WO2012060163A1 - 細胞分析装置 - Google Patents
細胞分析装置 Download PDFInfo
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- WO2012060163A1 WO2012060163A1 PCT/JP2011/071258 JP2011071258W WO2012060163A1 WO 2012060163 A1 WO2012060163 A1 WO 2012060163A1 JP 2011071258 W JP2011071258 W JP 2011071258W WO 2012060163 A1 WO2012060163 A1 WO 2012060163A1
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Classifications
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- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume, or surface-area of porous materials
- G01N15/10—Investigating individual particles
- G01N15/14—Electro-optical investigation, e.g. flow cytometers
- G01N15/1484—Electro-optical investigation, e.g. flow cytometers microstructural devices
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/483—Physical analysis of biological material
- G01N33/487—Physical analysis of biological material of liquid biological material
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- G01N15/10—Investigating individual particles
- G01N15/14—Electro-optical investigation, e.g. flow cytometers
- G01N15/1468—Electro-optical investigation, e.g. flow cytometers with spatial resolution of the texture or inner structure of the particle
- G01N2015/1472—Electro-optical investigation, e.g. flow cytometers with spatial resolution of the texture or inner structure of the particle with colour
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Definitions
- the present invention relates to a cell analyzer.
- cell differentiation 1) Visual morphological cell classification: For example, examination of bladder cancer and urethral cancer by examination of atypical cells appearing in urine, classification of atypical cells in blood, cancer examination by cytology in tissues, etc. Can give. 2) Cell classification by cell surface antigen (marker) staining by the fluorescent antibody method: Cell surface antigens generally called CD markers are stained with a specific fluorescently labeled antibody. Cell sorting by cell sorters, flow cytometers and tissues Used for cancer screening by staining. Of course, these are widely used not only for medical purposes but also for cell physiology research and industrial cell utilization.
- stem cells containing stem cells are roughly separated using a fluorescent dye incorporated into the cells as a reporter, and then the target stem cells are separated by actually culturing. This is because an effective marker for stem cells has not yet been established, and the target cells are substantially separated by using only those actually cultured and differentiated.
- the light absorption and birefringence properties are different, and this is imaged to visualize the intracellular state. There are a phase contrast optical microscope, a differential interference optical microscope, and the like as an observation technique.
- the nucleus is hematoxylin
- the cytoplasm is orange G, eosin Y, and light green SF (in order of decreasing molecular weight)
- those with a dense cytoplasmic structure are likely to contain small molecular weight pigments (eg, keratinized) Squamous epithelial cells: orange), both of which enter sparse cells, but pigments with large molecular weight are less mobile and charged, making them easily adsorbed inside cells (eg non-keratinized squamous cells, glandular cells: light) Green)
- lipids can be dyed with Bismarck Brown, and it is a method to identify malignant tumor cells and infectious diseases with different colors from dark blue green to dark red.
- Giemsa staining is a mixture of the basic dye methylene blue and its oxidized derivative methylene azur and eosin acid dye, but these dyes are molecules that penetrate the cell with differences in the terminal methyl group.
- the azure eosinate with relatively few methyl groups penetrates not only into the cytoplasm but also into the nucleus, binds to the phosphate group of the nucleic acid, and is stained in a red-purple nucleus.
- methylene blue which is a relatively large molecule and has a strong polarity, binds to cytoplasmic proteins and exhibits a blue tone. Therefore, cell information can be obtained depending on the degree of staining.
- the existing cell identification method by staining combines multiple dyes with different light absorption characteristics and visually determines the relative color development (absorption) degree of those dyes. It is an intuitive measurement method that targets the spectrum of the entire visible spectrum, depending on the information processing of the visual system, such as what color colors are combined with different intensities. is there. This is the most different part from the fluorescence measurement evaluation technique that only needs to measure the fluorescence of a specific one wavelength quantitatively.
- Such separation and recovery of specific cells in the culture medium is an important technique in biological and medical analysis.
- the cells When the cells are separated based on the difference in specific gravity of the cells, they can be separated by a velocity sedimentation method. However, if there is almost no difference in specific gravity between cells that distinguishes unsensitized cells from sensitized cells, the cells are separated one by one based on information stained with fluorescent antibodies or visual information. There is a need to.
- the cell sorter isolates and drops cells after fluorescence staining in a charged droplet in units of one cell, and based on the presence or absence of fluorescence in the droplet and the amount of light scattering, In the process of dropping, a high electric field is applied in any direction in the normal direction to the direction of drop, and the drop direction of the drop is controlled and fractionated into multiple containers placed underneath.
- Non-patent document 1 Kamarck, ME, Methods Enzymol. Vol. 151, p150-165 1987 (1987)).
- the cell sorter created using this microfabrication technique has a slow response speed of sample separation to the observation means, and in order to put it to practical use, a separation processing method that does not damage the sample and has a faster response is required. there were.
- the separation efficiency of the device cannot be sufficiently increased even at a dilute cell concentration. If the sample is concentrated in a separate device, it is difficult not only to recover the concentrated solution without loss, but also in the complicated pretreatment stage, the collected cells are contaminated. The problem is that an undesirable problem occurs in terms of use.
- Patent Document 1 JP 2003-107099; Patent Document 2: JP 2004-85323; Patent Document 3: WO 2004 / 101731).
- cancer cells metastasize to other organs by their ability to infiltrate blood vessels or lymph vessels from their own tissues.
- Malignant tumor cells that circulate in the peripheral blood are called peripheral blood circulating cancer cells (Circulating Tumor Cells: CTCs), and it is considered that there are several hundred cancer cells in 100,000 cells of blood cells (including red blood cells). ing.
- anticancer agents for specific targets have been developed one after another, and if the type of malignant tumor in the blood can be identified, it has become possible to select an anticancer agent that effectively destroys the cells.
- Non-patent Document 4 Harnett, ⁇ M). ., “Laser scanning cytometry: metryunderstanding the immune system in situ. Nature Review Immunology, Vol. 7, pp. 897-904 (2007)).
- This imaging cytometry is a concept of conventional optical microscope imaging and cytometry. A standard value is set for a specific index such as a cell shape or a fluorescent label from a conventional random microscopic imaging of a tissue, and all cells in the tissue are measured and statistically processed.
- fluorescence measurement etc., there is a scanning fluorescence imaging method that scans and measures focused laser light. And it has a flow.
- Non-Patent Document 5 Kinosita K Jr, Itoh H, Ishiwata S, Hirano K, Nishizaka T, Hayakawa T. “Dual-view microscopy with a single camera: real-time imaging of molecular orientations and calcium.”, J Cell Biol.
- the light source light for phase contrast microscope observation and the excitation light for fluorescence observation are introduced on the same optical path, and after observing the sample, the wavelength is divided by the dichroic mirror and the fluorescence image and the phase contrast image are obtained.
- a fluorescent image and a phase contrast image can be recorded simultaneously in one image frame.
- this combination is a technique that enables observation of two fluorescences, comparison of longitudinal and lateral polarization of one fluorescence, and the like. However, this technique is not used for applications such as absorption spectrum measurement.
