JP2017225354A - Method and apparatus for imaging cultured cells - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide optimized imaging conditions for cultured cells.SOLUTION: According to the present invention, there is provided a method for imaging cultured cells. Here, the method comprises: imaging cultured cells 11 while changing a current-setting value of an LED lamp 2 within a predetermined variation range with a predetermined increment; for each captured observation image, calculating an average luminance (L) and average saturation (S) of the observed image in the HLS color space; for each observation image, calculating an index (I) expressed by the following equation (1); and setting the current set value of the LED lamp 2 at the time of imaging the observation image whose index (I) is the maximum value as an optimum value. Index (I)=(255-L)×(L+S)....(1)SELECTED DRAWING: Figure 1

Description

  The present invention relates to a cultured cell imaging method and apparatus, and more particularly to an imaging method and apparatus capable of optimizing the imaging conditions of cultured cells.

  In cell culture, time-lapse imaging in which cells in culture are imaged every predetermined time may be performed. Image processing is performed on the captured observation image in order to extract various information about the cultured cells. In this case, the observation image needs to have an image brightness and focus suitable for image processing.

  Japanese Patent Application Laid-Open Publication No. 2004-228561 describes an imaging condition for adjusting the brightness of an observation image and a technique for imaging by adjusting a focal position. In the observation system described in Patent Literature 1, when performing time-lapse imaging, luminance control (S64 in FIG. 13) and autofocus control (S65 in FIG. 13) are performed on the observation image. In the luminance control, LED control or the like is performed so that the average luminance of the observation image falls within a predetermined range set in advance (FIG. 14).

JP 2011-141407 A

  By the way, when the cell culture is continued, the cell concentration is changed, and cell metabolites such as lactic acid are generated to change the pH of the culture solution, so that the turbidity and color tone of the culture solution are changed. As a result, the observed image may not be suitable for image processing even if the average luminance of the observed image is within a predetermined range.

  In addition, when the culture medium is fed in cell culture and the depth of the culture solution changes, the turbidity and color tone of the culture solution also change because the cell concentration and the pH of the culture solution change. As a result, the observed image may not be suitable for image processing even if the average luminance of the observed image is within a predetermined range. Moreover, since the culture solution is stirred after feeding the culture medium, the focal position of the observation image changes. In addition, imaging is performed with the cultured cells settled in the culture solution, but it is not necessarily located on the same plane in the culture solution, and it may be difficult to focus on all the cultured cells at the same time. Can occur.

  Thus, in the process of continuous cell culture, conditions such as color tone and liquid depth inside the culture vessel change. For this reason, the brightness and focus position of the observation image optimum for image processing sometimes differ between the initial stage of cell culture and after the passage of time.

  The present invention has been made in view of the above circumstances, and an object thereof is to provide a culture cell imaging method and apparatus capable of optimizing the imaging conditions of cultured cells.

  In order to achieve the above object, the first cultured cell imaging method of the present invention is an imaging method for imaging an observation image of a cultured cell in a culture vessel, and sets imaging conditions for adjusting the brightness of the observation image A brightness setting step for performing observation, and an image acquisition step for acquiring an observation image, wherein the brightness setting step changes an imaging condition for adjusting the brightness of the observation image within a predetermined change range with a predetermined step size. Imaging the cultured cells, calculating the average luminance and average saturation of the observation image in the HLS color space for each of the captured observation images, and calculating the average luminance and the average saturation for each observation image. And a step of setting an imaging condition for adjusting brightness when an observation image having the maximum value is taken as an optimum value.

  The second cultured cell imaging method of the present invention is an imaging method for imaging an observation image of a cultured cell in a culture container, and includes a focal position setting step for setting a focal position of the observation image, and an observation image. An image acquisition step of acquiring, and the focus position setting step includes: a step of imaging the cultured cells by changing the focus position within a predetermined change range with a predetermined step size; and observing each of the captured observation images. The method includes a step of measuring the number of particles in the image and a step of setting the focal position when an observation image having the maximum number of particles is captured as an optimum value.

