JP5745752B2 - Cell screening method, control software used in the method, cell screening device, image analysis device, and cell screening system - Google Patents

Description

  The present invention relates to a cell screening method, a cell screening method, control software used in the method, a cell screening device, an image analysis device, and a cell screening system.

  In recent years, scanning cells in each container arranged in a cell array plate such as a multiwell plate, imaging light such as fluorescence emitted from the surface or inside of the cell, analyzing the image of the captured cell, Cell screening devices that output analysis results have been used.

  For example, in Patent Document 1, in the HTS (high-throughput screening) mode, the entire region of the well to be scanned is screened, the features of the well are extracted from the entire well, The extraction result is compared with a predetermined threshold or intensity response to determine “hit” or “non-target”, store the position of the well determined as “hit”, and then HCS (High Content Screening: High Content A technique for measuring and analyzing the well in detail in the Screening mode is described.

  In addition, regarding cancer clinical tests using cell screening, especially colorectal cancer clinical tests, for example, Patent Document 2 discloses collecting colon cancer cells from a stool sample and fixing them in a cell observation container. Describes a method of immunostaining using a detection antibody that can specifically bind to the lysine, and a technique for finding colorectal cancer cells by acquiring and analyzing microscopic cell images after these steps using a test kit. Yes. In recent years, the invention of this method and test kit has made it possible to extract only desired cancer cells from a specimen, so that the combination of microscopic cell image acquisition technology and image analysis technology is about to be applied to clinical tests. Yes.

Japanese Patent No. 3466568 Japanese Patent Application No. 2008-33274

  The cell screening apparatus described in Patent Document 1 is obtained by performing a low resolution (low magnification) screening to determine whether a measurement target well is a “hit” well or not “many or all cells in each well. Alternatively, the “total signal output of a large amount of substance” is averaged, and the average value is used as a threshold value.

However, in the case of a clinical specimen, the number of cells to be measured (mainly cancer cells) present in a well varies as one or several, and in some cases, thousands. Therefore, as in the method described in Patent Document 1, when the average value is used as a threshold value for determining whether or not a “hit” well, the threshold value is accurate when the number of cells present in the well is small. It will be lacking. In addition, when the measurement target cell is very small, if the screening is performed at a low magnification as in the method described in Patent Document 1, the cell itself may not be found. For this reason, when the method described in Patent Document 1 is used, there is a possibility that a well that should be determined as “hit” is not determined as “hit”.
In the technique described in Patent Document 1, wells determined to be “hits” are screened again in the high resolution mode, so that it is troublesome twice in terms of switching of the optical system and stage operation. Inefficient.

  Patent Document 2 discloses that in a method for diagnosing colorectal cancer, a recovery antibody that specifically binds to a cell surface antigen that is commonly expressed in normal cells derived from colorectal mucosa and colorectal cancer cells in a stool sample is a linker. The solid phase carrier that is bound via the mixture is mixed to recover normal cells and colon cancer cells, and then separated from the solid phase carrier, and then the separated cells are specifically bound to the colon cancer cells. It describes that immunostaining is performed using the obtained antibody for detection, and then a microscopic image of the immunostained cell is obtained and image analysis is performed.

  However, there may be only a few cancer cells recovered by using the method described in Patent Document 2 in a well, or only one in some cases. If even one cancer cell exists in the well, the specimen provider will be diagnosed with cancer. However, the description in Patent Document 2 describes a specific method for efficiently searching for cancer cells in the well. There is no mention of the well scanning method.

  The present invention has been made in view of the above-described conventional problems, a cell screening method capable of efficiently detecting cells to be measured in a well, control software used for the method, a cell screening apparatus, An object of the present invention is to provide an image analysis apparatus and a cell screening system.

  The present applicant has an illumination optical system for irradiating a cell with illumination light, an imaging optical system and an imaging element for imaging light emitted from the cell, an autofocus means for focusing the cell for imaging, and a well storing the cell. A cell screening device equipped with a stage to be placed and transported to the scan position, and in conjunction with the cell screening device, the image captured immediately after imaging was analyzed, and the analysis result satisfied a predetermined condition In some cases, it has been found that cell screening can be performed efficiently by having an image analysis device and control software that transmits the information to the cell screening device and terminates the screening in the corresponding well, thereby completing the present invention. It was.

