JPWO2019088034A1 - Observation area setting device, imaging control device, operation method of observation area setting device, and observation area setting program - Google Patents

Abstract

In the observation area setting device, the imaging control device, the operation method of the observation area setting device, and the observation area setting program, it is possible to prevent an increase in the amount of data of the captured image and an increase in the imaging time. For the observation target housed in the container, a part of a new observation area adjacent to the previously set observation area is overlapped with respect to the previously set observation area, and new observation areas are sequentially set. When doing so, the degree of overlap of the overlapping overlapping areas is set larger as the degree of inclination of the bottom surface of the container located in the previously set observation area in the alignment direction of the previously set observation area and the new observation area is larger. To do.

Description

The present invention relates to a technique for setting each observation area in tiling photography.

Pluripotent stem cells such as ES (Embryonic Stem) cells and iPS (Induced Pluripotent Stem) cells have the ability to differentiate into cells of various tissues, such as regenerative medicine, drug development, and elucidation of diseases. It is attracting attention as something that can be applied in.

Then, a method has been proposed in which pluripotent stem cells such as ES cells and iPS cells, or cells in which differentiation has been induced are imaged with a microscope or the like, and the differentiation state of the cells is evaluated by capturing the characteristics of the images. ..

On the other hand, as described above, when imaging cells with a microscope, it has been proposed to perform so-called tiling imaging in order to acquire a high-magnification wide-field image. Specifically, for example, by moving a stage on which a well plate or the like is installed with respect to an imaging optical system, each observation area in the well is scanned to photograph each observation area, and then each observation area is photographed. A method of joining images to generate a composite image has been proposed.

Here, since the bottom surface of the culture vessel used for microscopic observation is distorted, it is necessary to perform autofocus control for each observation region to adjust the focus position. Patent Document 1 discloses a method in which the position of the bottom surface of the culture vessel is measured in advance and the bottom surface of the culture vessel is automatically focused for photographing.

On the other hand, for example, when the center of the observation area is focused in all the observation areas, the larger the degree of distortion of the bottom surface of the culture vessel, the more the peripheral part of the observation area is not focused and the peripheral part is blurred. It will be imaged. Therefore, when performing tiling shooting, a certain amount of shot images taken in other observation areas are overlapped in the out-of-focus area, and a shot image with better image quality is selected and combined for the overlapped area. An image is being generated.

JP-A-63-106615

However, if the overlapping area, that is, the overlapping area is too large, the number of captured images increases when all the observation targets are photographed, which may increase the amount of data and lengthen the photographing time.

The present invention has been made in view of the above circumstances, and by appropriately setting the degree of overlap of overlapping regions in adjacent observation regions, it is possible to prevent an increase in the amount of data in a captured image and a lengthening of the shooting time. The purpose.

The observation area setting device of the present invention overlaps a part of a new observation area adjacent to the observation area set earlier with respect to the observation area set earlier with respect to the observation target housed in the container. An observation area setting unit that sequentially sets new observation areas,
The degree of overlap of the overlapping overlapping areas is set as the degree of inclination of the bottom surface of the container located in the previously set observation area in the alignment direction of the previously set observation area and the new observation area increases. It has a setting unit.

In the present invention, the "previously set observation area" refers to the area set before the "new observation area" is set, and the "previously" has a time-series relationship. means. Further, in the present invention, the "arrangement direction" refers to a direction along a line connecting the centers of the previously set observation area and the new observation area.

Further, the observation area setting device of the present invention includes a shape information receiving unit that receives shape information of the bottom surface of the container.
The degree of overlap setting unit may acquire the degree of inclination based on the shape information input from the shape information receiving unit.

Further, in the observation area setting device of the present invention, the degree of inclination may be a value based on the difference in height between both ends in the alignment direction of the bottom surface of the container located in the observation area set earlier.

Further, in the observation area setting device of the present invention, the degree of inclination is a region including one side of the previously set observation area on the side where a new observation area is set, and has a size that matches the previously set observation area. It may be a value based on the difference in height between both ends in the alignment direction of the bottom surface of the container located in the area.

Further, the observation area setting device of the present invention may have an observation area smaller than that of the container.

Further, in the observation area setting device of the present invention, the container may be a dish, a well plate or a flask.

