JP3955115B2 - Confocal microscope focusing device and focusing method - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
  The present invention relates to a focusing device applied to a confocal microscope.And focusing methodIt is about.
[0002]
[Prior art]
2. Description of the Related Art Conventionally, as a focusing device widely used, a device that performs focus detection by treating a contrast value appearing in a high-frequency component of an image signal as a defocus amount is known. That is, in such a focusing apparatus, the captured image of the sample is captured by the CCD, and the contrast value appearing in the high-frequency component of the image signal at this time becomes the maximum at the in-focus position as shown in FIG. Focusing control is performed by a so-called hill-climbing servo system using the characteristic that the contrast value decreases as the amount increases.
[0003]
According to such a method, since the change in the contrast value becomes gentle in the vicinity of the in-focus position, the in-focus position is found in this gentle range. The accuracy will be high.
[0004]
[Problems to be solved by the invention]
However, in practice, it is difficult to determine the in-focus position from a gentle range of contrast value changes, and the in-focus position may vary to some extent. On the other hand, for example, in the case of an optical microscope using a CCD camera, since the depth of focus of the observation optical system is large, the variation in the focus position of the captured image of the CCD camera is absorbed, and the focused image However, in the case of a confocal microscope, since the depth of focus of the confocal image is very small, variations in focus position may not be completely absorbed, resulting in an out-of-focus image.
[0005]
  Further, conventionally, since the contrast value is used, there is a problem that a region having high contrast in the observation range is focused, and this does not always focus on a place where observation is desired. The present invention has been made in view of the above circumstances, and is a confocal microscope focusing device capable of realizing stable automatic focusing.And focusing methodThe purpose is to provide.
[0006]
[Means for Solving the Problems]
  According to the first aspect of the present invention, the sample is scanned with the light from the light source, and the confocal light that enables the confocal detection of the sample image corresponding to the scanningSpotlightIn a confocal microscope with an academic system,
  A detection optical system having a depth of focus deeper than the confocal optical system and capable of detecting the sample image;
  Sample image detected by this detection optical systemFromSetting means for setting an arbitrary position information on the sample image and a moving amount for each movement of the moving range of the sample in the optical axis direction;
  Storage means for storing image data corresponding to the arbitrary position information for the sample image detected by the confocal optical system for each movement according to the amount of movement set once in the setting means;
  The in-focus position calculating means calculates the in-focus position corresponding to the maximum luminance from the image data stored in the storage means.
[0007]
The invention described in claim 2 is the storage device according to claim 1, wherein the storage means stores the average value of the image data corresponding to the arbitrary position information and the image data in the vicinity of the image data, and the in-focus position calculation means includes: An in-focus position corresponding to the maximum luminance is calculated from the averaged image data stored in the storage means.
[0008]
  A third aspect of the present invention is the first aspect according to the first aspect, wherein the arbitrary position of the sample image detected by the confocal optical system for each movement according to a single movement amount set in the setting unit. A second storage means for storing a predetermined range of image data set together with the image data corresponding to the information, and processing the image data stored in the second storage means with a predetermined weight to obtain the arbitrary data Image data processing means for replacing with image data corresponding to the position information,
The image data replaced by the image data processing means is stored in the storage means as image data corresponding to the arbitrary position information, and the in-focus position corresponding to the maximum luminance is calculated from the image data stored in the storage means. Like to do.
  According to a fourth aspect of the present invention, in any one of the first to third aspects, the apparatus further includes arithmetic means for performing a polynomial operation on the discrete data output by the confocal optical system. The in-focus position at the maximum brightness is obtained from the calculation result of the calculation means.
  According to the fifth aspect of the present invention, the sample is scanned with light from the light source, and confocal detection of the sample image corresponding to the scanning is enabled.SpotlightA focusing method for a confocal microscope having an academic system, wherein the depth of focus is deeper than that of the confocal optical system.With detection optical system havingThe sample image is detected, and the detected sample imageFromArbitrary position information on the sample image and a movement amount for each movement of the movement range of the sample in the optical axis direction are set, and the confocal optical system for each movement according to the set one movement amount The image data corresponding to the arbitrary position information is stored for the sample image detected by the above, and the in-focus position corresponding to the maximum luminance is calculated from the stored image data.
