JP5034328B2 - Observation device and observation program - Google Patents

Description

  The present invention relates to an observation apparatus for observing an observation object, and an observation program.

  Non-patent document 1 discloses a cell observation method using a fluorescence method using a fluorescence microscope as follows. According to this method, the cells are irradiated with excitation light, and the fluorescence emitted from the cells is observed. Then, the observation position is set before the observation. At this time, the cell is irradiated with excitation light, and the observation position is set from the fluorescence image.

Understanding bioimaging 22-24 (issued in September 2005, Yodosha)

  However, in the conventional observation method, as the time for irradiating the cells with the excitation light becomes longer, the fluorescence is weakened due to photobleaching, or the cells are damaged due to phototoxicity. For this reason, when selecting cells to be observed and setting the observation position, if it takes time to select the cells, the cells will be adversely affected by photobleaching and phototoxicity at the selection stage. There is a possibility that accurate observation cannot be performed.

The observation apparatus according to the first aspect of the present invention performs the fluorescence observation before performing the fluorescence observation , the objective lens, the excitation light source that irradiates the excitation light that excites the fluorescent reagent added to the observation specimen for fluorescence observation, and the fluorescence observation. A transmission illumination device that performs transmission illumination on an observation specimen to perform preparatory observation to determine a position, an imaging device that acquires an image of the observation specimen, and the preparation that is obtained by irradiating the observation specimen with transmission illumination by the transmission illumination apparatus Position specifying means for performing observation and performing fluorescence observation on the acquired specimen image and specifying based on a navigation position input, and surroundings adjacent to the navigation position centered on the navigation position specified by the position specifying means the set navigation range related to fluorescent observation area to perform fluorescence observation in a predetermined range, the excitation light to the navigation range after being set Characterized in that it comprises a control means for performing control of the excitation light emitted from the.

  ADVANTAGE OF THE INVENTION According to this invention, the bad influence to the target object by irradiating light can be reduced.

  FIG. 1 is a diagram schematically showing the configuration of the cell observation device in the present embodiment. The cell observation device 100 includes a stage 101, a chamber 102, an objective lens 103, a camera 104, a control device 105, a transmitted light illumination device 106, a shutter 107, an excitation light source device 108, a personal computer (PC). ) 109, a monitor 110, and an environment control device 111. The stage 101, the chamber 102, the objective lens 103, the camera 104, the control device 105, the transmitted light illumination device 106, the shutter 107, and the excitation light source device 108 constitute a microscope.

  In this cell observation device 100, the user can issue various instructions to the cell observation device 100 by operating the PC 109, and the PC 109 sends a control signal corresponding to the instruction from the user to the control device 105. Output. The control device 105 executes various processes according to instructions from the user based on the control signal from the PC 109. In the present embodiment, a process when the user observes the cells in the cell culture container A set in the chamber 102 on the stage 101 will be described.

For the cell culture container A, for example, a 35 mm dish is used. The chamber 102 is hermetically sealed, and the internal environment is maintained by the environment control device 111 in an environment suitable for cell culture. For example, the CO ₂ concentration in the chamber 102 is maintained at 5% by the CO ₂ mixer 111a, the humidity is maintained at 95% by the humidity adjuster 111b, and the temperature is maintained at 37 ° C. by the temperature adjuster 111c.

  The excitation light source device 108 irradiates the cell culture container A in the chamber 102 with excitation light having a wavelength that excites the dye that fluorescently stains the cells. The excitation light output from the excitation light source device 108 can be blocked by the shutter 107. The transmitted light illumination device 106 includes an LED as a light source. When excitation light is blocked by the shutter 107, the transmitted light illumination device 106 causes the LED to emit light and transmits transmitted light (illumination light) to the cell culture container A in the chamber 102. Irradiation is also possible. The blocking of the excitation light by the shutter 107 and the light emission of the LED from the transmitted light illumination device 106 are all controlled by the control device 105.

  The microscope has a phase difference observation mode and a fluorescence observation mode, and can transmit the transmitted light from the transmitted light illumination device 106 to the cell culture vessel A in the chamber 102 to observe the phase difference of the cells. The cell culture vessel A in the chamber 102 can be irradiated with excitation light from the light source device 108 and the cells stained with the fluorescent reagent can be observed with fluorescence. The camera 104 includes an imaging device such as a CCD, and can capture a cell image (enlarged image) input through the objective lens 103 to obtain a microscope image, that is, a phase difference image and a fluorescence image.