  The third cultured cell imaging method of the present invention is an imaging method for capturing an observation image of a cultured cell in a culture vessel, and a brightness setting step for setting an imaging condition for adjusting the brightness of the observation image And a focus position setting step for setting the focus position of the observation image, and an image acquisition step for acquiring the observation image, wherein the brightness setting step changes the imaging condition for adjusting the brightness of the observation image with a predetermined change. The step of imaging the cultured cells with a predetermined step size within the range, the step of calculating the average luminance and the average saturation of the observation image in the HLS color space for each of the captured observation images, and the observation image A step of calculating an index based on the average luminance and the average saturation, and a step of setting an imaging condition for adjusting brightness when an observation image having the maximum value of the index is captured as an optimum value. The focal position setting step The step of imaging the cultured cells by changing the focal position by a predetermined step within a predetermined change range, the step of measuring the number of particles in the observed image for each of the captured observation images, and the maximum number of particles And a step of setting, as an optimum value, a focal position when an observation image is captured.

  The first cultured cell imaging apparatus of the present invention is an imaging apparatus that captures an observation image of a cultured cell in a culture container, and illuminates the cultured cell and images the cultured cell. An imaging unit that generates an observation image; and a brightness control unit that sets an imaging condition for adjusting the brightness of the observation image. The brightness control unit sets an imaging condition for adjusting the brightness of the observation image to a predetermined value. The imaging unit is caused to capture the cultured cells with a predetermined step size within a change range, and the average luminance and average saturation of the observation image in the HLS color space are calculated for each observation image captured by the imaging unit. For each of the observation images, an index is calculated based on the average luminance and the average saturation, and an imaging condition for adjusting brightness when the observation image having the maximum index is captured is set as an optimal value. It is characterized by that.

  The second cultured cell imaging device of the present invention is an imaging device that captures an observation image of a cultured cell in a culture container, and illuminates the cultured cell and images the cultured cell. An imaging unit that generates an observation image; and a focal position control unit that sets a focal position of the observation image. The focal position control unit changes the focal position within a predetermined change range with a predetermined step size and performs the imaging. Optimum focus position when the number of particles in the observed image is measured for each observation image captured by the imaging unit and the observation image with the maximum number of particles is captured. It is characterized by being set as a value.

  The third cultured cell imaging device of the present invention is an imaging device that captures an observation image of a cultured cell in a culture container, and illuminates the cultured cell and images the cultured cell. An imaging unit that generates an observation image, a brightness control unit that sets an imaging condition for adjusting the brightness of the observation image, and a focal position control unit that sets a focal position of the observation image, the brightness control unit The imaging condition for adjusting the brightness of the observation image is changed within a predetermined change range by a predetermined step size, and the cultured cell is imaged by the imaging unit, and an HLS color is obtained for each observation image captured by the imaging unit. When the average luminance and average saturation of the observation image in space are calculated, and for each observation image, an index is calculated based on the average luminance and the average saturation, and an observation image in which the index is the maximum value is captured Set the imaging conditions to adjust the brightness of The focal position control unit changes the focal position by a predetermined step size within a predetermined change range, causes the imaging unit to image the cultured cells, and for each observation image captured by the imaging unit, The number of particles is measured, and the focal position when an observation image having the maximum number of particles is captured is set as an optimum value.

  According to the first and third cultured cell imaging methods and apparatuses of the present invention, the average luminance and average color of the observed image in the HLS color space are obtained for each observed image captured by changing the imaging condition for adjusting the brightness of the observed image. An imaging condition for adjusting the brightness when an observation image having a maximum index based on the degree is captured is set as an optimal value, and the observation image is acquired with the optimal value. Thereby, even if it is a case where the turbidity and color tone of a culture solution change while continuing cell culture, the imaging conditions of a cultured cell can be optimized.

  In the second and third cultured cell imaging methods and apparatuses of the present invention, the focal position when the observation image with the maximum number of particles in the observation image captured by changing the focal position is captured is the optimum value. And an observation image is acquired with the optimum value. Thereby, even if it is a case where a cultured cell is not located on the same plane in a culture solution, the imaging condition of a cultured cell can be optimized.

