JP5206064B2 - Microinjection apparatus and microinjection method - Google Patents

Description

  The present invention relates to a microinjection apparatus, a microinjection method, an intermittent imaging apparatus, and an intermittent imaging method.

  Research that modifies the genetic information of a cell by injecting the gene into the cell clarifies the role of the gene and enables tailor-made medicine that performs gene therapy tailored to the individual's genetic characteristics. Such research has made it possible to treat diseases caused by genetic causes that could not be treated.

  Experiments using such cells generally involve changes in the position and shape of cells (hereinafter referred to as cells) while photographing cells every time a predetermined time elapses after injecting a target object such as a gene into the cells. This is done by observing the changes in the position and shape of the cell.

  Methods for injecting the target substance into cells include electrical methods (electroporation), chemical methods (lipofection), biological methods (vector method), mechanical methods (microinjection), and optics. Method (laser injection).

  Among the above methods, the electrical method causes a large current to flow and breaks the cell membrane, causing significant damage to the cells. The chemical method is inefficient and biological because there are restrictions on the genes that can be introduced. The system has the disadvantages that all materials cannot be introduced and safety cannot be confirmed. Therefore, at present, the mechanical method is attracting attention as the safest and most efficient method, and several microinjection apparatuses for performing microinjection have been proposed.

  As a method of photographing cells, there is a method of photographing using a microscope having a photographing function. In recent years, an intermittent photographing apparatus having an intermittent photographing function for automatically photographing cells at regular intervals has been proposed for the purpose of reducing labor of an experimenter. In general, when photographing cells, bright-field observations such as halogen lamps and LEDs (Light Emitting Diodes) are used to illuminate the cells, and fluorescence excitation light illumination such as mercury lamps is applied to the cells. Fluorescence observation is performed.

JP-A-5-192171 JP-A-6-343478 JP 2003-125750 A JP 2002-277754 A JP 2006-259701 A

  However, the above-described conventional technique has a problem that it is impossible to observe or photograph a cell change immediately after execution of microinjection. Specifically, in the above-described prior art, after performing microinjection on a cell, such a cell must be placed on the base surface of the imaging device and the focus of the imaging device must be set to the cell. did not become. For this reason, in the above-described prior art, it has been impossible to observe or photograph a cell change that occurs while moving a cell to the imaging apparatus or while the imaging apparatus is focused.

  Further, the above-described conventional intermittent imaging apparatus has a problem that damage to cells increases. Specifically, since the above-described conventional intermittent imaging apparatus images cells at a certain imaging interval, in order to image cells at a short imaging interval in a time zone in which the cells change frequently, the cells change little. I had to take a picture of the cells at short intervals even when I was not. The fluorescence excitation light illumination that illuminates cells during fluorescence imaging has a very strong light intensity, so in conventional intermittent imaging devices that need to capture cells at short imaging intervals even during the time when cells hardly change, Damage increased. In this regard, it may be possible to lengthen the imaging interval in consideration of damage to the cells, but it becomes impossible to observe the sudden change of cells, which is the original purpose.

  Also, even if the experimenter can change the shooting interval during intermittent shooting, the experimenter has to change the shooting interval while observing the change in cells, which increases the labor for the experimenter. Occurs. Further, if it takes time to adjust the photographing interval, there is a possibility that a sharp change in cells cannot be photographed.

  The disclosed technology has been made to solve the above-described problems caused by the prior art, and a microinjection device and a microinjection method capable of observing a cell change immediately after execution of microinjection, and damage to cells. It is an object of the present invention to provide an intermittent imaging apparatus and an intermittent imaging method that can capture steep changes in cells while reducing as much as possible.

  In order to solve the above-described problems and achieve the object, a microinjection device disclosed in the present application is a microinjection device that injects a target object by inserting a fine needle into a cell on a base surface, and the base surface Among the above cells, an injection execution means for performing an injection process that is a process for injecting a target object into an injection target that is a target cell to be injected, and a cell in which the target object has been injected by the injection execution means And an intermittent imaging means for executing an intermittent imaging process for imaging the injected cells at predetermined intervals.

  Further, the intermittent imaging device disclosed in the present application is an intermittent imaging device that images a test object on the base surface at a predetermined interval via an objective lens for obtaining a partial enlarged image on the base surface, Intermittent imaging condition storage means for storing a plurality of imaging interval times, which are intervals at which the subject is imaged, and the imaging interval time while changing the imaging interval time to any of the imaging interval times stored in the intermittent imaging condition storage means It is necessary to have intermittent photographing means for photographing the test object.

  It should be noted that a component, expression, or any combination of components of the microinjection device and the intermittent imaging device disclosed in the present application is applied to a method, device, system, computer program, recording medium, data structure, etc. It is effective as an embodiment.

  According to the microinjection apparatus disclosed in the present application, there is an effect that cells immediately after the microinjection can be intermittently photographed.

  Moreover, according to the intermittent imaging apparatus disclosed in the present application, it is possible to reduce damage to cells and to capture an abrupt change in cells.

  Hereinafter, embodiments of a microinjection device, a microinjection method, an intermittent imaging device, and an intermittent imaging method disclosed in the present application will be described in detail with reference to the drawings. Note that the microinjection device, the microinjection method, the intermittent imaging device, and the intermittent imaging method disclosed in the present application are not limited to this embodiment.

  First, the outline of the microinjection apparatus according to the first embodiment will be described. The microinjection apparatus according to the first embodiment has a function of executing microinjection and a function of intermittent shooting. Then, when performing microinjection, the position of the microinjected cell is stored in a predetermined storage unit, and at the time of intermittent shooting, imaging of the cell is performed based on the cell position information stored in the storage unit. Do.

  FIG. 1 is a diagram illustrating a schematic configuration of a microinjection apparatus 100 according to the first embodiment. As shown in the figure, the microinjection device 100 includes a microscope unit 110, a monitor 130, an illumination control device 140, a needle driving device 150, a stage driving device 160, a lens driving device 170, and a shutter driving device 180. And a control unit 190.

  The microscope unit 110 includes an illumination 111, a petri dish 112, a needle 113, a stage 114, an objective lens 115, an excitation light source 116, a shutter 117, a reflecting mirror 118, an imaging lens 119, and a camera 120. Have. The illumination 111 is a device for irradiating the cells on the petri dish 112 with bright field illumination from above. The petri dish 112 is a flat dish on which cells are placed. The needle 113 is a hollow glass needle whose tip is miniaturized, and injects a target object such as a gene into cells on the petri dish 112.

  When microinjection is performed, a plate having fine holes may be provided on the bottom surface of the petri dish 112, and the cells may be trapped in the fine holes for injection. In the following description, both the bottom surface of the petri dish when the plate is not used and the upper surface of the plate when the plate is used are referred to as a base surface.

  The stage 114 is an XY stage that can move in the horizontal direction, and is a stage on which the petri dish 112 is mounted. The objective lens 115 is a lens for obtaining an enlarged image of a part of the petri dish 112. The excitation light source 116 is a light source for irradiating the cell with fluorescence excitation light when photographing the cell. The shutter 117 is a shutter for opening and closing the optical path of the fluorescence excitation light emitted from the excitation light source 116.

  The reflecting mirror 118 is a mirror that reflects the image obtained by the objective lens 115 toward the imaging lens 119 and reflects the fluorescence excitation light emitted from the excitation light source 116 toward the petri dish 112. The imaging lens 119 is a lens for forming an image on the video element of the camera 120. The camera 120 is a device that captures an image reflected by the reflecting mirror 118, and outputs the captured image to the control unit 190.

  The monitor 130 is a display device that displays various types of information, and displays an image captured by the camera 120, an information setting GUI (Graphical User Interface) described later, and the like. The illumination control device 140 is a device that controls the time during which the illumination 111 emits bright field illumination, the intensity of the bright field illumination emitted by the illumination 111, and the like. The needle driving device 150 is a device that controls the needle 113. For example, the needle driving device 150 moves the needle 113 so that the tip of the needle 113 is positioned at a cell that is the execution target of the microinjection (hereinafter referred to as “injection target”) during the microinjection.

  The stage driving device 160 is a device that controls the stage 114. For example, the stage driving device 160 moves the stage 114 so that the cell is positioned at the tip of the needle 113 when performing microinjection. Further, for example, the stage driving device 160 moves the stage 114 so that the cells are positioned in the observation field range of the objective lens 115 at the time of cell imaging.

  The lens driving device 170 is a device that controls the objective lens 115. For example, the lens driving device 170 moves the objective lens 115 so that the injection target is included in the enlarged image obtained by the objective lens 115. For example, the lens driving device 170 moves the objective lens 115 so that the injection target is positioned on the focal plane of the objective lens 115.

  The shutter driving device 180 is a device that controls the shutter 117. For example, the shutter driving device 180 opens the shutter 117 when it is necessary to irradiate cells with fluorescence excitation light during cell imaging.

  The control unit 190 is a control unit that controls the microinjection apparatus 100 as a whole. Specifically, the control unit 190 controls the monitor 130 to display a captured image, and controls the camera 120, the illumination control apparatus 140, the needle drive apparatus 150, and the stage drive. The operations of the device 160, the lens driving device 170, and the shutter driving device 180 are controlled.

  Under such a configuration, in the microinjection apparatus 100 according to the first embodiment, when performing microinjection, the needle driving apparatus 150 moves the needle 113 and the stage driving apparatus 160 moves the stage 114 in accordance with an experimenter's instruction. The objective lens 115 is moved by the lens driving device 170, and the needle 113 injects the target object into the injection target on the petri dish 112. At this time, the microinjection apparatus 100 uses a combination of the position of the stage 114 when the microinjection is performed and the position of the objective lens 115 (hereinafter, a combination of the position of the stage 114 when the microinjection is performed and the position of the objective lens 115). Is called “injection execution position information”) in a predetermined storage unit. When an instruction to perform intermittent shooting is received from the experimenter, the stage driving device 160 moves the stage 114 according to the position information stored in the storage unit, and the lens driving device 170 causes the objective lens 115 to move. Then, the camera 120 captures the cells on the petri dish 112 at a predetermined interval (for example, every 5 minutes).

  As described above, since the microinjection apparatus 100 according to the first embodiment has the function of executing microinjection and the function of executing intermittent imaging, the cells to be observed are moved to the intermittent imaging apparatus after the microinjection is performed. You can save time. In addition, the microinjection apparatus 100 according to the first embodiment stores the injection execution position information in a predetermined storage unit after the microinjection execution, and performs intermittent shooting according to the injection execution position information stored in the storage unit. The work performed by the experimenter, such as installing the cells on the intermittent imaging device, moving the stage or objective lens of the intermittent imaging device, or setting the focus of the objective lens of the intermittent imaging device on the cells, etc. Can be omitted. For this reason, the microinjection apparatus 100 according to the first embodiment can reduce the work burden on the experimenter and can intermittently photograph cells immediately after the microinjection. As a result, the experimenter can observe the cell change immediately after execution of the microinjection without performing a laborious work.

