JP6534294B2 - Imaging apparatus and method, and imaging control program - Google Patents

Description

  The present invention relates to an imaging device and method for imaging a cell group multiple times over time and an imaging control program.

  In recent years, phase difference measurement has begun to be widely used as a method of observing cultured transparent cells such as stem cells without staining. And, as a device for performing such phase difference measurement, a phase contrast microscope using an imaging element such as a CCD (Charge-Coupled Device) image sensor or a CMOS (Complementary Metal-Oxide Semiconductor) image sensor is used. A phase contrast microscope produces an image of a cell by detecting the phase difference between diffracted light and direct light produced by the cell.

JP, 2014-76000, A JP, 2009-036808, A Japanese Patent Laid-Open No. 2000-13690 Japanese Patent Publication No. 2010-500573

  Here, when cell proliferation progresses, a cell group (also referred to as cell colony) is formed. Then, when the proliferation of this cell group progresses, the cells overlap and stack in a plurality of layers from the state of a monolayer. When the cell group is thus laminated, diffracted light generated by the cell causes multiple scattering in the cell group and propagates in the same optical path as the direct light. As a result, the intensity of light emitted from the layered cell group is increased, which is largely different from the intensity of light emitted from the cell group of a single layer.

  Therefore, when both monolayer cell groups and layered cell groups are included in the imaging region, if they are imaged under the same exposure conditions, for example, appropriate exposure is performed for monolayer cell groups. The image is taken under the conditions, but for the stacked cell group, the intensity of the light emitted from the cell group becomes equal to or higher than the detection upper limit level of the imaging device, and the imaging device is saturated to obtain an image of the appropriate cell group There is a problem that can not be done.

  Also, conventionally, before main imaging, automatic exposure control is performed in which cells are captured under a plurality of exposure conditions to obtain a plurality of cell images, and appropriate exposure conditions are set based on the plurality of cell images. However, when this automatic exposure control is performed, in order to search for appropriate exposure conditions, at least three or more imagings are required, and there is a problem that the time until the main photographing is finished becomes long.

  In Patent Document 1, in order to observe the movement of the myocardium more clearly, the cell group of the myocardium is imaged under a plurality of exposure conditions, and among them, the exposure condition which makes the area of the movement region the largest Although disclosed to be determined, nothing is proposed as to a method of determining an exposure condition capable of appropriately imaging stacked cell groups as described above.

  Further, Patent Document 2 discloses that the optimal exposure conditions are determined based on the pH (potential hydrogen) value of the culture medium and the absorption characteristics of the culture medium additive, but as described above, it is laminated. There is no suggestion of a method of determining an exposure condition that can appropriately image such cell groups.

  Further, Patent Document 3 and Patent Document 4 disclose that the exposure condition is optimized according to the brightness of each partial region, but also the exposure capable of appropriately imaging the laminated cell group Nothing has been proposed as to how to determine the conditions.

  SUMMARY OF THE INVENTION In view of the above problems, the present invention is an imaging device and method capable of imaging under appropriate exposure conditions without saturation even with respect to a cell group which is laminated as described above and the light intensity is increased. The purpose is to provide a control program.

  The imaging device of the present invention is detected within the detection limit range of the imaging device among an imaging unit that images a plurality of cells by the imaging device, and a cell or a cell group included in a cell image imaged by the imaging unit. And an exposure condition determining unit that determines a first exposure condition of the imaging device based on an image of the identified cell or cell group, and the imaging unit determines the first exposure condition. Based on the imaging of a plurality of cells, the exposure condition determination unit determines a region exceeding the detection limit range of the imaging element in the cell group included in the cell image imaged based on the first exposure condition. The cell group to be included is specified, the second exposure condition of the imaging device is determined based on the image of the specified cell group, and the imaging unit performs imaging of a plurality of cells based on the second exposure condition It is characterized by

  Further, in the imaging device of the present invention, the exposure condition determination unit can identify the cell group of the monolayer, and determine the first exposure condition based on the image of the cell group of the identified monolayer. .

  Further, in the imaging apparatus according to the present invention, the exposure condition determination unit determines the size of the image signal of the area exceeding the detection limit range based on the image of the cell group including the area exceeding the detection limit range of the imaging device. The second exposure condition can be determined based on the estimated magnitude of the image signal.

  Further, in the imaging apparatus of the present invention, the exposure condition determination unit can estimate the size of the image signal of the region exceeding the detection limit range using a Gaussian distribution.

  Further, in the imaging device of the present invention, the exposure condition determination unit can estimate the size of the image signal of the region exceeding the detection limit range using linear approximation.

  In the image pickup apparatus according to the present invention, the exposure condition determination unit estimates the image signal within the detection limit range among the image signals of the image of the cell group including the region exceeding the detection limit range of the image pickup element. Acquire the average value, the variance value, and the maximum value of the image signal with the image signal, and if the difference between the value obtained by adding the variance value to the average value and the maximum value is within the preset range, And determining the second exposure condition based on a value obtained by adding the dispersion value to the average value if the second exposure condition is determined and the difference is out of a preset range. it can.

  Further, in the above-described imaging apparatus of the present invention, the exposure condition determination unit sets a line segment crossing the cell group including the region exceeding the detection limit range, and based on the image signal on the line segment, the second. Exposure conditions can be determined.

  Further, in the imaging device of the present invention, the exposure condition determination unit can set, as the line segment, the major diameter of the cell group including the region exceeding the detection limit range.

