JP6499506B2 - Imaging apparatus and method, and imaging control program - Google Patents

Description

  The present invention relates to an imaging apparatus and method and an imaging control program for moving an imaging target range within an imaging region including a cell group and capturing an image for each imaging target range.

  Conventionally, pluripotent stem cells such as ES (Embryonic Stem) cells and iPS (Induced Pluripotent Stem) cells and differentiation-induced cells are imaged with a microscope, etc. A determination method has been proposed.

  Pluripotent stem cells such as ES cells and iPS cells have the ability to differentiate into cells of various tissues, and are attracting attention as being applicable in regenerative medicine, drug development and disease elucidation, etc. Yes.

  On the other hand, when a cell is imaged with a microscope as described above, in order to obtain a high-power wide-field image, for example, an imaging region such as a well is divided into a plurality of imaging target ranges, and each of the imaging target ranges at a high magnification It has been proposed to perform so-called tiling photography that combines images for each imaging target range after imaging (see Patent Document 1).

JP 2008-76088 A

  Here, when performing tiling photography, automatic exposure control may be performed for each imaging target range. Automatic exposure control refers to imaging of each imaging target range under a plurality of exposure conditions to obtain a plurality of images before actual shooting of each imaging target range, and based on the plurality of images, This is a method for setting appropriate exposure conditions.

  However, for example, when imaging a cell group (also referred to as a cell colony) in which a plurality of cells are densely populated, the cell group matures and a region where cells are stacked is present in the cell group or locally in the cell group. Cell differentiation may occur.

  When there is an area where cells are stacked in a cell group, for example, when imaging is performed with a phase contrast microscope, the diffracted light generated by the cell causes multiple scattering in the cell group and is the same as the direct light. As a result, the stacked area locally becomes a high-luminance area. Further, when cell differentiation occurs locally in the cell group, the region is locally a low luminance region.

  As described above, when local high-brightness areas and low-brightness areas exist in the cell group, when automatic exposure control is performed, these areas are compared with areas other than the high-brightness areas or low-brightness areas. There are cases where the exposure conditions become inappropriate. As a result, there is a problem that luminance unevenness occurs in the same cell group when a tiling image is generated by combining images for each imaging target range.

  In order to avoid the above problem, when performing tiling shooting, a method of manually setting an exposure time before shooting and imaging the entire imaging region under the same exposure condition can be considered.

  However, the degree of cell growth may be different for each cell group existing in the imaging region, that is, appropriate exposure conditions may be different for each cell group. In such a case, if the entire imaging region is imaged under the same exposure condition, it is difficult to image all cell groups under an appropriate exposure condition.

  In view of the above problems, the present invention provides an imaging apparatus and method, and an imaging control program capable of imaging each cell group under appropriate exposure conditions without causing luminance unevenness in an image of the same cell group. The purpose is to do.

  The imaging device of the present invention is included in an imaging unit that moves an imaging target range within an imaging region including a cell group and captures an image for each imaging target range, and a cell image of the imaging region captured by the imaging unit. A region information acquisition unit that acquires information on the region of the cell group, and an exposure condition acquisition unit that acquires an exposure condition for each cell group. A plurality of imaging target ranges are imaged according to exposure conditions for each cell group.

  In the imaging device of the present invention, the region information acquisition unit can acquire information on the region of the cell group based on the cell image.

  In the imaging device of the present invention, the exposure condition acquisition unit can determine the exposure condition for each cell group based on the cell image.

  In the imaging device of the present invention, the exposure condition acquisition unit can determine the exposure condition based on the histogram of the image signal of the cell image for each cell group.

  In the imaging device of the present invention, the exposure condition acquisition unit can determine the exposure condition based on the spatial frequency distribution of the cell image for each cell group.

  Further, in the imaging apparatus of the present invention, the exposure condition acquisition unit applies to the image signal of the cell image excluding the region that is included in the cell image for each cell group and the dispersion value of the image signal is equal to or greater than the threshold value. Based on this, the exposure conditions can be determined.

  In the imaging apparatus of the present invention, when the imaging target range includes a plurality of cell groups, the imaging unit can capture the imaging target range under the exposure condition of the cell group having the largest area.