  In addition, in the microinjection apparatus 100 according to the first embodiment, a period from when a cell is imaged until the next cell is imaged (hereinafter, from when a cell is imaged to when the cell is next imaged). It is also possible to execute microinjection on another cell different from the cell on which microinjection has already been executed. In such a case, the microinjection apparatus 100 stores the injection execution position information in the storage unit every time the microinjection is executed. Then, when performing intermittent imaging, the microinjection apparatus 100 images the cells after moving the stage 114 and the objective lens 115 in accordance with the first injection execution position information stored in the storage unit. Subsequently, the microinjection apparatus 100 images the cells after moving the stage 114 and the objective lens 115 in accordance with the second injection execution position information stored in the storage unit. In this way, the microinjection apparatus 100 images the cells present at the positions indicated by all the injection execution position information stored in the storage unit.

  That is, the microinjection apparatus 100 according to the first embodiment can execute microinjection even during intermittent shooting. For example, even if it takes 10 hours for all the intermittent imaging processes to be completed, if the microinjection apparatus 100 according to the first embodiment is used, other cells can be used without waiting for 10 hours until the intermittent imaging is completed. On the other hand, microinjection can be executed. Further, in the microinjection apparatus 100 according to the first embodiment, after the microinjection is performed, the position information at the time of the injection execution is stored in a predetermined storage unit at any time. Therefore, for the cells for which microinjection has been performed during intermittent imaging, Intermittent shooting can be started immediately after execution of microinjection.

  Next, the configuration of the control unit 190 shown in FIG. 1 will be described. FIG. 2 is a block diagram illustrating a configuration of the control unit 190 illustrated in FIG. As shown in the figure, the control unit 190 includes an input unit 10, a storage unit 20, a monitor 130, an illumination control device 140, a needle driving device 150, a stage driving device 160, a lens driving device 170, It is connected to the shutter driving device 180 by a bus or the like.

  The input unit 10 is an input device for inputting various information and operation instructions, and is, for example, a keyboard or a mouse. The storage unit 20 is a storage device for storing various types of information, and includes a setting storage unit 21, a position storage unit 22, an intermittent shooting condition storage unit 23, and an image storage unit 24.

  The setting storage unit 21 is a storage unit that stores various setting information used by each functional unit included in the microinjection apparatus 100. For example, the setting storage unit 21 stores the origins of the needle 113, the stage 114, and the objective lens 115. The origin of the needle 113 or the like indicates a reference position for moving the needle 113 or the like. For example, the needle driving device 150 or the like calculates the movement amount from the origin, and moves the needle 113 or the like by the calculated movement amount.

  The position storage unit 22 is a storage unit that stores injection execution position information and observation conditions. The observation conditions indicate the illumination intensity of bright field illumination by the illumination 111, the time for irradiating the fluorescence excitation light, and the like. The position storage unit 22 stores a plurality of injection execution position information when microinjection is performed on a plurality of cells.

  The intermittent shooting condition storage unit 23 is a storage unit for storing intermittent shooting conditions. Specifically, the bright field illumination intensity of the illumination 111, the exposure time of the camera 120, and the interval time for shooting cells (hereinafter referred to as cells). The interval time for shooting is referred to as “shooting interval time”), the total time for performing intermittent shooting (hereinafter, the total time for performing intermittent shooting is referred to as “total shooting time”), and the like. For example, when the total photographing time “5 hours” and the photographing interval time “10 minutes” are stored in the intermittent photographing condition storage unit 23, the intermittent photographing unit 198 described later photographs the cells every 10 minutes and performs intermittent photographing. When 5 hours have elapsed since the start of the intermittent shooting, the intermittent shooting is terminated.

  The image storage unit 24 is a storage unit that stores images taken by the camera 120. The captured image stored in the image storage unit 24 is displayed on the monitor 130 by a GUI display control unit 191 described later.

  The control unit 190 also includes a GUI display control unit 191, an origin determination unit 192, an altitude inclination calculation unit 193, a drive device control unit 194, a cell interval determination unit 195, a warning unit 196, and an injection execution unit 197. And an intermittent photographing unit 198.

  The GUI display control unit 191 performs a screen for inputting various setting information (for example, intermittent shooting conditions) used by each function unit, various operations (for example, an operation for moving the stage 114, and intermittent shooting). This is a processing unit that causes the monitor 130 to control display of a screen for accepting an operation for starting (or the like) and a screen for displaying an observation image. In this specification, a screen that the GUI display control unit 191 controls the display of the monitor 130 is referred to as an “information setting GUI”.

  The origin determining unit 192 is a processing unit that determines the origins of the needle 113, the stage 114, and the objective lens 115. Specifically, the origin determination unit 192 determines a predetermined position of the needle 113, the stage 114, and the objective lens 115 as the origin when an operation for determining the origin is performed on the information setting GUI. Thus, the determined origin is stored in the setting storage unit 21.

  The altitude tilt calculation unit 193 performs autofocus on an arbitrary cell on the petri dish 112, calculates the tilt of the petri dish 112, and stores the calculated tilt and the position of the objective lens 115 during autofocus in the setting storage unit 21. It is a processing part to be made. Here, the altitude gradient calculation processing by the altitude gradient calculation unit 193 will be specifically described with reference to FIG. FIG. 3 is a diagram for explaining altitude gradient calculation processing by the altitude gradient calculation unit 193 shown in FIG.

  The altitude gradient calculation unit 193 performs autofocus on any three cells on the petri dish 112. In the example illustrated in FIG. 3, the altitude gradient calculation unit 193 performs autofocus on the cells P1, P2, and P3. At this time, the altitude inclination calculation unit 193 measures the X position and Y position of the stage 114 and the Z position of the objective lens 115 when autofocusing is performed on the cell P1. Similarly, the X position and Y position of the stage 114 and the Z position of the objective lens 115 when the autofocus is performed on the cells P2 and P3 are measured. Subsequently, the altitude inclination calculation unit 193 calculates the inclination of the petri dish 112 based on the measured three XYZ positions.

A method for calculating the inclination of the petri dish 112 will be described below. Assuming that the three XYZ coordinates measured by the altitude gradient calculation unit 193 are P1 (x1, y1, z1), P2 (x2, y2, z2), and P3 (x3, y3, z3), respectively, the vector P1 → P2 And the normal vector N (Nx, Ny, Nz) of the vector P1 → P3 is expressed by the following equation.
Nx = (y2-y1) (y3-y1)-(y2-z1) (y3-y1) (1)
Ny = (z2-z1) (x3-x1)-(x2-x1) (z3-z1) (2)
Nz = (x2-x1) (y3-y1)-(y2-y1) (x3-x1) (3)

  From the above equations (1) to (3), the rotation angle θx around the X axis of the petri dish 112 and the rotation angle θy around the Y axis of the petri dish 112 are obtained by the following equations.

  The altitude inclination calculation unit 193 stores the positions z1, z2, and z3 and the inclinations θx and θy calculated in this way in the setting storage unit 21. By using the positions z1, z2, and z3 and the inclinations θx and θy, the distance between the objective lens 115 and an arbitrary cell on the petri dish 112 can be calculated. In the following, the positions z1, z2, and z3 are referred to as “stage altitude”, and the inclinations θx and θy are referred to as “petri dish inclination”.

  Returning to the description of FIG. 2, the drive device control unit 194 controls the illumination control device 140, the needle drive device 150, the stage drive device 160, the lens drive device 170, and the shutter drive device 180 to control the illumination intensity of the illumination 111. Is a processing unit that adjusts the angle, moves the needle 113, the stage 114, and the objective lens 115, and opens and closes the shutter 117.

  Specifically, the drive device control unit 194 receives an operation instruction to change the time and intensity of the bright field illumination irradiated by the illumination 111 on the information setting GUI via the input unit 10. The time and intensity of the bright field illumination by the illumination 111 are changed by controlling the illumination control device 140 according to the operation instruction.

  Further, when an operation instruction for moving the needle 113 is given, the driving device control unit 194 moves the needle 113 to a desired position by controlling the needle driving device 150 in accordance with the operation instruction. At this time, the drive device control unit 194 uses the stage height and the petri dish inclination stored in the setting storage unit 21 to lower the needle 113 so that the object can be injected into the injection target.

  In addition, when an operation instruction for moving the stage 114 is given, the driving device control unit 194 moves the stage 114 to a desired position by controlling the stage driving device 160 according to the operation instruction. At this time, the drive device control unit 194 uses the stage height and the petri dish inclination stored in the setting storage unit 21 to move the objective lens 115 so that the focal plane of the objective lens 115 is located on a cell on the petri dish 112. Move. This is because, when the petri dish 112 is tilted, if the stage 114 is moved, the focal plane of the objective lens 115 is shifted from the cells on the petri dish 112.

  In addition, when an operation instruction for moving the objective lens 115 is given, the drive device control unit 194 moves the objective lens 115 to a desired position by controlling the lens drive device 170 in accordance with the operation instruction.

  For example, the drive device control unit 194 receives an operation instruction for moving the needle 113 and the stage 114 via the input unit 10 when executing microinjection. When the driving device control unit 194 moves the stage 114 and the like according to the instruction, the experimenter can observe the injection target through the objective lens 115. Hereinafter, a range on the petri dish 112 that can be observed through the objective lens 115 when the stage 114 is stopped is referred to as an “observation field range”.

  In addition, for example, the drive device control unit 194 moves the stage 114 and the objective lens 115 according to the injection execution position information stored in the position storage unit 22 during intermittent shooting.

  The cell interval determination unit 195 is a processing unit that determines whether or not the injection target is sufficiently separated from the cells that have already been subjected to microinjection. Specifically, the cell interval determination unit 195 first determines the range of fluorescence excitation light irradiated by the excitation light source 116 (hereinafter referred to as “excitation light irradiation range”) to the length obtained by dividing 2 by the diagonal length of the observation visual field range. ”) Is added (hereinafter referred to as“ overlapping irradiation distance ”). Subsequently, the cell interval determining unit 195 determines whether or not the distance between the observation visual field range including the cells that have been subjected to microinjection and the observation visual field range including the injection target is equal to or greater than the overlapping irradiation distance.

  The cell interval determination unit 195 obtains the injection execution position information from the position storage unit 22, thereby grasping the observation field range of the cells that have been subjected to microinjection. Further, since the observation visual field range and the excitation light irradiation range are fixed, the cell interval determination unit 195 may calculate the overlapping irradiation distance only once and store it in a predetermined storage unit.

  The cell interval determination process by the cell interval determination unit 195 will be described using the example shown in FIG. FIG. 4 is a diagram for explaining cell interval determination processing by the cell interval determination unit 195. In the example shown in the figure, it is assumed that the cell C1 has been subjected to microinjection and the cell C2 is an injection target. The observation visual field range K1 indicates the observation visual field range when microinjection is performed on the cell C1. That is, the position storage unit 22 stores the injection execution position information in which the observation visual field range becomes the observation visual field range K1. The excitation light irradiation range S1 indicates the excitation light irradiation range when the observation visual field range is the observation visual field range K1.