[0009]
  As a result, according to the first aspect of the present invention, a detection optical system having a depth of focus deeper than that of the confocal optical system is used in advance, and a sample image obtained by the detection optical system.By thisArbitrary position information on the sample image and the movement amount for each movement of the movement range in the optical axis direction of the sample are set and detected by the confocal optical system for each movement according to the set movement amount. Image data corresponding to arbitrary position information is stored in the storage means, and the in-focus position corresponding to the maximum luminance is calculated from the stored image data. A plurality of points of data are captured as in-focus candidates, and automatic focusing with high accuracy can be realized at a position to be focused as compared with the case where only one point of image data is employed from the beginning.
[0010]
  According to the second aspect of the present invention, a stable luminance distribution in the direction of the optical axis can be obtained even when the sample surface is uneven, and stable automatic focusing can be realized.
  According to the third aspect of the present invention, even when the boundary of the step portion of the sample having a step is indicated, the upper step or the lower step of the step can be stably detected, and the stable automatic focusing can be performed. Can be realized.
According to the invention of claim 4, it has a calculation means for performing a polynomial calculation on the discrete data detected by the confocal optical system. From the calculation result of the calculation means, the maximum brightness is obtained. The in-focus position can be obtained, and stable automatic focusing can be realized.
According to the invention described in claim 5, the confocal microscope having the confocal detection optical system that scans the sample with the light from the light source and enables the confocal detection of the sample image according to the scan. The sample image having a depth of focus deeper than that of the confocal optical system is detected, and an arbitrary position information on the sample image and an optical axis direction of the sample are detected based on the detected sample image. Image data corresponding to the arbitrary position information is set for the sample image detected by the confocal optical system for each movement corresponding to the set one movement amount. Since the focus position corresponding to the maximum brightness is calculated from the stored image data, a plurality of points of data are captured as focus candidates in the image data at the position where the focus is desired. Only one point of image data from the beginning Compared to employ, it can be realized focus highly accurate automatic focus with respect to the position to be focused.
[0011]
DETAILED DESCRIPTION OF THE INVENTION
Hereinafter, embodiments of the present invention will be described with reference to the drawings.
(First embodiment)
FIG. 1 shows a schematic configuration of a focusing device of a confocal microscope to which the present invention is applied. In the figure, reference numeral 1 denotes a confocal microscope main body. The confocal microscope main body 1 includes a laser light source 2, a scanning optical system 3, an objective lens 4, a beam splitter 5, a mirror 6, a confocal optical system 7, and a non-confocal optical system. An optical system 8 and a stage 10 are provided.
[0012]
The laser light output from the laser light source 2 enters the scanning optical system 3 via the beam splitter 5, and irradiates the sample 9 on the stage 10 through the objective lens 4.
[0013]
Here, the scanning optical system 3 is for scanning a laser beam two-dimensionally, has two optical deflectors, the first optical deflector deflects the laser light in the X direction, and the second light By deflecting in the Y direction by a deflector, the laser light is deflected two-dimensionally along a plane perpendicular to the optical axis. Note that scanning control for the scanning optical system 3 is performed by an instruction from the computer 16 via a controller box 11 described later. As a result, the laser beam condensed on the sample 9 of the stage 10 through the objective lens 4 is made into a minute spot, and the sample 9 is set in the same manner as the TV raster scan in accordance with the scanning control of the scanning optical system 3. It moves in two dimensions.
[0014]
On the other hand, the laser beam reflected from the sample 9 is returned to the opposite direction of the incident optical path, reflected by the beam splitter 5 and incident on the mirror 6. The mirror 6 can be inserted into and removed from the optical path. When the mirror 6 is inserted into the optical path, the reflected light enters the confocal optical system 7 to obtain a confocal image, and the mirror 6 is removed from the optical path. In this case, the reflected light is incident on the non-confocal optical system 8 so that a non-confocal image can be obtained.
[0015]
A control box 11 is connected to the confocal microscope main body 1 configured as described above, and a computer 16 is connected to the control box 11. The control box 11 includes an image memory 12, a line memory 13, and an arithmetic circuit 14.
