Image acquisition method and image acquisition system

Abstract

This invention provides an image acquisition method that enables the rapid acquisition of phase-difference contrast images. [Solution] An image acquisition method for acquiring a phase contrast image of a sample containing cells in a liquid contained in a container, wherein the sample is divided into N regions, and of the N regions, M

Description

Technical Field

[0001] The present invention relates to an image acquisition method and an image acquisition system.

Background Art

[0002] In cell culture, the process of culturing may be observed using a microscope or the like. Since cells are generally colorless and transparent, it is possible to observe the growth by observing a phase-contrast image using a phase-contrast microscope. At this time, since the angle of view of a general phase-contrast microscope is small with respect to the bottom area of the culture vessel, the range to be observed may not fit within one field of view.

[0003] On the other hand, Patent Document 1 describes a method of acquiring images of small regions (hereinafter also referred to as tiles), and synthesizing them to obtain a large-region image (hereinafter also referred to as tiling).

Prior Art Documents

Patent Documents

[0004] [[ID=***********]] [[ID=***********]]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0005] In the method described in Patent Document 1, there are cases where the entire necessary observation range cannot be accommodated within one dynamic range of the camera, and it may be necessary to set appropriate exposure conditions for each tile. However, when obtaining a large-region image by determining appropriate exposure conditions for each tile, there is a problem that the image acquisition time becomes long.

[0006] The present invention has been made in view of the above problems, and an object thereof is to provide an image acquisition method and an image acquisition system that enable rapid acquisition of a phase-contrast image.

Means for Solving the Problem

[0007] An image acquisition method according to an aspect of the present invention is an image acquisition method for acquiring a phase contrast image captured by an imaging device using the phase contrast method for a sample containing cells in a liquid stored in a container, comprising: a step of acquiring an exposure acquisition image obtained by imaging M (M is an integer of 2 or more) regions satisfying M < N out of the N (N is an integer of 3 or more) regions when the range for the purpose of acquiring the phase contrast image for the sample is divided into N regions; a step of calculating appropriate exposure conditions for the exposure acquisition image; a step of determining exposure conditions for imaging each of the N regions based on the positions of the M regions in the target range and the appropriate exposure conditions for the exposure acquisition image; a step of determining an imaging order of the N regions based on the exposure conditions for imaging each of the N regions; a step of acquiring a composite image obtained by imaging each of the N regions by the phase contrast method based on the imaging order and the exposure conditions for imaging each of the N regions; a step of synthesizing the composite images; and characterized by comprising the above.

Advantages of the Invention

[0008] According to the present invention, there are provided an image acquisition method and an image acquisition system capable of quickly acquiring a phase contrast image.

Brief Description of the Drawings

[0009] [Figure 1] It is a schematic diagram showing the whole apparatus configuration according to the first embodiment. [Figure 2A] It is a block diagram showing the functional configuration of each apparatus according to the first embodiment. [Figure 2B]It is a block diagram showing the functional configuration of each device according to the first embodiment. [Figure 3] It is a flowchart showing the overall flow of the image acquisition method according to the first embodiment. [Figure 4] It is a flowchart showing a part of the flow of the image acquisition method according to the first embodiment. [Figure 5] It is a flowchart showing a part of the flow of the image acquisition method according to the first embodiment. [Figure 6] It is a flowchart showing a part of the flow of the image acquisition method according to the first embodiment. [Figure 7] It is a flowchart showing the overall flow of the image acquisition method according to the second embodiment. [Figure 8] It is a flowchart showing a part of the flow of the image acquisition method according to the second embodiment.

Mode for Carrying Out the Invention

[0010] During the culturing process, the cells are in the culture medium, which is a liquid filled in the culture vessel. When observed with a phase-contrast microscope, a phenomenon occurs where the peripheral wall surface of the culture vessel becomes bright. The cause of this phenomenon is considered to be that the optical distance changes due to the meniscus of the culture medium and the curvature (flatness) of the bottom surface of the culture vessel, resulting in a change in the phase-contrast image. Note that the liquid filled in the culture vessel when observed with a phase-contrast microscope can be any of liquids for different purposes such as a culture medium, a cell detachment solution, a washing solution, a buffer solution, etc. And the above problem can occur not only when the liquid filled in the culture vessel is a culture medium. Also, the process of observing cells with a phase-contrast microscope is not limited to the culturing process and can be a process such as detachment, washing, addition of a drug, etc.

[0011] Since the phase-contrast image varies depending on the configuration of the phase-contrast microscope, preparing a high-angle (macro) observation system different from the observation system for acquiring tile images and obtaining brightness changes thereby cannot address the above phenomenon.

[0012] Furthermore, the intensity and range of brightness that change due to the aforementioned phenomenon vary depending on the amount of the culture solution and the shape of the container. Since the amount of the culture solution varies among operators, it is necessary to determine appropriate exposure conditions for the actual object for which an image is to be acquired.

[0013] As a result of intensive studies, the inventors of the present application have found that by the image acquisition method according to the present invention described below, it is possible to quickly determine appropriate exposure conditions and acquire a large-area image. The image acquisition method according to the present invention is an image acquisition method for acquiring a phase-contrast image obtained by imaging a sample containing cells in a liquid contained in a container using an imaging device by the phase-contrast method. Here, the liquid contained in the container is not particularly limited and can be any of liquids for different uses such as a culture solution, a cell detachment solution, a washing solution, and a buffer solution. And the image acquisition method according to the present invention has a step of acquiring an image for exposure acquisition, a step of calculating exposure conditions, a step of determining exposure conditions, a step of determining an imaging order, a step of acquiring an image for synthesis, and a step of synthesizing the image for synthesis.

