JPWO2019180810A1 - Cell observation device - Google Patents

Abstract

After the entire culture plate has been photographed by the holographic microscope, the operator specifies a specific measurement position on the displayed hologram image. Then, the partial phase image and the partial intensity image are reconstructed from the hologram data at that position, and these images are drawn in the image display area (71) on the designated position image display screen (70) displayed on the display unit. When the operator sets the slice condition in the slice condition setting area (73) on the screen and clicks the button (74), the partial phase image at a plurality of focal positions according to the slice condition is reconstructed. The operator compares images having different focal positions by operating a slider or the like in the focal position selection operation area (75), finds an appropriate focal position, and clicks the button (76). Then, the focal position of the image at that time is set as an appropriate focal position for the well in which the measurement position exists, and a phase image of the entire plate at the focal position is created. As a result, the focus alignment can be performed immediately after the end of shooting without waiting for the creation of the phase image of the entire plate.

Description

The present invention relates to a cell observation device for observing the state of cells. More specifically, the present invention acquires a hologram in which interference fringes between an object wave and a reference wave are recorded by a holographic microscope, and obtains phase information and intensity information based on the hologram data. The present invention relates to a cell observation device that creates a phase image, an intensity image, etc. after calculation.

In the field of regenerative medicine, research using pluripotent stem cells such as iPS cells and ES cells has been actively conducted in recent years. Since cells are generally transparent and difficult to observe with an ordinary optical microscope, a phase-contrast microscope has been widely used for observing cells. However, since it is necessary to perform focusing when taking a microscopic image with a phase-contrast microscope, it takes time to measure when acquiring a microscopic image for each small area in which a wide observation target area is finely divided. There is a problem that it is too much. In order to solve this problem, in recent years, a digital holographic microscope using a digital holography technique has been developed and put into practical use (see Patent Documents 1 and 2, Non-Patent Document 1 and the like).

In a digital holographic microscope, an interference fringe (hologram) formed by an object light reflected or transmitted on the surface of an object and a reference light directly arriving from the same light source on a detection surface such as an image sensor is acquired. , The phase information and the amplitude (intensity) information are acquired by performing the back propagation arithmetic processing of the light wave based on the hologram, and the intensity image and the phase image are created as the reconstructed image.

Digital holographic microscopes include in-line type, off-axis type, and phase shift type. Each of these methods mainly differs in the configuration of the optical system for acquiring the hologram. In the off-axis type, the light emitted from the laser light source is usually divided into a reference light and an irradiation light irradiating the object, and the object light transmitted through the object and the reference light are incident differently from each other with respect to the irradiation light. It is incident on the image sensor with an angle. On the other hand, in the in-line type, the light emitted from the laser light source is irradiated to the object without being divided, and both the object light that has passed through the object and the reference light that has passed in the vicinity of the object without passing through the object are combined. It is incident on the image sensor approximately vertically. Further, in the phase shift type, a hologram having a phase difference in a plurality of stages is acquired by changing the optical path length of the divided reference light in a plurality of stages using a phase shift interferometer.

In any of the methods, in the digital holographic microscope, it is necessary to calculate the phase information and the amplitude information of the light wave based on the acquired hologram data and perform a process of imaging the information by a computer. For example, Patent Document 1, Non-Patent Document 2, and the like propose a phase retrieval method by iterative calculation of light wave propagation based on holograms for a plurality of wavelengths of light acquired by an in-line digital holographic microscope. With a digital holographic microscope, phase information at an arbitrary distance (focus position) can be obtained at the stage of arithmetic processing after acquiring a hologram, so it is not necessary to perform focusing each time during shooting, and the measurement time is shortened. This has the advantage of suppressing damage to living cells.

However, when observing the entire culture plate with such a cell observation device, it is not always possible to observe the entire plate well with a phase image at one focal position.
Usually, a culture plate used for cell culture has a plurality of recesses called wells, and cells are cultured on a medium contained in each well. The height of the bottom surface of a plurality of wells in one culture plate varies due to a molding error or the like when molding the culture plate. If the height of the bottom surface of the well varies, the height of the medium will differ even if the same amount of medium is added, and even if the thickness of the cells themselves is the same, the surface height will vary. In addition, the variation in the injection amount of the medium also causes the variation in the surface height of the cells. Although there are variations in the thickness of individual cells, this is sufficiently smaller than the variations in the bottom height of the wells and the injection amount of the medium.

Due to the above factors, in general, the focusing position may be different for each well in one culture plate, and an appropriate focus position may be set for each well to reconstruct the phase image of the entire culture plate. There is a need to do. Conventionally, in order to obtain such a phase image, first, a phase image reconstruction process at a common predetermined focal position is performed on the entire culture plate. After that, the operator checks the phase image for each well to determine whether the focal position is appropriate, and if not, changes the setting of the focal position to reconstruct the phase image of the entire culture plate. A method such as redoing is adopted.

However, since the amount of calculation of phase information and the calculation of image reconstruction processing is large, even if a computer having a certain degree of performance is used, the phase image of the entire culture plate is reconstructed to obtain a phase image. It takes time. According to the study by the present inventors, for example, the entire surface of the culture plate is divided into about 900 small regions, and holograms for a plurality of wavelengths are obtained for each small region at a high resolution (for example, 4000 × 3000 pixels / sheet). It takes one hour or more from the start of measurement to the end of the phase image reconstruction process in the device. That is, the focusing operation can be performed only after one hour or more has passed from the start of measurement. Further, if the phase image reconstruction process is repeated in order to obtain an appropriate focal position for each well, it takes a longer time to complete the focusing.

International Patent Publication No. 2016/0842420 Japanese Unexamined Patent Publication No. 10-268740

"Cell culture analyzer CS-1", [online], Shimadzu Corporation, [Search on March 6, 2018], Internet <URL: https://www.an.shimadzu.co.jp/bio /cell/cs1/index.htm ＞ Peng Bao and 3 others, "Lensless phase microscopy using phase retrieval with multiple illumination wavelengths", "Lensless phase microscopy using phase retrieval with multiple illumination wavelengths", Applied Optics, The Optical Society of America, 2012, Vol. 51, No. 22, pp. 5486-5494

The present invention has been made to solve the above problems, and an object of the present invention is to perform arithmetic processing such as phase retrieval based on a hologram acquired by a holographic microscope to perform arithmetic processing such as phase retrieval on a phase of a sample containing cells. It is an object of the present invention to provide a cell observation device for reconstructing an image, which can quickly adjust the focus position after the completion of imaging and can avoid wasting time and manpower.