That is, in the cell screening method according to the present invention, the cell screening apparatus performs a first scan in a predetermined well order for each well of the multi-well plate in which cells are stored, and performs the first scan. divided the entire area of the wells are in a plurality of shot areas, performs a second scan to each shot region in a predetermined order, each shot area captured, the image analysis apparatus, each shot area by the cell screening system each but to be imaged, a cell screening method for analyzing the image of the shot area captured by the cell screening system, the image analysis of the first shot area in the well which is performed the first scan through until the image analysis of the shot area, the number of cells detected in the detection target eye If it reaches number, the second scan for the next subsequent shot area of the shot area in the well, and terminates the imaging and image analysis, the first scan to the next well, the first The second scan, imaging, and image analysis are started for each shot area in the next well in which scanning is performed .

  In the cell screening method of the present invention, it is preferable that the cells to be detected are human or animal-derived cells to which at least one color fluorescent label is applied.

  In the cell screening method of the present invention, the detection target cell is preferably a colon cancer cell.

  In the cell screening method of the present invention, the detection target cell is preferably a uterine cancer cell.

In the cell screening method of the present invention, as a detection target number of cells and the detection target, it is capable of one or more settings, the first shot area in the well which is performed the first scan through the image analysis to the image analysis of the shot area, if the number of cells detected in the detection target has reached the target number, the second scan for the next subsequent shot area of the shot area in the well , exit imaging and image analysis, the first scan to the next well, the second scan for each shot region in the next well doing the first scan, the imaging and image analysis It started, through the image analysis of our Keru first shot area on the well until the image analysis of the shot area, to the detection target If the number of cells does not reach the target number, the for the next shot area of the shot area in the well the second scan, to perform imaging and image analysis, the cell screening system, and the image analyzer It is preferable to use control software to control.

In the cell screening method of the present invention, the control software arbitrarily sets a mode for controlling the cell screening apparatus so that the second scan is performed without overlapping and without gaps for each shot region. preferably, with respect to the shot area of the range it has to be selected a mode for controlling the cell screening system to perform the second scan was.

  In the cell screening method of the present invention, the cell screening apparatus images an illumination optical system for irradiating illumination light to the cells in the well located at the imaging position, and light emitted from the cells in the well. An imaging means; an autofocus means for focusing the imaging means on the cells in the well located at the imaging position; and a stage on which the multiwell plate is placed and the cells in each well are transported to the imaging position; It is preferable to have.

In the cell screening method of the present invention, as a result of analyzing the analysis software immediately after the image is analyzed by the image analysis device , the control software analyzes the first software. When the number of cells to be detected reaches the target number from the first shot area image analysis to the shot area image analysis in the well being scanned, the information is transmitted to the cell screening by the transmission means The cell screening apparatus transmits the second scan to the next shot area after the shot area in the well, and completes the imaging, and the first scan to the next well is transmitted to the apparatus by the control means. , Each shot in the next well performing the first scan The relative frequency second scan, to start imaging and image analysis, preferably controls the cell screening system, and the image analyzer.

  The control software of the present invention is control software used for the cell screening method of the present invention.

  The cell screening apparatus of the present invention is a cell screening apparatus used for the cell screening method of the present invention.

  The image analysis apparatus of the present invention is an image analysis apparatus used for the cell screening method of the present invention.

In addition, the cell screening system according to the present invention includes a stage on which a multiwell plate having a plurality of wells for storing cells is mounted, the position of the multiwell plate being adjustable with respect to the imaging position, and the imaging position An illumination unit that irradiates the cells in the well located in the well with an illumination light, an imaging unit that images light emitted from the cells in the well, and a focal point of the imaging unit in the well located in the imaging position. and autofocus means to fit the cell, the divided the entire region in each well of the multiwell plate to a plurality shot areas, so that each shot area is aligned with the imaging position at a predetermined pitch and order, said stage A cell screening system having a cell screening device having a stage drive unit to be driven, the detection pair A detection condition setting unit capable of setting the cell condition and the number of detection targets to be analyzed, and an analysis that analyzes the image captured through the imaging unit and detects cells that satisfy the conditions set in the detection condition setting unit And a scan control unit that controls driving of the stage by the stage driving unit and imaging by the imaging unit according to an analysis result in the analysis unit, and the scan control unit is detected by the analysis unit and when the number of cells reached a target number, which is set through the detection condition setting unit, the alignment and the to the imaging position by the stage driving unit for the next subsequent shot area of the shot area during this the wells terminate the imaging by the imaging unit, the positioning of the imaging position by the stage driving unit for each of the shot areas in the well where the next has a scan It is characterized in that to start imaging by the fine imaging unit.

