JPWO2018158810A1 - Cell observation device - Google Patents

Abstract

An image display frame (101) and a slider (102) capable of selecting a focal position are arranged in the focal position adjusting screen (100). On the phase image of the entire cell culture plate to be observed, a number of phase images with different focal positions in multiple stages corresponding to the range specified by the observer are created in advance, and one of the images is displayed in the image display frame. It is displayed in (101). When the observer moves the knob (103) of the slider (102), the focus processing unit selects a phase image at a focus position corresponding to the position of the knob (103) and displays the phase image in the image display frame (101). I do. The observer checks whether or not the displayed image is in focus by operating the slider (102) as appropriate, and if the image is in focus, clicks the "OK" button (104) to operate the focus position. Confirm. Thereby, an appropriate focal position can be determined efficiently.

Description

  The present invention relates to a cell observation device for observing the state of cells, and more specifically, a phase image and an intensity image of an object obtained by a digital holography microscope by performing arithmetic processing on a hologram that records interference fringes between an object wave and a reference wave. The present invention relates to a cell observation device that creates a reconstructed image such as the above.

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. In general, a phase contrast microscope is widely used for observing cells since cells are generally transparent and difficult to observe with a normal optical microscope.
However, since it is necessary to perform focusing when capturing a microscopic image in a phase contrast microscope, when acquiring a microscopic image of each small region obtained by finely dividing a wide observation target region, a great deal of measurement is required. There is a problem that it takes time and is not practical. In order to solve this, holographic microscopes using digital holography technology have been recently developed and put to practical use (see Patent Documents 1 and 2 and the like).

  In a holographic microscope, an interference fringe (hologram) formed on a detection surface of an image sensor or the like by an object light in which light from a light source is reflected or transmitted on an object surface and a reference light directly arriving from the same light source is acquired. By performing a predetermined arithmetic process based on the hologram, an intensity image or a phase image is created as a reconstructed image of the object. With such a holographic microscope, it is possible to form a reconstructed image at an arbitrary distance at the stage of arithmetic processing for phase recovery and the like after acquiring a hologram, so that it is not necessary to focus each time at the time of photographing, and the measurement time is reduced. Can be shortened.

  On the other hand, the above advantage of the holographic microscope means that an appropriate focus position needs to be determined at the stage of creating an image based on a hologram obtained by photographing. When observing a pluripotent cell in culture in a cell culture plate, it is necessary for the observer to visually determine at which position in the cell culture plate the cell is to be focused. For this purpose, for example, a plurality of images (for example, phase images) having different focal positions are created for an entire region of one cell culture plate or one or a plurality of wells in the plate, and the observer displays the plurality of images on a display unit. It is necessary to find an image in the most appropriate focus state by comparing the images on the screen.

However, even if a large number of images with different focuses are viewed in a list, or if the observer checks and selects images one by one by clicking with a mouse or the like, is the fine part focused? It is difficult and time-consuming to determine whether or not.
Further, since the size of the image of the entire region of one cell culture plate or one or a plurality of wells is large, it takes time to generate a plurality of images having different focal positions.
Furthermore, the focal position is not always the same for the entire region of one cell culture plate or one or a plurality of wells. Because the bottom surface of the culture plate is inclined, it may not be possible to determine one focus position for the entire observation target area.

International Patent Publication No. 2016/084420 JP-A-10-268740

  The present invention has been made to solve the above problems, and in a cell observation device that creates and displays a phase image or the like based on hologram data obtained by a holographic microscope, an observer confirms an image while maintaining an appropriate focus. The main object of the present invention is to facilitate visual judgment and improve workability in determining the position.

The present invention has been made in order to solve the above problems, by performing an arithmetic process based on hologram data obtained by measuring a sample containing cells with a holographic microscope, phase, intensity, or at least the pseudo phase A cell observation device that creates a reconstructed image showing any two-dimensional distribution and displays the reconstructed image on a display unit,
a) a focal position comparison image creating unit that creates a plurality of reconstructed images having different focal positions for the entire observation target region or a part thereof based on the obtained hologram data,
b) The image display frame on which one of a plurality of reconstructed images having different focal positions is displayed, and a screen on which a slider as an operator for changing the focal position within a predetermined range is displayed. A focus position alignment display processing unit to be displayed on the unit;
c) selecting a plurality of reconstructed images having different focal positions according to the operation of the slider by the user, and a focal position comparison image display processing unit that renders the image in the image display frame;
d) a focus position determining unit that determines that the focus position corresponding to one of the plurality of reconstructed images in which the focus position specified by the user is different is a focus position;
It is characterized by having.

