JPH116961A - Confocal microscope - Google Patents

Abstract

PROBLEM TO BE SOLVED: To realize a confocal microscope capable of automatically detecting the upper and the lower end faces of a sample. SOLUTION: In this confocal microscope, a confocal image is obtained by rotating a disk having plural apertures and condensing light passing through the apertures so that a sample may be scanned with the light. The microscope is equipped with a light source 1, an objective lens 4 condensing the input light on the sample and emitting the returning light from the sample, a confocal scanner 2 making the output light from the light source 1 and passing through the apperture incident on the lens 4 and outputting the returning light from the lens 4 so that the light may pass through the aperture again, a stage on which the sample 5 is placed, a driving means 9 moving the position of the stage 6, a photographing means 7 photographing the output light from the scanner 2, and a control means 8a controlling the driving means 9 to move the stage 6 and fetching the confocal image from the photographing means 7. The upper and the lower end faces of the sample 5 are automatically detected by detecting the fluctuation part of the differential signal of a gray level signal from a gray level detecting area set at a part of the light receiving surface of the photographing means 7.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a confocal microscope, and more particularly to a confocal microscope capable of automatically detecting upper and lower end surfaces of a sample.

[0002]

2. Description of the Related Art A conventional confocal microscope obtains a confocal image by rotating a disk having a plurality of openings, condensing light passing through the openings, and scanning a sample.

FIG. 5 is a block diagram showing an example of such a conventional confocal microscope. In FIG. 5, 1 is a light source such as a laser light source, 2 is a confocal scanner, 3 is an optical microscope, 4 is an objective lens, 5 is a sample, 6 is a stage, and 7 is C
Shooting means such as a CD (Charge Coupled Device) camera,
Reference numeral 8 denotes control means, and 9 denotes drive means for moving the stage 6 up and down.

[0004] Output light from a light source 1 is incident on a confocal scanner 2 via an optical fiber or the like, and output light from the confocal scanner 2 is applied to a sample 5 via an objective lens 4 of an optical microscope.

[0005] Return light such as reflected light and fluorescence from the sample 5 is again incident on the photographing means 7 via the confocal scanner 2 via the objective lens 4, and the output signal of the photographing means 7 is controlled by the control means 8
Connected to.

The control signal from the control means 8 is
The driving means 9 drives the stage 6 on which the sample 5 is set up and down.

Here, a conventional example shown in FIG. 5 will be described. A disk having a plurality of openings is rotating inside the confocal scanner 2, and the output light from the light source 1 passes through the plurality of openings and is condensed on the sample 5 by the objective lens 4.

The reflected light and fluorescent light from the sample 5 are incident on the confocal scanner 2 via the objective lens 4 as return light, and pass through the same one of the plurality of apertures previously passed to the photographing means 7. Incident.

That is, since the light passing through the same aperture is photographed, the image obtained by the photographing means 7 is an image having a resolution in the optical axis direction, and the whole specimen 5 is scanned by rotating the aperture. Therefore, a confocal image which is a slice image of the sample 5 can be obtained.

Then, the control means 8 controls the driving means 9 to move the stage 6 on which the sample 5 is set in the optical axis direction (up and down direction) shown by "a" in FIG. Then, the plurality of confocal images are subjected to image processing to obtain a three-dimensional image of the sample 5.

FIG. 6 is an explanatory diagram for explaining the scanning in the optical axis direction described above. 6, reference numeral 5 is the same as that in FIG.

The control means 8 controls the driving means 9 to move the scanning plane of the confocal image to the upper end face of the sample 5 shown by "a" in FIG.
A confocal image of the upper end surface of the sample 5 indicated by “a” in FIG. 6 is obtained.

Next, the control means 8 controls the drive means 9 to move the scanning plane of the confocal image to the plane indicated by "b" in FIG.
A confocal image of the surface shown in the middle "b" is obtained.

Similarly, the control means 8 controls the driving means 9 so that the scanning surface of the confocal image has the scanning surface of the sample 5 indicated by "c" in FIG.
The scanning surface is sequentially moved until it moves to the lower end surface of, and the output signal from the photographing means 7 is sequentially taken in to obtain a confocal image of each surface.

When the acquisition of all the confocal images is completed, the control means 8 processes the obtained plural confocal images to obtain a three-dimensional image of the sample 5.

As a result, the confocal scanner 2 is attached to the optical microscope 3 and the stage 6 on which the sample 5 is placed is moved in the direction of the optical axis, and a plurality of confocal images obtained are image-processed. Can be obtained.

[0017]

However, in the confocal microscope shown in FIG. 5, the upper and lower end surfaces of the sample 5 indicated by "a" and "c" in FIG. 6 are determined by the operator by visually moving the stage 6. Was.

On the other hand, the confocal microscope has a shallow depth of focus in the direction of the optical axis. In other words, since the image of the portion other than the focal plane cannot be seen, when the focal plane is displaced from the position of the specimen 5, There was a problem that it was difficult to find the sample 5 itself before determining the upper and lower end surfaces.

