WO2015097858A1 - 生体分子解析装置 - Google Patents
生体分子解析装置 Download PDFInfo
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- C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
- C12Q1/6837—Enzymatic or biochemical coupling of nucleic acids to a solid phase using probe arrays or probe chips
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- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/44—Sample treatment involving radiation, e.g. heat
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- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
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- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/645—Specially adapted constructive features of fluorimeters
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/52—Use of compounds or compositions for colorimetric, spectrophotometric or fluorometric investigation, e.g. use of reagent paper and including single- and multilayer analytical elements
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- G01N15/10—Investigating individual particles
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- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
- G01N2021/6439—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks
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- G02B21/16—Microscopes adapted for ultraviolet illumination ; Fluorescence microscopes
Definitions
- the present invention relates to a biomolecule analyzer.
- the cells can be isolated by cleaving the bonds between the cells by chemical treatment such as trypsin treatment.
- Non-Patent Documents 1 and 2 it is possible to cut out a specific cell group on a microscope image using a technique called laser microdissection or laser capture microdissection.
- Non-Patent Documents 1 and 2 in order to cut out a tissue section at least as thick as the cell size using a laser, A cutting margin of several ⁇ m or more is necessary. Since the margin of several ⁇ m is about the same as the cell size, it is difficult to isolate the cells without damaging the adjacent cells when the cells are close. In addition, by destroying the surrounding cells, biomolecules contained in the surrounding cells are mixed into the sample solution, thereby reducing the measurement accuracy. Furthermore, isolation of cells from a section composed of a plurality of layers is impossible because the conventional technique is a technique for cutting in a two-dimensional plane.
- an object of the present invention is to provide a biomolecule analyzer that can collect and analyze biomolecules in a single cell without damaging surrounding cells.
- the biomolecule analyzer of the present invention includes a means for obtaining an optical image of a plurality of cells, a means for destroying part or all of one or more cells of the plurality of cells, An array device in which regions for capturing biomolecules in cells released by the means for destroying are arranged, and a portion of the optical image corresponding to the cells destroyed by the means for destroying, the array device And a means for associating the region where the biomolecules are captured.
- biomolecules such as DNA, RNA, and protein in adherent cultured cells and cells in a tissue section are collected in an array device for each single cell, and the region on the collected array device and a microscope are collected.
- FIG. 1 is a configuration diagram of a biomolecule analysis apparatus according to a first embodiment of the present invention. It is a top view of the pore array sheet in a 1st embodiment of the present invention. It is a figure which shows the electrode structure in the case of carrying out dielectrophoresis of a biomolecule. It is a flowchart explaining operation
- the biomolecule analysis apparatus of the present invention includes a means for acquiring an optical image of a plurality of cells arranged on a plane, and a part or all of one or more cells of the plurality of cells.
- the cell to be measured is attached to the surrounding cells in order to destroy part of the intracellular tissue such as the cell membrane without isolating the cell by cutting it out.
- part of the intracellular tissue such as the cell membrane
- biomolecules such as genes and proteins obtained by destroying cells
- an optical image obtained without destroying the cells with a fluorescence microscope or a Raman microscope.
- This association is difficult to quantitatively evaluate many types of biomolecules, but the data obtained by imaging means that can measure cells alive and detailed quantitative data on biomolecules
- correlating data obtained by means that are possible but cannot assess the dynamic properties of cells to destroy them detailed and dynamic characterization of cells is made possible.
- mRNA extracted from a microscopically observed cell is captured on the device directly under the cell and reverse-transcribed, so that the gene expression analysis of the cell corresponding to the microscopic image of the cell is performed. It can be carried out without isolation. Therefore, the problem that the correspondence between the microscopic image of the cell and the target cell for gene expression analysis is lost at the time of cell isolation is avoided.
- the material to which the cultured cells adhere is limited to the material constituting the porous membrane.
- the array device is brought close to a target cell, the cell is destroyed with a laser or the like, and a biomolecule in the cell is collected for each single cell.
- Single cell analysis can be performed using a conventional petri dish used for cultured cells.
- the biomolecule analysis apparatus includes means for obtaining an optical image of a sample composed of adherent cultured cells using a laser fluorescence microscope, and means for destroying the cells using a laser light source.
- FIG. 1 shows a configuration diagram of a biomolecule analysis apparatus according to the present embodiment.
- This biomolecule analysis apparatus acquires an optical image, and has a function of destroying part or all of a cell at a predetermined position on the optical image, and a biomolecule (mRNA) released / diffused from the cell.
- mRNA biomolecule released / diffused from the cell.
- the microscope system 1 includes a laser light source 4 for a fluorescence microscope.
- a continuous wave 488 nm semiconductor laser with 50 mW output is used as the laser light source 4 for the fluorescence microscope.
- a laser having a wavelength of 405 nm or 633 nm may be used according to the variation of fluorescence to be observed, or laser light having a plurality of wavelengths is output from the laser light source 4 for the fluorescence microscope using a dichroic mirror or an optical filter. It is also possible to do so.
- the microscope system 1 includes a laser light source 5 for cell destruction.
- a pulse laser of 355 nm band (maximum average output 2 W, repetition frequency 5 kHz) is used as the cell light source 5 for cell destruction.
- the laser light source 4 for fluorescence microscope and the laser light source 5 for cell destruction are combined using a dichroic mirror 6 (edge wavelength 409 nm).
- Annexin V FITC and Hoechst Dye are used as fluorescent dyes for staining cells.
- the former emits light at 535 nm with 488 nm excitation, and the latter emits light at 465 nm with 355 nm excitation.
- Cell membranes in the process of apoptosis are stained with the former fluorescent dye, and nuclei are stained with the latter fluorescent dye.
- the dichroic mirror 7 is, for example, one having an edge wavelength of 505 nm for FITC fluorescence imaging, and one having an edge wavelength of 385 nm for Hoechst fluorescence imaging.
- the microscope system 1 includes an objective lens 8.
- the objective lens 8 for example, a lens having NA of 0.8 and a magnification of 40 times is used.