- the present inventors have already performed flow cytometry and cell sorting based on the imaging observation technique of cell shape or cell population that has already been proposed by the present inventors, and simultaneously with these techniques.
- the absorption spectrum analysis technology for cell imaging which is an existing cell inspection identification technology, is added to the cell and intracellular absorption spectrum imaging technology.
- a cell analyzer capable of identifying cells based on data and selectively collecting target cells as needed. This is because the conventional flow cytometry technology can acquire optical signals of trace cells in bulk solution containers when detecting signals from a sample where there is no background light, such as fluorescent light or scattered light.
- erythrocytes that flow in the blood, cancer cells that become cells other than white blood cells, and stem cells.
- a method of collecting blood cells such as known white blood cells and erythrocytes and collecting unstained cells as target cells, or A method of collecting different cells from known cell shapes is effective. That is, the cells are analyzed by flow cytometry and cell sorting based on the imaging observation technique of the cell shape or cell grouping proposed by the present inventors, and comparison analysis by simultaneously performing fluorescence detection in addition to these techniques.
- the absorption spectrum analysis technology for cell imaging the technology for identifying cells based on the absorption spectrum imaging data in cells and cells is known.
- the present invention proposes a new method for recovering unlabeled cells as candidate cells for cancer cells or stem cells, which are used as labels for red blood cells and white blood cells in blood.
- the identification by shape is particularly intended for immature cells in which the ratio of the nucleus size to the cell size is very large.
- a cell analyzer for measuring cells in a sample solution of cells from a subject and selectively separating and purifying the cells, A first flow path for flowing a cell sample solution containing cells including target cells, a cell detection unit for detecting an absorption spectrum image of the cells in the first flow path, and the target cells
- a cell sorter chip including a cell separation unit including at least two branch channels branched from the first channel, in which selective recovery is performed by separating other cells, and
- a light irradiating means for irradiating the cells flowing in the first flow path with light from a plurality of monochromatic light sources arranged so as to have a visible light region spectrum, and an image capturing rate of at least 200 frames / second;
- An optical system comprising: a high-speed camera that obtains an image of the cell as an image of each monochromatic light, the high-speed camera capable of determining the light absorption characteristics of the cell with a spatial resolution by comparing the monochromatic light images
- the apparatus according to any one of [1] to [3] above, wherein the cell is detected at a single cell level and a target cell is identified.
- the optical system further includes a fluorescence detector, and information on the fluorescence image of the cells is used as an additional indicator.
- a cell analyzer for measuring cells in a cell sample solution derived from a subject and detecting the cells
- a flow channel chip including a flow channel for flowing a cell sample solution containing cells including target cells, and a cell detection unit for detecting an absorption spectrum image of the cell in the flow channel
- a light irradiating means for irradiating the cells flowing in the flow path with light from a plurality of monochromatic light sources arranged so as to have a visible light region spectrum; and an image capturing rate of the cells at least at 200 frames / second.
- An optical system including a high-speed camera that obtains an image as an image of each monochromatic light, the high-speed camera capable of determining the light absorption characteristics of the cells with spatial resolution by comparing the monochromatic light images;
- a cell analyzer A light irradiating means for irradiating light from a plurality of monochromatic light sources arranged so as to have a visible light region spectrum on the cells flowing in the flow path, and a spectrum for obtaining the size of the light receiving surface of the photographing camera
- a high-speed camera that obtains an image of the cell as an image of each monochromatic light, and a high-speed camera that can determine the light absorption characteristics of the cells with spatial resolution by comparing the monochromatic light images.
- a cell analysis method for measuring cells in a cell sample solution derived from a subject and selectively separating and purifying the cells A first flow path for flowing a cell sample solution containing cells including target cells, a cell detection unit for detecting an absorption spectrum image of the cells in the first flow path, and the target cells
- the cell sorter chip including the cell separation unit including at least two branch channels branched from the first channel, which is selectively collected by separating other cells, and the first channel in the cell sorter chip Introducing a cell sample solution; Irradiating the cells flowing in the first flow path with light from a plurality of monochromatic light sources; A high-speed image capable of acquiring the cell image as an image of each monochromatic light at an image capture rate of at least 200 frames / second and comparing the monochromatic light images with a spatial resolution.
- a cell sample solution containing cells previously selected by cell shape and / or staining is used as the cell sample solution.
- a cell analyzer for identifying cells in a cell sample solution derived from a subject A pulsed light source including an array of a plurality of single-color light sources in a specific wavelength band continuous within a wavelength range covering a light wavelength band from 280 nm to 780 nm, and irradiating each single-color light source as pulsed light by a control program
- An image acquisition camera having a wavelength characteristic capable of acquiring an image of the cell in the light wavelength band of the monochromatic light source array;
- An image processing unit for discriminating and recording the light absorption characteristics with spatial resolution A cell analyzer.
- the light source includes an array of monochromatic light pulse light sources capable of emitting pulse light in a time shorter than an image acquisition time of one frame of the image acquisition camera.
- the cell according to [12] comprising a high-speed camera capable of acquiring 10,000 images per second as an image acquisition time of one frame of the image acquisition camera, and an array of monochromatic LED light sources having a light emission time of less than 0.1 ms. Analysis equipment.
- the target cell is discriminated using as an index the disappearance of the nucleus in the cell image obtained from the acquired image.
- the visual color of a small amount of cells to be examined is estimated by absorption, or the characteristics of the absorption spectrum are analyzed, and the cells are identified by the intracellular distribution of the color or absorption characteristics. It is possible to selectively collect and purify the target cells, and to realize accurate gene information and expression information analysis of the target cells.
- FIG. 5 is a diagram schematically showing a second example of an optical system and image imaging recording used in the present invention. It is a figure which shows an example of the image of the cell absorbed by the monochromatic light of the specific wavelength obtained in this invention.
- FIG. 1 is a conceptual diagram of an embodiment of a cell analyzer 1 of the present invention.
- the cell analyzer 1 of the present invention typically obtains image data of about 10,000 image cells per second from cells flowing through a microchannel formed on the substrate of the cell sorter chip 100. Acquired by the image detection type 1-cell separation / purification module 200 and the module 200 for purifying and collecting a cell population or a cell mass in real time based on the analysis result of the image information with a time processing capability of 10,000 cells per second at the maximum.
- a recording / control unit 300 that records / analyzes the various data and controls each unit of the module 200 is provided.
- the cell analyzer 1 of this invention may be provided with the display which displays the acquired image data, an analysis result, etc.
- FIG. 2 is a schematic diagram showing an example of a cell sorter chip 100 that separates cells used in the cell analyzer 1 of the present invention.
- the cell sorter chip 100 is configured to flow the flow channel 102 formed on the chip substrate 107, the external force generator 103 for applying external force to the cells flowing through the flow channel 102, and the separated cells. Branch flow paths (108, 109).
- the external force generator 103 is not necessarily integrated with the chip substrate 107.
- the cell sorter chip 100 used in the cell analyzer 1 of the present invention typically includes a cell separation / purification unit having a function of separating and purifying cells in the flow path 102 of the chip substrate 107, and a stage preceding the separation unit.
