JP5015381B2 - Photomicroscope - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a photomicrograph apparatus that takes microscopic images at regular time intervals.
[0002]
[Prior art]
Conventionally, as a method of observing a specimen with a microscope, a microscope image is taken at regular time intervals (hereinafter referred to as time lapse photography), and a series of photographed images are reproduced after the photographing is completed, and temporal changes in the form of the specimen are performed. Is observed as a moving image. Such a method is extremely effective as a method for observing a temporal change of a specific specimen, for example, a living cell, existing in a microscope visual field.
[0003]
[Problems to be solved by the invention]
By the way, recently, in research fields such as confirming the effects of multiple reagents on cells cultured under the same conditions, time-lapse photography is performed even for multiple photography outside the microscope's field of view, and the temporal morphology change of the specimen It is also required to observe at the same time.
[0004]
However, the conventional method for enabling time-lapse photography is limited to the microscope field-of-view range, and time-lapse photography cannot be performed for a plurality of photographing ranges extending outside the microscope field-of-view.
[0005]
The present invention has been made in view of the above circumstances, and an object of the present invention is to provide a microscopic photographing apparatus capable of performing time-lapse photographing in a plurality of photographing ranges without being limited to the observation visual field of the microscope.
[0006]
[Means for Solving the Problems]
First aspect of the present invention, a microscope body having an objective lens for condensing the specimen, a stage for mounting the specimen to allow selecting the field of view of the microscope body, with the stage and the objective lens A photographing means for photographing a specimen image within a plurality of microscope visual field ranges selected by moving the lens in a direction perpendicular to the optical axis of the objective lens , and a specimen image photographed by the photographing means. a monitor for displaying, in said plurality of microscopic fields selected range with the stage, and the shooting range selecting means for selecting a position of the plurality of imaging ranges by on the specimen image displayed on the monitor, the and position information of the selected plurality of the imaging range, the setting information of the microscope main body corresponding to the plurality of shooting ranges, shooting starting time of time-lapse shooting a plurality of imaging ranges And storage means for storing an imaging condition including a photographing interval, the position information corresponding to the plurality of imaging range stored in the storage means, based on the setting information and the imaging conditions, the plurality of capturing range Is captured at predetermined time intervals to obtain time-lapse images for each photographing range , and these time-lapse images are displayed on the same screen of the monitor independently for each photographing range. And a photographing control means for storing in the storage means in a state distinguished from each other.
[0007]
According to a second aspect of the present invention, in the first aspect of the invention, when the photographing control means determines the photographing order of a plurality of photographing ranges, the photographing range closest to the photographing range that has not been photographed is selected. It is characterized by the following shooting range.
[0008]
According to a third aspect of the present invention, in the first or second aspect of the present invention, the stage has means for allowing the microscope visual field to move in a three-dimensional direction, and the storage means is a three-dimensional image of the imaging range. The position control information is stored, and the photographing control means can reproduce the focus position of the photographing range based on the three-dimensional position information stored in the storage means.
[0009]
As a result, according to the present invention, it is possible to acquire a temporal morphological change at substantially the same time as a time-lapse image for cells and the like for each of a plurality of imaging ranges without being limited to the visual field range of the microscope.
[0010]
Further, according to the present invention, when photographing a photographing range arbitrarily selected by the observer, a difference in photographing time between photographing ranges at the same time can be reduced.
[0011]
Furthermore, according to the present invention, these shooting ranges can be shot in the same focus state as when each shooting range is selected.
[0012]
DETAILED DESCRIPTION OF THE INVENTION
Hereinafter, embodiments of the present invention will be described with reference to the drawings.
[0013]
(First embodiment)
FIG. 1 shows a schematic configuration of a micrograph apparatus to which the present invention is applied. In the figure, reference numeral 1 denotes a microscope main body, and an electric stage 2 and an objective lens 3 as a field-of-view range selection means are arranged opposite to each other. A specimen 4 is placed on the electric stage 2, and an observation image of the specimen 4 can be observed with the eyepiece 5 through the objective lens 3, and can be imaged by an imaging unit 6 having a CCD imaging device. I have to. A control unit 7 is connected to the imaging unit 6.
[0014]
The control unit 7 includes a photographing control unit 71 and a photographing information management unit 72, and a monitor 8, an input device 9, a stage driving unit 10, and a revolver driving unit 11 are connected to the control unit 7.
