JP3573508B2 - Confocal scanning optical microscope - Google Patents

Description

[0001]
[Industrial applications]
The present invention relates to a scanning optical microscope, in particular, illuminates an observation sample in a point-like manner with a point-like light source, and forms a transmitted light or reflected light from the illuminated sample into a point-like image again to form a pinhole opening. The present invention relates to a confocal optical microscope that obtains image density information with a detector having the same.
[0002]
[Prior art]
An optical microscope is a structure in which a sample on a slide placed on a stage is magnified and observed with an objective lens.In general, illumination of the sample is performed by observing the sample from a light source such as a lamp using a condenser lens. The structure was applied uniformly over the entire area.
[0003]
However, when such a structure is adopted as the illumination system, there is a problem such as flare, and there is a problem that it is very difficult to observe a low-contrast sample. A projection type (spot light projection type) optical microscope has been proposed. In this optical microscope, the observation sample is illuminated by a point light source in a point-like manner, whereby the light transmitted through the observation sample (transmitted light) is imaged again in a point-like manner, which is detected by a detector having a pinhole opening. This is to obtain image density information. However, this alone can obtain only the density of the point irradiated by the point light source, so that the XY scanning method or the optical path in which the sample is moved in the X-axis and Y-axis directions and mechanically moved in a two-dimensional plane An optical system that scans the image is adopted, and an image display device such as a CRT display performs XY scanning in synchronism with XY scanning by these, and displays brightness as a signal corresponding to the density information and observes an image. I can do it. This is a kind of scanning optical microscope.
[0004]
By the way, an optical system that guides a point light source to the sample surface via an objective lens and an optical system that guides light from the sample surface through the objective lens to a detector via a pinhole have a confocal relationship, and the objective lens in the sample is A device that guides the image at the in-focus position to the detector is called a confocal optical microscope.In this configuration, the out-of-focus light cannot be detected because the optical path is shifted from the pinhole before the pinhole. That is, only the image at the in-focus position can be obtained.
[0005]
As described above, the confocal optical microscope illuminates the observation sample in a point-like manner with the point-like light source, forms a point-like image of transmitted light or reflected light from the illuminated sample again, and performs detection with a pinhole aperture. FIG. 3 shows an example of the configuration of a microscope that obtains image density information using a container.
[0006]
FIG. 3A is a schematic diagram of a conventional confocal optical microscope, which includes a point light source 31, a half mirror 32, an objective lens 33, a pinhole plate 35, and a photodetector 36. The light emitted from the point light source 31 passes through the half mirror 32, and is imaged as a point on the sample 34 by the objective lens 33 on the emission optical path of the point light source 31 with good aberration, and illuminates the sample 34. . The light reflected by the sample 34 passes through the objective lens 33 again, is reflected by the half mirror 32, and is collected.
[0007]
A pinhole plate 35 in which a pinhole is located is disposed at a condensing position on the reflected light path, and light passing through the pinhole of the pinhole plate 35 is transmitted to a light provided on the back side of the pinhole plate 35. The light enters the detector 36 and is detected. Then, a two-dimensional image of the sample 34 can be obtained by two-dimensionally scanning the light of the point light source 31 irradiating the sample 34 in the same manner as the raster scanning of the television.
[0008]
By the way, in FIG. 3A, light in the optical path indicated by the solid line is light that is focused on the focal position of the objective lens 33, and the plane passing through the focal point is f1. The light on the optical path indicated by the dotted line indicates light from a position A shifted from the focal position of the objective lens 33. In this case, it is assumed that a plane passing through the position A is f2.
[0009]
Of these, light in the optical path indicated by the solid line is focused on the pinhole position of the pinhole plate 35, but light from the position A is not focused on the pinhole position of the pinhole plate 35. Therefore, the light from the position A cannot pass through the pinhole in the pinhole plate 35 and does not reach the photodetector 36.
[0010]
In such an optical system, it is possible to obtain an image only at the converging position of the objective lens, that is, only at the focusing position. That is, the confocal optical system can be said to be an optical system having a resolution in the optical axis direction. Therefore, as shown in FIG. 3B, for the sample 34 having different heights of A, B, and C, for example, when the focus position is provided on the surface of height A, When only the image is obtained, and when the surface at the height B is provided with the in-focus position, only the image at the surface at the height B is obtained, and when the surface at the height C is provided with the in-focus position. In this case, only the image of the plane having the height C is obtained.
[0011]
Here, generally, there is a tendency that the smaller the diameter of the pinhole, the higher the resolution in the optical axis direction.
Here, as shown in FIG. 3B, consider a case where the samples 34 having different heights are observed with a conventional general optical microscope including an objective lens and an eyepiece. In general optical microscopes, the higher the magnification of the objective lens, the shallower the depth of focus, and the observation plane is adjusted to the focal position. Therefore, when focusing on the A plane, the B and C planes with different heights from this are blurred. Would. Similarly, when focusing on the B surface, the A surface and the C surface are blurred, and when focusing on the C surface, the A surface and the B surface are blurred, and so on. If this is done, the portion at the same height as that position can be observed as a clear microscope image, but the images at other height positions are blurred and cannot be observed. Therefore, it was impossible to obtain an image focused on all surfaces A, B, and C with a general optical microscope.