[0014] The image for exposure acquisition is an image obtained by imaging M regions satisfying M < N out of the N regions when the range for the purpose of acquiring the phase-contrast image of the sample is divided into N regions by the phase-contrast method. Here, M is an integer of 2 or more because it is used to obtain the exposure conditions by interpolation for each of the N regions, which will be described in detail later. Also, N is an integer of 3 or more in order to satisfy the relationship M < N. In the present disclosure, the image for exposure acquisition means an image (image for acquiring exposure conditions) used to determine and acquire appropriate exposure conditions.

[0015] The step of calculating the exposure conditions is a step of calculating appropriate exposure conditions for the image for exposure acquisition. Also, the exposure conditions in the step of determining the exposure conditions are exposure conditions for imaging each of the N regions based on the positions of the M regions in the target range and the appropriate exposure conditions for the image for exposure acquisition.

[0016] The above imaging sequence is the order in which the N regions are imaged, based on the exposure conditions for imaging each of the N regions. Furthermore, the composite image described above is an image obtained by capturing each of the N regions using the phase difference contrast method, based on the above-mentioned imaging sequence and exposure conditions for imaging each of the N regions.

[0017] The image acquisition method according to the present invention can be particularly preferably applied when the cells are cells contained in a sheet-like cell culture.

[0018] In recent years, in the field of regenerative medicine and cell therapy, attempts have been made to culture cells in a sheet-like form and transplant the sheet-like cell culture (cell sheet) to the affected area in order to repair damaged tissue. When producing cell sheets using adherent cells, for example, cells are cultured in a sheet-like form on a culture vessel filled with culture medium, and then the sheet is peeled off and collected from the culture substrate. Furthermore, the collected cell sheets may be damaged, such as wrinkles, tears, or holes. To identify the causes of these damages and improve yield, it is necessary to observe the cell growth state while culturing the cells.

[0019] Since cell sheets grow and spread to the walls of the culture vessel, it is necessary to observe the entire culture vessel using a phase-contrast microscope. Conventional methods, which involve determining appropriate exposure conditions for each tile to acquire large-area images, require a long time to acquire the images. Since prolonged image acquisition can lead to cell damage, it is necessary to acquire images as quickly and efficiently as possible. Therefore, the image acquisition method according to the present invention is particularly suitable for obtaining observation images of cell sheets.

[0020] The cells whose images are acquired using the image acquisition method according to the present invention are not limited to the cell sheet described above. For example, seeded single cells, groups of cells before they form a sheet, or cell aggregates (spheroids) may also be used.

[0021] Hereinafter, exemplary embodiments of the present invention will be described with reference to the drawings. In the drawings, similar or corresponding elements are denoted by the same reference numerals, and their descriptions may be omitted or simplified.

[0022] [First Embodiment] Figure 1 is a schematic diagram showing an example of an apparatus configuration for acquiring phase contrast images of cells, which includes an image acquisition system according to the first embodiment of the present invention and an imaging device equipped with a phase contrast microscope. The apparatus configuration shown in Figure 1 is an example of an apparatus configuration for acquiring a phase contrast image of a cell sheet in a culture vessel as a high-resolution, large-size (wide-angle) digital image. In this embodiment, since the subject is a cell sheet, the range for acquiring the phase contrast image includes the entire area of ​​the vessel.

[0023] The system configuration shown in Figure 1 consists of an imaging device 101, a computer 102, a display device 103, and an input device 104. The imaging device 101 and the computer 102 are connected by a dedicated or general-purpose I / F cable, and the computer 102 and the display device 103 are connected by a general-purpose I / F cable. The input device 104 is also connected to the computer 102. Details of the imaging device 101 and the computer 102 will be described later.

[0024] The display device 103 is a display device that uses, for example, liquid crystal, electroluminescence (EL), or a cathode ray tube (CRT). The display device 103 and the computer 102 may be an integrated notebook PC. The input device 104 is a device for the user to input commands such as the start of imaging, and consists of a keyboard, mouse, touch panel, etc. Alternatively, a device that integrates the display device 103 and the input device 104, such as a tablet, may be used.

[0025] In this embodiment, the configuration shown in the figure will be used as an example for explanation, but for example, a notebook PC in which the computer 102 and the display device 103 are integrated, or a device that integrates all of these components may be used.

[0026] Figure 2A is a schematic diagram showing the functional configuration of the imaging device 101, and Figure 2B is a schematic diagram showing the functional configuration of the computer 102. The imaging device 101 acquires image data of phase difference contrast images at multiple different positions along two orthogonal axes in a plane. The imaging device 101 comprises an illumination unit 201, a phase difference illumination optical system 202, a stage 203, a stage control unit 204, a phase difference detection optical system 205, and an imaging unit 206.

[0027] The illumination unit 201, used in conjunction with the phase contrast illumination optical system 202, is a means of irradiating a cell sheet 2081 in a culture medium 2082 contained in a culture vessel 208 placed on the stage 203 with light for phase contrast observation. The illumination unit 201 consists of an illumination light source 2011 and a light source control system 2012. The light source control system 2012 controls the illumination light source 2011 based on instructions from the light source control unit 211.

[0028] The stage 203 is driven and controlled by the stage control unit 204, and is capable of moving in three directions: the X, Y, and Z axes, which are perpendicular to each other. A positioning jig (hereinafter referred to as the positioning jig) 207 for the culture vessel 208 is installed on the stage 203. By fitting the culture vessel 208 into the groove provided in the positioning jig 207, it becomes possible to reliably position the culture vessel 208 on the stage 203.