  Further, in the imaging device of the present invention, the exposure condition determination unit can detect a region exceeding the detection limit range, and set a line segment passing the center of gravity of the detected one region.

  Further, in the above-described imaging apparatus of the present invention, when a plurality of regions exceeding the detection limit range of the imaging device are included in one cell group, the exposure condition determination unit determines the image signal based on the respective regions. A plurality of exposure condition candidates of the imaging device can be determined, and a second exposure condition can be determined based on the determined plurality of exposure condition candidates.

  Further, in the imaging apparatus of the present invention, the exposure condition determination unit can determine an average value of the plurality of exposure condition candidates as the second exposure condition.

  Further, in the imaging apparatus according to the present invention, the exposure condition determination unit determines, of the plurality of exposure condition candidates, the second exposure condition candidate determined based on the image signal of the region where the estimated image signal is largest. It can be determined as the exposure condition.

  Further, in the imaging device according to the present invention, when a plurality of cell groups including a region exceeding the detection limit range of the imaging device is specified, the exposure condition determination unit picks up an image based on the image of each specified cell group A plurality of exposure condition candidates of the element can be determined, and a second exposure condition can be determined based on the determined plurality of exposure condition candidates.

  Further, in the imaging apparatus of the present invention, the exposure condition determination unit can determine an average value of the plurality of exposure condition candidates as the second exposure condition.

  Further, in the imaging apparatus according to the present invention, the exposure condition determination unit determines, of the plurality of exposure condition candidates, the second exposure condition candidate determined based on the image of the cell group having the largest estimated image signal. It can be determined as the exposure condition.

  Further, in the image pickup apparatus of the present invention, it is desirable that the first and second exposure conditions be at least one of an exposure time of the image pickup element and an incident light amount.

  The imaging method of the present invention is an imaging method for imaging a plurality of cells by an imaging device, and is detected within the detection limit range of the imaging device among cells or cell groups included in a cell image obtained by imaging a plurality of cells. The first exposure condition of the imaging device is determined based on an image of the identified cell or cell group, and imaging of a plurality of cells is performed based on the first exposure condition. And identifying a cell group including a region exceeding the detection limit range of the imaging device among the cell groups included in the cell image imaged based on the first exposure condition, and an image of the specified cell group The second exposure condition of the imaging device is determined based on the above, and imaging of a plurality of cells is performed based on the second exposure condition.

  The imaging control program of the present invention is an imaging control program that causes a computer to function as an imaging control unit that controls an imaging unit that images a plurality of cells with an imaging device, and an exposure condition determination unit that determines an exposure condition of the imaging device. The exposure condition determination unit identifies the cells or cell groups detected within the detection limit range of the imaging device among the cells or cell groups included in the cell image captured by the imaging unit, and the identified cells or cells The first exposure condition of the imaging device is determined based on the image of the cell group, and the imaging control unit controls the imaging unit to perform imaging of a plurality of cells based on the first exposure condition, The exposure condition determination unit specifies a cell group including a region exceeding the detection limit range of the imaging device among the cell groups included in the cell image captured based on the first exposure condition, and The second exposure condition of the imaging device is determined based on the image of the identified cell group, and the imaging control unit controls the imaging unit to image a plurality of cells based on the second exposure condition. It is characterized by doing.

  According to the imaging device and method of the present invention and the imaging control program, among the cells or cell groups included in the cell image obtained by imaging a plurality of cells, the cells or cell groups detected within the detection limit range of the imaging device The first exposure condition of the imaging device is determined based on the identification and the image of the identified cell or cell group. Next, imaging of a plurality of cells is performed based on the first exposure condition, and the detection limit range of the imaging device is exceeded among the cell groups included in the cell image imaged based on the first exposure condition. The cell group including the area to be photographed is specified, the second exposure condition of the imaging device is determined based on the image of the specified cell group, and the plurality of cells are imaged based on the second exposure condition . Therefore, since it is possible to set the second exposure condition suitable for the cell group including the region exceeding the detection limit range of the imaging device, the above-described stacked cell group is also not saturated. Appropriate cell images can be taken.

  In addition, unlike the conventional automatic exposure control, since it is not necessary to perform imaging at least three times before main photographing, it is possible to shorten the time until the final observation phase difference image is imaged and displayed. it can.

The figure which shows schematic structure of the cell observation system using one Embodiment of the imaging device of this invention The figure which shows schematic structure of the imaging part of the cell observation system shown in FIG. A diagram showing an example of a first phase difference image including a plurality of cell groups Enlarged view of cell group S1 and cell group S2 shown in FIG. 3 Diagram for explaining how to set line segments that cross cell groups Diagram for explaining a method of estimating an image signal of a region exceeding the detection limit range of the imaging device using a Gaussian distribution The figure for demonstrating the method to estimate the image signal of the area | region which exceeds the detection limit range of an image pick-up element using linear approximation. A diagram showing an example of the distribution of image signal values when there are isolated points in an image of a cell group Flow chart for explaining the operation of a cell observation system using an embodiment of the imaging device of the present invention Diagram to explain the case where there are multiple regions exceeding the detection limit in one cell group The figure for demonstrating the determination method of the 2nd exposure condition in case there exist multiple area | regions which exceed the detection limit range in one cell group.

