving the target capture molecules released from the surface of the analysis substrate 102 due to the stimulation by the stimulation supply device 130 toward the cells. By this device, the target capture molecules are more effectively brought into the cells.

[0168] To perform the binding step S105 described in Section 1 above, the analysis system 10 may further include an incubation device for promoting the binding of the cell constituent components with the target capture molecules released from the surface of the analysis substrate 102 due to the stimulation of the stimulation supply device 130. The incubation device may include, for example, a constant temperature chamber.

[0169] In order to perform the analysis step S106 described in the above section 1, the analysis system 10 may further include an analysis device for analyzing the conjugate of the cell constituent components and the target capture molecules released from the surface of the analysis substrate 102 due to the stimulation by the stimulation supply device 130. The analysis device 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.

[0170] The analysis system 10 may also include a two-dimensional mapping unit, which performs two-dimensional mapping based on the association result of the association unit by using the analysis result of the analysis device and the specimen image. The two-dimensional mapping unit can be set as a component of the control unit 120 and can perform, for example, the two-dimensional mapping steps described in the above part 1. The two-dimensional mapping unit can map information such as about the constituent components of the cell onto the specimen image. For example, the information about the constituent components of the cell can be the type or quantity of the constituent components of the cell. Based on the image obtained by mapping, it is possible to grasp which cell at which position contains which amount of which molecule. That is, the position information obtained by imaging can be combined with the quantitative information obtained by sequence analysis, and therefore the molecules in the tissue sample can be comprehensively analyzed while maintaining spatial information at single cell resolution.

[0171] 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 may cause the output unit to output an image obtained by executing the analysis method according to the present technology, such as a specimen image acquired by the imaging element 110 and a mapping image generated by the two-dimensional mapping unit. In addition, the control unit 120 may cause the output unit to output the analysis result of the analysis device (e.g., the result of the sequencing process, etc.).

[0172] 3. Third Embodiment (Analysis Surface)

[0173] The present technology also provides an analytical surface, wherein a target capture molecule having a cleavable linking group, a barcode sequence, and a target capture portion is fixed to the analytical surface via the linking group, and the barcode sequence is used to provide information about the position where the molecule having the barcode sequence is fixed. An example of an analytical surface is the surface 101 of the analytical substrate 102 described in Section 1 above, and the description of the surface also applies to the present embodiment.

[0174] It should be noted that the present technology may also have the following configurations.

[0175] [1] An analysis method comprising:

[0176] an imaging step of imaging the specimen in a state where the specimen overlaps with a surface, wherein a molecule having a linking group that can be cleaved by stimulation, a barcode sequence, and a target capture portion is fixed to the surface via the linking group;

[0177] an associating step of associating the position of a cell with the barcode sequence of the molecule at the position by using the specimen image obtained by the imaging;

[0178] a cleavage step of selectively stimulating a location of the cell to cleave a linker group of the molecule at the location; and

[0179] The binding step binds the molecule released from the surface by the lysis to a constituent component of the cell via the target capture portion of the molecule.

[0180] [2] The analysis method according to [1], wherein the specimen includes a tissue sample.

[0181] [3] The analysis method according to [1] or [2], wherein in the cleavage step, the location of the cell is selectively stimulated so as not to cleave the linking group of the molecule at a location other than the location of the cell.

[0182] [4] The analysis method according to any one of [1] to [3], wherein the stimulation is light stimulation.

[0183] [5] The analysis method according to any one of [1] to [4], wherein the binding step includes a movement step of moving the molecule toward the cell by applying an electric field, a magnetic field, or a centrifugal force.

[0184] [6] The analysis method according to any one of [1] to [4], wherein the binding step includes a movement step of moving the molecule toward the cell by natural diffusion.

[0185] [7] The analysis method according to any one of [1] to [6], wherein the binding step includes an incubation step for allowing the molecule to bind to a constituent component of the cell.

[0186] [8] The analysis method according to any one of [1] to [7], further comprising an analysis step of analyzing a conjugate of the molecule and the constituent component of the cell after the binding step.

[0187] [9] The analysis method according to [8], wherein the conjugate is subjected to sequencing in the analysis step.

[0188]

[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 result of the association in the association step by using the result of the analysis and the specimen image.

[0189]

[11] An analysis system comprising:

[0190] an analytical substrate having a surface, a molecule having a linking group cleavable by stimulation, a barcode sequence, and a target capture portion immobilized to the surface via the linking group;

[0191] an imaging device for imaging and analyzing the specimen overlapped with the substrate;

[0192] an associating unit that associates the position of the cell selected in the specimen image obtained by the imaging with the barcode sequence of the molecule at the position; and

[0193] A stimulation supply device, wherein the stimulation supply device selectively stimulates the position of the cells; wherein,

[0194] A conjugate in which the molecule released from the surface due to the stimulus is bound to a constituent component of the cell via the target capturing portion of the molecule is used as an analysis target.

[0195]

[12] The analysis system according to

[11] , wherein the specimen includes a tissue sample.

[0196]

[13] An analysis system according to

[11] or

[12] , wherein the stimulus supply device is configured to selectively stimulate the location of the cell without cleaving the linking group of the molecule at a location other than the location of the cell.