- An optical analysis unit or cell detection unit for identifying and judging cells to be separated and purified is included.
- the cell detection unit is sufficient, and the cell separation / purification unit is not necessarily required.
- the cell separation / purification unit includes at least two branch flow paths (108, 109). For example, in the flow path in which cells are flowing, an external force is applied to the cells in different directions, and the downstream flow paths (108, 109). In order to induce cells to be collected into one of the two branched channels and other cells into the other channel, an external force is applied oppositely by the cells to move the cell flow position, Cells can be induced in either of the two branched channels.
- the shape of the cell is determined by fine identification and evaluation of the shape based on the image of the image detection type 1-cell separation / purification module, and is continuously conveyed by the flow path. A small amount of cells can be recovered from a cell sample without staining in a manner that eliminates contamination and manipulations to a minimum.
- each part of the image detection type 1-cell separation / purification module 200 will be described in detail.
- an image detection type 1-cell separation / purification module 200 typically includes a cell sorter chip 100, a light source 201, a mirror 202, a condensing lens 203, a dichroic mirror 204, and a filter 205 as necessary.
- a high-sensitivity light detection element (or fluorescence detector) 206 and a high-speed camera 207 are included.
- the light source 201 includes, for example, a light source array for spectrum analysis in which a pulse laser that emits monochromatic light or a high-intensity LED is set for cells passing through the cell sorter chip 100.
- the light source 201 includes a monochromatic light source array for spectrum analysis that is a light source set that covers a wide band by combining a monochromatic pulse laser that emits a plurality of monochromatic lights and monochromatic high-intensity LEDs.
- the irradiation light of the light source continuous light may be irradiated, but preferably, pulse light is generated in synchronization with the shutter cycle of the high-speed camera 207 in order to improve the spatial resolution of the image without blurring. As a result, an image with higher time resolution can be acquired by light irradiation for a short time.
- the light source has a function capable of emitting light as a pulsed light source in a shorter time than the shutter cycle (imaging time resolution for acquiring one frame) of the high-speed camera 207 for acquiring images.
- the conventional xenon flash light source has a limit of about 400 flashes per second, for example, a high-speed camera capable of acquiring an image of 10,000 frames per second is at least less than 0.1 ms.
- a monochromatic light pulsed LED light source can be used in the current technology as a light source capable of emitting arbitrary monochromatic light in such a short time, but is not limited thereto.
- An absorption spectrum image in this wavelength band is obtained by acquiring and comparing a plurality of monochromatic light images at intervals for the wavelength region desired to be obtained by combining combinations of each acquired image image (image frame) and the monochromatic light source pulse. be able to.
- an acquired image set includes an absorption spectrum image having spatial information. It becomes.
- the high-sensitivity light detection element (or fluorescence detector) 206 includes a photomultiplier that detects fluorescence.
- the high-speed camera 207 typically has a light source for each wavelength so that the output on the light-receiving side is approximately the same in order to adjust the dependency of the photoelectron conversion efficiency of the light-receiving element constituting the light-receiving surface of the camera on the wavelength.
- an electronic device capable of performing amplification correction of each wavelength reception data in the electronic circuit or image processing program stage in consideration of the wavelength dependency of the photoelectric conversion efficiency of the light receiving element in advance while keeping the light source intensity of each wavelength the same. It may be a target two-dimensional imaging device, a computer, a camera having an amplification electronic circuit, for example, a CCD camera (video camera, digital camera) or the like.
- the apparatus of the present invention includes: (1) a plurality of single-color flash light sources as light sources, and a plurality of continuous single-color flash light sources for each image.
- a plurality of single-color flash light sources as light sources
- a plurality of continuous single-color flash light sources for each image.
- an imaging image reflecting the absorption wavelength characteristics of the cells is obtained, and the spatial distribution estimated by the combination of the absorption (or transmission) light intensity of each wavelength is obtained.
- It is equipped with a system that identifies the cell type by converting (analyzing) it into spectral characteristics or visual color information.
- Such irradiation of light from a pulsed light source, image capture, analysis, and the like can be realized by a program using a computer (for example, a PC).
- the light source includes a plurality of monochromatic flash light sources each having a different wavelength.
- an array of monochromatic light in the range of 280 nm to 780 nm, for example, 50 nm width can be used. That is, for example, a light source that emits monochromatic light only in the wavelength range of 280 nm to 330 nm, a light source that emits monochromatic light only in the wavelength range of 330 nm to 380 nm, a light source that emits monochromatic light only in the wavelength range of 380 nm to 430 nm, etc. By combining 10 monochromatic light sources with different wavelengths, a monochromatic light source array that can cover the entire wavelength range can be obtained.
- the wavelength width of 50 nm is described as an example, but the wavelength range of the monochromatic light source ranges from 10 nm to 100 nm in consideration of the balance between wavelength resolution and analysis speed (the number of monochromatic light sources for covering the entire area). It is desirable to select one of the wavelength widths (for example, every 10 nm, every 20 nm, 30 nm, etc.) according to the purpose.
- the light source is a monochromatic light array
- the wavelength range that can be captured by the image acquisition camera covers the entire wavelength range of the monochromatic light source array. Since each light source is monochromatic light, there is no need to add a filter to select the wavelength range of transmitted light, especially for image acquisition cameras, and all the light that has passed through the objective lens can be directly guided to the camera. it can. Therefore, it is possible to realize the simplest optical system configuration that does not require any absorption bandpass filter (that is, the flash light from the monochromatic light source array is irradiated to the cells in the flow path by the condenser lens and obtained.
- absorption bandpass filter that is, the flash light from the monochromatic light source array is irradiated to the cells in the flow path by the condenser lens and obtained.
- the cell image with monochromatic light is irradiated directly from the objective lens onto the imaging surface of the image acquisition camera). Therefore, when a conventional broadband wavelength light source is used, in order to obtain a plurality of monochromatic light images having the same effect as in the present invention, a plurality of band pass filter groups corresponding to the wavelength width are included in the optical system. A complicated device configuration is required to be incorporated, and corrections due to differences in the transmittance of each filter and measures such as attenuation of transmitted light due to the combination of a plurality of filters are required. If one such feature is used, a simple device configuration that does not require the use of any bandpass filter is possible.
- the prior art only needs to exchange and adjust the bandwidth of the monochromatic light source constituting the monochromatic light source array. It is possible to cope with wavelength bandwidths and regions that have been difficult to create, and spectral images with different wavelength widths can be easily obtained.
- the feature of the present invention it is possible to obtain a superimposed image of a plurality of monochromatic pulse lights having two or more wavelengths, which is impossible with the conventional bandpass filter structure. That is, by simultaneously irradiating a plurality of monochromatic pulse lights having two or more wavelengths in a monochromatic light source array, for example, an image obtained by superimposing absorption image images of specific two monochromatic lights is optically acquired as one image. I can do things.
- the apparatus configuration described above has been described as an example particularly when used for cell discrimination with a cell sorter.
