JPH1195119A - Scanning microscope - Google Patents

Abstract

PROBLEM TO BE SOLVED: To quickly obtain an observation image without discoloring a fluorescent sample by operating the spot light only to an observation object on the sample and fetching the observation image obtained after sensitivity is adjusted in a short time. SOLUTION: The image of the sample 9 placed on a mount 8 is picked up by a TV camera 19. Then, this observation image is fetched to a controller 15 and the distribution of luminance in the observation image is obtained based on the average of luminance values on respective lines and a previously set threshold. Based on the distribution of the luminance, the line on the sample 9 where the spot light is operated by a two-dimensional scanning unit 5 is decided. By scanning an object on the sample 9 with the required minimum spot light, the observation image can be fetched. Even when the sensitivity of an optical detector is repeatedly adjusted in order to fetch a clear observation image in a desired area on the sample 9, the observation image obtained after the sensitivity is adjusted can be fetched in a short time. Thus, the desired observation image can be quickly obtained without discoloring the fluorescent sample and operability is also enhanced.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a scanning microscope for observing a sample by scanning a spot light on a sample placed on a stage.

[0002]

2. Description of the Related Art As is well known, a scanning microscope irradiates a laser beam, which is a spot light, onto an observation sample through an objective lens, and returns reflected light from the sample again through the objective lens and an optical system. A point-shaped image is formed, and this is detected by a photodetector to obtain density information of the image.In this case, simply irradiating the sample with a laser beam can obtain only dot-shaped density information. Actually, by scanning the laser beam on the sample surface and displaying the density distribution information on the sample surface with this scanning on a monitor etc.
An image of the sample surface is obtained.

[0003] In this case, the observer confirms the image of the observation sample area scanned by the laser beam on the monitor, and adjusts the sensitivity of the photodetector so that a clear image can always be observed. They also perform operations.

[0004]

By the way, in such a scanning microscope, a galvanometer is used in order to continuously scan a laser beam on the surface of a sample. However, this galvanometer has a high speed due to its characteristics. Scanning cannot be performed, and it takes several seconds to scan a laser beam on the sample surface and capture an image for one screen.

[0005] Therefore, in actual observation of a sample, it is necessary to repeatedly adjust the sensitivity of the photodetector in order to capture a clearer observation image of a region of interest on the sample. It takes a lot of trouble and time until it is done. That is, in such a case, adjust the sensitivity of the photodetector by looking at the image captured with the predetermined sensitivity of the photodetector, and then wait a few seconds until the next image is obtained. It is necessary to confirm the change and adjust the sensitivity of the photodetector again. This requires repetition of the operation, which causes not only poor operability but also long time.

Therefore, there is conventionally a method of performing thinning scanning. According to this method, the number of scanning lines is reduced by half, so that the time required to capture one screen can be reduced by half, but the amount of image information on the sample surface is also reduced by half. As a result, a clear image cannot be obtained, and also in this case, the same scanning is performed for the region including the region of interest on the sample and the other region. Not resolved. The present invention has been made in view of the above circumstances, and an object of the present invention is to provide a scanning microscope capable of quickly obtaining a desired observation image of a sample.

[0007]

According to the first aspect of the present invention,
A scanning microscope for observing a sample by scanning a spot light on a sample mounted on a stage has an imager for imaging the sample on the stage, and captures an observation image imaged by the imager. Image capturing means for capturing, luminance distribution detecting means for obtaining a luminance distribution in the observation image from luminance information of at least one line of the observation image captured by the image capturing means, and a detection result of the luminance distribution detecting means Scanning line determining means for determining a line for scanning the spot light on the sample on the basis of the above.

According to a second aspect of the present invention, there is provided a scanning microscope for observing a sample by scanning a spot light on a sample mounted on a stage, wherein the scanning microscope has an imaging means for imaging the sample on the stage. Image capturing means for capturing an observation image captured by the image capturing means, luminance distribution detecting means for obtaining a luminance distribution in the observation image by binarizing the observation image captured by the image capturing means, And a scanning area determining means for determining an area for scanning the spot light on the sample based on the detection result of the luminance distribution detecting means.