[0012]
However, in the case of a scanning microscope having a confocal optical system, an image focused on the surface A is obtained and stored, and an image focused on the surface B is obtained and stored. After obtaining an image focused on the C plane and storing the obtained image, by adding these stored images, even if the sample 34 having the A, B, and C planes with different heights, Images focused on the entire surfaces B and C can be easily obtained, and the surface of a sample having a large difference in elevation can be observed as a clear microscope image.
[0013]
In use, the beam is condensed on one point of the sample, and scanning is performed at that point to change the focus position in the optical axis direction (Z-axis direction) of the beam (scanning in the Z-axis direction). Next, pixel data is obtained by moving the beam to another position on the sample and performing scanning in the Z-axis direction. At this time, height information (Z-axis information) when the maximum value of brightness is shown for each pixel is obtained. (Information), the surface shape of the sample can be measured. This is disclosed in "THEORT AND PRACTICE OF SCANNING OPTICAL MICROSCOPY" (P126 to P130).
[0014]
That is, the beam is focused on one point of the sample, the point is scanned in the optical axis direction (Z-axis direction), the Z position at which the brightness becomes maximum during the scanning is detected, and the information is stored. Next, the condensed light is moved in the X direction, scanning is performed in the Z-axis direction at that point, a Z-direction position where the luminance becomes maximum during scanning is detected, and the information is stored. In this way, the Z-axis scanning is performed for one point, and after that, the position is moved in the X-axis direction and the Y-axis direction, and the Z-axis scanning is performed at the next different X-axis and Y-axis positions. When the Z-axis position information indicating the maximum luminance at each of the X and Y positions is stored, the confocal optical system hardly detects reflected light from a height position out of the focus position of the objective lens. It can be understood that the Z-axis position indicating the luminance of the object is the focus position of the objective lens. Therefore, when the Z-axis position at that time is stored, the data stored in each pixel indicates the height information of the sample at that pixel position. Thus, the surface shape of the sample can be measured.
[0015]
[Problems to be solved by the invention]
The confocal optical microscope illuminates the observation sample in a point-like manner with a point-like light source, focuses the transmitted light or reflected light from the illuminated sample again in a point-like manner, and uses a detector having a pinhole aperture to detect the image density. With this confocal optical microscope, it is possible to obtain a microscope image that clearly captures the surface image of a sample with a large unevenness, and also obtain information on the height direction of the sample. The height of the sample can be measured and the height of the sample can be measured.
[0016]
As described above, in the confocal optical microscope, light is condensed on one point of the sample, the point is scanned in the optical axis direction (Z direction), the Z position at which the luminance becomes maximum during the scanning is detected and stored, and By moving the condensed light in the X direction, scanning in the Z direction at that point, and detecting and storing the Z position at which the luminance is highest during scanning and storing the same, by performing the scanning in the X and Y directions, Measure the surface shape of the sample.
[0017]
Therefore, in order to obtain information in the Z direction, that is, height information, the focus position of the objective lens and the sample must be relatively moved. However, since the distance (operating distance) WD from the tip of the objective lens 33 to the focal position f is determined for the objective lens 33 as shown in FIG. 4, depending on the shape of the sample 34 to be measured, At the time of setting, the objective lens 33 and the sample 34 may collide.
[0018]
For example, a configuration is considered in which the position of the sample 34 is fixed and the objective lens 33 is moved up and down. As described above, the position where the image disappears is the upper or lower limit of the measurement range. Therefore, when the difference dl between the highest position PH and the lowest position PL of the sample 34 is smaller than the operating distance WD of the objective lens 33 as shown in [I] of FIG. After scanning with the focal point f of the objective lens 33 positioned ([II] in FIG. 5A), even if the scanning is performed with the focal point f of the objective lens 33 positioned at the position of PL ([II] in FIG. 5A). III]), the objective lens 33 can perform observation and measurement without hitting the position of PH of the sample 34, and the upper limit position and the lower limit position of the measurement range can be set without any trouble.
[0019]
However, when the difference dl ′ between the highest position PH and the lowest position PL of the sample 34 is larger than the operating distance WD of the objective lens 33 as in [I] of FIG. After scanning with the focal point f of the objective lens 33 ([II] in FIG. 5B), when scanning is performed with the focal point f of the objective lens 33 positioned at the position of PL (see [III] in FIG. 5B). ]), The objective lens 33 collides with the position of PH of the sample 34, and before the lowermost surface of the sample 34 is detected, the objective lens 33 collides with the high position PH of the sample 34 and Or the sample 34 may be damaged.
[0020]
Further, when positioning the objective lens 33 with respect to the sample 34, it is not particularly determined which of the upper limit position and the lower limit position is to be determined first. In some cases, the objective lens 33 collides with the sample 34.