[0015]
In this case, the control unit 7 specifically includes a CPU, a RAM connected to the CPU bus, an input / output device, and an external storage device such as a hard disk. The CPU is connected to the imaging control unit 71 and the external storage device. A program for operating as the photographing information management unit 72 is stored. The program used here operates using, for example, Microsoft Windows as basic software, and all commands are given through a pointing device such as a mouse and an input device 9 such as a keyboard. In addition, the imaging control unit 71 causes the imaging unit 6, the stage driving unit 10, and the revolver driving unit 11 to perform operations necessary for imaging the sample 4, and the sample image captured by the imaging unit 6 is displayed on the monitor 8. It is made to display. The stage driving unit 10 is for driving the electric stage 2 in accordance with an instruction from the imaging control unit 71. The revolver driving unit 11 rotates the revolver 31 in accordance with an instruction from the imaging control unit 71 to position the objective lens 3 having a desired magnification on the optical path. In addition, the shooting information database 12 is connected to the shooting information management unit 72. The shooting information database 12 stores shooting information 50 for each shooting range (# 0, #K, and #N indicate shooting range numbers) as shown in FIG. In this case, each piece of shooting information 50 includes a position coordinate 51 of a shooting range 25 described later represented by (X0, Y0) (X1, Y1), a shooting condition 52, and a time-lapse shot image storage location 53. Further, it is known that a time-lapse captured image generally has an enormous capacity. For this reason, in the present invention, it is stored as a file on a hard disk. Each storage location 53 contains a file name used for storage. Further, the time-lapse captured images of the respective capturing ranges # 0 to #N are sequentially arranged for each capturing time as in the first captured image and the next captured image. The format at this time may be a Windows AVI file format or another format.
[0016]
The electric stage 2 has a mechanical origin. The imaging control unit 71 can recognize the current position coordinates of the electric stage 2 by internally managing the movement amount instructed to the stage driving unit 10 with the mechanical origin as a base point. Yes. That is, the imaging control unit 71 has a function as a position detection unit that detects the position of the optical axis of the objective lens 3 with respect to the electric stage 2, and outputs the current position coordinates of the electric stage 2 as the current position of the observation field of view. . (As another configuration, a position detecting unit for detecting the current position of the electric stage 2 may be provided separately, and the position detection unit may be configured to directly recognize the position coordinates of the electric stage 2.)
Next, the operation of the embodiment configured as described above will be described.
[0017]
First, prepare for time-lapse photography.
[0018]
First, the imaging control unit 71 of the control unit 7 displays on the screen of the monitor 8 an image display window 13 for displaying the microscope image shown in FIG. 3 and a control window 14 for setting the imaging range and the imaging time. In this case, the image display window 13 displays a plurality of cells 15 on the microscope image. This microscope image is obtained by capturing an observation image of the specimen 4 on the electric stage 2 with the imaging unit 6 via the objective lens 3. In addition, the control window 14 displays a drop-down list 16 for selecting the magnification of the objective lens currently used, and a drop-down list 17 for selecting the magnification of the objective lens at the time of shooting. An edit box 18 for designating whether to start, an edit box 19 for designating how many minutes of time-lapse photography from the start of photography, and an edit box 20 for designating an interval time from photography to the next photography are displayed. Further, a shooting range creation button 21 for creating a shooting range corresponding to the magnification of the objective lens at the time of shooting, a shooting range deletion button 22 for deleting the shooting range, a shooting start button 23 and a stage movement for controlling the movement of the electric stage 2. Button 24 is displayed.
[0019]
In this state, the shooting range is determined. First, the stage moving button 24 is operated to move the entire stage 2 and select a microscope field of view. The stage movement button 24 here has arrows in a total of eight directions of up / down, left / right, upper right, lower right, upper left, and lower left, and the stage 2 can be moved in accordance with the directions of these arrows. When the direction arrow is pushed, the stage 2 is moved in the X direction, and when the right or left arrow is pushed, the stage 2 is moved in the Y direction. The white circle button at the center is for moving the stage 2 to the home position.
[0020]
Next, the shooting range creation button 21 is pressed. Then, a rectangular shooting range 25 corresponding to the magnification of the objective lens 3 at the time of shooting is displayed at the center position of the microscope image in the image display window 13.