  In such a situation, the cell interval determination unit 195 calculates the overlapping irradiation distance “d + r” obtained by adding the radius “r” of the excitation light irradiation range to the distance “d” obtained by dividing 2 by the diagonal distance of the observation visual field range. calculate. Subsequently, the cell interval determining unit 195 determines that the distance between the observation visual field range K1 including the cell C1 that has been subjected to microinjection and the observation visual field range K2 including the cell C2 that is the target of microinjection is the overlapping irradiation distance “d + r”. It is determined whether or not. In the figure, an example in which the distance between the observation visual field range K1 and the observation visual field range K2 is equal to or greater than the overlapping irradiation distance “d + r” is shown, and thus the cell interval determination unit 195 includes the observation visual field range K1 including the cell C1. Then, it is determined that the distance from the observation visual field range K2 including the cell C2 is equal to or greater than the overlapping irradiation distance “d + r”.

  When the distance between the observation visual field range including the cells that have been subjected to the microinjection and the observation visual field range including the cells to be subjected to the microinjection is smaller than the overlapping irradiation distance by the cell interval determination unit 195, the warning unit 196 When it is determined, the processing unit displays a warning on the monitor 130 to that effect.

  Here, the reason why the cell interval determination unit 195 performs the overlap irradiation determination process and the warning unit 196 performs the warning display will be described with reference to FIGS. 5 and 6. FIG. 5 is a diagram illustrating a part of the petri dish 112 when the excitation light irradiation ranges overlap. FIG. 6 is a diagram illustrating a part of the petri dish 112 when the excitation light irradiation ranges do not overlap.

  In FIG. 5, a region R <b> 1 indicates a region on the petri dish 112 that can be observed as the stage 114 moves. Here, it is assumed that the cells C10 and C20 in the observation visual field ranges K10 and K20 have been subjected to microinjection. That is, the position storage unit 22 stores the injection execution position information in which the observation visual field range is the observation visual field range K10 and the injection execution position information in which the observation visual field range is the observation visual field range K20. . Excitation light irradiation ranges S10 and S20 indicate excitation light irradiation ranges when the observation visual field ranges are the observation visual field ranges K10 and K20, respectively.

  In such a situation, when intermittent imaging is performed, the microinjection apparatus 100 first moves the stage 114 or the like so that the observation visual field range becomes the observation visual field range K10 in order to capture the cell C10. Then, the observation visual field range K10 is photographed in a state in which the fluorescence excitation light is irradiated by the excitation light source 116. The excitation light irradiation range at this time is the excitation light irradiation range S10. Thereafter, the microinjection apparatus 100 moves the stage 114 and the like so that the observation visual field range becomes the observation visual field range K20 in order to photograph the cell C20. Then, the observation visual field range K20 is photographed in a state in which the fluorescence excitation light is irradiated by the excitation light source 116. The excitation light irradiation range at this time is the excitation light irradiation range S20.

  That is, in the example shown in FIG. 5, the cell C10 is irradiated with the fluorescence excitation light even when the cell C20 is photographed. Similarly, the cell C20 is irradiated with fluorescence excitation light when the cell C10 is photographed. As described above, the fluorescence excitation light illumination has a very strong light intensity, and thus damages the cells greatly. As in the example shown in FIG. 5, even when the subject itself is not photographed, if the fluorescent excitation light is irradiated, the damage to the cells C10 and C20 increases and may be killed. When the damage to the cells increases, it causes a problem that it is impossible to observe changes with time of the cells.

  On the other hand, when the distance between the observation visual field ranges K11 to K31 including cells that have been subjected to microinjection is equal to or less than the overlapping irradiation distance as in the example illustrated in FIG. In addition, the fluorescence excitation light is not irradiated to other cells to be observed. Thereby, damage to a cell can be reduced.

  That is, the reason why the cell interval determination unit 195 performs the overlap irradiation determination process and the warning unit 196 performs the warning display is that the experimenter who intends to perform the microinjection is subjected to fluorescence excitation light on the cells that have already been subjected to the microinjection. This is for notifying that irradiation is repeated. This allows the experimenter to select the injection target so that the fluorescence injection light is not repeatedly irradiated onto the microinjected cells without having to remember the position of the cells that have already been microinjected. Can do.

  Returning to the description of FIG. 2, the injection execution unit 197 is a processing unit that executes an injection process of injecting a target object into the injection target via the needle 113. In addition, the injection execution unit 197 stores position information at the time of injection execution in the position storage unit 22 after the injection processing.

  The intermittent photographing unit 198 causes the camera 120 to photograph the cells on the petri dish 112 at predetermined intervals based on various information stored in the position storage unit 22 and the intermittent photographing condition storage unit 23, and stores the photographed image as an image. A processing unit to be stored in the unit 24.

  Specifically, the intermittent shooting unit 198 follows the first injection execution position information stored in the position storage unit 22 when the shooting interval time stored in the intermittent shooting condition storage unit 23 has elapsed. Then, the driving device control unit 194 is instructed to move the stage 114 and the objective lens 115. After the stage 114 and the objective lens 115 are moved, the intermittent photographing unit 198 causes the camera 120 to photograph the cells on the petri dish 112.

  The intermittent photographing unit 198 performs photographing processing on the second and subsequent injection execution position information stored in the position storage unit 22 after moving the stage 114 according to the injection execution position information. The intermittent shooting unit 198 stores all injection execution position information stored in the position storage unit 22 every time the shooting interval time elapses until the total shooting time stored in the intermittent shooting condition storage unit 23 elapses. The stage is moved to the position indicated by, and the photographing process is performed.

  The intermittent shooting process by the intermittent shooting unit 198 will be specifically described using the example shown in FIG. Here, it is assumed that predetermined cells in the observation visual field range K11 to K31 shown in FIG. 6 have been subjected to microinjection. Here, it is assumed that the shooting interval time is “1 minute” and the total shooting time is “5 hours”.

  In such a situation, the intermittent imaging unit 198 captures an image of the observation visual field range K11 after moving the stage 114 so that the observation visual field range becomes the observation visual field range K11. Subsequently, the intermittent imaging unit 198 captures an image of the observation visual field range K21 after moving the stage 114 so that the observation visual field range becomes the observation visual field range K21. Subsequently, the intermittent imaging unit 198 captures an image of the observation visual field range K31 after moving the stage 114 so that the observation visual field range becomes the observation visual field range K31. Then, after “1 minute” has elapsed, the intermittent photographing unit 198 photographs images in the observation visual field ranges K11, K21, and K31 in the same manner as the above-described processing. The intermittent photographing unit 198 performs processing for photographing images in the observation visual field ranges K11, K21, and K31 every "1 minute" until the total photographing time "5 hours" elapses.

  Next, the procedure of the microinjection process performed by the microinjection apparatus 100 according to the first embodiment will be described. FIG. 7 is a flowchart illustrating a microinjection processing procedure performed by the microinjection apparatus 100 according to the first embodiment.

  As shown in the figure, when receiving an instruction to start the microinjection process via the input unit 10, the GUI display control unit 191 of the microinjection apparatus 100 causes the monitor 130 to display the information setting GUI (step S1). S101).

  With such an information setting GUI, an operation instruction for moving the needle 113 and the stage 114 and an input of intermittent shooting conditions are accepted. For example, when intermittent shooting conditions are input on the information setting GUI, the control unit 190 causes the intermittent shooting condition storage unit 23 to store the input intermittent shooting conditions.

  Subsequently, when an instruction to perform the origin determination process is input on the information setting GUI (Yes in step S102), the origin determination unit 192 sets the predetermined positions of the needle 113, the stage 114, and the objective lens 115 as the origin. And the determined origin is stored in the setting storage unit 21 (step S103). The origin determination process is necessary when the origin has not been determined immediately after the microinjection apparatus 100 is turned on. If the origin has already been determined, the origin determination unit 192 The determination process may not be performed.

  When the origin determination process is completed by the origin determination unit 192, or when an instruction to perform the origin determination process is not input because the origin has already been determined (No in step S102), the altitude inclination calculation unit 193 selects the petri dish. The three cells on 112 are autofocused to calculate the stage altitude and petri dish inclination, and the calculated stage altitude and petri dish inclination are stored in the setting storage unit 21 (step S104).

  Subsequently, when an instruction to move the needle 113, the stage 114, and the objective lens 115 is received via the input unit 10 (Yes in step S105), the driving device control unit 194 performs shooting processing by the intermittent shooting unit 198. It is determined whether or not it is being performed (step S106).

  When the shooting process is being performed by the intermittent shooting unit 198 (Yes at Step S106), the driving device control unit 194 enters a shooting standby state or waits until the total shooting time elapses and the intermittent shooting process ends. (Step S107).

  On the other hand, when the shooting process is not performed by the intermittent shooting unit 198 (No in Step S106), or as a result of waiting in Step S107, the shooting standby state is entered, or when the intermittent shooting process ends, the driving device The control unit 194 controls the needle driving device 150, the stage driving device 160, and the lens driving device 170 in accordance with the movement instruction received via the input unit 10, and moves the needle 113, the stage 114, and the objective lens 115 (step). S108).

  Subsequently, the cell interval determination unit 195 determines whether or not the distance between the observation visual field range determined in step S108 and the observation visual field range including cells that have already been subjected to microinjection is equal to or greater than the overlapping irradiation distance. (Step 109). When the cell interval determination unit 195 determines that the distance between the observation visual field ranges is smaller than the overlapping irradiation distance (No at Step S109), the warning unit 196 displays a warning to that effect on the monitor 130 (Step S110). .

  Subsequently, when a confirmation operation for executing microinjection is received via the input unit 10 (Yes at Step S111), the injection execution unit 197 executes an injection process (Step S112). Subsequently, the injection execution unit 197 stores the injection execution position information in the position storage unit 22 (step S113).

  On the other hand, when the confirmation operation not performing the microinjection is received (No at Step S111), the microinjection apparatus 100 is in a state of receiving a movement instruction for moving the needle 113 and the like (Step S105). In other words, the microinjection apparatus 100 is in a state of accepting an instruction to execute microinjection on another cell.

  Note that the warning unit 196 only displays a warning on the monitor 130, and even if the distance between the observation visual field ranges is equal to or less than the overlapping irradiation distance, the experimenter confirms that the microinjection is executed in step S111. Can be operated.

  After the injection process by the injection execution unit 197 is completed, when the intermittent shooting process is performed by the intermittent shooting unit 198, that is, when the shooting process is being performed or in the shooting standby state (Yes at step S114), microinjection is performed. The apparatus 100 is in a state of accepting a movement instruction for moving the needle 113 and the like (step S105).