- the optical imaging of cells can be performed as follows. First, cultured cultured cultured cells 21, 22, and 23 are placed on a petri dish 20 having a bottom surface with a cover glass thickness (0.18 mm) that is transparent and does not emit fluorescence, and a through hole for an optical path is provided at the center. The petri dish 20 is placed on the opened sample stage 10.
- the sample stage 10 is driven at a 50 nm pitch by a stage 9 that can move in the XYZ directions.
- the fluorescence light receiving system includes a bandpass filter 11 that removes light having a wavelength other than fluorescence, an imaging lens 12, a PMT (Photomultiplier tube) that is a light receiver 13, and a pinhole 14.
- Optical images of a plurality of cells are acquired by driving them in synchronization. That is, the fluorescence signal from one point where the excitation light is collected is acquired by the light receiver 13 while driving the XYZ stage 9, and an optical image is configured in the control system.
- the biomolecule collection system 2 includes an array device in which regions for capturing biomolecules such as mRNA released from cells are arranged.
- a cDNA library can be constructed by capturing mRNA in a plurality of regions of the array device for each single cell and performing a reverse transcription reaction in the array device.
- the array device is constructed from a porous membrane that is transparent and has a large number of through-holes formed perpendicular to the surface, which is hereinafter referred to as a pore array sheet 30.
- a structure in which a cDNA library is formed on the pore array sheet 30 is referred to as a cDNA library pore array sheet.
- a separation wall 31 can be formed on the pore array sheet 30 to separate regions that capture biomolecules.
- the separation wall 31 can be formed by a semiconductor process using polydimethylsiloxane (PDMS), for example, and can be brought into close contact with the pore array sheet 30 with a thickness of about 80 ⁇ m.
- PDMS polydimethylsiloxane
- FIG. 1 A top view of the pore array sheet 30 is shown in FIG.
- a region 301 for capturing a large number of biomolecules such as mRNA is formed in the pore array sheet 30 (size 2 mm ⁇ 2 mm, thickness 80 ⁇ m).
- the size of the region 301 is 100 ⁇ m on one side and 80 ⁇ m in interval (arranged at a cycle of 180 ⁇ m).
- the size of the region 301 can be freely designed from about 1 ⁇ m to about 10 mm according to the amount of biomolecules to be captured and the ease of diffusion in the surface (molecule size).
- an array device in addition to the pore array sheet 30 made of a porous membrane formed by anodizing aluminum, a device in which a large number of through holes are formed by anodizing a material such as silicon may be used. . Furthermore, an array device may be constructed by providing a large number of through holes in a silicon oxide or silicon nitride thin film using a semiconductor process. Furthermore, as will be described later, a pore array sheet may be formed by packing beads of various sizes in a box-shaped part, or a monolithic column used as a column for liquid layer chromatography is thinly formed. It can also be used as an array device.
- the separation wall 31 can be formed by combining a known semiconductor process using a semiconductor material such as silicon or another resin material in addition to the PDMS resin.
- the present invention relates to a biomolecule analysis apparatus incorporating the array device as described above.
- the array is formed by arranging a plurality of regions that can be associated with optical images of a plurality of cells.
- the device itself is provided as a kit for biomolecule analysis. By using this array device kit in combination with a means for destroying cells such as a laser by a user, it is possible to efficiently analyze a biomolecule of a single cell.
- a loop-shaped platinum electrode 32 is joined to the tip of a shield wire 33 as a means for guiding biomolecules released from cells to a specific region in the pore array sheet 30 by electrophoresis. .
- the diameter of the wire of the platinum electrode 32 is 30 ⁇ m.
- the loop side is processed into a circle with a diameter of 100 ⁇ m.
- Two such electrodes are prepared, arranged so as to sandwich the pore array sheet 30, and a direct current of 1.5 V is applied by the power source 35. Since the released mRNA 36 has a negative charge, the upper platinum electrode 32 is used as a positive electrode.
- a silver-silver chloride reference electrode 39 is provided, and 0.2 V is applied to the lower platinum electrode 32.
- mRNA 36 can be induced by electrophoresis inside the region 301 for capturing biomolecules.
- the diameter of the loop of the upper platinum electrode 32 may be set to 50 ⁇ m in order to realize concentration of mRNA by lateral electrophoresis.
- the wire has a diameter of 10 ⁇ m.
- an electrode structure as shown in FIG. 3 may be used for dielectrophoresis of microgranules such as exosomes, DNA fragments of 40 kb or more, and proteins of 10 kDa or more. That is, a through-hole 44 is formed by wet etching in the center of a square quartz substrate 42 having a thickness of 200 ⁇ m, and four gold electrodes 43 are formed on the inner wall and outer wall of the quartz substrate 42 to produce a quadrupole terminal.
- the gold electrode 43 has a thickness of 1 ⁇ m and a width of 40 ⁇ m (part a).
- the peptide or protein extracted from a single cell is once captured by the array device, so that the measurement sensitivity for subsequent mass spectrometry can be increased. That is, in mass spectrometry of tissue sections using MALDI, it is necessary to mix a sample and a chemical substance called a matrix at an appropriate ratio and irradiate a laser. If the ratio is not appropriate, the ionization efficiency of the target molecule is greatly reduced. However, in MALDI for tissue sections, a matrix material is only added from the surface, so that a uniform material with an appropriate ratio cannot be obtained. Therefore, in general, the ionization efficiency varies depending on the position and decreases.
- mass spectrometry of cell peptides and proteins in highly efficient tissue sections is performed by adsorbing and capturing target biomolecules for each single cell and then performing MALDI on the array device to which the target molecules have been adsorbed. Is possible. Moreover, since only the target biomolecule is selectively captured, the ion suppression effect due to impurities is reduced, and the ionization efficiency is further improved.
- FIG. 4 shows a flowchart.
- a sample composed of the adhesive cultured cells 21, 22, 23 is placed on the petri dish 20 (step 001).
- the measurement target is a cultured cell
- the cell is cultured in advance using the petri dish 20 so that the measurement target cell adheres to the bottom surface. If the sample is a frozen section, it is placed on the petri dish 20.