According to a third aspect of the present invention, there is provided a scanning microscope for observing a sample by scanning a spot light on a sample mounted on a stage, wherein the scanning microscope has an imaging means for imaging the sample on the stage. Image capturing means for capturing an observation image captured by the image capturing means, luminance distribution detecting means for obtaining a distribution of luminance in the observation image captured by the image capturing means, and detection results of the luminance distribution detecting means. Scanning area determining means for determining an area for scanning the spot light on the sample based on the scanning area; and scanning area determining means for determining whether the spot light scanning area determined by the scanning area determining means is within a predetermined scanning area. The spot light scanning area determined by the scanning area determining means based on the determination result of the scanning area determining means is located within the predetermined scanning area. It is constituted by a stage driving means for moving the stage of mounting the sample.

As a result, according to the first aspect of the present invention,
An observation image obtained by imaging the sample placed on the stage is taken in, a distribution of luminance in the observation image is obtained from luminance information of at least one line of the observation image, and the sample is determined by the scanning line determination unit based on the luminance distribution. Since the line for scanning the upper spot light is determined, the observation image can be captured by the minimum necessary spot light scanning for the observation target.

According to the second aspect of the present invention, an observation image obtained by capturing an image of a sample placed on the stage is taken in, and the distribution of luminance in the observation image is obtained by binarizing the observation image. Since the scanning area determining means determines the area to be scanned with the spot light on the sample based on the distribution, it is possible to capture the observation image of the observation target by scanning the minimum necessary spot light.

According to the third aspect of the present invention, an observation image obtained by capturing an image of a sample placed on a stage is taken in, and a luminance distribution in the observation image is obtained by binarizing the observation image. Based on the distribution, the scanning area determining means determines an area for scanning the spot light on the sample, and if the determined spot light scanning area is not within the predetermined scanning area, the stage is moved to change the spot light scanning area. Since spot light scanning on the sample can be performed so as to be positioned in a predetermined scanning area, an observation image can be captured by a minimum necessary spot light scanning even for an observation target outside the predetermined area.

[0013]

Embodiments of the present invention will be described below with reference to the drawings. (First Embodiment) FIG. 1 shows a schematic structure of a first embodiment of the present invention. Here, an example in which the present invention is applied to a scanning laser microscope system is shown.

In FIG. 1, reference numeral 1 denotes a scanning laser microscope main body, which comprises a laser light source 2, a mirror 3, a dichroic mirror 4, a two-dimensional scanning unit 5, a revolver 6, an objective lens 7, a stage 8, and a sample 9 , Lens 1
0, pinhole plate 11, confocal light detector 12, light source 13
And an optical path switching unit 14.

Here, the laser light source 2 is for generating laser light as spot light for scanning the surface of the sample 9, and the laser light from the laser light source 2 is reflected by the mirror 3 to form a dichroic mirror 4. Through to the two-dimensional scanning unit 5. The two-dimensional scanning unit 5 is for two-dimensionally scanning the laser light from the laser light source 2 given by the mirror 3 on the sample 9. For example, a galvanometer mirror for scanning in the X-axis direction and a scanning mirror for scanning in the Y-axis direction are provided. It has a galvanomirror, and the optical path of the spot light with respect to the objective lens 7 is oscillated in the XY directions by oscillating these galvanomirrors in the X-axis direction and the Y-axis direction. The revolver 6 holds a plurality of objective lenses 7 having different magnifications.
The objective lens 7 having a desired magnification among the plurality of objective lenses 7 is selectively inserted into the observation optical path of the microscope. Then, the sample light on the stage 8 is irradiated with the spot light two-dimensionally scanned by the two-dimensional scanning unit 5 through the objective lens 7 which is selectively inserted.