  On the other hand, when the intermittent shooting unit 198 is not performing the intermittent shooting process, that is, when the shooting process is not being performed and the shooting standby state is not set (No in step S114), the GUI display control unit 191 is displayed on the information setting GUI. An inquiry is made as to whether or not to start intermittent shooting (step S115).

  When an instruction to start intermittent shooting is received on the information setting GUI (Yes in step S115), the intermittent shooting unit 198 starts intermittent shooting processing (step S116). The intermittent shooting unit 198 starts the intermittent shooting process only when an instruction to start intermittent shooting is received in step S115. The intermittent photographing unit 198 always accepts an instruction to start intermittent photographing if it is not during the injection processing, and starts the intermittent photographing processing.

  On the other hand, when an instruction not to start intermittent shooting is received on the information setting GUI (No in step S115), the microinjection apparatus 100 is in a state of receiving a movement instruction for moving the needle 113 and the like (step S115). S105).

  Next, a procedure of intermittent shooting processing by the microinjection apparatus 100 according to the first embodiment will be described. FIG. 8 is a flowchart illustrating the intermittent imaging processing procedure performed by the microinjection apparatus 100 according to the first embodiment.

  As shown in the figure, when receiving an instruction to perform intermittent shooting, the GUI display control unit 191 of the microinjection apparatus 100 causes the monitor 130 to display-control the information setting GUI (step S201). When intermittent shooting conditions are input on the information setting GUI, the control unit 190 causes the intermittent shooting condition storage unit 23 to store the input intermittent shooting conditions (step S202). Note that, in step S101 illustrated in FIG. 7, when the intermittent shooting condition is input, the control unit 190 does not need to accept the input of the intermittent shooting condition.

  Subsequently, the intermittent photographing unit 198 acquires the injection execution position information from the position storage unit 22 (step S203). Subsequently, the intermittent photographing unit 198 moves the stage 114 and the objective lens 115 to the drive device control unit 194 according to the first injection execution position information among the acquired injection execution position information (step S204). . After the stage 114 and the objective lens 115 are moved, the intermittent photographing unit 198 causes the camera 120 to photograph cells on the petri dish 112 (step S205). Subsequently, the intermittent photographing unit 198 stores the photographed image in the image storage unit 24 (step S206).

  Subsequently, the intermittent photographing unit 198 has the second injection execution position information in the injection execution position information acquired from the position storage unit 22 (Yes in step S207), and the intermittent photographing unit 198 has the second one. For the injection execution position information, the photographing process shown in steps S204 to S206 is performed. In this manner, the intermittent photographing unit 198 performs photographing processing for all the injection execution position information acquired from the position storage unit 22.

  When all of the injection execution position information acquired from the position storage unit 22 has been subjected to the shooting process (No at Step S207) and the total shooting time has not elapsed since the start of the intermittent shooting condition process (Step S208). No), the intermittent photographing unit 198 waits for the photographing interval time (step S209). It should be noted that the microinjection apparatus 100 can perform an injection process during the photographing standby state.

  On the other hand, if the total shooting time has elapsed since the start of the intermittent shooting condition process (Yes at step S208), the intermittent shooting unit 198 ends the intermittent shooting process. Note that the experimenter can also pause, resume, or cancel the intermittent imaging process at any timing while the intermittent imaging process is being executed by the intermittent imaging unit 198.

  As described above, the microinjection apparatus 100 according to the first embodiment has a function of executing microinjection and a function of executing intermittent imaging, and the position storage unit 22 stores position information at the time of execution of injection after the execution of microinjection. In the intermittent shooting, intermittent shooting is performed according to the injection execution position information stored in the position storage unit 22, so that the work burden on the experimenter can be reduced and immediately after the microinjection is executed. Cells can be taken intermittently.

  In the microinjection apparatus 100 according to the first embodiment, the cell interval determination unit 195 determines whether or not the injection target is sufficiently separated from the cells that have already been subjected to microinjection, and the cells that are the target of microinjection are determined. When the microinjection cell is not sufficiently distant from the cell, the warning unit 196 displays a warning on the monitor 130. Therefore, when the experimenter attempts to perform microinjection on a predetermined cell, such a cell is displayed. If the microinjection is executed, it is possible to notify that the fluorescence excitation light is irradiated to other observation target cells during intermittent imaging. Thereby, the experimenter can select the injection target so that the fluorescence excitation light is not repeatedly irradiated to the microinjected cell, and as a result, the damage to the cell can be reduced.

  In the first embodiment, an example in which a plurality of cells are intermittently photographed at the same photographing interval time has been described. However, the photographing interval time may be different depending on the observation visual field position where intermittent photographing is performed. In such a case, the intermittent shooting condition storage unit 23 stores the shooting interval time and the total shooting time in association with the injection execution position information.

  By the way, in Example 1 described above, after the drive device control unit 194 moves the stage 114 and the like, the cell interval determination unit 195 performs the overlap irradiation determination process, and the warning unit 196 performs the warning process. The stage 114 may be controlled so that it cannot move to a range that is not sufficiently separated from cells that have already been subjected to microinjection. Therefore, in the second embodiment, a microinjection apparatus that controls the stage 114 so that it cannot move to a range that is not sufficiently separated from cells that have already undergone microinjection will be described.

  First, the configuration of the control unit included in the microinjection apparatus 200 according to the second embodiment will be described. FIG. 9 is a block diagram illustrating a configuration of the control unit 290 included in the microinjection apparatus 200 according to the second embodiment. In the following, parts having the same functions as the constituent parts shown in FIG. 2 are denoted by the same reference numerals, and detailed description thereof is omitted. Moreover, since the schematic configuration of the microinjection apparatus 200 according to the second embodiment is the same as the schematic configuration illustrated in FIG. 1, the description thereof is omitted here.

  As illustrated in FIG. 9, the control unit 290 includes an overlapping irradiation range calculation unit 291 instead of the drive device control unit 194, the cell interval determination unit 195, and the warning unit 196 included in the control unit 190 illustrated in FIG. And a driving device control unit 294.

  The overlapping irradiation range calculation unit 291 is a processing unit that calculates a range in which the origin is the center point of the observation visual field range including cells that have been subjected to microinjection and the radius is the overlapping irradiation distance. Specifically, the overlapping irradiation range calculation unit 291 determines whether or not there is a cell that has already been subjected to microinjection, based on the injection execution position information stored in the position storage unit 22. When there is already a cell that has been subjected to microinjection, the overlapping irradiation range calculation unit 291 has a circle range (hereinafter referred to as “overlapping”) whose radius is the overlapping irradiation distance centering on the observation visual field range that includes the cells that have been subjected to microinjection. "Irradiation range").

  Similar to the drive device control unit 194, the drive device control unit 294 is a processing unit that controls the needle drive device 150 and the like to move the needle 113 and the like. In addition, the driving device control unit 294 according to the second embodiment controls the stage driving device 160 so that the observation visual field range does not fall within the overlapping irradiation range calculated by the overlapping irradiation range calculation unit 291.

  That is, when moving the stage 114 to a desired position in accordance with an instruction to move the stage 114, the driving device control unit 294 follows the movement instruction if the observation visual field range is included in the overlapping irradiation range due to such movement. Without moving the stage 114. At this time, the drive control unit 294 may display a warning that the movement is impossible on the monitor 130.

  Next, the procedure of the microinjection process performed by the microinjection apparatus 200 according to the second embodiment will be described. FIG. 10 is a flowchart illustrating a microinjection processing procedure performed by the microinjection apparatus 200 according to the second embodiment. In addition, description is abbreviate | omitted here about the process sequence (step S301-S304, S311-S316) similar to the process sequence shown in FIG.

  As shown in FIG. 10, an instruction to move the needle 113, the stage 114, and the objective lens 115 is received via the input unit 10 (Yes in step S305), and the imaging process is performed by the intermittent imaging unit 198. If not (No at Step S306), the overlapping irradiation range calculation unit 291 determines whether there is a cell that has already been subjected to microinjection (Step S308). Specifically, the overlapping irradiation range calculation unit 291 determines whether or not the injection execution position information is stored in the position storage unit 22.

  When there is a cell that has been subjected to microinjection (Yes at Step S308), the overlapping irradiation range calculation unit 291 calculates the overlapping irradiation range (Step S309). Specifically, the overlapping irradiation range whose radius is the overlapping irradiation distance is calculated around the observation visual field range including the cells that have been subjected to microinjection.

  On the other hand, when there is no cell that has been subjected to microinjection (No in step S308), or when the overlapping irradiation range calculation processing by the overlapping irradiation range calculation unit 291 is completed, the drive device control unit 294 is connected via the input unit 10. According to the received movement instruction, the needle 113, the stage 114, and the objective lens 115 are moved within a range where the observation visual field range is not included in the overlapping irradiation range (step S310).

  As described above, the microinjection apparatus 200 according to the second embodiment can save time for moving cells to be observed to the intermittent imaging apparatus after execution of the microinjection as in the first embodiment. Can be reduced, and cell changes immediately after the microinjection can be taken intermittently. In addition, in the microinjection apparatus 200 according to the second embodiment, the overlapping irradiation range is automatically calculated and the movable range is limited to the stage 114. Therefore, the microinjection apparatus 200 can reliably perform the microinjection without taking the labor of the experimenter. It is possible to prevent the fluorescence-excited light from being repeatedly irradiated to the cells that have been injected, and as a result, it is possible to reduce damage to the cells.

  In the first and second embodiments, the example in which intermittent shooting is performed at a fixed shooting interval time has been described. However, the shooting interval time may be changed according to the amount of cell change. In the third embodiment, an intermittent imaging apparatus that performs intermittent imaging by changing the imaging interval time according to the amount of cell change will be described.

  First, problems in the case where the shooting interval time is constant will be described. 11 and 12 are diagrams illustrating an example of the relationship between the cell change amount and the imaging interval time. FIG. 11 shows an example in which the shooting interval time is set shorter than the shooting interval time shown in FIG. As shown in FIG. 11, when the imaging interval time is set to a short interval time, the cells are imaged with high frequency, so that it is possible to observe a steep change in the cells. On the other hand, since the imaging process is frequently performed even when the amount of cell change is small, the amount of fluorescence excitation light irradiated to the cell increases, and the damage to the cell increases.

  In addition, as shown in FIG. 12, when the imaging interval time is set to a long interval time, the number of fluorescence excitation lights irradiated to the cells decreases, so that damage to the cells is reduced. On the other hand, even when the amount of cell change is large, the cells are photographed at a low frequency, so that it is impossible to observe a sharp change in the cells.

  Therefore, in the intermittent imaging apparatus 300 according to the third embodiment, the imaging interval time can be stored in association with the passage of time. The experimenter predicts the amount of cell change corresponding to the passage of time based on the characteristics of the cell, and stores the imaging interval time in association with the passage of time. The intermittent imaging apparatus 300 performs the intermittent imaging process by changing the imaging interval time according to the elapsed time after the start of the intermittent imaging process. This will be specifically described with reference to FIG. FIG. 13 is a diagram for explaining the outline of the intermittent imaging process performed by the intermittent imaging apparatus 300 according to the third embodiment.