- a sample in which a plurality of cells are three-dimensionally arranged in a gel may be used as a sample.
- an optical image of a target cell group is acquired using the microscope system 1 (step 002), and a user determines a target cell from which a biomolecule is collected and measured (step 003).
- the control system 3 the user designates a cell or a cell part to be measured. In general, a user often uses a plurality of cells as measurement targets. In that case, the control system 3 determines the order of the cells that capture the biomolecules.
- the pore array sheet 30 In the vicinity of the first target cell (in the example of FIG. 1, directly above the cell), the pore array sheet 30.
- the specific area for example, the area 301 at the address (1, 1) is approached using the XYZ stage 34 (step 004).
- the distance between the lower surface of the pore array sheet 30 and the petri dish 20 is set to 300 ⁇ m, but this distance can be changed depending on the type of biomolecule to be collected and the electrode structure. For example, about 1 ⁇ m to 10 mm is preferable.
- the movement of the pore array sheet 30 by the XYZ stage 34 is automatically performed by the control system 3 in accordance with a prior program.
- a voltage is applied to the platinum electrode 32 for electrophoresis, and at the same time, in order to destroy the cell membrane of the cell to be measured, the laser light from the cell light source 5 for cell destruction is used.
- the cells are irradiated (step 005).
- the irradiation time can be, for example, 10 seconds
- the electrophoresis driving time can be 60 seconds.
- the pore array sheet 30 is specified in the vicinity of the next target cell registered in the control system 3 (directly above the cell in the configuration of FIG. 1). (For example, the area 301 at address (1,2)) is made to approach (step 004).
- the next cell registered in the control system 3 is irradiated with laser light from the cell light source 5 for cell destruction (step 005).
- a voltage is simultaneously applied to the platinum electrode 32 as described above.
- the cells sequentially move to designated cells, destroy the cells, capture biomolecules in the cells in a specific region 301 of the pore array sheet 30, and then perform a process for measuring the captured biomolecules. Execute (step 006).
- the portion corresponding to the destroyed cell in the optical image and the region 301 where the biomolecule is captured in the pore array sheet 30 are associated with each other and presented to the user (step 007).
- the cell to be destroyed is one cell.
- one cell 301 on the array device is released and electrophoresed when a plurality of cells are destroyed.
- mRNA may be captured.
- a plurality of cells may be destroyed at the same time, or the cells may be destroyed one by one without moving the array device.
- the pore array sheet 30 is a 2 mm ⁇ 2 mm chip, and is removed from the XYZ stage 34, and gene expression analysis is performed by performing the following processing in a reaction vessel (tube).
- mRNA is captured by a DNA probe immobilized inside the pore array sheet 30 (FIG. 5A), and cDNA transcription is performed by reverse transcription (first strand synthesis) inside the pore array sheet 30.
- Production of rally FIGG. 5 (b)
- subsequent second strand synthesis FIG. 6 (c), FIG. 6 (d)
- PCR amplification FIGS.
- the DNA probe 56 fixed inside the pore array sheet 30 is provided for each position of the pore array sheet 30.
- a cell recognition sequence is included as a different sequence, and mRNA 36 is captured by this DNA probe 56.
- different cell recognition sequences are fixed for each region 301 in the pore array sheet 30 in FIG.
- This DNA probe 56 for capturing mRNA has a PCR amplification common sequence (forward direction), a cell identification tag sequence, a molecule identification tag sequence, and an oligo (dT) sequence from the 5 ′ end direction.
- a cell identification tag sequence for example, 5 bases
- a molecular identification tag sequence for example, 15 bases
- 4 15 1.1 ⁇ 10 9 kinds of molecules can be recognized, so a huge amount of decoding data obtained by the sequencer Can be recognized from which molecule.
- the oligo (dT) sequence located 3 ′ most is hybridized with the poly A tail added to the 3 ′ side of the mRNA and used to capture the mRNA (FIG. 5 (a)).
- a poly-T sequence is used as a part of the DNA probe 56 to capture mRNA, but a sequence complementary to the sequence to be analyzed instead of the poly-T sequence for microRNA or genome analysis is used. Needless to say, some of them may be used.
- the through holes 55 formed in the pore array sheet 30 penetrate through the pore array sheet 30 in the thickness direction, and the through holes 55 are completely independent.
- the surface of the inner wall of the through-hole 55 is hydrophilic, and protein adsorption to the surface is extremely small, and the enzymatic reaction proceeds efficiently.
- a treatment such as silane coupling is performed on the surface of the pore array sheet 30 to fix the DNA probe 56 to the surface of the through hole 55.
- the fixed DNA probes 56 are fixed to the surface at a rate of, for example, an average of 30 to 100 nm 2 , so that 4 to 10 ⁇ 10 6 DNA probes 56 are fixed to one through hole 55.
- the surface is coated with a surface coating agent to prevent surface adsorption.
- This surface coating may be performed simultaneously with the fixation of the DNA probe.
- the density of the DNA probe 56 is set such that mRNA passing through this space can be captured with an efficiency of almost 100%.
- the negatively charged mRNA 36 released from the cells destroyed by the laser irradiation is electrophoresed by the platinum electrode 32 and guided into the through holes 55 of the pore array sheet 30.
- the poly A tail of mRNA 36 is captured by the oligo (dT) portion of the DNA probe 56.
- the first strand cDNA strand 59 is synthesized using the mRNA 36 captured by the DNA probe 56 as a template.
- the through-hole 55 is filled with a solution containing a reverse transcriptase and a synthetic substrate, and the temperature is slowly raised to 50 ° C., and a complementary strand synthesis reaction is performed for about 50 minutes (FIG. 5B).
- RNase is passed through the through-hole 55 to decompose and remove mRNA 36.
- a liquid containing an alkali modifier and a cleaning liquid are passed through the through-holes 55 to remove the residue and decomposition products.
- RNA molecules Is synthesized.
- 20 types of sequences ATP5B, GAPDH, GUSB, HMBS, HPRT1, RPL4, corresponding to 20 ⁇ 5 bases upstream of 109 ⁇ 8 bases from the poly A tail of the target gene are used as gene-specific sequences.