Image information such as reflected light and fluorescence from the sample 9 is returned to the two-dimensional scanning unit 5 through the objective lens 7, and is returned to the dichroic mirror 4 via the two-dimensional scanning unit 5. This dichroic mirror 4 is a laser light source 2 for a two-dimensional scanning unit 5.
A semi-transparent mirror provided on the outgoing optical path for guiding image information from the sample 9 given via the two-dimensional scanning unit 5 to the light detection system.

Image information obtained from the two-dimensional scanning unit 5 obtained through the dichroic mirror 4 is condensed by a lens 10 and provided to a pinhole plate 11. The pinhole plate 11 has a pinhole of a predetermined diameter, and is located at an image forming position on the front surface of the light receiving surface of the confocal light detector 12.
It is arranged so that the pinhole is located. The confocal photodetector 12 is composed of a photodetector that converts light obtained through the pinhole of the pinhole plate 11 into an electric signal corresponding to the light amount.

On the other hand, the light source 13 is turned on when used as a normal optical microscope, and illuminates the sample 9 on the stage 8 through the objective lens 7. For example, the optical path switching unit 14 having a half mirror below the mirror is provided between the two-dimensional scanning unit 5 and the revolver 6, and is inserted into the optical path as an optical microscope with the light source 13 turned on. The optical path for laser scanning is switched to the optical path for capturing an image by the TV camera 19 of the imaging means described later.

A controller 15 is connected to the confocal light detector 12 of the main body 1 of the scanning laser microscope. The controller 15 has an operation panel 16, a memory 17, an image monitor 18, a TV camera 19, and a stage driving device 20. Are connected.

The operation panel 16 includes a pointing device such as a trackball, a joystick or a mouse in addition to the keyboard, and outputs a scanning instruction or the like to the controller 15 according to an instruction from a user.

When a scanning instruction is input from the operation panel 16, the controller 15 outputs a switching command to the optical path switching unit 14 to switch to an optical path for image capture by the TV camera 19, and also sends a sample 9 signal to the TV camera 19. Is instructed to capture the observation image of the sample 9, and the TV image of the sample 9 captured from the TV camera 19 is transferred to the memory 17.
The scanning of the two-dimensional scanning unit 5 is determined based on the luminance information of the TV image. In this case, as a method of obtaining the distribution of luminance in the image from the luminance information of the TV image of the TV camera 19, means for obtaining the average of the luminance values on each line is employed. As a criterion for determining, a threshold value of luminance is set in advance, and the presence or absence of an observation target is determined based on the average of the threshold value and the luminance value, and scanning is determined based on the determination result. Like that.

The controller 15 outputs a command to switch to the optical path for laser scanning to the optical path switching unit 14, and outputs a scanning start position signal based on the determination of scanning by the two-dimensional scanning unit 5. Image information of the sample 9 of the photodetector 12 obtained by the scanning of the scanning unit 5 is transferred to the memory 17. Further, a movement command of the stage 8 is output to the stage driving device 20 as needed.

The TV camera 19 captures an image for one screen of the sample 9 on the stage 8 in accordance with an instruction from the controller 15, and outputs an image capture completion signal to the controller 15 when the capture of the image is completed. . The memory 17 includes a TV camera 19 and the photodetector 12.
The image information on the sample 9 transferred from the controller 9 via the controller 15 is stored. The image monitor 18
The image information of the sample 9 stored in the memory 17 is displayed. The stage driving device 20 moves the stage 8 in the X and Y directions according to an instruction from the controller 15.

Next, the operation of the embodiment configured as described above will be described. First, the sample 9 was observed by ordinary microscope observation.
The upper observation area is determined, and then a scan start command is output from the operation panel 16 to the controller 15.

Thus, the flowchart shown in FIG. 2 is executed. In this case, when a scan start command is output from the operation panel 16 to the controller 15, step 201
Then, a switching command is output from the controller 15 to the optical path switching unit 14, and the optical path is switched to the optical path for image capture by the TV camera 19.