- a superposition image of absorption image images by a plurality of monochromatic light pulses can be obtained as an image acquisition camera. May be obtained by Or although the cell sorter which has a cell separation function was described as the example in the said Example, the board
- the substrate may be a channel made of a material such as synthetic quartz that is optically transparent in the emission wavelength region of the monochromatic light source, or a flow path for flowing a solution containing cells formed on such a substrate.
- a material such as synthetic quartz that is optically transparent in the emission wavelength region of the monochromatic light source
- a flow path for flowing a solution containing cells formed on such a substrate may of course be used as an absorption imaging cytometry technique for continuously performing absorption imaging of a sample flowing through the flow path.
- each wavelength region image to be observed is divided by a filter such as a dichroic mirror, and these are simultaneously received by one CCD camera.
- the image is projected by dividing it by the number of divisions to be observed, and by comparing the projected images of the respective wavelength components, It is equipped with a system for identifying cell types by performing spectral characteristics with a spatial distribution estimated by a combination of absorbed (or transmitted) light intensity or conversion (analogue) into visual color information.
- image imaging data that has been wavelength-divided using a plurality of filters is divided into light-receiving surfaces of a single camera. It may be projected onto each of the obtained regions and acquired as spectral imaging data.
- a filter is obtained by combining the same spectral imaging data technique with the pulse emission of each monochromatic light source and one image imaging image.
- Each spectrum data can be acquired without using.
- moving objects are sequentially imaged with different monochromatic light, it is necessary to correct the position information.
- wavelength filters are not required, measurement is performed with many wavelength resolutions in particular. There is an advantage that the loss of the cumulative amount of light due to a large number of filters can be suppressed.
- the processing by the image and the processing by the fluorescence may be used in combination.
- a filter 205 for fluorescence observation it is necessary to add a filter 205 for fluorescence observation, and attention should be paid to this wavelength because absorption spectrum imaging cannot be acquired.
- the image data obtained by the high-speed camera 207 can not only be recorded, but also displayed on the monitor of the computer (300) in real time for user observation.
- the filter 205 is appropriately adjusted to transmit a plurality of excitation lights, and a wavelength that does not overlap with the fluorescence wavelength for fluorescence detection in the lower stage is selected to light the cells.
- the fluorescence measurement function of the cell analyzer of the present invention By using the fluorescence measurement function of the cell analyzer of the present invention, the presence or absence of the fluorescent label of the target cell is detected and confirmed at the single cell level, and in addition to the absorbance information, the fluorescently labeled cell
- the target cells can be selectively collected by comprehensive judgment based on the above judgment.
- a plurality of pieces of information can be detected simultaneously for the cells flowing through the flow path of the cell sorter chip 100. Therefore, according to the cell analyzer of the present invention, it is possible to accurately separate and purify cells based on an index that cannot be identified by the conventional scattered light detection type cell sorter technique.
- the recording / control unit 300 used in the cell analyzer 1 of the present invention includes, for example, a personal computer equipped with an electronic recording medium (eg, ROM, RAM, hard disk, USB, memory card, etc.), a program type It is composed of arithmetic elements, SPGA, etc. Note that the recording unit and the control unit are not necessarily integrated.
- an electronic recording medium eg, ROM, RAM, hard disk, USB, memory card, etc.
- a program type It is composed of arithmetic elements, SPGA, etc. Note that the recording unit and the control unit are not necessarily integrated.
- the cell recognition and separation algorithm used in the present invention has the following characteristics.
- a part of the cell sorter chip 100 for observing the flow path portion with a high-speed camera such as a CCD camera is provided. Perform more reliable cell separation. What is important at this time is the image capturing speed. With a camera with a typical video rate of 30 frames / second, there is a possibility that cell loss will occur in the image. With an uptake rate of at least 200 frames / second, cells that normally flow through the channel at a significant rate can be recognized.
- the image detection type 1-cell separation / purification module 200 when a cell is captured and evaluated as an image, a portion to be observed by a CCD camera is provided upstream of the flow path branching portion, and a cell separation region is provided downstream thereof as necessary. Install.
- the fluorescence can be detected with a photodetector. Also in this case, a separation channel point that becomes a cell separation region can be provided downstream of the detection unit.
- the external force generator 103 can move the cells by applying external force to the cells from the outside to the cell sorting unit.
- the external force for example, dielectric electrophoresis force, electrostatic force, ultrasonic radiation pressure, or the like can be used.
- a pair of comb electrodes When using a dielectrophoretic force, a pair of comb electrodes is installed, and a flow path that can separate and discharge cells is provided.
- electrostatic force a voltage is applied to the electrode to change the position of the cell in the flow path. At this time, since the cell is generally charged negatively, it moves toward the positive electrode.
- ultrasonic radiation pressure is used as an external force, cells can be guided to the sound pressure node.
- the sample solution is introduced into the microchannel 102 from the inflow port in the direction of 101 by a cell introduction means that does not generate a pulsating flow such as a syringe pump, air pressure, or liquid level difference (see FIG. 2). ).
- the sample liquid containing the cells introduced into the microchannel is measured in the cell detection region arranged in the front stage of the cell sorting unit, and after the type of each cell is determined, the external force generator 103 is used.
- the first outlet 105 and the second outlet 105 pass through the two flow paths (108, 109) branched in the cell sorting section depending on whether or not an external force in the direction perpendicular to the flow from upstream to downstream is applied. To one of the outlets 106.
- the introduction pressure of the cell sample solution into the chip can be used as a driving force for moving the solution.
- the image detection type 1-cell separation / purification module 200 for example, as primary detection, the presence or absence of fluorescence emission based on a fluorescent label at the 1-cell level is confirmed. This makes it possible to confirm whether the cell is a target cell by a conventional labeling technique using fluorescence.
- the image collected by the high-speed camera 207 for the target cells that emit fluorescence (1) what kind of light absorption characteristics the cells emitting fluorescence have, It is determined by imaging which distribution of light absorption characteristics is present, and (2) the fluorescence-emitting cell is in a healthy state, or is in a state such as apoptosis in which the cell nucleus and cell shape are deformed.
- M-phase to determine the shape of organelles such as whether or not, or to determine what kind of cancer cells are by comparing the ratio of the size of the nucleus to the cell, or to eliminate the nucleus Based on the analysis of information that can be obtained from the image, depending on the purpose, such as whether or not there are cells, recovering healthy isolated cells, cell populations or cell masses in the blood Alternatively, cells undergoing apoptosis or cells having specific absorption characteristics can be collected.
- the difference in cell size and the number of populations of cells is acquired as image information, and cells are purified based on the result.
- image acquisition a high-speed camera is used, the light emission of the light source is adjusted according to the shutter cycle of the high-speed camera to acquire a fine image, and the light from the light source for a certain period of time during which each shutter is released. To emit light.
- the shutter speed is 1 / 10,000th of a second
- illuminate the target cells with a light source capable of high-speed light emission control, such as an LED light source or a pulsed laser light source, for a period of 1 / 10th of the shutter speed.
- a light source capable of high-speed light emission control such as an LED light source or a pulsed laser light source
- the cells assumed as targets for detection, analysis, and / or separation are described mainly with blood cells here, but it goes without saying that they are not limited to blood cells.