The imaging control device of the present invention includes the above-mentioned observation area setting device and
It is provided with a control unit for causing the imaging unit to take an image of the observation target housed in the container for each observation area set by the observation area setting device.

The imaging control device of the present invention may include a shape information storage unit that stores shape information of the bottom surface of the container.

Further, in this case, the shape information storage unit may store a table in which the identification information of the container and the shape information of the bottom surface of the container are associated with each other.

The method of operating the observation area setting device of the present invention is as follows.
It is an operation method of an observation area setting device including an observation area setting unit and an overlap degree setting unit.
The observation area setting unit newly overlaps a part of the new observation area adjacent to the observation area set earlier with respect to the observation area set earlier with respect to the observation target housed in the container. Observation area is set sequentially,
The degree of overlap setting unit determines the degree of overlap of the overlapping overlapping areas, and the degree of inclination of the bottom surface of the container located in the previously set observation area in the alignment direction of the previously set observation area and the new observation area is large. Set as large as possible.

In addition, you may provide it as a program which causes a computer to execute the operation method of the observation area setting apparatus by this invention.

Another observation area setting device according to the present invention includes a memory for storing instructions to be executed by a computer and a memory.
The processor comprises a processor configured to execute a stored instruction.
For the observation target housed in the container, a part of the new observation area adjacent to the previously set observation area is overlapped with respect to the previously set observation area, and new observation areas are sequentially set. In doing so
The process of setting the degree of overlap of the overlapping overlapping areas to be larger as the degree of inclination of the bottom surface of the container located in the previously set observation area in the alignment direction of the previously set observation area and the new observation area is larger. Run.

According to the present invention, with respect to the observation target housed in the container, a part of a new observation area adjacent to the previously set observation area is overlapped with respect to the previously set observation area. When setting various observation areas in sequence, the degree of overlap of the overlapping overlapping areas is determined by the inclination of the bottom surface of the container located in the previously set observation area in the alignment direction of the previously set observation area and the new observation area. By setting the larger the degree, the degree of overlap of the overlapping areas in the adjacent observation areas can be appropriately set according to the shape of the container, so that the amount of data of the captured image is increased and the shooting time is lengthened. Can be prevented.

The figure which shows the schematic structure of one Embodiment of the microscope observation system which applied the image processing apparatus of this invention. The figure which shows the scanning locus of each observation area in a well plate Diagram for explaining shape information of the bottom surface of the culture vessel Schematic block diagram showing the configuration of the observation area setting unit The figure explaining the overlapping area for each observation area in a culture vessel. The figure which shows an example of the acquired photographed image Diagram showing multiple overlapping regions set for the culture vessel A partially enlarged view of the observation image in the upper left corner of FIG. A partially enlarged view of the observation image in the upper right corner of FIG. A diagram illustrating another method of calculating the degree of inclination Flowchart showing the processing performed in this embodiment

Hereinafter, embodiments of the present invention will be described. FIG. 1 is a diagram showing a schematic configuration of a microscope observation system to which the observation area setting device according to the embodiment of the present invention is applied. As shown in FIG. 1, the microscope observation system of the present embodiment includes a microscope device 1, a microscope control device 2, an input device 3, and a display device 4. The microscope control device 2 corresponds to the image pickup control device of the present invention.

In the present embodiment, the microscope device 1 is a phase-contrast microscope, and as an observation target, for example, a phase-contrast image of cultured cells is imaged as a captured image. Specifically, as shown in FIG. 1, the microscope device 1 includes an illumination light irradiation unit 10, an imaging optical system 30, a stage 61, and an imaging unit 40.

A culture container 60 containing an observation target S such as cells and a culture solution C is installed on the stage 61. A rectangular opening is formed in the center of the stage 61. The culture container 60 is installed on the member forming the opening, and the photographed image of the observation target S in the culture container 60 is configured to pass through the opening.

In the culture vessel 60 installed on the stage 61, a cultured cell group (cell colony) is arranged as an observation target S. The cultured cells include pluripotent stem cells such as iPS cells and ES cells, nerves induced to differentiate from stem cells, skin, myocardium and liver cells, and skin, retina, myocardium, blood cells, nerves and nerves extracted from the human body. There are cells of organs. As the culture container 60, for example, a well plate having a plurality of wells (corresponding to the container of the present invention) is used, but the culture dish 60 is not limited to this, and a petri dish, a flask, a dish or the like may be used. In this embodiment, a well plate in which a plurality of wells are arranged is used as the culture vessel 60.