- RPLP1, RPS18, RPL13A, RPS20, ALDOA, B2M, EEF1G, SDHA, TBP, VIM, RPLP0, RPLP2, RPLP27 and OAZ1, SEQ ID NOS: 3 to 22) are used. This is to unify the size of the PCR amplification product to about 200 bases in the subsequent PCR amplification step.
- PCR amplification is performed using the amplification common sequence (forward / reverse) to prepare PCR amplification products derived from a plurality of genes (FIG. 6 (e)).
- amplification bias problem in which the amplification rate differs depending on the gene, is known, but even if an amplification bias occurs between genes or molecules in this process, after obtaining data by a sequencer, Since the amplification bias can be corrected using the tag sequence, highly accurate quantitative data can be obtained.
- other amplification methods such as rolling circle amplification (RCA), NASBA, and LAMP method can be used.
- the surface of the through-hole 55 inside the pore array sheet is a surface on which DNA probes 56 are immobilized at a high density, and at the same time, nucleic acids such as mRNA and PCR amplification primers, and proteins such as reverse transcriptase and polymerase are not adsorbed.
- a silane coupling agent for immobilizing the DNA probe 56 and a silanized MPC polymer for preventing adsorption are simultaneously immobilized on the surface of the through-hole by a covalent bond at an appropriate ratio.
- an alumina porous membrane is immersed in an ethanol solution for 3 minutes, and then UVO3 treatment is performed for 5 minutes, followed by washing with ultrapure water three times.
- MPC 0.8 -MPTMSi 0.2 (MPC: 2-methacryloyloxyethyl phosphorylcholine / MPTMSi: 3-methacryloxypropyltrimethoxysilane) which is a silanized MPC polymer having an average molecular weight of 9700 (degree of polymerization 40) (Biomaterials 2009, 30, 4930-4938; and Lab Chip 2007, 7, 199-206) 3 mg / ml and 0.3 mg / ml silane coupling agent GTMSi (GTMSi: 3-glycidoxypropyltrimethoxysilane, Shin-Etsu Chemistry), and immersed in an 80% ethanol solution containing 0.02% acetic acid as an acid catalyst for 2 hours.
- GTMSi 3-glycidoxypropyltrimethoxysilane, Shin-Etsu Chemistry
- DNA probe After washing with ethanol, it is dried in a nitrogen atmosphere and heat-treated at 120 ° C. for 30 minutes in an oven. Next, in order to immobilize the DNA probe, 1 ⁇ M of 5 ′ amino group-modified DNA probe (SEQ ID NO: 1), 0.05 M borate buffer (pH 8) containing 7.5% glycerol and 0.15 M NaCl. .5) are discharged as DNA probes having different cell identification tag sequences (1024 types) for each region of 25 ⁇ m ⁇ 25 ⁇ m by 100 pl on the pore array sheet by the same technique as the ink jet printer. Then, it is made to react at 25 degreeC in a humidification chamber for 2 hours.
- SEQ ID NO: 1 1 ⁇ M of 5 ′ amino group-modified DNA probe (SEQ ID NO: 1), 0.05 M borate buffer (pH 8) containing 7.5% glycerol and 0.15 M NaCl. .5) are discharged as DNA probes having different cell identification tag sequences (1024 types) for
- a pore array sheet (for 100 regions) capturing mRNA was introduced into a 0.2 ml tube, and 58.5 ⁇ l of 10 mM Tris buffer (pH 8.0) containing 0.1% Tween 20 and 4 ⁇ l of 10 mM dNTP 5 ⁇ RT buffer (SuperScript III, Invitrogen) 22.5 ⁇ l, 0.1 M DTT 4 ⁇ l, RNaseOUT (Invitrogen) 4 ⁇ l, and Superscript III (reverse transcriptase, Invitrogen) 4 ⁇ l are mixed, and the above pore array sheet Dispense into the contained tube.
- 10 mM Tris buffer pH 8.0
- 10 mM dNTP 5 ⁇ RT buffer SuperScript III, Invitrogen
- RNaseOUT Invitrogen
- Superscript III reverse transcriptase, Invitrogen
- the temperature of the solution and the pore array sheet is raised to 50 ° C. and maintained for 50 minutes to complete the reverse transcription reaction, and the first strand cDNA strand 59 having a complementary sequence to mRNA is synthesized (FIG. 5B). ).
- the reverse transcriptase is inactivated by holding at 85 ° C. for 1.5 minutes, cooled to 4 ° C., and after discharging the solution, RNase and 0.1% Tween 20 are contained.
- RNase and 0.1% Tween 20 are contained.
- 0.2 ml of 10 mM Tris buffer (pH 8.0) into the tube containing the pore array sheet, the mRNA was decomposed, and the same amount of alkali denaturant was flowed in the same manner to remove the residue in the through-hole and Remove and wash the decomposition products.
- a complementary strand extension reaction is performed to synthesize a second strand cDNA strand 61 (FIG. 6D).
- the PCR amplification step is performed after cooling to 0 ° C. (FIG. 6E).
- a heat block with heater aluminum alloy or copper alloy
- a temperature controller can be used.
- the target portions of the 20 target genes are amplified, and the size of the PCR amplification product is almost uniform at 200 ⁇ 8 bases.
- the PCR amplification product solution accumulated in the solution inside and outside the through-hole of the pore array sheet is collected.
- PCR Purification Kit QIAGEN
- emPCR amplification or bridge amplification After applying emPCR amplification or bridge amplification to this solution, it is applied to a next generation sequencer and analyzed.
- FIG. 7 schematically shows a state in which data sequenced as the same sequence other than the molecular identification tag sequence is obtained (related portions of the obtained sequencing data are schematically shown as the same pattern).
- a, b, c, d, and e are the same sequences, including a molecular identification tag sequence that is a random sequence, and 1, 7, 4, 2, and 2 reads are obtained, respectively.
- These sequences are all one molecule at the time when the second strand cDNA strand 61 is synthesized in FIG. 6D, and the number of molecules increases at the same time as the number of molecules increases by subsequent PCR amplification.