- small cells include bacteria
- large cells include animal cells (eg, cancer cells)
- cancer cells include, but are not limited to, cell populations and cell masses.
- the cell sample size typically ranges from about 0.5 ⁇ m to about 100 ⁇ m. For this reason, when a channel incorporating a cell separation function is formed on one surface of a biochip substrate to continuously perform cell concentration and separation, the first problem is the channel width (cross-sectional shape).
- the channel is formed in a substantially two-dimensional plane using a space of about 10 to about 100 ⁇ m in the thickness direction of the substrate on one of the substrate surfaces.
- cell size about 5 to about 10 ⁇ m in the thickness direction for bacteria and about 100 to about 500 ⁇ m in the thickness direction for animal cells and cell populations / cell masses are the most typical sizes.
- human blood can be directly collected with a heparin-containing blood collection tube and used directly as a cell sample.
- treatment with 10 times volume of BD ⁇ Pharm Lyse at room temperature for 15 minutes may be used.
- the staining method used in conventional biopsy may be used as it is for each staining method.
- cancer cells that become cells other than white blood cells, stem cells, etc. those for which labeled antibodies and / or staining methods have already been determined
- These labeling / staining techniques may be used as they are, but there are also cells that cannot be identified and dealt with by staining or antibody reaction alone because of the diversity and / or undifferentiation of the target cells. In that case, it is only necessary to identify and identify known blood cells such as white blood cells, red blood cells, and platelet cells by shape or staining, and collect cells that could not be identified by these identifications as candidate target cells.
- the cells are analyzed by flow cytometry and cell sorting based on the imaging observation technique of the cell shape or cell grouping proposed by the present inventors, and comparison analysis by simultaneously performing fluorescence detection in addition to these techniques.
- Technology to identify cells based on the data of absorption spectra and imaging data inside cells in addition to existing cell inspection identification technology, absorption spectrum analysis technology of cell imaging And used as a label for red blood cells and white blood cells in the blood, and unlabeled cells may be recovered as candidate cells for cancer cells or stem cells.
- immature cells having a very large ratio of the nucleus size to the cell size may be discriminated and recovered.
- FIG. 3 is a schematic diagram for explaining an example of the image processing method.
- the cell for example, when each cell first appears in the image frame, the cell is numbered, and thereafter, management is performed with the same number until it disappears from the image frame. That is, the number is used to manage the situation in which the cell image moves in a plurality of consecutive frames.
- the cells in each frame move from the upstream side to the downstream side, and the cells between the frames are linked under the condition that the moving speed of the numbered specific cells recognized in the image falls within a certain range. Let In this way, each cell can be traced reliably even if the cell is overtaken. Moreover, by doing in this way, the absorption (or permeation
- the cell image is first binarized and its center of gravity determined.
- the luminance center of gravity, area, perimeter length, major axis, minor axis of the binarized cells are obtained, and each cell is numbered using these parameters. It is possible to automatically save each cell image as an image at this point because it is beneficial to the user.
- the separation index may be information such as the luminance center of gravity, area, circumference length, major axis, minor axis, or the like, or may be obtained by using fluorescence detection separately from the image.
- the cells obtained by the detection unit are separated according to the numbering. Specifically, the movement speed (V) of the numbered cells is calculated from the image captured every predetermined time, and the distance from the detection unit to the selection unit with respect to the cell movement speed (V) (L), By setting the application timing from (L / V) to (L / V + T) depending on the application time (T), the cells are electrically distributed and separated when the cells of the target number come between the electrodes.
- a cell image with first monochromatic light can be acquired first with the nth image.
- the next monochromatic pulsed light can be emitted and the image can be acquired as the (n + 1) -th image. Proceeds in the direction of flow velocity.
- the third monochromatic pulse light is emitted and the image of the cell at the incident light wavelength is acquired as the (n + 2) -th image, the cell is similarly further subjected to v ⁇ ⁇ in the image. Is proceeding in the direction of flow velocity.
- slits 401 that can narrow the image area so that the same image can be projected evenly divided into several minutes, the size of the image is adjusted, and then two or more that can be evenly wavelength-divided
- a dichroic mirror and a wavelength filter are arranged in parallel, and this is passed through an observation image with a narrowed area, and spectrally decomposed.
- the angle of the mirror 204 is finely adjusted so that the positions projected from the respective mirrors onto the light receiving surface of the high-speed camera are arranged side by side without overlapping with the images from the respective mirrors.
- similar cell images 403, 404, and 405 having different incident light wavelengths can be recorded side by side on a single light receiving surface 402, and the light intensity of the cells that are next to each other is relatively compared. By doing so, the spectral distribution of the cell image can be acquired.
- An example of the configuration of the cell separation and purification chip is as follows. A series of microfabricated channels arranged in a two-dimensional manner on a flat chip that performs everything from concentrating, arranging, and purifying cells in a sample solution, and acting on the cells incorporated in the chip. It consists of means to make.
- the micro flow channel 102 is provided inside the chip substrate, and an opening communicating with the flow channel is provided on the upper surface to serve as a supply port for a sample and a necessary buffer solution (medium).
- the flow path can be created by so-called injection molding in which a plastic such as PMMA is poured into a mold, or can be created by bonding a plurality of glass substrates.
- the size of the chip is, for example, 50 ⁇ 70 ⁇ 1 mm (t), but is not limited thereto.
- the cells envisaged in the present invention are bacteria such as small ones and animal cells such as large ones, such as cancer cells, and these cell populations or cell masses. Therefore, the cell sample size is typically a wide range of about 0.5 ⁇ m to 500 ⁇ m, but is not strictly limited to this range, and cells of any size can be used as long as the present invention is effectively used. Can be used.
- the first problem is the flow channel width (cross-sectional shape).
- the channel 102 is formed on one of the surfaces of the chip 100 in a substantially two-dimensional plane in a space typically 10 to 500 ⁇ m in the thickness direction of the substrate.
- the appropriate size for bacteria is 5 to 10 ⁇ m in the thickness direction, and for animal cells and cell populations / cell masses, the appropriate size is 10 to 500 ⁇ m in the thickness direction.
- FIG. 5 shows a part of an image of a characteristic wavelength portion of an example in which absorption images for various monochromatic lights of various cells are actually obtained using the cell analyzer according to the embodiment of the present invention. It is a thing. In this case, it is an image of Giemsa-stained cells, but as can be seen from this photograph, the absorption characteristics in the cells at each absorption wavelength differ depending on the cell type (the staining characteristics differ).
- the present invention is particularly useful for separating and purifying a very small amount of target cells in blood using an index called cell absorption characteristics, and performing accurate gene information, expression information analysis, re-culture, and the like of the target cells.