Next, in step 202, when the controller 15 instructs the TV camera 19 to capture the observation image of the sample 9, the observation image of the sample 9 on the stage 8 is captured by the TV camera 19, and the TV image is taken. Captured as In this case, the light source 13 is turned on, and the sample 9 is illuminated.

Then, in step 203, the TV camera 1
When the image capture of one screen of the sample 9 by the sample 9 is completed,
By the image capture completion signal to the controller 15, T
The V image is transferred to the memory 17 and stored.

Thereafter, when a switching command is output from the controller 15 to the optical path switching unit 14 in step 204, the optical path is switched to a laser scanning optical path. Next, in step 205, the scanning of the two-dimensional scanning unit 5 is determined by the controller 15 based on the luminance information of the TV image stored in the memory 17. In this case, first
Attention is paid to the first line of the TV image, and step 206
Then, the average value of the luminance on this one line is obtained. Then, in step 207, the average value of the obtained luminance values is compared with a preset threshold value.
If it is determined that the calculated average value is larger than the threshold value, the controller 15 outputs a scanning start position signal to the two-dimensional scanning unit 5 by the controller 15 in step 208.
Laser light from the laser light source 2 is irradiated onto the sample 9 for one line through the objective lens 7 by scanning of the two-dimensional scanning unit 5, and image information based on fluorescence from the sample 9 obtained by the laser light irradiation is obtained. It is taken into the photodetector 12 via the pinhole plate 11. Then, in step 209, the image information captured by the photodetector 12 is transferred to and stored in the memory 17. That is, when the average value of the luminance on one line is larger than the threshold value, it is determined that the object is an observation target, and scanning for this one line is determined.

On the other hand, if it is determined in step 207 that the average value of the luminance on one line is equal to or smaller than the threshold value, the actual scanning by the two-dimensional scanning unit 5 is not performed, and step 2
At 10, data for one line is set to 0, and at step 209, data 0 is stored in the memory 17. Then, in step 211, the image information of the sample 9 stored in the memory 17 is displayed on the image monitor 18.

Next, at step 212, it is determined whether the processing up to the last line of the TV image has been completed. If the processing up to the last line has not been completed, at step 213, the TV image stored in the memory 17 is stored. Is moved to the next line, and the process returns to step 206, where the processes from step 206 to step 213 are repeated. afterwards,
After the processing of the image data for one screen is completed, step 21
If it is determined in step 2 that the processing is for the last line, step 21
As a result of Step 4, the process returns to Step 205 and the processes from Step 205 to Step 214 are repeated until the observer instructs the end of scanning from the operation panel 16.

Therefore, according to this configuration, the sample 9 placed on the stage 8 is imaged by the TV camera 19, and the captured observation image is taken into the controller 15, and the brightness of each line of the observation image is measured. A distribution of luminance in the observation image is obtained from the average value and a preset threshold value, and a line for scanning the spot light on the sample 9 by the two-dimensional scanning unit 5 is determined based on the luminance distribution. Therefore, the observation image can be captured by the minimum necessary spot light scanning for the observation target on the sample 9. That is, in this case, only the portion of the observation target 32 on the sample 9 with respect to the TV image 31 captured by the TV camera 19 as shown in FIG.
Scanning of the spot light is performed only on the observation target 32, and an observation image can be obtained. In this way, even if the sensitivity of a photodetector or the like is repeatedly adjusted in order to capture a clear observation image of the region of interest on the sample, the observation image after the sensitivity adjustment can be captured in a short time.
The desired observation image can be obtained quickly without causing fading of the fluorescent sample, and the operability at this time can be improved. (Second Embodiment) Next, a second embodiment of the present invention will be described. In this case, the scanning laser microscope system to which the second embodiment is applied is the same as that of FIG. 1 described in the first embodiment, and the description is omitted here with reference to FIG. I do.

The controller 15 of this embodiment has a
As a method of calculating the luminance distribution of the TV image obtained from the V camera 19, it is assumed that a means for performing binarization processing is adopted and a threshold value for binarization is set in advance.

