WO2007040117A1

WO2007040117A1 - Method of simultaneously measuring a plural number of cell responses

Info

Publication number: WO2007040117A1
Application number: PCT/JP2006/319156
Authority: WO
Inventors: Ritsu Honda; Hiroyuki Kishi; Atsushi Muraguchi; Eiichi Kanaumi; Kihachiro Tohbo
Original assignee: National University Corporation University Of Toyama; Ns Materials Inc.; Toyama New Industry Organization
Priority date: 2005-09-30
Filing date: 2006-09-27
Publication date: 2007-04-12
Also published as: JP4089916B2; JP2007097411A

Abstract

[PROBLEMS] To provide a method whereby the states of a large number of cells held on a chip (for example, the reactivities of lymphocytes to an antigen stimulation) can be simultaneously measured and the states of the individual cells can be understood. [MEANS FOR SOLVING PROBLEMS] A method of measuring cell states which comprises independently holding a plural number of cells at a plural number of positions on a cell chip, detecting the fluorescence from at least a part of the a plural number of positions by using an image sensor, recording the intensities of the fluorescence thus detected for each of the positions, and indicating the fluorescent intensities or data converted from the fluorescent intensities thus recorded. At least the detection and recording as described above are repeated with the passage of time.

Description

Specification

Simultaneous measurement of multicellular responses

Technical field

[0001] The present invention relates to a multi-cell response simultaneous measurement method using an image sensor such as a CCD image scanner. The present invention is a method that allows the fluorescence emitted by a large number of cells to be tracked independently for each cell over time and in a massively parallel manner.

Background art

[0002] Attempts have been made to identify and sort cells at each level and use the sorted cells. For example, the antigen specificity of each lymphocyte is detected individually, and then the detected antigen-specific lymphocyte is recovered, and the recovered antigen-specific lymphocyte is used, for example, antibody Has been studied (Japanese Unexamined Patent Publication No. 2004-173681 and Japanese Unexamined Patent Publication No. 2004 187676).

Patent Document 1: Japanese Unexamined Patent Application Publication No. 2004-173681

Patent Document 2: Japanese Patent Application Laid-Open No. 2004-187676

Disclosure of the invention

Problems to be solved by the invention

[0003] Conventionally, the antigen specificity of each lymphocyte was individually detected by a manual, and the detected antigen-specific lymphocytes were collected. Patent Documents 1 and 2 describe that it has been confirmed that cells are identified on a cell-by-cell basis, and describe that the identified cells can also be collected. However, it was not easy to detect many lymphocyte forces from lymphocytes that actually reacted specifically to the antigen.

[0004] As a detection method, for example, an increase in calcium ion concentration in lymphocytes in response to an antigen is used, and a change in the calcium ion concentration is detected as fluorescence to identify an antigen-specific lymphocyte. However, depending on the type of cells (lymphocytes), the manner in which fluorescence is generated and the intensity, etc. are not always constant. In some cells, the calcium ion concentration increases after a certain period of time, resulting in an increase in fluorescence intensity. Ma In addition, it is necessary to measure the fluorescence intensity of about 1 to 200,000 lymphocytes held on the surface of a chip about several centimeters square at the same time, and the fluorescence is fluorescence caused by each cell. For this reason, it is also necessary to perform fluorescence detection with high sensitivity and low fluorescence intensity.

[0005] However, so far, there has been no method and apparatus that can simultaneously measure a large number of weak fluorescence intensities on the surface of such highly integrated chips.

[0006] As a conventional apparatus, there is a laser scanning 'cytometer, and when a laser single-scanning' cytometer is used, intracellular calcium concentration can be individually measured for several hundreds of thousands of cells. However, the detection of lymphocytes whose fluorescence intensity changes within a few minutes after an original stimulus, which takes 10 minutes or more for a single scan, lacks real-time properties and is unsuitable.

[0007] The fluorescence microscope 'imaging device has an imaging device in addition to a normal fluorescence microscope.