  Note that the microscope can perform fluorescence observation by switching a plurality of fluorescence filters. For example, in the case of multiple staining in which staining is performed with a plurality of fluorescent reagents, a plurality of fluorescent colors can be observed by performing fluorescence observation by switching a plurality of fluorescent filters. Thereby, the camera 104 can capture a plurality of fluorescent images for each fluorescent filter.

  A microscope image taken by the camera 104 is output to the control device 105 and further output to the PC 109 and then displayed on the monitor 110. As a result, the user can check the microscope image on the monitor 110.

  In cell observation apparatus 100 in the present embodiment, a user can operate PC 109 to instruct fluorescent image capturing conditions according to the type of cells to be observed. For example, an excitation light amount, a camera gain, an exposure time, a focal plane, and a filter type are specified as imaging conditions. This imaging condition may be set in advance for each cell type and stored in the memory provided in the PC 109 so that the user can select the setting contents according to the type of cell to be observed. The shooting conditions may be set each time.

  In general, when fluorescence observation is performed on a cell, as the time for which the cell is irradiated with excitation light becomes longer, the fluorescence is weakened by photobleaching, or the cell is damaged by phototoxicity. For this reason, when performing fluorescence observation (preparation fluorescence observation) for selecting cells to be observed as a pre-stage for performing actual fluorescence observation (main fluorescence observation), if it takes time to prepare fluorescence observation, There is a possibility that the observation results as expected may not be obtained, for example, the cells may be adversely affected by photobleaching or phototoxicity at the stage of preliminary fluorescence observation, and accurate experimental data cannot be obtained by this fluorescence observation.

  In the present embodiment, a method for suppressing the irradiation time of excitation light in the preparatory observation stage as much as possible in order to minimize the adverse effect on the cells in the preparatory observation stage will be described.

  In order to select a cell to be observed from the cell culture vessel A, the user executes navigation described later to set a range (navigation range) for taking a microscope image. For this purpose, the control device 105 first sets the microscope to the phase difference observation mode, closes the shutter 107 and blocks the excitation light, and then controls the transmitted light illumination device 106 to cause the LED to emit light. The transmitted light is irradiated to the cell culture container A. Then, the objective lens 103 is set at a high magnification, and the camera 104 is controlled to take an image input through the objective lens 103. The phase difference image of the cell culture vessel A thus photographed is output to the PC 109.

  The CPU provided in the PC 109 displays on the monitor 110 a GUI screen (navigation position designation screen) on which the LIVE screen 2a and the navigation screen 2b are displayed as shown in FIG. Then, a high-magnification phase difference image (real-time image) output from the control device 105 is displayed in the LIVE screen 2a. In the navigation screen 2b, the position of the phase difference image displayed in the LIVE screen 2a within the observable range when the entire range inside the cell culture vessel A is set as the observable range is displayed. That is, in the example shown in FIG. 2, the phase difference image currently displayed in the LIVE screen 2a is an image at a position corresponding to the range 2c within the observable range.

  Further, in the navigation position designation screen, a movement button 2d for moving the position of the phase difference image displayed in the LIVE screen 2a within the observable range is displayed. Then, the user can move the position of the phase difference image displayed in the LIVE screen 2a within the observable range by operating the mouse and operating the movement button 2d. In the example shown in FIG. 2, the movement button 2d is a circular button, and the movement direction is determined according to the position on the button clicked with the mouse. Further, the amount of movement is determined according to the number of times of pressing with the mouse or the pressing time.

  Specifically, when the movement button 2d is operated by the user, the operation signal is output to the control device 105, and the control device 105 responds to the number of times the button is pressed and the pressing time in the direction in which the button is pressed. The objective lens 103 is moved by the moving amount to change the photographing range by the objective lens 103. The CPU provided in the PC 109 acquires the position of the objective lens 103 at that time, for example, the coordinate value from the control device 105. Based on the position of the objective lens 103, the position within the observable range of the phase difference image currently displayed in the LIVE screen 2a is displayed in the navigation screen 2b. That is, the display content in the navigation screen 2 b is updated in accordance with the movement of the objective lens 103.

  Thus, the user can confirm the phase difference image at an arbitrary position within the observable range by operating the movement button 2d and moving the position of the objective lens 103 on the LIVE screen 2a. At the same time, it is possible to confirm at a glance which range within the observable range the range of the phase difference image currently being captured by the objective lens 103 by the display in the navigation screen 2b.