In the culture vessel 60 installed on the stage 61, the bottom surface of the culture vessel 60 is the installation surface P1 of the observation target S, and the observation target S is arranged on the installation surface P1. The culture container 60 is filled with the culture solution C. In the present embodiment, the cells cultured in the culture medium are set as the observation target S, but the observation target S is not limited to those in such a culture solution, and water, formalin, ethanol, methanol, etc. The cells fixed in the liquid of the above may be the observation target S.

The illumination light irradiation unit 10 irradiates the observation target S housed in the culture vessel 60 on the stage 61 with illumination light for so-called phase difference measurement, and in the present embodiment, the phase difference thereof. A ring-shaped illumination light is irradiated as an illumination light for measurement.

Specifically, the illumination light irradiation unit 10 of the present embodiment has a white light source 11 that emits white light for phase difference measurement and a ring-shaped slit, and the white light emitted from the white light source 11 is incident on the white light source 11. A slit plate 12 that emits ring-shaped illumination light, and a condenser lens 13 that receives ring-shaped illumination light emitted from the slit plate 12 and irradiates the incident ring-shaped illumination light onto the observation target S.

The slit plate 12 is provided with a ring-shaped slit that transmits white light to a light-shielding plate that blocks white light emitted from the white light source 11, and the white light passes through the slit to form a ring shape. Illumination light is formed. The condenser lens 13 converges the ring-shaped illumination light emitted from the slit plate 12 toward the observation target S.

The imaging optical system 30 forms an image of the observation target S in the culture vessel 60 on the imaging unit 40, and includes an objective lens 31, a phase plate 32, and an imaging lens 33.

The phase plate 32 is formed by forming a phase ring on a transparent plate that is transparent to the wavelength of ring-shaped illumination light. The size of the slit of the slit plate 12 described above has a conjugate relationship with this phase ring.

The phase ring is formed by forming a ring-shaped phase film that shifts the phase of the incident light by 1/4 wavelength and a dimming filter that dims the incident light. The direct light incident on the phase plate 32 passes through the phase ring, so that the phase shifts by 1/4 wavelength and the brightness thereof is weakened. On the other hand, most of the diffracted light diffracted by the observation target S passes through the transparent plate portion of the phase plate 32, and its phase and brightness do not change.

Direct light and diffracted light that have passed through the phase plate 32 are incident on the imaging lens 33, and these lights are imaged on the imaging unit 40.

The image pickup unit 40 includes an image pickup device that receives an image of the observation target S imaged by the imaging lens 33, images the observation target S, and outputs a phase difference image as an observation image. As the image pickup device, a CCD (charge-coupled device) image sensor, a CMOS (Complementary Metal-Oxide Semiconductor) image sensor, or the like can be used.

Here, the stage 61 is driven by the drive unit 62 and moves in the X and Y directions orthogonal to each other in the horizontal plane. By moving the stage 61, each observation area smaller than the well in each well of the well plate is scanned, and the captured image for each observation area is acquired by the imaging unit 40. At this time, each observation area is scanned by overlapping a part of the adjacent observation areas. The overlapping regions of adjacent observation regions will be described in detail later. Then, the captured image for each observation area is output to the microscope control device 2.

FIG. 2 is a diagram showing a scanning locus of each observation region as a solid line 77 when a well plate 70 having six wells 71 is used. As shown in FIG. 2, each observation region in the well plate 70 is scanned along the solid line 77 from the scanning start point 75 to the scanning end point 76 by the movement of the stage 61 in the X and Y directions.

In the present embodiment, the captured image for each observation area in the well is acquired by moving the stage 61, but the present invention is not limited to this, and the imaging optical system 30 is moved with respect to the stage 61. By doing so, the captured image for each observation area may be acquired. Alternatively, both the stage 61 and the imaging optical system 30 may be moved. Further, in the present embodiment, scanning is performed using the scanning locus shown in FIG. 2, but the present invention is not limited to this, and scanning may be performed using another scanning locus such as a spiral shape.

