JP3518925B2 - Automatic image forming equipment for scanning microscope - Google Patents

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an automatic image of a scanning microscope.
The present invention relates to an image forming apparatus .

[0002]

2. Description of the Related Art Conventionally, a point light source illuminates an observation sample in a point shape, and transmitted light or reflected light from the illuminated sample is imaged again in a point shape, and a detector having a pinhole opening is used. There have been microscopes with confocal optics to obtain image density information.

A microscope having this confocal optical system will be described below with reference to FIGS. 8 and 9.

As shown in the schematic diagram of FIG. 8, a point light source is constituted by a light source 1 and a pinhole 2, and this point light source is pointed on an observation sample 4 by an objective lens 3 whose aberration is well corrected. The sample 4 is imaged, and the sample 4 is illuminated.

Further, the point-like light on the sample 4 is imaged as a point on the pinhole 6 by the condenser lens 5 whose aberration is well corrected, and the imaged point-like light is passed through the pinhole 6 to a photodetector. It is designed to detect at 7.

Then, a two-dimensional image is obtained by two-dimensionally scanning the sample 4 in the same manner as the raster scanning of the television.

By the way, the light shown by the broken line in FIG. 8 indicates the light from the position L deviated from the condensing position of the objective lens 3. Since the shifted light is not condensed on the pinhole 6, most of the light does not pass through the pinhole 6 and does not reach the photodetector 7. From this, with the optical system as shown in FIG. 8, it is possible to obtain an image only at the focus position of the objective lens 3, that is, the focus position.

[0008]

Since the confocal optical system has a shallow depth of focus, it is effective when the observation surface of the sample 4 is flat, but as shown in FIG. 9, the observation surface has a stepped shape. In the case of a non-planar sample in which the portions having different heights have irregularities, it becomes impossible to obtain an image of the sample surface deviated from the focus position. The sample 11 in FIG. 9 is composed of a high-height portion B, a low-height portion C, and an intermediate height portion A. When, for example, the observation surface of A is focused, observation of B and C is performed. The surface is blurred. Therefore, A, B, C
It is impossible to obtain an image focused on all the observation planes.

Of course, when an image of the observation surface of the sample 11 of FIG. 9 is obtained using the microscope of FIG. 8, for example, an image obtained by focusing the observation surface of A is stored, and B obtained in the same manner. If the image of the observation surface of No. 1 and the image of the observation surface of No. C are stored and added together, an image focused on the entire surface can be obtained. In this case, in practice, the maximum brightness value may be held for each pixel.

However, the positioning of the A (one end side) observation surface and the B (center side) observation surface of the sample 11 in FIG. 9 was determined by the observer while observing the image. Therefore, when a large amount of the sample 11 as shown in FIG. 9 is observed, the same work is repeated repeatedly, which causes a heavy burden on the observer.

Therefore, in order to reduce the burden on the observer,
Japanese Patent Laid-Open No. 6-3083 discloses an automatic image forming apparatus capable of automatically setting both ends of an observation sample having different heights in the scanning direction in the scanning direction.
Proposed as disclosed in No. 93.

However, this Japanese Patent Laid-Open No. 6-308393
In this case, since only the brightness and pixels in the screen are judged, for example, when the observation between the BC planes in FIG.
The face may be detected as an end face during scanning.
This is because a sharp portion is detected as a black image in the confocal optical system, which causes a problem that a portion different from the desired range is observed.

Further, in Japanese Unexamined Patent Publication No. 6-308393, there is no restriction on the moving range of the stage supporting the sample in the detection of both ends, so that the sample and the objective lens may interfere with each other when the lower end is detected. .

The present invention has been made in view of the above circumstances, and it is possible to reliably perform automatic setting of the upper and lower end surfaces of an observation sample having different heights of the observation surface, and the sample and the objective lens during the automatic setting. Scanning type that can prevent the interference of
An object is to provide an automatic image forming apparatus for a microscope .

[0015]

The invention according to claim 1 is
A point light source illuminates the observation sample placed on the stage.
An objective that collects the light that is emitted from the observation sample and re-focuses it.
The lens and the observation sample collected by the objective lens.
Scan for relatively scanning the light on the material and the observation sample
Means and a light detection hand for detecting reflected light from the observation sample
Stage, the focus position of the objective lens and the phase of the observation sample
And a moving means for moving the opposite position in the optical axis direction.
A scanning microscope that has a detection frame that sets the number of detection frames.
Lame setting means and the level of the detection signal of the light detection means
Count the number of pixels where is below a preset signal level
However, if this number of pixels is greater than or equal to the preset number of pixels
Of the pixel number detecting means for determining whether
End face detection means for detecting the end face of the observation sample based on the result
And a memory for storing the position detected by the end face detecting means.
Means for moving in one direction by the moving means,
When the position detected by the end face detecting means is held,
And the stored position based on the number of detected frames.
Further in the same direction to move the signal from the photodetector.
Check the bell and use the saved position as the end face of the observation sample.
So that that.