  The user designates the navigation position on the phase difference image displayed in the LIVE screen 2a. Here, as shown in FIG. 3, as a result of the movement button 2d being operated, the position of the phase difference image displayed in the LIVE screen 2a corresponds to the range 2e within the observable range as shown in the navigation screen 2b. It is assumed that it is in the position to do. At this time, the user designates the position on the phase difference image currently displayed on the navigation screen 2b, that is, the range 2e, by clicking the decision button 3b.

  The CPU specifies the navigation position 2e designated by the user, and sets a predetermined range around the navigation position 2e around the navigation position 2e as the navigation range 3a. In addition, although the magnitude | size of the range set as the navigation range 3a shall be preset, you may change suitably in arbitrary magnitude | sizes. Thereby, the user can set the navigation range 3a including the surroundings only by designating an arbitrary navigation position 2e on the LIVE screen 2a. Therefore, the navigation range 3a can be set without performing preliminary fluorescence observation.

  Further, the CPU displays the designated navigation position 2e and the navigation range 3a set around the navigation position 2e in the navigation screen 2b. Thereby, the user can confirm the position within the observable range of the navigation position 2e and the navigation range 3a on the navigation screen 2b. Then, the CPU creates a recipe that stores information (navigation range information) for specifying the set navigation range 3 a and the above-described shooting conditions, and outputs the recipe to the control device 105.

  Here, the navigation range 3a is set based on the navigation position 2e for the following reason. That is, in general, the user often selects a cell to be observed by looking at the situation around the cell, for example, the situation of binding with an adjacent cell. For this reason, conventionally, after the user specifies a region where cells are grown in advance, it is necessary to slide the stage 101 and observe the surrounding situation.

  However, according to the present embodiment, a predetermined range including the navigation position 2e designated by the user is set as the navigation range 3a, and a microscope image in the navigation range 3a is presented to the user by a process described later. As a result, the user can grasp the surrounding situation only by specifying the point where the cell is growing as the navigation position 2e, and can easily select the observation target cell in consideration of the surrounding situation. It becomes like this.

  A plurality of navigation positions 2e can be specified. When the user specifies a plurality of navigation positions 2e, the CPU sets a navigation range 3a corresponding to the specified number of navigation positions 2e, The navigation range information of each navigation range 3a is stored in the recipe. And the control apparatus 105 performs the process mentioned later with respect to all the navigation ranges 3a stored in the recipe. Here, for simplification of description, an example in which one navigation position 2e is designated and one navigation range 3a is set will be described.

  When a recipe is input from the PC 109, the control device 105 reads the navigation range information and shooting conditions stored in the recipe, and executes navigation within the navigation range 3a. That is, the objective lens 103, the camera 104, the transmitted light illumination device 106, and the shutter 107 are controlled to take a phase difference image and a fluorescence image while moving the objective lens 103 within the navigation range 3a. Specifically, the control device 105 performs navigation as follows.

  The control device 105 first specifies the coordinate value (X, Y) of the start point of the navigation range 3a based on the navigation range information. For example, the coordinate value of the upper left end point of the navigation range 3a is specified as the coordinate value (X, Y) of the start point of the navigation range 3a. Then, the objective lens 103 is moved to the start point of the navigation range 3a specified by setting the microscope in the phase difference observation mode, the shutter 107 is closed to block the excitation light, and then the transmitted light illumination device 106 is controlled to control the position. The LED is caused to emit light for the time required for capturing the phase difference image, for example, the exposure time of the camera 104. Then, the objective lens 103 is set at a high magnification, and an image input through the objective lens 103 is photographed by controlling the camera 104 and stored. Thereby, a phase difference image at the start point of the navigation range 3a can be taken.

  Further, the control device 105 sets the microscope in the fluorescence observation mode, controls the transmitted light illumination device 106 to turn off the LED, and holds the shutter 107 for the time required for photographing the fluorescent image, for example, the exposure time of the camera 104. Open and irradiate with excitation light. Then, the objective lens 103 is set at a high magnification, and the camera 104 is controlled to capture and store an image input through the objective lens 103. Thereby, a fluorescence image at the start point of the navigation range 3a can be taken. At this time, when a plurality of types of fluorescent filters are designated as the imaging conditions, the fluorescent filters are switched and a fluorescent image is captured for each fluorescent filter.