It is a schematic diagram of the imaging device of the cultured cell which concerns on 1st Embodiment of this invention. It is a flowchart of the imaging method of the cultured cell which concerns on 1st Embodiment of this invention. It is a flowchart which sets the imaging condition which adjusts the brightness of an observation image. (A)-(e) is the observation image imaged by changing the imaging condition which adjusts the brightness of an observation image. It is a graph which shows the relationship between the imaging condition which adjusts the brightness of an observation image, and a parameter | index. It is a flowchart which sets the focus position of an observation image. (A)-(e) is the observation image imaged by changing a focus position. It is a graph which shows the relationship between a focus position and the number of particles. (A)-(c) is an observation image which shows the relationship between a focus position and the number of particles. It is a flowchart of the imaging method of the cultured cell which concerns on 2nd Embodiment of this invention. (A)-(e) are the observation images imaged by alternately repeating the brightness setting and the focus position setting. (A) is a graph which shows the relationship between the imaging condition and index which adjust the brightness in the 1st brightness setting process, (b) is the imaging which adjusts the brightness in the 2nd brightness setting process. It is a graph which shows the relationship between conditions and a parameter | index, (c) is a graph which shows the relationship between the imaging condition which adjusts the brightness in the brightness setting process of the 3rd time, and a parameter | index. (A) is a graph showing the relationship between the focal position and the number of particles in the first focal position setting step, and (b) shows the relationship between the focal position and the number of particles in the second focal position setting step. It is a graph.

Hereinafter, embodiments of the present invention will be described with reference to the drawings.
[First Embodiment]
(Imaging device)
First, with reference to FIG. 1, the cultured cell imaging device (hereinafter also referred to as an imaging device) of the first embodiment will be described. FIG. 1 is a schematic diagram of an imaging apparatus according to the present embodiment. The imaging device is a device that performs time-lapse imaging of the cultured cells 11 in the culture solution 10 accommodated in the culture vessel 1 every predetermined time (for example, 60 minutes).

  As shown in FIG. 1, the imaging device of the present embodiment is an imaging device that captures an observation image of a cultured cell 11 in a culture vessel 1, and an LED lamp 2 as an illumination unit that illuminates the cultured cell 11; A CCD camera 3 is provided as an imaging unit that images the cultured cells 11 and generates an observation image. The CCD camera 3 is disposed on the opposite side of the LED lamp 2 with the culture vessel 1 in between, and takes a transmission image.

  The imaging apparatus according to the present embodiment includes a dimmer 4 that adjusts the brightness of the LED lamp 2, an actuator 5 that moves the CCD camera 3, and a control unit 6 that is a computer that controls the dimmer 4 and the actuator 5. Is further provided. The control unit 6 controls the CCD camera 3 to automatically execute time-lapse imaging.

The control unit 6 also functions as a brightness control unit that sets an imaging condition for adjusting the brightness of the observation image. When the controller 6 controls the dimmer 4, the current value applied to the LED lamp 2 is adjusted, and the brightness of the observation image is adjusted. Therefore, in the present embodiment, the current value of the LED lamp 2 is an imaging condition for adjusting the brightness of the observation image.
Note that the brightness of the observation image may be adjusted not only by the current value of the LED lamp 2 but also by the aperture of the diaphragm and the shutter speed of the CCD camera, or may be adjusted in combination.

The control unit 6 also functions as a focal position control unit that sets the focal position of the observation image. The position of the CCD camera 3 is adjusted by the control unit 6 controlling the actuator 5. The distance between the CCD camera 3 and the culture vessel 1 is adjusted by moving the CCD camera 3 in the direction of the arrow in FIG. Therefore, in the present embodiment, the focal position is adjusted according to the position of the CCD camera 3.
The focal position may be adjusted not only by moving the CCD camera 3, but also by an optical system such as a lens of the CCD camera 3, or may be adjusted by moving the culture vessel 1, or a combination thereof. May be adjusted.