[0029] The stage control unit 204 consists of a drive control system 2041 and a stage drive mechanism 2042. The drive control system 2041 receives instructions from the stage control unit 212 and controls the drive of the stage 203. The stage drive mechanism 2042 drives the stage 203 according to the instructions from the drive control system 2041.

[0030] The phase difference detection optical system 205 is used in conjunction with the phase difference illumination optical system 202 to form a phase difference contrast image onto the light-receiving element surface of the imaging sensor 2061 in the imaging unit 206.

[0031] The imaging unit 206 consists of an imaging sensor 2061 and an imaging control system 2062, and performs imaging in response to instructions from the imaging control unit 210. The stage 203 is driven in directions along the X and Y axes, with the plane perpendicular to the optical axis being the XY plane, thereby capturing tile images and acquiring image data. The tile image data acquired by the imaging unit 206 (hereinafter also referred to as tile image data) is transmitted to the display data generation unit 220. The imaging sensor 2061 is a two-dimensional image sensor that converts a two-dimensional optical image into electrical physical quantities by photoelectric conversion, and for example, a CCD or CMOS device is used. The imaging control system 2062 controls the sensitivity (e.g., ISO sensitivity), exposure time, and execution of imaging of the imaging sensor 2061.

[0032] Computer 102 is an example of a device that functions as an image processing system according to the present invention, and includes a CPU (Central Processing Unit), RAM (Random Access Memory), storage device, data input / output interface, and an internal bus connecting these to each other. As described above, a display device 103 and an input device 104 are connected to computer 102. A program according to one embodiment of the present invention is installed in the storage device of the computer 102, and the CPU operates according to this program, thereby executing the processes in the imaging control unit 210, the display data generation unit 220, and the input information acquisition unit 230.

[0033] The imaging control unit 210 consists of a light source control unit 211, a stage control unit 212, an imaging sequence control unit 213, and an exposure control unit 214.

[0034] The light source control unit 211 receives an instruction from the exposure control unit 214, issues an instruction to the light source control system 2012 in the illumination unit 201, and controls the brightness of the illumination light source 2011. The stage control unit 212 issues an instruction to the drive control system 2041 of the stage control unit 204 and controls the position of the stage 203.

[0035] The imaging order control unit 213 issues an instruction to the stage control unit 212 based on the imaging order list described later generated by the imaging order determination unit 224, and controls the imaging order of the tile images. The exposure control unit 214 issues an instruction to the light source control unit 211 and the imaging control system 2062 in the imaging unit 206 so that the exposure is appropriate based on the exposure conditions determined by the exposure condition determination unit 223.

[0036] The display data generation unit 220 includes an exposure acquisition image acquisition unit 221, an exposure calculation unit 222, an exposure condition determination unit 223, an imaging order determination unit 224, a composite image acquisition unit 225, and an image composite unit 226, and performs processing related to each step in the image acquisition method according to the present invention.

[0037] The exposure acquisition image acquisition unit 221 acquires the position information of the positioning jig 207 transmitted from the stage control unit 212 and the exposure acquisition image transmitted from the imaging unit 206. That is, the exposure acquisition image acquired by the exposure acquisition image acquisition unit 221 is associated with the position information of the positioning jig 207. Here, the exposure acquisition image is an exposure acquisition tile image obtained by imaging M regions satisfying M < N out of the N regions when the range for the purpose of acquiring a phase contrast image of the culture vessel 208 is divided into N regions. The exposure acquisition tile image is imaged by the phase contrast method using the imaging device 101, and the exposure acquisition tile image transmitted from the imaging unit 206 can also be displayed on the display device 103.

[0038] The exposure calculation unit 222 analyzes the exposure acquisition image data acquired by the exposure acquisition image acquisition unit 221 to determine the exposure status of the image (correct, overexposed, or underexposed) and calculates the appropriate exposure conditions for the exposure acquisition image. One method for determining the exposure status is to calculate a histogram of the exposure acquisition image data.

[0039] The exposure condition determination unit 223 determines the exposure conditions for imaging each of the N regions based on the position of the M regions within the desired range and the appropriate exposure conditions for the exposure acquisition image. Here, the position of the M regions within the desired range can be determined from the position information of the positioning jig 207 associated with the exposure acquisition image. The exposure condition determination unit 223 transmits the information regarding the determined exposure conditions to the exposure control unit 214.

[0040] The imaging order determination unit 224 determines the imaging order of the N regions based on the exposure conditions for each region determined by the exposure condition determination unit 223. The imaging order determination unit 224 transmits information regarding the determined imaging order of the N regions to the imaging order control unit 213.

[0041] The image acquisition unit 225 acquires a composite image (composite tile image) by capturing each of the N regions using the phase difference contrast method. The composite image is captured by the imaging device 101 based on instructions issued by the exposure control unit 214 based on the information regarding the exposure conditions and instructions issued by the imaging sequence control unit 213 based on the information regarding the imaging sequence of the N regions.

[0042] The image synthesis unit 226 acquires data for all synthesis tile images from the synthesis image acquisition unit 225, and then synthesizes them to generate high-resolution, large-area (wide-angle) image data (synthesized image data). The synthesized image data is stored in the storage device of the computer 102 and is also output to the display device 103 to display the synthesized image.

[0043] The input information acquisition unit 230 receives information input from the user via the input device 104 and issues instructions to the imaging control unit 210. The user inputs information and makes various settings via a graphical user interface (GUI). Examples of these settings in this embodiment include the shape of the positioning jig 207, the initial settings of the light source control unit 211, and the initial settings of the sensitivity and exposure time of the imaging sensor 2061.