  Thereafter, the control device 105 moves the objective lens 103 so as to photograph the next area in the navigation range 3a. That is, the control device 105 moves the objective lens 103 so that the imaging range of the camera 104 at the objective lens position after the movement is adjacent to the imaging range of the camera 104 at the objective lens position before the movement, and the both ranges do not overlap. Control the amount. Then, the above-described processing is executed at the moved objective lens position, and the phase difference image and the fluorescence image at the moved objective lens position are photographed and stored. By executing this process for the entire range of the navigation range 3a, a phase difference image and a plurality of fluorescent images corresponding to the types of fluorescent filters can be taken for each of a plurality of small regions in the navigation range 3a. .

  Then, the control device 105 joins (tils) the phase difference images corresponding to the small regions that are photographed and stored by executing the navigation, and forms one combined image (macro position) photographing the navigation range 3a. Phase difference image). Further, the control device 105 similarly tiles the fluorescent image corresponding to each region for each fluorescent image of each fluorescent filter to generate a combined image (macro fluorescent image) for each fluorescent filter.

  A method of generating a macro phase difference image will be specifically described with reference to FIG. FIG. 4 is a diagram schematically illustrating a procedure for generating a macro phase difference image based on a phase difference image captured when navigation is performed on four small regions. Here, as a result of the navigation being performed on four small areas, four phase difference images 4a to 4d shown in FIG. The control device 105 performs tiling by arranging the four phase difference images side by side as shown in FIG. 4B while maintaining the positional relationship of the respective small regions within the navigation range. As a result, a macro phase difference image 4e as shown in FIG. 4C is generated.

  Next, a method for generating a macro fluorescent image will be specifically described with reference to FIG. FIG. 5 is a diagram schematically showing a procedure for generating a macro fluorescent image based on a fluorescent image photographed when navigation is performed using two types of fluorescent filters for four small regions. is there. Here, as a result of navigation performed on four small regions using the first fluorescent filter, four fluorescent images 5a to 5d shown in FIG. 5A are taken. Further, as a result of navigation performed on four small areas using the second fluorescent filter, four fluorescent images 5f to 5i shown in FIG. 5D are taken.

  First, the control device 105 joins the four fluorescent images 5a to 5d shown in FIG. 5A side by side as shown in FIG. 5B while maintaining the positional relationship of the respective small regions within the navigation range. Tiling by doing. As a result, a macro phase difference image 2e of the first fluorescent filter as shown in FIG. 5C is generated. Next, the four fluorescent images 5f to 5i shown in FIG. 5 (d) are aligned and joined as shown in FIG. 5 (e) while maintaining the positional relationship of the respective small regions within the navigation range. Do the ring. Thereby, a macro phase difference image 2j of the second fluorescent filter as shown in FIG. 5F is generated.

  The control device 105 outputs the macro phase difference image generated in this way and the macro fluorescence image for each fluorescence filter to the PC 109.

  In the PC 109, the CPU displays a GUI screen (observation target selection screen) as shown in FIG. The user operates the observation target selection screen on the monitor 110, and based on the macro phase difference image and the plurality of macro fluorescent images photographed using the plurality of fluorescent filters, the observation target is as follows. Select cells. Note that the user operates the observation object selection screen shown in FIG. 6 by operating the mouse provided in the PC 109. Moreover, only the item relevant to this Embodiment is demonstrated about the observation object selection screen of FIG. 6, and description is abbreviate | omitted about the other item.

  First, the user selects the phase difference image display button 6a on the observation object selection screen, and displays the macro phase difference image in the image display area 6d on the observation object selection screen. The user can confirm the shape of the cell from the macro phase contrast image. Then, on the basis of this macro phase difference image, a macro fluorescence image (filter image) photographed with an arbitrary fluorescence filter can be superimposed (superimposed) and displayed. For example, when a filter image obtained by photographing an image emitted in red by fluorescence observation is displayed superimposed on the macro phase difference image, the user selects the red filter image display button 6b on the observation target selection screen. That is, by setting both the phase difference image display button 6a and the red filter image display button 6b to be selected, the red filter image is superimposed on the base macro phase difference image in the image display area 6d. Can be displayed.

  Similarly, when a filter image obtained by capturing an image emitted in blue by fluorescence observation is displayed superimposed on the macro phase difference image, the user can use the blue filter together with the phase difference image display button 6a on the observation target selection screen. The image display button 6c is selected. Further, when it is desired to display both the red filter image and the blue filter image on the macro phase difference image, the phase difference image display button 6a, the red filter image display button 6b, and the blue filter image display button 6c are all selected. do it.