- reads having the same molecular identification tag sequence are derived from the same molecule and can be regarded as one molecule.
- the deviation in the number of molecules for each sequence caused by PCR amplification after the synthesis of the second strand cDNA strand 61 and adsorption inside the pore array sheet when taking out the solution to the outside is eliminated by the above identification.
- the prepared pore array sheet can be used repeatedly, and for gene groups that need to know the expression level, a target gene-specific sequence primer to which a PCR amplification common sequence primer (reverse, SEQ ID NO: 2) is added And preparing a second strand cDNA strand, PCR amplification, and emPCR in the same manner as described above, and analyzing with a sequencer. That is, by repeatedly using a cDNA library, it is possible to perform highly accurate expression distribution measurement for a necessary type of gene.
- the above-mentioned pore array sheet has a size of 2 ⁇ 2 mm and can be used as a reaction vessel with a 0.2 ml tube. Generally, the size of the pore array sheet is larger than 2 ⁇ 2 mm. It doesn't matter. However, in order to prepare the samples shown in FIGS. 5 and 6 at this time, it is necessary to divide the pore array sheet and introduce it into the reaction vessel. This method will be described with reference to FIG. On the pore array sheet 30, 16 chips 302 in which regions for capturing biomolecules are arranged are formed. In order to separate the chip 302 by cutting, for example, the output of the laser used for cell destruction can be increased 10 times.
- a tube identification tag sequence for identifying different reaction containers by sequence information may be introduced into the target gene-specific sequence primer 60 used in the second strand cDNA strand synthesis step of FIG. .
- the length of the tube identification tag sequence may be determined by the number of reaction vessels to be identified. However, if the same 5 bases as the cell identification tag sequence are used, ideally 1024 reaction vessels can be identified. Become.
- the insertion position of the tube identification tag sequence can be between the gene-specific sequence that hybridizes with the first strand cDNA strand 59 and the PCR amplification primer.
- the size of 2 ⁇ 2 mm is used as a unit, and it is cut from the pore array sheet 30 and introduced into the reaction vessel.
- the region 301 corresponding to one cell may be cut and introduced into the reaction vessel.
- the cell identification tag sequence for identifying the cell may be eliminated, and only the tube identification tag sequence may be used instead. Of course, both may be used.
- the differential interference microscope image only observes the shape without using a fluorescent reagent, but it is one of the measurement methods that has the least effect on cells when cells must be returned to the body in regenerative medicine or the like. If it is possible to associate the change in cell shape obtained from this image with the change in gene expression, a measurement system that can perform detailed cell classification with the least cell damage is obtained.
- the biomolecule analyzer shown in FIG. 9 includes a light source 1401.
- the light source 1401 is a halogen lamp.
- the biomolecule analysis apparatus shown in FIG. 9 further includes a polarizer 1402, a Wollaston filter 1403, a Wollaston prism 1404, a condenser lens 1405, an objective lens 1406, and an image sensor 801.
- the image sensor 801 for example, a 1024 ⁇ 1024 pixel CCD camera can be used.
- a focus adjustment lens 802 and two dichroic mirrors 803 having an edge wavelength of 370 nm are disposed in order to irradiate the center of the field of the differential interference image with the light from the cell light source 5 for cell destruction.
- the laser is set to be turned on / off by a control system (not shown) so that the cells are irradiated with the laser only when necessary.
- the pore array sheet 30 is movable, and when acquiring a high-resolution differential interference image, the biomolecule collection system 2 is arranged outside the petri dish 20 as shown in FIG. After acquiring the optical image and designating the cell to be measured, the pore array sheet 30 is moved using the XYZ stage 34 to bring the region 301 of the pore array sheet 30 close to the designated cell, and then Destroy cells.
- FIG. 10 shows an apparatus configuration diagram. Since the CARS microscope can obtain a spectrum corresponding to the chemical species of the laser-excited portion in the same manner as the Raman microscope and the IR microscope, it can increase the amount of information related to the state of the cell more than the differential interference microscope. In addition, CARS is a non-linear process, and has a merit that the signal intensity is higher than that of the Raman signal and a sufficient signal can be obtained with a relatively weak laser excitation intensity, so that damage to cells is small. With such an apparatus configuration, an optical image obtained by a CARS microscope and gene expression analysis data can be associated with each other, and cell classification information based on gene expression analysis can be given to measurement without using a label.
- CARS Coherent Anti-Stokes Raman Scattering
- a pulse laser microwave laser
- This is split into two by a beam splitter 1502, and one is introduced into a nonlinear fiber (photonic crystal fiber) 1503 to generate Stokes light.
- the other light is used as it is as pump light and probe light, and is condensed on the sample (in the cells 21, 22 and 23) by the water immersion objective lens 1504 to generate anti-Stokes light.
- Only the anti-Stokes light is transmitted through the high-pass filter 1505, condensed by the imaging lens 1508, and then the coherent anti-Stokes Raman spectrum is acquired by the spectroscope CCD camera 1507 through the spectroscope 1506.
- the configuration for irradiating the center of the optical image by CARS with the light from the cell light source 5 for cell destruction for cell destruction is the same as in the case of the differential interference microscope.
- cells can be identified by optical images obtained with a fluorescence microscope or the like, and gene expression data can be acquired in correspondence with cell images. Using this function, it is possible to confirm the dynamic characteristics of cells with high accuracy.
- a flowchart for executing such an analysis is shown in FIG. First, a cell sample is placed on a petri dish (step 001) and observed with a microscope to obtain an optical image (step 002).
- a reagent (RNA, differentiation inducer, etc.) according to the research application is introduced into the cell (step 003), and the change on the optical image of the cell is confirmed. For this purpose, an optical image may be acquired multiple times.
- a specific region of the array device is moved to the vicinity of the cell selected by the user (step 004), and the cell is destroyed.
- Intracellular biomolecules are captured on the array device (step 005), and the amount thereof is measured (step 006).
- the detailed cell state and type are identified by the quantification of the biomolecule, and the correspondence between the optical image and the cell state and type can be performed with high accuracy by taking correspondence with the optical image (step 007). It becomes possible.