  Hereinafter, a cell observation system using one embodiment of the imaging device and method of the present invention and an imaging control program will be described in detail with reference to the drawings. FIG. 1 is a block diagram showing a schematic configuration of a cell observation system 1.

  As shown in FIG. 1, the cell observation system 1 according to the present embodiment includes an imaging unit 10, an imaging control device 20, a display device 30, and an input device 40.

  The imaging unit 10 captures a phase difference image of the cultured cell group. In addition, the imaging unit 10 performs so-called time-lapse imaging in which a cell group is imaged a plurality of times over time. Specifically, the cells to be imaged include pluripotent stem cells such as iPS (induced pluripotent stem) cells and ES (embryonic stem) cells, nerves derived from stem cells, cells of skin, heart muscle and liver, and human body And skin of the retina, myocardium, blood cells, nerves and organs.

  FIG. 2 is a view showing a schematic configuration of the imaging unit 10. As shown in FIG. As shown in FIG. 2, the imaging unit 10 has a white light source 11 that emits white light, and a ring-shaped slit, and the white light emitted from the white light source 11 is incident to perform so-called phase difference measurement. Slit plate 12 for emitting ring-shaped illumination light L1 and ring-shaped illumination light L1 emitted from slit plate 12 are incident, and the incident ring-shaped illumination light L1 is imaged including cell group S And an objective lens 13 for irradiating the region.

  The slit plate 12 is provided with a ring-shaped slit for transmitting white light to a light shielding plate for shielding white light emitted from the white light source 11, and the white light passes through the slit to form a ring shape. Illumination light L1 is formed.

  The imaging unit 10 further includes a phase difference lens 14, an imaging lens 15, and an imaging element 16.

  The phase difference lens 14 includes an objective lens 14 a and a phase plate 14 b. The phase plate 14 b is formed by forming a phase ring on a transparent plate transparent to the wavelength of the illumination light L 1. The size of the slit of the slit plate 12 described above is in a conjugate relationship with this phase ring.

  In the phase ring, a phase film that shifts the phase of the incident light by 1⁄4 wavelength and a light reduction filter that attenuates the incident light are formed in a ring shape. The direct light incident on the phase ring has its phase shifted by 1⁄4 wavelength by passing through the phase ring, and its brightness is weakened. On the other hand, most of the diffracted light diffracted by the cell group passes through the transparent plate of the phase plate 14b, and its phase and brightness do not change.

  The phase difference lens 14 is moved in the Z direction (the direction of arrow A) by an optical system driving unit (not shown). The autofocus control is performed by the movement of the phase difference lens 14 in the Z direction, and the contrast of the image captured by the imaging element 16 is adjusted.

  The imaging lens 15 receives the diffracted light and the direct light that have passed through the phase difference lens 14 and forms an image on the imaging element 16.

  The imaging element 16 detects the diffracted light and the direct light imaged by the imaging lens 15 and captures a phase difference image. As the imaging device 16, a CCD image sensor, a CMOS image sensor, or the like is used. As an imaging device, an imaging device provided with a RGB (Red Green Blue) color filter may be used, or a monochrome imaging device may be used. In the present embodiment, the phase difference image corresponds to the cell image of the present invention.

  A stage 51 is provided between the objective lens 13 and the phase difference lens 14. Then, on this stage 51, the culture vessel 50 containing the cell group S and the culture solution C is placed. The stage 51 is moved in the X direction and the Y direction orthogonal to each other by a stage driving unit (not shown). A petri dish, a dish, a well plate or the like can be used as the culture vessel 50.

  Returning to FIG. 1, the imaging control device 20 includes a cell image acquisition unit 21, an exposure condition determination unit 22, an imaging control unit 23, and a display control unit 24.

  The imaging control apparatus 20 is one in which an imaging control program according to an embodiment of the present invention is installed in a computer.

  The imaging control device 20 includes a central processing unit, a semiconductor memory, a hard disk, and the like, and one embodiment of the imaging control program of the present invention is installed in the hard disk. Then, the program is executed by the central processing unit to operate the cell image acquisition unit 21, the exposure condition determination unit 22, the imaging control unit 23, and the display control unit 24 as shown in FIG.

  The cell image acquisition unit 21 acquires and stores phase difference images captured a plurality of times with time in the imaging unit 10.

  The exposure condition determination unit 22 determines an exposure condition when imaging each phase difference image in the imaging unit 10. The exposure condition determination unit 22 of the present embodiment determines at least one of the exposure time of the imaging device 16 and the amount of incident light as the exposure condition.

  Specifically, based on the initial exposure condition stored in advance in the exposure condition determination unit 22, the imaging control unit 23 controls the imaging unit 10 to capture a first phase difference image, and the cell image acquisition unit 21 It is acquired.

  The first phase difference image acquired by the cell image acquisition unit 21 is input to the exposure condition determination unit 22, and the exposure condition determination unit 22 identifies the cells or cell groups included in the first phase difference image. As a method of identifying a cell or a cell group, for example, after converting a phase difference image into a binarized image, a cell or a cell group may be automatically detected by template matching or the like. Further, the method of identifying cells or cell groups is not limited to the method described above, and other known methods may be used.

  Next, the exposure condition determination unit 22 specifies the cells or cell groups detected within the detection limit range of the imaging element 16 among the cells or cell groups included in the first phase difference image. The upper limit of the detection limit range is the same as the saturation level of the imaging device 16.