  For example, when the first fluorescent filter image 5e and the second filter image 5j shown in FIG. 5 are superimposed and displayed on the macro phase difference image 4e shown in FIG. 4, the composition as shown in FIG. An image (superimposed image) is displayed in the image display area 6d.

  By superimposing and displaying the synthesized macro phase difference image and the synthesized macro fluorescence image (filter image) in this way, the user can confirm the fluorescence state of the fluorescent reagent as well as the shape of the cell while observing the object. The cells to be selected can be selected. Specifically, the user moves the cursor 6e displayed on the macro image in the image display area 6d by operating the movement button 6f with the mouse and hits the cell to be observed in the main observation. By clicking the selection button 6g with a mouse, a cell to be observed in this observation is selected. When the user wants to select a plurality of cells as the observation target of the main observation, this process is repeated.

  In the observation target selection screen, the movement button 6f is a circular button as in the navigation position designation screen described above with reference to FIGS. Then, the movement direction of the cursor 6e on the screen is determined according to the position on the button clicked with the mouse, and the movement amount of the cursor 6e on the screen is determined according to the number of times the mouse is pressed or the pressing time. The

  When the user selects a cell to be observed in the main observation, the CPU adds information specifying the position of the cursor 6e at the time when the selection button 6g is clicked in the main observation target display area 6h.

  As a result, the user can select a cell to be observed as a preparatory stage before the main observation while observing the macro image displayed on the monitor 110, and in order to select the cell, transmitted light or excitation light is applied to the cell. It will not be necessary to continue irradiation for hours. That is, since the irradiation time of the excitation light only needs to be a time necessary for taking a fluorescent image, damage due to photobleaching or phototoxicity of cells in the preparation stage can be minimized. Further, since the irradiation time of the transmitted light is only the time necessary for taking the phase difference image, even if the cell is adversely affected by the transmitted light, this adverse effect can be minimized.

  Thereafter, when the user operates the PC 109 to instruct the start of the main observation, the CPU outputs a main observation execution instruction signal to the control device 105 together with the information displayed in the main observation target display area 6h. For example, the user instructs the start of the main observation on the selected cells displayed in the main observation target display area 6h by clicking the time lapse execution button 6i shown in FIG.

  When the execution instruction signal for the main observation is input, the control device 105 performs, for example, a time lapse process on the observation target cell to observe the cell, that is, the main observation. The result of this observation is output to the PC 109 via the control device 105, and observation data (test data) is recorded on the hard disk in the PC 109 and displayed on the monitor 110 at the same time.

  FIG. 8 is a flowchart showing processing of the control device 105 in the present embodiment. The process shown in FIG. 8 is executed by the control device 105 as a program that is activated when the user operates the PC 109 to instruct start of test preparation and receives a test start signal from the PC 109.

  In step S10, it is determined whether or not the operation signal for the movement button 2d on the navigation position designation screen described above has been received from the PC 109. If it is determined that the operation signal of the movement button 2d has not been received, the process proceeds to step S30 described later. On the other hand, if it is determined that the operation signal for the movement button 2d has been received, the process proceeds to step S20. In step S20, the objective lens 103 is moved based on the operation signal of the movement button 2d, and the process proceeds to step S30.

  In step S30, the microscope is set in the phase difference observation mode, the objective lens 103, the camera 104, and the shutter 107 are controlled to output a phase difference image photographed at a high magnification to the PC 109, and the process proceeds to step S40. In step S40, it is determined whether or not the above-described recipe is input from the PC 109. If it is determined that no recipe has been input, the process returns to step S10 and the process is repeated. On the other hand, if it is determined that the recipe has been input, the process proceeds to step S50.

  In step S50, the shooting conditions and navigation range information stored in the recipe are read, and the process proceeds to step S60. In step S60, the start point of the navigation range 3a is specified, and the objective lens 103 is moved to the start point of the specified navigation range 3a.

  Thereafter, the process proceeds to step 70, and navigation is performed by controlling the objective lens 103, the camera 104, the transmitted light illumination device 106, and the shutter 107 as described above while switching between the phase difference observation mode and the fluorescence observation mode of the microscope. A phase difference image and a fluorescence image are taken for each small area. At this time, when a plurality of fluorescent filter types are designated as the imaging conditions, the plurality of fluorescent filters are switched and a fluorescent image is captured for each type of filter. Thereafter, the process proceeds to step S80.