The microscope control device 2 is composed of a computer including a CPU (Central Processing Unit) 20, a primary storage unit 24, a secondary storage unit 25, an external I / F (Interface) 28, and the like. The CPU 20 includes a control unit 21, an observation area setting device 22, and an image processing unit 23, and controls the entire microscope observation system. The primary storage unit 24 is a volatile memory used as a work area or the like when executing various programs. An example of the primary storage unit 24 is a RAM (Random Access Memory). The secondary storage unit 25 is a non-volatile memory in which various programs, various parameters, and the like are stored in advance, and the shape information 26, which is an example of the shape information storage unit of the present invention, is stored. Further, an embodiment of the observation area setting program 27 of the present invention is installed in the secondary storage unit 25. The observation area setting device 22 functions when the observation area setting program 27 is executed by the CPU 20. Examples of the secondary storage unit 25 include EEPROM (Electrically Erasable Programmable Read-Only Memory), flash memory, and the like. The external I / F 28 controls the transmission and reception of various information between the microscope device 1 and the microscope control device 2. The CPU 20, the primary storage unit 24, and the secondary storage unit 25 are connected to the bus line 29. The external I / F 28 is also connected to the bus line 29.

The observation area setting program 27 is recorded and distributed on a recording medium such as a DVD (Digital Versatile Disc) and a CD-ROM (Compact Disc Read Only Memory), and is installed in a computer from the recording medium. Alternatively, the observation area setting program 27 is stored in a storage device or network storage of a server computer connected to the network in a state of being accessible from the outside, and is downloaded to the computer in response to a request from the outside. It may be installed.

The shape information 26 is the shape information of the bottom surface of the culture container 60, that is, the installation surface P1. The shape information of the bottom surface of the culture vessel 60 is measured in advance using, for example, a laser displacement meter. FIG. 3 is a diagram for explaining the shape information of the bottom surface of the culture vessel 60. As shown in FIG. 3, the shape information of the present embodiment is information obtained by measuring the bottom surface 60a of the culture vessel 60 in the XY directions with a spatial resolution of 10 μm × 10 μm. The spatial resolution of the shape information is not limited to this. Further, the method of measuring the shape information of the bottom surface 60a of the culture vessel 60 is not limited to the measurement by the laser displacement meter, and the measurement may be performed by using other methods such as a confocal method and a spectral interference method.

Further, the shape information of the bottom surface 60s of the culture vessel 60 may be measured by a laser displacement meter provided in the microscope device 1 when the culture vessel 60 is installed on the stage 61, or the culture vessel 60 may be measured. A table in which the identification information of the above is associated with the shape information measured in advance may be stored in the secondary storage unit 25. Then, the user sets and inputs the identification information of the culture container 60 installed on the stage 61 using the input device 3, and reads out the shape information of the culture container 60 having the identification information from the secondary storage unit 25. May be good. Further, the identification information of the culture container 60 may be obtained by assigning a bar code or the like to the culture container 60 and reading the bar code instead of inputting the setting by the user. The identification information of the culture container 60 may be, for example, the model number of the manufacturer or the serial number.

Further, although the case where the general-purpose computer functions as the microscope control device 2 has been described above, it may be carried out by the dedicated computer. The dedicated computer may be firmware that executes a program recorded in the non-volatile memory such as a built-in ROM (Read Only Memory) or a flash memory. Furthermore, dedicated circuits such as ASICs (Application Specific Integrated Circuits) and FPGAs (field programmable gate arrays) that permanently store programs for executing at least some of the functions of the microscope control device 2. May be provided. Alternatively, the program instructions stored in the dedicated circuit may be combined with the program instructions executed by the general-purpose CPU programmed to use the program of the dedicated circuit. As described above, program instructions may be executed in any combination of computer hardware configurations.

The control unit 21 controls the drive of the illumination light irradiation unit 10, the drive unit 62 for driving the stage 61, the imaging optical system 30, and the imaging unit 40 to acquire a captured image of the observation target S. The control unit 21 also functions as a display control unit that displays a single composite retardation image generated by combining the phase difference images of each observation position captured by the microscope device 1 on the display device 4. .. Further, the control unit 21 causes the imaging unit 40 to image the observation target S. In the present embodiment, since the culture vessel 60 is a well plate in which a plurality of wells are arranged, the control unit 21 causes the imaging unit 40 to image each observation region in each well.