According to a second aspect of the present invention, in the first aspect of the invention, the storage means is constituted by the moving means.
Direction, and detected by the signal from the end face detection means
First storage means for storing a position, and the first storage means
Moves in the opposite direction from the position stored in
A second memory for storing the detected position by a signal from the means
It has a step . In addition, according to claim 3,
In the invention, in the invention according to claim 1, the moving means
One direction moved by is the observation sample and the objective lens
When moving in the direction opposite to the one direction
Is the range of relative movement between the observation sample and the objective lens.
I am trying to limit. The invention according to claim 4
Then, in the invention according to claim 3, the observation sample and
The range of relative movement with respect to the objective lens is
Make sure it is within the range of the distance from the tip position to the focus position.
ing.

[0017]

As a result, according to the first and second aspects of the present invention, the in-focus position is moved and the upper and lower ends of the observation sample are reliably detected even with respect to observation samples having different heights on the observation surface. Can be set automatically.

According to the inventions of claims 3 and 4 , in addition to the effect of the invention of claim 1, when the focus position is moved to automatically set the upper and lower ends of the observation sample, the sample and the objective lens Interference with can be prevented.

[0019]

DESCRIPTION OF THE PREFERRED EMBODIMENTS An embodiment of the present invention will be described below with reference to the drawings.

FIG. 1 shows the system configuration of a scanning confocal microscope. In the figure, 21 is a scanning confocal microscope, and 22 is a laser light source that serves as a light source for scanning light. The laser light as the spot light generated from the laser light source 22 is totally reflected by the mirror 23, and is sent to the two-dimensional scanning mechanism 25 after passing through the half mirror 24.

The two-dimensional scanning mechanism 25 has, for example, a galvanometer mirror for X-axis direction scanning and a galvanometer mirror for Y-axis direction scanning, and is controlled by an XY scanning drive control circuit 34 described later. The spot light is scanned by XY scanning by swinging two galvanometer mirrors in the X-axis direction and the Y-direction. The spot light scanned by the two-dimensional scanning mechanism 25 passes through the objective lens 27 selected by the revolver 26. The sample 29 placed on the stage 28 is irradiated.

The revolver 26 holds a plurality of objective lenses having different magnifications. Among these objective lenses 27, the objective lens 27 having a desired magnification is selected in the observation optical path of the microscope by rotating the revolver 26. By inserting
The spot light from the two-dimensional scanning mechanism 25 is transmitted to the sample 29 on the stage 28 via the selected objective lens 27.
Irradiation can be performed while performing dimension scanning.

The reflected light from the sample 29 due to this irradiation returns to the two-dimensional scanning mechanism 25 via the objective lens 27 and returns from the two-dimensional scanning mechanism 25 to the half mirror 24. The half mirror 24 is provided on the emission optical path of the laser light source 22 with respect to the two-dimensional scanning mechanism 25, and is provided in the two-dimensional scanning mechanism 25.
The reflected light from the sample 29 obtained via the half mirror 24 is condensed by the lens 30 and is guided to the detection system. It is sent to the photodetector 32 via the plate 31.

That is, the photodetector 32 has a lens 30 and a pinhole plate 31 arranged on the front surface of its light-receiving surface, and the pinhole plate 31 is located at the focal position of the lens 30.
The light obtained through the pinhole plate 31 is converted into an analog electric signal corresponding to the amount of light and sent to the image processing unit 33.

The image processing unit 33 has two image memories 33A and 33B and an image determination circuit 33C, the detailed structure of which will be described later, and the XY scan drive control circuit 34 and the Z scan drive control circuit. 35 is connected.

The Z scanning drive control circuit 35 is a circuit controlled by the computer 36 or the image determination circuit 33C to move the revolver 26 in the height direction, that is, the Z axis direction, in units of reference width. It also has a counting function of counting the position information each time the revolver 26 is moved in the reference width unit, and a function of sending the count value to the image processing unit 33.

The image memories 33A and 33B provided in the image processing unit 33 each have a storage capacity for one frame.
The image is stored with 2 pixels × 512 pixels × 8 bits (256 gradations) as one frame.