Further, there is another problem that it is difficult to find the sample itself even in the case of a dark sample which cannot be visually recognized without image processing. Therefore, an object of the present invention is to realize a confocal microscope capable of automatically detecting upper and lower end surfaces of a sample.

[0020]

According to a first aspect of the present invention, a disk having a plurality of openings is rotated to collect light passing through the openings and scan a sample. In a confocal microscope that obtains a confocal image by performing, a light source, an objective lens that focuses input light on the sample and emits return light from the sample, and outputs light of the light source that has passed through the opening. A confocal scanner for entering the objective lens and passing the return light from the objective lens through the aperture again for output, a stage on which the sample is placed, a driving unit for moving the position of the stage, and the confocal A photographing means for photographing the output light of the scanner; and a control means for controlling the driving means to move the stage and take in a confocal image from the photographing means. It is characterized in that the automatic detection of the upper and lower end surfaces of the sample by detecting the variable portion of the differential signal of the gray level signal from the gray-level-detection area set in.

According to a second aspect of the present invention, there is provided a gray level signal from a gray level detecting area set on a part of a light receiving surface of the photographing means. Detecting a variation portion of the differential signal of the above automatically detects the side edge of the sample.

In order to achieve the above object, a third aspect of the present invention is characterized in that, in the first aspect of the present invention, return light from the sample is reflected light.

According to a fourth aspect of the present invention, there is provided the first aspect of the present invention, wherein the return light from the sample is fluorescence.

[0024]

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described below in detail with reference to the drawings. FIG. 1 is a configuration block diagram showing one embodiment of a confocal microscope according to the present invention.

In FIG. 1, reference numerals 1 to 7 and 9 denote the same reference numerals as in FIG. 5, and reference numeral 8a denotes control means. The connection relation is the same as in FIG.

The operation of the embodiment shown in FIG. 1 will now be described with reference to FIG.
This will be described with reference to FIGS. FIG. 2 is a plan view showing details of the light receiving surface of the photographing unit 7, FIG. 3 is a characteristic curve diagram showing a detection signal and its differential signal, and FIG. 4 is a flowchart for explaining the operation of the control unit 8a.

"A" in FIG. 2 is the entire light receiving surface of the photographing means 7, and the control means 8a is provided on an arbitrary part of the light receiving surface in FIG.
A region for gray level detection as shown in "b" is set.

When automatically detecting the upper end face of the sample 5, the control means 8a determines the average value, the maximum value, the median value, etc. of a plurality of pixels in the gray level detection area by using the gray level signal "I (z)". ".

For example, FIG. 3A is a characteristic curve diagram showing an example of the gray level signal "I (z)".
The positions of “a” and “b” are the upper end face of sample 5 and “Zs”.
And the lower end surface “Ze”. In FIG. 3, "C" and "D" are mechanical upper movement limits of the stage 6 "Zu".
And the lower movement limit “Zb”.

As can be seen from FIG. 3 (A), the gray level signal "I (z)" is the same as that shown in FIG.
The background signal level is as shown in the middle "e", and the signal level rises sharply when it reaches the upper end surface "Zs" of the sample 5 shown in the "a" in FIG. When the lower end surface of the sample 5 shown is "Ze", the signal level sharply drops.

The differential signal "ΔI / Δz" of the gray level signal "I (z)" is as shown in FIG.
The upper end surface "Zs" of the sample 5 shown in "a" and "b" in FIG.
And the signal level fluctuates rapidly at the lower end surface “Ze”.

Therefore, the control means 8a determines the positions of the upper and lower end surfaces of the sample 5 by detecting the fluctuation of the signal level shown in FIG.

First, as shown in FIG. 4 (a), the control means 8a sets a threshold "T" for discriminating the fluctuation at the upper and lower end surfaces of the sample, and as shown in FIG. 4 (b). Drive means 9
To move the position of the stage 6 to the upper movement limit “Zu”.

As shown in FIG. 4C, the control means 8a receives the gray level signal "I (z)", and as shown in FIG. Is moved by “Δz” by the slice interval.

Then, as shown in FIG. 4E, the control means 8a determines whether or not the position after the movement has reached the lower limit "Zb", and if the position after the movement is already "Zb".
If it has reached b ", necessary error processing is performed as shown in FIG.

If the position after the movement has not reached "Zb", the control means 8a fetches the gray level signal "I (z)" as shown in FIG. The difference “ΔI” from the signal is calculated.

Next, as shown in FIG.
“a” calculates “ΔI / Δz” and determines whether or not it is equal to or larger than the previously set threshold value “T”.

If ".DELTA.I / .DELTA.z <T", the control means 8a returns to the processing of FIG. 4C and ".DELTA.I / .DELTA.z.gtoreq.T".
If so, the control means 8a determines the current position as the upper end face "Zs" as shown in FIG. 4 (i) and stores the current position.

