JP2005234435A - Microobservation apparatus, microobservation method and microobservation program - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a microobservation apparatus capable of displaying an image of an observation sample of which the change accompanying the lapse of time is detected, together with the effect that the change is detected when the image of the sample gained by a microscope is observed, and to provide a microobservation method and a microobservation program which are executed in the microobservation apparatus. <P>SOLUTION: The microobservation apparatus is provided with an image gain means for gaining image data on a predetermined observation region of the sample, an image data storing means for storing image data gained by the image gain means, a change detecting means for detecting the change of the image data accompanying the lapse of time with respect to the image data on the same observation region stored by the image data storing means and a sample display means for displaying the image of the sample corresponding to the image data of which the change is detected by the change detecting means together with change detection information of presenting the effect that the change is detected. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a microscopic observation apparatus for observing an image of an observation sample acquired by a microscope, a microscopic observation method and a microscopic observation program executed in the microscopic observation apparatus, and in particular, the image changes with time. The present invention relates to a microscopic observation apparatus, a microscopic observation method, and a microscopic observation program.

  For example, in various tests in the biochemical field, a microscopic observation operation of observing a biological sample such as a plant cell or a microorganism with a microscope and capturing a microscopic image of the biological sample is frequently performed. In such microscopic observation, there is an increasing demand for a method for observing and comparing various samples at multiple points in time series.

Usually, a biological sample to be observed is stored in a container such as a microplate. When observing and capturing a microscopic image of a biological sample stored in such a microplate, a reference point defined in the container is used. Positioning for imaging the plurality of sample storage units by controlling the movement of the stage based on the position coordinates of the plurality of sample storage units with the origin as the origin and the relative position coordinates of the observation position with the position coordinates as the origin Thus, multipoint samples are observed and imaged efficiently (see, for example, Patent Document 1).
JP 2002-303801 A

  However, in the conventional microscopic observation, even if the observation and imaging can be efficiently performed at multiple points, it is observed whether or not the image at each point has changed and how much has changed. There is a problem that a person has to make a judgment by visually observing each image, and trouble is required every time to determine a change from the image acquired by the observer.

  The present invention has been made in view of the above-described drawbacks of the prior art, and when observing an image of an observation sample acquired by a microscope, the change was detected in the image of the observation sample in which a change with the passage of time was detected. It is an object of the present invention to provide a microscopic observation apparatus that can be displayed together with the effect, a microscopic observation method and a microscopic observation program that are executed in the microscopic observation apparatus.

The present invention employs the following configuration in order to solve the above problems.
That is, according to one aspect of the present invention, the microscopic observation apparatus of the present invention is a microscopic observation apparatus that observes an image of an observation sample acquired by a microscope, and acquires image data of a predetermined observation region of the observation sample. Image data storage means for storing the image data acquired by the image acquisition means, and image data in the same observation area stored in the image data storage means, the change of the image data over time And an observation sample display means for displaying an image of the observation sample corresponding to the image data whose change has been detected by the change detection means together with change detection information indicating that the change has been detected. It is characterized by providing.

Thereby, the observer who observes the image of the observation sample acquired by the microscope can easily know the observation area where the image data changes and the observation area where there is no change.
Further, in the microscopic observation apparatus of the present invention, the change in the image data is a change in shading information of the image data, and the change detection information has a plurality of indexes corresponding to the amount of change in the shading information. Is desirable.

Thereby, the observer who observes the image of the observation sample acquired by the microscope can easily know the observation area where the density information of the image data is changed and the observation area where there is no change.
The microscopic observation apparatus of the present invention further includes a graph creating unit that creates a graph indicating the amount of change detected by the change detecting unit, and the observation sample display unit displays the graph created by the graph creating unit. Further display is desirable.

Thereby, the observer who observes the image of the observation sample acquired with the microscope can easily know the change in the image data with the passage of time in the observation region where there is a change.
In addition, the microscopic observation apparatus of the present invention performs control so as to stop storing image data by the image data storage unit thereafter when the amount of change detected by the change detection unit is within a predetermined range within a predetermined time. It is desirable to further include control means for

This eliminates the need to store image data that does not need to be stored, and improves memory efficiency without using extra memory.
The microscopic observation apparatus of the present invention controls to stop the image acquisition by the image acquisition unit thereafter when the change amount detected by the change detection unit is within a predetermined range within a predetermined time. It is desirable to further comprise means.