  In the example of the cell shown in the figure, the cell change amount gradually increases in the time zone A immediately after the microinjection execution, the cell change amount increases rapidly in the time zone B, and the cell change amount in the time zone C. Will become smaller and eventually will not change. In such a case, the experimenter sets a shooting interval time with a long interval time in the time zones A and C where the cell change amount is small, and sets a shooting interval time with a short interval time in the time zone B where the cell change amount is large. Set.

  Accordingly, the intermittent imaging apparatus 300 performs intermittent imaging with the imaging interval time a having a long interval time in the time zone A in which the cell change amount is small, and the imaging interval time in which the interval time is short in the time zone B where the cell variation amount is large. In the time zone C in which the amount of cell change is small, intermittent imaging is performed with the imaging interval time c having a long interval time. Hereinafter, a shooting interval time having a long interval time is referred to as “low frequency shooting interval time”, and a shooting interval time having a short interval time is referred to as “high frequency shooting interval time”.

  As described above, in the intermittent imaging apparatus 300 according to the third embodiment, intermittent imaging is performed with the low frequency imaging interval time in the time zone where the cell change amount is small, and the high frequency imaging interval time is used in the time zone where the cell change amount is large. Since it is possible to perform intermittent imaging, the number of fluorescence excitation lights to be irradiated on the cells can be reduced in the time zone where the cell change amount is small, and the cells change sharply in the time zone where the cell change amount is large. Can be taken. That is, in the intermittent imaging apparatus 300 according to the third embodiment, it is possible to reduce the damage to the cells and to capture a sharp change in the cells.

  Next, a schematic configuration of the intermittent imaging apparatus 300 according to the third embodiment will be described. FIG. 14 is a diagram illustrating a schematic configuration of the intermittent imaging apparatus 300 according to the third embodiment. Here, parts having the same functions as the constituent parts shown in FIG. 1 are denoted by the same reference numerals, and detailed description thereof is omitted.

  As shown in the figure, the intermittent imaging device 300 includes a monitor 130, an illumination control device 140, a stage driving device 160, a lens driving device 170, a shutter driving device 180, a microscope unit 310, and a control unit 390. Have

  The microscope unit 310 includes an illumination 111, a petri dish 112, a stage 114, an objective lens 115, an excitation light source 116, a shutter 117, a reflecting mirror 118, an imaging lens 119, and a camera 120.

  The control unit 390 is a control unit that controls the intermittent imaging device 300 as a whole. Specifically, the control unit 390 controls the camera 120, the illumination control device 140, the stage driving device 160, the lens driving device 170, the shutter driving device 180, and the like. .

  Next, the configuration of the control unit 390 shown in FIG. 14 will be described. FIG. 15 is a block diagram showing the configuration of the control unit 390 shown in FIG. As shown in the figure, the control unit 390 includes an input unit 10, a storage unit 30, a monitor 130, an illumination control device 140, a stage driving device 160, a lens driving device 170, a shutter driving device 180, and a bus. Etc. are connected.

  The storage unit 30 includes a setting storage unit 21, a position storage unit 22, an image storage unit 24, and an intermittent shooting condition storage unit 33.

  The intermittent shooting condition storage unit 33 is a storage unit that stores shooting interval times in association with the passage of time. Specifically, the intermittent shooting condition storage unit 33 stores the shooting interval time in association with the shooting time at the shooting interval time. For example, the intermittent shooting condition storage unit 33 stores the shooting interval time “5 minutes” in association with the shooting time “1 hour”, and stores the shooting interval time “1 minute” in association with the shooting time “3 hours”. Then, the shooting interval time “10 minutes” is stored in association with the shooting time “1 hour”.

  The control unit 390 also includes a GUI display control unit 391, an origin determination unit 392, an altitude inclination calculation unit 193, a drive device control unit 394, a correction unit 395, an intermittent shooting unit 398, and an image processing unit 399. Have

  The GUI display control unit 391 receives a screen for inputting injection execution position information, a screen for inputting various setting information for operating each functional unit of the intermittent imaging device 300, and various operations. And an information setting GUI that is a screen for displaying an observation image on the monitor 130.

  When an observation condition is input to the information setting GUI, the control unit 390 causes the setting storage unit 21 to store the input observation condition. Note that the experimenter determines the illumination intensity of the bright field illumination by adjusting the illumination intensity of the illumination 111 via the illumination control device 140, or simply inputs a numerical value of the illumination intensity. In addition, the experimenter opens and closes the shutter 117 via the shutter driving device 180 and temporarily performs fluorescence observation to determine the irradiation time of the fluorescence excitation light, or simply inputs the numerical value of the illumination time.

  When the injection execution position information is input to the information setting GUI, the control unit 390 causes the position storage unit 22 to store the input injection execution position information. Note that the experimenter moves the stage 114 and the like to the drive device controller 394 via the input unit 10 and performs a predetermined determination operation after moving the stage 114 to a desired observation position. Determine information.

  When the shooting interval time associated with the passage of time is input to the information setting GUI, the control unit 390 causes the intermittent shooting condition storage unit 33 to store the shooting interval time associated with the passage of time. Note that the experimenter inputs the imaging interval time corresponding to the time change into the information setting GUI in consideration of the characteristics of the cells to be microinjected. For example, as in the example shown in FIG. 13, when the cell change amount transitions, the experimenter sets a low frequency imaging interval time in a time zone in which the cell change amount is expected to be small, In a time zone where the amount is expected to be large, a high-frequency shooting interval time is set.

  The origin determining unit 392 is a processing unit that calculates the origins of the stage 114 and the objective lens 115. The driving device control unit 394 acquires position information at the time of injection execution stored in the position storage unit 22 during intermittent shooting, and moves the stage 114 and the objective lens 115 according to the acquired position information at the time of injection execution. It is.

  The correction unit 395 is a processing unit that corrects the position information of the objective lens 115 stored in the position storage unit 22 in the shooting standby state. Specifically, the correction unit 395 first performs autofocus on any three cells on the petri dish 112. Subsequently, the correction unit 395 calculates the inclination of the petri dish 112 based on the three XYZ positions obtained by autofocus. Subsequently, the correcting unit 395 displays the position information of the objective lens 115 stored in the position storage unit 22 based on the XYZ position and the inclination of the petri dish 112, and the focal plane of the objective lens 115 is a cell on the petri dish 112. Correct it so that it is located at.

  The reason why the correction unit 395 corrects the position information of the objective lens 115 will be described. During intermittent imaging, the focus of the objective lens 115 may be deviated from the observation target cell due to a change in the optical system or a change in the inclination of the petri dish 112. In other words, the inclination of the petri dish 112 calculated by the altitude inclination calculation unit 193 before intermittent shooting may vary during intermittent shooting. When the inclination of the petri dish 112 fluctuates, the focal plane of the objective lens 115 is not positioned on the cell on the petri dish 112. If the focal plane of the objective lens 115 is deviated, the photographed image is blurred, and detailed data cannot be obtained. In the intermittent imaging apparatus 300 according to the third embodiment, during the intermittent imaging, the correction unit 395 corrects the positional information of the objective lens 115 so that the focal plane of the objective lens 115 is always positioned on the cell on the petri dish 112. It is possible to prevent the photographed image from being blurred.

  The intermittent photographing unit 398 is a processing unit that causes the camera 120 to photograph the cells on the petri dish 112 based on various information stored in the position storage unit 22 and the intermittent photographing condition storage unit 33. Specifically, the intermittent shooting unit 398 performs the intermittent shooting process while changing the shooting interval time to the shooting interval time stored in the intermittent shooting condition storage unit 33 according to the elapsed time after the start of the intermittent shooting process. Do.

  For example, as in the above example, the intermittent shooting condition storage unit 33 stores the shooting interval time “5 minutes” in association with the shooting time “1 hour”, and the shooting interval time in association with the shooting time “3 hours”. It is assumed that “1 minute” is stored, and the shooting interval time “10 minutes” is stored in association with the shooting time “1 hour”. In such a case, the intermittent photographing unit 398 performs intermittent photographing with the photographing interval time “5 minutes” until one hour has elapsed after starting the intermittent photographing processing, and four hours have elapsed since the intermittent photographing processing was started. Until then, intermittent shooting is performed at the shooting interval time “1 minute”, and thereafter, intermittent shooting is performed at the shooting interval time “10 minutes” until 5 hours have elapsed since the start of the intermittent shooting processing. Then, the intermittent imaging apparatus 300 ends the intermittent imaging process after 5 hours have elapsed from the start of the intermittent imaging process.

  The image processing unit 399 is a processing unit that generates an interpolated image so that images captured at unequal time intervals by the intermittent image capturing unit 398 are at equal time intervals, and stores them in the image storage unit 24. Further, the image processing unit 399 generates moving image data based on the captured image stored in the image storage unit 24, and causes the monitor 130 to display and control the generated moving image data.

  For example, as in the above example, the intermittent shooting unit 398 performs intermittent shooting with the shooting interval time “5 minutes” for the first hour, and performs intermittent shooting with the shooting interval time “1 minute” for the next three hours. In the next hour, it is assumed that intermittent shooting is performed with the shooting interval time “10 minutes”. In such a case, the image processing unit 399 generates, for example, four interpolated images obtained by duplicating the photographed image every time the first one hour is photographed with the photographing interval time “5 minutes”. Then, the image processing unit 399 stores one captured image and four interpolated images in the image storage unit 24. Subsequently, the image processing unit 399 causes the image storage unit 24 to store an image photographed at the photographing interval time “1 minute” for the next three hours. At this time, the image processing unit 399 does not generate an interpolation image. Subsequently, the image processing unit 399 generates nine interpolated images every time the next one hour is photographed with the photographing interval time “10 minutes”, and generates one captured image, four interpolated images, and so on. Is stored in the image storage unit 24.

  As described above, by generating the interpolated image, even if the shooting interval time fluctuates during intermittent shooting, the number of images generated at a predetermined time becomes equal. As a result, the image processing unit 399 can generate moving image data that makes it easy for an experimenter to grasp changes in cells over time.

  Next, a procedure of intermittent shooting preparation processing by the intermittent shooting device 300 according to the third embodiment will be described. FIG. 16 is a flowchart illustrating the intermittent shooting preparation processing procedure performed by the intermittent shooting apparatus 300 according to the third embodiment.

  As shown in the figure, when receiving an instruction to start the intermittent imaging process via the input unit 10, the GUI display control unit 391 of the intermittent imaging apparatus 300 causes the monitor 130 to display-control the information setting GUI ( Step S401).

  Subsequently, when an instruction to perform the origin determination process is input on the information setting GUI (Yes in step S402), the origin determination unit 392 determines a predetermined position of the stage 114 and the objective lens 115 as the origin, and determines The set origin is stored in the setting storage unit 21 (step S403).