[0021]
Therefore, it is an object of the present invention to prevent a collision between a sample and an objective lens when setting an area of a measurement range, thereby preventing damage to the sample and the objective lens. A focus scanning optical microscope is provided.
[0022]
[Means for Solving the Problems]
In order to solve the above problems and achieve the above object, the confocal scanning optical microscope of the present invention is configured as follows. That is, the light emitted from the light source isAn objective lens for converging on the sample, means for two-dimensionally scanning the emitted light with respect to the sample, a photodetector for detecting light obtained from the sample via the objective lens, and What is claimed is: 1. A scanning microscope comprising: a moving mechanism that moves a focal position of an objective lens and a sample relatively in an optical axis direction, until an upper limit of the relative movement in the optical axis direction by the moving mechanism is determined. Is controlled so that the focal position of the objective lens can be moved only in a direction in which the objective lens is separated from the sample, and after the upper limit is determined, a range in which the moving amount based on the upper limit does not exceed the operating distance of the objective lens. Control means for controlling the moving mechanism so as to be movable in a direction in which the focus position of the objective lens and the sample approach each other.It is characterized by:
[0023]
In this case, the control means may control to stop the moving mechanism when the moving amount based on the upper limit becomes about 90% of the operating distance of the objective lens. Further, an alarm may be issued when an operation contrary to the restriction on the relative movement in the optical axis direction by the moving mechanism is performed.
[0024]
[Action]
As described above, the present apparatus allows movement only in a direction in which the distance between the objective lens and the sample is reduced so that the focus of the objective lens is aligned with the top surface of the sample, and the focus of the objective lens is set at the top of the sample. If it fits on the surface, thenOperating distance (Focal length)Movement in the direction in which the objective lens comes into contact with and separates from the sample within the range of, the movable distance in the contact and separation direction between the objective lens and the sample is set to the operating distance of the objective lens. (Focal length), and the collision between the objective lens and the sample can be prevented.
[0025]
【Example】
Hereinafter, embodiments of the present invention will be described with reference to the drawings.
FIG. 1 shows a system according to a first embodiment of the present invention.
In FIG. 1, 1 is a confocal scanning optical microscope, 2 is a laser light source, 3 is a mirror, 4 is a half mirror, 5 is a two-dimensional scanning mechanism, 6 is a revolver, 7 is an objective lens, 8 is a sample, 9 Is a stage, 10 is a coarse movement stage, 11 is a lens, 12 is a pinhole plate, 13 is a photodetector, 14 is a mirror, 15 is a lens, 16 is a half mirror, 17 is a white light source, 18 is a TV as an imaging device. A camera, 19 is a computer, 20 is a display, 22 is an image processing unit, 22A and 22B are image memories, 23 is a Z movement drive control circuit, 24 is a monitor, and 25 is an operation unit.
[0026]
The operation unit 25 is for giving various commands and operation commands to the computer 19 and for setting a mode, and includes a pointing device such as a trackball, a joystick, or a mouse in addition to a keyboard. .
[0027]
The stage 9 places the sample 8 thereon, and enables the XY-axis alignment and the Z-axis alignment. The coarse movement stage 10 holds the stage 9 and moves it in the Z-axis direction. Can be manipulated.
[0028]
The laser light source 2 is a laser light source for generating laser light as spot light for scanning the surface of the sample 8, and the mirror 3 is a reflecting mirror for guiding the laser light from the laser light source 2 to a two-dimensional scanning mechanism 5. It is. The two-dimensional scanning mechanism 5 is a mechanism for performing two-dimensional scanning (XY scanning) of the laser light from the laser light source 2 obtained via the mirror 3, and is mounted on the stage 9 via the objective lens 7 attached to the revolver 6. The sample 8 can be irradiated with laser light while scanning it two-dimensionally.
[0029]
The half mirror 4 is provided on an emission optical path of the laser light source 2 with respect to the two-dimensional scanning mechanism 5, and is a mirror for guiding reflected light from the sample 8 obtained through the two-dimensional scanning mechanism 5 to a detection system. It is a transparent mirror. The lens 11 is a lens for condensing the reflected light from the two-dimensional scanning mechanism 5 obtained through the half mirror 4, and the pinhole plate 12 has a pinhole of a required diameter, and a photodetector 13. The pinhole is located at the focal position of the lens 11 on the front surface of the light receiving surface of the lens 11. The photodetector 13 is a photodetector that converts light obtained through the pinhole into an electric signal corresponding to the light amount.
[0030]
Each of the image memories 22A and 22B is an image memory having a capacity of one frame, for example, a memory having a configuration of 512 pixels × 512 pixels × 8 bits for one frame.
[0031]
The image processing unit 22 incorporates two image memories 22A and 22B having a capacity for one frame, receives an output signal from the photodetector 12, and outputs, for example, the image memory 22A among the image memories 22A and 22B. To the pixel position corresponding to the current XY scanning position of the spot light, the signal value is stored as 8-bit data, and the current scanning information in the Z-axis direction given from the Z movement drive control circuit 23 is processed. In addition to the processing of storing the received value as 8-bit data at the pixel position corresponding to the XY scanning position of the current spot light in the image memory 22B, the data stored in the image memories 22A and 22B are read and provided to the computer 19. Is performed. The current XY scanning position information of the spot light with respect to the image processing unit 22 is given via the computer 19.