The pseudo phase is a value corresponding to a phase difference in a phase contrast microscope, that is, phase information including an intensity element.
The holographic microscope may be of any type, such as an in-line type, an off-axis type, and a phase shift type.

  In the cell observation device according to the present invention, typically, the sample is a cell culture plate, and the observation target area is an area including the entire cell culture plate or one or more wells formed in the plate. It can be. That is, the cell observation device according to the present invention is a device suitable for observing living cells being cultured in a cell culture plate.

  In the cell observation device according to the present invention, for example, the focal position comparison image creating unit differs in the focal position for the entire observation target area based on hologram data acquired for a cell culture plate in which cells are being cultured. A plurality of phase images are created as reconstructed images. The focus position display processing unit displays a screen on which an image display frame of a predetermined size and a slider are arranged on the display unit, and the focus position comparison image display processing unit responds to the position of the slider knob. The selected phase image at the focal position is selected from the plurality of created phase images, and is drawn in the image display frame. Therefore, when the user performs an operation of appropriately moving the slider knob with the pointing device, phase images of different focal positions with respect to the same area are sequentially displayed according to the operation. Thereby, the user can visually confirm the change of the phase image due to the change of the focal position, that is, the state of the image blur while moving the slider knob.

  For example, the user performs a predetermined operation after moving the knob of the slider so that the part of interest is seen most clearly, that is, in a focused state. In response to this operation, the focus position determination unit determines that the focus position corresponding to the phase image displayed at that time is the focus position. Of course, the same operation may be performed using an intensity image or a quasi-phase image instead of the phase image. Through such a series of operations, the user can easily and accurately determine the focus position of the reconstructed image.

In order to accurately adjust the focus position, it is desirable that the pitch of the change of the focus position assigned to the slider is narrow, that is, the focus position changes little by little according to the movement of the slider knob. The range of the corresponding change in the focus position becomes narrow, and the focus position may deviate from the range.
Therefore, the cell observation device according to the present invention preferably has a configuration in which a focus position adjustment parameter setting unit for setting a range of change of the focus position assigned to the slider and a pitch of the change by a user is preferable. .

  According to this configuration, the user can freely set the range of change of the focus position and the pitch of the change assigned to the slider by the focus position parameter setting unit. After setting the focus position roughly after setting the pitch (narrow), it is possible to search for the focus position more finely by resetting the pitch of the change of the focus position wide (the change range is narrow). This makes it easier to find a more accurate focus position.

In the cell observation device according to the present invention,
The user specifies the same focal position applicable range in which the same focal position can be applied on a wide range display image that is a reconstructed image of the entire observation target region or a part of the observation target region displayed on the display unit. Further comprising a range designating section for performing
The focus position determination unit may determine the focus position for each of the same focus position application ranges specified by the range specification unit.

Here, for example, the range designating unit is a pointing device capable of designating a range of an arbitrary shape and an arbitrary size on a displayed image, and an operation of recognizing the range designated by the pointing device as the same focal position applicable range. And a receiving unit.
According to this configuration, for example, even when an appropriate focal position differs for each well in one cell culture plate as described above, the focal position can be determined for each well, and conversely, in the same well. Then, the focal position can be made common.

In the cell observation device having the above configuration,
An enlarged observation range designation unit for the user to designate a partial range on the wide range display image as an enlarged observation range,
An enlarged image creating unit that creates an enlarged image at a magnification higher than at least the wide range display image for the specified enlarged observation range and displays the enlarged image on the screen of the display unit.
The configuration may further include

  The enlarged observation range designation unit is, for example, a pointing device capable of designating a range of any shape and any size on a displayed image, and a range designated by the enlarged device. And an operation receiving unit that recognizes the range.