  In the imaging apparatus of the present invention, the imaging unit can capture an imaging region including a cell group a plurality of times over time, and the exposure condition acquisition unit is the Nth time when N is a natural number of 1 or more. Based on the cell images of each past cell group imaged so far, the exposure condition for each of the (N + 1) th cell groups can be determined.

  Further, in the imaging apparatus of the present invention, the imaging unit has a magnification for performing imaging according to exposure conditions for each cell group, rather than a magnification for capturing a cell image for acquiring information on a region of the cell group. The image can be taken at a high level.

  In the imaging apparatus of the present invention, it is desirable that the exposure condition is at least one of an exposure time and an incident light amount of an imaging element included in the imaging unit.

  The imaging device of the present invention may further include an image combining unit that generates a cell image of the imaging region by combining images for each imaging target range.

  An imaging method of the present invention is an imaging method for moving an imaging target range within an imaging region including a cell group and imaging an image for each imaging target range, and the cell group included in a cell image obtained by imaging the imaging region And acquiring exposure conditions for each cell group, and imaging a plurality of imaging target ranges in the cell group based on the exposure conditions for each cell group based on the information on the cell group area. And

  The imaging control program of the present invention controls the imaging unit to move the imaging target range within the imaging region including the cell group, and the imaging control unit captures an image for each imaging target range, and the imaging unit captures the image. An imaging control program for causing a computer to function as an area information acquisition unit that acquires information on a region of a cell group included in a cell image of an imaging region and an exposure condition acquisition unit that acquires an exposure condition for each cell group, The imaging control unit is configured to image a plurality of imaging target ranges in the cell group based on exposure conditions for each cell group based on information on the region of the cell group.

  According to the imaging apparatus and method and the imaging control program of the present invention, an imaging area including a cell group is imaged, information on the area of the cell group included in the cell image of the imaging area is acquired, and each cell group Get exposure conditions. Then, when the imaging target range is moved within the imaging region and an image for each imaging target range is taken, a plurality of imaging target ranges in the cell group are determined for each cell group based on the information on the region of the cell group. Therefore, each cell group can be imaged under appropriate exposure conditions without causing luminance unevenness in an image of the same cell group.

The block diagram 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. Schematic diagram showing an example of an imaging region including a cell group and an imaging target range in the imaging region The figure which shows an example of the imaging target range in the case of low magnification imaging, and the imaging target range in the case of high magnification imaging The flowchart for demonstrating the effect | action of the cell observation system using one Embodiment of the imaging device of this invention. The figure for demonstrating the method of estimating the image signal value of the cell group imaged next time from the image signal value of the cell group imaged in the past

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

  As shown in FIG. 1, the cell observation system 1 of this 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. The imaging unit 10 performs so-called time-lapse imaging in which a cell group is imaged a plurality of times over time. Specific examples of cells to be imaged include pluripotent stem cells such as iPS cells and ES cells, nerves, skin, myocardium and liver cells derived from stem cells, and skin, retina and myocardium extracted from the human body. , Blood cells, nerves and organ cells.

  FIG. 2 is a diagram illustrating a schematic configuration of the imaging unit 10. As shown in FIG. 2, the imaging unit 10 includes 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. The ring-shaped illumination light L1 emitted from the slit plate 12 and the ring-shaped illumination light L1 emitted from the slit plate 12 are incident, and the incident ring-shaped illumination light L1 is imaged including the cell group S. And an objective lens 13 for irradiating the region.

  The slit plate 12 is provided with a ring-shaped slit that transmits white light to the light-shielding plate that blocks the white light emitted from the white light source 11, and the ring shape is obtained when the white light passes through the slit. Illumination light L1 is formed.

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

  The phase difference lens 14 includes an objective lens 14a and a phase plate 14b. The phase plate 14b is obtained by forming a phase ring on a transparent plate that is transparent with respect to the wavelength of the illumination light L1. Note that 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 incident light by ¼ wavelength and a neutral density filter that attenuates incident light are formed in a ring shape. When the direct light incident on the phase ring passes through the phase ring, the phase is shifted by a quarter wavelength, and the brightness is weakened. On the other hand, most of the diffracted light diffracted by the cell group S 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 (arrow A direction) by an optical system driving unit (not shown) based on a control signal output from an imaging control unit 25 described later. 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 image sensor 16 is adjusted.