Of these, the image memory 33A stores the brightness information of the reflected light obtained by the photodetector 32 as 8-bit data at a pixel position corresponding to the current XY scanning position of the spot light, thereby generating an image signal. Is to remember.
When the brightness information of this time is larger (brighter) than the brightness information of the previous scan position, this memory is updated and saved as the image information of the scan pixel position, so that the images of different height positions are stored. It is something that can be added.

In the image memory 33B, information about the Z scanning direction, specifically, how many times the revolver 26 is moved from the Z scanning drive control circuit 35 to the pixel position corresponding to the current XY scanning position of the spot light. The value of the number of times is counted.

In this case, the image processing unit 33 refers to the image data in the image memory 33A, and when the level of the brightness information of this time is higher than the brightness information of the position at the previous time, the image processing unit 33 sends to the image memory 33B. A function of updating the value of the number of times as data and storing and storing is provided.

In this way, when the image memory 33B has information indicating the maximum brightness at each pixel position in each pixel position, the revolver movement count value is information in the Z scanning direction (this information indicates the height position). Will be stored).

Here, the current XY scanning position information of the spot light for the image processing unit 33 is given from the XY scanning drive control circuit 34. The image determination circuit 33C receives an output signal from the photodetector 32 and automatically sets the automatic range during automatic image formation, transmits the set value to the computer 36, controls the Z scan drive control circuit 35, and an end face described later. Detection, movement control of the in-focus position and the like are performed, and data stored in the image memories 33A and 33B are read out and given to the computer 36.

The computer 36 centrally controls the XY scan drive control circuit 34, the Z scan drive control circuit 35 and the image processing unit 33, and at the same time controls the entire process such as storing, reproducing and editing image data. The monitor 37 displays and outputs necessary information and images.

First, the overall operation of the scanning confocal microscope system having the above configuration will be described.

That is, when observing the sample 29, a so-called extended focus operation is performed in which a microscope image (luminance signal) and height information are simultaneously acquired.

This is the XY of the laser beam for the sample 29.
Luminance information is collected by performing scanning for each height position with respect to the unevenness of the sample 29, and the obtained luminance information for each height is compared for each same XY scanning position to store the maximum luminance. Create and display a single image.

Further, each time the height position is changed, the count is advanced by one, and when the value of the luminance signal at the same XY scanning position is higher than that of the previous time, the count value is updated to the position corresponding to the XY scanning position. Remember.

That is, to give an example, when the height position Z1 (count value: 1), the luminance signals at the X1 and Y1 positions are "0", and when the height position Z2 (count value: 2) is the same. X
The luminance signals at positions 1 and Y1 are also "0", and the height position is Z.
If the luminance signal at the X1 and Y1 positions is 3 when the count value is 3 (count value: 3), the height position Z3 (count value: 3) is stored as height information at the X1 and Y1 positions. It's like doing.

In performing this extended focus, it is necessary to specify the range in the height direction of the sample 29. There are two methods for this, that is, the observer uses the computer 36 to set the upper and lower ends of the range. And when
In some cases, it may be automatically set according to the judgment of the image judgment circuit 33C.

The XY scanning of spot light is performed as follows, and an image and height information are acquired. That is, when the power is turned on at the start of use, the laser light source 22 oscillates laser light. This laser light is mirror 2
It is reflected by 3, passes through the half mirror 24, and enters the two-dimensional scanning mechanism 25. The two-dimensional scanning mechanism 25 uses the XY scanning drive control circuit 3 based on a command from the computer 36.
The operation is started by the XY scanning XY scanning control signal generated by No. 4.

Therefore, here, the laser light is deflected in the X-axis and Y-axis directions similarly to the raster scanning of the television, and the revolver 2
6. Passing through the objective lens 27, the sample 2 on the stage 28
The light is focused on a small spot on the sample 9, and the spot position moves on the sample 29 by the XY scanning.

The light reflected from the sample 29 follows the incident optical path in the opposite direction, is reflected by the half mirror 24, and is reflected by the lens 3
It is focused at 0. A pinhole plate 31 is arranged at the light collecting position by the lens 30, and a photodetector 32 is provided behind it.
Is provided. Therefore, the light passing through the pinhole of the pinhole plate 31 is incident on the photodetector 32, and the photodetector 32 uses the light passing through the pinhole as reflected light of the sample 29 and generates electricity according to its brightness. Convert to signal.

Here, in the confocal microscope, when the diameter of the pinhole is sufficiently small, only the reflected light from the sample 29 from the in-focus position of the objective lens 27 passes through the pinhole. Therefore, only the reflected light on the sample surface at the in-focus position of the objective lens 27 will be detected by the photodetector 32.