  For example, even if the user views a wide range of phase images, such as the entire cell culture plate or the entire well, it is difficult to determine whether the image is in focus. On the other hand, according to the above configuration, the user can determine whether or not the image is in focus by checking the enlarged image in the narrow range specified on the wide-range display image. As a result, the accuracy of determining whether or not the camera is at the in-focus position is improved, and the workability for that purpose is also improved.

  Further, even when the user designates an enlarged observation range on the displayed wide-range display image and displays the enlarged image, the enlarged observation range may not always be a desired portion. In some cases, the user may want to check the state of the image in other parts.

Therefore, in the cell observation device according to the present invention,
An operation unit for a user to perform an operation of moving or enlarging or reducing the enlarged observation range on the wide display image displayed on the display unit,
It is preferable that the enlarged image creation unit creates and displays an enlarged image after movement or enlargement or reduction by an operation via the operation unit.
Thus, the user can check the enlarged image at an arbitrary part in the observation target area and determine whether the focus position is appropriate.

  Further, in the cell observation device according to the present invention, preferably, a focus position adjustment target range designation for a user to designate a focus position adjustment target range used for determining a focus position in one or a plurality of the same focus position application ranges. The configuration may further include a unit.

  Furthermore, in the cell observation device according to the present invention, the focus position comparison image creating unit may be configured to create a plurality of reconstructed images having different focus positions in the focus alignment target range.

  According to such a configuration, the user can narrow down the focus position adjustment target range, so that the data amount of a reconstructed image such as a phase image created by the focus position comparison image creating unit can be reduced. This makes it possible to speed up the process of calculating phase information and the like and the process of creating an image using the obtained phase information and the like. As a result, the time required for the operation of determining the focus position can be reduced, and the operation of cell observation can be performed efficiently.

  ADVANTAGE OF THE INVENTION According to the cell observation apparatus which concerns on this invention, when an observer (user) performs the operation which determines an appropriate focus position, visually confirming a reconstructed image, such as a phase image, it is determined whether it is in focus. Easy and accurate. As a result, the efficiency of the cell observation operation itself can be improved, and highly accurate cell observation using a good reconstructed image can be performed.

FIG. 1 is a configuration diagram of a main part of a cell observation device according to an embodiment of the present invention. FIG. 4 is an explanatory diagram of an image creation process in the cell observation device of the present embodiment. 6 is a flowchart showing operations and processing at the time of focusing in the cell observation device of the present embodiment. FIG. 7 is an explanatory diagram of a display method of a focus position comparison image at the time of focusing in the cell observation device of the present embodiment. FIG. 4 is a schematic diagram of a focus position adjustment screen in the cell observation device of the present embodiment. FIG. 4 is an explanatory diagram at the time of focusing on the cell observation device of the present embodiment. FIG. 4 is an explanatory diagram at the time of focusing on the cell observation device of the present embodiment. FIG. 4 is an explanatory diagram at the time of focusing on the cell observation device of the present embodiment.

Hereinafter, an embodiment of the cell observation device according to the present invention will be described with reference to the accompanying drawings.
FIG. 1 is a configuration diagram of a main part of the cell observation device of the present embodiment.

The cell observation device of the present embodiment includes a microscopic observation unit 1, a control / processing unit 2, an input unit 3 and a display unit 4, which are user interfaces.
The microscopic observation unit 1 is an in-line holographic microscopy (IHM), and includes a light source unit 10 including a laser diode and the like and an image sensor unit 11. Then, a cell culture plate 12 including cells 13 to be observed is arranged. The cell culture plate 12 is movable in two axial directions of an X axis and a Y axis by a moving unit 14 including a drive source such as a motor.

The control / processing unit 2 controls the operation of the microscopic observation unit 1 and processes data acquired by the microscopic observation unit 1. The control / processing unit 2 includes a photographing control unit 20, a measurement data storage unit 21, a phase information calculation unit 22, A functional block includes an entire image creating unit 23, a focus position comparison image creating unit 24, an image data storage unit 25, a focusing processing unit 26, a focus position information storage unit 27, a display processing unit 28, an operation reception processing unit 29, and the like.
The entity of the control / processing unit 2 is a personal computer or a higher-performance workstation. By running dedicated control / processing software installed on such a computer on the computer, the functions of the functional blocks described above are realized. Is realized. Therefore, the input unit 3 includes a pointing device such as a keyboard and a mouse. Further, as will be described later, the function of the control / processing unit 2 may be shared by a plurality of computers connected via a communication network, instead of a single computer.