Thereby, acquisition and storage of image data that do not need to be observed can be omitted, the observation work efficiency is improved, and the memory efficiency is improved without using an extra memory.
In the microscopic observation apparatus according to the present invention, the image acquisition unit acquires image data of a plurality of observation regions, and the observation sample display unit displays the images of the plurality of observation regions together with the respective change detection information. It is desirable to display.

Thereby, the observer who observes the image of the observation sample acquired by the microscope can easily know simultaneously the observation area where the image data has changed and the observation area where there is no change.
Further, according to one aspect of the present invention, the microscopic observation method of the present invention is a microscopic observation method executed by a computer that observes an image of an observation sample acquired by a microscope, wherein a predetermined observation region of the observation sample is observed. Acquire image data, store the acquired image data in an image data database, detect changes in the image data over time for image data in the same observation area stored in the image data database, and An image of the observation sample corresponding to the image data in which the change is detected is displayed together with change detection information indicating that the change has been detected.

Thereby, the observer who observes the image of the observation sample acquired by the microscope can easily know the observation area where the image data changes and the observation area where there is no change.
According to another aspect of the present invention, the microscopic observation program of the present invention is a procedure for acquiring image data of a predetermined observation region of the observation sample with respect to a computer that observes an image of the observation sample acquired by a microscope. A procedure for storing the acquired image data in an image data database; a procedure for detecting a change in image data over time for image data in the same observation area stored in the image data database; and the change This is a microscope observation program for executing a procedure for displaying an image of an observation sample corresponding to image data in which the change is detected together with change detection information indicating that the change has been detected.

  Thereby, the observer who observes the image of the observation sample acquired by the microscope can easily know the observation area where the image data changes and the observation area where there is no change.

According to the present invention, an observer who observes an image of an observation sample acquired by a microscope can easily know an observation area where image data changes and an observation area where there is no change.
In addition, according to the present invention, an observer who observes an image of an observation sample acquired by a microscope can easily know an observation area where there is a change in density information of image data and an observation area where there is no change.

In addition, according to the present invention, an observer who observes an image of an observation sample acquired by a microscope can easily know a change in image data with the passage of time in an observation region having a change.
In addition, according to the present invention, it is possible to omit the storage of image data that does not need to be stored, and the memory efficiency is improved without using an extra memory.

In addition, according to the present invention, it is possible to omit the acquisition and storage of image data that does not need to be observed, thereby improving the observation work efficiency and improving the memory efficiency without using an extra memory.
In addition, according to the present invention, an observer who observes an image of an observation sample acquired by a microscope can easily simultaneously know an observation area where there is a change in image data and an observation area where there is no change.

  That is, according to the present invention, the observer is only required to set each observation point at the start of observation, and is freed from performing complicated work for searching for a change point during or after observation. The

Embodiments of the present invention will be described below with reference to the drawings.
FIG. 1 is a diagram showing an outline of a microscopic observation system to which an embodiment of the present invention is applied.
As shown in FIG. 1, the microscope observation system 100 according to the present embodiment includes a microscope observation apparatus 110 and a laser scanning microscope 120 connected via a connection interface 7. The microscopic observation apparatus 110 is a general-purpose personal computer, for example, and includes a monitor 1, a CPU 2, a storage medium 3, a keyboard 4, a mouse 5, and the like.

  The control unit 10 of the laser scanning microscope 120 outputs a scanning control signal to the scanning unit 11 when a scanning instruction command is input from the microscope observation apparatus 110. The control unit 10 is connected to a laser light source (Ar laser) 25, a laser light source (HeNe-G laser) 26, and a laser light source (HeNe-R laser) 27, and selects a laser to be used for scanning.

  The scanning unit 11 emits from a laser light source (Ar laser) 25, a laser light source (HeNe-G laser) 26, or a laser light source (HeNe-R laser) 27 selected by the control unit 10, and a mirror 24, a dichroic for synthesis. Based on the scanning control signal from the control unit 10, the X scanning galvanometer mirror and the Y scanning galvanometer mirror in the scanning unit 11 are driven by the laser light collected by the mirror 22 or 23 and input through the mirror 21. Then, XY scanning is performed as spot light on the observation specimen placed on the electric stage 9 through the objective lens 8.