  In the cell screening system of the present invention, it is preferable that the cell to be detected is a human or animal-derived cell to which at least one color fluorescent label is applied.

  In the cell screening system of the present invention, it is preferable that the detection condition setting unit can set one or more detection target numbers of cells to be detected.

In the cell screening system of the present invention, the stage driving unit, each shot area in the each well, without overlapping, to and without gaps, so as to be aligned to the imaging position, drives the stage Is preferred.

In the cell screening system of the present invention, the cell screening system has a scan range setting unit for setting a scan range of each shot region in each well of the multi-well plate , and the stage driving unit is provided in each well of the multi-well plate. wherein within a set range in the scanning range setting unit, so that each shot area in each well is aligned with the imaging position at a predetermined pitch and order, it is preferable to drive the stage.

  According to the present invention, a cell screening method capable of efficiently detecting target cells in a well, control software used in the method, a cell screening device, an image analysis device, and a cell screening system are obtained.

It is explanatory drawing which shows schematic structure of the cell screening system used for the cell screening method concerning one Embodiment of this invention. It is a flowchart which shows an example of the process sequence of the cell screening method of this embodiment using the cell screening system of FIG. It is explanatory drawing which shows the scanning order with respect to the well in a multiwell plate by the cell screening apparatus of this embodiment. It is explanatory drawing which shows notionally the number of imaging shots with respect to 1 well in the cell screening apparatus of this embodiment. It is explanatory drawing which shows an example of the scanning order with respect to the shot area | region in 1 well in the cell screening apparatus of this embodiment. It is explanatory drawing which shows an example of the detection condition parameter setting screen displayed on the monitor of the cell screening system of this embodiment. FIG. 7 is an explanatory diagram illustrating an example of a “Water Shed” function that is one setting item on the detection condition parameter setting screen of FIG. 6, and (a) illustrates a “Water Sheed” function for a captured image that is adjacent to or adjacent to two cells. (B) shows the state of cell recognition when the image is analyzed with the image analysis device with “OFF” set to “OFF”, and “Water Shed” function is set to “ON” for the captured image that is adjacent to or adjacent to two cells. It is a figure which shows the recognition state of the cell when set to "" and analyzed with the image analyzer. It is explanatory drawing which shows the state which started screening using shot cell (i, g) as a starting position using the cell screening system of this embodiment, and advanced to shot field (iv, j). In the cell screening system of the present embodiment, when advancing to the shot area (iv, j), a target number of positive cells satisfying the conditions set in various parameter setting items is detected from the image of the shot area (iv, j). It is explanatory drawing which shows an example of the image recognition state recognized by the image analysis apparatus at the time. It is explanatory drawing which shows an example of the well by which the target number of the detection object cell was detected as a result of having screened all the wells of a multiwell plate using the cell screening system of this embodiment. It is explanatory drawing which shows an example of GUI provided with the recall function for reconfirming the positive cell after a screening and the arbitrary shot area | regions of each well displayed on the monitor of the cell screening system of this embodiment.

As in the present invention, through, for example, by setting the number of cells needed for analysis prior to screening, from the image analysis of the first shot area in the well which is scanning to image analysis of the shot area, detection target If the number of cells detected to have reached the target number (minimum one) second scan for the next subsequent shot area of the shot area in the well, and terminates the imaging and image analysis, the following The time spent for screening can be saved by switching to the first scan of the well, the second scan for each shot region in the well performing the first scan, imaging, and image analysis. . In particular, in a cell-based diagnosis in which the number of cells extracted from a biological sample is very small and even one cell is detected as a positive diagnosis, the screening time saving effect is greatly increased.

Hereinafter, embodiments of the present invention will be described with reference to the drawings.
FIG. 1 is an explanatory diagram showing a schematic configuration of a cell screening system used for a cell screening method according to an embodiment of the present invention, and FIG. 2 is a processing procedure of the cell screening method of the present embodiment using the cell screening system of FIG. It is a flowchart which shows an example.
The cell screening system of the present embodiment includes a cell screening device, an image analysis device, and control software.