(Imaging method)
Next, with reference to FIG. 2, the imaging method by the imaging device of this embodiment is demonstrated. As shown in the flowchart of FIG. 2, in setting the imaging conditions for capturing the observation image of the cultured cell 11, first, the current value of the LED lamp 2 is set as the imaging condition for adjusting the brightness of the observation image. Is set (S1), and then the CCD camera 3 is moved to set the focal position (S2). And the observation image imaged on the imaging condition which optimized the electric current value of LED lamp 2, and the focus position of CCD camera 3 is acquired (S3).

(Brightness setting process)
A method of setting the current value of the LED lamp 2 in order to set the brightness of the observation image in the brightness setting step (S1) of FIG. 2 will be described with reference to the flowchart of FIG.
First, as an imaging condition for adjusting the brightness of the observation image, the cultured cell 11 is imaged by changing the current setting value of the LED lamp 2 within a predetermined change range at a predetermined step size to generate a plurality of observation images ( S11). The current setting value of the LED lamp 2 of the present embodiment is “0” as the minimum value and “100” as the maximum value. Here, the dimmer 4 sets the current setting value to “10” in increments of “10”. The CCD camera 3 images the cultured cells 11 at each current setting value, and a total of 10 observation images are generated.

FIG. 4A to FIG. 4E show five observation images when the current set value is “10” to “50” among the ten observation images. Each observation image in FIG. 4 is a black-and-white image, but in the actual image, the culture solution 10 is yellow.
As can be seen from these observation images, if the current setting value of the LED lamp 2 is too low, it becomes difficult to distinguish the particles of the cultured cells 11 in the observation image as shown in FIG. On the other hand, even if the current setting value of the LED lamp 2 is too high, as shown in FIG. 4E, the entire observation image becomes white and small particles disappear, making it difficult to distinguish the particles. Although not shown in FIG. 4, when the current setting value of the LED lamp 2 is further increased, the entire observation image becomes whiter and particle discrimination becomes more difficult.

  Next, for each of the captured observation images including the observation images shown in FIGS. 4A to 4E, the control unit 6 causes the average luminance (L) and average saturation of the observation image in the HLS color space. (S) is calculated (S12).

  The HLS color space (also referred to as HSL color space) refers to a color space represented by three components of hue (hue), luminance (lightness / luminance), and saturation (saturation). Hue represents the hue with an angle of 360 degrees. Brightness has a different meaning from “luminance” described in Patent Document 1, and represents the brightness of a color with white as a maximum value and black as a minimum value, and when the brightness is half of the maximum value and the minimum value. It becomes a pure color. Saturation represents the vividness of a color, with a pure color as the maximum value and gray as the minimum value.

  The average luminance (L) and average saturation (S) of the observation image are values obtained by averaging the data for each pixel of the RGB color system representing the received light intensity of the RGB light receiving elements of the CCD camera 3 over the entire observation image. Is converted into the HLS color system. Table 1 below shows the average luminance (L) and average saturation (S) of the entire observation image for each current setting value of the LED lamp 2. In Table 1, the average luminance (L) and the average saturation (S) are represented by 256 gradations from 0 to 255.

Next, for each observation image, the control unit 6 calculates an index based on the average luminance (L) and the average saturation (S) (S13).
Here, the index is the difference (255−L) between the maximum luminance (255), which is the maximum value of the luminance gradation, and the average luminance (L) of the observation image, and the average luminance as in the following equation (1). It includes the product of (L) and the sum (L + S) of average saturation (S).
Index (I) = (255−L) × (L + S) (1)
The above formula (1) has been found empirically by the present inventor through various experiments and studies.

  Table 1 shows the value of the index (I) for each current setting value of the LED lamp 2. As shown in Table 1, when the current setting value of the LED lamp 2 is “20”, the index (I) is the maximum value “25652”.

  Further, the index (I) for each current setting value of the LED lamp 2 is shown in the graph of FIG. FIG. 5 shows that the index (I) is maximum when the current setting value of the LED lamp 2 is “20”.

  Next, the current setting value of the LED lamp 2 when the observation image having the maximum index (I) is captured is set as the optimum value (S14). Here, the current set value “20” of the LED lamp 2 is set as the optimum value of the imaging condition for adjusting the brightness.