The first aspect of the present invention made to solve the above problems is a cell observation device using a holographic microscope.
a) Light source and
b) A detection unit that acquires a hologram that is an interference fringe between an object wave and a reference wave when a sample containing cells is irradiated with light emitted from the light source unit.
c) A moving unit that moves one or both of the light source unit, the detection unit, and the sample so that the measurement position on the sample moves.
d) Measurement that controls the light source unit, the detection unit, and the moving unit so as to repeatedly acquire holograms at each measurement position in a predetermined observation target region while moving the measurement position on the sample by the moving unit. Control unit and
e) Reconstructed image creation unit that creates partial phase images and / or partial intensity images at multiple focal positions based on hologram data obtained at one or more specific measurement positions or ranges specified by the user. When,
f) A display processing unit that simultaneously or switchably displays a partial phase image and / or a partial intensity image at a plurality of focal positions created by the reconstructed image creation unit on the display unit.
g) When the partial phase image and / or the partial intensity image is displayed from the display processing unit, the partial phase image and / or the partial intensity image displayed at that time is operated by the user. A focus position setting unit that stores the focal position in the storage unit as the focal position in the focused state when creating a phase image and / or an intensity image of a predetermined region on the sample including the measurement position or range corresponding to the image. When,
It is characterized by having.

In the cell observation apparatus according to the present invention, for example, by moving the light source unit and the detection unit integrally with respect to the culture plate whose position is fixed, the light emitted from the light source unit on the culture plate is emitted. The measurement position to be irradiated is changed. The measurement control unit is formed on the detection surface of the detection unit by irradiating one measurement position in the culture plate with coherent light while, for example, the light source unit and the detection unit are integrally and stepwise moved by the moving unit. The operation of acquiring data (hologram data) showing the two-dimensional distribution of light intensity by the hologram is repeated.

The reconstructed image creation unit provides phase information and phase information based on hologram data obtained at a specific one or more measurement positions specified by the user, or within a predetermined range within one measurement position or across a plurality of measurement positions. / Or the intensity information is calculated, and at that time, the phase information and / or the intensity information at a plurality of focal positions is calculated. The plurality of focal positions may be those specified by the user or may be predetermined. Then, the reconstructed image creating unit creates a partial phase image and / or a partial intensity image for each focal position based on the obtained information. The range in which the partial phase image and / or the partial intensity image is created is limited to a region much smaller than the entire observation target region. That is, the partial phase image or the partial intensity image is an image corresponding to a small part of the entire observation target area.

In general, it takes a considerable amount of time to reconstruct a phase image or an intensity image for the entire observation target area based on hologram data for the entire observation target area. On the other hand, the amount of hologram data to be processed by the reconstructed image creation unit is much smaller than the amount of hologram data for the entire observation target area. Therefore, the reconstructed image creating unit can create a partial phase image or a partial intensity image within a relatively short time from the time when the measurement is completed. A plurality of partial phase images and partial intensity images having different focal positions, which are created in this way, are simultaneously or switchably displayed on the display unit by the display processing unit. This allows the user to compare partial phase images and the like at different focal positions and determine which focal position is appropriate for observing the cells.

For example, the user compares the partial phase images and performs a predetermined operation in a state where the partial phase image determined to be appropriate is displayed. Then, in response to this user's operation, the focal position setting unit sets the focal position of the partial phase image displayed at that time to the phase of a predetermined region on the sample including the measurement position or range corresponding to the image. It is stored in the storage unit as a condition of the focal position for creating an image or an intensity image. In this way, the appropriate focal position for reconstructing the phase image or intensity image of the entire observation target area is determined by using the partial phase image or partial intensity image displayed within a relatively short time after the measurement is completed. Can be done. When reconstructing the phase image or the intensity image of the entire observation target area, it is sufficient to create an image at least at the focal position stored in the storage unit, but the focal range of a predetermined plurality of stages including the focal position An image may be created.

In the present invention, the holographic microscope method such as in-line type, off-axis type, and phase shift type is not limited, but the structure of the optical system is simple and the distance between the sample and the light source portion is changed in a plurality of steps. An in-line type configuration using holograms at a plurality of wavelengths is desirable in that the drive mechanism is simplified because it is not necessary.

In the first aspect of the present invention, preferably
A hologram image creating unit that creates a hologram image based on the hologram data obtained at each measurement position in the predetermined observation target area and displays it on the screen of the display unit.
An instruction for the user to instruct the measurement position or range corresponding to the partial phase image and / or the partial intensity image created by the second reconstructed image creating unit on the hologram image displayed by the hologram image creating unit. Input section and
It is preferable that the configuration further includes.

Unlike a phase image or the like, a hologram image can be displayed immediately after the measurement is completed. Further, even during measurement, a partial hologram image corresponding to the measurement position can be displayed each time the measurement for one measurement position is completed. Since this hologram image has a two-dimensional distribution of light intensity corresponding to the hologram, it is often not sufficiently visible for living cells, but it is a sufficiently visible image for the edges of wells with large steps. .. Therefore, the user instructs, for example, which part of which well the partial phase image or the partial intensity image is desired to be viewed on the displayed hologram image by the instruction input unit. As a result, the user can confirm the partial phase image and the partial intensity image of the desired measurement position and range with a short waiting time.

In addition to allowing the user to specify one or more specific measurement positions on the hologram image, for example, the coordinate position that the user wants to see is specified by referring to an optical microscopic image separately acquired for the sample, and the coordinate position is specified. By inputting the information of the coordinate position into the apparatus according to the present invention, it may be possible to determine a specific one or a plurality of measurement positions.

Further, when the cell observation device according to the first aspect of the present invention is a cell observation device for observing cells being cultured in each recess of the culture plate having a plurality of recesses formed therein. ,
In response to the user's operation, the focal position designation receiving unit sets the focal position of the partial phase image and / or the partial intensity image displayed at that time to the phase in the recess including the measurement position or range corresponding to the image. The focal position is stored in the storage unit as a focal position for creating an image and / or an intensity image, and the focal position for independently creating a phase image and / or an intensity image in the recesses for each of the plurality of recesses. It is good to have a configuration that memorizes.