[0021]
When the mouse cursor is moved to the shooting range 25 and a mouse button (not shown) is pressed, small square portions 25a are displayed at the four corners of the shooting range 25, and the mouse cursor changes to a cross shape 26. In this state, by moving the mouse cursor while keeping the mouse button pressed, the shooting range 25 can be moved to the position where the cell 15 to be shot is included, and then when the mouse button is released, the shooting range 25 The position is fixed.
[0022]
Next, the position of the imaging range 25 created in this way in the stage coordinate system is calculated. The position coordinates in this case are calculated from the current coordinates of the stage 2, the pixel coordinates of the imaging range 25, and the size of one pixel in the stage coordinate system. Note that the upper left (X0, Y0) and lower right (X1, Y1) of the position coordinates of the photographing range 25 at this time are calculated as rectangular information.
[0023]
Here, the current coordinates of the stage 2 correspond to the position of the observation field when the imaging range is designated. The pixel coordinates of the shooting range 25 correspond to the position of the specified shooting range 25 in the observation field. The size of one pixel in the stage coordinate system is a distance on the stage corresponding to one pixel of the display screen. Therefore, if an XY coordinate system with the origin of the optical axis center of the observation visual field is used as the pixel coordinate, the pixel coordinate of the imaging range 25 is converted into a distance on the stage and added to the position of the observation visual field. The position of the imaging range 25 thus made can be replaced with the position of the objective optical axis on the stage.
[0024]
The position coordinates of the shooting range 25 calculated in this way are position information of “shooting range of number # 0”. The calculated position coordinates (X0, Y0) and (X1, Y1) are stored as position information 51 constituting the photographing information 50 of # 0. Further, the objective lens magnification at the time of photographing specified by the drop-down list 17 and other observation condition setting information of the microscope main body are stored as photographing conditions 52 constituting the photographing information 50 of # 0. Further, a file name for storing image data obtained by imaging this imaging range is stored as a storage location constituting the imaging information 50 of # 0.
[0025]
Hereinafter, the shooting information 50 of each shooting range # 0 to #N is stored in the shooting information database 12 by selecting the desired shooting range 25 in order and performing the same operation.
[0026]
Further, when it is desired to select an imaging range in a region outside the range of different microscope visual fields, the stage moving button 24 is operated to move the entire stage 2. Then, as the stage 2 moves, the observation image displayed on the monitor 8 also moves. Therefore, after reselecting the microscope field of view while viewing the monitor 8, the photographing range is selected in the same manner as described above.
[0027]
If it is desired to delete the imaging range 25 once formed, the imaging range 25 to be deleted is selected with the mouse cursor, and when the imaging range deletion button 22 is pressed in this state, the corresponding imaging range 25 is deleted. In this case, the shooting information 50 of the shooting range 25 selected from the shooting information database 12 is deleted, and the shooting information 50 of the subsequent shooting range 25 is stored in a vacant part.
[0028]
Finally, in the edit box 18, data on how many minutes to start shooting is written, and in the edit box 19, data on how many minutes of time-lapse shooting is taken after writing is written, and further in the edit box 20. The data for the interval time from the first shooting to the next shooting is written, and the preparation for time-lapse shooting is completed.
[0029]
Next, actual time-lapse shooting is performed based on such shooting preparation.
[0030]
In this case, when the shooting start button 23 is pressed on the control window 14 of the monitor 8 in the flowchart shown in FIG. 5, a counter J indicating the number of the shot image is initialized (Step 1). The counter J indicates the number (# 0 to #N) of the photographic image in the photographic range shown in FIG. Then, a timer (not shown) is started by the photographing control unit 71 (Step 2). When the time written in the edit box 18 elapses (Step 3), a counter K indicating the number of the photographing information 50 is initialized to K = 0 (Step 4). Next, shooting information 50 corresponding to K = 0 is first read from the shooting information database 12 (Step 5), the stage 2 moves based on the position coordinates 51 of the shooting range 25 at this time, and the shooting range 25 Is positioned in the microscope visual field range (Step 6). Further, the photographing condition 52 corresponding to K = 0 is read, and optical system conditions such as the magnification of the objective lens 3 of the microscope body 1 are set based on the photographing condition 52 at this time (Step 7). From this state, an observation image in the imaging range 25 of K (initially K = 0) is captured by the imaging unit 6 via the objective lens 3, and the first time-lapse captured image J (initially J = 1) As A1) in FIG. 4, it is stored in the photographing information database 12 (Step 8).
