JPH0667095A - Scanning type laser microscope - Google Patents

Abstract

PURPOSE:To obtain a three dimensional information of a sample before carrying out observation of a sliced image of a sample and to greatly improve operation efficiency when obtaining a sample image. CONSTITUTION:In the scanning type laser microscope where the sample image is formed by reflected light or translucent light from the sample, a piezoelectric element 25 to adjust the focal position of an objective lens 23, a means to detect the sample 24's outline based on the luminance value from its cross sectional image and a means to construct the three dimensional image of the sample from the outline data from a multiple number of cross sectional images derived by outline detecting means are provided.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a scanning laser microscope which scans a sample with laser light and obtains a sample image by using reflected light or transmitted light from the sample.

[0002]

2. Description of the Related Art The light from a light source is narrowed down to a minute spot by an objective lens, a spot is scanned on the sample, the transmitted light and the reflected light from the sample are detected by a photodetector and converted into an electric signal, and the CRT There is known a scanning laser microscope that displays a sample image on a display. This scanning laser microscope has an advantage that an image with little flare and good contrast can be obtained, and special detection such as confocal point and differentiation can be easily performed by devising a detection optical system. Such a scanning laser microscope has been proposed in Japanese Patent Laid-Open No. 61-219919 and will be described below as an example.

FIG. 5 is a diagram showing the structure of such a scanning laser microscope. In this scanning laser microscope, a laser beam 2 from a light source 1 passes through a beam splitter 3 and enters a galvanometer mirror 4 of an optical deflector provided at a position conjugate with the pupil position of the objective lens 10. Here, the laser beam 2 is scanned in the Y direction. Next, the light is incident on the galvanometer mirror 7 provided at a position conjugate with the pupil position of the objective lens 10 by the pupil transmission lenses 5 and 6. Here, the laser beam 2 is deflected and scanned in the X direction.
The two-dimensionally scanned laser beam 2 passes through a pupil projection lens 8 and an imaging lens 9 and enters an objective lens 10. Then, a laser spot limited by diffraction is generated on the sample 11, and the sample 11 is scanned by XY with the laser spot.

If the sample 11 is a transparent object, the laser beam 2 passes through the condenser lens 12 and the detectors 13 and 14
Detected by.

On the other hand, if the sample 11 is a reflecting object, the reflected light reflected by the sample 11 returns to the original optical path and enters the beam splitter 3, and the light reflected here passes through the condenser lens 15. It is detected by the detectors 16 and 17.

In the scanning optical system configured as described above,
Set the galvanometer mirrors 4 and 7 in the X direction and Y, respectively.
By scanning in the X and Y directions, the sample 11 is scanned in the X and Y directions by the beam spots formed thereon. Then, the reflected light or the transmitted light of the scanning beam is detected by the detectors 13 and 14 or the detectors 16 and 17, respectively. The electric signals output from these detectors are converted into B-mode and C-mode image data and a sample image is displayed on the CRT.

[0007]

However, in the above-mentioned scanning laser microscope, in order to obtain the shape of the entire specimen, the slice image is observed at each Z position of the sample 11 according to preset slice conditions. It is possible to grasp the sample shape only after performing all the above and constructing a stereoscopic image from these slice images.

Therefore, since the outer shape of the sample is unknown at the initial stage, the slice range (upper and lower limit values) and the interval between slices are set to approximate values for observation. Therefore, there is a possibility that unnecessary data is included in the obtained slice image, and a process of deleting such data in a later process is required. If the necessary data is not included, the slice conditions must be set again and the observation must be repeated. Therefore, there is a problem that the operation efficiency in obtaining the sample image is poor and it takes time.

The present invention has been made in view of the above circumstances, and three-dimensional information of a sample can be obtained before observing a sample slice image, and the operation efficiency at the time of obtaining a sample image is greatly improved. It is an object of the present invention to provide a scanning laser microscope that can be improved.

[0010]

In order to achieve the above object, in the scanning laser microscope according to the present invention, light from a light source is condensed on a sample by an objective lens to form a beam spot on the sample. Then, the beam spot is two-dimensionally scanned on the sample by an acousto-optic polarization element arranged on the optical path between the light source and the objective lens, and the light reflected or transmitted from the sample by this scanning is converted into an electric signal. Then, in the scanning laser microscope that forms a sample image from this electric signal, a piezoelectric element for adjusting the focal position of the objective lens, and means for detecting the contour of the sample based on the brightness value from the cross-sectional image. And a means for constructing a stereoscopic image of the sample from the contour data of a plurality of cross-sectional images obtained by this means.