  Subsequently, the altitude inclination calculation unit 193 performs autofocus on any three cells on the petri dish 112, calculates the stage altitude and petri dish inclination, and stores the calculated stage altitude and petri dish inclination in the setting storage unit 21. Store (step S404).

  Subsequently, the GUI display control unit 391 receives input of injection execution position information and observation conditions on the information setting GUI (step S405). The control unit 390 stores the input injection execution position information and observation conditions in the position storage unit 22 (step S406).

  Subsequently, the GUI display control unit 391 inquires whether to input other injection execution position information and observation conditions on the information setting GUI (step S407). When the input of other injection execution position information and observation conditions is received via the input unit 10 (Yes at Step S407), the control unit 390 performs the processing procedure of Steps S405 and S406.

  When the input of other injection execution position information and observation conditions is not accepted (No at Step S407), the GUI display control unit 391 sets the shooting interval time and the shooting time at the shooting interval time on the information setting GUI. An input is accepted (step S408). In addition, as an example of receiving the combination of the shooting interval time and the shooting time on the information setting GUI, “(a) 120 seconds interval for 10 minutes, (b) 20 seconds interval for 5 minutes, (c) 120 seconds interval” For 15 minutes ". Subsequently, the control unit 390 stores the input shooting interval time and shooting time in the intermittent shooting condition storage unit 33 (step S409).

  Next, a procedure of intermittent shooting processing by the intermittent shooting device 300 according to the third embodiment will be described. FIG. 17 is a flowchart illustrating the intermittent imaging processing procedure performed by the intermittent imaging apparatus 300 according to the third embodiment.

  As shown in the figure, when an instruction to perform intermittent shooting is received after completion of the intermittent shooting preparation process, the intermittent shooting unit 398 of the intermittent shooting device 300 receives position information and observation conditions during injection from the position storage unit 22. And the interval time and the imaging time are acquired from the intermittent imaging condition storage unit 33 (step S501).

  Subsequently, the intermittent photographing unit 398 adjusts the illumination height of the illumination 111 according to the acquired observation conditions. Subsequently, the intermittent imaging unit 398 moves the stage 114 and the objective lens 115 to the driving device control unit 394 according to the first injection execution position information among the injection execution position information acquired from the position storage unit 22. (Step S502). After the stage 114 and the objective lens 115 are moved, the intermittent photographing unit 398 causes the camera 120 to photograph cells on the petri dish 112 (step S503). Subsequently, the image processing unit 399 stores the image photographed by the intermittent photographing unit 398 and the interpolated image generated from the image in the image storage unit 24 (step S504).

  Subsequently, the intermittent imaging unit 398, when the injection execution position information acquired from the position storage unit 22 includes the second injection execution position information (No in step S505), the intermittent imaging unit 398 For the injection execution position information, the photographing process shown in steps S502 to S504 is performed. In this way, the intermittent photographing unit 398 performs photographing processing for all the injection execution position information acquired from the position storage unit 22.

  For all injection execution position information acquired from the position storage unit 22, when the shooting process is completed (Yes at step S505), the intermittent shooting unit 398 calculates the shooting interval time based on the elapsed time after the start of the intermittent shooting process. It is determined whether or not to change (step S506).

  When the elapsed time after the start of the intermittent shooting process exceeds the shooting time stored in the intermittent shooting condition storage unit 33, that is, when it is necessary to change the shooting interval time (Yes in step S506), the intermittent shooting unit 398 is performed. Changes the shooting interval time to the shooting interval time stored in the intermittent shooting condition storage unit 33 in association with the elapsed time (step S507). Subsequently, the intermittent photographing unit 398 waits until the next photographing timing according to the photographing interval time changed in step S507 (step S508). In such a shooting standby state, the correction unit 395 performs position correction processing (step S509), and then the intermittent shooting unit 398 acquires position information of the objective lens 115 from the intermittent shooting condition storage unit 33 (step S510). The position correction process by the correction unit 395 will be described later.

  On the other hand, if it is not necessary to change the shooting interval time (No at Step S506), the intermittent shooting unit 398 calculates the total shooting time stored in the intermittent shooting condition storage unit 33 as the elapsed time after the start of the intermittent shooting process. It is determined whether it has been exceeded (step S511).

  If the elapsed time after the start of the intermittent shooting process does not exceed the total shooting time (No at step S511), the intermittent shooting unit 398 waits until the next shooting timing (step S508). On the other hand, when the elapsed time after the start of the intermittent shooting process exceeds the total shooting time (Yes in step S511), the intermittent shooting unit 398 ends the intermittent shooting process.

  Next, a procedure of position correction processing by the correction unit 395 shown in FIG. 15 will be described. FIG. 18 is a flowchart showing a position correction processing procedure by the correction unit 395 shown in FIG.

  As shown in the figure, when an instruction to start correction processing is received, the correction unit 395 of the intermittent imaging device 300 first determines whether or not imaging processing is being performed by the intermittent imaging unit 398 ( Step S601). When photographing processing is performed by the intermittent photographing unit 398 (Yes in step S601), the correction unit 395 waits until the photographing standby state is set (step S602).

  On the other hand, when the image capturing process is not performed by the intermittent image capturing unit 398 (No in step S601), or when the image capturing standby state is set as a result of waiting in step S602, the correcting unit 395 causes the drive device control unit 394 to On the other hand, the stage 114 and the objective lens 115 are moved to arbitrary positions (step S603).

  After the stage 114 and the objective lens 115 are moved, the correction unit 395 performs autofocus on an arbitrary cell within the observation visual field range (step S604). Subsequently, the correction unit 395 stores the X position and Y position of the stage during autofocus and the Z position of the objective lens 115 in, for example, a storage unit such as an internal buffer (step S605).

  Subsequently, when the autofocus is not performed on the three cells (No at Step S606), the correction unit 395 repeats the processing procedure of Steps S603 to S605. When autofocus is performed on three cells (Yes in step S606), the correction unit 395 calculates the inclination of the petri dish 112 based on the three XYZ positions (step S607).

  Subsequently, the correcting unit 395 displays the position information of the objective lens 115 stored in the position storage unit 22 based on the XYZ position and the inclination of the petri dish 112, and the focal plane of the objective lens 115 is a cell on the petri dish 112. (Step S608).

  Subsequently, the correction unit 395 waits until the next shooting standby state is reached (step S609). Subsequently, when the intermittent photographing process by the intermittent photographing unit 398 is not completed (No at Step S610), the correcting unit 395 performs the processing procedure of Steps S603 to S609. On the other hand, when the intermittent shooting process by the intermittent shooting unit 398 has been completed (Yes at step S610), the correction unit 395 ends the position correction process.

  As described above, in the intermittent imaging apparatus 300 according to the third embodiment, the imaging interval time can be changed according to the passage of time, so that the experimenter is in a time zone where the amount of cell change is expected to be small. If the shooting interval time is set to the low frequency shooting interval time and the cell change amount is expected to be large, if the shooting interval time is set to the high frequency shooting interval time, the cell change amount time is small. In the band, the imaging process can be performed with the low frequency imaging interval time, and the imaging process can be performed with the high frequency imaging interval time in the time zone with a large amount of cell change. Thereby, in the intermittent imaging apparatus 300 according to the third embodiment, damage to the cells can be reduced and a steep change of the cells can be captured.

  In the intermittent imaging apparatus 300 according to the third embodiment, during intermittent imaging, the correction unit 395 displays position information of the objective lens 115 stored in the position storage unit 22 and the focal plane of the objective lens 115 is the petri dish 112. Since the correction is performed so that the cell is positioned on the upper cell, the focal plane of the objective lens 115 can always be positioned on the cell on the petri dish 112 even when the inclination of the petri dish 112 fluctuates during intermittent imaging. . As a result, the image photographed by the intermittent photographing unit 398 can be prevented from being blurred.

  By the way, in the said Example 3, an experimenter estimates the cell variation | change_quantity according to a time change, sets an intermittent imaging condition, and shows the example which performs intermittent imaging | photography processing according to the intermittent imaging condition which the intermittent imaging | photography part 398 applies. However, the intermittent photographing apparatus may calculate the cell change amount and automatically change the photographing interval time. Therefore, in the fourth embodiment, an intermittent imaging apparatus that calculates a cell change amount and automatically changes an imaging interval time will be described.

  First, an overview of intermittent shooting processing by the intermittent shooting device according to the fourth embodiment (referred to as the intermittent shooting device 400) will be described. FIG. 19 is a diagram for explaining the outline of the intermittent imaging process performed by the intermittent imaging apparatus 400 according to the fourth embodiment.

  The intermittent imaging apparatus 400 according to the fourth embodiment performs cell change amounts (position change amounts in the observation visual field range of cells and cell shapes) based on two images obtained by intermittent shooting during intermittent shooting. Change amount). Then, as illustrated in FIG. 19, the intermittent imaging apparatus 400 changes the imaging interval time when the calculated cell change amount exceeds a predetermined threshold. In the example shown in the figure, the intermittent imaging apparatus 400 performs intermittent imaging with the low frequency imaging interval time a in the time zone A in which the cell change amount is smaller than the threshold, and the time when the cell change amount exceeds the threshold. In the band B, the frequency is changed to the high frequency imaging interval time b, and in the time zone C in which the amount of cell change is smaller than the threshold, the frequency is changed to the low frequency imaging interval time c.

  As described above, in the intermittent imaging apparatus 400 according to the fourth embodiment, since the cell change amount is calculated and the imaging interval time is automatically changed, even if the experimenter cannot predict the cell change amount, the experimenter In addition, it is possible to reduce the damage to the cells without performing a setting operation or the like, and it is possible to reliably photograph a sharp change of the cells.

  Next, the configuration of the control unit 490 included in the intermittent imaging apparatus 400 according to the fourth embodiment will be described. FIG. 20 is a block diagram illustrating a configuration of the control unit 490 included in the intermittent imaging apparatus 400 according to the fourth embodiment. The schematic configuration of the intermittent imaging apparatus 400 according to the fourth embodiment is the same as the schematic configuration illustrated in FIG.

  As shown in FIG. 20, the control unit 490 includes an input unit 10, a storage unit 40, a monitor 130, an illumination control device 140, a stage driving device 160, a lens driving device 170, a shutter driving device 180, and a bus. Etc. are connected.

  The storage unit 40 includes a setting storage unit 21, a position storage unit 22, an image storage unit 24, and an intermittent shooting condition storage unit 43. The intermittent shooting condition storage unit 43 stores a plurality of shooting interval times, threshold values, and total shooting times. The plurality of shooting interval times indicate shooting interval times such as “10 seconds”, “1 minute”, “5 minutes”, “10 minutes” and the like. The threshold indicates a threshold of cell change amount and is associated with the imaging interval time. In the following description, the intermittent shooting condition storage unit 43 stores two shooting interval times, a low frequency shooting interval time (for example, “20 minutes”) and a high frequency shooting interval time (for example, “20 seconds”). Is stored.