(Focus position setting process)
Next, a method for setting the focal position in the focal position setting step (S2) of FIG. 2 will be described with reference to the flowchart of FIG.
First, the cultured cell 11 is imaged while changing the focal position within a predetermined change range with a predetermined step size, and a plurality of observation images are generated (S21). Here, the actuator 5 moves the CCD camera 3 to change the Z coordinate (arbitrary unit) representing the focal position in increments of “250” from “0” to “9750”. The cultured cells 11 were imaged at the coordinates, and a total of 40 observation images were generated.

  FIG. 7A to FIG. 7E show 5 when the Z coordinate of the focal position is “0”, “3000”, “4250”, “6000”, and “9750” among the 40 observation images. A representative observation image is shown.

Next, for each captured observation image including the observation images shown in FIGS. 7A to 7E, the number of particles in the observation image is measured (S22). Individual particles in the observation image are measured by image processing.
The number of particles for each set value of the Z coordinate is shown in the graph of FIG. FIG. 8 shows that the number of particles is maximum when the Z coordinate is “4250”.

  Table 2 shows the number of particles measured from the observed image when the Z coordinate of the focal position is “3000” to “6000”. As shown in Table 2, when the Z coordinate is “4250”, the number of particles is the maximum value “6110”.

  9A and 9C show an observation image when the focus is not achieved, and FIG. 9B shows an observation image when the focus is achieved. As shown in FIGS. 9 (a) and 9 (c), when the focus is not achieved, the contours of the individual particles are unclear and integrated with the adjacent particles. For this reason, it becomes difficult to separate and extract individual particles during image processing, and the number of particles to be measured is reduced. In addition, as shown in FIGS. 7A and 7C, when the focus is not at all, it is difficult to determine the particles themselves in the observation image, and thus the number of particles to be measured is large. descend.

  On the other hand, as shown in FIG. 9B, when the image is in focus, the outline of each particle becomes clear, and each particle can be extracted from adjacent particles during image processing. For this reason, the number of particles to be measured increases. Therefore, when the most particles are measured, the most particles are in focus.

  Next, the focal position when an observation image having the maximum number of particles is captured is set as an optimum value (S23). Here, the Z coordinate “4250” is set as the optimum value of the focal position of the CCD camera 3.

(Image acquisition process)
Next, an image captured under the imaging condition of the optimal current setting value of the LED lamp 2 and the optimal focal position of the CCD camera 3 is acquired (S3 in FIG. 2).
In acquiring this image, an observation image may be taken again under the optimized imaging conditions, or in the process of setting the imaging conditions, the observation image captured under the same conditions as the optimized imaging conditions may be captured. Data may be selected and used.

  As described above, in the present embodiment, for each observation image captured by changing the current setting value of the LED lamp 2, an index based on the average luminance (L) and average saturation (S) of the observation image in the HLS color space ( An imaging condition was set so that I) was a maximum value. Furthermore, in this embodiment, an imaging condition is set that maximizes the number of particles in the observed image captured by changing the focal position. Thereby, even if the color tone of the culture solution 10 is changed or the transparency is lowered while the cell culture is continued, the cultured cells 11 are not located on the same plane in the culture solution 10. Even in this case, the imaging conditions of the cultured cell 11 can be optimized.

[Second Embodiment]
Since the imaging apparatus of the second embodiment is the same as that of the first embodiment, a detailed description thereof will be omitted.
In the imaging method of the present embodiment, as shown in the flowchart of FIG. 10, an LED lamp is used as the imaging condition for adjusting the brightness of the observation image when setting the imaging condition when imaging the observation image of the cultured cell 11. The brightness setting step (S1, S3 and S5) for setting the current value of 2 and the focus position setting step (S2 and S4) for setting the focus position by moving the CCD camera 3 are repeated alternately. And the observation image imaged on the imaging condition optimized with the electric current value of the LED lamp 2 set last and the focus position of the CCD camera 3 is acquired (S6).