Fluorescence microscope · The imaging device has no problem in terms of speed, but with an ordinary microscope, the imaging range is narrow, and up to 1000 cells cannot be detected at a time, and tens of thousands to hundreds of thousands of cells cannot be detected. It was impossible to detect strong fluorescence at once.

[0008] A cell chip detection device based on a DNA microarray scanner has been developed to detect cell fluorescence arrayed on a cell chip, and can detect intracellular or extracellular fluorescence. However, since this system uses a laser to excite the fluorescent dye and detect the excitation light, a detection system that tracks changes over time cannot simultaneously detect a large number of cell regions with a slow line scan speed.

[0009] In the method described in Patent Documents 1 and 2 described above, in order to detect a cell that specifically reacts with an antigen, in the reaction detection system, Because of the real-time nature that can capture changes in force over time and the need to detect very few (reactive) cells in a large number of cells, parallelism that can analyze many cells individually and simultaneously , Is required.

[0010] Therefore, the object of the present invention is to simultaneously measure the state of a large number of cells retained on the chip, more than 10,000, preferably more than 100,000, for example, lymphocyte reactivity to antigen stimulation, The object is to provide a method for individually grasping the state of each cell.

Means for solving the problem The present invention is as follows.

[1] Fluorescence having at least a part of the position of the plurality of positions on a cell chip that holds a plurality of cells independently at a plurality of positions is detected by an image sensor,

Record the detected fluorescence intensity for each position,

Including displaying the recorded fluorescence intensity or the converted value from the fluorescence intensity

A cell state measurement method,

A method wherein at least the detection and recording are repeated over time.

[2] The method according to [1], wherein the plurality of cells are lymphocytes, and the cell state is a state of lymphocytes before and after stimulation with an antigen.

[3] The method according to [2], wherein the state of the lymphocyte is measured by a change in fluorescence intensity of an increase in intracellular calcium caused by a signal via a receptor possessed by the lymphocyte.

[4] The method according to claim 3, wherein the lymphocyte is a B lymphocyte and the receptor is a B lymphocyte receptor.

[5] The method according to any one of [1] to [4], wherein the plurality of cell forces are at least 10,000 cells.

[6] The image sensor is a CCD image scanner or a CMOS image scanner

The method according to any one of [I] to [5].

[7] The method according to any one of [1] to [6], wherein the fluorescence is detected by the image sensor at least once every 60 seconds.

[8] The method according to any one of [3] to [7], wherein the converted value of fluorescence intensity is calcium concentration.

[9] The method according to any one of [1] to [8], wherein the recorded fluorescence intensity or the converted value of fluorescence intensity force is displayed over time in parallel with detection and recording.

[10] The cell chip that holds a plurality of cells independently at a plurality of positions has a spot or a hole at a position for holding the cell on one surface of the substrate, and at least the spot or the hole The method according to any one of [1] to [9], wherein some cells are independently retained.

[II] An address is attached to the plurality of positions, and the firefly detected in association with the address The method according to any one of [1] to [10], wherein recording of light intensity is performed.

The invention's effect

[0012] According to the method of the present invention, a stimulus response that an antigen exerts on lymphocytes can be rapidly detected on a large scale and quantitatively for individual cells. As a result, it becomes easier to produce an antibody having a high affinity for a specific antigen.

[0013] Furthermore, it is known that lymphocytes of patients with B cell leukemia (leukemia) have a reduced calcium response to stimulation by anti-IgM antibodies. In clinical settings, it is possible to quickly and quantitatively measure and evaluate the responsiveness of B cells to anti-IgM antibodies by stimulating lymphocytes derived from patients with anti-IgM antibodies over time. If possible, the power of being able to diagnose Beel 1 Leukemia (leukemia) The method of the present invention can also be applied in this way, thereby enabling rapid diagnosis of leukemia, life expectancy tests, and the like. . As a result, the necessary treatment can be performed accurately, and it can be used effectively in clinical examinations.

BEST MODE FOR CARRYING OUT THE INVENTION

[0014] The method of the present invention comprises:

(1) Fluorescence of at least a part of the position force of the plurality of positions on the cell chip holding a plurality of cells independently at a plurality of positions is detected by an image sensor (fluorescence detection step),

(2) Record the detected fluorescence intensity for each position (fluorescence intensity recording step),

(3) A method of measuring a cell state including displaying (recording) fluorescence intensity or a converted value from fluorescence intensity (display step). Then, at least the detection and recording are repeated over time.