The cell screening apparatus has an electric stage 101, an illumination unit, an imaging unit, an autofocus (AF) unit 109 as an autofocus unit, and a stage controller 115 as a stage drive unit.
The electric stage 101 is connected to a stage controller 115 and is configured to place the multiwell plate 1 and be adjustable with respect to the imaging position.
In the present embodiment, a 96-well multiwell plate is used as the multiwell plate 1. The multi-well plate 1 is a detection target of human or animal-derived colon cancer cells or uterine cancer cells collected from, for example, different subjects for each well, for example, having at least one color fluorescent label. Samples that may contain the cells are stored. If the stored specimen belongs to a negative subject, the target cell does not exist in the well. If the stored specimen belongs to a positive subject, the target cell does not exist in the well. There is a high probability that one or more exist.

The illumination unit includes a mercury xenon lamp 102, a lamp power supply 103, a shutter unit 104, and a collimator lens 105.
The mercury xenon lamp 102 is connected to a lamp power source 103 and illuminates fluorescently stained cells stored in each well of the multiwell plate 1. The shutter unit 104 includes a shutter blade 104a, and blocks illumination light in addition to a necessary exposure time in order to prevent discoloration when capturing an image of a cell. The collimator lens 105 converts the light from the mercury xenon lamp 102 into parallel rays and guides it to the fluorescent cube 107.

  The fluorescent cube 107 includes a dichroic mirror 107A, an excitation filter 107B, and an absorption filter 107C. In addition, the cell screening apparatus according to the present embodiment includes a turret (not shown) in which a plurality of fluorescent cubes 107 can be incorporated in order to support a plurality of fluorescent dyes. The fluorescent cube 107 can be switched by rotating the turret via a rotation mechanism (not shown).

The imaging unit includes an imaging lens 108 and a CCD camera 110.
The imaging lens 108 images light emitted from the cells in the well as a cell image on the CCD element inside the CCD camera 110. The CCD camera 110 is connected to the camera controller 111 and is controlled by the camera controller 111. In the present embodiment, a 12-bit CCD camera is used as the CCD camera 110.

  The camera controller 111 transmits an image captured through the CCD camera 110 to the personal computer 112.

  The autofocus (AF) unit 109 is a generally well-known pupil division type AF unit, and includes an AF controller 109A, a semiconductor laser element 109B, a polarization beam splitter 109C, and a two-divided photodetector 109D. It is configured. The autofocus unit 109 irradiates the multiwell plate 1 with the AF laser beam emitted from the semiconductor laser element 109B and via the polarization beam splitter 109C via the prism 117 and the objective lens 118. The reflected light passing through the objective lens 118 and the prism 117 is received by the two-divided photodetector 109D via the polarization beam splitter 109C, and the AF controller 109A selects an objective lens drive unit (not shown) according to the light reception result. Then, the objective lens 118 is moved up and down to adjust the focus.

  The stage controller 115 is connected to a personal computer (PC) 112. Then, the electric stage 101 is driven so that the imaging area of each shot in each well of the multi-well plate 1 is aligned with the imaging position in a predetermined pitch and order by control by control software described later provided in the PC 12. Let

  The image analysis apparatus includes a camera controller 111 and a personal computer (PC) 112. Then, under the control of the control software described later included in the PC 12, the image captured through the imaging unit is analyzed, and the detection target cell set by the detection condition setting unit described later is detected for each well to be scanned. .

The PC 112 is connected to a camera controller 111, a stage controller 115, and a microscope controller (not shown). Further, the PC 112 performs the above-described AF control, fluorescent cube switching control, image capture control from the CCD camera 110, drive control of the electric stage 101 according to the analysis result in the image analysis apparatus, and the like via these controllers. Control software programmed to function as the controlling means to perform is provided.
In FIG. 1, 113 is a keyboard, 114 is a monitor, and 116 is a mouse. In the cell screening apparatus of this embodiment, the monitor 114, the keyboard 113, and the mouse 116 function as a detection condition setting unit, and are configured to be able to set the conditions of the cells to be detected and the number of detection targets.