FIG. 11A to FIG. 11E show observation images picked up under the image pickup conditions optimized in steps S1 to S5 in FIG.
FIG. 11A shows an observation image taken with the current setting value of the LED lamp 2 optimized in the first brightness setting step (S1 in FIG. 10). At this stage, the particles cannot be identified because they are not yet in focus.

  The graph of FIG. 12A shows the index (I) for each current setting value of the LED lamp 2 in the first brightness setting step (S1). In the first brightness setting step (S1), the current setting value of the LED lamp 2 is changed from “2” to “40” in increments of “2”, and the cultured cells 11 are imaged at each current setting value. A total of 20 observation images are generated.

  As shown in FIG. 12A, when the current setting value of the LED lamp 2 is “14”, the index (I) is maximized. The observation image shown in FIG. 11A is an image taken under an imaging condition where the current set value is “14”.

  Next, FIG. 11B shows an observation image captured at the focus position optimized in the first focus position setting step (S2 in FIG. 10). At this stage, the particles in the observed image can be distinguished because they are in focus.

  The graph of FIG. 13A shows the number of particles for each focal position of the CCD camera 3 in the first focal position setting step (S2). In the first focus position setting step (S2), the Z coordinate representing the focus position is changed from “0” to “9750” in increments of “250”, and the cultured cells 11 are imaged at each Z coordinate, for a total of 40 sheets. The observed image is generated.

  As shown in FIG. 13A, when the Z coordinate is “3500”, the number of particles is maximum. The observation image shown in FIG. 11B is an image taken under an imaging condition in which the Z coordinate is “3500” while the current setting value is “14”.

  Next, FIG. 11C shows an observation image captured with the current setting value of the LED lamp 2 optimized in the second brightness setting step (S3 in FIG. 10). Even if the brightness of the observation image is not optimal as a result of the first focus adjustment, the brightness of the observation image is optimized again by the second brightness adjustment.

  In the graph of FIG. 12B, the index (I) for each current setting value of the LED lamp 2 in the second brightness setting step (S3) is shown. Similarly to the first brightness setting process, the second brightness setting process (S3) is also performed by changing the current setting value of the LED lamp 2 from “2” to “40” in increments of “2”. The cultured cells 11 are imaged with the current setting value, and a total of 20 observation images are generated.

  As shown in FIG. 12B, when the current setting value of the LED lamp 2 is “14”, the index (I) is maximized. The observation image shown in FIG. 11C is an image taken under an imaging condition in which the current set value is “14” while the Z coordinate is “3500”.

  FIG. 11D shows an observation image captured at the focus position optimized by the second focus adjustment in the second focus position setting step (S4 in FIG. 10).

  The graph of FIG. 13B shows the number of particles for each focal position of the CCD camera 3 in the second focal position setting step (S4). In the second focus position setting step (S4), the step size of the Z coordinate representing the focus position is set to “100” units narrower than the first time, and the change range of the Z coordinate is “3500, which is the first optimum value. ”And a range narrower than the first time,“ 2800 ”to“ 4200 ”, the cultured cells 11 are imaged at each Z coordinate, and a total of 15 observation images are generated.

As shown in FIG. 13B, in the second focus position setting step (S4), the number of particles is maximum when the Z coordinate is “3600”. The observation image shown in FIG. 11D is an image taken under an imaging condition in which the Z coordinate is “3600” while the current setting value is “14”.
As described above, the step size in the second focus position setting is made narrower than that in the first time, and the change range in the second focus position setting is made narrower than that in the first time. The accuracy of setting the imaging conditions for the focal position of the CCD camera 3 can be improved.

  Next, FIG. 11E shows an observation image captured with the current setting value of the LED lamp 2 optimized in the third brightness setting step (S5 in FIG. 10).

  FIG. 12C shows an index (I) for each current setting value of the LED lamp 2 in the third brightness setting step (S5). In the third brightness setting step (S5), the step size of the current setting value of the LED lamp 2 is set to “1” units narrower than the first time, and the change range of the current setting value is the optimum value for the second time. From “9” to “18” including “14”, the cultured cells 11 are imaged at each current setting value, and a total of ten observation images are generated.