[0015] (Fluorescence detection step)

The measurement target in the method of the present invention is a cell chip that holds a plurality of cells independently at a plurality of positions. If the cell chip is to hold a plurality of cells independently at a plurality of positions, the method of holding the cell, the number of held cells, the density of the held cells on the chip surface, etc. are particularly limited. There is no. As an example of the cell chip, for example, a plurality of microchips for storing cells on one surface of the substrate described in Patent Documents 1 and 2 above. A microwell array chip having a clowell may contain one cell (independently) in each microwell. In this cell chip example, a plurality of microwells (holes) correspond to the “plural positions”. In the microwell in which the cells are stored, the culture solution can be stored together with the cells.

[0016] Alternatively, the cell chip may be one in which cells are individually fixed on the chip surface using a thermosensitive polymer described in JP-A-2005-102628. In this cell chip example, the fixed positions of a plurality of cells on the chip surface correspond to the “plural positions”.

[0017] The cell chip can be held in a plurality of 1S clusters that can hold cells uniformly (at equal intervals) throughout. For example, if one cluster is a group of 10 to 1000 cells X 10 to 1000 cells (positions), a plurality of clusters, for example, n cells x m (n and m are independently 2 to 50, for example) ) On the substrate.

[0018] The number of cells held on the cell chip is not particularly limited. However, since the object of the present invention is to simultaneously measure the state of a large number of cells, for example, at least 10,000 cells. Yes, preferably 50,000 or more, more preferably 100,000 or more, and even more preferably 200,000 or more. In particular, from the viewpoint of searching for lymphocytes that are antigen-specific and low in frequency, the number of cells retained on the cell chip is preferably at least 200,000. The condition for the number of cells held on the cell chip can be determined appropriately in consideration of the purpose of measurement and the sensitivity of the image sensor, which is a measuring instrument, and the resolution required for fluorescence detection. For example, about 1 million cells And preferably 500,000. However, the upper limit of the number of cells retained on the cell chip can be further increased if the capabilities of the image sensor and the equipment required for recording processing of the detected fluorescence intensity improve as the technology advances. It is a spear.

[0019] The cells retained in the cell chip are not particularly limited! /, But examples include lymphocytes, and examples of lymphocytes include B lymphocytes and T lymphocytes. When lymphocytes are used as cells in the method of the present invention, the state before and after stimulation with an antigen for lymphocytes can be measured. More specifically, measurement of the state before and after stimulation with antigen is based on intracellular calcium generated by a signal through a receptor possessed by lymphocytes. Can be measured by a change in fluorescence intensity using a Ca ion-dependent fluorescent dye. More specifically, for example, an increase in intracellular calcium caused by a signal through the B cell receptor can be measured by a change in fluorescence intensity. The same applies to T cell receptors.

[0020] Fluorescence from at least some of the plurality of positions is detected by an image sensor. Fluorescence from the position where the cell is fixed is generated when the cell reacts with the antigen by adding a Ca ion-dependent fluorescent dye to the fixed cell in advance. When an antigen binds to the antigen receptor (immunoglobulin) of B lymphocytes, intracellular signal transduction occurs first, and as a result, the concentration of intracellular Ca ions changes, and when Ca ion-dependent fluorescent dyes coexist, Fluorescence is generated. As the fluorescent dye, for example, Fura-2, Fluo_3 or Fluo-4 can be used. However, the fluorescent dye is not particularly limited as long as it is a Ca ion-dependent fluorescent dye. In addition, the fluorescence may be a deviation of intracellular fluorescence, fluorescence on the cell membrane surface, or fluorescence emitted from a cell! ,.

[0021] In the method of the present invention, for example, when detecting an antigen-specific cell that is very rarely present from a large number of cells, a cell chip containing 200,000 cells is placed in the measurement range. In addition, use an image scanner that has sufficient sensitivity to fully examine cell responses.