  In step S80, it is determined whether or not navigation has been completed for the entire range of the navigation range 3a. If it is determined that the execution of navigation has not been completed, the process returns to step S60 and the objective lens 103 is moved in order to photograph the adjacent small area, and the process is repeated. On the other hand, if it is determined that the execution of navigation is completed, the process proceeds to step S90.

  In step S90, the phase difference image for each small region captured by navigation and the fluorescence image captured by at least one type of fluorescent filter are tiled, and the above-described macro phase difference image and macro fluorescence image are obtained. create. Then, it progresses to step S100, the created macro phase difference image and macro fluorescence image are output to PC109, and it progresses to step S110.

  In step S110, it is determined whether an execution instruction signal for main observation is received from the PC 109 or not. When it is determined that the execution instruction signal for the main observation has been received, the process proceeds to step S120, and the time lapse process is executed on the observation target cell selected by the user on the PC 109. Thereafter, the process proceeds to step S130, the result of the time lapse process is output to the PC 109, and the process ends.

  FIG. 9 is a flowchart showing the processing of the PC 109 in the present embodiment. The processing shown in FIG. 9 is executed by the CPU of the PC 109 as a program that is activated when a phase difference image captured by the objective lens 103 from the control device 105, that is, a phase difference image displayed in the above-described LIVE screen 2a is input. Is done.

  In step S200, the navigation position designation screen described above is displayed on monitor 110, and the flow proceeds to step S210. In step S210, it is determined whether or not the movement button 2d on the navigation position designation screen has been operated. If it is determined that the move button 2d has not been operated, the process proceeds to step S240 described later. On the other hand, if it is determined that the move button 2d has been operated, the process proceeds to step S220.

  In step S220, the operation signal of the movement button 2d is output to the control device 105, and the process proceeds to step S230. In step S230, the display contents in the LIVE screen 2a and the navigation screen 2b are updated according to the position of the objective lens 103 after the movement, and the process proceeds to step S240.

  In step S240, it is determined whether or not the navigation position 2e has been designated on the LIVE screen 2a by the user. If it is determined that the navigation position 2e is not designated, the process returns to step S210 and the process is repeated. On the other hand, if it is determined that the navigation position 2e is designated, the process proceeds to step S250. In step S250, the navigation range 3a is set to a predetermined adjacent range centered on the designated navigation position 2e, and the process proceeds to step S260. In step S260, a recipe storing the navigation range information and shooting conditions of the set navigation range 3a is created, the recipe is output to the control device 105, and the process proceeds to step S270.

  In step S270, it is determined whether or not the above-described macro phase difference image and macro fluorescence image are input from the control device 105. If it is determined that these images have been input, the process proceeds to step S280, the observation target selection screen shown in FIG. 6 is displayed on the monitor 110, and the process proceeds to step S290. In step S290, it is determined whether display of the macro phase difference image is instructed by the user selecting the phase difference image display button 6a on the observation target selection screen. If it is determined that the display of the macro phase difference image has been instructed, the process proceeds to step S300, and the macro phase difference image is displayed in the image display area 6d on the observation target selection screen. Thereafter, the process proceeds to step S310.

  In step S310, it is determined whether the display of the macro fluorescent image is instructed by selecting the fluorescent image display button corresponding to the type of the fluorescent filter on the observation target selection screen by the user. If it is determined that the display of the macro fluorescent image is not instructed, the process proceeds to step S330 described later. On the other hand, if it is determined that the display of the macro fluorescent image is instructed, the process proceeds to step S320, and the macro fluorescent image of the fluorescent filter indicated in the macro phase difference image displayed in the image display area 6d is displayed. Overlay and display. Thereafter, the process proceeds to step S330.

  In step S330, it is determined whether a cell to be observed has been selected by the user. If it is determined that the observation target cell is not selected, the process returns to step S310 and the process is repeated. On the other hand, if it is determined that the cell to be observed has been selected, the process proceeds to step S340. In step S340, it is determined whether or not the user has instructed execution of the main observation. If it is determined that the execution of the main observation has been instructed, the process proceeds to step S350, the execution instruction signal of the main observation is output to the control device 105, and the process is terminated.