[0016]
The image memory 12 stores image signals from the confocal optical system 7 and the non-confocal optical system 8 as luminance data. The luminance image in this case is updated every time the XY scanning described above is performed. The output data of the image memory 12 is sent to the computer 16 and displayed on the display 17 as the luminance signal of the sample 9, and a part of this output data is sent to the line memory 13.
[0017]
The line memory 13 fetches luminance data from the image memory 12 and saves it every time the sample 9 moves in the optical axis direction. The movement of the sample 9 in the optical axis direction here is realized by moving the stage 10 in the optical axis direction by the Z drive circuit 15 according to an instruction from the computer 16. In addition, the stage 10 is automatically driven in accordance with a certain process during automatic focusing.
[0018]
The arithmetic circuit 14 calculates the in-focus position based on the luminance data of the line memory 13 and the movement data in the optical axis direction from the computer 16, and sends the calculation result to the computer 16 for the Z drive circuit 15. By outputting a driving signal, the stage 10 is moved to the in-focus position.
[0019]
Here, the stage 10 can be freely controlled by the observer from the computer 16 except during automatic focusing.
Reference numeral 18 denotes a mouse connected to the computer 16.
[0020]
Next, the operation of the embodiment configured as described above will be described.
First, the sample 9 is first placed on the stage 10 and a laser scanning instruction is issued from the computer 16. In this case, it is set in advance that the mirror 6 is removed from the optical path, the reflected light from the sample 9 is incident on the non-confocal optical system 8, and an image from the non-confocal optical system 8 is displayed on the display 17. . This is because the non-confocal optical system 8 can obtain an image with a deeper depth of focus than the image of the confocal optical system 7, so that the position on the sample 9 to be automatically focused can be easily set. Is also necessary.
[0021]
In this state, when the laser beam from the laser light source 2 is irradiated onto the sample 9 on the stage 10 through the beam splitter 5, the scanning optical system 3, and the objective lens 4 by starting the laser scanning, the reflected light from the sample 9 is incident on the optical path. In reverse, the light is incident on the non-confocal optical system 8 via the beam splitter 5. As a result, the image signal from the non-confocal optical system 8 is stored in the image memory 12, and the output data of the image memory 12 is sent from the computer 16 to the display 17.
[0022]
However, if the sample 9 is greatly deviated from the in-focus position, the output data of the image memory 12 is almost zero, so that nothing is displayed on the display 17.
[0023]
Therefore, the observer gives an instruction to the computer 16 to move the stage 10 or rotate the coarse movement handle of the stage 10 (not shown) to move the sample 9 to a rough focus position.
[0024]
In this state, for example, if the surface of the sample 9 has A, B, and C surfaces with different heights as shown in FIG. As shown in FIG. 3, the image of the sample 9 displayed on the surface B is substantially in focus on the B surface, and is not in focus on the A surface and C surface in the shaded area in the figure. .
[0025]
From this point, in order to focus on the out-of-focus C-plane, first, in order to indicate a new focusing position, the C-plane to be focused is indicated by an arrow M as shown in FIG. Specifically, the observer operates the mouse 18 connected to the computer 16 to move the arrow M to the position X to be focused on the C plane, and when the arrow M has moved to the target position. A click operation is performed with the mouse 18, and the information on the position X is stored in the computer 16.
[0026]
In this case, the position of the image displayed on the display 17 has a one-to-one correspondence with the pixel of the image memory 12 of the control box 11, so the information of the position X input by the click operation of the mouse 18 is the control box. 11, the pixel data of the image memory 12 corresponding to the position X is output to the line memory 13.
[0027]
Next, in order to obtain the luminance distribution in the optical axis direction at the position X, the sample 9 is moved in the optical axis direction. In this case, as described above, the luminance distribution data is stored in the line memory 13 every time the sample 9 is moved, so that the sample 9 is moved discretely. Here, if the amount of movement of the sample 9 per time is made finer, the amount of information increases, so the accuracy of the calculation result of the subsequent in-focus position increases. However, it takes time to collect data and a quick in-focus operation is performed. I can't hope. Therefore, the observer sets the movement range and the movement amount in one optical axis direction while taking this point into consideration.