As described above, when a plurality of wells (recesses) are formed in the culture plate, the height of the bottom surface of the wells and the height of the medium in the wells are different for each well, and therefore the focusing position is different for each well. Often there is. On the other hand, according to the above configuration, before creating a phase image or an intensity image of the entire culture plate, a partial phase image or a portion of each of the plurality of wells formed on the culture plate at a plurality of different focal positions is provided. By creating an intensity image and determining an appropriate focal position for each recess, the user can collect information on an appropriate focal position for each well. As a result, it is only necessary to create a phase image or an intensity image at the in-focus position of each well at the stage of creating a phase image or an intensity image of the entire culture plate, which saves time and effort for creating an image of an unnecessary focal position. It can be mitigated.

Further, in this configuration, the focus position designation reception unit further displays the focus position information set for each of the plurality of recesses on the screen of the display unit, and the value of the focus position by the user on the display. It may be configured to include a focus position editing unit that accepts editing of.

According to this configuration, when the user has some prior information or knowledge about an appropriate focal position, the focal position editing unit can appropriately correct the value of the focal position. Further, the user may appropriately input the focal position by the focal position editing unit before executing the reconstruction process of the partial phase image or the like.

A second aspect of the present invention made to solve the above problems is a cell observation device using a holographic microscope for observing cells being cultured in one or more containers. ,
a) A light source that emits coherent light,
b) A detection unit that acquires a hologram that is an interference fringe between an object wave and a reference wave when the sample, which is one or more containers containing cells, is irradiated with light emitted from the light source unit.
c) A moving unit that moves one or both of the light source unit, the detection unit, and the sample so that the measurement position on the sample moves.
d) Measurement that controls the light source unit, the detection unit, and the moving unit so as to repeatedly acquire holograms at each measurement position in a predetermined observation target region while moving the measurement position on the sample by the moving unit. Control unit and
e) A height measuring unit that non-contactly measures the relative height of the inner bottom of the one or more containers or the surface of the medium contained in the container.
f) A focal position setting unit that sets the focal position for each container based on the relative height measured by the height measuring unit.
g) Under the control of the measurement control unit, the focus position setting unit for each region corresponding to the container is based on the hologram data obtained at each measurement position in the predetermined observation target region on the sample. Create a reconstructed image that calculates the phase information and / or intensity information at the set focal position and creates a phase image showing the two-dimensional distribution of the phase information and / or an intensity image showing the two-dimensional distribution of the intensity information. Department and
It is characterized by having.

Here, when only one container is used, the easy is a petri dish, a flask, or the like. On the other hand, when a plurality of containers are used, the containers are generally wells formed on the culture plate.

In this second aspect, instead of the user visually checking the partial phase image or the like to find the in-focus position, the height measuring unit is placed on the inner bottom of one or more containers or the medium contained in the container. Measure the relative height of the surface in a non-contact manner. As the height measuring unit, for example, a laser range finder or the like can be used. Then, the focal position setting unit sets the focal position for each container based on the relative height value obtained by the measurement by the height measuring unit. When the height of the inner bottom of the container is measured by the height measuring unit, the height of the medium surface is estimated based on information such as the amount of the medium contained in the container, and the culture is further carried out from that height. The height of the cell surface inside can be estimated. Further, when the height of the medium surface in the container can be measured by the height measuring unit, the height of the cell surface during culturing can be estimated from the height.

In any case, since the focal position is determined using the value actually measured by the height measuring unit, it depends on the judgment of the operator even if the height of the inner bottom is different for each of a plurality of wells, for example. It is possible to focus with high accuracy. Further, since it is not necessary for the operator to confirm the partial phase image and the partial intensity image, it is possible to create an automatic observation image that further saves manpower.

According to the present invention, the operator can quickly determine the focal position after the measurement is completed without waiting for the phase image or intensity image created through complicated calculations based on the hologram data acquired by the measurement to be displayed. You can do the work of matching. Further, in particular, according to the first aspect of the present invention, at the same time as the work of confirming whether the shooting state or the like is appropriate with the partial phase image or the partial intensity image displayed immediately after the measurement is completed, the work of focusing is aligned. It can be performed. As a result, it is possible to improve the efficiency of the work that the worker should perform when observing the cells.

The whole block diagram of the cell observation apparatus which is an Example of this invention. The schematic block diagram of the measurement terminal in the cell observation apparatus of this Example. The conceptual diagram for demonstrating the image reconstruction processing in the cell observation apparatus of this Example. The explanatory view of the characteristic processing operation in the cell observation apparatus of this Example. The figure which shows an example of the screen displayed on the measurement terminal after the completion of imaging in the cell observation apparatus of this Example. The figure which shows an example of the IHM phase image and the IHM intensity image of the designated measurement position displayed by the measurement terminal in the cell observation apparatus of this Example. The schematic block diagram of the measurement terminal in the cell observation apparatus which is another Example of this invention.

Hereinafter, an embodiment of the cell observation apparatus according to the present invention will be described with reference to the accompanying drawings.
FIG. 1 is an overall configuration diagram of the cell observation device of this embodiment, and FIG. 2 is a schematic configuration diagram of a measurement terminal in the cell observation device of this embodiment.

The cell observation device of this embodiment includes a measurement terminal 1, a browsing terminal 3, and a server 5 connected via a communication network 4 such as the Internet or an intranet. Although FIG. 1 shows one measurement terminal 1 and one viewing terminal 3, each of these can be provided in an appropriate number.

The server 5 is a high-performance computer, and as functional blocks embodied by dedicated software installed in the computer, a data transmission / reception unit 51, a hologram data storage unit 52, a first phase retrieval calculation unit 53, and a second phase recovery calculation unit 53. It includes a phase retrieval calculation unit 54, a first image reconstruction unit 55, a second image reconstruction unit 56, an image data storage unit 57, and the like.

The measurement terminal 1 includes a microscopic observation unit 10 and a control / processing unit 20. In the cell observation apparatus of this embodiment, the microscopic observation unit 10 is an in-line digital holographic microscope, and as shown in FIG. 2, includes a light source unit 11 including a laser diode and the like and an image sensor 12, and the light source unit 11 and A culture plate 13 containing the cells 14 to be observed is arranged between the image sensor 12 and the image sensor 12. The light source unit 11 and the image sensor 12 can be integrally moved in the biaxial directions of the X-axis and the Y-axis orthogonal to each other by a moving unit 15 including a drive source such as a motor.

In addition, in FIG. 2, in order to avoid complicating the drawings, the light source unit 11 and the image sensor 12 are shown one by one, that is, only one set, but in reality, the light source unit 11 and the image sensor 12 are shown. Is provided in four sets with one culture plate 13 sandwiched between them, and as will be described later, the four sets of light source units 11 and the image sensor 12 provide holograms of different measurement positions of one culture plate 13. It is possible to acquire in parallel.