- SYMBOLS 1 Cell analyzer 100 Cell sorter chip 101 Cell sample introduction path 102 Channel 103 External force generator 104 Direction of external force 105 Flow of recovered cells (1) 106 Flow of recovered cells (2) 107 Chip Substrate 108, 109 Branching Channel 200 Image Detection Type 1 Cell Separation / Purification Module 201 Laser 202 Mirror 203 Condensing Lens 204 Dichroic Mirror 205 Wavelength Filter 206 Photomultiplier 207 for Fluorescence Detection High Speed Camera 300 Recording / Control Unit 301 Cell 401 Slit 402 Photosensitive surface 403, 404, 405 of high-speed camera Cell image by each monochromatic light
Abstract
Description
1)目視による形態学的な細胞分類:例えば尿中に出現する異型細胞検査による膀胱がんや尿道のがんなどの検査や血中の異型細胞分類、組織中における細胞診によるがん検査などをあげることができる。
2)蛍光抗体法による細胞表面抗原(マーカー)染色による細胞分類:一般にCDマーカーと呼ばれる細胞表面抗原を、それに特異的な蛍光標識抗体で染色するもので、セルソーターによる細胞分離やフローサイトメーターや組織染色によるがん検査などに用いられている。もちろんこれらは、医療面のみならず、細胞生理研究用や、工業的な細胞利用の上でも多用されている。
3)あるいは、幹細胞の分離に関しては、細胞内に取り込まれる蛍光色素をレポーターとして幹細胞を含む細胞を大まかに分離し、更にその後で実際に培養を行うことで目的の幹細胞を分離する例がある。これは、幹細胞の有効なマーカーがまだ確立されていないので、実際に培養し、分化誘導したもののみを利用することで、実質的に目的細胞を分離しているのである。
4)また、細胞の中の微小器官、細胞内抗生物質の種類に応じて、その光の吸収特性、複屈折特性が異なることを利用して、これを画像化することで細胞内の状態を観察する技術として、位相差光学顕微鏡や微分干渉光学顕微鏡などが存在しているが、ともに同時に複数の波長の光を用いると色収差のために像がぼけるため単色光を用いて観察を行う。
5)あるいは、特定の細胞あるいは細胞内小器官が、特異的に染まる染色法、例えば、グラム染色では、クリスタルバイオレットやゲンチアナバイオレットで染色し、ヨウ素溶液で媒染した後、アルコールで脱色し、その後フクシンまたはサフラニンで対比染色を行うことで、グラム陽性菌は暗い青や青紫に染まり、グラム陰性菌は対比染色によって赤やピンクに染まるため、細胞が染色された色を識別することで、細菌のグラム陽性か陰性を判断することができる。また、ロマノフスキー染色では、還元したエオシンとメチレンブルー(時にその酸化物であるアズールAとアズールBを含む)の組み合わせで染色することによって、全て骨髄生検や骨髄穿刺液・末梢血液塗沫の検体を診るのに使われ、異なった種類の白血球を容易に区別できる。あるいは、パパニコロー染色では、核はヘマトキシリン、細胞質はオレンジG、エオジンY、ライトグリーンSF(分子量の小さい順)が、細胞質構造が緻密なものには分子量の小さい色素が入りやすく(例:角化型扁平上皮細胞:オレンジ)、疎なものには両方が入り込むが、分子量の大きい色素は移動性が少なく荷電が大きいので細胞内に吸着されやすく(例:非角化型扁平上皮細胞、腺細胞:ライトグリーン)、類脂質はビスマルクブラウンで染めることができ、悪性腫瘍細胞や感染症などを濃青緑色から暗赤色までの色のちがいで同定する方法である。ギムザ染色は、塩基性色素のメチレン青と、その酸化誘導体であるメチレンアズールとエオジンの酸性色素との混合液であるが、これらの色素は、末端のメチル基の違いや細胞内へ浸透する分子の影響によって染色性が異なり、比較的メチル基の少ないアズール・エオジネートは細胞質のみならず核内にも浸透し、核酸のリン酸基と結合し赤紫色調の核に染色される。他方、比較的分子の大きく極性の強いメチレン青は、細胞質のタンパク質と結合し青色調を呈することから、この染色の程度によって細胞の情報を得ることができる。ここで重要なことは、既存の染色による細胞同定法は、異なる複数の吸光特性を持った色素を組み合わせて、それらの色素の発色(吸収)の程度の相対的な組み合わせから視覚的にどのように異なる強度で各色成分が組み合わされるかで、どのような色に見えるかという視覚系の情報処理に依存した直観的でかつ可視スペクトル全体領域のスペクトルを対象とした計測法となっていることである。このことは、特定の1波長の蛍光を定量的に計測すればよい蛍光計測評価技術と最も異にする部分である。
[1]被験体由来の細胞試料液(a sample solution of cells from a subject)中の細胞を測定して、該細胞を選択的に分離・精製する細胞分析装置であって、
対象細胞を含む細胞を含有する細胞試料液を流す第1の流路と、該第1の流路内に細胞の吸収光スペクトル・イメージング像を検出するための細胞検出部と、上記対象細胞とそれ以外の細胞とを分離して選択的回収が行われる、第1の流路から分岐する少なくとも2本の分岐流路を含む細胞分離部とを含むセルソーターチップと、
上記第1の流路中を流れる上記細胞に、可視光領域スペクトルとなるように配置された複数の単色光光源からの光を照射する光照射手段と、少なくとも200フレーム/秒の画像取り込みレートで上記細胞の像を各単色光の像として取得する高速カメラであって、上記各単色光像を比較することで上記細胞の吸光特性を空間分解能をもって判別することができる高速カメラとを含む光学系と、
上記少なくとも2本の分岐流路のうちいずれかの流路に上記対象細胞を誘導するための外力を細胞に選択的に印加する機構と、
を備える、細胞分析装置。
[2]上記外力が、超音波放射圧、重力、静電力、または誘電電気泳動力である、上記[1]に記載の装置。
[3]上記細胞の上記各単波長について得られた画像を組み合わせて視覚的に観察される色を推定し、該細胞内での空間分布の結果に基づいて、各上記細胞を一細胞レベルで検出し、対象となる細胞を識別する、上記[1]または[2]に記載の装置。
[4]上記細胞の全体の光学的イメージング像を2値化し、該2値化画像の輝度重心、面積、周囲長、長径、および短径からなる群から選択される少なくとも1つの指標によって、各上記細胞を一細胞レベルで検出し、対象となる細胞を識別する、上記[1]~[3]のいずれかに記載の装置。
[5]上記細胞試料液中の上記細胞が蛍光標識されており、上記光学系が、蛍光検出器をさらに含み、上記細胞の蛍光画像の情報が追加的な指標として利用される、上記[4]に記載の装置。
[6]被験体由来の細胞試料液中の細胞を測定して、該細胞を検出する細胞分析装置であって、