The electric signal detected by the photodetector 32 is input to the image processing unit 33. As described above, the image processing unit 33 is provided with two image memories 33A and 33B each having a storage capacity of 512 pixels × 512 pixels × 8 bits (256 gradations) as one frame.

One of them, the image memory 33A stores the electric signal of the reflected light, and after the electric signal obtained by the photodetector 32 is A / D converted into a digital value, the spot light is emitted. Is stored and saved as data at a pixel position corresponding to the XY scanning position of.

This memory is updated and stored as the memory information of the pixel position when the level of the luminance signal of this time is higher than the luminance signal of the pixel position of the last time, and by doing so, the height position You will be able to add different images.

Further, in the image memory 33B, the number of times the revolver 26 moves from the Z scan drive control circuit 35 to the information on the Z scan direction to the pixel position corresponding to the current XY scan position of the spot light. The value of the number of times is given to the image processing unit 33, and the value of the number of times is stored in the image memory 33B as data when the level of the luminance signal at this time is higher than the luminance signal at the position at the previous time. Update and store.

However, the movement of the revolver 26 in the optical axis direction (Z-axis direction) is performed in reference width units every time XY scanning is completed based on a command from the computer 36 or the image determination circuit 33C. Then, every time the command is received, the Z scanning drive control circuit 35 generates a Z-axis direction control signal for the reference width unit to drive a revolver Z-axis direction scanning mechanism (not shown), and causes the revolver 26 to move by the reference width unit by Z. Move in the axial direction.

The moving amount of the revolver 26 is only the reference width unit for each command in this way, and the computer 36 or the image judging circuit 33C is required every time it is necessary to move the revolver 26.
Will issue a command from.

Then, when the extended focus is completed, the image focused on all the observation surfaces of the sample 29 and the information of the unevenness of the sample 29 stored in the image memories 33A and 33B are transferred to the computer 36, and the image processing is performed. And is displayed on the monitor 37.

Next, the schematic configuration of the image determination circuit 33C in the image processing unit 33, which is a feature of the present invention, will be described with reference to FIG.

As shown in FIG. 2, the image determination circuit 33C moves the in-focus position by changing the relative position between the observation sample 29 and the objective lens 27, and the in-focus position moving unit 4 moves.
The Z scan drive control circuit 35 as 7 is connected to the outside, and the end face detection unit 41 that detects the upper and lower end faces of the sample 29 by the detection signal of the photodetector 32 and the focus position of the sample 29 are moved to the focus position. The first when the end face detection signal from the end face detection unit 41 is received while moving in one direction by the unit 47
From the end face detector 41 while moving the focus position of the sample 29 in the opposite direction from the first focus position by the focus position moving unit 47. Storage unit 46 for storing the second focus position when the end face detection signal of the above is received, and a CPU for integrally controlling these
And 44.

The end face detector 41 further includes the photo detector 3
The number-of-pixels detection unit 42 that counts the number of pixels in which the detection signal No. 2 is equal to or lower than a predetermined level and determines whether the number of pixels is equal to or greater than a preset number of pixels in the screen; It is composed of an end face recognition section 43 which receives the judgment result and detects the end face of the sample 29.

FIG. 3 shows a specific circuit configuration of the image determination circuit 33C.

In FIG. 3, the pixel number detection unit 42 of the end face detection unit 41 includes a comparator 51, an AND circuit 52,
53, an OR circuit 54, a pixel counter 55, and a preset circuit 56 including a D flip-flop.

The comparator 51 inputs the brightness signal Vin obtained by the photodetector 32 to the minus input terminal and the preset setting voltage Vref to the plus input terminal, and its output is "Vin <Vref". Is "H" level, and when "Vin>Vref", it is "L" level and is input to the AND circuit 53. The setting voltage Vref is
When the position 9 is out of the in-focus position of the objective lens 27, the brightness signal Vin ideally becomes zero, but since it actually has a noise component, it is set as a constant voltage that is not zero. It is something.

The AND circuit 52 is supplied with the sampling clock SCLK and the data enable signal DE indicating the effective period of image capturing, and its output is supplied to the AND circuit 53.

However, the output of the AND circuit 53 is input to the clock terminal of the pixel counter 55 via the OR circuit 54.

The preset circuit 56 is composed of a D flip-flop, and its delay input terminal D receives a vertical synchronizing signal VD indicating the start of scanning in the vertical scanning direction, and its clock terminal receives start and end of an image in the horizontal scanning direction. Is input to the clock terminal of the pixel counter 55 via the OR circuit 54 as a preset signal.