The entity of the control / processing unit 20 controls the operation of the microscopic observation unit 10, sends the data acquired by the microscopic observation unit 10 to the server 5, and further receives and displays the data processed by the server 5. It is a personal computer (PC). As functional blocks embodied by the dedicated software installed in this PC, a shooting control unit 21, a hologram data storage unit 22, a data transmission / reception unit 23, a hologram image creation unit 24, a confirmation position designation reception unit 25, and an image data storage unit. A unit 26, a display processing unit 27, a focus designation reception unit 28, and the like are provided. Further, the control / processing unit 20 is connected to an input unit 201, which is a pointing device such as a keyboard or a mouse, and a display unit 202.

The browsing terminal 3 is a general PC in the same manner as the control / processing unit 20 in the measuring terminal 1. Then, the dedicated software installed on the PC can receive the data from the server 5 and display an appropriate image formed based on the data.

Next, the operation of the cell analyzer of this embodiment when acquiring hologram data will be described with reference to FIG. FIG. 3 is a conceptual diagram for explaining the image reconstruction process in the cell observation device of this embodiment.

FIG. 3A is a schematic top view of the culture plate 13 used in the cell observation apparatus of this example. Six wells 13a having a circular shape in the top view are formed on the culture plate 13, and cells are cultured in each of the wells 13a. Here, the entire culture plate 13, that is, the entire rectangular area including the six wells 13a is the observation target area. The microscopic observation unit 10 includes four sets of a light source unit 11 and an image sensor 12, and each of the light source unit 11 and the image sensor 12 of each set has a total culture plate 13 of 4 mag as shown in FIG. 3A. It is responsible for collecting hologram data of the four divided four-division ranges 81. That is, the four sets of the light source unit 11 and the image sensor 12 share the collection of hologram data over the entire culture plate 13.

As shown in FIGS. 3 (b) and 3 (c), the range in which the set of the light source unit 11 and the image sensor 12 can be photographed at one time includes the well 13a in the four division range 81. It is a range corresponding to one imaging unit 83 obtained by dividing the square-shaped range 82 into 10 equal parts in the X-axis direction and 12 equal parts in the Y-axis direction. One 4-division range 81 includes a predetermined number of imaging units 83, for example, 15 × 12 = 180, which are designated in advance by the operator. The four light source units 11 and the four image sensors 12 are arranged in the XY plane including the light source unit 11 and the image sensor 12, respectively, near the four vertices of a rectangle having the same size as the four division range 81. , Acquire holograms for four different imaging units 83 on the culture plate 13 at the same time.

When collecting hologram data for the culture plate 13, the operator first sets the culture plate 13 in which the cells 14 to be observed are cultured at a predetermined position of the microscopic observation unit 10 and identifies the culture plate 13. After inputting information such as a number and a measurement date and time from the input unit 201, the measurement execution is instructed. In response to this measurement instruction, the imaging control unit 21 controls each unit of the microscopic observation unit 10 to execute imaging.

That is, one light source unit 11 irradiates a predetermined region (one imaging unit 83) of the culture plate 13 with coherent light having a spread of a minute angle of about 10 °. The coherent light (object light 17) transmitted through the culture plate 13 and the cells 14 reaches the image sensor 12 while interfering with the light (reference light 16) transmitted through the region close to the cells 14 on the culture plate 13. The object light 17 is light whose phase changes when it passes through the cell 14, while the reference light 16 is light that does not pass through the cell 14 and therefore does not undergo the phase change caused by the cell 14. Therefore, on the detection surface (image surface) of the image sensor 12, an interference image of the object light 17 whose phase has been changed by the cell 14 and the reference light 16 whose phase has not changed, that is, a hologram is formed, and this hologram is formed. The two-dimensional light intensity distribution data (hologram data) corresponding to the above is output from the image sensor 12.

As described above, coherent light is emitted from the four light source units 11 toward the culture plate 13 at substantially the same time, and the four image sensors 12 acquire hologram data of regions corresponding to different imaging units 83 on the culture plate 13. Will be done. Each time the measurement at one measurement position is completed, the light source unit 11 and the image sensor 12 are moved in the X-axis direction and the Y-axis direction by the moving unit 15 by a distance corresponding to one imaging unit 83 in the XY plane. It is moved sequentially in steps. As a result, the measurement is performed by 180 imaging units 83 included in the 4-division range 81, and the measurement of the entire culture plate 13 is executed by the four sets of the light source unit 11 and the image sensor 12 as a whole. The hologram data thus obtained by the four image sensors 12 of the microscopic observation unit 10 is stored in the hologram data storage unit 22 together with the attribute information such as the measurement date and time.

After the measurement (shooting) of all the imaging units 83 is completed, when a predetermined operation described later is performed by the operator, the data transmission / reception unit 23 measures the hologram data stored in the hologram data storage unit 22 such as the measurement date and time. The data is sequentially transferred to the server 5 via the communication network 4 together with the attribute information of. It is sufficient to send raw, that is, unprocessed hologram data from each measurement terminal 1 to the server 5, but if necessary, processing that corrects an error factor peculiar to each measurement terminal 1. The processed hologram data may be sent to the server 5.

In the server 5, the data transmission / reception unit 51 receives the hologram data sent from the measurement terminal 1, and identifies the identification information for identifying the measurement terminal 1, the identification information of the culture plate input at the time of photographing, the measurement date and time, and the like. Hologram data is stored in the hologram data storage unit 52 together with the attribute information. After collecting the hologram data, the first phase recovery calculation unit 53 reads the hologram data for each imaging unit from the hologram data storage unit 52 and performs light wave propagation calculation processing to restore the phase information and the intensity (amplitude) information. Ask for. Since the spatial distribution of the phase information and the intensity information is obtained for each imaging unit 83, if the phase information and the intensity information of all the imaging units 83 are obtained, the first image reconstruction unit 55 will perform the phase information and the intensity information. A phase image or an intensity image of the entire observation target area is formed based on the above.

More specifically, the first image reconstruction unit 55 reconstructs the phase image of each imaging unit 83 based on the spatial distribution of the phase information calculated for each imaging unit 83, and joins the phase images in a narrow range thereof. By performing the tying process (see FIG. 3D), a phase image of the observation target region, that is, the entire culture plate 13 is formed. Of course, during the tiling process, it is advisable to perform an appropriate correction process so that the phase images at the boundary of the imaging unit 83 are smoothly connected. When calculating such phase information and reconstructing an image, an algorithm disclosed in known documents such as Patent Documents 1 and 2 may be used.