  Next, the operation and processing of the observer when performing cell observation in the cell observation apparatus of the present embodiment will be described with reference to FIGS. FIG. 2 is an explanatory diagram of an image creation process in the cell observation device of the present embodiment, FIG. 3 is a flowchart showing an operation and a process at the time of focusing, and FIG. FIG. 5 and FIG. 5 are schematic diagrams of the focus position adjustment screen, and FIGS. 6 to 8 are explanatory diagrams at the time of focus position adjustment.

  The observer sets a cell culture plate 12 in which cells (pluripotent cells) 13 to be analyzed are cultured in a predetermined position, and inputs information such as an identification number for identifying the cell culture plate 12 and the measurement date and time. 3 and then instruct measurement execution. In this example, as shown in FIG. 2A, six wells 50 each having a circular shape in a top view are formed in the cell culture plate 12, and cells are cultured in each of the wells 50. Therefore, the entire one cell culture plate 12, that is, the entire rectangular range including the six wells 50 is the observation target region, that is, the imaging target range. Upon receiving the measurement instruction, the imaging control unit 20 controls the microscopic observation unit 1 to acquire data on the imaging target range as described below.

  Although not shown in FIG. 1, four CMOS image sensors are provided on the same XY plane of the image sensor unit 11. The four CMOS image sensors respectively take pictures of four quadrants 51 obtained by dividing the entire cell culture plate 12 shown in FIG. 2A into four. As shown in FIGS. 2B and 2C, a range in which one CMOS image sensor can photograph at one time is a rectangular range 52 including only one well 50 in a four-division range 51. This is a range corresponding to the imaging unit 53 obtained by dividing the image into 10 equal parts in the X-axis direction and 12 equal parts in the Y-axis direction. Accordingly, the quadrant 51 includes 15 × 12 = 180 imaging units 53. The four CMOS image sensors each have a rectangular long side having a long side corresponding to 15 imaging units in the X-axis direction and a short side having a length corresponding to 12 imaging units in the Y-axis direction. It is arranged near each of the vertices, and simultaneously photographs four different imaging units of the cell culture plate 12.

  Under the control of the imaging control unit 20, the light source unit 10 irradiates a predetermined area of the cell culture plate 12 with coherent light having a small angle spread of about 10 °. The light (object light 16) transmitted through the cell culture plate 12 and the cells 13 reaches the image sensor unit 11 while interfering with light (reference light 15) transmitted through an area on the cell culture plate 12 close to the cells 13. . The object light 16 is light whose phase has changed when transmitting through the cell 13, while the reference light 15 is light which does not receive a phase change due to the cell 13 because it does not transmit through the cell 13. Therefore, the interference between the object light 16 whose phase has been changed by the cell 13 and the reference light 15 whose phase has not changed is detected on the detection surfaces (image surfaces) of the four CMOS image sensors arranged in the image sensor unit 11. An image (hologram) is formed, and two-dimensional light intensity distribution data (hologram data) corresponding to the hologram is output from the image sensor unit 11.

  The cell culture plate 12 is moved stepwise by the moving unit 14 by a distance corresponding to the size of the imaging unit 53 in the XY plane. Thereby, the irradiation region of the coherent light emitted from the light source unit 10 moves on the cell culture plate 12, and each CMOS image sensor in the image sensor unit 11 can acquire hologram data corresponding to one imaging unit 53. it can. The cell culture plate 12 is moved by the moving unit 14 in 180 steps corresponding to the number of imaging units 53 included in one quadrant 51, and hologram data is acquired for each movement. In this manner, hologram data for the entire cell culture plate 12 can be obtained without omission.

  As described above, the hologram data obtained by the image sensor unit 11 of the microscopic observation unit 1 is sequentially sent to the control / processing unit 2 and stored in the measurement data storage unit 21. When the measurement of the entire cell culture plate 12 is completed, in the control / processing unit 2, the phase information calculation unit 22 reads the hologram data for each of the imaging units 53 from the measurement data storage unit 21 and executes the back propagation calculation of light. Calculates phase information and intensity information reflecting the optical thickness of the cell 13. The whole image creating unit 23 performs a tiling process (see FIG. 2D) for joining the phase images in a narrow range based on the phase information calculated for each imaging unit 53, thereby obtaining the observation target region, that is, the cell culture. A phase image of the entire plate 12 is created. The display processing unit 28 displays the phase image of the entire observation area on the screen of the display unit 4 (Step S1).