- FIG. PC in the figure is an abbreviation for principal-component, where PC1 indicates the first main factor and PC2 indicates the second main factor.
- Each point corresponds to gene expression data for one cell. In many cases, it is divided into a plurality of clusters (in this example, 6 clusters) corresponding to the state and type of cells.
- principal factor analysis is used for clustering based on gene expression of cells, but various methods such as hierarchical clustering and k-means method can be applied. Also, as a method of machine learning, various methods used for data mining such as a support vector machine are known, and any of them may be used.
- the DNA probe 80 fixed inside the pore array sheet 51 includes a T7 promoter sequence from the 5 ′ end direction, a common sequence for emPCR amplification (forward direction, SEQ ID NO: 24), a cell identification tag sequence, and a molecular identification tag sequence. And oligo (dT) sequences.
- a molecular identification tag sequence for example, 15 bases
- 4 15 1.1 ⁇ 10 9 molecules can be recognized, so a huge amount of decoding data obtained by a next-generation sequencer
- the oligo (dT) sequence located at the most 3 ′ side hybridizes with the poly A tail added to the 3 ′ side of the mRNA and is used to capture the mRNA (FIG. 13 (a)).
- mRNA 54 is captured by an 18-base poly-T sequence that is complementary to the poly-A sequence at the 3 'end of the mRNA, as in the first embodiment.
- the first strand cDNA strand 59 is synthesized to construct a cDNA library (FIG. 13B).
- plural ( ⁇ 100) target gene-specific sequence primers 60 corresponding to the gene to be quantified are annealed to the first strand cDNA strand 59 (FIG. 14 (c)), and the second strand cDNA strand 61 is obtained by complementary strand extension reaction. Is synthesized (FIG. 14D).
- the second strand cDNA strand 61 is synthesized under multiplex conditions.
- double-stranded cDNA having a common sequence for amplification (forward / reverse) at both ends and including a cell identification tag sequence, a molecular identification tag sequence, and a gene-specific sequence therein is synthesized.
- there are 20 types (ATP5B, GAPDH, GUSB, HMBS, HPRT1, RPL4, RPLP1, RPS18, RPL13A, RPS20) that are 20 ⁇ 5 bases upstream of 109 ⁇ 8 bases from the poly A tail of the target gene.
- cRNA 63 is synthesized. Further, in order to synthesize double-stranded DNA for emPCR, a plurality of ( ⁇ 100 types) target gene-specific sequence primers 71 to which a common sequence for PCR amplification (reverse) is added using the amplified cRNA 63 as a template. Are hybridized (FIG. 15 (f)), and cDNA 72 is synthesized (FIG. 15 (g)). Further, as in the first embodiment, the cRNA 63 is degraded using an enzyme, and then a second strand is synthesized using a forward common primer to synthesize a double-stranded DNA 73 for emPCR (FIG. 15 (h)). ).
- This amplification product has the same length, and can be directly applied to emPCR and next-generation sequencer. Even if an amplification bias occurs between genes or molecules in this process, the amplification bias can be corrected by using the molecular identification tag sequence after data acquisition by the next-generation sequencer. Data can be obtained as in the first embodiment (see FIG. 7).
- the reverse transcriptase was inactivated by holding at 85 ° C. for 1.5 minutes, cooled to 4 ° C., and then 10 mM Tris buffer containing RNase and 0.1% Tween 20 (pH 8. 0) By injecting 10 ml from the inlet and discharging it from the outlet, the RNA was decomposed, and the same amount of alkali denaturing agent was flowed in the same manner to remove and wash the residue and decomposition products in the through holes.
- the temperature of the solution and the pore array sheet is raised to 37 ° C. and maintained for 180 minutes to complete the reverse transcription reaction and perform cRNA amplification.
- the target portions of the 20 target genes are amplified, and the size of the cRNA amplification product is almost uniform at 200 ⁇ 8 bases.
- the cRNA amplification product solution accumulated in the solution inside and outside the through-hole is collected.
- it is purified using PCR Purification Kit (QIAGEN) and suspended in 50 ⁇ l of sterile water.
- This solution is mixed with 10 ⁇ l of 10 mM dNTP mix and 30 ⁇ l of 50 ng / ⁇ l random primer, heated to 94 ° C. for 10 seconds, lowered to 30 ° C. at 0.2 ° C./second, heated at 30 ° C. for 5 minutes, Further decrease to 4 ° C. Thereafter, 20 ⁇ l of 5 ⁇ RT buffer (Invitrogen), 5 ⁇ l of 0.1M DTT, 5 ⁇ l of RNase OUT, and 5 ⁇ l of SuperScript III are mixed, heated at 30 ° C. for 5 minutes, and heated to 40 ° C. at 0.2 ° C./second. Raise to °C. For the purpose of removing residual reagents such as enzymes contained in this solution, it is purified using a PCR Purification Kit (QIAGEN), applied to emPCR amplification, and then applied to a next-generation sequencer for analysis.
- QIAGEN PCR Purification Kit
- FIG. 16 shows an apparatus configuration diagram of this embodiment.
- the needle 1001 is manufactured using a semiconductor process, the length of the needle is, for example, 10 ⁇ m, and the diameter of the tip portion is 1 ⁇ m.
- a silicon oxide film can be used as the material of the needle portion. It can be manufactured using a manufacturing method similar to that of a cantilever of an atomic force microscope.
- a silicon substrate is used as the needle holding member 1002.
- This silicon substrate is driven by the Z stage 1003, and the tip of the needle 1001 is pierced into the cell membrane to perforate the cell membrane, and the leaked mRNA is guided to the pore array sheet 30 by electrophoresis.
- the position in the plane of the needle 1001 is arranged and fixed at the center of the platinum electrode 32.
- the needle 1001 may be changed to a hollow needle so that biomolecules in the cell are released through the hollow needle.
- a method of perforating the cell membrane and destroying the cells a method of concentrating ultrasonic waves, a method of concentrating charged particles or electron beams, etc. can be used in addition to the above method.