[0028]
Thereafter, when the setting of the movement range of the sample 9 and one movement amount in the optical axis direction is completed, the observer gives an instruction for automatic focusing to the computer 16.
In this case, the flow shown in FIG. 4 is executed by the computer 16. First, in step 401, the contents of the line memory 13 are cleared. Next, in step 402, the confocal optical system 7 is selected by inserting the mirror 6 into the optical path and causing the reflected light from the sample 9 to enter the confocal optical system 7. In this case, since the confocal optical system 7 can obtain an image by an optical system common to the non-confocal optical system 8, the position X set in the non-confocal optical system 8 is completely the same even in the confocal optical system 7. At the same position.
[0029]
Next, in step 403, the sample 9 is moved to the upper limit position or the lower limit position of the movement range. Here, it is assumed to move to the upper limit position.
From this state, in step 404, laser scanning is started. Then, the reflected light from the sample 9 is incident on the confocal optical system 7 this time via the beam splitter 5 and the mirror 6. In this case, in the confocal optical system 7, since the reflected light is blocked by the pinhole unless the sample 9 is in the in-focus position, data having a value close to zero is stored in the image memory 12.
[0030]
Thereafter, when one laser scan is completed in step 405, pixel data corresponding to the position X in the image memory 12 is output to the line memory 13 and stored in step 406. Then, in step 408, the stage 10 is moved in the lower limit direction along the optical axis by a preset amount of movement. When this movement is completed, the process returns to step 404 and laser scanning is performed again. Such an operation is repeatedly executed until it is detected in step 407 that the stage 10 reaches the lower limit position.
[0031]
When the stage 10 reaches the lower limit position, all operations are temporarily stopped and the in-focus position is calculated based on the data in the line memory 13.
In this case, since the confocal optical system 7 has a very shallow depth of focus, the luminance intensity changes rapidly in the vicinity of the in-focus position. Therefore, if the position having the maximum luminance intensity is obtained, the in-focus position can be detected.
[0032]
FIG. 5 shows an example of changes in luminance data in the line memory 13 when the horizontal axis represents the movement distance of the stage 10 in the optical axis direction. In this case, as apparent from the figure, the luminance data of the line memory 13 is discrete, so that when this data is used as it is, for example, there is true maximum luminance data between the luminance data a and the luminance data b. In this case, the luminance data a is determined as the maximum luminance. That is, if an in-focus position is obtained only from the luminance data of the line memory 13, a deviation from the true in-focus position may occur.
[0033]
Therefore, in the present invention, in order to obtain the true in-focus position, in step 409, the arithmetic circuit 14 further calculates a quadratic or cubic polynomial approximation on the discrete data in the line memory 13, and this calculation is performed. From the result, in step 410, the stage position (focus position) at the maximum luminance is obtained.
[0034]
In this case, the calculation result of the calculation circuit 14 is as shown in FIG. 6, and the true maximum luminance is obtained and the true in-focus position is determined as is apparent from the figure. Here, as a polynomial algorithm calculated by the arithmetic circuit 14, a method generalized by a known optimization method using spline interpolation or the like is used. Thus, the arithmetic circuit 14 can be realized by a one-chip microcomputer.
[0035]
Then, the true in-focus position information obtained by the arithmetic circuit 14 is sent to the computer 16, and the computer 16 outputs a movement command to the Z drive circuit 15 in step 411 based on the sent information. . As a result, the stage 10 moves to the in-focus position, so that the sample 9 is accurately focused.
[0036]
Accordingly, in this way, a non-confocal optical system 8 having a depth of focus deeper than that of the confocal optical system 7 is prepared in advance, and a position to be focused from the sample image obtained by the non-confocal optical system 8 X and the movement distance of the sample 9 in the direction of the optical axis are set for each movement, and then the sample is switched to the confocal optical system 7 for each movement according to the movement amount set in advance. Since the image data corresponding to the position X for the image is stored in the line memory 13 and the in-focus position corresponding to the maximum luminance is calculated from the stored image data by the arithmetic circuit 14, the in-focus position X to be focused is calculated. As a result, multiple points of data can be captured as in-focus candidates, and automatic focusing with higher accuracy is possible for the position to be in focus than when only one point of image data is used from the beginning. This makes it possible for microscopic observation Always allowing accurate observation by eliminating the influence of the cement displacement.