  In calculating such phase information and creating a phase image, a well-known algorithm disclosed in Patent Documents 1 and 2 and the like may be used. In addition, in addition to the phase information, intensity information and quasi-phase information may also be calculated based on the hologram data, and an intensity image and a quasi-phase image based on these may be created and displayed.

The observer visually recognizes the phase image of the entire observation target area on the screen of the display unit 4 and inputs a range in which the same focal position can be applied on the phase image (corresponding to the same focal position applicable range in the present invention). It is designated by an operation using the unit 3 (pointing device). Specifically, when the observer specifies a rectangular frame having an arbitrary size both vertically and horizontally on the phase image with the pointing device and performs a predetermined operation, the operation reception processing unit 29 is surrounded by the specified frame. Is recognized as the same focal position applicable range (step S2). By repeating such operations, a plurality of the same focal position application ranges can be designated. Of course, the same focus position may be applied to the entire observation target area without such designation.
FIG. 6 is a schematic diagram illustrating an example in which the confocal position applicable range 210 is specified for a region corresponding to two wells 201 on the phase image 200 of the entire cell culture plate 12.

  Next, as shown in FIG. 7, the observer designates a local focus position adjustment target range 220 used for focus position adjustment for each of the same focus position application ranges 210 on the phase image 200 of the entire cell culture plate 12. (Step S3). This designation method may be the same as in the same focus position application range 210, and appropriately designate a site to be observed clearly or a site expected to be easily focused, such as a site where a cell of interest of the observer exists. You can specify it. However, since it is difficult to see the outline of each cell in the phase image 200 of the entire cell culture plate 12, it is difficult to search for the cell of interest. Therefore, when the observer performs a predetermined enlargement operation after specifying an appropriate focus position adjustment target range 220, the focusing processing unit 26 that has received the instruction via the operation reception processing unit 29, as shown in FIG. Next, an enlarged image 240 of the phase image corresponding to only the designated range is created and displayed on the screen of the display unit 4 (step S4).

  In addition, when the observer performs an operation of moving, enlarging, or reducing the range indicated by the enlarged image 240 on the phase image 200 of the entire cell culture plate 12 or on the enlarged image 240, the focusing is performed. The processing unit 26 creates an enlarged image after movement or enlargement / reduction by the operation, and updates the displayed enlarged image 240. This allows the observer to find an appropriate focus alignment target range 220 while searching for the cell of interest and confirming in the enlarged image 240 whether or not the cell is clearly observable. After the appropriate focus position adjustment target range 220 is determined in this way, the input unit 3 may perform a predetermined operation to determine the focus position adjustment target range 220.

  After specifying the same focus position application range 210 and the focus position adjustment target range 220 as described above, the observer instructs execution of the focus position adjustment from the input unit 3 (step S5). In response to this instruction, the focusing processing unit 26 creates a phase image having different focal positions in a plurality of stages as a focal position comparison image for one or a plurality of designated focal position adjustment target ranges 220 (see FIG. 4). ), And stores the image data constituting the image in the image data storage unit 25 (step S6). Since the size of the focus alignment target range 220 can be freely set by the observer, the focus alignment target range 220 may fall within one imaging unit 53 or may extend over a plurality of imaging units 53. In some cases. In the latter case, the above-described tiling processing is performed when a phase image is created. In this way, by preparing in advance all the focus position comparison images that may be viewed later, a smooth display is performed at the time of continuous confirmation of a plurality of focus position comparison images as described later. be able to.

  The range and pitch of the focal position of the focal position comparison image created in step S6 may be appropriately determined in advance by the manufacturer that provides this apparatus. These parameters may be appropriately changed by a user (observer or an administrator of the apparatus). Since the range and pitch of the change are restricted, it is desirable that the range of the focal position is not too narrow and the pitch of the focal position is not too wide.