[0022] This device 1) is a cell derived from a biological host regularly arranged on a microwell array chip !, or puts all the intracellular or extracellular fluorescence of a subcultured cell line into the field of view, 2) By allowing time-lapse photography, the reaction of cells after drug loading can be performed for each of several hundreds of thousands of cells. This can be analyzed.

[0023] In order to detect cells that react with an antigen, the resolving power of the image sensor does not necessarily have to be remarkably high, and may be appropriate. Usually, to obtain high resolution, it is possible to use a CMOS type image scanner that is slightly inferior in resolution if it has the ability to detect fluorescence of cells that use CCD type image scanners.

Rather, it is appropriate that the design is such that a stimulus response in the detection range can be detected in real time for each individual cell. In such a case, the image scanner is, for example, It is appropriate that the imaging area has a resolution of 3 million pixels or more and has a function of capturing fluorescence per cell (position) by 4 pixels or more of the imaging element. The number of pixels required for the image scanner is determined appropriately by the product of the number of pixels that capture fluorescence per cell (position) and the number of cells (positions) that include the imaging area. Is done.

In the method of the present invention, a fluorescence measuring device including the image sensor can be used. An explanatory diagram of this fluorescence measuring apparatus is shown in FIG. It consists of an image sensor 10, a split filter 20, a telecentric optical system 30, a light source 40, a shutter 50, and a computer 60 that controls them.

The image sensor 10 receives fluorescence emitted from the cell chip 70 and converts it into digital data. In addition, the image sensor 10 has a sufficient number of pixels necessary for data acquisition, and it is appropriate that the image sensor 10 has a function of photographing fluorescence per sphere with four or more pixels of the image sensor. 9 pixels or more is desirable to stabilize From this, the number of lymphocytes that can be handled by the device is determined by the total number of image sensors.

[0027] The split filter 20 reflects and transmits the excitation light wavelength selected from the light emitted from the light source. In addition, it has a function to select and pass the fluorescence wavelength emitted from the sample.

The telecentric optical system 30 has a function of efficiently irradiating the sample with excitation light and efficiently condensing the fluorescent light emitted from the sample onto the image sensor 10. Usually, when observing under a microscope, there is a phenomenon that the peripheral part of the field of view is abnormally distorted. This is called distortion, but in this scanner, it is preferable to use a telecentric optical system (lens system) as the optical system in order to minimize the distortion of the peripheral area. By using a telecentric optical system (lens system), distortion at the periphery is minimized.

[0029] The light source 40 has a function of generating light necessary for excitation with a sufficient output.

The shutter 50 has a function of irradiating the cell chip 70 with excitation light only when necessary in order to minimize damage to the cell chip 70, and is controlled by the computer 60.

The computer 60 has a function of controlling the image sensor 10 and the shutter 50, and has a function of acquiring, analyzing, and storing fluorescence data output from the image sensor 10. It also monitors and controls the entire device. [0031] The cycle of fluorescence detection by the image sensor can be appropriately determined according to the state of the cell to be measured. For example, if the cell is a lymphocyte and the cell condition is the lymphocyte's reactivity to antigen stimulation, fluorescence detection by the image sensor should be performed at least once every 60 seconds, preferably every 30 seconds. Is appropriate. The fluorescence detection cycle is limited by the data processing capability of the device used for fluorescence detection and recording. With the fluorescence measurement device shown in Fig. 1, fluorescence detection and recording is performed once every 10 seconds. Is possible. However, the shortest cycle time can be further shortened by a fluorescence measuring device. One fluorescence detection and recording (1 cycle) includes a series of processes including all fluorescence detection, including fluorescence detection, transfer of image data to a computer, and image correction on a personal computer. .

[0032] (fluorescence intensity recording step),

The fluorescence detected by the image sensor 10 is digitized by the image sensor 10 and recorded in the computer 60. Data digitized by the image sensor 10 is digitized in proportion to the amount of fluorescence emitted. The data that can be stopped here is the relative fluorescence.