  FIG. 3 shows an example of the first phase contrast image, which includes a plurality of cells and cell groups. Among the cell groups included in the first phase difference image shown in FIG. 3, the cell group in which the cell group not including the area where the brightness is saturated and white is detected within the detection limit range of the imaging element 16 For example, cell group S1 corresponds. On the other hand, a cell group including a region of cells in which the brightness is saturated and white is a cell group including a region (saturation region) exceeding the detection limit range of the imaging element 16, and for example, cell group S2 is applicable. Do. 4I is an enlarged view of cell group S1 shown in FIG. 3, and FIG. 4II is an enlarged view of cell group S2 shown in FIG.

  As a method of identifying the cell group detected within the detection limit range of the imaging device 16, for example, a region of an image signal equal to or more than a preset threshold value is detected in the first phase difference image of each cell group If a region where the area of the region is equal to or greater than a preset threshold is detected as a saturated region, and a cell group not including the saturated region is specified as a cell group detected within the detection limit range. Good. It should be noted that not including the saturated region here does not necessarily mean that the saturated image signal is not zero. For example, the isolated point where the brightness is saturated and white where the brightness is saturated due to the occurrence of halo and the like. A streak or the like may be included, that is, the part other than the cell may include a part saturated in luminance.

  Also, in the cell group consisting of monolayers, the cell area does not exceed the detection limit range, so by identifying the cell groups in the monolayer, the cell groups being detected within the detection limit range should be specified. May be As a method of identifying cell groups in a monolayer, for example, nucleoli appear relatively clearly in phase contrast images of cell populations in monolayer, but in phase contrast images of layered cell populations, nucleoli appear Becomes unclear. Therefore, the nucleoli contained in each cell group of the first phase difference image is detected, the sharpness of the image of the nucleolus is calculated, and the sharpness is equal to or greater than a preset threshold value. It may be specified as a cell group of layers.

  In addition, the cell group of the monolayer includes the above-described white streaks, and the stacked cell group has a relatively small number of white streaks which are included in the cell group of the monolayer due to the lamination. Therefore, white stripes included in each cell group may be detected, and a cell group having an area or density of the white stripes equal to or more than a preset threshold value may be specified as a cell group of a single layer.

  Further, in the above description, the cell group of the monolayer is specified based on the features of the first phase difference image of each cell group, but the present invention is not limited to this, and the first position as shown in FIG. The display control unit 24 causes the display control unit 24 to display the phase difference image, and the user specifies the cell group of the single layer using the input device 40 on the first phase difference image displayed on the display unit 30. May be

  Note that the exposure condition determination unit 22 in the present embodiment specifies a cell group of a single layer as a cell or a cell group detected within the detection limit range of the imaging device 16.

  After identifying the cell group of the monolayer as described above, the exposure condition determination unit 22 determines the first exposure condition based on the first phase difference image of the cell group of the identified monolayer. The first exposure condition is an exposure condition capable of imaging a monolayer cell group more appropriately. As a method of determining the first exposure condition, for example, the maximum value of the image signal of the first phase difference image of the monolayer cell group is 90% to 95% of the upper limit value of the detection limit range of the imaging element 16 The exposure condition may be determined as the first exposure condition, and the average value of the image signals of the first phase difference image of the cell group of the single layer is 40 of the upper limit value of the detection limit range of the imaging device 16. The exposure condition of% to 60% may be determined as the first exposure condition.

  In addition, when the first exposure condition is determined based on the maximum value of the image signal of the monolayer cell group as described above, the luminance is obviously higher than that in the monolayer cell group as compared with the surroundings. There may be isolated points that are elevated. In such a case, when the first exposure condition is determined based on the maximum value of the image signal of the monolayer cell group as described above, the first exposure condition is determined based on the image signal value of the isolated point. It may be determined, and appropriate exposure conditions may not be set.

  Therefore, when the average value, the dispersion value and the maximum value of the image signals of the cell group in a single layer are obtained, and the difference between the maximum value and the value obtained by adding the dispersion value to the average value is within a preset range. That is, when the difference between these values is small, the first exposure condition is determined using the maximum value of the image signal value, and the difference between the maximum value and the value obtained by adding the dispersion value to the average value is previously determined. If it is out of the set range, that is, if it is recognized that the difference between these values is large and the maximum value is clearly the image signal value of the isolated point, the value obtained by adding the variance value to the average value It may be used to determine the first exposure condition. Note that the variance value added to the average value here is the variance value on the positive side.

  Then, based on the first exposure condition determined by the exposure condition determination unit 22, the imaging control unit 23 controls the imaging unit 10 to capture a second phase difference image, which is acquired by the cell image acquisition unit 21. Ru.

  The second phase difference image acquired by the cell image acquisition unit 21 is input to the exposure condition determination unit 22, and the exposure condition determination unit 22 specifies a cell group included in the second phase difference image. The method of identifying the cell group is as described above.

  Next, the exposure condition determination unit 22 specifies a cell group including a region exceeding the detection limit range of the imaging element 16 among the cell groups included in the second phase difference image. That is, a cell group including a region exceeding the saturation level of the imaging device 16 is identified. As a method of identifying a cell group exceeding the detection limit range of the imaging device 16, for example, as in the above, a saturated region is detected in the second phase difference image of each cell group, and the saturated region is included. A group of cells may be identified as a group of cells exceeding the detection limit range.