[0011]

According to the present invention, the contour of the specimen is detected from the sectional image of the specimen, and the contour of the entire specimen is obtained by changing the focal position of the objective lens by the piezoelectric element. Therefore, the observer can refer to this stereoscopic image
Optimal slice conditions can be set easily, unnecessary data processing and resetting of slice conditions can be eliminated, and the observation efficiency is greatly improved.

[0012]

Embodiments will be described below with reference to the drawings.

FIG. 1 shows a schematic structure of a scanning laser microscope according to an embodiment of the present invention. In this scanning laser microscope, laser light emitted from a laser light source 20 passes through a dichroic mirror 21 and is incident on an acousto-optic polarization element 22. The acousto-optical polarization element 22 deflects incident light in the X direction and the Y direction orthogonal to each other. In this embodiment, the projection direction of the light from the objective lens 23 to the sample 24 is the Z direction, and the plane orthogonal to this is the XY plane. Therefore, the two directions orthogonal to each other on the XY plane are the X direction and the Y direction, and the scanning direction by the acousto-optic polarizing element 22 corresponds to these two directions. The laser light polarized by the acousto-optic polarization element 22 enters the objective lens 23, and the sample 2 placed near the focal point of the objective lens 23.
A beam spot is formed on the laser beam 4 by the laser beam. The objective lens 23 is attached to the microscope main body through a cylindrical piezoelectric element 25 that matches the aperture diameter of the objective lens. When a voltage is applied to the piezoelectric element 25 from a controller described later, the piezoelectric element 25 extends in the optical axis direction of the objective lens to adjust the focus position. The piezoelectric element 25 may not be directly attached to the objective lens 23, but may be indirectly moved by a cam or the like.

The scanning optical system shown in the figure is constructed by taking the case where the sample 24 is a reflecting object as an example. When the sample 24 is a transparent object, as shown in FIG. 5, a condenser lens and a photodetector are provided on the back side of the sample.

The reflected light reflected by the sample 24 is the objective lens 2
3, the light enters the dichroic mirror 21 through the acousto-optic polarization element 22. The light reflected by the dichroic mirror 21 passes through the confocal pinhole 26 and is photoelectrically converted by the photodetector 27. The electric signal generated by photoelectric conversion in the photodetector 27 is input to the controller 28. The controller 28 has a contour extraction mode for extracting the contour of the sample 24 from this electric signal, and a function of generating a sample image from the electric signal obtained under the set slice condition.
A display device 29 for visualizing the stereoscopic image of the sample obtained in the contour extraction mode and the sample image finally obtained is connected to the controller 28.

Next, the operation of this embodiment configured as described above will be described.

A beam spot is formed on the sample 24 by the light emitted from the laser light source 20, and the acousto-optical polarization element 22 is controlled to scan the sample surface with the beam spot in XY scanning. The reflected light is converted into an electric signal by the photodetector 27 and input to the controller 28. The controller 28 uses the sample 24
The image data of the XY scanning range including the cross-sectional image at the predetermined Z position is generated. The controller 28 extracts the contour data of the sample 24 at the Z position based on this image data.

Now, the contour extraction mode by the controller 28 will be described with reference to FIG. First, when a cross section (XY cross section) at a predetermined Z position is obtained, the Y direction is fixed and the brightness value of each pixel is determined for one line (scan line) in the X direction of the cross section to determine the contour portion. Extract. In the cross-sectional image, the brightness value changes abruptly at the boundary between the background portion and the object. Therefore, a point P ₁ where the brightness value changes abruptly in one line scan is detected. If the point P ₁ is detected, the scanning is interrupted and the position information P at that point is detected.
₁ (X ₁ , Y ₁ , Z ₁ ) is stored ((a) in the figure).

Next, the scanning line is moved in the Y direction by a preset Y _STEP . This causes the scan line to shift from Y ₁ to Y ₂ = (Y ₁ + Y _STEP ). Then, the scanning line Y ₂ is scanned again in the X direction. The scanning width at this time is (X ₁ ± Xα). Xα is a scan width that is preset so that the scan width includes the boundary portion. In this scanning, the point P where the brightness value changes abruptly as in the above
₂ (X ₂ , Y ₂ , Z ₁ ) is detected and stored ((b) in the same figure).