Next, the operation of the embodiment configured as described above will be described. First, the sample 9 was observed by ordinary microscope observation.
The upper observation area is determined, and then a scan start command is output from the operation panel 16 to the controller 15.

Thus, the flowchart shown in FIG. 4 is executed. In this case, when a scan start command is output from the operation panel 16 to the controller 15, step 401
Then, a switching command is output from the controller 15 to the optical path switching unit 14, and the optical path is switched to the optical path for image capture by the TV camera 19.

Next, in step 402, when the controller 15 instructs the TV camera 19 to take in the observation image of the sample 9, the observation image of the sample 9 on the stage 8 is picked up by the TV camera 19, and the TV image is taken. Captured as In this case, the light source 13 is turned on, and the sample 9 is illuminated.

Then, at step 403, the TV camera 1
When the image capture of one screen of the sample 9 by the sample 9 is completed,
By the image capture completion signal to the controller 15, T
The V image is transferred to the memory 17 and stored.

Thereafter, when a switching command is output from the controller 15 to the optical path switching unit 14 at step 404, the optical path is switched to the laser scanning optical path. Up to this point, the operation is the same as in the first embodiment.

Next, in step 405, a binarization process is performed on the TV image stored in the memory 17 based on a preset threshold value. Then, step 406
Attention is paid to a portion remaining as an image in the data obtained by the binarization process, a rectangle circumscribing this image portion is obtained, and this rectangular range is determined as a region to be scanned. The information of the scanning area is stored in the memory 17 in step 407.

Next, at step 408, the controller 1
5 for an area where scanning is not performed outside the scanning area,
Write 0 data for one screen to the memory 17. Then
At step 409, attention is paid to the first scanning area information stored in the memory 17, and at step 410, the scanning area corresponding to this scanning area information is scanned by the two-dimensional scanning unit 5 with laser light. In this case, when the controller 15 outputs a scanning start position signal to the two-dimensional scanning unit 5, the laser light from the laser light source 2 is scanned by the two-dimensional scanning unit 5 via the objective lens 7 on the sample 9. The area is irradiated, and image information based on fluorescence from the sample 9 obtained by the irradiation of the laser light is taken into the photodetector 12 via the pinhole plate 11.

At step 411, the image information captured by the photodetector 12 is transferred to and stored in the memory 17. Then, in step 412, the memory 17 is stored in the image monitor 18.
Is displayed.

Next, in step 413, it is determined whether or not the processing has been completed for the scanning area corresponding to all pieces of scanning area information.
Then, the processing target is moved to the scanning area corresponding to the next scanning area information stored in the memory 17, and the process returns to step 410, and the processing from step 410 to step 414 is repeated. Thereafter, when the processing corresponding to all the scanning area information for one screen is completed, the process returns to step 409 until the observer instructs the end of the scanning from the operation panel 16 in step 415, and then returns to step 409. 4
The processing up to 15 is repeated.

Accordingly, even in this case, the sample 9 placed on the stage 8 is imaged by the TV camera 19, and the captured observation image is taken into the controller 15, and the observation image is binarized. The distribution of luminance in the observed image is obtained, and the two-dimensional scanning unit 5 is determined based on the luminance distribution.
Since the region for scanning the spot light on the sample 9 is determined, the observation image can be captured by the minimum necessary spot light scanning on the observation target on the sample 9. That is, in this case, as shown in FIG.
The spot light scanning area 37 is provided for each of the observation objects 35 and 36 on the sample 9 with respect to the TV image 34 captured by the camera 9.
And an area 37 including these observation objects 35 and 36
Is scanned with only the spot light, and an observation image can be obtained. Thus, the same effect as in the first embodiment can be expected. (Third Embodiment) Next, a third embodiment of the present invention will be described. In this case, the scanning laser microscope system to which the third embodiment is applied is the same as that of FIG. 1 described in the first embodiment, and thus the description is omitted here with reference to FIG. I do.