  In addition, the imaging unit 10 of the present embodiment is configured to be able to replace a plurality of phase difference lenses 14 having different magnifications. The phase difference lens 14 may be replaced automatically according to an instruction input from the user, or may be replaced manually by the user.

  In the present embodiment, low-magnification imaging for specifying a region of a cell group included in the imaging region and high-magnification imaging for acquiring a phase difference image for observation are performed. 1 to 4 times the phase difference lens 14 is used, and 10 to 20 times the phase difference lens 14 is used for high magnification imaging. However, the low magnification imaging and the high magnification imaging only need to have relatively different magnifications, and are not limited to these magnifications.

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

  The imaging device 16 detects a diffracted light and direct light imaged by the imaging lens 15 and images a phase difference image. As the imaging element 16, a CCD (Charge-Coupled Device) image sensor, a CMOS (Complementary Metal-Oxide Semiconductor) image sensor, or the like is used. As the image sensor, an image sensor provided with an RGB (Red Green Blue) color filter may be used, or a monochrome image sensor 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, a culture vessel 50 in which the observation object S and the culture medium C are accommodated is installed. The stage 51 is moved in the X direction and the Y direction orthogonal to each other by a stage driving unit (not shown) based on a control signal output from the imaging control unit 25 described later. As the culture vessel 50, a petri dish, a dish, a well plate, or the like can be used.

  Then, when the stage 51 moves in the X direction and the Y direction, the imaging target range is moved and changed within the imaging region including the observation target S. FIG. 3 is a diagram schematically illustrating an example of the imaging region R including the cell groups S1 to S3 as the observation target S and the imaging target range IR1 in the imaging region R. The position of the imaging target range IR1 is changed by the movement of the stage 51 in the X direction and the Y direction, and a phase difference image for each imaging target range IR1 is captured.

  In the present embodiment, as described above, low-magnification imaging and high-magnification imaging are performed, but the imaging target range in the case of high-magnification imaging is narrower than the imaging target range in the case of low-magnification imaging. It becomes. FIG. 4 shows an example of an imaging target range in the case of low magnification imaging and an imaging target range in the case of high magnification imaging. In FIG. 4, a rectangular range indicated by a solid line is an imaging target range IR1 in the case of low magnification imaging, and a rectangular range indicated by a dotted line is an imaging target range IR2 in the case of high magnification imaging.

  Returning to FIG. 1, the imaging control device 20 includes a cell image acquisition unit 21, an image combination unit 22, a region information acquisition unit 23, an exposure condition acquisition unit 24, an imaging control unit 25, and a display control unit 26. I have.

  The imaging control device 20 is obtained by installing an embodiment of an imaging control program of the present invention on a computer.

  The imaging control device 20 includes a central processing unit, a semiconductor memory, a hard disk, and the like, and an embodiment of the imaging control program of the present invention is installed on the hard disk. When this program is executed by the central processing unit, a cell image acquisition unit 21, an image combination unit 22, an area information acquisition unit 23, an exposure condition acquisition unit 24, an imaging control unit 25, and the like as shown in FIG. The display control unit 26 operates.

  The cell image acquisition unit 21 acquires and stores phase difference images captured a plurality of times over time in the imaging unit 10. In addition, the imaging unit 10 according to the present embodiment moves the imaging target ranges IR1 and IR2 in the imaging region R as described above, and captures phase difference images for the imaging target ranges IR1 and IR2. The cell image acquisition unit 21 adds position information in the imaging region R to the phase difference images for the imaging target ranges IR1 and IR2 in the imaging region R, and a plurality of imagings included in the same imaging region R. The phase difference images of the target range IR1 or the plurality of imaging target ranges IR2 are stored in association with each other.

  The image combining unit 22 generates a single phase difference image of the entire imaging region by combining the phase difference images for each imaging target range.

  The region information acquisition unit 23 acquires information on the region of the cell group included in the phase difference image captured by the imaging unit 10. In addition, a cell group is a cell assembly in which a plurality of cells are gathered together, and is also referred to as a cell colony.