[0033] The image sensor 10 includes a plurality of light receiving units, and each light receiving unit has a respective light receiving characteristic. In order to treat each characteristic as a uniform characteristic, a correction map for the light intensity of each light receiving unit is created to correct the input light quantity and obtain an accurate fluorescence amount. enable. Fluorescence data digitized by the image sensor is captured by a plurality of pixels for one lymphocyte. By providing an addition processing method that handles the total amount of fluorescence data emitted by one lymphocyte, a method that handles the average value, and a method that handles the maximum value, various patterns can be analyzed.

[0034] The converted value of the fluorescence intensity can be a calcium concentration. In this case, by generating table data for converting the fluorescence emission intensity into the calcium concentration, the fluorescence emission amount can be converted into the calcium concentration and the calcium concentration can be quantitatively measured.

The above fluorescence detection step and fluorescence intensity recording step are repeated over time, and data is accumulated in the computer ₆₀ . The converted value from the fluorescence intensity is also stored in the computer 60 in the same manner. [0036] (Display step)

Display of recorded fluorescence intensity or converted value of fluorescence intensity force may be performed at the appropriate time after being stored over time in parallel with the above detection and recording, or after being temporarily stored in the computer. You can also Each position where the cell is held is assigned an address, and the fluorescence intensity detected in association with the address is recorded. Therefore, the acquired fluorescence intensity or calcium concentration can be displayed on a computer monitor in real time for each location (cell). The addresses can be numbered in the direction of the entire raster, the number of the cluster, and the lymphocytes in the cluster.

[0037] In the method of the present invention, the fluorescence intensity and the calcium concentration can be displayed in time series. It is also possible to display data of only lymphocytes selected arbitrarily. In addition, fluorescence intensity and calcium concentration can be compared individually for the time acquired. The change in overall brightness can be confirmed at a glance. In addition, any range of data can be selected and displayed on the graph. The computer display in Fig. 1 shows the changes over time in the amount of fluorescence reflecting the intracellular calcium concentration in individual cells.

Example

Hereinafter, the present invention will be described in more detail with reference to examples.

[0039] When evaluating the mobilization of intracellular calcium caused by an anti-IgM antibody or antigen to B cells, it is carried out as follows.

[0040] Cell preparation

For human subjects, lymphocyte fractions are collected from peripheral blood B lymphocytes using a known method (density gradient centrifugation). The B lymphocyte fraction is preferably collected using a commercially available kit. The collected B lymphocytes should be preliminarily introduced with a calcium indicator that increases or decreases the amount of fluorescence when excited by binding to calcium. The amount of fluorescent dye introduced is controlled by the amount of fluorescent dye in the liquid in which the cells are reacted, and is preferably between 0.1 micromole / liter and a concentration force of 5 micromole Z liter. The action time is preferably 15 minutes or 60 minutes to maintain cell activity. For temperature, room temperature Or it is desirable to do it at the temperature of body temperature.

[0041] When whole blood derived from mammals such as mice is used, the procedure is similar to that for humans. When cells derived from the mouse spleen are used, lymphocytes are isolated from the spleen after splenectomy, and isotonic solution After washing well, suspend in a culture solution such as RPMI1640 containing 10% FCS or a buffer solution that retains cell function.

Samples shall be arrayed on cell chips.

[0042] Cell chip conditions for arraying

Cell chips are mainly 10x5 clusters (45000 pieces: 900 quenore per cluster) as experimental chips! / ヽ i¾15xl 5 (20¾ "2500) {@

[0043] As the substrate of the cell chip, it is necessary to use a silicon chip that has been surface-treated, or polyethylene glycolanol 2000 (PEG2000) and polyethyleneglycolole 6000 (PEG6000) in a ratio of about 2 to 1. Mix, dissolve in pure water so that it is about 5% of the total, soak in it for a while, and use ultrasonically wash in water, buffer solution, or cell culture medium before use. Use a surface that does not cause excessive adsorption of cells on the surface of the cell chip.