[0031]
Next, K is counted up (Step 9), and by returning to Step 5 (Step 10), the shooting information 50 of the next shooting range 25 is read (Step 5), and in the same manner as described above (Step 6, Step 7), An observation image of the next shooting range 25 is picked up by the image pickup unit 6 through the objective lens 3, and the first time-lapse shot image J (initially, J = K) of the shooting range of K (K is counted up, 1). Since it is 1, it is stored in the photographing information database 12 as B1) in FIG. 4 (Step 8).
[0032]
Thereafter, similarly, the shooting information 50 of each shooting range 25 is read in order, so that the first time-lapse shooting image (J = 1) is acquired up to the shooting range 25 of the #N shooting information 50 (Step 10). 12 is stored. Next, by counting up the counter J, the location where the time-lapse image is stored next is designated (Step 12). Such time-lapse shooting is repeated at the time interval written in the edit box 20 (Step 13). As a result, the time-lapse captured images A1, A2, A3,..., The time-lapse captured images B1, B2 of the # 1 shooting range 25 in the # 0 shooting range 25 are stored in the shooting information database 12. , B3,..., #N, and time-lapse captured images C1, C2, C3,. Such an operation is continued until the time-lapse shooting time written in the edit box 19 elapses (Step 11).
[0033]
Then, time-lapse captured images A1, A2, A3,..., # 0-capture range 25 of # 0, and time-lapse captured images B1, B2, B3,. If the time-lapse captured images C1, C2, C3... For each shooting time in the N shooting range 25 are continuously displayed on the monitor 8 via the shooting control unit 71, the # 0 to #N shooting ranges 25 are displayed. Each can be observed as moving images A, B, and C.
[0034]
Therefore, in this way, the plurality of imaging ranges 25 are selected while moving the electric stage 2 and selecting the microscope visual field range, and the imaging information 50 of these imaging ranges 25 is stored in the imaging information database 12, and thereafter Since the photographing of these photographing ranges 25 is repeated at a predetermined time interval and the time-lapse photographing image for each photographing range is obtained, each photographing range 25 is not limited within the scope of the microscope field of view. The temporal morphological change of the cells 15 at almost the same time can be acquired as an image. Also, shooting conditions such as the objective lens magnification can be arbitrarily set for each shooting range.
[0035]
(Second Embodiment)
In the second embodiment, the difference in shooting time in a plurality of shooting ranges is reduced by reducing the amount of movement from the shooting range to the next shooting range. In this case, it is the same as that described in the first embodiment except that the order in which the electric stage 2 is moved to each photographing range is changed. Therefore, the above-described FIGS. 1 to 3 are used. In other words, in the second embodiment, when shooting is performed at each time interval, the closest shooting range is searched from the database 12 when shooting of the first shooting range ends and when moving to the next shooting range. Then, the next shooting range is set, and the process of selecting the closest shooting range from the shooting ranges that have not been shot in the same manner and shooting is repeated.
[0036]
In this way, when shooting the shooting range selected by the observer, the difference in shooting time between the shooting ranges at the same time can be reduced, so highly accurate time-lapse shooting can be performed in each shooting range. It can be carried out.
[0037]
Here, when shooting, the next shooting range is searched. However, the order of the shooting information 50 in the shooting information database 12 may be rearranged in the order of the shooting range.
[0038]
(Third embodiment)
In the third embodiment, the electric stage 2 in the first embodiment is configured to be able to move further in the vertical (Z) direction, and the moving amount can be controlled by the imaging control unit 71. ing. Others are the same as those described in the first embodiment, and therefore, FIG. 1 to FIG. 3 described above are used.
[0039]
In the third embodiment, when the shooting range is selected, the position of the stage 2 in the Z direction is also recorded as the coordinates of the shooting range. That is, in the first embodiment, the imaging range is two-dimensional information in the XY direction, but is recorded as three-dimensional position information by adding information in the Z direction.
[0040]
In this way, since the focus state can be recorded when the shooting range is selected, the stage coordinates including the Z axis can be reproduced when shooting is actually performed for each shooting range. You can shoot in the same focus state as when you selected.
[0041]
Embodiments of the present invention also include the following inventions.