対象細胞を含む細胞を含有する細胞試料液を流す流路と、該流路中に細胞の吸収光スペクトル・イメージング像を検出するための細胞検出部とを含む流路チップと、
上記流路中を流れる上記細胞に、可視光領域スペクトルとなるように配置された複数の単色光光源からの光を照射する光照射手段と、少なくとも200フレーム/秒の画像取り込みレートで該細胞の像を各単色光の像として取得する高速カメラであって、上記各単色光像を比較することで上記細胞の吸光特性を空間分解能をもって判別することができる高速カメラとを含む光学系と、
を備える、細胞分析装置。
[7]上記流路中を流れる上記細胞に可視光領域スペクトルとなるように配置された複数の単色光光源からの光を照射する光照射手段と、撮影カメラの受光面のサイズを取得したいスペクトル数分に分割するために得られた像を絞り込むためのスリットと、均等に画像スペクトルを分割することができる2個以上のダイクロイックミラーと光学フィルターの組と、少なくとも200フレーム/秒の画像取り込みレートで該細胞の像を各単色光の像として取得する高速カメラであって、上記各単色光像を比較することで上記細胞の吸光特性を空間分解能をもって判別することができる高速カメラとを含む光学系と、
を備える、上記[1]~[6]のいずれかに記載の細胞分析装置。
[8]被験体由来の細胞試料液中の細胞を測定して、該細胞を選択的に分離・精製する細胞分析方法であって、
対象細胞を含む細胞を含有する細胞試料液を流す第1の流路と、該第1の流路内に細胞の吸収光スペクトル・イメージング像を検出するための細胞検出部と、上記対象細胞とそれ以外の細胞とを分離して選択的回収が行われる、第1の流路から分岐する少なくとも2本の分岐流路を含む細胞分離部とを含むセルソーターチップの上記第1の流路に上記細胞試料液を導入する工程、
上記第1の流路中を流れる上記細胞に、複数の単色光光源からの光を照射する工程、
少なくとも200フレーム/秒の画像取り込みレートで上記細胞の像を各単色光の像として取得し得、上記各単色光像を比較することで上記細胞の吸光特性を空間分解能をもって判別することができる高速カメラを用いて、上記細胞の画像を撮像する工程、
上記撮像した画像を分析した結果に基づいて、上記少なくとも2本の分岐流路のうちいずれかの流路に上記対象細胞を誘導するための外力を細胞に選択的に印加する工程、および
上記いずれかの流路に選別された上記対象細胞を回収する工程、
を含む、方法。
[9]上記細胞試料液中の上記細胞が予め抗体または色素により標識されている、上記[8]に記載の方法。
[10]上記細胞試料液として、予め細胞の形状および/または染色により選別された細胞を含む細胞試料液を用いる、上記[8]または[9]に記載の方法。
[11]被験体由来の細胞試料液中の細胞を同定するための細胞分析装置であって、
波長280nmから780nmの光波長帯域をカバーする波長域内の連続する特定の波長帯の複数の単色光光源のアレイを含むパルス光光源であって、各単色光光源を制御プログラムによってパルス光として照射することが可能なパルス光光源と、
対象細胞を含む細胞を含有する細胞試料液を保持し、上記光源アレイの波長領域について光学的に透明な材料で構成された細胞保持部と、
上記単色光光源アレイの光波長帯域での前記細胞の画像取得ができる波長特性を有する画像取得カメラと、
上記取得した画像を記録するための記録部と、
上記パルス光光源の単色光の波長と取得画像のフレームの相関を関連づけて該画像を前記記録部に記録し、これを用いて上記各単色光像の画像を比較することで上記細胞の細胞内部の吸光特性を空間分解能をもって判別して記録する画像処理部と、
を備える、細胞分析装置。
[12]上記[11]記載の細胞分析装置において、上記光源が、上記画像取得カメラの1フレームの画像取得時間より短い時間でのパスル光を発光することができる単色光パスル光源のアレイを含む、上記[11]記載の細胞分析装置。
[13]上記画像取得カメラの1フレームの画像取得時間として毎秒1万画像を取得できる高速カメラと、発光時間が0.1ms未満の単色光LED光源のアレイを備える、上記[12]記載の細胞分析装置。
[14]上記[11]記載の細胞分析装置において、上記単色光光源アレイの2つ以上の単色光光源を同時にパルス発光させることで画像取得カメラでの取得画像が単色光光源の発光に応じた任意の2波長以上の波長での吸光像として取得することができるようにプログラムされている、上記[11]記載の細胞分析装置。
[15]上記[11]記載の細胞分析装置において、上記観察用光源波長帯域について光学的に透明な材料で構成された細胞保持部が、試料溶液を連続して流す機能を有するフローセルである、上記[11]記載の細胞分析装置。
[16]上記試料溶液を連続して流す機能を有するフローセルが、観察領域の下流に対象細胞を分離精製するための細胞分離・精製部を備える、上記[15]記載の細胞分析装置。
[17]上記制御プログラムにより、上記単色光源アレイ中の各単色光の照射順序を任意に設定できる、上記[11]記載の細胞分析装置。
[18]上記取得画像から得られた細胞像の中の核消失を指標にして対象細胞を判別する、上記[11]記載の細胞分析装置。
[19]上記取得画像から得られた細胞像の中の細胞サイズと核サイズの比を指標にして対象細胞を判別する、上記[11]記載の細胞分析装置。
[20]上記取得画像から得られた細胞像から細胞の集団化の程度を指標にして対象細胞を判別する、上記[11]記載の細胞分析装置。
図5は、実際に本発明の一実施形態に係る細胞分析装置を用いてさまざまな細胞のさまざまな単色光に対する吸光イメージを取得したものの一例について、特徴的な波長部分のイメージの一部を示したものである。この場合は、ギムザ染色した細胞についてのイメージであるが、この写真からもわかるように、細胞の種類の違いによって各吸収波長での細胞内での吸収特性が異なっており(染色の特性が異なっている)、このような取得画像を用いて、上でも述べたように(1)細胞内の核の有無あるいは細胞内の核の状態(M期)の区別、(2)細胞内の核のサイズの細胞サイズに対する割合、(3)特定の吸収波長での細胞内オルガネラの染色特性とその量(あるいは積算面積の細胞サイズに対する割合)、(4)細胞内での同一位置(同一オルガネラ)の複数の波長での吸収特性(吸収スペクトル)比較に基づいたオルガネラ種類と数の同定、(5)細胞のクラスター化の有無とクラスター中での各細胞の上記(1)(2)(3)の指標の特性等についての情報を得る事ができる。
100 セルソーターチップ
101 細胞サンプルの導入路
102 流路
103 外力発生器
104 外力の向き
105 回収細胞の流れ(1)
106 回収細胞の流れ(2)
107 チップ基板
108,109 分岐流路
200 画像検出型1細胞分離・精製モジュール
201 レーザー
202 ミラー
203 集光レンズ
204 ダイクロイックミラー
205 波長フィルター
206 蛍光検出用フォトマルチプライヤー
207 高速カメラ
300 記録・制御部
301 細胞
401 スリット
402 高速カメラの受光面
403、404、405 各単色光による細胞イメージ
Claims (20)
- 被験体由来の細胞試料液(a sample solution of cells derived from a subject)中の細胞を測定して、該細胞を選択的に分離・精製する細胞分析装置であって、