(Second Embodiment)
FIG. 7 shows a schematic configuration of the second embodiment, and the same components as those in FIG.
[0037]
In this case, a TV image is used for the confocal microscope main body 1 to obtain the first in-focus position. Here, in order to obtain a TV image, a white light source 60, a half mirror 61, a switching mirror 62, and an image sensor 63 are provided instead of the non-confocal optical system 8. The white light source 60 is the same as that used in a conventional optical microscope such as a halogen lamp. The illumination light from the white light source 60 passes through the half mirror 61 and is reflected by the switching mirror 62 inserted between the scanning optical system 3 and the objective lens 4 to illuminate the sample 9 via the objective lens 4. ing. Further, the reflected light from the sample 9 is reflected by the switching mirror 62 and further reflected by the half mirror 61 so as to be condensed on the image sensor 63. Here, the switching mirror 62 is retracted from the optical path when automatic focusing is started.
[0038]
A CCD is used for the image sensor 63 and captures an image of the sample 9. Then, a signal from the image sensor 63 is taken into the computer 16 via the control box 11 and displayed on the display 17.
[0039]
Thus, the image obtained by the image sensor 63 is the same as the non-confocal optical system 8 in the first embodiment, and is an image having a deeper focal depth than the confocal optical system 7. As described in the description of the optical system 8, it can be used as an image when determining the first in-focus position. In this case, if a color CCD is used as the image sensor 63, the color information of the sample 9 can also be obtained, so that the position setting is further facilitated.
[0040]
In the case of the second embodiment, the in-focus position is set by a TV image, and the in-focus processing is performed by the confocal optical system 7, and two different optical systems are used. Each positional relationship needs to be adjusted in advance. For example, a sample such as a scale is displayed by superimposing a TV image and an image of the confocal optical system 7 by image processing, and the scanning optical system is swung so that the image of the confocal optical system 7 matches the TV image. The width may be adjusted.
[0041]
Accordingly, even in this way, the same effect as described in the first embodiment can be expected.
In the second embodiment, an average processing circuit 64 is further arranged between the image memory 12 and the line memory 13.
[0042]
That is, in the first embodiment, information on one point on the image is stored in the line memory 13. In this case, if the surface of the sample 9 is a mirror surface, there is no problem with information on one point, but if there are fine irregularities on the entire surface, the amount of reflected light may become unstable depending on the state of the sample surface. In such a state, luminance distribution data as shown in FIG. 5 cannot be obtained.
[0043]
Therefore, in the second embodiment, the luminance data at the position X designated in FIG. 3 and the luminance data of the surrounding image are averaged by the average processing circuit 64 are used. Thereby, even when the sample surface has irregularities, a stable amount of reflected light can be secured, so that accurate luminance data can be secured.
[0044]
The range around the position X for averaging can be freely set by the observer using the computer 16.
Therefore, in this way, a stable luminance distribution in the direction of the optical axis can be obtained even if the surface of the sample 9 is uneven, and stable automatic focusing can be realized.
(Third embodiment)
In the first embodiment, one point of image data at the position X input and instructed by the mouse 18 is sequentially stored in the line memory 13, and in the second embodiment, minute data is stored. The average value of the image signal of the area is stored in the line memory 13 so that stable luminance data can be obtained even when the amount of reflected light is unstable. For example, there is a step on the sample 9. In some cases, when the instructed sample ascending position is at the step boundary, it may be unstable which position of the upper or lower step is detected as a luminance peak.
[0045]
Therefore, in the third embodiment, even when a boundary portion of a sample having a step is indicated, the upper or lower step of the step can be stably detected.
In this case, FIG. 8 shows a main part of the third embodiment, and a spatial memory 70 and a filter operation mechanism 71 are inserted between the image memory 12 and the line memory 13. Others are the same as FIG.