The pixel counter 55 is loaded (LOAD).
By inputting the vertical synchronizing signal VD to the terminal, the number of detection pixels P is set in advance, and thereafter, by counting down, the luminance signal Vin, which is the detection signal from the photodetector 32, becomes less than or equal to the set voltage Vref. The number of pixels is counted, and the pixel detection signal 62 is input to the delay input terminal D of the frame detection circuit 58 which is a D flip-flop.

The end face recognizing unit 43 of the end face detecting unit 41 is
This frame detection circuit 58, an inverter 57 and a frame counter 59 are included.

The frame detection circuit 58 inputs a signal obtained by inverting the vertical synchronizing signal VD by the inverter 57 to the clock terminal, and indicates whether or not the inverted output is counted by the set number of pixels P or more in the previous frame. Enable terminal EN of the frame counter 59 as the surface detection signal 61
And a load (LOAD) terminal and the CPU 44.

The frame counter 59 is loaded (LOA
D) By inputting the surface detection signal 61 to the terminal, the preset detection frame number F is read, and thereafter, the surface detection signal 61 is used as an enable signal to perform the down count by the vertical synchronization signal VD input to the clock terminal. , The upper and lower end detection signal 60 indicating whether or not the preset number of pixels P is continuously counted by the preset number of pixels P or more
Output to PU44.

The CPU 44 uses the Z scanning drive control circuit 3 described above.
5 for giving a drive command at a predetermined timing for a predetermined time.
Is moved in the Z-axis direction to move the focus position of the objective lens 27 away from the sample 29, and the focus position at the time when the surface detection signal 61 is received from the frame detection circuit 58 is stored in the first storage unit 45. To the objective lens 27
The second storage unit 46 stores the focus position at the time when the in-plane detection signal 61 is received while moving the focus position of (1) toward the sample 29.

The operation of the embodiment configured as described above will be described. Here, in the sample 29 having the shape shown in FIGS. 9 and 5, the B (uppermost end) observation surface is first detected. Then, the operation of detecting the C (lowermost end) observation surface will be assumed and described. The observation surface of B and A
The observation is started from the position D between the observation planes.

FIG. 4 is a flow chart showing the processing operation of the CPU 44. When an extended focus start command is issued at the beginning of the processing, the scanning of the sample 29 is started (step S1) and the pixel counter 55 is preset. The detected pixel number P is written in the frame counter 59, and the preset detected frame number F is written in the frame counter 59 (step S2).

Here, the detection frame number F is "F> H / d", where H is the maximum step between the surfaces of the sample 29 adjacent in the height direction and d is the reference width unit for moving the focus position. It will be an integer.

To write the detected pixel number P, the preset circuit 56 based on the vertical synchronizing signal VD and the horizontal synchronizing signal HD is used.
The preset signal 63 output by is used.

The vertical synchronizing signal VD is input to the load terminal of the pixel counter 55, so that the pixel counter 55 can read the preset value (the number of detected pixels P). The preset signal 63, which is the output from the preset circuit 56, passes through the OR circuit 54 and enters the clock terminal of the pixel counter 55, so that the preset value (the number of detected pixels P) set in advance is taken into the pixel counter 55. Be done.

Further, the vertical sync signal VD and the surface detection signal 61 output from the frame detection circuit 58 are used for writing the detected frame number F.

The surface detection signal 61 is at the "L" level at the beginning of the frame because the pixel detection signal 62 is at the "H" level immediately after the start of scanning, and is input to the load terminal of the frame counter 59. As a result, the frame counter 59 is made ready to read the preset value (the number of detected frames F). When the vertical synchronizing signal VD enters the clock terminal of the frame counter 59, the preset value (the number of detected frames F) set in advance is taken into the frame counter 59.

FIG. 6 shows the vertical synchronizing signal VD, the pixel detection signal 62, the surface detection signal 61 and the frame counter 5 at this time.
9 shows the count value of 9, and it can be seen that the count value of the frame counter 59 shown in FIG. 6 (4) is set to the preset value (the number of detected frames F) at the timing indicated by I in the figure.

Next, the image signal Vin photoelectrically converted by the photodetector 32 is input to the minus input terminal of the comparator 51. As described above, the preset voltage Vref set in advance is input to the plus input terminal of the comparator 1.
The comparator 51 determines whether "Vin <Vref" (step S3).

If “Vin <Vref”, the output of the comparator 51 is “H” level, and if “Vin> Vref”, the output of the comparator 51 is “L” level. Is at the "H" level, that is, when "Vin <Vref", the AND circuit 53 is in the gate open state.