  Specifically, for example, when a phase difference image of the imaging region R as illustrated in FIG. 3 is captured, the region information acquisition unit 23 acquires region information of the cell groups S1 to S3 included in the phase difference image. To do. The region information acquisition unit 23 of the present embodiment acquires cell group region information included in the phase difference image acquired by low-magnification imaging. Since low-magnification imaging can be performed at a higher speed than high-magnification imaging, it is possible to acquire cell group region information earlier.

  The cell group region information may be information that can identify the cell group region, and may be a coordinate value of a pixel representing the boundary of the cell group, or a coordinate value of all the pixels included in the cell group region. But you can. Further, the position information in the imaging region R of the imaging target range IR1 including the cell group region may be used.

  As a method for specifying the regions of the cell groups S1 to S3, for example, after the phase difference image is converted into a binarized image, the cell group may be automatically detected by template matching or the like. Further, the method for specifying the cell group region is not limited to the method described above, and other known methods may be used. In the present embodiment, the area information of the cell group is automatically acquired based on the characteristics of the phase difference image. However, the present invention is not limited to this, and the phase difference image acquired by low magnification imaging is displayed. The user may specify the region of the cell group included in the displayed phase difference image by using the input device 40. That is, the user may manually acquire the region information of the cell group.

  The exposure condition acquisition unit 24 acquires the exposure condition for each cell group specified by the region information acquisition unit 23. Specifically, the exposure condition acquisition unit 24 of the present embodiment acquires a phase difference image acquired by low magnification imaging and region information of each cell group included in the phase difference image, and low magnification for each cell group. Based on the phase difference image, exposure conditions suitable for each cell group are acquired. The exposure condition acquisition unit 24 of the present embodiment acquires at least one of the exposure time of the image sensor 16 and the incident light amount as the exposure condition.

  For example, the exposure conditions are determined based on statistical values such as the average value, maximum value, minimum value, mode value, and combination of average value and variance value of the image signal of the phase difference image of each cell group. What is necessary is just to determine the exposure time or incident light quantity of the image pick-up element 17 suitable for imaging of a group. More specifically, for example, an exposure condition in which the average value of the image signal of the phase difference image of the cell group falls within a preset range is determined, or the maximum value of the image signal of the phase difference image of the cell group is determined in advance. What is necessary is just to determine the exposure conditions which become below the set threshold value.

  In addition, there may be isolated points in the phase difference image of the cell group that are clearly brighter than the surroundings, such as when the cell group contains dust such as dead cells. . In such a case, if the exposure condition is acquired including the image signal of the isolated point, an appropriate exposure condition may not be acquired. Therefore, for example, a histogram of the image signal of the phase difference image of the cell group is generated, the image signal that is out of the distribution set of the image signal is specified as the image signal of the isolated point, and the exposure condition is determined by excluding the specified image signal. May be obtained. Alternatively, the exposure condition may be determined using only an image signal within a preset range in the histogram.

  In addition, as the proliferation of the cell group progresses, the cells are stacked, thereby generating a region that is brighter than the surroundings in the cell group. In such a case, for example, when the exposure condition is acquired based on the average value of the image signal of the cell group and imaging is performed, the stacked region may be saturated white and the cell state may not be observed. Therefore, the exposure condition may be acquired based on the maximum value of the image signal of the stacked region as described above. As for the stacked region, for example, when image signals equal to or higher than a preset signal threshold value are distributed in a region equal to or higher than a preset area threshold value, the region is detected as a stacked region. You can do it.

  In addition, when the differentiation of cells included in the cell group proceeds, a darker hole than the surrounding may be formed in the cell group. In such a case, for example, when the exposure condition is acquired based on the average value of the image signal of the cell group and imaging is performed, the hole region may be too dark to observe the cell state. Therefore, the exposure condition may be acquired based on the minimum value of the image signal of the hole area as described above. As for the hole region, for example, when image signals equal to or smaller than a preset signal threshold value are distributed in a region equal to or larger than a preset area threshold value, the region may be detected as a hole region.