In addition, the reconstructed image obtained by normal processing is the image with the highest resolution that can be obtained in principle from the acquired hologram data, but not only that, the resolution is adjusted by binning processing or the like based on the phase image with the highest resolution. A phase image having a plurality of reduced resolutions (magnifications) may be created. Then, the image data constituting the phase image and the intensity image created in this way is stored in the image data storage unit 57.

Although it depends on the performance of the server 5, since the amount of hologram data obtained from one culture plate 13 is enormous, it takes a considerable amount of time for the first phase retrieval calculation unit 53 and the first image reconstruction unit 55 to process. Needs. Therefore, in general, even if all the measurements are completed on the measurement terminal 1 side, the phase image and the intensity image of the entire observation target region cannot be immediately viewed. Further, as described above, when calculating the phase information and the intensity information, the phase information and the intensity information at an arbitrary focal position can be obtained, but it is confirmed whether or not the focal position is in focus and the focus is in focus. If not, the focusing work must wait until a phase image or an intensity image of the entire observation target area is obtained. Therefore, in the cell observation apparatus of this example, in order to avoid such a situation, the characteristic processing described below is executed.

FIG. 4 is an explanatory diagram of this characteristic processing operation, FIG. 5 is a diagram showing an example of a screen displayed on the measurement terminal after shooting is completed, FIG. 6 is a phase image of a designated measurement position displayed on the measurement terminal, and FIG. It is a figure which shows an example of an intensity image.

When the operator performs a predetermined operation on the input unit 201 before executing the measurement, the display processing unit 27 displays the captured image display screen 60 as shown in FIG. 5 on the display unit 202. An image display area 61 and a plate information display area 62 are arranged on the captured image display screen 60, and a “stop” button 63 is further arranged at the lower right. In the plate information display area 62, attribute information such as the name (plate name) and the identification number (plate ID) of the culture plate 13 during or after the measurement is displayed is displayed. However, the screen shown in FIG. 5 is after the measurement is completed, and no substantial image is displayed in the image display area 61 before the measurement is started.

When the measurement is started and hologram data for the region corresponding to each imaging unit 83 on the culture plate 13 is obtained as described above, the hologram image creating unit 24 is based on the obtained data for each imaging unit 83. To create a hologram image showing a two-dimensional distribution of light intensity. The hologram image created at this time is a thumbnail image having the lowest resolution. The display processing unit 27 attaches and displays the created hologram thumbnail image at a position corresponding to each imaging unit in the image display area 61. That is, every time the hologram data of one imaging unit is newly obtained, the thumbnail image of the hologram based on the data is additionally displayed in addition to the image in the image display area 61 in almost real time. Then, when the measurement for all the imaging units is completed, the hologram image of the entire observation target region as shown in FIG. 5 is displayed. When the operator clicks the "Create image" button 65 on the captured image display screen 60 after the measurement is completed, the collected hologram data is transferred to the server 5, and the full-screen image reconstruction process is performed as described above. Will be executed.

Although it is difficult to observe the state of cells in a hologram image, most of the phenomena caused by device malfunctions such as damage to the light source unit 11 and the image sensor 12 and inadequate measurement can be grasped on the image. Is. Therefore, the operator monitors the hologram image displayed in the image display area 61 during the measurement execution, and clicks the "stop" button 63 when it can be determined that there is some problem. Then, the imaging control unit 21 receives this operation and stops the measurement. In this way, by promptly stopping the measurement when there is any problem in the measurement, it is possible to avoid wasting time on the remaining unnecessary measurement.

When it is desired to confirm the phase image or the intensity image immediately after the measurement is completed, the operator uses a pointing device such as a mouse which is a part of the input unit 201 to display the image display area 61 in the captured image display screen 60 shown in FIG. Move the cursor over one of the many thumbnail images that make up the hologram image displayed in. The confirmation position designation reception unit 25 accepts this operation and displays a rectangular mark 67 indicating the selected imaging unit superimposed on the image. Then, when the operator performs a double-click operation with the pointing device, the confirmation position designation reception unit 25 recognizes one imaging unit selected at that time as a target for creating a partial phase image, and responds accordingly. The display processing unit 27 displays the designated position image display screen 70 as shown in FIG. 6 on the screen of the display unit 202. The designated position image display screen 70 is provided with an image display area 71.

At the same time, the confirmation position designation reception unit 25 reads the hologram data obtained for one designated imaging unit from the hologram data storage unit 22, and the data transmission / reception unit 23 reads the read data through the communication network 4 to the server 5 Transfer to. The hologram data transfer operation for this one imaging unit may be performed in parallel with the transfer operation of the hologram data of the entire imaging target area to the server 5 as described above, or may be performed in parallel with the transfer operation, and has priority over that. You may go. In the latter case, if the operation of transferring the hologram data of the entire imaging target area has already started, it may be interrupted once and the hologram data of one imaging unit instructed to be selected may be transferred.

In the server 5, the data transmission / reception unit 51 receives the hologram data for one imaging unit sent from the measurement terminal 1 as described above, and temporarily stores the data in the hologram data storage unit 52. Since the amount of data transferred at this time is much smaller than the amount of hologram data in the entire observation target area, the transfer time is also much shorter (see FIG. 4).

The second phase retrieval calculation unit 54 reads out the hologram data of one imaging unit stored in the hologram data storage unit 22 and performs a light wave propagation calculation process to restore the phase information and obtain the intensity information. Subsequently, the second image reconstruction unit 56 forms a partial phase image and a partial intensity image for one imaging unit based on the calculated phase information and intensity information. When calculating the phase information and the intensity information from the hologram data, the information of the arbitrary focal position can be calculated, but the focal position set at this point is the default value specified from the measurement terminal 1. (For example, the focal position set at the most recent past time for the culture plate and well with the same identification number). The image data constituting the partial phase image and the partial intensity image is stored in the image data storage unit 57 and sent from the data transmission / reception unit 51 to the measurement terminal 1. Since the arithmetic processing and the image reconstruction processing are only required for one imaging unit and no tiling processing or the like is required, the processing time until the partial phase image and the partial intensity image are formed is also the phase of the entire observation target area. It is much shorter than the time required to form an image and an intensity image (see FIG. 4).