Similarly, as shown in FIG. 4 (j), the control means 8a takes in the gray level signal "I (z)",
As shown in FIG. 4K, the control means 8a controls the driving means 9 to move the position of the stage 6 by "Δz" by the slice interval.

Then, as shown in FIG. 4 (l), the control means 8a determines whether the position after the movement has reached the lower limit "Zb", and if the position after the movement is already "Zb". ", The necessary error processing is performed as shown in FIG.

If the position after the movement has not reached "Zb", the control means 8a takes in the gray level signal "I (z)" as shown in FIG. The difference “ΔI” from the signal is calculated.

Next, as shown in FIG.
“a” calculates “−ΔI / Δz” to determine whether or not it is equal to or larger than the previously set threshold “T”.

If "-.DELTA.I / .DELTA.z <T", the control means 8a returns to the processing of FIG.
If it is T ", the control means 8a determines the current position as the lower end surface" Ze "as shown in FIG. 4 (o) and stores the current position.

As a result, by detecting a fluctuation portion of the differential signal of the gray level signal from the gray level detection area set in a part of the light receiving surface, the upper end surface “Z” of the sample 5 is detected.
s "and the lower end surface" Ze "can be automatically detected.

Further, even in the case of a dark sample which cannot be visually recognized without image processing, the upper and lower end surfaces of the sample can be automatically detected.

Note that the gray level detecting area in FIG. 2 is set on an arbitrary part of the light receiving surface of the photographing means 7, but it is only a setting in the processing of the control means 8 a, and special setting is performed on the photographing means 7. It is not done.

In the flow chart shown in FIG.
From the upper movement limit “Zu” shown in FIG.
Although it is moved toward the lower movement limit "Zb" shown in the middle "d", it is of course possible to move it in the opposite direction.

Although the stage 6 is moved only in the direction of the optical axis in the embodiment shown in FIG. 1, the center position and the like of the sample are determined by moving the stage 6 on a plane perpendicular to the optical axis. Is also possible.

That is, by moving the stage 6 in the direction of the optical axis and moving the stage 6 on a plane perpendicular to the optical axis at that time, and detecting a change in the differential signal of the gray level signal at that time, the upper and lower end surfaces of the sample are obtained. In addition, since the end of the side surface of the sample can be detected, a positional relationship on a plane perpendicular to the optical axis of the sample can be obtained.

The light source 1 is exemplified by a laser light source, but may be a white light source, an LED, a lamp light source having a bright line spectrum or a continuous spectrum, or the like.

Although the CCD means is exemplified as the photographing means 7, a SIT (Silicom Intensified Target) camera, a PCD (Plasma Charge-couped Device) camera or the like may be used.

[0052]

As is apparent from the above description,
According to the present invention, the following effects can be obtained. Confocal that can automatically detect the top and bottom surfaces of the sample by detecting the variation of the differential signal of the gray level signal from the gray level detection area set on a part of the light receiving surface A microscope can be realized.

[Brief description of the drawings]

FIG. 1 is a configuration block diagram showing an embodiment of a confocal microscope according to the present invention.

FIG. 2 is a plan view showing details of a light receiving surface of a photographing unit.

FIG. 3 is a characteristic curve diagram showing a detection signal and its differential signal.

FIG. 4 is a flowchart illustrating an operation of a control unit.

FIG. 5 is a configuration block diagram showing an example of such a conventional confocal microscope.

FIG. 6 is an explanatory diagram illustrating scanning in the optical axis direction.

[Explanation of symbols]

 DESCRIPTION OF SYMBOLS 1 Light source 2 Confocal scanner 3 Optical microscope 4 Objective lens 5 Sample 6 Stage 7 Imaging means 8, 8a Control means 9 Driving means

Claims (4)

[Claims] 1. A confocal microscope for obtaining a confocal image by rotating a disk having a plurality of apertures, condensing light passing through the apertures, and scanning a specimen, wherein a light source and input light are supplied to the specimen. An objective lens that condenses the light and emits return light from the sample; an output light of the light source that has passed through the opening is incident on the objective lens; and a return light from the objective lens passes through the opening again and is output. A confocal scanner, a stage on which the sample is placed, a driving unit for moving the position of the stage, a photographing unit for photographing output light of the confocal scanner, and controlling the driving unit to control the stage. Control means for moving and taking in the confocal image from the photographing means, the control means comprising: a fine level detector for detecting a gray level signal from a gray level detection area set on a part of the light receiving surface of the photographing means. A confocal microscope characterized in that upper and lower end surfaces of the sample are automatically detected by detecting a change portion of the minute signal. 2. The method according to claim 1, wherein the edge of the sample is automatically detected by detecting a change in a differential signal of a gray level signal from a gray level detection area set on a part of a light receiving surface of the photographing means. The confocal microscope according to claim 1, wherein: 3. The confocal microscope according to claim 1, wherein the return light from the sample is reflected light. 4. The confocal microscope according to claim 1, wherein the return light from the sample is fluorescence.