[0032]
The XY scanning drive control unit 21 is for controlling the scanning of the two-dimensional scanning mechanism 5, and the computer 19 receives these commands and operation commands from the operation unit 25 and operates the XY scanning drive control unit 21 and the image processing unit 22. , And plays a central role in control and processing such as saving, reproducing, and editing image data.
[0033]
Further, in the measurement range setting mode, the computer 19 operates to move the stage 9 upward or downward based on the operation of the operation unit 25 by the observer. Until this operation is performed, control is performed with a restriction that the stage 9 can be moved only in a direction away from the objective lens 7, and when the observer performs an operation contrary thereto, an alarm is issued, and the observer operates the operation unit 25. When the upper limit position is determined based on the operation, the position of the stage 9 at that position is registered as the upper limit position, and the stage 9 is operated in the direction approaching the objective lens 7 based on the operation of the operation unit 25 by the observer. When the lower limit position is determined based on the operation of the operation unit 25 by the observer, the position of the stage 9 at that position is registered as the lower limit position. Certain stage 9 as a configuration for controlling so as to move in the Z-axis direction at a predetermined pitch between from the upper limit position when gathering point image to the lower limit position. The monitor 20 is an image display terminal of the computer 19, and is used for displaying necessary information, displaying images, and the like.
[0034]
The revolver 6 holds a plurality of objective lenses 7 having different magnifications, the stage 9 holds a sample 8, and switches the revolver 6 to one of the plurality of objective lenses 7 having a desired magnification. Thus, by setting the position in the observation optical path of the microscope, the sample 8 on the stage 9 can be irradiated with the spot light from the two-dimensional scanning mechanism 5 via the set objective lens 7. Further, the reflected light from the sample 8 passes through the objective lens 7, returns to the two-dimensional scanning mechanism 5, and returns from the two-dimensional scanning mechanism 5 to the half mirror 4.
[0035]
Further, in the present apparatus, a mirror 14 is provided which can move forward and backward with respect to the optical path between the objective lens 7 and the two-dimensional scanning mechanism 5. The white light source 7 is a light source that generates white light, and the half mirror 16 is arranged to face the mirror 14 and reflects the light from the white light source 7 to the optical path to the mirror 14 and emits the light. .
[0036]
Therefore, when observing the confocal image, the mirror 14 is retracted from the optical path between the objective lens 7 and the two-dimensional scanning mechanism 5 and the confocal image cannot be obtained when the mirror 14 is inserted in the optical path. It is a mechanism. When the mirror 14 is inserted in the optical path, the light from the white light source 7 is reflected by the half mirror 16 to the mirror 14 and guided, and can be sent to the objective lens 7. The reflected light from the sample 8 incident on the mirror 8 can be reflected by the mirror 14 and guided to the half mirror 16.
[0037]
The TV camera 18 is disposed to face the mirror 14 via the half mirror 16 and converts an image of the sample 8 into a TV image signal. The reflected light is transmitted through the half mirror 16 and guided to the TV camera 18. The mirror 14 is inserted in the optical path between the objective lens 7 and the two-dimensional scanning mechanism 5 when observing an image by the TV camera 18, and is used when observing and measuring a confocal image. 5 out of the light path.
[0038]
The monitor 24 is a TV monitor that displays a TV image signal obtained by imaging with the TV camera 18 as an image.
The two-dimensional scanning mechanism 5 performs XY scanning of the spot light under the control of the scanning drive control unit 21, and includes, for example, a galvanometer mirror for scanning in the X-axis direction and a galvanometer mirror for scanning in the Y-axis direction. By rotating these galvanometer mirrors in the X-axis direction and the Y-axis direction, the optical path of the spot light with respect to the objective lens 7 can be swung in the XY directions.
[0039]
The Z movement drive control circuit 23 is controlled by the computer 19, and is a circuit for controlling the stage 9 to drive and move the stage 9 in the height direction, that is, the Z-axis direction in units of a reference width. The Z-movement drive control circuit 23 also has a function of incrementing the count by one each time the stage 9 is driven and controlled by the reference width in the Z-axis direction, and a function of giving the count value to the image processing unit 22.
[0040]
In the image processing unit 22, for example, each of the image memories 22A and 22B having a size of 512 pixels × 512 pixels × 8 bits (256 gradations) is prepared. Of these, the electric signal (luminance signal) of the reflected light is stored in the image memory 22A, and the electric signal detected by the photodetector 13 is stored in the image memory 22A. Control is performed so that data is stored and stored in the pixel position corresponding to the scanning position. This storage is a value obtained by adding the storage information of the pixel position. By doing so, it is possible to add images having different height positions.