  The control unit 490 newly includes a correlation value calculation unit 491 and an intermittent imaging unit 498 instead of the intermittent imaging unit 398 included in the control unit 390 illustrated in FIG. The correlation value calculation unit 491 is a processing unit that calculates the amount of cell change as a correlation value based on the image photographed by the intermittent photographing unit 498 and the image stored in the image storage unit 24.

  Specifically, the correlation value calculation unit 491, when an image (captured image I) is captured by the intermittent capturing unit 498, is an image captured before the captured image I (template image T). ) Is acquired from the image storage unit 24. Subsequently, the correlation value calculation unit 491 performs template matching between the captured image I and the template image T, and calculates a correlation value (cell change amount).

  Template matching is a process of searching for a position where the correlation value with the template image T is maximized while shifting the image position of the captured image I. Here, an example in which a cross-correlation coefficient is obtained as a correlation value will be described as using the cross-correlation method. When the search target image is I (m, n) and the template image is T (m, n) for the photographed image size “M” × “N”, the cross-correlation coefficient C is obtained by the following equation.

  However, φ (i, j) in the above equation (6) is defined by the following equation.

  “I” and “j” in the above formulas (6) to (8) are search coordinates of two-dimensional spatial coordinates. The cross-correlation coefficient C indicates that the larger the value, the higher the similarity between images. When the two images completely match, the value of the cross correlation coefficient C is “1”.

  The intermittent photographing unit 498 sends a petri dish to the camera 120 based on the cross-correlation coefficient C calculated by the correlation value calculating unit 491 and various information stored in the position storage unit 22 and the intermittent photographing condition storage unit 43. 112 is a processing unit that causes the cells on 112 to be photographed.

  Specifically, the intermittent shooting unit 498 sets the shooting interval time to a high frequency when the cross-correlation coefficient C calculated by the correlation value calculation unit 491 is equal to or greater than the threshold stored in the intermittent shooting condition storage unit 43. Change to the shooting interval time to perform intermittent shooting processing. Further, the intermittent photographing unit 498 sets the photographing interval time to the low-frequency photographing interval time when the cross-correlation coefficient C calculated by the correlation value calculating unit 491 is smaller than the threshold value stored in the intermittent photographing condition storage unit 43. Change to, and perform intermittent shooting.

  Next, a procedure of intermittent shooting processing by the intermittent shooting device 400 according to the fourth embodiment will be described. FIG. 21 is a flowchart illustrating the intermittent imaging processing procedure performed by the intermittent imaging apparatus 400 according to the fourth embodiment. Here, the description of the processing procedure (steps S701 to S704) similar to the processing procedure shown in FIG. 17 is omitted.

  As shown in the figure, when the shooting processing is completed for all the injection execution position information acquired from the position storage unit 22 (Yes in step S705), the correlation value calculation unit 491 is shot by the intermittent shooting unit 498. A template image T photographed immediately before the image (captured image I) is acquired from the image storage unit 24. Subsequently, the correlation value calculation unit 491 performs template matching between the captured image I and the template image T, and calculates a correlation value (cell change amount) (step S706). Here, it is assumed that the cross-correlation coefficient C is obtained as the correlation value.

  Subsequently, when the cross-correlation coefficient C calculated by the correlation value calculation unit 491 is equal to or greater than the threshold value stored in the intermittent shooting condition storage unit 43 (Yes in step S707), the intermittent shooting unit 498 sets the shooting interval time. Is changed to the high frequency imaging interval time (step S708). On the other hand, when the cross-correlation coefficient C calculated by the correlation value calculation unit 491 is smaller than the threshold value stored in the intermittent shooting condition storage unit 43 (No in step S707), the intermittent shooting unit 498 sets the shooting interval time. The frequency is changed to the low frequency photographing interval time (step S709).

  Subsequently, when the elapsed time after the start of the intermittent shooting process exceeds the total shooting time stored in the intermittent shooting condition storage unit 43 (Yes in step S710), the intermittent shooting unit 498 ends the intermittent shooting process. On the other hand, if the elapsed time after the start of the intermittent shooting process does not exceed the total shooting time (No at Step S710), the intermittent shooting unit 498 waits until the next shooting timing (Step S711). In such a shooting standby state, the correction unit 395 performs position correction processing (step S712), and then the intermittent shooting unit 498 acquires position information of the objective lens 115 from the position storage unit 22 (step S713).

  As described above, in the intermittent imaging apparatus 400 according to the fourth embodiment, since the cell change amount is calculated and the imaging interval time is automatically changed, even if the experimenter cannot predict the cell change amount, Without causing the experimenter to perform setting work, damage to the cells can be reduced, and a sharp change of the cells can be reliably imaged.

  In the fourth embodiment, the correlation value calculation unit 491 performs template matching and calculates the cell change amount using the images shot at the predetermined shooting interval time by the intermittent shooting unit 498. The correlation value calculation unit 491 may calculate the cell change amount using an image captured at a time interval different from the imaging interval time captured by the intermittent imaging unit 498.

  In the fourth embodiment, the correlation value calculation unit 491 uses the cross-correlation method to calculate the cross-correlation coefficient as the cell change amount. However, the correlation value calculation unit 491 uses other methods ( For example, the amount of cell change may be calculated using a residual method.

  Hereinafter, an example using the remaining work method will be described as an example of another method. The residual R that is a correlation value in the residual cropping method is as follows when the search target image is I (m, n) and the template image is T (m, n) with respect to the captured image size “M” × “N”. It is expressed by a formula.

  However, the residual R indicates that the smaller the value, the higher the similarity between images. When the two images are completely coincident with each other, the value of the residual R is “0”. That is, the threshold value determination described above is opposite to the case where the cross-correlation coefficient C is used.

  In the fourth embodiment, the example in which the two shooting interval times of the low frequency shooting interval time and the high frequency shooting interval time are stored in the intermittent shooting condition storage unit 43 has been described. 43 may store three or more shooting interval times. For example, the intermittent shooting condition storage unit 43 stores the shooting interval time “10 seconds” in association with the threshold “H1”, stores the shooting interval time “1 minute” in association with the threshold “H2”, and stores the threshold “ Assume that the shooting interval time “5 minutes” is stored in association with H3, and the shooting interval time “10 minutes” is stored in association with the threshold “H4”. In such a case, if the cell change amount is less than the threshold “H1”, the intermittent imaging unit 498 performs intermittent imaging with the imaging interval time “10 seconds”, and the cell change amount is equal to or greater than the threshold “H1”. If it is less than “H2”, intermittent shooting is performed with a shooting interval time “1 minute”.

  Further, in Example 4 described above, an example in which the amount of change in position and the amount of change in cell shape within the observation field of view of the cell is used as the amount of cell change is shown. However, the amount of light emitted from the cell when irradiated with fluorescence excitation light is shown. The amount of change may be the amount of cell change. A combination of the positional change amount, the cell shape change amount, and the light emission amount change amount may be the cell change amount.

  In the intermittent imaging apparatuses 300 and 400 shown in the third and fourth embodiments, the example in which the imaging target is a cell has been described. However, the intermittent imaging apparatuses 300 and 400 shown in the third and fourth embodiments are It can also be used for intermittent imaging of test objects other than cells.

  Further, the processing procedures, control procedures, specific names, information including various data and parameters shown in the above-mentioned documents and drawings can be arbitrarily changed unless otherwise specified. Further, each component of each illustrated apparatus is functionally conceptual, and does not necessarily need to be physically configured as illustrated. That is, the specific form of distribution and integration of each device is not limited to the one shown in the figure, and all or a part thereof is functionally or physically distributed in arbitrary units according to various loads or usage conditions. Can be integrated and configured. Further, all or any part of each processing function performed in each device may be realized by a CPU and a program analyzed and executed by the CPU, or may be realized as hardware by wired logic.

  The following appendices are further disclosed with respect to the embodiments including the above Examples 1 to 4.

(Appendix 1) A microinjection device for injecting a target object by inserting a fine needle into a cell on a basal plane,
An injection execution means for executing an injection process that is a process of injecting a target object into an injection target that is a target cell into which the target object is to be injected, among the cells on the basal plane;
A microinjection device comprising: intermittent imaging means for executing intermittent imaging processing for imaging an injected cell, which is a cell into which an object has been injected by the injection execution means, at a predetermined interval.

(Appendix 2) an objective lens for obtaining a partial enlarged image on the basal plane;
A basal plane drive unit for driving the basal plane;
A lens driving unit for driving the objective lens;
Drive unit control means for controlling the basal plane drive unit and the lens drive unit;
The injection execution unit is moved by the drive unit control unit so that the base surface is positioned within an observation field range that is a range of an enlarged image obtained by the objective lens, and the drive After the objective lens is moved so that the injection target is located on the focal plane of the objective lens by the unit control means, the injection processing is executed,
The intermittent photographing means performs the intermittent photographing process after the driving unit control means moves the basal plane and the objective lens to the position of the basal plane and the position of the objective lens when the injection process is executed. The microinjection device according to appendix 1, wherein the microinjection device is executed.

(Additional remark 3) The said injection execution means is other than the said injection-completed cell at the time of the imaging | photography standby state after it is until the said injection-completed cell is image | photographed after the said injection-completed cell is image | photographed by the said intermittent imaging means. 3. The microinjection apparatus according to appendix 1 or 2, wherein an injection process is performed on another injection target.

(Additional remark 4) It further has a position storage means which memorizes the combination of the position of the base and the position of the objective lens when the injection process is executed,
The intermittent photographing means performs an intermittent photographing process after the driving unit control means moves the basal plane and the objective lens to the position of the basal plane and the position of the objective lens stored in the position storage means. The microinjection device according to appendix 2 or 3, wherein the microinjection device is executed.

(Additional remark 5) The cell space | interval determination means which determines whether the distance of the observation visual field range in which the said injection object is included, and the observation visual field range in which the said injected cell is contained is more than predetermined value,
Additional notes 1 to 4, further comprising warning means for displaying a warning when the distance between the observation visual field ranges is determined to be smaller than a predetermined value by the cell interval determination means. The microinjection apparatus as described in any one.

(Additional remark 6) The fluorescence excitation light irradiation means to irradiate the said fluorescence injection light to the said injected cell is further provided,
The cell interval determination means is configured such that the distance between the observation visual field range including the injection target and the observation visual field range including the injected cells is a value obtained by dividing the diagonal distance of the observation visual field range by two. The micro as set forth in appendix 5, wherein it is determined whether or not the overlapping irradiation distance is a value obtained by adding the radii of the excitation light irradiation range, which is a range irradiated with the fluorescence excitation light by the excitation light irradiation means. Injection device.