The control software is programmed so as to control the detection condition setting unit so that one or more settings can be made as the detection target number of cells to be detected.
The control software, for each shot area overlapped without and no gap and the mode of controlling the cell screening system to perform the second scanning, the range set in any shot area with respect to the second It is programmed to select a mode for controlling the cell screening device to perform scanning.

In addition, when the number of cells to be detected as a detection target reaches the target number from the image analysis of the first shot area to the image analysis of the shot area in the well performing the first scan, the control software Each shot in the well in which the second scan, imaging, and image analysis for the shot area following the shot area in the well are completed, the first scan to the next well, and the first scan are performed. When the number of cells to be detected as the detection target does not reach the target number from the first shot area image analysis to the shot area image analysis in the well starting the second scan, imaging and image analysis for the area Is a second scan for the shot region next to the shot region in the well. , So as to perform imaging and image analysis, it is programmed to control the cell screening device and an image analyzer.
Specifically, the control software uses the analysis unit to analyze the image captured by the cell screening apparatus immediately after the image is analyzed, and as a result of the analysis, the first shot region in the well performing the first scan is analyzed. When the number of cells to be detected as the detection target reaches the target number from the image analysis of the shot area to the image analysis of the shot area, the information is transmitted to the cell screening apparatus by the transmission means, and the cell screening apparatus is Then, the stage drive unit aligns the shot area following the shot area in the well with the imaging position and the imaging unit finishes imaging, and drives the stage for each shot area in the well to be scanned next. Of the imaging position by the imaging unit and the imaging unit To start an image, it is programmed to control the screening device and an image analyzer.

FIG. 3 is an explanatory diagram showing the scan order of the wells in the multi-well plate , which is the first scan, by the cell screening apparatus of this embodiment.
In the cell screening apparatus of the present embodiment, the control software changes the direction of the row direction for each well arranged in the same row position (scanning of the electric stage 101 via the stage controller 115 and imaging by the imaging unit). It is programmed to do. For example, as shown in FIG. 3, each well arranged in the A row is scanned sequentially from the well in the first column to the well in the 12th column, and then each well arranged in the B row is scanned from the well in the 12th column to one column. Scan sequentially into eye wells. Thereafter, the same scanning operation is repeated until the last well (in the example of FIG. 3, the first column well in the H row). When all the scans are performed in this order, the transfer operation of the wells of the electric stage 101 is minimized, and all the wells arranged on the plate can be efficiently scanned, and the time can be reduced. .

FIG. 4 is an explanatory diagram conceptually showing the number of shots for imaging one well in the cell screening apparatus of the present embodiment. In FIG. 4, a circle represents a well, and a small square represents a region imaged in one shot. Each shot region includes at least a partial region of the well.
In the screening apparatus of the present embodiment, the control software is programmed so as to control the drive of the electric stage 101 by the stage controller 115 so that the areas imaged in these shots are arranged without gaps. The entire region of the well can be imaged.

FIG. 5 is an explanatory diagram showing an example of the scan order for the shot region in one well , which is the second scan, in the cell screening apparatus of the present embodiment.
In the cell screening apparatus of this embodiment, the control software changes the direction of the column direction for each shot region arranged in the same column position from one region farthest in the horizontal direction from the center of the well toward the other region. Programmed to scan. For example, as shown in FIG. 5, the shot areas arranged in the i-th column are sequentially conveyed from the shot area in the g-th row to the shot area in the j-th row to the imaging position, and then each image is arranged in the ii-th row. The shot area is sequentially conveyed from the m-th shot area to the e-th shot area to the imaging position and imaged. Thereafter, the same scanning operation is repeated up to the last shot region (in the example of FIG. 5, the mth row well in the XVii column).

FIG. 6 is an explanatory diagram showing an example of a detection condition parameter setting screen displayed on the monitor 114 of the cell screening system of the present embodiment.
In the cell screening system of the present embodiment, the control software displays the detection condition parameters on the monitor 114, and can set the conditions of the cells to be detected and the target number of detection via the keyboard 113 and the mouse 116. Is programmed.
In FIG. 6, “Intensity” is a setting item of the minimum value of the brightness of the pixel to be detected among pixels in the shot to be imaged, and “Maximum Cell Size” is “Intensity” in the object to be detected. “Minimum Cell Size” is a setting item for the maximum value of the area having a luminance value that is equal to or greater than the minimum luminance value set in Step 1. The luminance value that is equal to or higher than the minimum luminance value set in “Intensity” in the object to be detected. This is a setting item for the minimum value of the area to have. “Water Shed” is an ON / OFF setting item for a function that prevents a plurality of cells from being erroneously recognized as one cell when the cell density in the well is high and the cells are crowded.