As shown in FIG. 12C, in the third brightness setting step (S5), when the current setting value of the LED lamp 2 is “15”, the index (I) is maximized. The observation image shown in FIG. 11E is an image taken under an imaging condition where the current set value is “15” while the Z coordinate is “3600”.
As described above, the current setting value in the third brightness setting step (S5) is set while the step size of the current setting value of the LED lamp 2 in the third brightness setting step (S5) is made narrower than that in the second time. As a result of making the change range of the value narrower than that of the second time, it is possible to improve the setting accuracy of the imaging condition of the current setting value of the LED lamp 2 while avoiding an increase in the number of captured images.

  In this way, the observation image shown in FIG. 11E is acquired as the optimum image (S6 in FIG. 10).

  As mentioned above, although embodiment of this invention was described, this invention can perform a various change and deformation | transformation. For example, in each of the above-described embodiments, an example in which a CCD camera as an imaging unit is arranged on the opposite side of the LED lamp with the culture vessel interposed therebetween and an observation image in which transmitted light is imaged is generated has been described. Then, the observation image is not limited to the transmitted light image, and may be a reflected light image, for example.

  In the above-described embodiment, the example in which the LED lamp current value is set as the imaging condition for adjusting the brightness of the observation image has been described. However, in the present invention, the imaging condition for adjusting the brightness of the observation image is However, the present invention is not limited to this. For example, the brightness may be adjusted by the aperture of the aperture, or the brightness may be adjusted by the shutter speed of the CCD camera.

  The index may be calculated using the HSV color system. The HSV color system is represented by three components of hue, saturation, and value. Hue represents the hue with an angle of 360 degrees. Saturation represents the vividness of a color, with a pure color as the maximum value and gray as the minimum value. The brightness represents the brightness of the color.

  In the above-described embodiment, the example in which the CCD camera as the imaging unit is moved in changing the focal position has been described. However, in the present invention, the method for adjusting the focal position is not limited to this, and the CCD camera is not limited to this. The focal position may be adjusted by moving the optical system, or the focal position may be adjusted by moving the culture vessel.

  In the above-described embodiment, an example in which an LED lamp is used as the illumination unit has been described. However, in the present invention, the illumination unit is not limited to this. In the above-described embodiment, an example in which a CCD camera is used as the imaging unit has been described. However, in the present invention, the imaging unit is not limited to this.

  The present invention is suitable for application to automatic time-lapse imaging of cultured cells.

DESCRIPTION OF SYMBOLS 1 Culture container 2 LED lamp 3 CCD camera 4 Dimmer 5 Actuator 6 Control part 10 Culture solution 11 Culture cell

Claims (9)