[0044] Coating that makes it difficult for cells on the surface of the cell chip to be adsorbed is also possible by the above-mentioned other methods. When lipid membrane coating agent called lipidid is used, in the case of ethanol solvent, use a commercially available 10X or 50X solution and apply it to the array area on the cell chip. If this happens, the subsequent operation may be affected, so the ethanol may be washed twice or three times with more ethanol than the amount applied before it dries. This removes excess coating agent and prevents it from peeling off during measurement or adversely affecting cells. When using aqueous type lipidids, in a solution diluted 10 to 50 times, vibrate evenly inside the well on the cell chip in an ultrasonic cleaner. After leaving it for a while, clean it with water again in water to remove the excess coating agent.

[0045] When using the coating agent for cell chips as described above, it is preferable to remove as much as possible the excess of the coating agent that may peel off during use.

[0046] Alternatively, use a Teflon or silicon coating that has been industrially made. Therefore, it is possible to prevent strong adsorption of cells.

[0047] When the cells were arrayed on the cell chip, the cell suspension obtained by suspending the cells in a culture solution containing 10% FCS or a serum-free medium having a function of maintaining the cell function was replaced with ethanol. Fills a buffer with the same composition as the cell suspension with the vacuum expelling the air in the well on the cell chip, and gently places a sufficient amount of the cell suspension on it.

[0048] After 1 to 2 minutes, the cells have settled and a thin white layer of cells is visible outside the cell tip. With a P200 or P20 micropipette, Stir the cells that have settled on the chip surface with a soft discharge force that prevents the cells from going outside. In the case of a raw silicon surface without the above-mentioned coating agent, the efficiency of stirring at this time is significantly reduced, and the cells on the surface may not be sufficiently stirred. By repeating this operation 2-3 times, the cell array ratio of the cell chip can be dramatically increased.

[0049] A rubber spacer is placed on the cell chip after the array work, and a thin glass (such as a cover glass) cut to an appropriate size is placed on the spacer. The cover glass and the surface of the cell chip are filled with the same culture solution as the cell suspension or a buffer with a composition having an appropriate function of maintaining cell responsiveness.

[0050] Alternatively, a rubber or other glass, plastic or metal spacer is placed directly on a glass, plastic or metal base to which the cell chip is fixed, and a bridge is formed on the spacer. Install the cover glass so that Hereinafter, the cell arrayed on the cell chip is referred to as a prep.

[0051] The light source of the scanner is activated and the warm-up operation is performed until the temperature inside the casing reaches equilibrium. By performing the warm-up operation, it is possible to perform highly accurate measurement while suppressing fluctuations in the amount of excitation light.

[0052] The prep is placed on the moving stage of the array scanner, and set so that the optical axis and the cell chip surface are vertical. By making the optical axis and the cell chip surface perpendicular to each other and preventing tilting, accurate measurement is possible.

[0053] The dedicated data capture software is started up on the computer for analysis that has been started up. Under the red LED ring illumination light, data is collected for the part to be measured on the prep. Adjust the XY stage under the scanner receiver so that it is within the field of view of the insertion software, and focus the optical system on the chip surface. At this time, the ΧΥ stage does not drift easily, and precise position adjustment is possible.

[0054] Fluorescence shooting

Cell fluorescence is photographed on a computer screen.

First, in order to specify the cell position, exposure is usually performed for 100 ms under red ring LED illumination, and a well position detection image is acquired. In the position detection image, if the cell area of the prep is narrow, other parts may interfere with detection. Therefore, the cell area must be specified by computer mouse input (click and drag), and the execution button must be clicked. Start with.

For recognition, the position detection image is digitally processed, noise is removed, enhancement processing and rotation correction are performed, and the positions of the wells are arranged so as to form individual grids. The pixel of each image sensor constituting each cell is 2x2 or more, preferably 4x4 pixels or more. This cell is large enough to contain a single cell, and is used for detection as if the cell is in this position. A desirable example that fits within the field of view of an image sensor in the case of a 15x15 cluster prep would be a 4x4 pixel comprising a single cell area.

[0056] These images showing cell regions created from the position detection images are called fluorescence detection matrixes. The creation of the fluorescence detection matrix is performed fully automatically by the automatic image processing process on the computer as shown in FIG. 2 after the position detection image is collected. In order to detect the fluorescence of cells, we automatically create a matrix for fluorescence detection that corresponds one-to-one with the number of cells as many as the number of cells using our digital image processing technology.