  FIG. 7 shows an example of the “Water Sheed” function. For example, in the case where a plurality of cells are adjacent or stuck together, when the “Water Shed” function setting item is set to “OFF” on the detection condition parameter setting screen displayed on the monitor 114, FIG. As shown in a), two cells are misrecognized as one cell. However, when the “Water Shed” function setting item is set to “ON”, as shown in FIG. 7B, the cell can be correctly divided by recognizing the constriction and the edge of the cell image. Normally, it is preferable to set the “Water Shed” function setting item to “ON”. If the cell density is clearly low, individual cells can be correctly recognized without using the “Water Shed” function. If this function is set to “OFF”, the image processing speed is increased accordingly. Can be increased.

“Ce11 Count” is a setting item for the detection target number of cells to be detected in one well. In the example of FIG. 6, it is set to one.
In the cell screening system of this embodiment, the control software sets the number of cells to be detected in one well detected by the image analysis apparatus as “Ce11 Count”, with the PC 112 functioning as a scan control unit. When the target number is reached, the second scan, imaging and image analysis for the well are finished, and the first scan to the next well and the respective shot regions in the well performing the first scan Programmed to initiate a second scan, imaging and image analysis .

Cell screening using the cell screening system of the present embodiment configured as described above is performed as follows.
The user activates the system, places the multiplate well 1 storing the specimen in each well on the electric stage 101, and operates the PCl 12, the keyboard 113, the monitor 114, and the mouse 116 to perform the first scan. A well to perform is selected (step S1).
Next, as shown in FIG. 6, the operator sets various parameters (conditions of cells to be detected and the number of detection targets) on the detection condition parameter setting screen displayed on the monitor 114. In addition, the monitor 114, PCl 12, keyboard 113, monitor 114, and mouse 116 are operated to set the imaging conditions, illumination conditions, autofocus conditions, and the number of wells to be imaged in one plate (step S2). .
Next, the user starts screening (step S3).

When the user starts the screening, as shown in FIG. 8, the second scan (from the first well (A1 well in the case of the present embodiment)) is started with the shot region (i, g) as the start position. The conveyance to the imaging position by the electric stage 101 and the imaging by the imaging unit are started (steps S4 to S6). At the same time as the imaging by the imaging unit, analyzes the image analyzer is an image of each shot area, in the image of each shot region, whether positive cells satisfying the set detection condition parameter setting screen condition exists Recognize (step S7). In the cell screening system of this embodiment, the control software may be configured to perform processing such as background removal at that time. The detection condition parameters for cell recognition include the circularity factor, circumferential perimeter, elongation factor, etc. in addition to those shown in FIG. 6, and the control software is configured to perform analysis according to the parameters. Then it is still better.

Then, through the image analysis of the first shot area by the image analysis device to the image analysis of the shot area, the number of detecting the positive cells that satisfy the conditions set in various parameters setting items described above reaches the target number Taka not Is checked (step S8). If the number of positive cells detected does not reach the target number, it is checked whether or not the shot area analyzed by the image analyzer is the last shot area in the well (step S9). If it is not the last shot area in the well, the electric stage 101 is driven so that the next shot area in the same well is located at the imaging position (step S10), and the next shot area in the same well is screened. The processing is switched (processing after step S5). If it is the last shot area in the well, it is checked whether this last shot area is in the last well in the multi-well plate 1 (step S11). When this well is not the last well, the electric stage 101 is driven so that the next well is located at the imaging position (step S12), and the process is switched to the next well screening (from step S5 onward). processing). When this well is in the last well, a result to be described later is displayed through the monitor 114 (step S13).