  Then, after the exposure condition determination unit 22 identifies the cell group including the region exceeding the detection limit range as described above, a second phase difference image of the identified cell group is used. Determine the exposure conditions. The second exposure condition is an exposure condition in which the image signal value of the region exceeding the detection limit range of the image sensor 16 is less than the upper limit value of the detection limit range. Hereinafter, the method of determining the second exposure condition will be described in detail.

  First, as shown in FIG. 5, the exposure condition determination unit 22 obtains the center of gravity G of the cell group S3 including the region SR exceeding the detection limit range, and includes a line segment L including the center of gravity G and crossing the cell group S3. Set The line segment L is preferably the major axis of the cell group S3. The major axis is a line segment having the longest distance between the intersections of the contour of the cell group S3 and the line segment among the line segments crossing the cell group S3. By setting the major axis in this manner, it is possible to improve the accuracy of estimation of the image signal in the saturation region described later.

  Then, the exposure condition determination unit 22 plots the value of the image signal on the line segment L, as shown in the upper part of FIG. The vertical axis in the upper part of FIG. 6 is the magnitude of the image signal value, and the horizontal axis is the position of the pixel on the line segment L. Further, LV shown in FIG. 6 is the upper limit value of the detection limit range of the imaging device 16. As shown in the upper part of FIG. 6, all the image signals in the region SR shown in FIG. 5 are saturated and have the same value as the upper limit value LV.

Next, the exposure condition determination unit 22 estimates the original image signal value SV on the line segment L in the region SR based on the image signal value on the line segment L, as shown in the lower part of FIG. The image signal value SV is
It is an image signal value estimated to be acquired when light emitted from the region SR is detected when it is considered that there is no upper limit of the detection limit range of the imaging device 16. The image signal value SV is estimated, for example, such that the distribution of the value of the image signal on the line segment L becomes a Gaussian distribution. The method of estimating the image signal value SV on the line segment L in the region SR is not limited to the method using a Gaussian distribution, but, for example, as shown in FIG. 7, based on the distribution of values of image signals other than the region SR. The value of the image signal of the region SR may be estimated by linear approximation. As described above, by using the Gaussian distribution or the linear approximation, the image signal can be estimated by a simple process.

  Then, the exposure condition determination unit 22 determines the second exposure condition using the image signal value SV estimated in the region SR as described above. Specifically, for example, the maximum value of the estimated image signal value SV in the region SR and the image signal value on the line segment L other than the region SR is 90% to 90% of the upper limit value LV of the detection limit range of the imaging device 16 An exposure condition which is 95% is determined as a second exposure condition. This can suppress saturation of the image signal of the region SR. In addition, the exposure condition in which the average value of the estimated image signal value SV in the region SR and the image signal value on the line segment L other than the region SR is 40% to 60% of the upper limit LV of the detection limit range. It may be determined as the second exposure condition.

  Since the second exposure condition is determined based on the image signal value on the line segment L set to the cell group as described above, the second exposure condition may be determined by a simple calculation process. it can.

  Further, as described above, the second exposure condition or exposure condition candidate is determined based on the estimated image signal value SV in the region SR and the maximum value of the image signal value on the line segment L other than the region SR. In such a case, there may be an isolated point on the line segment whose luminance is obviously higher than that of the surroundings. Such an isolated point appears when, for example, dead cells exist in the cell group. The image signal value IP at this isolated point is considered to have a delta function distribution as shown in FIG. In such a case, when the image signal value IP exceeding the upper limit LV is estimated also for the image signal value IP of the isolated point, the estimated image signal value may diverge to infinity. Therefore, when the second exposure condition or exposure condition candidate is determined based on the maximum value of the image signal value as described above, an appropriate condition is set under the influence of the image signal value estimated for the isolated point. It may not be.

  Therefore, the average value, the variance value and the maximum value of the image signal value estimated for the isolated point, the estimated image signal value SV in the region SR, and the image signal value on the segment L other than the isolated point and the region SR are respectively If the difference between the maximum value and the value obtained by adding the variance value to the average value is within a preset range, that is, if the difference between these values is small, the maximum value of the image signal value is used. Second exposure condition or exposure condition candidate is determined, and if the difference between the maximum value and the value obtained by adding the variance value to the average value is out of the preset range, that is, the difference between these values If it is recognized that the maximum value is clearly an image signal value of an isolated point, the second exposure condition or exposure condition candidate is determined using a value obtained by adding the variance value to the average value. Just do it. Note that the variance value added to the average value here is the variance value on the positive side.

  The imaging control unit 23 controls the imaging operation of the imaging unit 10. Specifically, the imaging control unit 23 controls the amount of light emitted from the white light source 11, the movement of the stage 51 in the XY direction, the movement of the phase difference lens in the Z direction, and the exposure operation of the imaging element 16. is there. In particular, the imaging control unit 23 determines the amount of light emitted from the white light source 11 and the image pickup device 16 based on the preset exposure condition, the first exposure condition and the second exposure condition determined by the exposure condition determination unit 22. Control the exposure time of the

  The exposure conditions include the exposure time of the imaging device 16 and the incident light amount to the imaging device 16 as described above, but in the present embodiment, the emission light amount from the white light source 11 is controlled as described above. The amount of light incident on the imaging device 16 is controlled. The control of the amount of light incident on the imaging device 16 is not limited to the control of the amount of emitted light from the white light source 11. For example, the imaging unit 10 may be a stop for limiting the amount of white light emitted from the white light source 11. When the light amount limiting unit is provided, the light amount limiting unit may be controlled to control the amount of light incident on the imaging device 16. Both the exposure time of the imaging device 16 and the amount of light incident on the imaging device 16 may be controlled.