Therefore, since the AND circuit 52 is also in the gate open state when the data enable signal corresponding to the effective period of the image is at the "H" level, the sampling clock SCLK passes through these AND circuits 52 and 53. Pixel counter 5 is extracted and passed through OR circuit 54.
5 is input to the clock terminal, and as a result, "Vin <Vre
The number of pixels when "f" is reached is counted by the pixel counter 55 (step S4).

The count value of the pixel counter 55 is constantly compared with the number of detected pixels P during scanning to determine whether or not the number of detected pixels P has been reached (step S5).

In this case, the pixel counter 55 counts down each time the sampling clock SCLK is input from the preset value (the number of detected pixels P) as described above, and when the count value becomes "0". , That is, the sampling clock SCLK for the number of detected pixels P
At the time of counting, the output pixel detection signal 62 is changed from “H” level to “L” level, the counting operation is automatically stopped, and the state of the pixel detection signal 62 is maintained.

If the pixel detection signal 62 is not output when the scanning of one screen is completed (step S1)
0, S11 or step S9), the CPU 44 outputs an operation command to the Z scanning drive control circuit 35 so that the in-focus position moves to the upper direction of the sample 29 (step S14), and the next scanning position is set from step S2. The pixel detection process of is repeatedly executed.

On the other hand, the pixel detection signal 62 and the inverter 57
The vertical synchronizing signal VD inverted by the frame detection circuit 58
Is input to the frame, the state of the pixel detection signal 62 is checked at the beginning of each frame.

When the pixel detection signal 62 is input, the frame detection circuit 58 changes the surface detection signal 61 from the "L" level to the "H" level at the beginning of the next frame. Due to this change, the frame counter 59, which has been in the state of reading the preset value until then, becomes the operating state and counts the input vertical synchronizing signal VD (step S7).

The count value of the frame counter 59 is constantly compared with the detected frame number F to determine whether or not the detected frame number F has been reached (step S8).

Number of detected frames F and frame counter 59
If the count values are not equal to each other (steps S12 and S13), the CPU 44 outputs an operation command to the Z scanning drive control circuit 35 so that the focus position moves to the upper direction of the sample 29 (step S14). ), The pixel detection process from step S2 is repeatedly performed at the next scanning position.

By the way, the surface detection signal 61 is inputted to the CPU 44 to inform whether or not the previous frame has reached the number of detected pixels, and the pixel detection signal 6 is continuously supplied every frame.
If 2 is output, it remains at "H" level, but if it remains at "H" level in a certain frame and the pixel detection signal 62 is interrupted even once, it changes from "H" level to "L" level ( Step S6).

This change means that the observation surface was still present while the focus position was moved and the end face was being detected. At the same time frame counter 59
Will be in the preset state again, and the count value up to that point will be abandoned.

Therefore, in response to this change, the CPU 44 determines that the position immediately before the current in-focus position is the upper end face and stores it in the first storage section 45. In the above FIG. 5, D
A pixel detection signal 62 is output at the timing indicated by II in FIG. 6 (2) during the scanning starting from the surface, and the surface detection signal 61 is outputted at the timing indicated by III in FIG. 6 (3) at the beginning of the next frame. Is output and the frame counter 59 enters the operating state.

The D surface, D + d surface, D + 2d surface, ...
When the reference width unit d is scanned, the pixel detection signal 62 is output for each frame, and the frame counter 59 counts the input vertical synchronization signal VD.

However, the number of detected frames is initially F
Since it is impossible for the frame counter 59 to count up to the number F of detection frames in the middle of the step of the sample 29, the pixel detection signal 62 remains at the “H” level when it reaches the observation surface B. And will not be output.
Therefore, at the beginning of the scanning of the B + d surface, IV in FIG.
The surface detection signal 61 changes from the “H” level to the “L” level at the timing shown by.

At the same time, the frame counter 59 is again in the preset state, and the count value up to that point is abandoned. At this time, the CPU 44 sets the current focus position to 1
The previous one, that is, the B side as the upper end side, is updated and stored in the first storage section 45.

Then, scanning is continuously performed, and B + d
The pixel detection signal 62 is output again on the surface. FIG. 7 shows the vertical synchronizing signal VD, the pixel detection signal 62, the surface detection signal 61, the count value of the frame counter 59, and the upper and lower end detection signals 60 at this time, at the timing indicated by I in FIG. 7 (2). The pixel detection signal 62 is output, the surface detection signal 61 is output at the timing of II shown in FIG. 7 (3) at the beginning of the next frame, and the frame counter 59 enters the operating state as shown in FIG. 7 (4). .

This time, since the frame counter 59 is in operation for each frame, it counts down each time the vertical synchronizing signal VD is input from the number of detected frames F which is a preset value, and the E plane shown in FIG. When the count value reaches “0”, the CPU 44 is notified that the upper end surface of the sample 29 has been exceeded.