  Further, when the proliferation of the cell group proceeds, halo occurs in the cell group at the peripheral portion, and white stripes brighter than the surroundings may occur. Halo is generated due to diffracted light passing between cells. In such a case, when the exposure condition is determined including the image signal of the white stripe region, an appropriate exposure condition may not be acquired. Therefore, the exposure condition may be acquired by excluding the image signal of the white stripe region as described above. Note that the white streak region may be detected by detecting pixels having an image signal equal to or greater than a preset threshold value, or a white line may be detected by filter processing or the like.

  Further, the spatial frequency distribution of the phase difference image of the cell group may be calculated, and the exposure condition may be acquired based on the spatial frequency distribution. Specifically, when dust such as white streaks or dead cells as described above is present in the cell group, these become bright image signals and appear as a distribution of high-frequency components. Therefore, the exposure condition may be acquired by excluding an image signal in a region where the spatial frequency component is equal to or higher than a preset high frequency component threshold. Further, when there is a stacked region in the cell group, this region appears as a distribution of low frequency components. Therefore, an image signal in an area where the spatial frequency component is equal to or lower than a preset low frequency component threshold value may be extracted, and the exposure condition may be acquired based on the maximum value of the image signal in the area. .

  Further, the dispersion value of the image signal of the phase difference image for each predetermined area in the cell group is calculated, and when the dispersion value is equal to or larger than a preset dispersion threshold, Assuming that there is a high-intensity isolated point generated due to the above, the exposure condition may be acquired by excluding the image signal in that region. Note that the variance value may be calculated for each imaging target range, for example.

  Further, the exposure condition determination method is not limited to the above method, and other known methods may be used. In the above description, the exposure condition of each cell group is automatically acquired based on the phase difference image for each cell group. However, the present invention is not limited to this, and the user can use the input device 40 to input each cell group. The area information and the exposure conditions may be manually set and input.

  The imaging control unit 25 controls the imaging unit 10 to perform the above-described low magnification imaging and high magnification imaging. When performing low-magnification imaging, the imaging control unit 25 captures a phase difference image for each imaging target range IR1 in the imaging region R based on preset initial exposure conditions. In addition, when performing high-magnification imaging, the imaging control unit 25 acquires area information of each cell group and exposure conditions of each cell group, and controls the imaging unit 10 based on the exposure conditions. . Specifically, in response to a control signal from the imaging control unit 25, the imaging unit 10 images a plurality of imaging target ranges including a cell group region using exposure conditions corresponding to the cell group.

  In the case of the example shown in FIG. 4, for example, when imaging the imaging target range IR2 including the region of the cell group S1, imaging is performed using the exposure conditions corresponding to the cell group S1. Similarly, when imaging the imaging target range IR2 (dotted line rectangular range) including the region of the cell group S2, imaging is performed using the exposure conditions corresponding to the cell group S2, and the region of the cell group S3 When imaging an imaging target range IR2 (dotted line rectangular range) including, imaging is performed using the exposure conditions corresponding to the cell group S3. In addition, as an exposure condition for regions other than the cell groups S1 to S3, for example, an average value of the exposure conditions acquired for each of the cell groups S1 to S3 may be used, or when performing low magnification imaging. The used initial exposure conditions may be used as they are.

  The imaging target range including the cell group region is specified based on the cell group region information acquired by the region information acquisition unit 23. At this time, the coordinate value of the pixel representing the boundary of the cell group or the coordinate value of all the pixels included in the region of the cell group may be used, or the imaging target range IR1 at the time of low magnification imaging. The imaging target range IR2 included in the imaging target range IR1 including the cell group region may be specified.

  Further, for example, when a plurality of cell group regions are included in one imaging target range IR2, it is desirable to perform imaging under an exposure condition corresponding to the cell group having the largest area.

  The exposure conditions include the exposure time of the image sensor 16 and the amount of light incident on the image sensor 16 as described above. In this embodiment, the image sensor is controlled by controlling the amount of light emitted from the white light source 11. The amount of light incident on 16 is controlled. Note that the control of the amount of light incident on the image sensor 16 is not limited to the control of the amount of light emitted from the white light source 11, but, for example, a diaphragm that restricts the amount of white light emitted from the white light source 11. In the case where the light amount limiting unit is provided, the amount of light incident on the image sensor 16 may be controlled by controlling the light amount limiting unit. Both the exposure time of the image sensor 16 and the amount of light incident on the image sensor 16 may be controlled.