  Then, the imaging control unit 23 according to the present embodiment controls the second phase difference image based on the first exposure condition until the cell group including the area exceeding the detection limit range of the imaging element 16 is detected. When a cell group including a region exceeding the detection limit range of the imaging device 16 is detected, the third phase difference image is captured based on the second exposure condition. The third phase-contrast image can be made free of the saturated region also for the layered cell group, and each cell of the cell group can be appropriately observed.

  The display control unit 24 causes the display device 30 to display the phase difference image acquired by the cell image acquisition unit 21. In addition, the first and second exposure conditions determined in the exposure condition determining unit 22 and the estimated image signal value SV shown in FIGS. 6 and 7 may be displayed on the display device 30. In addition, a warning message or the like may be displayed on the display device 30 to notify the user that a cell group including a region exceeding the detection limit range of the imaging device 16 has been detected.

  The display device 30 displays the phase difference image and the like acquired by the cell image acquisition unit 21, and includes, for example, a liquid crystal display. Further, the display device 30 may be configured by a touch panel and used as the input device 40.

  The input device 40 includes a mouse, a keyboard, and the like, and receives various setting inputs from the user. The input device 40 of the present embodiment receives, for example, the setting input of the above-described initial exposure condition.

  Next, the operation of the cell observation system 1 of the present embodiment will be described with reference to the flowchart shown in FIG.

  First, initial exposure conditions are set by the user using the input device 40 (S10). The imaging control unit 23 controls the imaging unit 10 based on the initial exposure condition, and the first phase difference image imaged by the imaging unit 10 is acquired by the cell image acquisition unit 21 (S12).

  The first phase difference image acquired by the cell image acquisition unit 21 is acquired by the exposure condition determination unit 22, and the exposure condition determination unit 22 specifies the cell group of the monolayer included in the first phase difference image (FIG. S14). Then, the exposure condition determination unit 22 determines, based on the first phase difference image of the monolayer cell group, the first exposure condition capable of imaging the monolayer cell group more appropriately (S16). .

  The first exposure condition determined by the exposure condition determination unit 22 is acquired by the imaging control unit 23, and the imaging control unit 23 controls the imaging unit 10 based on the first exposure condition, and the second imaging condition is determined by the imaging unit 10. The second phase difference image is acquired by the cell image acquisition unit 21 (S18).

  The second phase difference image acquired by the cell image acquisition unit 21 is input to the exposure condition determination unit 22, and the exposure condition determination unit 22 identifies a cell group included in the second phase difference image, and identifies the cell group. Among the group of cells, a group of cells including a region exceeding the detection limit of the imaging device 16 is specified.

  Then, when the second phase difference image includes a cell group including a region exceeding the detection limit range, the exposure condition determination unit 22 determines the image signal value of the cell group based on the image signal value of the cell group. A second exposure condition is determined (S20, YES).

  Specifically, the exposure condition determination unit 22 sets the major axis passing through the center of gravity of the identified cell group, plots the image signal value on the major axis, and makes the above description based on the plotted image signal value. Then, the image signal value in the saturated region is estimated (S22), and the second exposure condition is determined using the image signal value in the estimated saturated region (S24).

  The second exposure condition determined by the exposure condition determination unit 22 is acquired by the imaging control unit 23. The imaging control unit 23 controls the imaging unit 10 based on the second exposure condition, and the imaging unit 10 performs a third operation. The third phase difference image is acquired by the cell image acquisition unit 21 (S26). The third phase difference image acquired by the cell image acquisition unit 21 is output to the display control unit 24, and the display control unit 24 causes the display device 30 to display the third phase difference image (S28).

  When the second phase difference image captured by the imaging unit 10 based on the first exposure condition does not include a cell group including a region exceeding the detection limit range of the imaging element 16. (S20, NO), the second phase difference image is output to the display control unit 24, and the display control unit 24 causes the display device 30 to display the second phase difference image (S30).

  According to the cell observation system 1 of the above embodiment, among the cells or cell groups included in the cell image obtained by imaging a plurality of cells, the cells or cell groups detected within the detection limit range of the imaging device 16 are specified Based on the image of the specified cell or cell group, the first exposure condition of the imaging device 16 is determined. Next, imaging of a plurality of cells is performed based on the first exposure condition, and the detection limit range of the imaging element 16 in the cell group included in the cell image imaged based on the first exposure condition is A cell group including a region that exceeds the area is identified, a second exposure condition of the imaging device 16 is determined based on the image of the identified cell group, and imaging of a plurality of cells is performed based on the second exposure condition. I do. Therefore, since the second exposure condition suitable for the cell group including the region exceeding the detection limit range of the imaging device can be set, an appropriate cell image can be obtained without saturation even for the laminated cell group. Can be taken.

  Further, unlike the conventional automatic exposure control, since it is not necessary to perform imaging at least three times before main imaging, the time until the final phase difference image for observation can be displayed can be shortened. .