  In this way, the fluorescence or reflected light generated from the specimen by the spot light that scans the observation specimen returns back to the incident optical path, and the fluorescent component is reflected by the spectral filter 12 to the detection optical path. 13 and 14 are used to select a long wavelength and a short wavelength, and each wavelength is further selected by the barrier filters 15 and 16 and converted into an electric signal by the two photodetectors 17 and 18 to be controlled. Is input.

Then, the control unit 10 transfers the image information (image data) of the observation specimen obtained from the photodetectors 17 and 18 obtained above to the microscopic observation device 110, and the microscopic observation device 110 transfers them to the storage medium 3. At the same time, the image is displayed on the monitor 1.
Note that the storage medium 3 of the microscopic observation device 110 has a memory area for storing conditions necessary for scanning and a memory area for storing image information.

Further, in the microscopic observation apparatus 110, a microscopic observation processing program for controlling a laser scanning microscope 120 described later, storing image data, processing, and the like is executed.
Next, an example of acquiring time-lapse images in a plurality of observation regions (observation points) on a specimen using the laser scanning microscope configured as described above will be described.

FIG. 2 is a diagram illustrating an example in which observation points on the microplate are displayed.
The microplate has a plurality of multipoint observation samples, and the laser scanning microscope can set a plurality of observation points in each multipoint observation sample.
First, the observer determines a plurality of observation points on the specimen while operating the electric stage 9, and obtains image data such as a focus position at each observation point, a range on the Z axis to be scanned, and the number of slices. The conditions are set, and the sensitivity of the photodetectors 17 and 18 and the intensity of each laser 25, 26, and 27 are adjusted. These set values are stored in the storage medium 3 together with the position information for each observation point.

  Thereafter, when the observer gives an instruction to start scanning, the microscopic observation apparatus 110 first reads the setting conditions for the first observation point stored in the storage medium 3, and sends the moving position of the electric stage 9 to the control unit 10. The sensitivity, laser intensity, and three-dimensional data acquisition conditions of each detector are set. When the setting is completed, a scanning start instruction is sent to the control unit 10 and the image data acquired as a result is held in the storage medium 3.

  Next, the microscope observation apparatus 110 reads the setting of the second observation point stored in the storage medium, and acquires the movement position of the electric stage 9, the sensitivity of each detector, the laser intensity, and image data to the control unit 10. When the conditions are set and the setting is completed, a scanning start instruction is sent to the control unit 10 and the image data obtained as a result is held in the storage medium 3.

As described above, image acquisition and storage in the storage medium 3 are performed at all designated observation points.
When the image acquisition at all the observation points and the storage to the storage medium 3 are completed (the first end), the microscopic observation device 110 then waits for the elapse of a preset interval time, By performing image acquisition at the observation point and holding in the storage medium 3, time-lapse observation is performed for each observation point (end of the second time).

Then, the image data of the first and second observations at each observation point obtained as described above are compared, and the observed observation point is displayed on the monitor 1.
In this way, in subsequent observations, the data acquired two times before and after the observation are compared, and the observation points determined to have changed from the comparison result are displayed on the monitor 1.

FIG. 3 is a diagram illustrating a display example of observation points with changes.
As described above, when displaying a changed observation point on the monitor 1, in order to determine whether or not the observation point has changed, for example, a change in luminance of the observation target is used.
Next, a specific change determination method using the data acquired in the first and second experiments will be described.

At each observation point, a plurality of pieces of image data acquired as observation results are held in the storage medium 3.
Therefore, in the method for determining the change in luminance, when the image acquisition at each observation point is completed, the average value of luminance values at each observation point is calculated based on the data. Next, the difference from the first average brightness value of the experiment is taken. The solution obtained as a result is compared with the threshold value set by the observer before the experiment, and when the threshold value is exceeded, it is determined that there is a change in luminance, and an index on the observation point on the monitor 1 is as shown in FIG. indicate.

  In the example shown in FIG. 3, as an index, an observation point having no change is represented by a white square, an observation point having a small amount of change is represented by a double square, and an observation point having a medium amount of change is represented by a double. The rectangle is painted black on the inside, and the observation points with a large amount of change are represented by black rectangles. However, the indicators are not limited to these, and the display colors may be different, or only two types of observation points that do not change and observation points that change.

After obtaining the luminance change of the observation point as described above, the presence / absence of these changes is then displayed on the monitor 1 as shown in FIG. Can know.
Next, a second embodiment to which the present invention is applied will be described.