  On the other hand, the display processing unit 28 displays a focus position adjustment screen 100 as shown in FIG. 5 on the screen of the display unit 4 (step S7). On the focus position adjustment screen 100, an image display frame 101 for displaying a focus position comparison image and a focus position change slider 102, which is one of GUI components, are arranged. When a new focus position adjustment screen 100 is displayed, the focus position change range and the initial value of the focus position change pitch, which are defined by default, are assigned to the focus position change slider 102. A focus position comparison image corresponding to the maximum or minimum focus position within the range is selected from the image data storage unit 25 and displayed in the image display frame 101.

  The observer performs an operation of appropriately moving the knob 103 of the focus position changing slider 102 by using the input unit 3 while watching the focus position comparison image displayed in the image display frame 101 of the focus position adjustment screen 100. Then, the focus processing unit 26 that has received the instruction through the operation reception processing unit 29 selects a focus position comparison image associated with a focus position corresponding to the position of the knob 103 from the image data storage unit 25, and The display image in the display frame 101 is updated (step S8).

  In each of the focus position comparison images having different focus positions, the state of the image blur is slightly different. Therefore, the observer determines whether or not a focused part such as a contour or a pattern of a cell is most clearly visible, that is, whether or not the focused state is present (step S9). The position of the knob 103 of the focus position changing slider 102 is adjusted so as to find the position (step S10). Then, if it is determined that the in-focus state is obtained when the knob 103 is at a certain position (Yes in step S9), the user clicks the "OK" button 104 arranged at the bottom of the focus position adjustment screen 100. Thus, an instruction to determine the focal position is issued (step S12). Upon receiving this instruction, the focus processing unit 26 sets the focus position corresponding to the position of the knob 103 at that time as the focus position for the same focus position applicable range 210 including the focus position adjustment target range 220 at that time. Is stored in the focal position information storage unit 27 (step S13).

  However, as described above, when the pitch of the change of the focus position defined by default is too coarse, or when the actual focus position is out of the range of the change of the focus position defined by default, In some cases, even if the observer adjusts the position of the knob 103 of the focus position changing slider 102, the state may not be determined to be in focus. In this case, the observer appropriately changes the range and pitch of the change of the focal position to be assigned to the focal position changing slider 102 on a parameter setting dialog screen that is pop-up displayed, for example, in response to a predetermined operation by the input unit 3. (Step S11). When such a change is made, the display processing unit 28 assigns the focus position change range and pitch after the change to the focus position change slider 102. Then, for example, a focus position comparison image corresponding to the maximum or minimum focus position within the change range of the focus position is selected from the image data storage unit 25 and displayed in the image display frame 101 (step S7). By repeating steps S7 to S11 described above, the observer can determine an appropriate focus position while viewing the focus position comparison image.

When a plurality of the same focus position application ranges 210 are set, the operation and processing shown in FIG. 3 may be performed for each focus position adjustment target range 220 included in the same focus position application range 210. This makes it possible to determine the focal position for the entire observation target region, that is, the phase image of the entire cell culture plate 12. Then, once the focal position is determined, a process of newly creating a phase image of the entire cell culture plate 12 or a part thereof corresponding to the focal position may be performed.
Note that the focus position obtained for the phase image may be used for the intensity image or the quasi-phase image, or the focus position may be determined for the intensity image or the quasi-phase image in the same manner as the phase image. .

  In the cell observation device of the above embodiment, different focus position comparison images are displayed one after another in response to the observer manually operating the slider, but a plurality of focus position comparison images having different focus positions are automatically displayed. May be added with an automatic playback function that is displayed one after another. In this case, a button for instructing automatic reproduction of the focus position comparison image is prepared, and when the button is operated, the focus position comparison image within the focus position change range determined at that time is automatically generated. It is good to reproduce like a moving image by displaying in order. At this time, the playback speed may be adjusted. Such automatic reproduction is particularly convenient for an observer to determine a rough focus position.