- the cells to be destroyed with a needle or the like may be one cell or several cells. Further, the cells may be perforated one by one, or a plurality of cells may be destroyed at a time. Select according to the target sampling resolution.
- a configuration for analyzing a protein-derived peptide as a biomolecule will be described.
- An antibody having a protein or peptide to be measured as an antigen instead of a DNA probe is immobilized in the pore array sheet using a silane coupling agent.
- the fixed conditions may be the same. Since proteins and peptides do not always have a negative charge, they cannot be induced to the inside of the pore array sheet using electrophoresis. Therefore, a nozzle for aspirating the solution is arranged on the opposite side of the cell to be measured with the pore array sheet as the symmetry plane. The solution sucked by the nozzle can be returned to the inside of the petri dish and circulated.
- the nozzle inner diameter is, for example, 0.1 mm, and the suction speed can be 500 ⁇ l / second. This creates a flow of solution in the pore array sheet and in the area between the pore array sheet and the cells, and biomolecules including proteins and peptides released when the cell membrane is broken with a laser. Can be guided to.
- the cells to be destroyed may be one cell or several cells. Select according to the target sampling resolution.
- a bead array having beads packed on the surface is used as an array device instead of a pore array sheet made of a porous membrane.
- the configuration of the biomolecule analysis apparatus is the same as that of the example of FIG. 1 except that a bead array as shown in FIG. 17 is used instead of the pore array sheet 30.
- regions 1702 holding beads are arranged in the bead array 1701.
- a region 1702 corresponds to the region 301 in FIG.
- the bead array 1701 can be manufactured as follows. First, the alumina porous membrane used in the pore array sheet of the first embodiment is cut into a size of 2 mm ⁇ 2 mm. A PDMS resin film having a thickness of 100 ⁇ m in which through holes of 50 ⁇ m square are formed at a pitch of 100 ⁇ m is bonded onto this sheet. Thereafter, a solution of beads 1703 to which a DNA probe including a cell identification tag sequence different for each region 1702 is fixed is driven by a piezo injector used in an ink jet printer. Since the solution is sucked toward the porous membrane by the capillary effect and then dried, only the beads 1703 are packed. One region 1702 can be packed with 10 4 to 10 5 beads 1703.
- the beads 1703 for example, magnetic beads having a diameter of 1 ⁇ m coated with streptavidin can be used.
- a DNA probe whose end is biotin-modified is used, and is immobilized on the bead surface via streptavidin.
- Such beads are commercially available from a number of manufacturers.
- the array device thus fabricated is placed with the opening side of the region 1702 facing the cell and facing down on the XYZ stage 34.
- the present invention is not limited to the above-described embodiment, and includes various modifications. For example, with respect to a part of the configuration of the embodiment, it is possible to add, delete, or replace another configuration.
Abstract
Description
本実施形態に係る生体分子解析装置は、レーザー蛍光顕微鏡を用いて接着系培養細胞からなるサンプルの光学イメージを得る手段と、レーザー光源を用いて細胞を破壊する手段とを備え、細胞中のmRNAをアレイデバイスに捕捉することで遺伝子発現解析を行うことができる装置である。図1に本実施形態に係る生体分子解析装置の構成図を示す。この生体分子解析装置は、光学イメージを取得し、光学イメージ上の所定の位置の細胞の一部又は全部を破壊する機能を有する顕微鏡システム1と、細胞から放出・拡散される生体分子(mRNA)を捕捉するための領域が配列したアレイデバイス、これを駆動する機構、及びサンプル細胞を移動させる機構を有する生体分子採取システム2と、これら2つのシステムの動きを制御する制御システム3とから構築される。