The observation area setting device 22 sets an observation area for the culture vessel 60. FIG. 4 is a schematic block diagram showing the configuration of the observation area setting device 22. As shown in FIG. 4, the observation area setting device 22 includes an observation area setting unit 50, an overlap degree setting unit 51, and a shape information reception unit 52. The shape information receiving unit 52 reads and acquires the shape information of the bottom surface of the culture container 60 from the shape information 26 stored in the secondary storage unit 25. Although the observation area setting device 22 of the present embodiment is provided with the shape information receiving unit 52, the present invention is not limited to this, and the shape information receiving unit 52 may not be provided. In this case, the shape information 26 is read out from the secondary storage unit 25 as needed by a command from the CPU 20.

The observation area setting unit 50 newly overlaps a part of a new observation area adjacent to the observation area set earlier with respect to the observation target S housed in the culture container 60. The observation area is set sequentially. Here, FIG. 5 shows a diagram for explaining the overlapping region for each observation region in the culture vessel 60, and FIG. 6 shows a diagram showing an example of the captured image acquired. In the present embodiment, since the culture vessel 60 is a well plate in which a plurality of wells are arranged, an observation area is set in each well. In FIG. 5, both ends of the bottom surface of the well are defined as ends A and B, respectively, and the center of the bottom surface is defined as the center O.

Normally, when the depth of field is shallow, for example, when the center f is focused in one observation area from a to b as shown in FIG. 5, the captured image captured by the photographing unit 40 is shown in FIG. As shown by the diagonal lines, the peripheral parts on the a side and the b side become blurred images.

Therefore, by overlapping adjacent observation areas with the observation area corresponding to the peripheral portion that is easily blurred in the captured image and sequentially setting the observation area, the photographing unit 40 is made to shoot a plurality of captured images including the overlapping region. It is possible to select the captured image with better image quality in the overlapping area.

Here, FIG. 7 shows a diagram showing a plurality of overlapping regions set for the culture vessel 60. In the present embodiment, as described above, each observation area is scanned by overlapping a part of the adjacent observation areas. Therefore, as shown in FIG. 7, when the culture vessel 60 is the well plate 70, a plurality of captured images corresponding to each observation region of one well 71 are acquired, but each observation region Gi (i is The number of observation regions) includes overlapping regions that overlap with adjacent observation regions. In FIG. 7, the observation areas G1 to G5 are in order from the left end of the upper row (first row) along the scanning direction, G6 to G12 are in order from the right end of the second row, and G13 is in order from the left end of the third row. ~ G21, G22 to G30 in order from the right end of the 4th line, G31 to G39 in order from the left end of the 5th line, G40 to G48 in order from the right end of the 6th line, G49 to G57, 8th line in order from the left end of the 7th line. G58 to G64 are set in order from the right end of, and G65 to G69 are set in order from the left end of the 9th line. In FIG. 7, the widths d of the overlapping regions are all shown to have the same size on the drawing, but they are actually set to different sizes by the overlapping degree setting unit 51.

Here, a method of setting the degree of duplication by the degree of duplication setting unit 51 will be described. 8 is an enlarged view of the observation image in the upper left corner of FIG. 7, and FIG. 9 is an enlarged view of the observation image in the upper right corner of FIG. 7. As shown in FIG. 8, the overlapping area between the observation area G1 in the upper left corner of FIG. 7 and the observation area G2 adjacent to the right thereof is the overlapping area K1, and the overlapping area between the observation area G2 and the observation area G3 adjacent to the right side thereof. When the region is K2, the observation region G1 becomes the observation region set earlier in the present invention, the observation region G2 becomes a new observation region with respect to the observation region G1, and the observation region G2 becomes the observation region set earlier. Then, the observation area G3 becomes a new observation area with respect to the observation area G2.