[0041]
In this apparatus, two options are available for obtaining an image of the sample 8. One is a method of illuminating the sample 8 using light from the white light source 17 and using an image captured by the TV camera 18 through the objective lens 7, the mirror 14, the lens 15, and the half mirror 16 in the reflected light. And the other, two-dimensional scanning of the laser beam from the laser light source 2 by the two-dimensional scanning mechanism 5 to give the sample 8 and the reflected light of the objective lens 7, the two-dimensional scanning mechanism 5, the half mirror 4, the lens 11, In this method, the light is incident on the photodetector 13 through the pinhole plate 12, the output from the photodetector 13 is supplied to the image processing unit 22 to obtain an image, and the image is displayed on the display 20 for observation.
[0042]
Next, the operation of the confocal scanning optical microscope 1 having such a configuration will be described. Laser light emitted from the laser 2 is reflected by the mirror 3, passes through the half mirror 4, and enters the two-dimensional scanning mechanism 5. The two-dimensional scanning mechanism 5 starts operating according to a scanning control signal generated from the scanning control unit 21 based on a command from the computer 19. Therefore, the laser beam is deflected in the X and Y directions here, as in the raster scanning of the TV.
[0043]
The laser beam passes through the revolver 6 and the objective lens 7 and is focused on the sample 8 on the stage 9 in a minute spot, and the spot moves on the sample 8. The light reflected from the sample 8 traces the incident light path in reverse, is reflected by the half mirror 4 and is collected by the lens 11. A pinhole plate 12 having a pinhole located therein is disposed at the light condensing position of the lens 11.
[0044]
The light passing through the pinhole of the pinhole plate 12 enters the photodetector 13. The photodetector 13 converts the reflected light of the sample into an electric signal. The electric signal is input to the image processing unit 22. The image processing unit 22 is provided with image memories 22A and 22B, and one of the image memories 22A stores an electric signal of reflected light. The movement of the stage 8 in the optical axis direction (Z direction) is performed by issuing a movement command from the computer 19 to the Z movement drive control circuit 23. The image memory 22B stores, from the Z movement drive control circuit 23, a value of the number of times the stage has moved.
[0045]
The image obtained by the laser scanning is sent from the image processing unit 22 to the computer 19 and displayed on the display 20 of the computer 19. In addition, the setting of the measurement range, the setting of the movement amount in the measurement range, the display of the image, and the control of the system are performed on the display 20 of the computer 19.
[0046]
As described above, in the present apparatus, acquisition of a microscope image (luminance signal) and acquisition of height information are performed simultaneously. That is, the XY scanning of the sample 8 with the laser beam is performed for each height position with respect to the unevenness of the sample 8 to collect the luminance information, and the obtained luminance information for each height is converted to the same XY scanning position. , An image is created and displayed at the same time. Each time the height position is changed, the count is incremented by one. If the value of the luminance signal at the same XY scanning position is higher than the previous value, the count value is updated and recorded corresponding to the XY scanning position. That is, as an example, when the luminance signal at the X1 and Y1 positions is “0” at the height position Z1 (count value “1”), and when the luminance signal is at the height position Z2 (count value “2”), Assuming that the luminance signal at the X1 and Y1 positions is “0” and the luminance signal at the X1 and Y1 positions is “1” at the height position Z3 (count value “3”), The height position Z3 (the count value “3”) is stored as the height position Z3 at the X1 and Y1 positions.
[0047]
The XY scanning of the spot light is performed as follows, and an image and height information are obtained. That is, in this apparatus, the power is turned on at the start of use, whereby the laser light source 2 oscillates a laser beam. This laser light is reflected by the mirror 3, passes through the half mirror 4, and enters the two-dimensional scanning mechanism 5. The two-dimensional scanning mechanism 5 starts operation based on an XY scanning control signal generated from the XY scanning control unit 21 based on a command from the computer 19. Therefore, the laser light is deflected in the X and Y-axis directions in the same manner as in the raster scanning of the TV.
[0048]
The laser beam passes through the revolver 6 and the objective lens 7 and is focused on the sample 9 on the stage 8 into a minute spot, and the spot light moves on the sample 8 by performing the XY scanning. be able to. The reflected light from the sample 8 follows the incident optical path in reverse, is reflected by the half mirror 4, and is collected by the lens 11. A pinhole plate 12 is disposed at a position where light is condensed by the lens 11, and a photodetector 13 is provided thereafter. Therefore, the light passing through the pinhole in the pinhole plate 13 enters the photodetector 13. The photodetector 13 converts the light passing through the pinhole into an electric signal as reflected light of the sample. become.
[0049]
Here, since this apparatus is a confocal microscope, if the pinhole diameter is sufficiently small, only the light reflected from the sample 8 from the focus position of the objective lens 7 passes through the pinhole. Therefore, only the reflected light from the sample surface at the in-focus position of the objective lens 7 is detected by the photodetector 13.
[0050]
The electric signal detected by the light detector 13 is input to the image processing unit 22. In the case of the present embodiment, the image processing unit 22 is provided with two 512 × 512 pixels × 8 bits (256 gradations) as image memories. The electric signal of the reflected light is stored in one of the image memories 22A, and the electric signal detected by the photodetector 13 is transmitted to the image memory 22A by the current XY scanning of the spot light. The data is stored and stored in the pixel position corresponding to the position as data. This storage is a value obtained by adding the storage information of the pixel position. By doing so, it is possible to add images having different height positions.