  Then, based on the exposure conditions for each cell group as described above, the phase difference images for each imaging target range acquired by high-magnification imaging are combined by the image combining unit 22, and one level of the entire imaging region is displayed. A phase difference image is generated.

  The display control unit 26 causes the display device 30 to display the phase difference image of the entire imaging region acquired by high magnification imaging as described above as an observation phase difference image. The display control unit 26 may also cause the display device 30 to display a phase difference image of the entire imaging region acquired by low magnification imaging. In addition, the exposure condition for each cell group acquired by the exposure condition acquisition unit 24 is displayed on the display device 30 together with information for specifying the cell group such as a name added to the cell group and region information of the cell group. Also good.

  The display device 30 displays the phase difference image generated by the image combining unit 22 and the exposure conditions for each cell group as described above, and includes a liquid crystal display, for example. Further, the display device 30 may be configured by a touch panel and may also be used as the input device 40.

  The input device 40 includes a mouse, a keyboard, and the like, and accepts various setting inputs by the user. The input device 40 according to the present embodiment receives, for example, an input of setting exposure conditions for each cell group described above.

  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. The imaging control unit 25 controls the imaging unit 10 based on the initial exposure conditions, performs low-magnification imaging, and acquires a phase difference image for each imaging target range IR1 in the imaging region R (S10).

  The phase difference image for each imaging target range IR1 acquired by low magnification imaging is stored in the cell image acquisition unit 21 and then input to the image combining unit 22. The image combining unit 22 combines the phase difference images for each imaging target range IR1 and generates one phase difference image of the entire imaging region R.

  Then, one phase difference image generated by the image combining unit 22 is input to the region information acquisition unit 23, and the region information acquisition unit 23 acquires information on the region of the cell group included in the phase difference image. (S12).

  The cell group region information acquired by the region information acquisition unit 23 is output to the exposure condition acquisition unit 24. The exposure condition acquisition unit 24 extracts the phase difference image of each cell group from the phase difference image acquired by low magnification imaging based on the area information of each cell group, and the phase difference image of each cell group is extracted. Based on this, exposure conditions for each cell group are acquired (S14).

  Then, the exposure condition for each cell group acquired by the exposure condition acquisition unit 24 and the region information of each cell group are input to the imaging control unit 25. The imaging control unit 25 controls the imaging unit 10 based on the area information of each cell group and the exposure condition of each cell group, and the imaging unit 10 selects a plurality of imaging target ranges including the cell group area of the cell group. Imaging is performed at a high magnification using the exposure conditions (S16).

  The phase difference image for each imaging target range IR2 acquired by high-magnification imaging is stored in the cell image acquisition unit 21, and then input to the image combining unit 22. The image combining unit 22 combines the phase difference images for each imaging target range IR2 and generates one phase difference image of the entire imaging region R.

  The high-magnification phase difference image generated by the image combining unit 22 is output to the display control unit 26, and the display control unit 26 causes the display device 30 to display the high-magnification phase difference image (S18).

  According to the cell observation system 1 of the above-described embodiment, information on the area of the cell group included in the cell image obtained by imaging the imaging area is acquired, and the exposure condition for each cell group is acquired. Then, when the imaging target range is moved within the imaging region and an image for each imaging target range is taken, a plurality of imaging target ranges in the cell group are determined for each cell group based on the information on the region of the cell group. Therefore, each cell group can be imaged under appropriate exposure conditions without causing luminance unevenness in an image of the same cell group.

  In addition, although the cell observation system 1 of the said embodiment performs time-lapse imaging as mentioned above, you may make it perform acquisition of the exposure conditions for every cell group mentioned above every time of each imaging | photography. For example, until the Nth time (N is a natural number of 1 or more), low-magnification imaging is performed as in the cell observation system 1 of the above embodiment to determine the exposure condition for each cell group. An image signal for each cell group after the (N + 1) th time is estimated based on an image signal for each cell group captured up to the Nth time, and an exposure condition for high-magnification imaging is determined based on the estimated image signal. May be. That is, low-magnification imaging may not be performed after the (N + 1) th time.