[0044] Each step in the operation of the imaging device 101 and the computer 102 in this embodiment will be explained using the flowcharts shown in Figures 3 to 6. In this embodiment, a culture vessel 208 with a nearly circular bottom is used. Examples of culture vessels 208 include dishes. Furthermore, it is assumed that the positional relationship between the X and Y axis coordinates of the stage 203 and the culture vessel 208 is known, based on measurements taken separately when installing the positioning jig 207 on the stage 203.

[0045] Figure 3 is a flowchart showing the overall process. Step S301 involves initializing the imaging device. The flowchart in Figure 4 shows the process for initializing the imaging device.

[0046] In step S401, the user inputs imaging condition information such as information from the positioning jig 207, the area (field of view) that can be acquired by the imaging unit 206, the initial exposure time of the imaging sensor 2061, and the initial sensitivity, via the input device 104. The input information is stored in the memory device of the computer 102 via the input information acquisition unit 230.

[0047] In step S402, the input information acquisition unit 230 acquires the information entered in step S401 and transmits it to the imaging control unit 210 and the display data generation unit 220. In step S403, based on the information of the positioning jig 207 obtained in step S402, the coordinate values of the X-axis and Y-axis on the stage 203 of each tile are obtained. At the same time, initialization of the imaging sensor 2061, the stage 203, the illumination light source 2011, etc. is performed.

[0048] In step S302, an exposure parameter table is generated. Briefly, the data of the exposure acquisition image (exposure acquisition image data) described above is acquired, and an interpolation formula for calculating parameters (exposure parameters) for specifying specific exposure conditions is obtained. Subsequently, after obtaining the exposure parameters to be set for each of the N tiles, this is output as an exposure parameter table. Note that the exposure conditions preferably include at least one selected from the group consisting of the intensity (brightness) of the illumination light source 2011, the sensitivity of the imaging sensor 2061, and the exposure time of the imaging sensor 2061.

[0049] In the present embodiment, the phenomenon in which the brightness of the phase contrast image described above changes is utilized for the characteristic that when the bottom surface of the culture vessel 208 is generally circular, it is rotationally symmetric about the center of the bottom surface of the culture vessel 208. Thereby, when the number of tiles to be acquired as described above is, for example, a total of 9 tiles of 3 in the vertical direction × 3 in the horizontal direction (that is, N = 9), the interpolation formula described later can be obtained by acquiring at least 2 exposure image acquisition image data (that is, M = 2), and a parameter table can be created. That is, M is an integer of 2 or more, and N is an integer of 3 or more since it satisfies the relationship of M < N. Of course, not limited to this example, in view of accuracy and acquisition time, the value of M may be increased from 2. For example, when N is 15 × 15 = 225, M may be set to a value of 2 or more and less than 7.

[0050] The steps related to generating the exposure parameter table are shown in FIG. 5. Among the steps shown in FIG. 5, the steps of step S501 to step S503 are included in the exposure acquisition image acquisition process executed by the exposure acquisition image acquisition unit 221. Further, the steps of step S504 to step S507 are included in the exposure calculation process executed by the exposure calculation unit 222. Further, the steps of step S508 and step S509 are included in the exposure condition determination process executed by the exposure condition determination unit 223.

[0051] In step S501, the exposure acquisition image acquisition unit 221 determines the conditions for acquiring the exposure acquisition tile image and the synthesis tile image. Specifically, according to the information acquired in step S402, from the area of the entire container (the area in which the entire bottom surface of the culture container 208 is contained) and the viewing angle (FOV) of the imaging device 101, the value of the number N of all tiles to be acquired is determined. Also, the number M of exposure acquisition tile images is determined. Regarding the method of determining the number M of exposure acquisition tile images, there is no particular limitation as long as the relationship M < N is satisfied. The value of M may be automatically determined by the computer 102 according to a predetermined standard, for example, or the value determined by the user according to the value of N may be input from the input device 104.

[0052] In step S502, the exposure acquisition image acquisition unit 221 instructs the imaging sequence control unit 213 to move the imaging field to the center of the culture container 208 in order to acquire the first exposure acquisition image. The movement is performed by driving the stage 203. The center position of the culture container 208 is determined based on the information of the positioning jig 207 input in step S401.

[0053] In step S503, the exposure acquisition image acquired by the imaging device 101 is acquired. In step S504, based on the data of the exposure acquisition image, the exposure calculation unit 222 calculates the exposure parameter that results in an appropriate exposure. In step S505, the exposure parameter calculated in step S504 is recorded in the storage device in the computer 102.

[0054] In step S506, it is determined whether the imaging field of view has reached the outer edge of the culture vessel 208. If it has, the process proceeds to step S508; otherwise, it proceeds to step S507. The determination of whether the outer edge of the culture vessel 208 has been reached is made by comparing the positioning jig 207 information input in step S401 with the current coordinates of the stage 203.

[0055] In step S507, the imaging field of view is moved radially around the culture vessel 208 by a predetermined distance. The distance the imaging field of view is moved can be determined, for example, based on the size of the area from which to acquire N composite tile images and the value of M. The distance the imaging field of view is moved may be constant or may vary depending on the position of the exposure acquisition tile image. The change in brightness of the phase contrast image in the exposure acquisition image tends to be small in the center of the bottom of the vessel and increases as it approaches the vessel wall. By utilizing this, exposure acquisition image data can be acquired sparsely in the center and densely as it approaches the wall, thereby shortening the acquisition time while maintaining accuracy. After moving the imaging field in step S507, return to step S503.