本実施形態では、PCR増幅の代わりにT7プロモーターを用いた場合の例を説明する。第1実施形態との相違点は、シーケンシングサンプルの調製方法にある。図5及び図6に対応するサンプル調製の手順を図13、図14及び図15に示す。細孔アレイシート51の内部に固定されたDNAプローブ80は、5’末端方向からT7プロモーター配列、emPCR増幅用共通配列(フォワード方向、配列番号24)、細胞識別用タグ配列、分子識別用タグ配列、及びオリゴ(dT)配列で構成される。T7プロモーター配列をDNAプローブ80へ導入することで、後続のIVT(In Vitro Transcription)によるcRNA63増幅工程(図6(e))におけるターゲット配列の増幅が可能となる。すなわち、T7プロモーター配列はT7RNAポリメラーゼにより認識され、その下流配列から転写(cRNA63増幅)反応が開始される。同様にPCR増幅用共通配列を導入することで、後続のemPCR増幅工程において共通プライマーとして利用することができる。また、細胞識別用タグ配列を例えば5塩基としてDNAプローブ80に導入することによって、45=1024個の単一細胞を識別することが可能となることは第1実施形態と同様である。さらに、分子識別用タグ配列(例えば15塩基)をDNAプローブ80へ導入することにより、415=1.1×109分子を認識することができるため、次世代シーケンサーで得られる膨大な解読データが、どの分子由来であるかを識別することが可能となることも第1実施形態と同様である。すわなち、IVT/emPCR等の増幅工程で生じた遺伝子間の増幅バイアスを修正することができるため、始めに細胞中に存在していたmRNA量を高い精度で定量することが可能となる。最も3’側に位置するオリゴ(dT)配列は、mRNAの3’側に付加されているポリAテールとハイブリダイズし、mRNAを捕捉するために利用される(図13(a))。
第1及び第2実施形態においては、細胞を破壊する手段としてレーザー光を用いたが、様々な材料からなるニードルを用いても良い。図16に本実施形態の装置構成図を示す。ニードル1001は半導体プロセスを用いて作製し、針の長さは例えば10μmであり、先端部分の直径は1μmとする。針部分の材料は、シリコン酸化膜を用いることができる。原子間力顕微鏡のカンチレバーと同様の作製方法を用いて作製することができる。ニードルの保持部材1002としてはシリコン基板を用いる。このシリコン基板はZステージ1003により駆動し、ニードル1001の先端部分を細胞膜に突き刺すことによって、細胞膜に穿孔し、漏れ出たmRNAを細孔アレイシート30まで電気泳動にて誘導する。ニードル1001の面内での位置は白金電極32の中心に配置して固定する。ニードル1001を中空のニードルに変えて、細胞中の生体分子が中空ニードル内を通って放出されるようにしても良い。
本実施形態では、生体分子としてタンパク質由来のペプチドを解析するための構成について説明する。細孔アレイシートの内部に、シランカップリング剤を用いて、DNAプローブではなく計測対象とするタンパク質やペプチドを抗原とする抗体を固定する。固定条件は同じで良い。タンパク質やペプチドは、マイナスの電荷を持つとは限らないので、電気泳動を用いて細孔アレイシートの内部までこれらを誘導することはできない。そこで、細孔アレイシートを対称面として、計測対象とする細胞の反対側に、溶液を吸引するためのノズルを配置する。ノズルにより吸引した溶液はシャーレの内部に戻して循環させることができる。ノズル内径は例えば0.1mmであり吸引速度は500μl/秒とすることができる。これによって、細孔アレイシートの内部及び細孔アレイシートと細胞の間の領域に溶液の流れが生じ、レーザーで細胞膜を破壊したときに放出されるタンパク質やペプチドを含む生体分子を細孔アレイシートに誘導することができる。なお、破壊する細胞は1細胞でも数細胞程度でも良い。目標とするサンプリング分解能に応じて選択する。
本実施形態では、アレイデバイスとして、多孔質メンブレンからなる細孔アレイシートの代わりに、表面にビーズがパッキングされたビーズアレイを用いる場合について説明する。生体分子解析装置の構成は図1の例と同じであり、細孔アレイシート30に代えて、図17に示すようなビーズアレイを用いた点のみが異なる。ビーズアレイ1701には、ビーズが保持された領域1702が配列している。領域1702が、図1における領域301に対応する。
2 生体分子採取システム
3 制御システム
4 蛍光顕微鏡用レーザー光源
5 細胞破壊用レーザー光源
6 ダイクロイックミラー
7 ダイクロイックミラー
9 ステージ
10 試料ステージ
11 バンドパスフィルタ
12 結像レンズ
13 受光器
14 ピンホール
20 シャーレ
21 接着系培養細胞
22 接着系培養細胞
23 接着系培養細胞
30 細孔アレイシート
31 分離壁
32 白金電極
33 シールド線
34 XYZステージ
35 電源
36 mRNA
39 参照電極
41 電源
42 石英基板
43 金電極
44 貫通孔
51 細孔アレイシート
54 mRNA
55 貫通孔
56 DNAプローブ
59 ファーストストランドcDNA鎖
60 ターゲット遺伝子特異的配列プライマー
61 セカンドストランドcDNA鎖
62 2本鎖cDNA
63 cRNA
71 ターゲット遺伝子特異的配列プライマー
72 cDNA
73 2本鎖DNA
80 DNAプローブ
301 領域
302 チップ
801 撮像素子
802 フォーカス調整レンズ
803 ダイクロイックミラー
1001 ニードル
1002 保持部材
1003 Zステージ
1401 光源
1402 偏光子
1403 ウォラストンフィルタ
1404 ウォラストンプリズム
1405 コンデンサレンズ
1406 対物レンズ
1501 光源
1502 ビームスプリッタ
1503 非線形ファイバ
1504 水浸対物レンズ
1505 ハイパスフィルタ
1506 分光器
1507 分光器用CCDカメラ
1508 結像レンズ
1701 ビーズアレイ
1702 領域
1703 ビーズ
Claims (13)
- 複数の細胞の光学イメージを得る手段と、
前記複数の細胞のうちの1つ以上の細胞の一部又は全部を破壊する手段と、
前記破壊する手段によって放出される細胞中の生体分子を捕捉するための領域が配列したアレイデバイスと、
前記光学イメージにおける、前記破壊する手段によって破壊された細胞に相当する部分に対し、前記アレイデバイスにおける生体分子を捕捉した領域を対応付ける手段と、
を備える生体分子解析装置。 - 破壊する手段によって細胞を破壊する前に、アレイデバイスの領域と、破壊する細胞とを接近させる手段をさらに備える請求項1に記載の生体分子解析装置。
- 放出される細胞中の生体分子を、アレイデバイスの領域に吸引し又は泳動させる手段をさらに備える請求項1又は2に記載の生体分子解析装置。
- アレイデバイスが、多孔質メンブレン、又は表面にビーズがパッキングされたビーズアレイである請求項1~3のいずれかに記載の生体分子解析装置。
- アレイデバイスの表面又は内部に、細胞中の生体分子を選択的に捕捉するためのプローブ分子が固定されている請求項1~4のいずれかに記載の生体分子解析装置。
- 細胞中の生体分子がmRNAであり、プローブ分子がDNAプローブである請求項5に記載の生体分子解析装置。
- DNAプローブが、アレイデバイスの位置ごとに異なる配列を有する請求項6に記載の生体分子解析装置。
- 細胞中の生体分子がタンパク質又はペプチドであり、プローブ分子が抗体である請求項5に記載の生体分子解析装置。
- 破壊する手段が、レーザーである請求項1~8のいずれかに記載の生体分子解析装置。
- 破壊する手段が、ニードルである請求項1~8のいずれかに記載の生体分子解析装置。
- 破壊する手段が、中空ニードルであり、細胞中の生体分子は前記中空ニードル内を通って放出される請求項1~8のいずれかに記載の生体分子解析装置。
- 破壊する手段が、電子線又は荷電粒子線である請求項1~8のいずれかに記載の生体分子解析装置。
- 複数の細胞が、ゲル中に3次元的に配置されている請求項1~12のいずれかに記載の生体分子解析装置。
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WO2017056274A1 (ja) * | 2015-09-30 | 2017-04-06 | 株式会社日立製作所 | 細胞解析装置およびそれを用いた細胞解析方法 |
JP2018057342A (ja) * | 2016-10-06 | 2018-04-12 | 株式会社東芝 | 細胞分取装置および細胞分取システム |
CN109313326A (zh) * | 2016-05-19 | 2019-02-05 | 株式会社尼康 | 显微镜 |
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