  In the cell observation system 1 of the above-described embodiment, a plurality of regions exceeding the detection limit range may be included in the cell group identified in the second phase contrast image. Specifically, as shown in FIG. 10, there may be a case where a region SR1 and a region SR2 exceeding the detection limit range are included in the cell group S4. FIG. 11 is a diagram in which image signal values on the line segment L set to the cell group S4 are plotted. In this case, the exposure condition determination unit 22 estimates the image signal value SV1 of the region SR1 and the image signal value SV2 of the region SR2 using the Gaussian distribution or the like as described above, and these estimated image signals The second exposure condition may be determined based on the values SV1 and SV2. Specifically, for example, an exposure condition in which the maximum value among the image signal values of both the image signal value SV1 and the image signal value SV2 is 90% to 95% of the upper limit value LV of the detection limit range of the imaging device 16 It may be determined as the second exposure condition. The average value of the image signal value SV2 of the region SR1, the image signal value SV2 of the region SR2, and the image signals on the line segment L other than the region SR1 and the region SR2 is 40% to 60% of the upper limit LV of the detection limit range. The exposure condition of% may be determined as the second exposure condition.

  Moreover, in the cell observation system 1 of the said embodiment, two or more cell groups including the area | region which exceeds the detection limit range may be contained in a 2nd phase contrast image. In such a case, the exposure condition determination unit 22 determines exposure condition candidates corresponding to each cell group based on the phase difference image of each identified cell group, and sets the determined multiple exposure condition candidates. Based on the above, the second exposure condition may be determined.

  More specifically, the exposure condition determination unit 22 estimates the image signal value of the saturated region based on the image signal value of each cell group, and exposes each cell group based on the estimated image signal value. Determine condition candidates. Then, for example, the average value of the plurality of exposure condition candidates determined may be determined as the second exposure condition. Further, the minimum value may be used instead of the average value of the plurality of exposure condition candidates. Alternatively, the exposure condition candidate determined based on the image of the cell group having the largest maximum value of the image signal value of the estimated saturated region among the plurality of exposure condition candidates is determined as the second exposure condition. It is also good.

  Further, in the cell observation system 1 of the above embodiment, when the second exposure condition is determined, the major axis passing through the center of gravity of the cell group is set as a line segment crossing the cell group including the region exceeding the detection limit range. However, the present invention is not limited to this. For example, a region exceeding the detection limit range may be detected, and a line segment passing the center of gravity of the detection limit range may be set. As a method of detecting the detection limit range, a region where the image signal value has reached the saturation level may be detected. Also in this case, it is preferable to set the major axis passing through the center of gravity of the detection limit range. In addition, when there are a plurality of regions exceeding the detection limit range in one cell group, it is preferable to set a line segment passing the center of gravity of the region having the largest area of the detection limit range.

  Further, in the cell observation system 1 of the above embodiment, when determining the second exposure condition, one line segment is set for one cell group, but the number is not limited to one, and a plurality of line segments are used. A line segment of a book may be set. In that case, the image signal values on the respective line segments are plotted, and the image signal values in the region exceeding the detection limit range are estimated based on the image signal values on the respective line segments as in the above embodiment. Just do it. Then, as in the above embodiment, the second exposure condition is determined based on the maximum value or the average value of the image signal values excluding the saturated region on each line segment and the estimated image signal values. do it.

  Further, in the cell observation system 1 of the above embodiment, when the second exposure condition is determined, the maximum value of the image signal value estimated for the region exceeding the detection limit range is the upper limit value of the detection limit range. Although the exposure condition of 90% to 95% is determined as the second exposure condition, the ratio to the upper limit value may be changed according to the area of the region exceeding the detection limit range. Good. That is, since the estimation accuracy of the estimated image signal value increases as the area of the area exceeding the detection limit decreases, the ratio to the upper limit value increases as the area of the area exceeding the detection limit decreases. You may Specifically, for example, when the area of the region exceeding the detection limit range is equal to or less than a preset threshold value, the maximum value of the estimated image signal value is 95% of the upper limit value of the detection limit range. If the area of the region exceeding the detection limit range is less than the threshold, the maximum value of the estimated image signal value is the upper limit value of the detection limit range. The exposure condition which becomes 90% may be determined as the second exposure condition.

  Further, in the cell observation system 1 of the above embodiment, the imaging based on the second exposure condition is performed only once, but the imaging based on the second exposure condition is performed multiple times, and the imaging is performed multiple times. The plurality of third phase difference images acquired by the above may be added and displayed. As a result, the regions other than the region exceeding the detection limit range can be clearly observed without becoming dark.

  Further, in the cell observation system 1 according to the above embodiment, the imaging unit 10 captures a phase difference image, but not limited to the phase difference image, for example, a bright field image, a differential interference image or a fluorescence image as a cell image The image may be taken.