[0056] Step S508 is a process of obtaining an approximate function from the exposure acquisition image that represents the relationship between the position of the sample within the imaging range and the exposure conditions. In this embodiment, step S508 uses the exposure parameters recorded in step S505 to obtain an interpolation formula for the radius of the culture vessel 208. The interpolation formula is given by, when r is the distance from the center of the culture vessel 208 and W(r) is the exposure parameter, W(r)=a n r n +a n―1 r n―1 +···+a1r+a0 This can be expressed as a polynomial such that n is a natural number. nn is the coefficient of the polynomial and is determined by known methods such as the least squares method. Depending on the value of M, n is preferably around 3 or 4. Note that the interpolation formula is not limited to polynomials; known formulas such as exponential functions, trigonometric functions, and sigmoid functions can be used. These may also be combined as appropriate.

[0057] Step S509 is the process of determining the exposure conditions for imaging each of the N regions (tiles) using the approximation function obtained in step S508. In step S509, the exposure parameters to be set for each tile are calculated according to the interpolation formula obtained in step S508 and output as an exposure parameter table. The output exposure parameter table is recorded in the storage device of the computer 102.

[0058] In step S303, the imaging order of the N regions (composite tiles) is determined based on the exposure conditions for imaging each of the N regions (exposure parameter table generated in step S302). In essence, tiles with similar exposure parameters are grouped from the exposure parameter table, the imaging order of the groups is determined, and then the imaging order of the tiles within each group is determined. The determined imaging order is output as an imaging order list.

[0059] Figure 6 shows the process for determining the imaging order. Each step shown in Figure 6 is performed by the imaging order determination unit 224. In step S601, the exposure parameter table generated in step S509 is obtained and loaded.

[0060] In step S602, the exposure parameter table is scanned, and composite tiles with similar exposure parameters are grouped together. One method of grouping is to perform cluster analysis using a known method such as the K-means method. When using the K-means method, it is preferable to keep the number of clusters to three or less, as small as possible. When grouping, it is preferable to prioritize the exposure time, the brightness of the illumination light source 2011, and the sensitivity of the image sensor 2061 in that order. This is because the exposure time directly affects the entire acquisition time, so it is preferable to give it the highest priority, and generally, switching the brightness of the illumination light source 2011 takes more time than changing the sensitivity of the image sensor 2061. In step S602, for N regions, regions with the same exposure conditions may be grouped together.

[0061] In step S603, the imaging order between the groups generated by grouping in step S602 is determined. In step S603, it is preferable to determine the imaging order between the groups such that the sum of the distances spanning between the groups is minimized. In this embodiment, the order is determined so that the group at the center of the bottom surface of the culture vessel 208 is scanned first, and the groups at the edges are scanned last.

[0062] In step S604, the imaging order between the regions included in each group is determined. In step S604, it is preferable to determine the imaging order between the regions (composite tiles) included in each of the above groups so that imaging can be performed in a single continuous stroke. In this case, it is preferable to determine the order within the same group based on the imaging order between groups determined in step S603 so as to minimize the distance across the groups. In this embodiment, it is preferable to determine the imaging order of the N regions in step S604 such that the trajectory of the imaging order of the N regions forms a spiral shape. In particular, for the synthesis tiles in the group located at the center of the bottom surface of the culture vessel 208, the imaging order is determined so that it spirals outwards from the center.

[0063] In step S605, based on the results of steps S603 and S604, an imaging sequence list describing the imaging order of the composite tiles is output. The imaging sequence list is recorded in the storage device of the computer 102.

[0064] Steps S304 to S310 are steps included in the composite image acquisition process performed by the composite image acquisition unit 225. In step S304, based on the imaging sequence list generated in step S303, the stage 203 is driven to move the imaging field to the tile for the first image. In step S305, the exposure parameter table generated in step S302 is referenced to obtain the exposure parameter for the current tile.

[0065] In step S306, the exposure parameters obtained in step S305 are compared with the exposure parameters used when imaging the previous tile in the imaging sequence list. If the exposure parameters have changed, the process proceeds to step S307; otherwise, it proceeds to step S308. In step S307, the exposure parameters acquired in step S305 are applied to the illumination unit 201 and the imaging unit 206.

[0066] In step S308, imaging is performed by the imaging unit 206 to acquire tile image data. The acquired tile image data is transmitted to the display data generation unit 220 as described above. Focusing may be performed immediately before imaging. Focusing can be performed by known methods such as contrast detection. The user may also manually focus.

[0067] In step S309, it is determined whether imaging has been performed to the end of the imaging sequence list generated in step S303. If it has been performed to the end, the process proceeds to step S311; otherwise, it proceeds to step S310. In step S310, the stage 203 is driven to move the imaging field of view to the next tile based on the imaging sequence list generated in step S303. After moving the imaging field in step S310, return to step S305.

[0068] Step S311 is a step included in the image synthesis process performed by the image synthesis unit 226. In step S311, the tile image data acquired so far is synthesized to generate high-resolution and large-size (wide-angle) digital image data. As described above, in this embodiment, exposure acquisition image data is acquired before acquiring all tile image data, and exposure parameters are calculated to ensure appropriate exposure for each tile. This allows for the rapid acquisition of a composite image in which phase difference contrast images are obtained with appropriate exposure conditions for all tiles.