[0046]
That is, in the third embodiment, the line memory 13 of the first embodiment described above is a one-dimensional one that stores the contents of one point of image data that is the designated position in the order of observation height. However, in addition to the line memory 13, a three-dimensional space memory 70 is used as shown in FIG.
[0047]
The space memory 70 can simultaneously store the contents of the image data at the designated position and the contents of the image data in the vicinity thereof. That is, the value of the image data at the designated position at the first observation height position is stored in e of the first layer of the space memory 70, and the image data in the vicinity thereof is stored in a, b, c, d, f, g, The h and i are stored corresponding to the respective positions.
[0048]
Then, as in the case of the line memory 13, the observation position is changed in order, and the image data at each height position is stored in order from the second hierarchy to the third hierarchy of the space memory 70. At this time, the image data at the designated position is stored in e as in the first layer, and the image data in the vicinity thereof is stored in positions a, b, c, d, f, g, h, i. I am doing so.
[0049]
The filter calculation mechanism 71 performs calculation processing for determining a representative value in each layer of the space memory 70. This filter operation mechanism 71 can be easily realized by incorporating a one-chip microcomputer, and can also be realized by performing arithmetic processing using an arithmetic circuit.
[0050]
That is, calculation processing is executed by the filter calculation mechanism 71 on the image data corresponding to the indicated position stored in the spatial memory 70 and the observation height position in the vicinity thereof, and each value after the filter processing as shown in FIG. The representative values of the hierarchies are obtained, the representative values of the respective hierarchies are stored in the line memory 13, and the upper and lower steps of the steps are selectively detected by the same processing as described in the first embodiment. .
[0051]
In FIG. 10, 72 represents image data of each layer stored in the spatial memory 70, and this is processed by the image filter coefficient of the filter 73 of the filter calculation mechanism 71 to obtain the output representative value 74 of each layer. I have to.
[0052]
Each image filter coefficient in the filter 73 here is called an X-direction Sobel filter. When the luminance changes from left to right in the X direction, the output representative value increases, and conversely, the luminance from left to right. The output representative value becomes larger in the negative direction when the value changes to a lower value.
[0053]
Thus, now, in the case of the sample 9 having a step as shown in FIG. 11, the observation images when the respective positions (1), (2), and (3) in the Z direction of the sample 9 are observed are images ( Assuming 1), image (2), and image (3), the representative values obtained by using the image filter coefficients of the filter 73 for these image data are those shown at the right end of the figure.
[0054]
The value of the image data here is calculated when one pixel of the image memory 12 is 8 bits, and shows the result when the bright part of (1) and (3) is 256 and the dark part is 0, In the middle of (2), all image data are calculated as 128.
[0055]
As a result, the maximum value is 1024 when changing from dark to bright from the left, and the minimum value is -1024 when changing from light to dark.
When focusing on the upper right side using this property, it is only necessary to detect the position where the representative value is maximized. When focusing on the lower left side, the value obtained by inverting the sign of the representative value is maximized. What is necessary is just to detect a position.
[0056]
Note that the image filter coefficient of the filter 73 can be used by changing the value of the filter coefficient so as to detect the luminance change in the Y direction in accordance with the luminance change of the image data. Up to this point, the data range to be processed in each layer of the spatial memory 70 has been set to 9 × 3 × 3. However, in order to detect a peak position based on information on a wider area, 5 × 5 , 7 × 7 can achieve the same effect as described above.
[0057]
Therefore, in this way, even when the boundary portion of the step portion of the sample having a further step is indicated, the upper or lower step of the step can be stably detected, and stable automatic focusing can be realized.
[0058]
【The invention's effect】
As described above, according to the present invention, high-precision automatic focusing can be realized at a position where focusing is desired, and high-precision observation can be realized by eliminating the influence of focus shift in confocal microscope observation. In addition, a stable luminance distribution in the optical axis direction can be obtained even if the sample surface has irregularities, and stable automatic focusing can be realized. Furthermore, even when the boundary of the step portion of the sample having a step is indicated, the upper step or the lower step of the step can be stably detected, and stable automatic focusing can be realized.
[Brief description of the drawings]
FIG. 1 is a diagram showing a schematic configuration of a focusing device of a confocal microscope according to a first embodiment of the present invention.