Here, in the cell screening system of the present embodiment, when the shot started from the shot area (i, g) proceeds to the shot area (iv, j) as shown in FIG. 8, the shot area (iv, j) ), The target number of positive cells satisfying the conditions set in the above-mentioned various parameter setting items is detected.
FIG. 9 shows an example of an image recognition state recognized by the image analysis apparatus at that time. In the example of FIG. 9, three positive cells are detected, and a square essence indicating that they are recognized is displayed surrounding the three cells. In the cell screening system of the present embodiment, at this stage, the screening for this well is finished, and it is checked whether this last shot region is in the last well in the multiwell plate 1 (step S14). When this well is not the last well, the electric stage 101 is driven so that the next well is located at the imaging position (step S12), and the process is switched to the next well screening (from step S5 onward). processing). When this well is in the last well, the result is displayed as shown as an example in FIG. 10 via the monitor 114 (step S15).

  In the example of FIG. 10, wells containing positive cells are colored in the monitor 114. At this time, the cell screening apparatus of the present embodiment is configured to display an analysis screen as shown in FIG. 9 when the well displayed in color is clicked with the mouse 116.

  In the cell screening system of this embodiment, the control software may further include a recall function for reconfirming positive cells and arbitrary shot regions of each well after screening. For example, a GUI (Graphical User Interface) as shown in FIG. 11 may be displayed on the monitor 114 of the PCl 12 so that positive cells can be reconfirmed visually. In FIG. 11, 201 is an imaging screen, 202 is a screen schematically showing a well to be screened, and a well number (example: A1) is displayed at the center. Reference numeral 203 denotes a shot area in which positive cells are found in the well displayed on the screen 202. Reference numeral 204 denotes a shot area in the well currently displayed on the imaging screen 201. A scan button 205 is used to move the display area of the imaging screen. The imaging stage in the well can be moved by driving the electric stage 101 by a predetermined amount in a predetermined direction with the up / down / left / right buttons. Reference numeral 206 denotes an illumination setting item, which allows selection of Intensity (brightness) and Filter (fluorescence filter). 207 is an objective lens switching setting item, which sets a predetermined objective lens such as a magnification larger than the magnification at the time of screening (for example, 60 ×, etc.) when the image of positive cells is further enlarged and examined in detail. Can be done.

Cell screening system of the present embodiment, according to the cell screening methods and cell screening device, if the number of cells detected in the detection target while scanning has reached the target number, the first scan to the next well Since switching to the second scan, imaging, and image analysis for each shot area in the well in which the first scan is performed , efficient screening can be performed and time spent for screening can be saved. Can do. In particular, in a cell-based diagnosis in which the number of cells extracted from a biological sample is very small and even one cell is detected as a positive diagnosis, the screening time saving effect is greatly increased.

  Scan cells in each container arrayed on a cell array plate such as a multi-well plate, image fluorescence or other light emitted from the surface or inside of the cell, analyze the captured cell image, and analyze results This is particularly useful in the field of clinical examinations that are required to output.

DESCRIPTION OF SYMBOLS 1 Multiwell plate 101 Electric stage 102 Mercury xenon lamp 103 Lamp power supply 104 Shutter unit 104a Shutter blade 105 Collimator lens 107 Fluorescent cube 107A Dichroic mirror 107B Excitation filter 107C Absorption filter 108 Imaging lens 109 Autofocus (AF) unit 109A AF controller 109B Semiconductor laser element 109C Polarization beam splitter 109D Two-divided photodetector 110 CCD camera 111 Camera controller 112 Personal computer (PC)
113 Keyboard 114 Monitor 115 Stage Controller 116 Mouse 117 Prism 118 Objective Lens

Claims (16)