The overlap degree setting unit 51 sets the overlap degree of the overlapping region, that is, the overlap region K1 and the overlap region K2 in FIG. It is preferable that the overlapping region is set at a ratio of, for example, 20 to 30% with respect to one observation region. First, from the shape information of the bottom surface of the culture vessel 60 (well 71 in this embodiment) located in the observation region G1, the overlap degree setting unit 51 determines the alignment direction of the observation region G1 and the observation region G2, that is, in FIGS. 7 and 8. The height of the bottom surface corresponding to the center position of the sides of both ends of the observation area G1 in the direction of the arrow M1 (X direction) is acquired, and the difference in height between both ends is calculated. In the present embodiment, the value of this height difference is defined as the degree of inclination of the overlapping region K1. Here, the height of the bottom surface corresponding to the center position of the sides of both ends of the observation area G1 was acquired, but the present invention is not limited to this, and the average value of the heights of the bottom surfaces of the positions corresponding to each of the sides of both ends is obtained. The difference in the average height may be the difference in height at both ends. The degree of inclination is not limited to using the value of the height difference itself at both ends of the observation region, and for example, a new value may be set based on the value of the height difference in the overlapping region K1.

The degree of inclination of the overlapping region K2 is also calculated in the same manner as in the overlapping region K1. That is, the heights of both ends in the direction of the arrow M1 of the observation area G2 are acquired, and the difference between the heights of both ends is calculated and used as the degree of inclination. Here, assuming that the bottom surface of the culture container 60 (well 71) of the present embodiment has the shape shown in FIG. 5, the bottom surface is inclined when the bottom surface is further away from the center of the culture container 60 in the direction of arrow M1. Is large, so that the overlap region K1 is larger than the overlap region K2 in the calculated inclination degree.

Therefore, the overlap degree setting unit 51 sets the width d2 of the overlap region K2 to be smaller than the width d1 of the overlap region K1 so that the overlap region K1 is larger than the overlap region K2. Similarly, the widths d1 to d4 of the overlapping regions K1 to K4 shown in FIG. 7 are set. The initial width, that is, the width d1 of the overlapping region K1, is predetermined so that the overlapping region K1 is set at a ratio of, for example, 20 to 30% with respect to the observation region G1, and is stored in the secondary storage unit 25, for example. You may read the default value that has been set. Further, the secondary storage unit 25 may store a correspondence table in which the relationship between the height difference and the width d is determined, and the width d1 may be set based on the correspondence table.

Next, the overlap degree setting unit 51 sets the overlap degree of the overlap region K5 and the overlap region K6 in FIG. 9. First, from the shape information of the bottom surface of the culture vessel 60 (well 71 in this embodiment) located in the observation region G5, the overlap degree setting unit 51 determines the alignment direction of the observation region G5 and the observation region G6, that is, in FIGS. 7 and 8. The height of the bottom surface corresponding to the center position of the sides of both ends of the observation area G5 in the direction of the arrow M2 (Y direction) is acquired, and the difference in height between both ends is calculated. In the present embodiment, the value of this height difference is defined as the degree of inclination of the overlapping region K5. Further, the degree of inclination of the overlapping region K6 is also calculated in the same manner as in the overlapping region K5. That is, the height of the bottom surface corresponding to the center position of the sides of both ends of the observation area G6 in the direction of the arrow M2 of the observation area G6 is acquired, and the difference between the heights of both ends is calculated and used as the degree of inclination.

Here, assuming that the bottom surface of the culture container 60 (well 71) of the present embodiment has the shape shown in FIG. 5, the bottom surface is inclined when the bottom surface is further away from the center of the culture container 60 in the direction of arrow M2. Is large, so that the overlap area K5 is larger than the overlap area K6 in the calculated inclination degree.

Then, the overlap degree setting unit 51 sets the width d6 of the overlap region K6 to be smaller than the width d5 of the overlap region K5 so that the overlap region K5 is larger than the overlap region K6. Regarding the observation areas G6 to G12 observed by scanning in the direction of the arrow M3, the overlapping area from the overlapping area K8 between the observation area G7 and the observation area G8 to the overlapping area K11 between the observation area K10 and the observation area K11 is The same widths as the widths d1 to d4 of the overlapping regions K1 to K4 shown in FIG. 7 are set respectively. The width d7 of the overlapping region K7 between the observation region G6 and the observation region G7 and the width d12 of the overlapping region K12 between the observation region G11 and the observation region G12 are set in the same manner as in the above method. As described above, the width d of the overlapping region K is set for each column and row in the direction of the arrow M1 in FIG. 7 (including the one scanning the direction of the arrow M3) and the direction of the arrow M2, respectively.