FIG. 2 is a view for explaining a sample surface used in the first embodiment.
FIG. 3 is a view for explaining a state of focus on a sample surface used in the first embodiment.
FIG. 4 is a flowchart for explaining the operation of the first embodiment;
FIG. 5 is a diagram illustrating an example of changes in luminance data in the line memory used in the first embodiment.
FIG. 6 is a diagram showing a calculation result in an arithmetic circuit used in the first embodiment.
FIG. 7 is a diagram showing a schematic configuration of a second embodiment of the present invention.
FIG. 8 is a diagram showing a schematic configuration of only a main part of a third embodiment of the present invention.
FIG. 9 is a diagram for explaining a spatial memory used in the third embodiment;
FIG. 10 is a diagram for explaining a representative value determination method according to the third embodiment;
FIG. 11 is a view for explaining a sample having a step in the third embodiment;
FIG. 12 is a diagram for explaining an example of a conventional automatic focusing method.
[Explanation of symbols]
1 ... Confocal microscope body,
2 ... Laser light source,
3. Scanning optical system,
4 ... Objective lens,
5 ... Beam splitter,
6 ... mirror,
7 ... Confocal optical system,
8 ... Non-confocal optics
9 ... Sample,
10 ... stage,
11 ... Controller box,
12: Image memory,
13: Line memory,
14 ... arithmetic circuit,
15 ... Z drive circuit,
16 ... computer,
17 ... Display,
18 ... mouse,
60 ... white light source,
61 ... Half mirror,
62 ... Switching mirror,
63 ... Image sensor,
64: Average processing circuit,
70: Spatial memory,
71: Filter operation mechanism,
72 ... image data of each layer,
73 ... Filter,
74: Output representative value of each layer.

Claims (5)

Scanning the light from the light source to the sample, in a confocal microscope having a confocal optical science system that enables confocal detection of sample images corresponding to the scanning,
A detection optical system having a depth of focus deeper than the confocal optical system and capable of detecting the sample image;
From the sample image detected by the detection optical system, and setting means for setting a movement amount for each one arbitrary position information and the moving range of the optical axis direction of the sample on the sample image,
Storage means for storing image data corresponding to the arbitrary position information for the sample image detected by the confocal optical system for each movement according to the amount of movement set once in the setting means;
A focusing apparatus for a confocal microscope, comprising: a focusing position calculation unit that calculates a focusing position corresponding to the maximum luminance from the image data stored in the storage unit. The storage means
Storing the average value of image data corresponding to the arbitrary position information and image data in the vicinity of the image data;
2. A focusing apparatus for a confocal microscope according to claim 1, wherein the focusing position calculation means calculates a focusing position corresponding to the maximum brightness from the averaged image data stored in the storage means. . Further, the sample image detected by the confocal optical system for each movement corresponding to one movement amount set in the setting unit is set in a predetermined range together with image data corresponding to the arbitrary position information. Second storage means for storing image data;
Image data processing means for processing the image data stored in the second storage means with a predetermined weight and replacing it with image data corresponding to the arbitrary position information,
The image data replaced by the image data processing means is stored in the storage means as image data corresponding to the arbitrary position information,
2. A focusing apparatus for a confocal microscope according to claim 1, wherein a focusing position corresponding to the maximum luminance is calculated from the image data stored in the storage means. Furthermore, it has a calculation means for performing a polynomial calculation on the discrete data output by the confocal optical system,
4. A focusing apparatus for a confocal microscope according to claim 1, wherein a focusing position at a maximum luminance is obtained from a calculation result of the calculation means. Scanning the light from the light source to the sample, a confocal microscope of the focusing method with the confocal optical science system that enables confocal detection of sample images corresponding to the scanning,
Detecting the sample image by a detection optical system having a depth of focus deeper than the confocal optical system;
From the detected sample image, set the amount of movement each time an arbitrary position information and the moving range of the optical axis direction of the sample on the sample image,
Storing image data corresponding to the arbitrary position information with respect to the sample image detected by the confocal optical system for each movement according to the set one movement amount;
A focusing method for a confocal microscope, wherein a focusing position corresponding to the maximum luminance is calculated from the stored image data.

Images