An imaging method for imaging an observation image of cultured cells in a culture vessel,
A brightness setting step for setting imaging conditions for adjusting the brightness of the observation image;
An image acquisition step of acquiring an observation image,
The brightness setting step includes
Changing the imaging condition for adjusting the brightness of the observed image at a predetermined step size within a predetermined change range, and imaging the cultured cells;
A step of calculating an average luminance and an average saturation of the observation image in the HLS color space for each captured observation image;
For each observation image, calculating an index based on the average luminance and the average saturation;
And a step of setting, as an optimum value, an imaging condition for adjusting brightness when an observation image having the maximum index value is captured. An imaging method for imaging an observation image of cultured cells in a culture vessel,
A focus position setting step for setting the focus position of the observation image;
An image acquisition step of acquiring an observation image,
The focal position setting step includes
Changing the focal position within a predetermined change range at a predetermined step size and imaging cultured cells;
For each captured image, a step of measuring the number of particles in the observed image;
And a step of setting, as an optimum value, a focal position when an observation image having the maximum number of particles is captured. An imaging method for imaging an observation image of cultured cells in a culture vessel,
A brightness setting step for setting imaging conditions for adjusting the brightness of the observation image;
A focus position setting step for setting the focus position of the observation image;
An image acquisition step of acquiring an observation image,
The brightness setting step includes
Changing the imaging condition for adjusting the brightness of the observed image at a predetermined step size within a predetermined change range, and imaging the cultured cells;
A step of calculating an average luminance and an average saturation of the observation image in the HLS color space for each captured observation image;
For each observation image, calculating an index based on the average luminance and the average saturation;
A step of setting an imaging condition for adjusting brightness when an observation image having the maximum value as the index is captured as an optimum value,
The focal position setting step includes
Changing the focal position within a predetermined change range at a predetermined step size and imaging cultured cells;
For each captured image, a step of measuring the number of particles in the observed image;
And a step of setting, as an optimum value, a focal position when an observation image having the maximum number of particles is captured. The brightness setting step and the focus position setting step are alternately repeated,
In the second and subsequent brightness setting steps,
The imaging condition changing range of the imaging condition for adjusting the brightness includes the imaging condition for adjusting the brightness set in the previous brightness setting step, and the imaging condition for adjusting the brightness in the previous brightness setting step Narrower than the change range of
4. The culture according to claim 3, wherein the step size of the change in the imaging condition for adjusting the brightness is narrower than the step size of the change in the imaging condition for adjusting the brightness in the previous brightness setting step. Cell imaging method. The brightness setting step and the focus position setting step are alternately repeated,
In the second and subsequent focal position setting steps,
The focal position change range includes the focal position set in the previous focal position setting step, and is narrower than the focal position change range in the previous focal position setting step,
5. The method for imaging cultured cells according to claim 3, wherein the step size of the focal position change is narrower than the step size of the focal position change in the previous focal position setting step. The index includes a product of a difference between a maximum luminance which is a maximum value of luminance gradation and an average luminance of the observation image, and a sum of the average luminance and the average saturation of the observation image. The imaging method of the cultured cell as described in any one of Claim 1, 3-5. An imaging device for imaging an observation image of cultured cells in a culture vessel,
An illumination unit for illuminating the cultured cells;
An imaging unit that images the cultured cells and generates an observation image;
A brightness control unit for setting imaging conditions for adjusting the brightness of the observation image,
The brightness control unit changes the imaging condition for adjusting the brightness of the observation image at a predetermined step size within a predetermined change range, causes the imaging unit to image the cultured cells, and each image captured by the imaging unit For the observation image, the average luminance and the average saturation of the observation image in the HLS color space are calculated. For each observation image, an index is calculated based on the average luminance and the average saturation, and the index is the maximum value. An imaging device for cultured cells, wherein an imaging condition for adjusting brightness when an observation image is captured is set as an optimum value. An imaging device for imaging an observation image of cultured cells in a culture vessel,
An illumination unit for illuminating the cultured cells;
An imaging unit that images the cultured cells and generates an observation image;
A focal position control unit for setting the focal position of the observation image,
The focal position control unit changes the focal position at a predetermined step size within a predetermined change range, causes the imaging unit to image the cultured cells, and for each observation image captured by the imaging unit, An apparatus for imaging cultured cells, wherein the number of particles is measured and a focal position is set as an optimum value when an observation image having the maximum number of particles is captured. An imaging device for imaging an observation image of cultured cells in a culture vessel,
An illumination unit for illuminating the cultured cells;
An imaging unit that images the cultured cells and generates an observation image;
A brightness control unit for setting imaging conditions for adjusting the brightness of the observation image;
A focal position control unit for setting the focal position of the observation image,
The brightness control unit changes the imaging condition for adjusting the brightness of the observation image at a predetermined step size within a predetermined change range, causes the imaging unit to image the cultured cells, and each image captured by the imaging unit For the observation image, the average luminance and the average saturation of the observation image in the HLS color space are calculated. For each observation image, an index is calculated based on the average luminance and the average saturation, and the index is the maximum value. Set the imaging condition to adjust the brightness when the observation image is captured as the optimal value,
The focal position control unit changes the focal position at a predetermined step size within a predetermined change range, causes the imaging unit to image the cultured cells, and for each observation image captured by the imaging unit, A culture cell imaging apparatus, wherein the number of particles is measured, and a focal position when an observation image having the maximum number of particles is captured is set as an optimum value.