  For example, as shown in FIG. 6, until the third imaging, the low-magnification imaging is performed to determine the exposure condition for each cell group as in the above embodiment, and the fourth and subsequent imaging is performed from the first to the third time. Based on the average value a1 to a3 of the image signal values of the cell group acquired by the high-magnification imaging up to 4, the average value a4 of the image signal value of the fourth cell group is estimated, and based on the estimated value of a4 You may make it determine the exposure conditions of the 4th high magnification imaging.

  For the estimation of the image signal for the fourth and subsequent imaging, for example, an approximate function f (t) is determined based on the average values a1 to a3 of the image signal values of the cell group, and based on this approximate function f (t). The image signal after the fourth time may be estimated.

  As the approximate function f (t), for example, an even function such as a quadratic function or a quartic function may be used, a linear function may be used, or an exponential function may be used. Good. Further, the approximation function f (t) may be obtained using polynomial approximation. As a method for obtaining the approximate function f (t), other known methods can be used.

For the approximate function f (t), the initial setting function fa (t) is stored in advance in the exposure condition acquisition unit 24, and the initial setting function is based on the phase difference images captured up to the Nth time. The approximate function f (t) may be obtained by changing fa (t). Specifically, for example, a quadratic function fa (t) = at ² + bt + c is stored in advance as the initial setting function fa (t), and the coefficients a and b are based on the phase difference images captured up to the Nth time. The approximate function f (t) may be obtained by changing the coefficient c. The initial setting function is not limited to a quadratic function, and an even function such as a quartic function and an exponential function may be used.

  Further, the initial setting function may be stored for each type of cell constituting the cell group or for each culture condition. Since the degree of cell growth varies depending on the cell type or culture condition, as described above, by storing the initial setting function in advance for each cell type or each culture condition, an image of the cell group captured at the (N + 1) th time The signal can be estimated with higher accuracy. More specifically, for example, a quadratic function is used in the case of cells or culture conditions where the cell growth rate is relatively fast, and an exponential function is used in the case of cells or culture conditions where the cell growth rate is relatively slow. You may make it use. Alternatively, the coefficient set in the initial setting function may be changed for each cell type or each culture condition. The culture conditions include the type of medium, the type or amount of growth factor, and the culture period, but other conditions may be used as long as they affect the cell growth rate.

  In the cell observation system 1 of the above embodiment, the phase difference image for specifying the region of the cell group is captured at a low magnification, and the phase difference image is captured using the exposure condition for each cell group at a high magnification. However, the imaging may be performed at the same magnification.

  Moreover, in the cell observation system 1 of the said embodiment, although the phase difference image was imaged by the imaging part 10, it is not restricted to a phase difference image, For example, a bright field image, a differential interference image, or a fluorescence image is made into a cell image. The image may be taken.

DESCRIPTION OF SYMBOLS 1 Cell observation system 10 Imaging part 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 17 Imaging element 20 Imaging control apparatus 21 Cell image acquisition part 22 Image coupling part 23 Area information acquisition unit 24 Exposure condition acquisition unit 25 Imaging control unit 26 Display control unit 30 Display device 40 Input device 50 Culture vessel 51 Stage

Claims (11)