As described above, the time required for transferring the hologram data for one imaging unit instructed by the operator and forming the partial phase image and the partial intensity image based on the data is short. Therefore, within a relatively short time from the time when the operator double-clicks the thumbnail image corresponding to one imaging unit as described above in the measurement terminal 1, the portion from the server 5 to the measurement terminal 1 is partially displayed. Image data constituting a phase image and a partial intensity image is sent. The sent image data is temporarily stored in the image data storage unit 26.

The display processing unit 27 creates a partial phase image and a partial intensity image based on the image data, and displays the two images side by side in the image display area 71 in the designated position image display screen 70. The type of image to be displayed in the image display area 71 can be selected by putting a check mark in the display image selection check box 72, and only one of the partial phase image and the partial intensity image can be selected in the image display area 71. It can also be displayed in. In the example of FIG. 6, the focal positions of the displayed partial phase image and partial intensity image are 6820 μm.

The focal position set at the beginning is not always the focusing position in cell observation, and it is necessary to change the focal position when observing a foreign substance having a height different from that of the cell. Therefore, when it is desired to observe a partial phase image or a partial intensity image at different focal positions, the operator sets a condition for determining a plurality of stages of the focal position in the slice condition setting area 73 in the designated position image display screen 70. That is, when the operator inputs the range of the focal position and the slice width (step width of the focal position) with the sliders, the number of slices (the number of steps of the focal position) is automatically calculated and displayed. These numbers can also be left at their defaults. Then, when the operator clicks the "slice image creation" button 74 after setting the slice condition, the focus designation reception unit 28 accepts this operation and instructs the server 5 of the set slice condition. When the slider in the slice condition setting area 73 is operated, the focal range of the "focus position set in the well" slider 75 below the slider is set in conjunction with the slider.

In the server 5, the second phase retrieval calculation unit 54 and the second image reconstruction unit 56 perform phase information at a plurality of focal positions according to instructed slice conditions based on the hologram data for one selected imaging unit described above. And intensity information is calculated to form a plurality of partial phase images and partial intensity images having different focal positions from each other. The image data constituting the partial phase image and the partial intensity image at the plurality of focal positions is stored in the image data storage unit 57 and sent from the data transmission / reception unit 51 to the measurement terminal 1 through the communication network 4. In the control / processing unit 20 of the measurement terminal 1, the image data received through the data transmission / reception unit 23 is temporarily stored in the image data storage unit 26.

When the image data constituting the partial phase image and the partial intensity image at different focal positions are prepared in this way, the "focus position set in the well" slider 75 in the designated position image display screen 70 shown in FIG. 6 is effective. become. That is, the knob of the slider 75 can be moved by the pointing device.

When wanting to see images with different focal positions, the operator moves the knob of the "focus position set to well" slider 75 to the left or right by the pointing device. Then, the focus designation reception unit 28 obtains the focus position corresponding to the position of the knob on the slider 75, and the display processing unit 27 displays the partial phase image and the partial intensity image displayed in the image display area 71 as images at the focal position. Update to. In the example of FIG. 6, since the number of slices is 11, the partial phase image and the partial intensity image at 11 different focal positions can be confirmed by operating the slider 75. By comparing the partial phase images or the partial intensity images at different focal positions, the operator can find the focal position where the cell or the like to be observed can be observed most clearly.

When a clear partial phase image and partial intensity image are obtained in this way, the operator confirms these images to see if there are any problems in the culture such as abnormalities in the cells, and further, photography. Judge whether there is any problem with the image. If it is determined that it is not necessary to create a phase image or intensity image of the entire observation target area due to a defect or the like, the full-screen phase image or intensity image reconstruction process based on the hologram data is executed. To avoid. Alternatively, even when the transfer of all hologram data from the measurement terminal 1 to the server 5 is started by the operation of the "execute image creation" button 65 in FIG. 5, the data can be obtained by performing a predetermined operation. The transfer can also be stopped (see FIG. 4). Since such a determination can be made within a relatively short time from the end of shooting, it is not necessary to wait a long time until a phase image is created by the full-screen image reconstruction process.

Further, if an appropriate focal position is found by comparing a plurality of partial phase images and partial intensity images at different focal positions as described above, the operator can perform "setting" in the designated position image display screen 70. Click button 76 to operate. Then, the focus designation reception unit 28 accepts this operation, and through the data transmission / reception unit 23, the value of the instructed focus position and the identification number of the well in which the imaging unit such as the partial phase image displayed at that time exists. The information is sent to the server 5. In the server 5, the designated focal position is stored in the hologram data storage unit 52 as the focal position in the focused state in association with the identification number of the well.

As described above, the focal position that can be determined based on the partial phase image and the partial intensity image for one imaging unit is only for one well in which the imaging unit exists. Therefore, when it is desired to acquire information on the focal position in the focused state for all of the six wells 13a formed on the culture plate 13, the above operation is repeated.

That is, one imaging unit located in another well on the hologram image displayed in the image display area 61 of the captured image display screen 60 as shown in FIG. 5 is selected, and the partial phase of the imaging unit is selected. The image and the partial intensity image are displayed on the designated position image display screen 70 as shown in FIG. Then, the slice condition is set on the designated position image display screen 70, and the creation of the slice image is instructed to create the partial phase image and the partial intensity image at a plurality of focal points. Then, the operator finds an appropriate focal position by comparing the partial phase images and the like at the created plurality of focal points, and clicks and operates the "setting" button 76 to determine the focal position. By repeating this operation for each well, information on different focal positions can be obtained for each of the six wells.

The value of the focal position set for each well as described above is reflected in the focal position list 66 arranged in the plate information display area 62 of the captured image display screen 60 shown in FIG. In the focal position list 66, "1: 5000 μm" means that the focal position of the well having the identification number 1 is 5000 μm. The focal position list 66 displays the latest focal position values set for each well at that time. The value of the focal position displayed in the focal position list 66 can also be changed by using the keyboard which is a part of the input unit 201. Therefore, the operator can determine the value of the focal position for each well by the above procedure, check the value in the focal position list 66, and manually correct the value if necessary. .. When such a modification is made, the information is sent to the server 5, and the value of the focal position associated with the identification number of each well stored in the hologram data storage unit 52 is also changed.