[0069] [Modified example 1 of the first embodiment] In the first embodiment, movement to the tiles to be imaged was performed by driving the stage 203, but the method of moving the imaging field of view is not limited to this. For example, the three-dimensional position of the culture vessel 208 may be fixed using a jig or the like without using the stage 203. Then, the tile image may be acquired by moving the illumination unit 201, the phase-contrast illumination optical system 202, the phase-contrast detection optical system 205, and the imaging unit 206 as an integrated structure.

[0070] [Modified example 2 of the first embodiment] In the first embodiment, the exposure parameters were calculated by determining an interpolation formula. However, if the container shape variations are predetermined, a method of referring to multiple pre-calculated tables may be used. In this case, the exposure parameters obtained from the exposure acquisition image data are compared with each table, and the closest table is adopted as the interpolated exposure parameters. This makes it possible to save computation time and resources such as memory required to calculate the interpolation formula.

[0071] [Second Embodiment] The overall apparatus configuration in the second embodiment is the same as in the first embodiment, so a description will be omitted. Each step in the operation of the imaging device 101 and the computer 102 in this embodiment will be explained using the flowcharts shown in Figures 7 and 8.

[0072] In this embodiment, a culture vessel 208 with a rectangular base is used. The shape of the base of the culture vessel 208 is not limited to a rectangle; the four corners may have any angle. Also, the number of corners is not limited to four; it may be any polygon with three or more corners. Examples of specific culture vessels 208 include flasks and trays. Furthermore, it is assumed that the positional relationship between the X and Y axis coordinates of the stage 203 and the culture vessel 208 is known, based on measurements taken separately when installing the positioning jig 207 on the stage 203.

[0073] Furthermore, in this embodiment, the exposure parameter described later is the exposure time of the image sensor 2061. Of course, either the brightness of the illumination light source 2011 or the sensitivity of the image sensor 2061 may be used as the exposure parameter illumination, or a combination of these may be used.

[0074] Figure 7 shows a flowchart of the entire process. The initialization of the imaging device in step S301 is the same as in the first embodiment, and is as shown in the flowchart in Figure 4.

[0075] Step S701 generates an exposure parameter table. In summary, it acquires exposure data, determines a curved surface in 3D space (exposure parameter surface) for calculating exposure parameters, determines the exposure parameters to be set for each tile, and then outputs this as an exposure parameter table.

[0076] The processes related to the generation of the exposure parameter table are shown in FIG. 8. Among the processes shown in FIG. 8, the processes of steps S801 to S803 are included in the exposure acquisition image acquisition process executed by the exposure acquisition image acquisition unit 221. Further, the processes of steps S804 to S807 are included in the exposure calculation process executed by the exposure calculation unit 222. Also, the processes of steps S808 and S809 are included in the exposure condition determination process executed by the exposure condition determination unit 223.

[0077] In step S801, the exposure acquisition image acquisition unit 221 determines the conditions for acquiring the exposure acquisition tile image and the synthesis tile image. Specifically, first, the value of the number N of all tiles to be acquired is determined in the same manner as described in step S501. Also, the number M of positions (hereinafter referred to as sampling points) for sampling the exposure acquisition tile image for obtaining the information necessary for calculating the aforementioned exposure parameter surface is determined and made into a list (hereinafter referred to as a sampling list). Regarding the method of determining the number M of exposure acquisition tile images as in the first embodiment, there is no particular limitation as long as the relationship M < N is satisfied. When the number of synthesis tiles to be acquired is, for example, 9 in total of 3 vertically and 3 horizontally (that is, N = 9), the exposure parameter surface can be obtained by acquiring 5 in total, that is, M = 5, at the center of the bottom surface of the container and at the corners of the exposure image acquisition image data. Similar to the first embodiment, the value of M may be increased or decreased in view of accuracy and acquisition time.

[0078] The determination of the sampling points may be made based on the information of the positioning jig 207, or may be input by the user via the input device 104. When using the rectangular culture container 208 as in this embodiment, it is preferable to set the center of the bottom surface of the culture container 208 and the corners of the bottom surface of the culture container 208 as the sampling points. If higher accuracy is required, one or more arbitrary positions on the sides of the bottom surface of the culture container 208 may be additionally set as the sampling points as described above.

[0079] In step S802, based on the list of sampling points created in step S801, the stage 203 is driven to the first sampling point in the list to move the imaging field of view. In step S803, an exposure acquisition image captured by the imaging device 101 is acquired.

[0080] In step S804, the exposure calculation unit 222 calculates the appropriate exposure parameters based on the exposure acquisition image data. As mentioned above, in this embodiment, the exposure parameter is the exposure time of the image sensor 2061. In step S805, sampling data is recorded. The sampling data consists of coordinate values ​​where U is the X-coordinate of the sampling point stage 203, V is the Y-coordinate, and W is the exposure parameter that results in proper exposure at the sampling point. The sampling data is recorded in the storage device of the computer 102.

[0081] In step S806, it is determined whether sampling data has been recorded at all sampling points in the aforementioned sampling list. If data has been recorded at all sampling points, the process proceeds to step S808; otherwise, it proceeds to step S807. In step S807, the stage 203 is driven to the next sampling point based on the list of sampling points created in step S801, thereby moving the imaging field of view. After moving the imaging field in step S807, return to step S803.

[0082] In step S808, the exposure parameter surface is determined from the sampling data. The exposure parameter surface is an approximate surface that passes near all of the following coordinates, where U is the X-coordinate value of the stage at the sampling point, V is the Y-coordinate value, and W is the exposure parameter that results in proper exposure at the sampling point. (U0,V0,W0),(U1,V1,W1),...,(U M ,V M ,W M )

[0083] Approximate surfaces can be obtained using known methods such as surface regression analysis. Alternatively, approximate surfaces can be found by assuming and fitting polynomial surfaces such as parabolas. In addition to methods that find a surface passing through all points in one step, there is also a two-step method in which two or more polynomials that pass through multiple points are found, and the surface passing through these is found.