An imaging unit that moves an imaging target range within an imaging region including a cell group and captures an image in which each pixel is represented by a luminance value for each imaging target range;
One of a coordinate value of a pixel representing a boundary of the region of the cell group included in the cell image of the imaging region captured by the imaging unit and a coordinate value of all the pixels included in the region of the cell group and area information acquisition unit that acquires information as the information of the area of the cell group,
Exposure in which the average value of the luminance values of the cell group area falls within a preset range for each cell group area specified by the cell group area information acquired by the area information acquisition unit. An exposure condition acquisition unit that acquires any one of the conditions and an exposure condition in which the maximum value of the luminance value of the region of the cell group is equal to or less than a preset threshold value ,
The imaging unit includes the cell group for each region of the cell group specified by the information of the cell group region based on the information on the region of the cell group and the exposure condition acquired by the exposure condition acquisition unit. An image pickup apparatus that picks up an image of a plurality of image pickup target ranges in accordance with an exposure condition for each region of the cell group. The exposure condition acquisition unit specifies a luminance value that is out of the distribution set of luminance values as a luminance value of an isolated point based on a histogram of luminance values of the cell image for each cell group , and the specified luminance value or negative to acquire the exposure condition, or pre-set imaging device according to claim 1, wherein for obtaining the exposure condition by using only the luminance value within the range of the histogram. Based on the spatial frequency distribution of the cell image for each cell group, the exposure condition acquisition unit excludes the brightness value of a region where the spatial frequency component is equal to or higher than a predetermined high frequency component threshold value, and sets the exposure condition. A brightness value of an area that is acquired or whose spatial frequency component is equal to or less than a preset low frequency component threshold value is extracted, and the exposure condition is acquired based on a maximum value of the brightness value in the area. The imaging apparatus according to 1 . The exposure condition acquisition unit determines the exposure condition based on a brightness value of a cell image excluding a region that is included in the cell image for each cell group and the brightness value variance value is equal to or greater than a threshold value. The imaging device according to any one of claims 1 to 3 . The imaging unit, if included multiple cell populations in the imaging target range, claim 1, area for imaging the imaging target range in the exposure condition of the largest cell group 4 according to any one of Imaging device. The imaging unit images the imaging region including the cell group multiple times over time,
The said exposure condition acquisition part determines the exposure condition for every said N + 1 cell group based on the cell image for every past cell group imaged by the Nth time, when N is set to 1 or more natural numbers. Item 6. The imaging device according to any one of Items 1 to 5 . The imaging is performed by increasing the magnification when imaging is performed according to the exposure condition for each cell group, compared to the magnification when the imaging unit captures the cell image for acquiring information on the region of the cell group. imaging device according to claim 1 to 6 any one of claims carried out. The exposure conditions, is at least one of the imaging apparatus of claims 1 to 7 any one wherein one of the exposure time and the amount of light incident on the image pickup device the image pickup unit has. Imaging device of the image combining unit according to claim 1 to 8 to any one of claims having a for generating a cell image of the imaging area by combining the image of each of the imaging target range. An imaging method in which an imaging target range is moved within an imaging region including a cell group, and an image in which each pixel is represented by a luminance value is captured for each imaging target range,
Information on either the coordinate value of a pixel representing the boundary of the region of the cell group included in the cell image obtained by imaging the imaging region or the coordinate value of all the pixels included in the region of the cell group is stored in the cell group. acquired as information of the area,
An exposure condition in which an average value of luminance values of the cell group region falls within a preset range for each cell group region specified by the acquired cell group region information , and the cell group Acquiring either one of the exposure conditions in which the maximum value of the luminance value of the area is equal to or less than a preset threshold ,
Based on the information on the area of the cell group and the acquired exposure conditions , for each area of the cell group specified by the information on the area of the cell group, a plurality of the imaging target ranges in the cell group are An imaging method comprising imaging according to exposure conditions for each region of a cell group. By controlling the imaging unit, the imaging target range is moved within the imaging region including the cell group,
An imaging control unit that captures an image in which each pixel is represented by a luminance value for each imaging target range;
One of a coordinate value of a pixel representing a boundary of the region of the cell group included in the cell image of the imaging region captured by the imaging unit and a coordinate value of all the pixels included in the region of the cell group and area information acquisition unit that acquires information as the information of the area of the cell group,
An exposure condition in which an average value of luminance values of the cell group region falls within a preset range for each cell group region specified by the acquired cell group region information , and the cell group An imaging control program that causes a computer to function as an exposure condition acquisition unit that acquires any one of the exposure conditions in which the maximum value of the luminance value of the area is equal to or less than a preset threshold value ,
The imaging control unit, based on the information on the area of the cell group and the acquired exposure conditions , for each area of the cell group specified by the information on the area of the cell group, a plurality of in the cell group An imaging control program that images the imaging target range according to an exposure condition for each region of the cell group.