[0057] The fluorescence value data recorded on the memory is recorded on a hard disk of a personal computer as a text file or binary data. The recorded data is a general-purpose format and can be read using a commercially available program. The fluorescence value data stored in the memory can be used for calculation without any special operation, and analysis can be performed at high speed.

[0058] Fluorescence of cells after stimulation Cell stimulation is performed by temporarily stopping imaging. Open the shading door and remove the semi-fixed prep on the imaging table, or pull out the imaging table without removing it, making it accessible for the experimenter to absorb water between the cell area and the cover glass. Bring in a neutral paper and absorb the cell culture medium or buffer. Or mechanically slowly suck and remove

[0059] Before the very small amount of the remaining cell culture solution or buffer solution is dried, gently remove the same cell culture solution or buffer solution that contains an antigen, a cell stimulating substance similar to the antigen, or a chemical substance. Place on cells. Be careful not to mix any bubbles.

[0060] Immediately after placing the cell culture solution containing the stimulating substance, return the prep to the imaging table, or return the drawn imaging table to its original position, close the light shielding door, and then Start.

[0061] When shooting is started, the program starts shooting immediately, and is released from a calcium indicator combined with intracellular free calcium excited under the excitation light of a mercury lamp for a limited time before and after each shooting cycle. Take the captured fluorescence with an image sensor.

[0062] The fluorescence imaging is performed every 10 seconds, and the image acquisition and analysis process is completed by the time the next imaging sequence starts. Even if image processing is prolonged, shooting, image data transfer by high-speed serial bus, and image analysis processing are processed in separate processes, and after the data transfer, the next shooting process is not affected.

[0063] Thereafter, the fluorescence image including the fluorescence of the photographed cell is compared with the fluorescence detection matrix as electronic information, and the information of the pixels in the target cell region is integrated, or the cell region (16 pieces) is integrated. It is recorded as the maximum value or average value or median value of the pixels. Images taken over time are processed before the next shooting, and the output numerical values are recorded on a memory or hard disk as electronic information in time series.

[0064] Cell fluorescence analysis work

Cell fluorescence analysis mainly uses at least one, preferably three of the following three filters (see Fig. 4). Figure 4-a shows the distribution of cell fluorescence using a histogram filter and selects a uniform cell population. Fig. 4-b is a scatter plot filter, showing the distribution of the fluorescence values of cells at two time points before and after, and the cells with changed fluorescence values can be detected at positions away from the population. Figure 4- c is a time-series filter, which shows a change with time for each cell.

[0065] The histogram filter shown in Fig. 4a is mainly used to separate cell fluorescence from background fluorescence. The fluorescence of cells before stimulation also varies depending on intracellular calcium. A very well-prepared prep is divided into a group of empty wells that contain nothing and a group of cells that contain fluorescence. Cells that appear scattered at positions with higher fluorescence values than this population are generally activated cells from the beginning, and are inappropriate as the initial cell state. The histogram 'filter is very useful for extracting a group of cells to be analyzed and facilitating the subsequent sorting operation.

[0066] The scatter diagram filter shown in Fig. 4b displays on the XY axis the fluorescence value at one time point before stimulation and the fluorescence value at an arbitrary time point after stimulation. When the time point displayed on the Y-axis is changed along the time series, the change in intracellular calcium concentration of cells activated in response to antigens or stimulating substances in the cell group selected on the histogram In response, it can be recognized as a separated cell population compared to the cell population. This cell group is set as an area connecting arbitrary points on the screen of the personal computer, and only the information on the cells contained in the enclosed area is selected. The screen can be enlarged or reduced to select a more accurate group.

[0067] The time-series filter shown in Fig. 4c shows a time-series fluorescence change on the horizontal axis for each individual cell, and among them, a cell having a desired intracellular calcium mobilization pattern can be selected. It is a filter that can. By adding analysis to the cells selected with other filters, it is possible to operate more efficiently.