As described above, the first phase retrieval calculation unit 53 calculates the phase information and the intensity information based on the hologram data for each imaging unit. At that time, the identification number of the well in which each imaging unit is located is referred to, and the phase information and the intensity information are calculated. Information on the focal position in the focused state associated with the identification number is acquired from the storage unit 52. Then, the phase information and the intensity information at the focal position are calculated. That is, as for the focal position for each well when creating a phase image or an intensity image of the entire imaging target area, the operator confirms a plurality of partial phase images and partial intensity images having different focal positions on the measurement terminal 1. Is determined by. Alternatively, it is determined by the operator directly inputting the value of the focal position in the focal position list 66 on the captured image display screen 60. In any case, the phase image and intensity image of the entire culture plate 13 are created instead of determining the focal position after once creating and displaying the phase image and intensity image of the entire culture plate 13 as in the conventional case. The appropriate focal position can be determined for each well before it is done. Further, when creating a phase image or an intensity image of a wide range of the entire culture plate 13, it is not necessary to create images at various focal positions that are not in the focused state, so that the time for image reconstruction processing can be saved. As a result, the efficiency of cell observation work can be improved.

Of course, even if the focusing position is determined for each imaging unit at one location for each well, the focusing position may be slightly deviated at other locations in the well. Therefore, a phase image or an intensity image at a plurality of focal positions in a predetermined focal range including the focal position as the in-focus position may be created. As a result, it is possible to observe a phase image and an intensity image at a focal position slightly deviated from the in-focus position.

Further, since the information on the focal position in the focused state is stored in the hologram data storage unit 52, for example, when the same culture plate is measured on another day, the storage is performed without performing the work of searching for the focused position. The full-screen phase image and intensity image can be reconstructed using the focal position information stored in the unit 52. Moreover, even if they are not the same, the heights of the wells are uniform to some extent if they are the same type of culture plates. Therefore, even when observing another culture plate, the focal position stored in the hologram data storage unit 52 is set as the initial position, and the most focused position is searched for in a predetermined focal position range for a short time. There is also the advantage that the focus position can be found with.

In the above embodiment, the partial phase image and the partial intensity image in the imaging unit were reconstructed from the hologram data of one imaging unit designated by the operator, but a part of the imaging unit. The range may be appropriately designated by the operator so that the partial phase image and the partial intensity image in the range can be reconstructed from the hologram data included in the designated range. As a result, the amount of data transferred from the measurement terminal 1 to the server 5 is further reduced, so that the data transfer time and processing time can be further shortened.

Further, after displaying the partial phase image or the intensity phase image for one imaging unit on the designated position image display screen 70, the range designated by the operator in the image may be enlarged and displayed. Further, when the operator appropriately specifies a part range in one imaging unit on the hologram image displayed on the captured image display screen 60, the second image reconstruction unit 56 of the server 5 After reconstructing the partial phase image or partial intensity image for that one imaging unit, only the image in the specified range is cut out, and only the image data in the cut out range is sent to the measurement terminal 1 and displayed on the screen. It may be displayed.

In addition, it is possible to specify not only one but multiple imaging units on the hologram image, or arbitrarily specify a range having a size larger than one imaging unit, and the specified plurality of imaging units or ranges. The partial phase image and the partial intensity image corresponding to the above may be reconstructed and displayed. However, as the number of designated imaging units increases or the designated range increases, the amount of data to be processed increases, so that it takes a long time to display a partial phase image or the like. Therefore, due to this time constraint, it is advisable to determine in advance the number of imaging units that can be specified and the upper limit of the range.

Next, the cell observation device of the second embodiment according to the present invention will be described with reference to FIG. 7. FIG. 7 is a schematic configuration diagram of the measurement terminal 1 as in FIG. In FIG. 7, the same components as or corresponding to the components in the measurement terminal 1 shown in FIG. 2 are designated by the same reference numerals.

In the cell observation apparatus of the second embodiment, the microscopic observation unit 10 in the measurement terminal 1 includes a laser light emitting unit that emits a pulsed laser beam for distance measurement, a detector that detects the reflected laser beam, and a detector. A ranging unit 18 including a signal processing unit that accurately recognizes the direction (angle) of light incident on the detector and calculates the distance to the inner bottom surface of the well of the culture plate 13 or the surface of the medium by a triangular ranging method. It is provided with a focus position calculation unit 19 that calculates the height of the surface of the medium contained in the well of the culture plate 13 from the determined distance and derives the focus position from the height. In this cell observation device, before collecting hologram data, the distance measuring unit 18 and the focal position calculation unit 19 measure the distance to the inner bottom or the surface of the medium for each well of the culture plate 13, and from the measured values. Obtain the relative height of the medium surface and calculate the appropriate focal position. Then, the information of the focal position for each well is input to the control / processing unit 20.

When the focus position information as described above is input from the focus position calculation unit 19, the focus designation reception unit 28 in the control / processing unit 20 sets the value as the focus position in the focused state of each well. That is, for example, the input focal position value is automatically input to the focal position list 66 in the captured image display screen 60, and the information is notified to the server 5. As a result, an appropriate focus position for each well is automatically set without the operator performing a manual input operation or a work of finding a focus position while checking the image as described above.

Although a distance measuring unit using a laser beam is used here, the distance measuring method is not particularly limited as long as the height of the inner bottom surface of the well or the height of the medium surface can be measured without contact. ..

Further, in the above embodiment, the microscopic observation unit 10 is an in-line type digital holographic microscope, but the microscopic observation unit 10 is limited to the in-line type as long as it acquires a hologram for each measurement position in the observation target area. Instead, it may be an off-axis type or phase shift type digital holographic microscope.

Further, in the above embodiment, arithmetic processing such as phase retrieval was performed on the server 5 connected to the measurement terminal 1 via the communication network 4, but as a configuration in which all the processing is performed by the stand-alone device. Naturally, it is also good.

Furthermore, the above-mentioned Examples and the above-mentioned modifications are both examples of the present invention, and even if appropriate changes, modifications, and additions are made within the scope of the present invention, they are included in the claims of the present application. That is natural.