In the present embodiment, the width d of the overlapping region K is set for each column and row in the direction of arrow M1 (including the one scanning the direction of arrow M3) and the direction of arrow M2 in FIG. 7, respectively. The invention is not limited to this, and the width d of the overlapping region K may be set for each of the column and the row, and the direction of the arrow M1 and the direction of the arrow M2 are overlapped for each observation region. The width d of the area K may be set.

Further, in the present embodiment, when the degree of inclination is acquired, the difference in height of the observation area set earlier, that is, the observation area scanned first when the observation area is scanned is calculated. Is not limited to this. Here, FIG. 10 shows a diagram illustrating another method of calculating the degree of inclination.

As shown in FIG. 10, the overlap degree setting unit 51 includes a side h of the observation region G1 on the side where the observation region G2 is set, and is located in a region Gh having a size corresponding to the observation region G1 ( In the present embodiment, the height of the bottom surface corresponding to the center position of the sides of both ends of the region Gh in the alignment direction of the observation area G1 and the observation area G2 is obtained from the shape information of the bottom surface of the well 71), and the heights of the bottom surfaces are obtained. The difference in the height of the bottom surface corresponding to the center position may be calculated and used as the degree of inclination. The position of the area Gh can be appropriately changed by the user.

The observation area setting device 22 is configured as described above, and the observation area setting unit 50 observes the observation target S housed in the culture vessel 60 based on the degree of overlap set by the overlap degree setting unit 51. Set the area sequentially. In the present embodiment, the degree of overlap of the overlapping regions in the adjacent observation regions can be preferably set according to the shape of the culture vessel 60, so that it is possible to prevent an increase in the amount of data of the captured image and a lengthening of the imaging time. can do.

Next, returning to FIG. 1, the image processing unit 23 performs various processing such as gamma correction, luminance / color difference conversion, and compression processing on the image signal acquired by the imaging unit 16. Further, the image processing unit 23 outputs an image signal obtained by performing various processes to the control unit 21 for each frame at a specific frame rate. Further, the image processing unit 23 generates one composite image by combining the phase difference images of each observation region captured by the microscope device 1. As for the overlapping area, the image of the overlapping portion having better image quality is selected. For example, the captured image having the highest contrast may be used as the image of the overlapping region, or the overlapping portion of the captured image on the side closest to the center of the culture vessel 60 may be used as the image of the overlapping region. In order to generate a composite image with no blurred image in the peripheral portion of the observation area, that is, with good image quality, the overlapping area may be set large. However, if the duplicate image is set large, the number of shots increases, so that it takes time to shoot. By setting the degree of overlap to be larger as the inclination of the container is larger as in the present invention, it is possible to maintain the high image quality of the composite image and prevent an increase in the amount of data of the photographed image and a lengthening of the photographing time.

The input device 3 includes a mouse, a keyboard, and the like, and accepts various setting inputs by the user.

The display device 4 displays a composite image generated by the image processing unit 53, and includes, for example, a liquid crystal display or the like. Further, the display device 4 may be configured by a touch panel and may also be used as the input device 3.

Next, the processing performed by the observation area setting device 22 in the present embodiment will be described. FIG. 11 is a flowchart showing the processing performed in the present embodiment. First, the shape information receiving unit 52 acquires the shape information of the bottom surface of the culture container 60 (step S1). Next, the overlap degree setting unit 51 calculates the difference in height of the bottom surface of the culture vessel 60 located in the observation region G1 in the observation region set earlier, that is, the observation region G1 in FIG. 8 from the shape information acquired in step S1 ( Step S2), the difference in height is acquired as the degree of inclination (step S3).

Then, the overlap degree setting unit 51 determines the overlap degree for each observation area as described above (step S4), and the observation area setting unit 50 sequentially sets the observation area based on the overlap degree determined by the overlap degree setting unit 51. Set (step S5) and end the process. The observation area setting device 22 sets the observation area as described above.

In the microscope control device 2, the control unit 21 causes the imaging unit 40 to take an image for each observation area set by the observation area setting device 22, acquires images captured for each of a plurality of observation areas, and a plurality of image processing units 23 A composite image Gs is generated by joining the captured images of. Then, the generated composite image Gs is displayed on the display device 4 and is used for observation.