As a result, the CPU 44 can recognize that the focus position finally stored in the first storage unit 45, that is, the position of the surface B in FIG. 5 is the uppermost end surface of the sample 29. .

Further, when the scanning is started from the G plane located above the B plane in FIG. 5, that is, even if the focusing position is moved upward, the observation plane appears once within the range of F × d. When the surface detection signal 61 has not changed from the “H” level to the “L” level but the upper and lower end detection signals 60 have changed from the “H” level to the “L” level, the scanning start G is started. It is determined that the surface position has already exceeded the uppermost position of the sample 29, and the position where the surface detection signal 61 changes from the “H” level to the “L” level while approaching the sample 29 from the G surface, that is, If the focus position is moved to a position recognized as the observation surface, the uppermost B surface can be detected.

At this time, since it may be considered that the distance between the G surface and the B surface is larger than the range of F × d, the CPU 44 ignores the upper and lower end detection signal 60 without considering it.

The above description is a method of detecting the B surface which is the uppermost end of the sample 29, and the detection of the C surface which is the lowermost end uses the drive command to the Z scan drive control circuit 35 as a command in the opposite direction. If the scanning is performed while sequentially moving the focus position downward from the detected uppermost B surface according to the flowchart of FIG. 4, the lowermost C surface can be detected in the same manner. Then, the explanation is omitted.

However, in the case of detecting the lowermost end, it is not always possible to move the focus position indefinitely, and within the range of the distance WD from the lower end position of the objective lens 27 to the focus position. Set a limit to do.

As a result, the number of steps that can be moved for detection is within "WD / d". If detection is not possible within this range, the position from the uppermost end to WD is set as the lowermost end. Or issue a warning and stop the operation.

Finally, while the CPU 44 sequentially moves the in-focus position from the maximized end to the uppermost end in a step different from the above d specified by the CPU 44 for the Z scan drive control circuit 35,
By holding the value at the maximum brightness for each pixel as the brightness information, it is possible to obtain the image of the sample 29 in which any pixel is in focus.

Further, the height information can be obtained by holding the amount of movement of the Z scan drive control circuit 35 until the brightness of each pixel becomes maximum for each pixel.

In the above embodiment, the Z scanning drive control circuit 35 is described as moving the revolver 26 in the Z axis direction to move the in-focus position, but the objective lens 27 is used.
Since it suffices to move the relative positions of the sample 29 and the sample 29 in the Z-axis direction, the objective lens itself or the stage 28 may be moved in the Z-axis direction in addition to the revolver 26. The stage 28 may be moved in the Z-axis direction.

According to the above-described embodiment, the photodetector 32 is used in the confocal optical system when the sample is located at a position other than the in-focus position, or when the sample is standing even at the in-focus position.
The following is based on the fact that the signal level from is zero.

That is, the focus position is moved up and down by the Z scanning drive control circuit 35, and the signal level from the photodetector 32 is continuously zero over a plurality of frames in addition to the ratio of the focus in the screen. by confirming the one in which the last case was confirmed image focus position can be reliably determined that the uppermost end or outermost lower end of the sample 29.

Therefore, even if the sample has a shape which is difficult to judge by the confocal optical system, the automatic setting of the uppermost end portion and the lowermost end portion can be surely performed.

Further, the uppermost end is first detected, and then the lowermost end is detected by limiting to the range of the distance WD from the front end position of the objective lens to the focal position. Can be surely prevented from interfering with each other.

The above description is based on the embodiments, but the present invention includes the following inventions.

(1) The observation sample supported on the stage is illuminated by a point light source through an objective lens, and the light illuminated on the observation sample is condensed through the objective lens and is again formed into a point shape. In a scanning confocal microscope that obtains an image and obtains density information of an image by a detector, a moving unit that moves a focusing position by changing a relative position between the observation sample supported on the stage and the objective lens, When the end face detection means for detecting the upper and lower end faces of the observation sample by the detection signal of the detector and the end face detection signal from the end face detection means while moving the focusing position of the observation sample in one direction by the moving means. First storage means for storing the first focus position of the first sample and the focus position of the observation sample by the moving means.
Second storage means for storing a second focus position when the end face detection signal from the end face detection means is received while moving in the opposite direction from the focus position Automatic image forming device for confocal microscope.

By doing so, it is possible to move the focus position even with respect to the observation samples having different observation surface heights, reliably detect the upper and lower ends thereof, and perform automatic setting.