The microscopic observation system according to the second embodiment is the same as that of the first embodiment described with reference to FIG.
The method described in the first embodiment is effective only when the presence or absence of a change in the observation target is known. However, it is insufficient to know the temporal change of the observation point.

Therefore, as a second embodiment, a method for displaying the change amount of the observation point with time as a graph is shown.
FIG. 4 is a diagram illustrating a graph display example of observation points with changes.
The operation performed by the observer before the observation and the observation are started, the setting conditions are read from the storage medium 3 for each observation point, and the data acquired at the first and second observations are compared to determine the change point. A series of operations such as acquisition is the same as in the first embodiment.

  In the second embodiment, in addition to the processing in the first embodiment, the fact that there is a change displayed on the monitor 1 is displayed during the image acquisition at each observation point or after the image acquisition ends. When the mouse 5 is held over the observation point, a luminance change graph is displayed as shown in FIG.

Here, detailed information of the graph will be described. In the luminance change graph, the vertical axis represents the luminance value and the horizontal axis represents the time axis, and the average luminance value at each observation point obtained by each observation is displayed.
As described above, the observer can know the temporal change of the observation point.

Next, a third embodiment to which the present invention is applied will be described.
The microscopic observation system according to the third embodiment is the same as the first embodiment described with reference to FIG.
According to the first and second embodiments described above, it is possible to know the presence / absence of change and temporal change for each observation point.

Therefore, as a third embodiment, when the observation point does not change with the passage of time, the observation of the observation point is stopped, and thereafter no image storage or image acquisition is performed.
FIG. 5 is a diagram illustrating a display example of observation points at which observation is stopped.

The operation performed by the observer before the observation and the observation are started, the setting conditions are read from the storage medium 3 for each observation point, and the data acquired at the first and second observations are compared to determine the change point. A series of operations such as acquisition is the same as in the first embodiment.
In the third embodiment, in addition to the processing in the first embodiment, the observation point is monitored during image acquisition at each observation point, and the observation set by the observer before observation is performed. If the observation point remains unchanged for the number of times, the observation at the observation point is stopped and the image is not acquired or stored.

Then, a specific mark is displayed on the stopped observation point as shown in FIG. In the example shown in FIG. 5, an index with a cross mark (×) added to a quadrangle is used as the observed observation point.
As described above, it is possible to omit the process of acquiring an image that does not change, or it is not necessary to manage unnecessary data generated by the acquisition, thereby preventing an increase in unnecessary data and saving resources of the storage medium 3. It can be used effectively.

The first to third embodiments of the present invention have been described above. Next, the flow of microscopic observation processing that realizes these embodiments will be described with reference to flowcharts.
FIG. 6 is a flowchart showing the flow of the microscopic observation process.
In order to simplify the explanation, explanation will be focused on observation at one observation point.

First, in step S1, image data of an observation sample acquired by scanning with a microscope is acquired, and in step S2, the acquired image data is stored in a memory.
In step S3, the image data acquired in step S1 and stored in the memory in step S2 is compared with the image data acquired and stored in the previous scan, and the image data at the same observation point stored in the memory is compared. The change of the image data with the passage of time is detected.

As a result of the comparison in step S3, in step S4, it is determined whether or not there is a change in image data due to previous and subsequent scanning. However, in the case of image data acquired and stored by the first scan, there is no object to be compared, and therefore it is determined that there is no change by comparing it with its own data.
If it is determined in step S4 that there is a change (step S4: Yes), an indicator that there is a change is displayed in step S5 (see FIG. 3).

Next, in step S6, it is determined whether or not there is a graph display instruction such as holding the mouse cursor over an observation point where there is a change indication.
If it is determined in step S6 that there is a graph display instruction (step S6: Yes), in step S7, the image data of all the scans acquired in step S1 are read from the memory.

In step S8, a graph is created based on the read image data. In step S9, the created graph is displayed.
On the other hand, if it is determined in step S4 that there is no change (step S4: No), an indicator that there is no change (open square in FIG. 3) is displayed in step S10.

Then, in step S11, it is determined whether or not a state in which no change has continued even after a predetermined number of scans is performed. If no change has continued (step S11: Yes), the next scan is not performed. The process ends.
If it is determined in step S6 that there is no instruction to display a graph (step S6: No), after the graph display is ended in step S9, the no-change state is still within the predetermined number of scans in step S11 ( In step S11: No), in step S12, it is determined whether or not the next scanning is performed.