対象細胞を含む細胞を含有する細胞試料液を流す第1の流路と、該第1の流路内に細胞の吸収光スペクトル・イメージング像を検出するための細胞検出部と、前記対象細胞とそれ以外の細胞とを分離して選択的回収が行われる、第1の流路から分岐する少なくとも2本の分岐流路を含む細胞分離部とを含むセルソーターチップと、
前記第1の流路中を流れる前記細胞に、可視光領域スペクトルとなるように配置された複数の単色光光源からの光を照射する光照射手段と、少なくとも200フレーム/秒の画像取り込みレートで前記細胞の像を各単色光の像として取得する高速カメラであって、前記各単色光像を比較することで前記細胞の吸光特性を空間分解能をもって判別することができる高速カメラとを含む光学系と、
前記少なくとも2本の分岐流路のうちいずれかの流路に前記対象細胞を誘導するための外力を細胞に選択的に印加する機構と、
を備える、細胞分析装置。 - 前記外力が、超音波放射圧、重力、静電力、または誘電電気泳動力である、請求項1に記載の装置。
- 前記細胞の前記各単波長について得られた画像を組み合わせて視覚的に観察される色を推定し、該細胞内での空間分布の結果に基づいて、各前記細胞を一細胞レベルで検出し、対象となる細胞を識別する、請求項1または2に記載の装置。
- 前記細胞の全体の光学的イメージング像を2値化し、該2値化画像の輝度重心、面積、周囲長、長径、および短径からなる群から選択される少なくとも1つの指標によって、各前記細胞を一細胞レベルで検出し、対象となる細胞を識別する、請求項1~3のいずれかに記載の装置。
- 前記細胞試料液中の前記細胞が蛍光標識されており、前記光学系が、蛍光検出器をさらに含み、前記細胞の蛍光画像の情報が追加的な指標として利用される、請求項4に記載の装置。
- 被験体由来の細胞試料液中の細胞を測定して、該細胞を検出する細胞分析装置であって、
対象細胞を含む細胞を含有する細胞試料液を流す流路と、該流路中に細胞の吸収光スペクトル・イメージング像を検出するための細胞検出部とを含む流路チップと、
前記流路中を流れる前記細胞に、可視光領域スペクトルとなるように配置された複数の単色光光源からの光を照射する光照射手段と、少なくとも200フレーム/秒の画像取り込みレートで該細胞の像を各単色光の像として取得する高速カメラであって、前記各単色光像を比較することで前記細胞の吸光特性を空間分解能をもって判別することができる高速カメラとを含む光学系と、
を備える、細胞分析装置。 - 前記流路中を流れる前記細胞に可視光領域スペクトルとなるように配置された複数の単色光光源からの光を照射する光照射手段と、撮影カメラの受光面のサイズを取得したいスペクトル数分に分割するために得られた像を絞り込むためのスリットと、均等に画像スペクトルを分割することができる2個以上のダイクロイックミラーと光学フィルターの組と、少なくとも200フレーム/秒の画像取り込みレートで該細胞の像を各単色光の像として取得する高速カメラであって、前記各単色光像を比較することで前記細胞の吸光特性を空間分解能をもって判別することができる高速カメラとを含む光学系と、
を備える、請求項1~6のいずれかに記載の細胞分析装置。 - 被験体由来の細胞試料液中の細胞を測定して、該細胞を選択的に分離・精製する細胞分析方法であって、
対象細胞を含む細胞を含有する細胞試料液を流す第1の流路と、該第1の流路内に細胞の吸収光スペクトル・イメージング像を検出するための細胞検出部と、前記対象細胞とそれ以外の細胞とを分離して選択的回収が行われる、第1の流路から分岐する少なくとも2本の分岐流路を含む細胞分離部とを含むセルソーターチップの前記第1の流路に前記細胞試料液を導入する工程、
前記第1の流路中を流れる前記細胞に、複数の単色光光源からの光を照射する工程、
少なくとも200フレーム/秒の画像取り込みレートで前記細胞の像を各単色光の像として取得し得、前記各単色光像を比較することで前記細胞の吸光特性を空間分解能をもって判別することができる高速カメラを用いて、前記細胞の画像を撮像する工程、
前記撮像した画像を分析した結果に基づいて、前記少なくとも2本の分岐流路のうちいずれかの流路に前記対象細胞を誘導するための外力を細胞に選択的に印加する工程、および
前記いずれかの流路に選別された前記対象細胞を回収する工程、
を含む、方法。 - 前記細胞試料液中の前記細胞が予め抗体または色素により標識されている、請求項8に記載の方法。
- 前記細胞試料液として、予め細胞の形状および/または染色により選別された細胞を含む細胞試料液を用いる、請求項8または9に記載の方法。
- 被験体由来の細胞試料液中の細胞を同定するための細胞分析装置であって、
波長280nmから780nmの光波長帯域をカバーする波長域内の連続する特定の波長帯の複数の単色光光源のアレイを含むパルス光光源であって、各単色光光源を制御プログラムによってパルス光として照射することが可能なパルス光光源と、
対象細胞を含む細胞を含有する細胞試料液を保持し、前記光源アレイの波長領域について光学的に透明な材料で構成された細胞保持部と、
前記単色光光源アレイの光波長帯域での前記細胞の画像取得ができる波長特性を有する画像取得カメラと、
前記取得した画像を記録するための記録部と、
前記パルス光光源の単色光の波長と取得画像のフレームの相関を関連づけて該画像を前記記録部に記録し、これを用いて前記各単色光像の画像を比較することで前記細胞の細胞内部の吸光特性を空間分解能をもって判別して記録する画像処理部と、
を備える、細胞分析装置。 - 請求項11記載の細胞分析装置において、前記光源が、前記画像取得カメラの1フレームの画像取得時間より短い時間でのパスル光を発光することができる単色光パスル光源のアレイを含む、請求項11記載の細胞分析装置。
- 前記画像取得カメラの1フレームの画像取得時間として毎秒1万画像を取得できる高速カメラと、発光時間が0.1ms未満の単色光LED光源のアレイを備える、請求項12記載の細胞分析装置。
- 請求項11記載の細胞分析装置において、前記単色光光源アレイの2つ以上の単色光光源を同時にパルス発光させることで画像取得カメラでの取得画像が単色光光源の発光に応じた任意の2波長以上の波長での吸光像として取得することができるようにプログラムされている、請求項11記載の細胞分析装置。
- 請求項11記載の細胞分析装置において、前記観察用光源波長帯域について光学的に透明な材料で構成された細胞保持部が、試料溶液を連続して流す機能を有するフローセルである、請求項11記載の細胞分析装置。
- 前記試料溶液を連続して流す機能を有するフローセルが、観察領域の下流に対象細胞を分離精製するための細胞分離・精製部を備える、請求項15記載の細胞分析装置。
- 前記制御プログラムにより、前記単色光源アレイ中の各単色光の照射順序を任意に設定できる、請求項11記載の細胞分析装置。
- 前記取得画像から得られた細胞像の中の核消失を指標にして対象細胞を判別する、請求項11記載の細胞分析装置。
- 前記取得画像から得られた細胞像の中の細胞サイズと核サイズの比を指標にして対象細胞を判別する、請求項11記載の細胞分析装置。
- 前記取得画像から得られた細胞像から細胞の集団化の程度を指標にして対象細胞を判別する、請求項11記載の細胞分析装置。
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