(Additional remark 7) The overlap irradiation range calculation means which calculates the overlap irradiation range which is a circle which makes the origin the center point of the observation visual field range in which the injected cell is contained, and makes the overlap irradiation distance a radius,
The drive unit control means moves the basal plane and the objective lens so that the observation visual field range is outside the overlap irradiation range calculated by the overlap irradiation range calculation means. Microinjection device.

(Supplementary note 8) A microinjection method using a microinjection device for injecting a target object by inserting a fine needle into a cell on a basal plane,
Microinjection device
An injection execution step of performing an injection process that is a process of injecting a target object into an injection target that is a target cell into which the target object is injected, among the cells on the basal plane;
A microinjection method, comprising: an intermittent imaging step of performing an intermittent imaging process of imaging an injected cell, which is a cell into which an object has been injected by the injection execution step, at a predetermined interval.

(Supplementary note 9) An intermittent imaging apparatus that images a test object on the base surface at a predetermined interval via an objective lens for obtaining a partial enlarged image on the base surface,
Intermittent imaging condition storage means for storing a plurality of imaging interval times that are intervals for imaging the test object;
An intermittent photographing apparatus comprising: intermittent photographing means for photographing the subject while changing the photographing interval time to any of the photographing interval times stored in the intermittent photographing condition storage means.

(Supplementary Note 10) The intermittent shooting condition storage unit stores a shooting interval time in association with the passage of time,
The intermittent imaging apparatus according to appendix 9, wherein the intermittent imaging unit images the test object at an imaging interval time stored in the intermittent imaging condition storage unit as time elapses.

(Additional remark 11) The correlation value calculation means which compares the two images image | photographed by the said intermittent imaging means, and calculates the correlation value showing the similarity degree of these two images is further provided,
The intermittent photographing means has a short photographing interval time out of photographing interval times stored in the intermittent photographing condition storage means when the correlation value calculated by the correlation value calculating means is equal to or greater than a predetermined threshold. A low-frequency shooting interval in which intermittent shooting is performed with a high-frequency shooting interval time, and the correlation value calculated by the correlation value calculation unit is less than a predetermined threshold, the shooting interval time is longer than the high-frequency shooting interval time. The intermittent photographing apparatus according to appendix 9, wherein intermittent photographing is performed according to time.

(Additional remark 12) The base surface drive part which drives the base surface,
A lens driving unit for driving the objective lens, a driving unit control means for controlling the basal plane driving unit and the lens driving unit;
The position of the basal plane when the test object is located within an observation field range that is a range of an enlarged image obtained by the objective lens, and the objective when the test object is located on the focal plane of the objective lens Position storage means for storing a combination with the position of the lens;
Based on the inclination of the basal plane obtained by performing autofocus on three or more test objects on the basal plane, the position of the test object is positioned on the focal plane of the objective lens. Correction means for correcting the position of the objective lens stored in the storage means,
The drive unit control means moves the base surface and the objective lens to the position of the base surface and the position of the objective lens stored in the position storage means,
The intermittent imaging apparatus according to any one of appendices 9 to 11, wherein the intermittent imaging unit performs intermittent imaging after the base surface and the objective lens are moved by the drive unit control unit.

(Additional remark 13) The said correction means is memorize | stored in the said position memory | storage means at the time of the imaging | photography waiting state which is after the said test subject is image | photographed by the said intermittent imaging | photography means until this test subject is image | photographed next. The intermittent photographing apparatus according to appendix 12, wherein the position of the objective lens is corrected.

(Additional remark 14) The image processing means which produces | generates an interpolation image so that it may become an equal time interval from the image image | photographed by the said intermittent imaging means at equal time intervals, and produces | generates moving image data based on the produced | generated image further The intermittent photographing apparatus according to any one of supplementary notes 9 to 13, wherein the intermittent photographing apparatus is provided.

(Supplementary Note 15) An intermittent imaging method using an intermittent imaging apparatus that images a test object on the basal plane at a predetermined interval via an objective lens for obtaining a partial enlarged image on the basal plane,
The intermittent photographing device is
An acquisition step of acquiring an imaging interval time from intermittent imaging condition storage means for storing a plurality of imaging interval times that are intervals of imaging the test object;
An intermittent imaging method, comprising: an intermittent imaging step of imaging the subject while changing an imaging interval time to any of the imaging interval times acquired by the acquisition step.

It is a figure which shows schematic structure of the microinjection apparatus which concerns on Example 1. FIG. It is a block diagram which shows the structure of the control part shown in FIG. It is a figure for demonstrating the altitude inclination calculation process by the altitude inclination calculation part shown in FIG. It is a figure for demonstrating the cell space | interval determination process by a cell space | interval determination part. It is a figure which shows a part of petri dish in case an excitation light irradiation range overlaps. It is a figure which shows a part of petri dish when excitation light irradiation ranges do not overlap. 3 is a flowchart illustrating a microinjection processing procedure by the microinjection apparatus according to the first embodiment. 3 is a flowchart illustrating an intermittent imaging processing procedure by the microinjection apparatus according to the first embodiment. It is a block diagram which shows the structure of the control part which the microinjection apparatus which concerns on Example 2 has. 10 is a flowchart illustrating a microinjection processing procedure by the microinjection apparatus according to the second embodiment. It is a figure which shows an example of the relationship between cell variation | change_quantity and imaging | photography interval time. It is a figure which shows an example of the relationship between cell variation | change_quantity and imaging | photography interval time. FIG. 10 is a diagram for explaining an overview of intermittent shooting processing by the intermittent shooting device according to the third embodiment. FIG. 6 is a diagram illustrating a schematic configuration of an intermittent imaging apparatus according to a third embodiment. It is a block diagram which shows the structure of the control part shown in FIG. 10 is a flowchart illustrating an intermittent shooting preparation processing procedure performed by the intermittent shooting apparatus according to the third embodiment. 10 is a flowchart illustrating an intermittent imaging processing procedure performed by the intermittent imaging apparatus according to the third embodiment. It is a flowchart which shows the position correction process procedure by the correction | amendment part shown in FIG. FIG. 10 is a diagram for explaining an overview of intermittent shooting processing by the intermittent shooting device according to the fourth embodiment. FIG. 10 is a block diagram illustrating a configuration of a control unit included in an intermittent imaging apparatus according to a fourth embodiment. 10 is a flowchart illustrating an intermittent imaging processing procedure by the intermittent imaging apparatus according to the fourth embodiment.

Explanation of symbols

DESCRIPTION OF SYMBOLS 10 Input part 20, 30, 40 Storage part 21 Setting storage part 22 Position storage part 23, 33, 43 Intermittent imaging condition storage part 24 Image storage part 100, 200 Microinjection apparatus 110, 310 Microscope part 111 Illumination 112 Petri dish 113 Needle 114 Stage 115 Objective lens 116 Excitation light source 117 Shutter 118 Reflector 119 Imaging lens 120 Camera 130 Monitor 140 Illumination control device 150 Needle drive device 160 Stage drive device 170 Lens drive device 180 Shutter drive device 190, 290, 390, 490 Control unit 191 , 391 GUI display control unit 192, 392 Origin determination unit 193 Altitude tilt calculation unit 194, 294, 394 Drive device control unit 195 Cell interval determination unit 196 Warning unit 197 Injection execution unit 198 398, 498 Intermittent imaging unit 291 Overlapping irradiation range calculation unit 300, 400 Intermittent imaging device 395 Correction unit 399 Image processing unit 491 Correlation value calculation unit

Claims (4)

A microinjection device for injecting a target object by inserting a fine needle into a cell on the basal plane,
An objective lens for obtaining a partial enlarged image on the basal plane;
A basal plane drive unit for driving the basal plane;
A lens driving unit for driving the objective lens;
Drive unit control means for controlling the basal plane drive unit and the lens drive unit;
An injection target that is a target cell for injecting a target object among cells on the base surface within the observation visual field range that is a range of an enlarged image obtained by the objective lens by the drive unit control means. After the objective lens is moved by the drive unit control means so that the injection target is positioned on the focal plane of the objective lens, an object is placed on the injection target. An injection execution means for executing an injection process which is a process to be injected;
Fluorescence excitation light irradiating means for irradiating fluorescence-excited light to injected cells, which are cells into which the target object has been injected by the injection execution means,
Fluorescence excitation by the fluorescence excitation light irradiation means is performed by dividing the distance between the observation visual field range including the injection target and the observation visual field range including the injected cells by the diagonal distance of the observation visual field range by two. A cell interval determination means for determining whether or not the overlapping irradiation distance is a value obtained by adding the radii of the excitation light irradiation range which is a range irradiated with light;
Warning means for displaying a warning when the distance between the observation visual field ranges is determined to be smaller than the overlapping irradiation distance by the cell interval determination means;
After the basal plane and the objective lens are moved to the position of the basal plane and the position of the objective lens when the injection process is performed by the driving unit control unit, the injected cells are photographed at a predetermined interval. A microinjection apparatus comprising: intermittent shooting means for performing intermittent shooting processing. A double irradiation range calculation means for calculating a double irradiation range, which is a circle having the origin as the center point of the observation visual field range including the injected cells and a radius of the overlapping irradiation distance,
The said drive part control means moves the said base surface and the said objective lens so that an observation visual field range may become other than the overlap irradiation range calculated by the said overlap irradiation range calculation means, The said base lens and the said objective lens are moved. Microinjection device.   The injection execution means includes other injections other than the injected cells during the imaging standby state from when the injected cells are imaged by the intermittent imaging means to when the injected cells are next imaged. The microinjection apparatus according to claim 1, wherein an injection process is performed on a target. It is the microinjection method by the microinjection apparatus in any one of Claims 1-3 ,
A drive unit control step for controlling a base surface drive unit for driving the base surface and a lens drive unit for driving an objective lens for obtaining a partial enlarged image on the base surface;
By the drive unit control step, the basal plane is an injection target that is a target cell for injecting a target object among cells on the basal plane within an observation field range that is a range of an enlarged image obtained by the objective lens. After the objective lens is moved so that the injection target is positioned on the focal plane of the objective lens by the driving unit control step, the target object is placed on the injection target. An injection execution process for executing an injection process that is an injection process;
A fluorescence excitation light irradiation step of irradiating fluorescence injection light to injected cells, which are cells into which the target object has been injected by the injection execution step;
Fluorescence excitation by the fluorescence excitation light irradiation step is performed by dividing the distance between the observation visual field range including the injection target and the observation visual field range including the injected cells by a value obtained by dividing 2 by the diagonal distance of the observation visual field range. A cell interval determination step of determining whether or not the overlapping irradiation distance is a value obtained by adding the radii of the excitation light irradiation range, which is a range irradiated with light,
A warning step of displaying a warning when the distance between the observation visual field ranges is determined to be smaller than the overlapping irradiation distance by the cell interval determination step;
After the basal plane and the objective lens are moved to the position of the basal plane and the position of the objective lens when the injection process is performed by the driving unit control step, the injected cells are photographed at a predetermined interval. microinjection wherein the including that the intermittent photographing step of performing the intermittent photographing processing for.

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