If it is determined in step S12 that the next scanning is to be performed (step S12: Yes), the process returns to step S1, and if it is determined that the next operation is not to be performed (step S12: No), the process ends. To do.
As described above, the flow of the microscopic observation process has been described with reference to FIG. 6, but the flow of the microscopic observation process is not limited to the flow of the flowchart of FIG. For example, when it is determined in step S11 that the state without change has continued (step S11: Yes), instead of ending the process, the process returns to step S1 to perform scanning, but the storage in step S2 is not performed. It is good.

  Although the embodiments of the present invention have been described above, the microscope observation apparatus, the microscope observation method, and the microscope observation program to which the present invention is applied are limited to the above-described embodiments as long as their functions are executed. However, various configurations, shapes, or means can be taken without departing from the scope of the present invention.

It is a figure which shows the outline of the microscope observation system to which embodiment of this invention is applied. It is a figure which shows the example which displays the observation point on a microplate. It is a figure which shows the example of a display of the observation point with a change. It is a figure which shows the example of a graph display of the observation point with a change. It is a figure which shows the example of a display of the observation point from which observation was stopped. It is a flowchart which shows the flow of a microscopic observation process.

Explanation of symbols

1 Monitor 2 CPU
DESCRIPTION OF SYMBOLS 3 Storage medium 4 Keyboard 5 Mouse 7 Connection interface 8 Objective lens 9 Electric stage 10 Control unit 11 Scan unit 12, 13, 14 Spectral filter 15, 16 Barrier filter 17, 18 Photo detector 21 Mirror 22, 23 Synthesis dichroic mirror 24 Mirror 25 Laser light source (Ar laser)
26 Laser light source (HeNe-G laser)
27 Laser light source (HeNe-R laser)
DESCRIPTION OF SYMBOLS 100 Microscopic observation system 110 Microscopic observation apparatus 120 Laser scanning microscope

Claims (8)

In a microscopic observation apparatus for observing an image of an observation sample acquired by a microscope,
Image acquisition means for acquiring image data of a predetermined observation region of the observation sample;
Image data storage means for storing the image data acquired by the image acquisition means;
For the image data in the same observation area stored in the image data storage means, a change detection means for detecting a change in the image data over time,
An observation sample display means for displaying an image of the observation sample corresponding to the image data whose change has been detected by the change detection means, together with change detection information indicating that the change has been detected;
A microscopic observation apparatus comprising:   The change in the image data is a change in shading information of the image data, and the change detection information has a plurality of indices corresponding to the amount of change in the shading information. Microscopic observation device. Further comprising a graph creating means for creating a graph indicating the amount of change detected by the change detecting means;
The microscopic observation apparatus according to claim 1, wherein the observation sample display unit further displays a graph created by the graph creation unit.   When the amount of change detected by the change detection unit within a predetermined time is within a predetermined range, the control unit further includes a control unit that controls to stop storing image data by the image data storage unit thereafter. The microscope observation apparatus according to any one of claims 1 to 3.   The apparatus further comprises a control unit that controls to stop the image acquisition by the image acquisition unit thereafter when the amount of change detected by the change detection unit within a predetermined time is within a predetermined range. Item 4. The microscope observation device according to any one of Items 1 to 3. The image acquisition means acquires image data of a plurality of observation areas,
The microscopic observation apparatus according to any one of claims 1 to 5, wherein the observation sample display means displays an image of the plurality of observation regions together with each change detection information. A microscopic observation method executed by a computer for observing an image of an observation sample acquired by a microscope,
Obtaining image data of a predetermined observation region of the observation sample;
Storing the acquired image data in an image data database;
For image data in the same observation region stored in the image data database, detecting changes in image data over time,
A microscopic observation method comprising displaying an image of an observation sample corresponding to image data in which the change is detected, together with change detection information indicating that the change has been detected. For a computer that observes an image of an observation sample acquired by a microscope,
A procedure for acquiring image data of a predetermined observation region of the observation sample;
Storing the acquired image data in an image data database;
A procedure for detecting changes in image data over time for image data in the same observation region stored in the image data database;
A procedure for displaying an image of the observation sample corresponding to the image data in which the change is detected, together with change detection information indicating that the change has been detected;
Microscopic observation program to execute.

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