In the second embodiment, when the scanning area is determined, the scanning area information of the determination result is stored in the memory 17, but in the third embodiment, the scanning area determination result is stored. An overlay display is provided on the TV image to enable correction and addition of a scanning area.

In this case, when a scanning start command is output to the controller 15, the flowchart shown in FIG. 6 is executed. First, when a scanning start command is output from the operation panel 16 to the controller 15, a switching command is output from the controller 15 to the optical path switching unit 14 in step 601, and the optical path is switched to the image capturing optical path by the TV camera 19.

Next, in step 602, when the controller 15 instructs the TV camera 19 to take in the observation image of the sample 9, the observation image of the sample 9 on the stage 8 is picked up by the TV camera 19, and the TV image is taken. Captured as In this case, the light source 13 is turned on, and the sample 9 is illuminated.

Then, in step 603, the TV camera 1
When the image capture of one screen of the sample 9 by the sample 9 is completed,
By the image capture completion signal to the controller 15, T
The V image is transferred to the memory 17 and stored.

Thereafter, at step 604, when a switching command is output from the controller 15 to the optical path switching unit 14, the optical path is switched to the laser scanning optical path. Next, in step 605, binarization processing is performed on the TV image stored in the memory 17 based on a preset threshold. Then, in step 606, focusing on a portion remaining as an image of the data obtained by the binarization processing, a rectangle circumscribing this image portion is obtained, and this rectangular range is determined as a region to be scanned.

Next, at step 607, the result of the scan area determination is displayed as an overlay on the TV image on the image monitor 18, and at step 608, correction and addition of the scan area are enabled. In this case, the observer operates the image monitor 1
While viewing the screen 8, a change such as correction and addition of a scan area is performed using the operation panel 16.

Then, the information of the changed scanning area is stored in the memory 17 in step 609. Next, in step 610, the memory 1 is used for an area where the controller 15 does not perform scanning outside the scanning area.
7, 0 data for one screen is written.

Next, in step 611, attention is paid to the first scanning area information stored in the memory 17, and in step 61
2, the scanning area corresponding to this scanning area information is 2
The scanning of the laser beam by the dimensional scanning unit 5 is performed.
In this case, when the controller 15 outputs a scanning start position signal to the two-dimensional scanning unit 5, the laser light from the laser light source 2 is scanned by the two-dimensional scanning unit 5 via the objective lens 7 on the sample 9. The area is irradiated, and image information based on fluorescence from the sample 9 obtained by the irradiation of the laser light is taken into the photodetector 12 via the pinhole plate 11.

At step 613, the image information captured by the photodetector 12 is transferred to and stored in the memory 17. Then, in step 614, the memory 17 is stored in the image monitor 18.
Is displayed.

Next, at step 615, it is determined whether or not the processing has been completed for the scanning area corresponding to all pieces of scanning area information.
Then, the processing target is moved to the scanning area corresponding to the next scanning area information stored in the memory 17, the process returns to step 612, and the processes from step 612 to step 616 are repeated. Thereafter, when the processing corresponding to all the scanning area information for one screen is completed, the process returns to step 611 until the observer instructs the end of the scanning from the operation panel 16 in step 617, and returns to step 611 from step 611. 6
The processing up to 17 is repeated.

Therefore, according to this configuration, the same effect as that of the second embodiment can be expected, and it is possible to easily respond to changes such as correction and addition of the scanning area in the observation image. (Fourth Embodiment) Next, a fourth embodiment of the present invention will be described. In this case, the scanning laser microscope system to which the fourth embodiment is applied is the same as that of FIG. 1 described in the first embodiment, and thus the description is omitted here with reference to FIG. I do.

In general, the scanning area of the LSM (laser scanning microscope) cannot cover the entire field of view of the microscope due to the characteristics of the galvanometer. Therefore, when there is an object to be observed outside the scanning area of the LSM, the sample is left as it is. No observations can be made. In the fourth embodiment, the LSM
Focusing on the fact that the TV image can observe a wider area than the scanning area, the observation of an observation object outside the scanning area of the LSM is also enabled.