[0068] By using these filters properly, they can be used for various purposes. Furthermore, by using a plurality of filters, a more detailed evaluation can be performed on the cells selected by the previous filter.

[0069] As the fluorescence data used for these three types of filters, RATIO indicating the degree of response of the cell, which is not just the absolute value data of fluorescence, or a primary differential value for sensitively detecting fluorescence changes, or the like is used. This makes it possible to examine qualitative changes in intracellular calcium, and to distinguish calcium mobilization caused by a specific antigen from calcium fluctuations as part of normal cellular activity.

[0070] The fluorescence information selected by the narrowing operation of the fluorescence information includes the fluorescence information. The position information on the obtained image is attached. Therefore, based on this information, by referring to the cluster address and the address of the well in the prep cell area, cells can be collected by a micromanipulator, or the address can be passed automatically. This makes it possible to collect cells with an automatic cell collection device.

FIG. 3 shows an example of actual cell fluorescence acquisition. As shown in Fig. 3, the calcium response of each cell can be analyzed with parallelism of 200,000 or more simultaneous processes. Stimulation is an anti-IgM antibody that stimulates the B cell receptor itself. (Displayed! /, The data for 250 samples randomly selected to improve visibility: Create images in Excel) 1% of mouse splenocytes that actually react with antigen were mixed When antigen stimulation was performed on a prep of a cell group (0.3-0.7% as reactive B cells), a clear cellular response that was considered to be antigen-specific was observed (bottom).

[0072] These series of operations can be performed very quickly, and the creation of calcium mobilization profiles over 200,000 individual cells over time, which could not be done with the prior art, can be done within minutes. It is a very useful method.

[0073] This method is not limited to antibody production techniques, and it is possible to examine more detailed cell profiles by measuring cell calcium mobilization on a prep and then staining the cell membrane antigen on the chip. . Using this technology, it can be expected to be used in fields such as clinical diagnosis.

Industrial applicability

[0074] The present invention is very useful in the case where it is necessary to grasp the state of a large number of cells, particularly lymphocytes at once, and is useful for preparing antigen-specific antibodies and diagnosing diseases involving lymphocytes, etc. Useful.

Brief Description of Drawings

FIG. 1 is an explanatory diagram of a fluorescence detection apparatus.

FIG. 2 is an explanatory diagram of the operation of the automatic recognition function of the CCD scanner shown in FIG.

[Figure 3] Example of actual cell fluorescence acquisition.

FIG. 4 is an explanatory diagram of three filters used for cell fluorescence analysis.

Claims

The scope of the claims

[I] Fluorescence from at least a part of the plurality of positions on a cell chip holding a plurality of cells independently at a plurality of positions is detected by an image sensor,

Record the detected fluorescence intensity for each position,

A cell state measurement method,

A method wherein at least the detection and recording are repeated over time.

[2] The method according to [1], wherein the cells are a plurality of cell lymphocytes, and the cell state is a state of lymphocytes before and after stimulation with an antigen.

[3] The method according to claim 2, wherein the state of the lymphocyte is measured by a change in fluorescence intensity of an increase in intracellular calcium caused by a signal through a receptor possessed by the lymphocyte.

[5] The method according to any one of claims 1 to 4, wherein the plurality of cells are at least 10,000 cells.

[6] The method according to any one of claims 1 to 5, wherein the image sensor is a CCD image scanner or a CMOS image scanner.

[8] The method according to any one of claims 3 to 7, wherein the converted value from the fluorescence intensity is a calcium concentration.

[9] The method according to any one of [1] to [8], wherein the display of the recorded fluorescence intensity or the converted value of the fluorescence intensity force is performed over time in parallel with the detection and recording.

[10] A cell chip that holds a plurality of cells independently at a plurality of positions has a spot or a hole at a position for holding the cell on one surface of the substrate, and at least the spot or the hole The method according to any one of claims 1 to 9, wherein a part of the cells is independently retained.

[II] Addresses are assigned to the plurality of positions, and fluorescence intensity detected in association with the addresses is detected. The method according to any one of claims 1 to: LO, wherein the degree of recording is performed.