(2) In the automatic image forming apparatus for a scanning confocal microscope according to (1), the one direction is a direction in which the observation sample moves away from the objective lens, and the observation sample is moved in the opposite direction. An automatic image forming apparatus for a scanning confocal microscope, further comprising: movement limiting means for limiting a range of relative positions between the objective lens and the objective lens.

In this way, in addition to the effect of item (1), when the focus position is moved to automatically set the upper and lower ends of the observation sample, interference between the sample and the objective lens can be prevented. it can.

[0109]

According to the present invention, it is possible to reliably detect the upper and lower ends of an observation sample having an observation surface having a different height, and automatically set the observation sample. Further, it is possible to prevent interference between the objective lens and the sample. Even when a large amount of specimens are observed to obtain focused images of each specimen, it is possible to prevent the occurrence of setting errors due to individual differences of the observers and to significantly reduce the burden on the observers due to repetitive work. Scanning type
An automatic image forming apparatus for a microscope can be provided.

[Brief description of drawings]

FIG. 1 is a block diagram showing a configuration of an entire system of an automatic image forming apparatus of a scanning confocal microscope according to an embodiment of the present invention.

FIG. 2 is a block diagram showing a conceptual configuration in the image determination circuit of FIG.

3 is a diagram showing a specific circuit configuration of FIG.

FIG. 4 is a flowchart showing the processing content according to the embodiment.

FIG. 5 is a view for explaining control of a focus position for an observation sample according to the same embodiment.

6 is a timing chart showing each signal waveform in the circuit of FIG.

7 is a timing chart showing each signal waveform in the circuit of FIG.

FIG. 8 is a schematic configuration diagram of a conventional scanning microscope having a confocal optical system.

9 is a perspective view of an observation sample for explaining the problem of FIG. 8. FIG.

[Explanation of symbols]

21 ... Scanning confocal microscope 22 ... Laser light source 23 ... Mirror 24 ... Half mirror 25 ... Two-dimensional scanning mechanism 26 ... Revolver 27 ... Objective lens 28 ... Stage 29 ... Observation sample 30 ... Lens 31 ... Pinhole plate 32 ... Photodetector 33 ... Image processing unit 33A, 33B ... Image memory 33C ... Image determination circuit 34 ... XY scanning drive control circuit 35 ... Z scan drive control circuit 36 ... Computer 37 ... Monitor 41 ... End face detector 42 ... Pixel number detection unit 43 ... Edge recognition unit 44 ... CPU 45 ... First storage unit 46 ... Second storage unit 47 ... Focus position moving unit 51 ... Comparator 52, 53 ... AND circuit 54 ... OR circuit 55 ... Pixel counter 56 ... Preset circuit 57 ... Inverter 58 ... Frame detection circuit 59 ... Frame counter

Claims (4)

(57) [Claims] 1. An observation sample placed on a stage is spotted.
Irradiate with a light source to re-illuminate the light irradiated on the observation sample.
And an objective lens that collects light and the above-mentioned observation sample on the observation sample that is collected by the objective lens.
A scanning unit that relatively scans the light and the observation sample, a light detection unit that detects reflected light from the observation sample, a relative focus position of the objective lens and the observation sample.
Scanning microscope having a moving means for moving the position in the optical axis direction
A microscope, which is a detection frame setting means for setting the number of detection frames and a signal for which the level of the detection signal of the light detection means is preset.
The number of pixels below the signal level is counted, and this number of pixels is calculated in advance.
Pixel count detection to determine whether the number of pixels is greater than or equal to the set number
The end surface of the observation sample based on the result of the output means and the pixel number detection means.
An end face detection means for detecting, storing means for storing the detected position by the end face detecting means
And is moved in one direction by the moving means to move the end face detecting hand.
The position detected by the step is held and the detection frame is
Based on the number of dome
Move and check the signal level from the photo detector before
An automatic image forming apparatus for a scanning microscope, characterized in that the stored position is the end surface of the observation sample . 2. The storage means is stored by the moving means.
Direction, and detected by the signal from the end face detection means
Moving a first storage means for storing the position, from the stored position to said first memory means in the opposite direction
The detection position is recorded by the signal from the end face detecting means.
A contract comprising a second storage means for storing
An automatic image forming apparatus for a scanning microscope according to claim 1 . 3. One direction moved by said moving means
Is the direction in which the observation sample and the objective lens move away from each other.
In the case of moving in the opposite direction to the one direction,
Characterized by limiting the range of relative movement with the objective lens
The automatic image forming apparatus for a scanning microscope according to claim 1. 4. The relative between the observation sample and the objective lens
The range of movement is from the tip position of the objective lens to the focus position.
4. The range of the distance up to
Image forming apparatus for scanning microscope.