1 ... Measurement terminal 10 ... Microscopic observation unit 11 ... Light source unit 12 ... Image sensor 13 ... Culture plate 13a ... Well 14 ... Cell 15 ... Moving unit 16 ... Reference light 17 ... Object light 20 ... Control / processing unit 21 ... Imaging control Unit 22 ... Hologram data storage unit 23 ... Data transmission / reception unit 24 ... Hologram image creation unit 25 ... Confirmation position designation reception unit 26 ... Image data storage unit 27 ... Display processing unit 28 ... Focus designation reception unit 201 ... Input unit 202 ... Display unit 3 ... Browsing terminal 5 ... Server 4 ... Communication network 51 ... Data transmission / reception unit 52 ... Hologram data storage unit 53 ... First phase recovery calculation unit 54 ... Second phase recovery calculation unit 55 ... First image reconstruction unit 56 ... First 2 Image reconstruction unit 57 ... Image data storage unit 60 ... Photographed image display screen 61 ... Image display area 62 ... Plate information display area 63 ... "Stop" button 65 ... "Execute image creation" button 66 ... Focus position list 67 ... Mark 70 ... Designated position image display screen 71 ... Image display area 72 ... Display image selection check box 73 ... Slice condition setting area 74 ... "Slice image creation" button 75 ... "Focus position to be set in well" Slider 76 ... "Setting" button

The second aspect of the present invention made to solve the above problems, for monitoring cell is in culture in a container of multiple, a cell observation apparatus utilizing a holographic microscope,
a) A light source that emits coherent light,
The b) cells including container as a sample, a detection unit for acquiring a hologram is an interference fringe between the object wave and the reference wave when the emitted light is irradiated to the sample from the light source unit,
c) a movement measurement position on the sample to move one or both of the light source unit and the detection unit and said sample to move,
d) each for a plurality of samples, the light source unit to repeat the acquisition of the hologram at each measurement location of the mobile unit by the predetermined observation area while moving the measurement position on the sample, the detector, and the A measurement control unit that controls the moving unit and
For each container of the sample e) before Kifuku number, the height measuring unit for measuring the relative height of the surface of the medium contained in the inner bottom portion or container of the container in a non-contact,
f) A focal position setting unit that sets the focal position for each container based on the relative height measured by the height measuring unit.
g) each of the plurality of samples, under the control of the measurement control unit, based on the hologram data obtained at each measurement position of a predetermined observation target area on the sample, before Symbol focus position setting unit The phase information and / or the intensity information at the focal position set for each container is calculated, and a phase image showing the two-dimensional distribution of the phase information and / or an intensity image showing the two-dimensional distribution of the intensity information is created. Reconstructed image creation department and
It is characterized by having.

Here, containers multiple, for example, a well formed in the culture plate.

In this second embodiment, instead of the user to find the focus position to check visually the partial phase image or the like, medium surface height measurement unit is accommodated in the inner bottom or the container of the container of multiple Measure the relative height of the non-contact. As the height measuring unit, for example, a laser range finder or the like can be used. Then, the focal position setting unit sets the focal position for each container based on the relative height value obtained by the measurement by the height measuring unit. When the height of the inner bottom of the container is measured by the height measuring unit, the height of the medium surface is estimated based on information such as the amount of the medium contained in the container, and the culture is further carried out from that height. The height of the cell surface inside can be estimated. Further, when the height of the medium surface in the container can be measured by the height measuring unit, the height of the cell surface during culturing can be estimated from the height.

Claims (5)

A cell observation device that uses a holographic microscope.
a) Light source and
b) A detection unit that acquires a hologram that is an interference fringe between an object wave and a reference wave when a sample containing cells is irradiated with light emitted from the light source unit.
c) A moving unit that moves one or both of the light source unit, the detection unit, and the sample so that the measurement position on the sample moves.
d) Measurement that controls the light source unit, the detection unit, and the moving unit so as to repeatedly acquire holograms at each measurement position in a predetermined observation target region while moving the measurement position on the sample by the moving unit. Control unit and
e) Reconstructed image creation unit that creates partial phase images and / or partial intensity images at multiple focal positions based on hologram data obtained at one or more specific measurement positions or ranges specified by the user. When,
f) A display processing unit that simultaneously or switchably displays a partial phase image and / or a partial intensity image at a plurality of focal positions created by the reconstructed image creation unit on the display unit.
g) When the partial phase image and / or the partial intensity image is displayed from the display processing unit, the partial phase image and / or the partial intensity image displayed at that time is operated by the user. A focus position setting unit that stores the focal position in the storage unit as the focal position in the focused state when creating a phase image and / or an intensity image of a predetermined region on the sample including the measurement position or range corresponding to the image. When,
A cell observation device comprising. The cell observation device according to claim 1.
A hologram image creating unit that creates a hologram image based on the hologram data obtained at each measurement position in the predetermined observation target area and displays it on the screen of the display unit.
An instruction for the user to instruct the measurement position or range corresponding to the partial phase image and / or the partial intensity image created by the second reconstructed image creating unit on the hologram image displayed by the hologram image creating unit. Input section and
A cell observation device characterized by further comprising. The cell observation device according to claim 1, which is a cell observation device for observing cells being cultured in each recess of the culture plate having a plurality of recesses formed therein.
In response to a user operation, the focal position setting unit sets the focal position of the partial phase image and / or partial intensity image displayed at that time as a phase image in a recess including a measurement position or range corresponding to the image. And / or the focal position to be stored in the storage unit as the focal position for creating the intensity image, and the focal position for creating the phase image and / or the intensity image in the recess independently for each of the plurality of recesses. A cell observation device characterized by memorizing. The cell observation device according to claim 3.
The focus position setting unit displays the focus position information set for each of the plurality of recesses on the screen of the display unit, and also accepts the user to edit the focus position value on the display. A cell observation device comprising. A cell observation device using a holographic microscope for observing cells being cultured in one or more containers.
a) A light source that emits coherent light,
b) A detection unit that acquires a hologram that is an interference fringe between an object wave and a reference wave when the sample, which is one or more containers containing cells, is irradiated with light emitted from the light source unit.
c) A moving unit that moves one or both of the light source unit, the detection unit, and the sample so that the measurement position on the sample moves.
d) Measurement that controls the light source unit, the detection unit, and the moving unit so as to repeatedly acquire holograms at each measurement position in a predetermined observation target region while moving the measurement position on the sample by the moving unit. Control unit and
e) A height measuring unit that non-contactly measures the relative height of the inner bottom of the one or more containers or the surface of the medium contained in the container.
f) A focal position setting unit that sets the focal position for each container based on the relative height measured by the height measuring unit.
g) Under the control of the measurement control unit, the focus position setting unit for each region corresponding to the container is based on the hologram data obtained at each measurement position in the predetermined observation target region on the sample. Create a reconstructed image that calculates the phase information and / or intensity information at the set focal position and creates a phase image showing the two-dimensional distribution of the phase information and / or an intensity image showing the two-dimensional distribution of the intensity information. Department and
A cell observation device comprising.