[0051]
Further, information on the Z-axis scanning direction, specifically, a value of the number of times the stage has been moved is given from the Z movement drive control circuit 23 to another image memory 22B. If the level of the current luminance signal is higher than the previous luminance signal at this position, the value of the number of times is updated and stored as data in the pixel position corresponding to the current XY scanning position of the spot light in the image memory 22B. Let me save.
[0052]
On the other hand, the movement of the stage 9 in the optical axis direction (Z-axis direction) is performed in units of the reference width every time the XY scanning is completed based on a command from the computer 19. Each time a command is received from the computer 19, the Z-movement drive control circuit 23 generates a Z-axis direction drive control signal for a reference width unit to drive a stage Z-axis direction drive operation mechanism (not shown). The stage 9 is moved in the Z-axis direction.
[0053]
The moving amount of the stage 9 is the reference width unit for each command, and a command is issued from the computer 19 every time the stage 9 needs to be moved.
The setting of the measurement range, the setting of the reference width of the stage movement amount in each measurement range, the display of the image, and the control of the system are performed by the observer of the microscope using the keyboard or the mouse, joystick, trackball, etc. The coarse movement stage 10 can be operated while operating the pointing device and watching the display 20 or by inserting the mirror 14 into the laser beam path between the objective lenses 7 and 5 while observing the image displayed on the monitor 24. Is roughly adjusted to perform the following.
[0054]
In this system, the observer places the sample 8 on the stage 9 and operates the computer 19 to generate a control command from the computer 19, thereby starting XY scanning of the spotlight to start observation and measurement. First, since it is necessary to focus on the sample 9, the stage 9 can also be moved up and down using the computer 19, so that the focus of the objective lens 7 can be adjusted to a desired height position of the sample 8.
[0055]
In this case, whether or not the subject is in focus is determined while looking at the image displayed on the display 20. In other words, the laser light source 2 is excited to generate a laser beam, and the laser beam is XY-scanned to perform XY scanning of the spot light on the sample 8, and the reflected light obtained by this is detected by the photodetector 13. By obtaining the image data by the reflected light, storing the image data in the image memory 22A corresponding to the pixel position, reading the image data and displaying the image on the display 20, the image in the current focus state can be observed. By adjusting the height position of the stage 9 while watching, it is possible to focus on the target height position of the sample 8.
[0056]
The following describes how to determine the measurement range using the confocal image. The user operates the computer 19 while observing the image, and moves the stage 9 downward from the focused position.
[0057]
Although the image is displayed as long as the sample 8 crosses the focus position, the image is not displayed when the uppermost surface of the sample 8 is below the focus position. Here, one side (upper limit position) of the measurement range is determined. This time, the stage 9 is moved up and the position where the image is no longer displayed (the lower limit position) is similarly determined.
[0058]
As described above, the measurement range in the Z-axis direction using the confocal image can be determined. However, since the depth of focus is extremely shallow in the confocal image, there are some points that are difficult to use. For this reason, in this system, focusing using a TV image can be performed.
[0059]
In order to determine the measurement range using the TV image, the observer first inserts the mirror 14 on the optical path connecting the objective lenses 7 and 5 so that the TV camera 18 can be used. Then, the incandescent light source 17 is turned on, and the light is sent to the objective lens 7 via the half mirror 16, the lens 15, and the mirror 14, and is irradiated on the stage 9 side. The reflected light from the stage 9 follows the reverse path, passes through the half mirror 16, is captured by the TV camera 18, is converted into a video signal, and the video signal is provided to the monitor 24 and displayed as an image. . The lens 15 also has a function of forming an image on the TV camera 18.
[0060]
Therefore, when the sample 8 is placed on the stage 9, an image of the sample 8 is captured by the TV camera 18, and the captured sample image is displayed on the monitor 24. While roughly adjusting the Z-axis position of the coarse movement stage 10, rough focusing is performed.
[0061]
After the rough focusing is completed, the mirror 14 is removed from the laser beam path connecting the objective lenses 7 and 5. As a result, an optical path is secured for the laser optical path that has been blocked by the mirror 14, and laser scanning of the sample 8 can be started. Therefore, the XY scanning of the laser beam is started by a command operation of the observer or by a command from the computer 19 generated by retracting the mirror 14 from the laser beam path. When the laser scanning is started, an image obtained by the laser scanning is obtained on the image memory 22A of 22. By displaying the image on the image memory 22A on the display 20, a confocal image can be observed.
[0062]
When the confocal image is displayed, the observer operates the operation unit 25 while watching the displayed image to set the measurement range. The measurement range is set by operating the operation unit 25 and moving the stage 9 up and down while searching for a position where the image is completely invisible.