[0042]
(1) a stage on which a specimen is placed;
Visual field selection means for selecting an observation visual field by moving the optical axis of the objective lens relative to the stage in a plane perpendicular to the optical axis;
Position detecting means for detecting a position in the plane of the optical axis of the objective lens with respect to the stage;
Photographing means for photographing an observation image within the range of the observation visual field;
In a photomicrograph apparatus comprising an image recording means for recording an image acquired by the photographing means,
An imaging range designating unit for designating an imaging range for imaging by the imaging unit within the range of the observation visual field;
Based on the position of the specified imaging range within the observation range and the position of the observation field of view where the imaging range is specified, the position of the specified imaging range is within the plane of the objective lens optical axis with respect to the stage. Photographing position calculation means for converting and recording the position in
By driving the visual field selection unit according to the position recorded in the shooting position calculation unit, a plurality of shooting ranges specified by the shooting range specifying unit are shot at predetermined time intervals and the acquired images are recorded in the image recording unit. And a photomicrograph photographing apparatus comprising a photographing control means for recording in the camera.
[0043]
【Effect of the invention】
As described above, according to the present invention, it is possible to provide a microscopic photographing apparatus capable of performing time-lapse photographing in a plurality of photographing ranges without being limited to the observation field of the microscope.
[Brief description of the drawings]
FIG. 1 is a diagram showing a schematic configuration of a first embodiment of the present invention.
FIG. 2 is a diagram showing a schematic configuration of a database of photographing ranges used in the first embodiment.
FIG. 3 is a diagram showing a display example of a monitor used in the first embodiment.
FIG. 4 is a diagram for explaining the operation of the first embodiment;
FIG. 5 is a flowchart for explaining the operation of the first embodiment;
[Explanation of symbols]
DESCRIPTION OF SYMBOLS 1 ... Microscope main body 2 ... Electric stage 3 ... Objective lens 31 ... Revolver 4 ... Sample 5 ... Eyepiece 6 ... Imaging part 7 ... Control part 71 ... Shooting control part 72 ... Shooting information management part 8 ... Monitor 9 ... Input device 10 ... Stage drive unit 11 ... Revolver drive unit 12 ... Shooting information database 50 ... Shooting information 51 ... Position coordinates 52 ... Shooting conditions 53 ... Storage location 13 ... Image display window 14 ... Control window 15 ... Cells 16, 17 ... Drop-down list 18, 19, 20 ... Edit box 21 ... Shooting range creation button 22 ... Shooting range delete button 23 ... Shooting start button 24 ... Stage move button 25 ... Shooting range 25a ... Square part 26 ... Cross shape

Claims (3)

A microscope body having an objective lens for focusing on the specimen ;
A stage for mounting the specimen to allow selecting the field of view of the microscope main body,
An imaging means for imaging a specimen image within a plurality of microscope visual field ranges selected by relatively moving the stage and the objective lens in a direction orthogonal to the optical axis of the objective lens ;
A monitor for displaying a specimen image photographed by the photographing means;
An imaging range selection means for selecting positions of a plurality of imaging ranges on the sample image displayed on the monitor within the plurality of microscope visual field ranges selected using the stage ;
Imaging comprising a position information of a plurality of imaging ranges said selected setting information of the microscope main body corresponding to the plurality of shooting ranges, the imaging start time and the imaging interval when the plurality of imaging ranges to time lapse photography Storage means for storing conditions ;
The position information corresponding to the plurality of imaging range stored in the storage means, on the basis of the setting information and the imaging conditions, for each shooting range by repeating the imaging of said plurality of imaging range at predetermined time intervals Shooting control for acquiring time-lapse photographed images and storing these time-lapse photographed images in the storage means in a state of being distinguished for each photographing range so that they can be displayed independently for each photographing range on the same screen of the monitor Means,
A photomicrograph apparatus comprising: Said imaging control means, wherein when determining the imaging order of the imaging range, the closest shooting range from the shooting range is not completed the shooting to claim 1, characterized in that a next shooting range Microscope photography device. The stage has means for enabling the microscope field of view to move in a three-dimensional direction, the storage means stores three-dimensional position information of the imaging range, and the imaging control means stores in the storage means 3. The microscopic photographing apparatus according to claim 1, wherein the focus position of the photographing range can be reproduced based on the three-dimensional position information.

Images