According to the present embodiment described above, the following operational effects can be obtained.
(1) The LIVE screen 2a and the navigation screen 2b are displayed on the navigation position designation screen, the phase difference image output from the control device 105 is displayed in the LIVE screen 2a, and the LIVE screen within the observable range is displayed. The position of the phase difference image displayed in 2a is displayed in the navigation screen 2b. As a result, the user can simultaneously confirm the phase difference image captured by the objective lens 103 and the position of the captured phase difference image within the observable range, improving the convenience for the user. To do.

(2) When the movement button 2d is operated by the user on the navigation position designation screen and the position of the phase difference image displayed in the LIVE screen 2a is moved, the display content in the navigation screen 2b is changed to the phase difference. Updated to match the movement of the image position. Thus, the user can always grasp the latest position within the observable range of the phase difference image displayed in the LIVE screen 2a.

(3) When the navigation position 2e is designated by the user on the LIVE screen 2a in the navigation position designation screen, a predetermined range around the navigation position 2e with the navigation position 2e as the center is set as the navigation range 3a. I set it. As a result, the user can set the navigation range 3a including the surroundings only by designating an arbitrary navigation position 2e on the phase difference image displayed in the LIVE screen 2a, which is convenient for the user. improves. Further, the user can grasp the surrounding situation only by designating the point where the cell is growing as the navigation position 2e, so that the cell to be observed can be easily selected in consideration of the surrounding situation. become. Furthermore, since it is possible to irradiate the excitation light up to the setting of the observation region in consideration of the surrounding situation and set it without acquiring the fluorescence image, the adverse effect of the excitation light on the cells can be reduced.

(4) The navigation position 2e designated in the LIVE screen 2a and the navigation range 3a set around the navigation position 2e are displayed in the navigation screen 2b. Thereby, the user can confirm the position within the observable range of the navigation position 2e and the navigation range 3a on the navigation screen 2b.

(5) A macro in which a fluorescence image is captured by irradiating cells with excitation light for a time required to capture an image for each small region in the navigation range 3a, and the fluorescence image for each captured small region is tiled A fluorescent image was provided to the user. As a result, damage due to photobleaching and phototoxicity of cells at the preparation stage can be minimized. Further, the user can select a cell to be observed over time on the macro phase contrast image without worrying about the adverse effect on the cells caused by continuing to irradiate the excitation light.

(6) For each small region in the navigation range 3a, a cell is irradiated with transmitted light for a time required for capturing an image to capture a phase difference image, and the phase difference image for each captured small region is tiled. The macro phase contrast image is provided to the user. As a result, even when observing cells that may be adversely affected by irradiating transmitted light as well as excitation light, the phase difference image can be displayed with minimal adverse effects on the cells caused by irradiating transmitted light. You can shoot.

(7) The objective lens 103 is configured to perform fluorescence observation by switching a plurality of fluorescence filters, and the camera 104 captures a fluorescence image for each fluorescence filter. Thereby, even in the case of multiple staining with a plurality of fluorescent reagents, a fluorescent image for each fluorescent color can be taken.

(8) Tiling the phase difference image and the fluorescence image for each small area in the navigation range 3a photographed by executing navigation to generate one macro image photographing the navigation range, and this tiling image Is displayed on the monitor 110. As a result, the user can select cells to be observed from the entire navigation range.

(9) The user superimposes and displays the macro phase difference image and each of the plurality of macro fluorescence images in the image display area 6d on the observation target selection screen displayed on the monitor 110 to display the cells. Enabled selection. Thereby, the user can select a cell to be observed while confirming the fluorescence state of the fluorescent reagent together with the shape of the cell.

-Modification-
In addition, the cell observation apparatus of embodiment mentioned above can also be deform | transformed as follows.
(1) In the above-described embodiment, at least one of a plurality of fluorescent images is superimposed and displayed on the phase difference image in the image display area 6d on the observation target selection screen displayed on the monitor 110. Allowed to select cells. However, the present invention is not limited to this, and two or more of the plurality of fluorescent images may be superimposed. Further, the macro image displayed in the image display area 6d may be enlarged. As a result, the user can further enlarge the macro image and easily select cells to be observed.

(2) In the above-described embodiment, the macro phase difference image and the macro fluorescence image are generated by synthesizing the phase difference image and the fluorescence image for each small area obtained by executing the navigation process on the navigation range. An example was described. However, only one of the phase difference image and the fluorescence image may be taken. In this case, the user only has to select the cells to be observed while confirming only one of the macro phase difference image and the macro fluorescence image on the observation object selection screen.