[0063]
That is, the user operates the operation unit 25 to set the measurement range setting mode for the computer 19, and further operates the operation unit 25 to move the stage 9 upward or downward. Until the upper limit position is determined, the computer 19 controls the stage 9 so that the stage 9 moves only in the direction away from the objective lens 7 (S2 in FIG. 2).
[0064]
As a result, the stage 9 can be moved downward from the initial position where the focus is roughly adjusted, but if the stage 9 is to be moved upward, the stage 9 does not move, and the computer 19 issues a warning message. Is output (S9 in FIG. 2).
[0065]
In this way, when the observer operates the operation unit 25 to set the measurement range setting mode and performs the measurement range setting operation, while performing laser scanning in the X and Y directions, the computer 19 responds to the operation by the stage. 9 is controlled in a direction away from the objective lens 7. Then, the observer operates the operation unit 25 at an appropriate position while observing the sample image on the display 20 to issue a command to complete the setting of the upper limit position, and gives it to the computer 19.
[0066]
That is, as long as there is a portion on the display 20 where the sample 8 crosses the focus position of the objective lens 7, an image is displayed, but when the top surface of the sample 8 comes below the focus position of the objective lens 7, Since the image is no longer displayed on the display 20, one side (upper limit position) of the measurement range is determined when the image is no longer displayed. At this point, the observer operates the operation unit 25. Then, an upper limit position setting completion command is issued to notify the computer 19 (S3 in FIG. 2). Thereby, the computer 19 stops further lowering of the stage 9 and holds the information on the height position of the stage 9 at this time (registers the value of the height position of the stage 9) (see FIG. 2). S4).
[0067]
When one side of the measurement range is determined, the observer next operates the operation unit 25 to start work for setting the lower limit position. This is by giving an operation command for moving the stage 9 in the vertical direction from the operation unit 25, and the computer 19 having received this command can move the stage 8 in both the vertical direction up to the height position. This is performed by changing the regulation so that the movement is controlled in the vertical direction in response to the operation command (S5 in FIG. 2).
[0068]
That is, in the stage of setting the upper limit position, the stage 9 is moved only downward, so that the stage 9 can be moved upward with the upper limit position set as the upper limit. Then, while observing the display 20, the observer operates to move the stage 9 in the vertical direction, and searches for a position (lower limit position) where the image is not displayed as before.
[0069]
Here, in the movement control in the lower limit direction, the computer 19 compares the movement amount of the stage 9 with reference to the set upper limit position and the operating distance (focal length) WD of the objective lens 7 currently used. Then, control is performed such that the stage 9 is moved up and down in response to the operation from the operation unit 25 within a range in which the moving amount of the stage 9 does not exceed the range of the operating distance (focal length) WD (S6 in FIG. 2).
[0070]
As a result of the above-described comparison based on WD, the computer 19 determines that the moving amount of the stage 9 is within the operating distance of the objective lens 7 and that the operation unit 25 continuously gives a moving operation command for moving the stage 9 up and down. If this is the case, the movement of the stage 9 in the up-down direction is subsequently permitted. However, when the moving amount of the stage 9 becomes equal to the operating distance of the objective lens 7, if the stage 9 is further moved downward, the objective lens 7 and the sample 8 will collide with each other. Even if a downward movement operation command has been given from the operation unit 25 to the stage 9, the Z movement drive control circuit 23 is controlled to stop the movement of the stage 9 (S10 in FIG. 2). Thereby, it is possible to prevent the objective lens 7 and the sample 8 from colliding with each other by force.
[0071]
Further, if the sample image on the display 20 is not displayed before the moving amount of the stage 9 becomes equal to the operating distance of the objective lens 7, the observer knows that the lower limit position has been reached at that point, and the operation unit 25 The upward movement operation of the stage 9 by the operation is stopped. As long as there is a portion on the display 20 where the sample 8 crosses the focus position of the objective lens 7, an image is displayed. However, when the top surface of the sample 8 comes below the focus position of the objective lens 7, the display 20 Since the image is not displayed on the upper side, the other side (lower limit position) of the measurement range is determined at the time when the image is not displayed. The operation of moving up the stage is stopped, and the operation section 25 is operated to issue a lower limit position setting completion command to notify the computer 19 (S7 in FIG. 2). As a result, the computer 19 stops the further elevation of the stage 9 and holds the information on the height position of the stage 9 at this time (registers the value of the height position of the stage 9) (see FIG. 2). S8).
[0072]
In this way, if the lower limit position can be detected within the range in which the stage 9 can be moved, the lower limit position can be set by ending the setting of the measurement range at that stage.