  Further, in the configuration of the embodiment shown in FIG. 1, all the processes are performed in the control / processing unit 2. However, in general, calculation of phase information based on hologram data and imaging of the calculation result are enormous. Quantitative calculations are required. Therefore, it takes a lot of time to calculate with a personal computer that is usually used, and it is difficult to perform an efficient analysis operation. Therefore, a personal computer connected to the microscope observation unit 1 is used as a terminal device, and a computer system in which this terminal device and a server which is a high-performance computer are connected via a communication network such as the Internet or an intranet is used. Good. In this case, complicated processing such as calculation of phase information and creation of a phase image based on the hologram data is performed on the server side, and the terminal device receives the image data created thereby, and performs phase image processing based on the image data. Is preferably performed on the terminal device side. In such a configuration, the functional blocks of the control / processing unit 2 shown in FIG. 1 are separated into the terminal device side and the server side. As described above, the functions of the control / processing unit 2 may be shared by a plurality of computers.

  Further, in the cell observation apparatus of the above embodiment, an in-line holographic microscope was used as the microscopic observation unit 1. However, if the microscope is capable of obtaining a hologram, another type of holographic microscope such as an off-axis type or a phase shift type may be used. Obviously, it can be replaced with a microscope.

  Furthermore, the above-described embodiment and the above-described modified examples are all examples of the present invention, and even if appropriate changes, modifications, and additions are made within the spirit of the present invention, they are also included in the claims of the present application. That is natural.

DESCRIPTION OF SYMBOLS 1 ... Microscopic observation part 10 ... Light source part 11 ... Image sensor 12 ... Cell culture plate 13 ... Cell 14 ... Moving part 15 ... Reference light 16 ... Object light 2 ... Control / processing part 20 ... Imaging control part 21 ... Measurement data storage part 22 ... Phase information calculation unit 23 ... Overall image creation unit 24 ... Focal position comparison image creation unit 25 ... Image data storage unit 26 ... Focus processing unit 27 ... Focal position information storage unit 28 ... Display processing unit 29 ... Operation acceptance processing unit 3 Input unit 4 Display unit 100 Focus position adjustment screen 101 Image display frame 102 Focus position change slider 103 Knob 104 ... "Enter" button

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. In general, a phase contrast microscope is widely used for observing cells since cells are generally transparent and difficult to observe with a normal optical microscope.
However, when the phase contrast microscope because it is necessary to perform focusing when taking a microscopic image, a so that Tokusu preparative microscopic images for each small area finely divide the wide observation area, great to measure It takes a long time and is not practical. In order to solve this, holographic microscopes using digital holography technology have been recently developed and put to practical use (see Patent Documents 1 and 2 and the like).

The present invention has been made in order to solve the above problems, in a cell observation device that creates and displays a phase image and the like based on holographic data obtained by a holographic microscope, while the observer confirms the image The main object of the present invention is to facilitate visual judgment and to improve the workability when determining an appropriate focus position.

The present invention has been made in order to solve the above problems, by performing arithmetic processing based on hologram data obtained by measuring a sample containing cells with a holographic microscope, phase, intensity, or at least the pseudo phase A cell observation device that creates a reconstructed image showing any two-dimensional distribution and displays the reconstructed image on a display unit,
a) By connecting a plurality of reconstructed images created based on hologram data corresponding to an imaging unit of a predetermined size obtained by one shot with the holographic microscope, a reconstructed image of the entire observation target area is formed. Creating and displaying an entire image on the display unit;
b) on the reconstructed image of the entire observation target area displayed on the display unit, a focus alignment target range designation unit that allows the user to specify a focus alignment target range,
c) based on the hologram data corresponding to the focus alignment target range specified by the focus alignment target range specifying unit, if the focus alignment target range spans a plurality of imaging units, the plurality of imaging units A focus position comparison image creating unit that creates a plurality of reconstructed images having different focus positions in the focus alignment target range by joining the corresponding reconstructed images ,
d) the image display frame on which one of a plurality of reconstructed images having different focal positions is displayed, and a screen on which a slider that is an operation element for changing the focal position within a predetermined range is displayed. A focus position alignment display processing unit to be displayed on the unit;
e) a focus position comparison image display processing unit that selects a plurality of reconstructed images having different focal positions in accordance with the operation of the slider by a user and causes the image to be drawn in the image display frame;
f) a focus position determining unit that determines that the focus position corresponding to one of the plurality of reconstructed images in which the focus position specified by the user is different is a focus position;
It is characterized by having.