[0084] In step S809, the exposure parameters to be set for each composite tile are calculated from the exposure parameter surface and output as an exposure parameter table. The output exposure parameter table is recorded in the storage device of the computer 102. Step S303 is the same as in the first embodiment and is as shown in the flowchart in Figure 6.

[0085] Steps S304 to S311 are the same as in the first embodiment, so their explanation will be omitted. As described above, in this embodiment, an exposure parameter surface is determined, and exposure parameters that result in appropriate exposure for each tile are calculated using this exposure parameter surface. This allows for the rapid acquisition of a composite image in which phase contrast images are obtained under appropriate exposure conditions for all tiles.

[0086] [Modified version of the second embodiment] In the second embodiment, the sampling points were set to the center of the bottom surface of the culture vessel 208 and the corner of the bottom surface of the culture vessel 208. However, if the shape of the bottom surface of the culture vessel 208 is symmetrical, the change in brightness of the phase difference contrast image described above will also be symmetrical. Therefore, if the shape of the bottom surface of the culture vessel 208 is symmetrical, it is possible to obtain the exposure parameter surface with fewer sampling points by sampling only one side of the pair. In the example above, if there were a total of 9 tiles to be acquired in a 3x3 grid (i.e., N=9), the number of images to be acquired for exposure acquisition can be reduced to a total of 3 images (i.e., M=3), one from the center of the bottom surface of the container and one corner.

[0087] [Other embodiments] In the first and second embodiments, the positional relationship between the stage 203 and the culture vessel 208 was ensured by the positioning jig 207. However, instead of the positioning jig 207, the positional relationship may be determined using a combination of an observation system capable of observing the entire stage 203 (e.g., a bright-field optical system and an imaging device) and image recognition.

[0088] In the first and second embodiments, the functionality was implemented as software installed on computer 102. However, similar functionality may also be implemented as a dedicated board using, for example, an FPGA (Field Programmable Gate Array), and integrated into computer 102. Furthermore, the present invention may be implemented by a circuit (e.g., an ASIC) that performs one or more functions.

[0089] In the first and second embodiments, the imaging device 101 and the computer 102 were connected by a dedicated or general-purpose I / F cable, but instead, the WEB (Internet) may be used for the connection between them. Furthermore, in this case, the computer 102 may be a virtual computer on a cloud network.

[0090] It should be noted that the embodiments described above are merely examples of how the present invention can be implemented, and the technical scope of the present invention should not be interpreted as being limited by them. In other words, the present invention can be implemented in various forms without departing from its technical concept or its main features. For example, the object of the present invention may be realized by combining the first and second embodiments described above with the other embodiments described above. That is, embodiments to which the present invention can be applied should be understood as embodiments to which the present invention can be applied in which a part of the configuration of one embodiment is added to another embodiment, or in which a part of the configuration of another embodiment is replaced.

[0091] The disclosure of embodiments of the present invention includes the following configurations and methods. (Method 1) An image acquisition method for obtaining a phase contrast image captured by an imaging device by the phase contrast method for a sample containing cells in a liquid contained in a container, comprising: Among the N regions (N is an integer of 3 or more) obtained by dividing the range for the purpose of obtaining the phase contrast image for the sample into N regions, obtaining exposure acquisition images obtained by imaging M regions (M is an integer of 2 or more) satisfying M < N by the phase contrast method; Calculating an appropriate exposure condition for the exposure acquisition image; Based on the positions of the M regions in the target range and the appropriate exposure condition for the exposure acquisition image, determining an exposure condition for imaging each of the N regions; Determining an imaging order of the N regions based on the exposure condition for imaging each of the N regions; Based on the imaging order and the exposure condition for imaging each of the N regions, obtaining a composite image obtained by imaging each of the N regions by the phase contrast method; Composite the composite images; An image acquisition method characterized by comprising the above. (Method 2) Further comprising the step of obtaining an approximation function representing the relationship between the position within the range for imaging the sample and the exposure condition from the exposure acquisition image, The image acquisition method according to Method 1, wherein the exposure condition for imaging each of the N regions is determined using the approximation function. (Method 3) The image acquisition method according to Method 1 or 2, wherein the cells are cells contained in a sheet-like cell culture. (Method 4) The image acquisition method according to any one of Methods 1 to 3, wherein the target range includes the entire area of the container. (Method 5) The image acquisition method according to any one of Methods 1 to 4, characterized in that the value of N is determined from the area of the entire area of the container and the field of view angle (FOV) of the imaging device. (Method 6) The image acquisition method according to any one of methods 1 to 5, characterized in that the exposure conditions include at least one selected from the group consisting of exposure time, sensitivity of the image sensor, and intensity of the illumination light source. (Method 7) The step of determining the imaging order of the N regions is as follows: The process involves grouping together regions with the same exposure conditions from among the aforementioned N regions, A step of determining the imaging order among the groups generated by the aforementioned grouping, A step of determining the imaging order between regions included in each of the aforementioned groups, An image acquisition method according to any one of methods 1 to 6, characterized by including the following: (Method 8) The image acquisition method according to Method 7, characterized in that, in the step of determining the imaging order of the N regions, the imaging order between the groups is determined such that the sum of the distances spanning between the groups is minimized. (Method 9) The image acquisition method according to Method 7, characterized in that, in the step of determining the imaging order of the N regions, the imaging order between the regions included in each of the groups is determined so that imaging can be performed in a single continuous line. (Method 10) An image acquisition method according to any one of methods 1 to 6, characterized in that, in the step of determining the imaging order of the N regions, the imaging order of the N regions is determined such that the trajectory of the imaging order of the N regions takes the shape of a spiral. (Composition 1) A program that causes a computer to execute one of the image acquisition methods described in Methods 1-10. (Configuration 2) An image acquisition system that acquires phase-contrast images of a sample containing cells in a liquid contained in a container, using an imaging device with phase-contrast imaging, When the range for the purpose of obtaining the phase contrast image for the sample is divided into N regions (N is an integer of 3 or more), among the N regions, an exposure acquisition image acquisition unit that acquires M exposure acquisition images (M is an integer of 2 or more that satisfies M < N) of M regions imaged by the phase contrast method, an exposure calculation unit that calculates appropriate exposure conditions for the exposure acquisition image, an exposure condition determination unit that determines exposure conditions for imaging each of the N regions based on the positions of the M regions in the target range and the appropriate exposure conditions for the exposure acquisition image, an imaging order determination unit that determines the imaging order of the N regions based on the exposure conditions in each of the N regions, a composite image acquisition unit that acquires composite images obtained by imaging each of the N regions by the phase contrast method based on the imaging order and the exposure conditions in each of the N regions, an image composite unit that composites the composite images, An image acquisition system characterized by comprising the above.