[0073]
As described above, the present apparatus focuses the emitted light from the light source on the sample via the objective lens, and detects the collected light and the light from the sample obtained by relatively two-dimensionally scanning the sample. A confocal scanning optical microscope that obtains an image by storing it in an image memory corresponding to the scanning position of the two-dimensional scanning, wherein the focal position of the objective lens and the position of the sample are relatively The objective lens is moved at a required pitch by a moving mechanism that scans in the axial direction, and when the light from the sample is detected higher by referring to the held information in the image memory and the detection output of the light detecting means. In a confocal scanning optical microscope equipped with a function of measuring a change in the optical axis direction of the sample by storing the relationship between the focus position and the sample position in storage means, illumination light is applied to the sample via an objective lens. give A source is provided, imaging means for capturing an image of the sample by light from the light source, and a monitor device for displaying an image obtained by the imaging means are provided. When scanning the focal position and the position of the sample relatively in the optical axis direction, when determining the scanning range, coarsely adjust the focus of the objective lens with respect to the sample, and then move the objective lens away from the sample in a direction away from the sample. When the image disappears while observing the image by the imaging means while controlling the movement direction so that only the movement is possible, the position is registered as the upper limit of the movement with respect to the objective lens, and then the direction in which the sample and the objective lens come and go with each other. When the moving range does not exceed the upper limit and is within the working distance of the objective lens, the position is adjusted when the image by the imaging means disappears. Registered as moving limit for the lens so as to obtain a confocal image by scanning the Z direction movement range of the moving limit this movement limit.
[0074]
In this way, when determining the scanning range, after coarsely adjusting the focus of the objective lens with respect to the sample, they are separated from each other while controlling the moving direction only in a direction in which the objective lens is moved away from the sample, and the objective lens and the sample are separated. Then, the sample and the objective lens are operated in a direction approaching each other to determine the lower limit of the movement of the objective lens within the operating distance of the objective lens, and Z is determined in the range of the upper limit of the movement and the lower limit of the movement. Since the confocal image is obtained by scanning in the directional movement, the sample and the objective lens do not collide, and the sample and the objective lens can be prevented from being damaged. In addition, since the sample and the objective lens are prevented from being damaged, the reliability of the apparatus is remarkably improved.
[0075]
In the above embodiment, the case where the stage 9 is moved has been described. However, the revolver 6 is supported by the Z-direction moving stage, and the objective lens 7 controlled by the computer 19 through the Z-direction drive control circuit 23 is moved up and down. Even in a configuration in which collision prevention is performed, collision prevention control can be performed based on the same concept.
[0076]
In the above example, the movement of the stage is prohibited when the movement amount of the stage matches the operation distance of the objective lens. However, since the operating distance of the objective lens varies depending on the selected objective lens, the movable range of the stage may be limited to 90% of the operating distance of the objective lens for safety. .
[0077]
Further, by using the image data of the image memory 22A, it is possible to determine whether or not the image is out of the confocal point by computer processing. Therefore, the computer 19 automatically determines the lower limit position based on the manual operation of the observer. It is also possible to use a device that performs this.
[0078]
【The invention's effect】
As described in detail above, in the confocal optical microscope, when determining the relative movement scanning range of the sample and the objective lens in obtaining a confocal image, by regulating the movement direction and limiting the movement amount, This makes it possible to prevent collision of the objective lens, thereby preventing damage to the sample and the objective lens, and at the same time, to obtain a highly reliable confocal optical microscope.
[Brief description of the drawings]
FIG. 1 is a schematic block diagram showing the overall configuration of an embodiment of the present invention.
FIG. 2 is a flowchart for explaining the operation of the present invention.
FIG. 3 is a diagram for explaining a conventional technique.
FIG. 4 is a diagram for explaining a conventional technique.
FIG. 5 is a diagram for explaining a conventional technique.
[Explanation of symbols]
1. Confocal scanning optical microscope
2 ... Laser light source
3 ... Mirror
4: Half mirror
5. Two-dimensional scanning mechanism
6 ... Revolver
7 Objective lens
8 ... sample
9 ... Stage
10 Coarse movement stage
11, 14, 15 ... Lens
12 ... Pinhole plate
13 ... Photodetector
16 Half mirror
17 ... White light source
18 ... TV camera
19. Computer
20 ... Display
22 ... Image processing unit
22A, 22B ... Image memory
23 ... Z movement drive control circuit
24 ... Monitor
25 Operation unit

Claims (3)

An objective lens for condensing the light emitted from the light source on the sample,
Means for two-dimensionally scanning the emitted light with respect to the sample;
A light detector for detecting light obtained from the sample via the objective lens,
A scanning microscope having a moving mechanism that relatively moves the focal position of the objective lens and the sample in the optical axis direction,
Until the upper limit of the relative movement in the optical axis direction by the moving mechanism is determined, the focus position of the objective lens is controlled to be movable only in a direction to be separated from the sample, and after the upper limit is determined. A control means for controlling the moving mechanism so that the moving amount based on the upper limit does not exceed the operating distance of the objective lens so that the focal position of the objective lens and the sample can be moved in a direction approaching. confocal scanning optical microscope according to. 2. The control device according to claim 1, wherein the control unit controls the moving mechanism to stop when a moving amount based on the upper limit becomes about 90% of an operating distance of the objective lens. Confocal scanning optical microscope. The apparatus according to claim 1, wherein the control unit issues an alarm when an operation is performed that is contrary to a limitation on the relative movement of the moving mechanism in the optical axis direction. 1 A confocal scanning optical microscope as described.

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