For example, the user performs a predetermined operation after moving the knob of the slider so that the part of interest is seen most clearly, that is, in a focused state. In response to this operation, the focus position determination unit determines that the focus position corresponding to the phase image displayed at that time is the focus position. Of course, the same operation may be performed using an intensity image or a quasi-phase image instead of the phase image. Through such a series of operations, the user can easily and accurately determine the focus position of the reconstructed image.
Further, according to the above configuration, the user can narrow down the focus position adjustment target range, so that the data amount of a reconstructed image such as a phase image created by the focus position comparison image creating unit can be reduced. This makes it possible to speed up the process of calculating phase information and the like and the process of creating an image using the obtained phase information and the like. As a result, the time required for the operation of determining the focus position can be reduced, and the operation of cell observation can be performed efficiently.

In the cell observation device according to the present invention,
The display unit on the wide display images in a reconstructed image of the observation target area total that is displayed on the same focal position application for users of the same focal position coverage is applicable to specify the same focal position A range designating section,
The focus position adjustment target range designation unit may be configured to allow a user to designate a focus position adjustment target range for each of the same focus position application ranges designated by the same focus position application range designation unit .

Here, the same-focal-position application range specifying unit includes, for example, a pointing device capable of specifying a range of an arbitrary shape and an arbitrary size on a displayed image, and the same focus position application range. And an operation accepting unit that recognizes as.
According to this configuration, for example, even when an appropriate focal position differs for each well in one cell culture plate as described above, the focal position can be determined for each well, and conversely, in the same well. Then, the focal position can be made common.

Claims (8)

By performing arithmetic processing based on hologram data obtained by measuring a sample containing cells with a holographic microscope, a reconstructed image showing at least one of a two-dimensional distribution of phase, intensity, or pseudo phase is created. A cell observation device for displaying on a display unit,
a) a focal position comparison image creating unit that creates a plurality of reconstructed images having different focal positions for the entire observation target region or a part thereof based on the obtained hologram data,
b) The image display frame on which one of a plurality of reconstructed images having different focal positions is displayed, and a screen on which a slider as an operator for changing the focal position within a predetermined range is displayed. A focus position alignment display processing unit to be displayed on the unit;
c) selecting a plurality of reconstructed images having different focal positions according to the operation of the slider by the user, and a focal position comparison image display processing unit that renders the image in the image display frame;
d) a focus position determining unit that determines that the focus position corresponding to one of the plurality of reconstructed images in which the focus position specified by the user is different is a focus position;
A cell observation device comprising: The cell observation device according to claim 1,
The cell observation device, wherein the sample is a cell culture plate, and the observation target region is an entire cell culture plate or a region including one or a plurality of wells formed in the plate. The cell observation device according to claim 1,
A cell observation apparatus further comprising a focus position adjustment parameter setting unit for allowing a user to set a range of a change in a focus position assigned to the slider and a pitch of the change. The cell observation device according to claim 1,
The user specifies the same focal position applicable range in which the same focal position can be applied on a wide range display image that is a reconstructed image of the entire observation target region or a part of the observation target region displayed on the display unit. Further comprising a range designating section for performing
The cell observation apparatus, wherein the focus position determination unit determines a focus position for each same focus position application range designated by the range designation unit. The cell observation device according to claim 4,
An enlarged observation range designation unit for the user to designate a partial range on the wide range display image as an enlarged observation range,
An enlarged image creating unit that creates an enlarged image at a magnification higher than at least the wide range display image for the specified enlarged observation range and displays the enlarged image on the screen of the display unit.
A cell observation device, further comprising: The cell observation device according to claim 5,
An operation unit for a user to perform an operation of moving or enlarging or reducing the enlarged observation range on the wide display image displayed on the display unit,
The cell observation device, wherein the enlarged image creating unit creates and displays an enlarged image after movement or enlarged or reduced by an operation via the operation unit. The cell observation device according to claim 4,
A cell observation apparatus, further comprising a focus position adjustment target range designation unit for allowing a user to designate a focus position adjustment target range used for determining a focus position in one or a plurality of the same focus position application ranges. The cell observation device according to claim 7,
The cell observation device, wherein the focus position comparison image creating unit creates a plurality of reconstructed images having different focus positions in the focus alignment target range.

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