[0020] Incidentally, in the scanning width in the scanning on the scan lines _{Y n (X n-1 ±} α), if the luminance change as the contour is not detected, the process returns to the scanning line Y _n-1, obtained previously In addition to the change point (X _n-1 , Y _n-1 , Z ₁ ), a point at which the brightness value suddenly changes is detected. Then, the shift direction of the scanning line is reversed again to the original direction, and the boundary point is detected and stored in the same manner as above while shifting the scanning line. The sample 2 is stored according to all the contour position information stored in this way.
The contour data on the Z ₁ cross section of _No. 4 is obtained. Then, from the obtained contour data, the barycentric position (Xg ₁ , Yg ₁ ,
Z ₁ ) is calculated.

Next, the contour extraction target cross section is moved by a preset Z _{STEP to} move from Z ₁ to Z ₂ , and scanning is started for each line from the scanning line Yg ₁ on the cross section, and the same as above. To obtain contour position information. In this way, similar contour extraction processing is repeated for each cross section while changing the Z position of the cross section.

Then, when the sudden change point of the luminance is not detected in the cross section at the Z position after the movement, it is assumed that the upper limit of the sample in the Z direction is exceeded, and the target cross section is changed by Z _{STEP in the} reverse direction from the first Z _1. Then, the contour extraction processing in each cross section is repeated until the lower limit in the Z direction is exceeded.

From the contour information of the sample 24 thus obtained, the shape of the entire sample is displayed on the display device 29 by wire frame representation or the like as shown in FIG. At this time, information on the Z direction is also displayed together with the display of the entire specimen shape.

The observer judges from the overall shape of the sample 24 displayed on the display device 29, to what extent the slice image should be observed based on the positional information in the Z direction, and sets the slice condition. Set.

The observation under the slice condition set in this way is the same as the conventional one, and therefore the explanation is omitted here.

As described above, according to the present embodiment, the sample cross-sectional image is scanned line by line, the contour portion is determined from the brightness value, the contour positions are sequentially stored, and Z is completed when one cross section is completed. _Since the contour extraction processing in each cross section is repeated by changing each step, the entire shape of the sample 24 can be obtained from this contour data, and the entire image of the sample can be displayed on the display device 29 together with the position information in the Z direction. . Therefore, the observer can easily determine in what range the slice image should be observed based on the position information in the Z direction, and the operation efficiency can be significantly improved.

In the above embodiment, the horizontal cross section (X
Although the contour extraction is performed for each Y plane), the vertical section (XZ plane or YZ plane) may be used as the contour extraction target section and the Z direction may be used as the scanning line to perform the contour extraction processing based on the brightness value. , The contour information of the whole sample can be obtained.

Further, instead of detecting only one luminance change point in one scanning line and interrupting the scanning of that line, even if the first change point is detected as shown in FIG. Further, the scanning is continued to perform another contour extraction process for detecting another change point.

By repeating the above-mentioned processing for each scanning line, the outer circumference information of each cross section is obtained, and the processed cross section is shifted to obtain the outer circumference information of the entire specimen. And
When the change in brightness in the target cross section is no longer detected, the shift from the first cross section is made in the opposite direction.

According to such a modification, it is possible to reduce the total number of line scans until the entire sample shape is obtained.

[0031]

As described above in detail, according to the present invention, three-dimensional information of a specimen can be obtained before observing the specimen slice image, and the operation efficiency in obtaining the specimen image is significantly improved. A scanning laser microscope capable of being provided can be provided.

[Brief description of drawings]

FIG. 1 is a schematic configuration diagram of a scanning laser microscope according to an embodiment of the present invention.

FIG. 2 is a diagram illustrating the operation of the embodiment.

FIG. 3 is a perspective view showing a sample shape whose contour has been detected.

FIG. 4 is an operation explanatory diagram of a modified example of the embodiment.

FIG. 5 is a configuration diagram of a conventional scanning laser microscope.

[Explanation of symbols]

20 ... Laser light source, 21 ... Dichroic mirror, 22
... Acousto-optic polarization element, 23 ... Objectives, 24 ... Specimen, 2
5 ... Piezoelectric element, 26 ... Confocal pinhole, 27 ... Photodetector, 28 ... Controller, 29 ... Display device.

Claims (1)

[Claims] 1. The light from a light source is condensed on a sample by an objective lens to form a beam spot on the sample, and the acousto-optical polarization element arranged on an optical path between the light source and the objective lens is used to form the beam spot. 2 beam spots on the specimen
In a scanning laser microscope that performs two-dimensional scanning, converts the light reflected or transmitted from the sample by this scanning into an electric signal, and forms a sample image from the electric signal, a piezoelectric element for adjusting the focal position of the objective lens. An element, a means for detecting the contour of a cross-sectional image of the sample based on a luminance value, and a means for constructing a stereoscopic image of the sample from the contour data of a plurality of cross-sectional images obtained by this means. Characteristic scanning laser microscope.