Although the above embodiment applies the present invention to a phase-contrast microscope, the present invention can be applied not only to a phase-contrast microscope but also to other microscopes such as a differential interference microscope and a bright-field microscope. ..

Hereinafter, the action and effect of this embodiment will be described.

With respect to the observation target S housed in the culture vessel 60, a part of a new observation area adjacent to the observation area set earlier with respect to the observation area set earlier is overlapped to form a new observation area. The degree of overlap of the overlapping overlapping areas is large in the degree of inclination of the bottom surface of the container located in the previously set observation area in the alignment direction of the previously set observation area and the new observation area. By setting the size as large as possible, the degree of overlap of the overlapping areas in the adjacent observation areas can be appropriately set according to the shape of the container, so that it is possible to prevent an increase in the amount of data of the captured image and a long shooting time. be able to.

1 Microscope device 2 Microscope control device 3 Input device 4 Display device 10 Illumination light irradiation unit 11 White light source 12 Slit plate 13 Condenser lens 21 Control unit 22 Image processing device 30 Imaging optical system 31 Objective lens 32 Phase plate 33 Imaging lens 40 Imaging unit 50 Observation area setting unit 51 Overlap degree setting unit 60 Culture vessel 60a Bottom surface 61 Stage 62 Area 70 Well plate 71 Well 75 Scanning end point 76 Scanning start point 77 Solid line showing scanning trajectory A0, A1 Arrow C Culture solution Gi Observation area Gs composite image K Overlapping area d Overlapping area width P1 Installation surface (bottom surface)
S Observation target M1, M2, M3 Arrangement direction

Claims (11)

For the observation target housed in the container, the observation area in which the new observation area is sequentially set by overlapping a part of the new observation area adjacent to the observation area with respect to the previously set observation area. Setting part and
The degree of overlap of the overlapping overlapping regions is such that the greater the degree of inclination of the bottom surface of the container located in the previously set observation region in the arrangement direction of the previously set observation region and the new observation region. An observation area setting device including an overlap degree setting unit for setting a large amount. A shape information receiving unit for receiving shape information on the bottom surface of the container is provided.
The observation area setting device according to claim 1, wherein the overlap degree setting unit acquires the inclination degree based on the shape information input from the shape information receiving unit. The observation area setting device according to claim 1 or 2, wherein the degree of inclination is a value based on the difference in height between both ends of the bottom surface of the container located in the previously set observation area in the alignment direction. The degree of inclination is located in a region including one side of the previously set observation region on the side where the new observation region is set, and in a region having a size corresponding to the previously set observation region. The observation area setting device according to claim 1 or 2, which is a value based on the difference in height between both ends in the alignment direction of the bottom surface of the container. The observation area setting device according to any one of claims 1 to 4, wherein the observation area is smaller than the container. The observation area setting device according to any one of claims 1 to 5, wherein the container is a dish, a well plate, or a flask. The observation area setting device according to any one of claims 1 to 6 and
An imaging control device including a control unit that causes an imaging unit to image an observation target housed in the container for each observation area set by the observation area setting device. The imaging control device according to claim 7, further comprising a shape information storage unit that stores shape information of the bottom surface of the container. The imaging control device according to claim 8, wherein the shape information storage unit stores a table in which the identification information of the container and the shape information of the bottom surface of the container are associated with each other. It is an operation method of an observation area setting device including an observation area setting unit and an overlap degree setting unit.
The observation area setting unit overlaps a part of a new observation area adjacent to the observation area with respect to the previously set observation area with respect to the observation target housed in the container, and the new observation. Set the area sequentially,
The overlap degree setting unit sets the overlap degree of the overlapping overlapping regions of the container located in the previously set observation region in the alignment direction of the previously set observation region and the new observation region. How to operate the observation area setting device that sets the larger the inclination of the bottom surface. Computer,
For the observation target housed in the container, the observation area in which the new observation area is sequentially set by overlapping a part of the new observation area adjacent to the observation area with respect to the previously set observation area. Setting means and
The degree of overlap of the overlapping overlapping regions is such that the greater the degree of inclination of the bottom surface of the container located in the previously set observation region in the alignment direction of the previously set observation region and the new observation region. An observation area setting program for functioning as a means for setting a large degree of overlap.