(3) In the above-described embodiment, the example in which the LIVE screen 2a, the navigation screen 2b, and the movement button 2d are arranged on the navigation position designation screen as shown in FIGS. 2 and 3 has been described. It is not limited to. That is, the LIVE screen 2a, the navigation screen 2b, and the movement button 2d may be arranged in other layouts.

(4) In the above-described embodiment, the example has been described in which the user selects cells to be observed on the observation target selection screen shown in FIG. 6, but the layout of the observation target selection screen is also shown in FIG. 6. Not limited to.

(5) In the above-described embodiment, the example in which the control device 105 executes the process shown in FIG. 8 and the PC 109 executes the process shown in FIG. 9 has been described. However, either the control device 105 or the PC 109 may perform all of the processes in FIGS.

(6) In the above-described embodiment, an example in which time-lapse processing is performed on cells to be observed as the main observation has been described. However, the present invention is a preparation for performing observations and tests by other observation methods. The present invention is also applicable when selecting target cells.

(7) In the above-described embodiment, the case where a cell is an observation target has been described. However, the present invention can be observed by irradiating light, and adverse effects can be caused by irradiating light. It is also applicable to other observation objects such as minerals.

(8) In the above-described embodiment, the example in which the phase difference image captured by the camera 104 is displayed on the LIVE screen 2a on the navigation position designation screen has been described. However, the present invention is not limited to this, and an image captured by a contrast generation method other than fluorescence, such as a differential interference contrast method, may be displayed.

  Note that the present invention is not limited to the configurations in the above-described embodiments as long as the characteristic functions of the present invention are not impaired.

It is a figure which shows the structure of a cell observation apparatus typically. It is a 2nd figure which shows the specific example of the screen for navigation position designation | designated. It is a figure which shows the example of designation | designated of the navigation position 2e, and the example of a setting of the navigation range 3a. It is the figure which showed typically the preparation procedure of a macro phase difference image. It is the figure which showed typically the preparation procedure of a macro fluorescence image. It is a 2nd figure which shows the specific example of the screen for observation object selection. It is a figure which shows the specific example at the time of superimposing a macro phase contrast image and a macro fluorescence image. It is a flowchart figure which shows the process of the control apparatus 105. It is a flowchart figure which shows the process of PC109.

Explanation of symbols

100 cell observation device, 101 stage, 102 chamber, 103 objective lens, 104 camera, 105 control device, 106 transmitted light illumination device, 107 shutter, 108 excitation light source device, 109 personal computer (PC), 110 monitor, 111 environmental control Equipment, 111a CO ₂ mixer, 111b humidity controller, 111c temperature controller

Claims (4)

An objective lens;
An excitation light source for irradiating excitation light for exciting a fluorescent reagent added to an observation specimen for fluorescence observation;
A transmission illumination device that performs transmission illumination on the observation specimen in order to perform preparatory observation to determine a position to perform fluorescence observation before performing the fluorescence observation;
An imaging device for obtaining an image of an observation specimen;
A position specifying means for irradiating the observation specimen with the transmission illumination by the transmission illumination device, performing the preliminary observation, and specifying based on an input of a navigation position for performing fluorescence observation on the acquired specimen image ;
A navigation range related to a fluorescence observation target area for performing fluorescence observation is set in a predetermined range around the navigation position centered on the navigation position specified by the position specifying means, and after the setting, the navigation range is set in the navigation range. An observation apparatus comprising: control means for performing control to irradiate excitation light emitted from an excitation light source . The observation apparatus according to claim 1,
A display device that simultaneously displays both the specimen image obtained by the preliminary observation and the navigation image of the specimen image;
The display device displays the navigation range in the navigation image displayed on the display device. The observation apparatus according to claim 1 or 2,
An observation apparatus, comprising: an image combining unit that captures an image of the navigation range for each small region and generates a combined image obtained by joining a plurality of images of the captured small regions . On the computer,
  A position specifying procedure for irradiating the observation specimen with the transmitted illumination by the transmission illumination device and performing a preparatory observation, and specifying a navigation position for performing fluorescence observation on the acquired specimen image based on an input from the user
  A procedure for setting a navigation range related to a fluorescence observation target region for performing fluorescence observation in a predetermined range adjacent to the navigation position centered on the navigation position specified in the position specifying procedure;
  An observation program for executing a procedure of irradiating the navigation range with excitation light emitted from an excitation light source after the navigation range is set.

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