[0197]

[14] An analysis system according to any one of

[11] to

[13] , wherein the stimulus supply device is a light irradiation device.

[0198]

[15] An analysis system according to any one of

[11] to

[14] , wherein the analysis system includes an electric field application device, a magnetic field application device or a centrifugal force application device, wherein the electric field application device, the magnetic field application device or the centrifugal force application device applies an electric field, a magnetic field or a centrifugal force to move molecules released from the surface due to stimulation by the stimulation supply device toward the cells.

[0199]

[16] The analysis system according to any one of

[11] to

[15] further includes an incubation device, which promotes the binding of molecules released from the surface due to stimulation by the stimulation supply device to the constituent components of the cells.

[0200]

[17] The analysis system according to any one of

[11] to

[16] further includes an analysis device that analyzes the conjugate of the molecule and the constituent components of the cell released from the surface due to stimulation by the stimulation supply device.

[0201]

[18] The analysis system according to

[17] , wherein the analysis device is a sequencer.

[0202]

[19] The analysis system according to

[17] or

[18] further includes a two-dimensional mapping unit that performs two-dimensional mapping based on the correlation result of the correlation unit by using the analysis result of the analysis device and the specimen image obtained by imaging.

[0203]

[20] An analysis surface, wherein

[0204] A molecule having a cleavable linker, a barcode sequence, and a target capture portion is immobilized to the analytical surface via the linker; and

[0205] The barcode sequence is used to provide information about the position where the molecule having the barcode sequence is immobilized.

[0206] Reference numerals list

[0207] 100 molecules

[0208] 101 Analyzing Surfaces

[0209] 102 substrate

[0210] 103 specimens

[0211] 104 substrate

[0212] 110 Imaging Components

[0213] 120 Control unit

[0214] 130 Stimulus supply device.

Claims

1. An analysis method comprising: an imaging step of imaging the specimen in a state where the specimen overlaps with a surface, wherein a molecule having a barcode sequence, a target capture portion, and a linker group that can be cleaved by stimulation is fixed to the surface via the linker group; an associating step, by using the specimen image obtained by imaging, associating the position of the cell with the barcode sequence of the molecule at the position; a cleavage step of selectively stimulating the location of the cell to cleave the attachment group of the molecule at the location; and a binding step of causing the molecule released from the surface by cleavage to bind to a constituent component of the cell via the target capture portion of the molecule, wherein, in the cleavage step, the position of the cell is selectively stimulated so that the linking group of the molecule at a position other than the position of the cell is not cleaved, Wherein, the stimulation is light stimulation.

2. The analysis method according to claim 1, wherein The specimen includes a tissue sample.

3. The analysis method according to claim 1, 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.

4. The analysis method according to claim 1, wherein The binding step includes a moving step of moving the molecule toward the cell by natural diffusion.

5. The analysis method according to claim 1, wherein The binding step comprises an incubation step to allow the molecule to bind to the constituents of the cell.

6. The analysis method according to claim 1, further comprising after the binding step: An analyzing step of analyzing the conjugate of said molecule with said constituent component of said cell.

7. The analysis method according to claim 6, wherein: In the analysis step, the conjugate is subjected to sequencing processing.

8. The analysis method according to claim 6, wherein: The analyzing step includes a two-dimensional mapping step of performing two-dimensional mapping based on a result of the correlation in the correlating step by using the result of the analysis and the specimen image.

9. An analysis system comprising: An analytical substrate having a surface, wherein a molecule having a barcode sequence, a target capture portion, and a linking group that can be cleaved by stimulation is immobilized to the surface of the analytical substrate via the linking group; an imaging device for imaging a specimen overlapping the analysis substrate; an associating unit that associates the position of the cell selected in the specimen image obtained by the imaging with the barcode sequence of the molecule at the position; and A stimulation supply device selectively stimulates the location of the cell; wherein, using as an analysis target a conjugate obtained by binding of the molecule released from the surface due to the stimulus to a constituent component of the cell via the target capturing portion of the molecule, wherein the stimulation supply device is configured to selectively stimulate the location of the cell without cleaving the linking group of the molecule at locations other than the location of the cell, Wherein, the stimulus supply device is a light irradiation device.

10. The analysis system according to claim 9, wherein: The specimen includes a tissue sample.

11. The analysis system according to claim 9, wherein: The analysis system includes an electric field applying device, a magnetic field applying device or a centrifugal force applying device for applying an electric field, a magnetic field or a centrifugal force, wherein the electric field, the magnetic field or the centrifugal force is used to move the molecules released from the surface due to the stimulation of the stimulation supply device toward the cells.

12. The analysis system according to claim 9, further comprising an incubation device that causes the molecules released from the surface due to the stimulation of the stimulation supply device to bind to the constituent components of the cell.

13. The analysis system according to claim 9, further comprising an analysis device that analyzes a conjugate of the molecule released from the surface due to the stimulation of the stimulation supply device and the constituent component of the cell.

14. The analysis system according to claim 13, wherein: The analytical device is a sequencer. 15 . The analysis system according to claim 13 , further comprising a two-dimensional mapping unit that performs two-dimensional mapping based on a result of the correlation by the correlation unit by using an analysis result by the analysis device and the specimen image obtained by the imaging.

Images