The cell screening apparatus performs a first scan in a predetermined well order for each well of the multi-well plate in which cells are stored, and a plurality of shots of the entire region of the well in which the first scan is performed Each of the shot areas is divided into areas, a second scan is performed in a predetermined order, and each shot area is imaged. Each time the shot area is imaged by the cell screening apparatus, the cell screening apparatus performs the cell screening. A cell screening method for analyzing an image of the shot region imaged by an apparatus,
When the number of cells to be detected as a detection target reaches the target number from image analysis of the first shot region to image analysis of the shot region in the well performing the first scan, the shot in the well Each shot in the next well in which the first scan to the next well and the first scan are performed after finishing the second scan, imaging and image analysis for the shot area after the area A cell screening method characterized by starting the second scan, imaging and image analysis for an area. 2. The cell screening method according to claim 1, wherein the cell to be detected is a cell derived from a human or an animal, to which a fluorescent label of at least one color is applied. The cell screening method according to claim 2, wherein the cell to be detected is a colon cancer cell. The cell screening method according to claim 2, wherein the cell to be detected is a uterine cancer cell. The detection target number of cells to be detected can be set to one or more, and from the first shot area image analysis to the shot area image analysis in the well performing the first scan. When the number of cells to be detected as the detection target has reached the target number, the second scan, imaging and image analysis for the shot area following the shot area in the well are finished, and the next well is completed. The first scan, the second scan, imaging and image analysis for each shot region in the next well performing the first scan are started, and from the image analysis of the first shot region in the well Through the image analysis of the shot area, if the number of cells to be detected as the detection target does not reach the target number, The control software for controlling the cell screening device and the image analysis device is used so as to perform the second scan, imaging, and image analysis on the shot region next to the shot region in the well. Item 2. The cell screening method according to Item 1. The control software includes a mode for controlling the cell screening apparatus so as to perform the second scan for each shot area without overlapping and without a gap, and the second software for a shot area within an arbitrarily set range. The cell screening method according to claim 5, wherein a mode for controlling the cell screening apparatus so as to perform scanning is selectable. The cell screening device comprises:
An illumination optical system for irradiating illumination light to the cells in the well located at the imaging position;
Imaging means for imaging light emitted from the cells in the well;
Autofocus means for focusing the imaging means on the cells in the well located at the imaging position;
The cell screening method according to claim 1, further comprising a stage on which the multi-well plate is placed and the cells in each well are transported to an imaging position. The control software causes the analysis unit to analyze the image captured by the cell screening device immediately after the image is analyzed, and as a result of the analysis, the first shot in the well performing the first scan is analyzed. When the number of cells to be detected as the detection target reaches the target number from the image analysis of the region to the image analysis of the shot region, the information is transmitted to the cell screening device by the transmission unit, and the cell is The screening apparatus completes the second scan and imaging for the shot areas following the shot area in the well, and performs the first scan and the first scan to the next well. Second scan, imaging and image analysis for each shot area in the next well To start, cell screening method according to any one of claims 7 wherein the cell screening system and claim 5 or the 6 and controls the image analyzer, depending on claim 5 or 6. Control software used for the cell screening method according to claim 8. The cell screening apparatus used for the cell screening method of Claim 8. An image analyzer used in the cell screening method according to claim 8. A stage in which a multiwell plate having a plurality of wells for storing cells is placed, the position of the multiwell plate can be adjusted with respect to the imaging position, and the cells in the well located at the imaging position are illuminated. An illumination unit that irradiates light; an imaging unit that images light emitted from cells in the well; an autofocus unit that focuses the imaging unit on cells in the well located at the imaging position; A cell screening apparatus having a stage driving unit that drives the stage so that the entire region in each well of the well plate is divided into a plurality of shot regions and each shot region is aligned with an imaging position in a predetermined pitch and order. A cell screening system,
A detection condition setting unit capable of setting the conditions of the cells to be detected and the detection target number;
An analysis unit that analyzes an image captured through the imaging unit and detects cells that satisfy a condition set by the detection condition setting unit;
A scan control unit that controls driving of the stage by the stage driving unit and imaging by the imaging unit according to an analysis result in the analysis unit;
When the number of cells detected by the analysis unit has reached a target number set via the detection condition setting unit, the scan control unit is configured to perform the stage for the shot region subsequent to the shot region in the well. The positioning to the imaging position by the driving unit and the imaging by the imaging unit are terminated, the positioning to the imaging position by the stage driving unit for each shot area in the well to be scanned next, and the imaging unit A cell screening system characterized by starting imaging. The cell screening system according to claim 12, wherein the cells to be detected are human or animal-derived cells to which at least one color fluorescent label is applied. The cell screening system according to claim 12, wherein the detection condition setting unit can set one or more detection target numbers of cells to be detected. The cell screening according to claim 12, wherein the stage driving unit drives the stage so that each shot region in each well is aligned with an imaging position without overlapping and without a gap. system. A scan range setting unit for setting a scan range of each shot region in each well of the multi-well plate;
The stage drive unit is positioned within the range set by the scan range setting unit in each well of the multi-well plate so that each shot region in each well is aligned with an imaging position in a predetermined pitch and order. The cell screening system according to claim 12, wherein the stage is driven.