Reference Signs List 1 cell observation system 10 imaging unit 11 white light source 12 slit plate 13 objective lens 14 phase difference lens 14a objective lens 14b phase plate 15 imaging lens 16 imaging element 20 imaging control device 21 cell image acquisition unit 22 exposure condition determination unit 23 imaging control Unit 24 Display control unit 30 Display device 40 Input device 50 Culture vessel 51 Stage

Claims (12)

An imaging unit for imaging a plurality of cells by an imaging device;
Among the cells or cell groups included in the cell image captured by the imaging unit, a monolayer cell or cell group is identified, and the maximum value of the image signal of the identified monolayer cell or cell group image , An exposure condition determination unit that determines a first exposure condition in which at least one of the average values falls within the detection limit range of the image sensor ;
The imaging unit performs imaging of the plurality of cells based on the first exposure condition;
The exposure condition determination unit identifies a cell group including a region exceeding the detection limit range of the imaging element among the cell groups included in the cell image captured based on the first exposure condition, and Based on the image of the identified cell group, the magnitude of the image signal on the line segment crossing the region exceeding the detection limit range is estimated using either Gaussian distribution or linear approximation, Determining a second exposure condition in which at least one of the maximum value of the image signal and the average value falls within the detection limit range of the image sensor ;
An imaging apparatus, wherein the imaging unit performs imaging of the plurality of cells based on the second exposure condition. An average value of an image signal within the detection limit range and the estimated image signal among image signals of images of a cell group including a region exceeding the detection limit range of the imaging device, the exposure condition determination unit; When the dispersion value and the maximum value are acquired, and the difference between the value obtained by adding the dispersion value to the average value and the maximum value is within a preset range, the second value is calculated based on the maximum value. of determining the exposure conditions, if the difference is outside the range of the preset based on the value obtained by adding the variance value to the average value, according to claim 1, wherein determining the second exposure condition Imaging device. The exposure condition determination unit sets a line segment crossing a cell group including a region exceeding the detection limit range, and determines the second exposure condition based on an image signal on the line segment. The imaging device according to 1 or 2 . The imaging apparatus according to claim 3, wherein the exposure condition determination unit sets a major axis of a cell group including a region exceeding the detection limit range as the line segment. The imaging device according to 3 or 4, wherein the exposure condition determination unit detects an area exceeding the detection limit range, and sets the line segment passing the center of gravity of the detected one area. When a plurality of areas exceeding the detection limit range of the imaging device are included in one cell group, the exposure condition determining unit performs a plurality of exposures of the imaging device based on the image signal estimated for each of the areas. The imaging apparatus according to any one of claims 1 to 5 , wherein a condition candidate is determined, and one of an average value and a minimum value of the plurality of determined exposure condition candidates is determined as the second exposure condition. When a plurality of areas exceeding the detection limit range of the imaging device are included in one cell group, the exposure condition determining unit performs a plurality of exposures of the imaging device based on the image signal estimated for each of the areas. determining the candidate conditions, among the plurality of exposure conditions candidates the determined billing for determining the exposure condition candidate which is determined based on the image signal of the estimated image signal is the largest area as the second exposure condition The imaging device according to any one of Items 1 to 5 . When the exposure condition determination unit identifies a plurality of cell groups including a region exceeding the detection limit range of the imaging element, a plurality of exposure condition candidates of the imaging element are determined based on the images of the identified cell groups The imaging apparatus according to any one of claims 1 to 5 , wherein one of the average value and the minimum value of the plurality of exposure condition candidates determined is determined as the second exposure condition. When the exposure condition determination unit identifies a plurality of cell groups including a region exceeding the detection limit range of the imaging element, a plurality of exposure condition candidates of the imaging element are determined based on the images of the identified cell groups determining the among the plurality of exposure conditions candidates said determined claims to determine the exposure condition candidates determined based on the image of the estimated image signal is the largest group of cells as the second exposure condition 1 5. An imaging device according to any one of 5 ). The imaging apparatus according to any one of claims 1 to 9, wherein the first and second exposure conditions are at least one of an exposure time and an incident light amount of the imaging element. An imaging method for imaging a plurality of cells by an imaging device, comprising:
Among the cells or cell groups included in the cell image obtained by imaging the plurality of cells, a monolayer cell or cell group is identified,
Determining a first exposure condition in which at least one of the maximum value and the average value of the image signal of the image of the cell or cell group of the specified monolayer is within the detection limit of the imaging device ;
Imaging the plurality of cells based on the first exposure condition;
Among the cell groups included in the cell image imaged based on the first exposure condition, a cell group including a region exceeding the detection limit range of the imaging device is specified,
Based on the image of the identified cell group, the magnitude of the image signal on the line segment crossing the region exceeding the detection limit range is estimated using either Gaussian distribution or linear approximation, and the estimation is performed. Determining a second exposure condition in which at least one of the maximum value of the image signal and the average value falls within the detection limit range of the image sensor ;
An imaging method comprising: imaging the plurality of cells based on the second exposure condition. An imaging control program that causes a computer to function as an imaging control unit that controls an imaging unit that images a plurality of cells by an imaging device, and an exposure condition determination unit that determines an exposure condition of the imaging device,
The exposure condition determining unit, of the cell or cells included in the captured cell image by the imaging unit, the monolayer cells or to identify groups of cells, of the specific monolayer of cells or cell populations image Determining a first exposure condition in which at least one of the maximum value of the image signal and the average value falls within the detection limit range of the image sensor ;
The imaging control unit performs imaging of the plurality of cells based on the first exposure condition by controlling the imaging unit.
The exposure condition determination unit identifies a cell group including a region exceeding the detection limit range of the imaging element among the cell groups included in the cell image captured based on the first exposure condition, and Based on the image of the identified cell group, the magnitude of the image signal on the line segment crossing the region exceeding the detection limit range is estimated using either Gaussian distribution or linear approximation, Determining a second exposure condition in which at least one of the maximum value of the image signal and the average value falls within the detection limit range of the image sensor ;
An imaging control program, wherein the imaging control unit performs imaging of the plurality of cells based on the second exposure condition by controlling the imaging unit.