In this case, when a scanning start command is output to the controller 15, the flowchart shown in FIG. 7 is executed. First, when a scanning start command is output from the operation panel 16 to the controller 15, a switching command is output from the controller 15 to the optical path switching unit 14 in step 701, and the optical path is switched to the image capturing optical path by the TV camera 19.

Next, in step 702, when the controller 15 instructs the TV camera 19 to take in the observation image of the sample 9, the observation image of the sample 9 on the stage 8 is picked up by the TV camera 19, and the TV image is taken. Captured as In this case, the light source 13 is turned on, and the sample 9 is illuminated.

Then, in step 703, the TV camera 1
When the image capture of one screen of the sample 9 by the sample 9 is completed,
By the image capture completion signal to the controller 15, T
The V image is transferred to the memory 17 and stored.

Thereafter, when a switching command is output from the controller 15 to the optical path switching unit 14 at step 704, the optical path is switched to the laser scanning optical path. Next, in step 705, a binarization process is performed on the TV image stored in the memory 17 based on a preset threshold value. Then, in step 706, focusing on a portion remaining as an image in the data obtained by the binarization processing, a rectangle circumscribing this portion is obtained, and this rectangular range is determined as a region to be scanned.

Next, in step 707, the result of the scan area determination is displayed as an overlay on the TV image on the image monitor 18, and in step 708, correction and addition of the scan area are enabled. In this case, the observer operates the image monitor 1
While viewing the screen 8, a change such as correction and addition of a scan area is performed using the operation panel 16.

The information of the changed scanning area is stored in the memory 17 in step 709. Next, in step 710, the memory 1 is used for an area where the controller 15 does not perform scanning outside the scanning area.
7, 0 data for one screen is written.

Next, in step 711, the first scanning area information stored in the memory 17 is noted, and
At 2, it is determined whether the scan area of the observation target is outside the scan area of the LSM. In this case, for example, as shown in FIG.
Is located outside the scanning area 40 and inside the TV image 41, it is determined in step 712 that the scanning area is outside the scanning area of the LSM.

Then, in step 713, the stage driving device 20 is driven by the controller 15, and the stage 8 is moved until the scanning area to be observed is inside the scanning area of the LSM. Further, in step 714, a scanning region in consideration of the amount of movement of the stage 8 is obtained, and in step 715, the two-dimensional scanning unit 5 scans this region with laser light. In this case, the controller 1
When the scanning start position signal is output to the two-dimensional scanning unit 5 by the scanning unit 5, the laser light from the laser light source 2 is irradiated onto the scanning area on the sample 9 via the objective lens 7 by the scanning of the two-dimensional scanning unit 5. Then, image information based on the fluorescence from the sample 9 obtained by the irradiation of the laser light is taken into the photodetector 12 via the pinhole plate 11.

Thereafter, in step 716, the stage 8 is returned to the original position. In step 717, the image information captured by the photodetector 12 is transferred to the memory 17 and stored. On the other hand, if it is determined in step 712 that the scanning area is inside the scanning area of the LSM, step 718
Then, the scanning area is scanned by the two-dimensional scanning unit 5 with laser light, and the scanning is performed by the photodetector 12.
The image information taken into the memory 1 is stored in the memory 1 in step 717.
7 and stored.

Then, in step 719, the image monitor 1
8 displays the image information stored in the memory 17 Next, in step 720, it is determined whether or not the processing has been completed for the scanning areas corresponding to all the scanning area information, and if the processing has not been completed, At step 721, the memory 1
The processing target is moved to the scan area corresponding to the next scan area information stored in 7, and the process returns to step 712, and the processes from step 712 to step 721 are repeated.
Thereafter, when the processing corresponding to all the scanning area information for one screen is completed, next, in step 722, the operation panel 16
Step 711 until the observer instructs the end of scanning.
And the processing from step 711 to step 722 is repeated.