Explanation of Signs

[0092] 101: Imaging device 102: Computer 103: Display device 104: Input device 210: Imaging control unit 220: Display data generation unit 221: Exposure acquisition image acquisition unit 222: Exposure calculation unit 223: Exposure condition determination unit 224: Imaging order determination unit 225: Composite image acquisition unit 226: Image composite unit 230: Input information acquisition unit

Claims

1. An image acquisition method for acquiring a phase contrast image of a sample containing cells in a liquid contained in a container, using an imaging device with a phase contrast method, The process of acquiring exposure acquisition images by imaging M regions (M being an integer of 2 or more) from among the N regions where the range for acquiring the phase difference contrast image of the sample is divided into N regions (N being an integer of 3 or more), and M < N regions being captured by the phase difference contrast method. A step of calculating appropriate exposure conditions for the exposure acquisition image, A step of determining exposure conditions for imaging each of the N regions based on the position of the M regions within the target range and the appropriate exposure conditions for the exposure acquisition image, A step of determining the imaging order of the N regions based on the exposure conditions for imaging each of the N regions, A step of acquiring a composite image by imaging each of the N regions using the phase difference contrast method, based on the imaging sequence and exposure conditions for imaging each of the N regions. The process of synthesizing the aforementioned composite images, An image acquisition method characterized by having the following features.

2. The process further includes determining an approximate function from the exposure acquisition image that represents the relationship between the position of the sample within the imaging range and the exposure conditions, The image acquisition method according to claim 1, wherein exposure conditions for imaging each of the N regions are determined using the approximation function.

3. The image acquisition method according to claim 1, characterized in that the cells are cells contained in a sheet-like cell culture.

4. The image acquisition method according to claim 1, characterized in that the scope of the objective includes the entire area of ​​the container.

5. The image acquisition method according to claim 1, characterized in that the value of N is determined from the total area of ​​the container and the field of view (FOV) of the imaging device.

6. The image acquisition method according to claim 1, characterized in that the exposure conditions include at least one selected from the group consisting of exposure time, sensitivity of the image sensor, and intensity of the illumination light source.

7. The step of determining the imaging order of the N regions is as follows: The process involves grouping together regions with the same exposure conditions from among the aforementioned N regions, A step of determining the imaging order among the groups generated by the aforementioned grouping, A step of determining the imaging order between regions included in each of the aforementioned groups, The image acquisition method according to claim 1, characterized by including the following:

8. The image acquisition method according to claim 7, characterized in that, in the step of determining the imaging order of the N regions, the imaging order between the groups is determined such that the sum of the distances spanning between the groups is minimized.

9. The image acquisition method according to claim 7, characterized in that, in the step of determining the imaging order of the N regions, the imaging order between the regions included in each of the groups is determined so that imaging can be performed in a single continuous line.

10. The image acquisition method according to claim 1, characterized in that, in the step of determining the imaging order of the N regions, the imaging order of the N regions is determined such that the trajectory of the imaging order of the N regions takes the shape of a spiral.

11. A program for causing a computer to execute the image acquisition method described in any one of claims 1 to 10.

12. An image acquisition system that acquires phase-contrast images of a sample containing cells in a liquid contained in a container, using an imaging device with phase-contrast imaging, An exposure acquisition image acquisition unit acquires exposure acquisition images by imaging M regions (M is an integer of 2 or more) from among the N regions where the range for acquiring the phase difference contrast image of the sample is divided into N regions (N is an integer of 3 or more), satisfying M < N, using the phase difference contrast method. An exposure calculation unit that calculates appropriate exposure conditions for the exposure acquisition image, An exposure condition determination unit determines the exposure conditions for imaging each of the N regions based on the position of the M regions within the target range and the appropriate exposure conditions for the exposure acquisition image, An imaging order determination unit determines the imaging order of the N regions based on the exposure conditions in each of the N regions, A composite image acquisition unit acquires a composite image obtained by capturing each of the N regions using the phase difference contrast method based on the imaging sequence and the exposure conditions in each of the N regions. The image synthesis unit synthesizes the aforementioned images for synthesis, An image acquisition system characterized by having the following features.

Images