Accordingly, in this case, the sample 9 placed on the stage 8 is imaged by the TV camera 19, and the captured observation image is taken into the controller 15, and the observation image is binarized. The distribution of luminance in the observed image is obtained, and the two-dimensional scanning unit 5 is determined based on the luminance distribution.
The area to scan the spot light on the sample 9 by the
If the spot light scanning area thus determined is not within the LSM scanning area, the stage 8 is moved by the stage driving device 20 so that the spot light scanning area is positioned within the LSM scanning area. Since scanning can be performed, an effect similar to that of the first embodiment can be expected. Further, it is possible to capture an observation image by a minimum necessary spot light scanning for an observation target outside the LSM scanning area. it can.

[0066]

As described above, according to the present invention, spot light scanning is performed only on the observation target on the sample,
Observation images can be obtained, so even if the sensitivity of a photodetector or the like is repeatedly adjusted to capture a clear observation image of the region of interest on the sample, the observation image after the sensitivity adjustment can be captured in a short time. In addition, a desired observation image can be promptly obtained without causing fading of the fluorescent sample, and operability at this time can be improved.

[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 flowchart for explaining the operation of the first embodiment.

FIG. 3 is a diagram for explaining the first embodiment.

FIG. 4 is a flowchart for explaining the operation of the second embodiment of the present invention.

FIG. 5 is a diagram illustrating a second embodiment.

FIG. 6 is a flowchart for explaining the operation of the third embodiment of the present invention.

FIG. 7 is a flowchart for explaining the operation of the fourth embodiment of the present invention.

FIG. 8 is a diagram illustrating a fourth embodiment.

[Explanation of symbols]

 DESCRIPTION OF SYMBOLS 1 ... Scanning laser microscope main body, 2 ... Laser light source, 3 ... Mirror, 4 ... Dichroic mirror, 5 ... 2D scanning unit, 6 ... Revolver, 7 ... Objective lens, 8 ... Stage, 9 ... Sample, 10 ... Lens, 11: Pinhole plate, 12: Confocal photodetector, 13: Light source, 14: Optical path switching unit, 15: Controller, 16: Operation panel, 17: Memory, 18: Image monitor, 19: TV camera, 20: Stage Drive.

Claims (3)

[Claims] 1. A scanning microscope for observing a sample by scanning a spot light on a sample mounted on a stage, comprising: an image pickup unit for picking up an image of the sample on the stage, and an image picked up by the image pickup unit. Image capturing means for capturing the observed image, brightness distribution detecting means for obtaining a luminance distribution in the observed image from luminance information of at least one line of the observed image captured by the image capturing means, A scanning line microscope comprising: a scanning line determining unit that determines a line for scanning the spot light on the sample based on a detection result of the distribution detecting unit. 2. A scanning microscope for observing a sample by scanning a spot light on a sample placed on a stage, comprising: an image pickup means for picking up an image of the sample on the stage, and an image picked up by the image pickup means. An image capturing means for capturing the observed image, a luminance distribution detecting means for obtaining a luminance distribution in the observed image by binarizing the observed image captured by the image capturing means, and a luminance distribution detecting means. A scanning region determining means for determining a region for scanning the spot light on the sample based on the detection result. 3. A scanning microscope for observing a sample by scanning a spot light on a sample placed on a stage, wherein the scanning microscope has an imager for imaging the sample on the stage and is imaged by the imager. Image capturing means for capturing the observed image, luminance distribution detecting means for obtaining a distribution of luminance in the observed image captured by the image capturing means, and a luminance distribution detecting means on the sample based on the detection result of the luminance distribution detecting means. Scanning area determining means for determining an area to be scanned with spot light; scanning area determining means for determining whether the spot light scanning area determined by the scanning area determining means is within a predetermined scanning area; The sample is placed so that the spot light scanning area determined by the scanning area determining means based on the determination result of the means is positioned within the predetermined scanning area. Scanning microscope characterized by comprising a stage driving means for moving the stage.