JP4544850B2 - Microscope image photographing device - Google Patents

Description

  The present invention relates to a microscope image capturing apparatus that inputs microscope image information and processes the image information to form a high-resolution and wide-field image.

  Conventionally, there is a method of observing a microscope image as a digital image. In general, when a specimen is observed using a microscope, the range that can be observed at a time is mainly determined by the magnification of the objective lens. Then, the higher the magnification of the objective lens, the narrower the observation range, and the image is limited to a very small part of the sample, but a high-definition image can be obtained instead.

  By the way, when the microscope is used for pathological diagnosis such as cytodiagnosis and histological diagnosis, it is necessary to grasp the entire elephant in order to prevent oversight of the diagnosis part. Further, with the development of information processing technology in recent years, there is an increasing demand for high-resolution images similar to those of conventional silver salt films in microscopic observation images used for pathological diagnosis.

Conventionally, various techniques have been developed in order to form high-resolution or wide-field images in taking microscopic images. As one example, for example, the entire specimen image is divided into small sections, and in each of the small sections, a high-definition microscope image corresponding to the magnification of the objective lens is taken and taken in while controlling the position in consideration of the overlapping portion. There has been proposed a microscope system that reconstructs a whole image of a specimen having a high resolution and a wide field of view by image synthesis in which the captured images are arranged and sequentially arranged. (For example, refer to Patent Document 1.)
By the way, in such image capturing for each small section, because of an error due to the surface accuracy of the specimen stage and the thickness of the specimen, the focus shifts when the optical axis of the objective lens moves through the small section. Need to be corrected. Therefore, there is a problem that it takes time to capture the image of all the small sections and form the entire specimen image.

As a solution to this problem, in order to correct the focal position with respect to the horizontal movement of the specimen stage, a plurality of focal positions on the specimen are examined in advance to obtain the inclination of the specimen. There has been proposed a method for shortening the time by performing correction in the height direction. (For example, see Patent Document 2.)
Further, in order to increase the accuracy of the focal position, it is necessary to perform autofocus again after correcting in the height direction. When re-executing this autofocus, the re-execution condition is to limit the number of times auto-focus is executed by limiting the horizontal movement by a certain distance from the position where auto-focus was previously executed. Means for shortening the time for acquiring the entire image have been proposed. (For example, refer to Patent Document 3.)
Japanese Patent Laid-Open No. 09-281405 (page 5, FIG. 3, FIG. 4) JP-A-11-231228 (paragraphs [0043], [0044], [0047], FIG. 1) JP 2001-91846 A (paragraphs [0051], [0052], FIG. 11)

  However, in the method of correcting the height direction at the same time as the horizontal movement after examining the multiple focal positions on the sample in advance, it is necessary to perform the operation for obtaining the sample tilt manually. In addition, when acquiring the in-focus position on the sample, if autofocus is used, an error will occur if there is no specimen, so it is necessary to select a location where autofocus is possible in advance, and these operations are complicated. There is a problem that there is.

  In addition, the method of reducing the number of auto focus executions by limiting the auto focus execution to a certain distance horizontally moved can reduce the number of auto focus executions even if the number of auto focus executions can be reduced. Since autofocus cannot be executed until the corrective movement in the vertical direction is completed, there is a problem in that it is not possible to shorten the time from horizontal movement of a small section until it becomes possible to capture an image. .

  In view of the above-described conventional situation, an object of the present invention is to provide a microscope image photographing apparatus that automatically performs a process of obtaining a correction amount in the height direction and forms an entire specimen image in a shorter time.

  The microscope image capturing apparatus of the present invention includes, for example, a sample image region extraction unit that extracts a region where a sample image exists from an image of the entire sample, and a sample image region extracted by the sample image region extraction unit. A height coordinate acquisition position setting unit that automatically sets a plurality of XY direction positions for acquiring the height coordinate Z, and a height coordinate that is a focal position at the XY direction position set by the height coordinate acquisition position setting unit The position of the focus correction at an arbitrary position in the sample image area using the coordinate reading unit that reads and the height coordinate data read by the coordinate reading unit at the position set by the height coordinate acquisition position setting unit A focus correction position calculation unit for calculating the sample, and the sample at the correction focus position calculated by the focus correction position calculation unit when the sample is moved horizontally. And the sample moving unit to move the height, and a.

For example, the coordinate reading unit is configured to execute AF processing in a state where the sample is horizontally moved to a set position, and read the height position of the sample moving unit after the AF is completed as a height coordinate.
The height coordinate acquisition position setting unit sets, for example, the position of the lattice point where the sample image is present among the lattice points of the section obtained by dividing the sample image area into the lattice-shaped sections at predetermined intervals as the position for acquiring the height coordinate. Configured to do.

  In addition, the microscope image photographing apparatus of the present invention includes, for example, a specimen image region extraction unit that extracts a region where a specimen image exists from an image of the whole specimen, and automatically detects a focal position following the horizontal movement of the specimen. An automatic focusing unit that starts detection of the focal position when moving horizontally to the position where the sample image extracted by the sample image area extraction unit exists, and the presence of the sample image It is configured to stop detecting the focal position when moving horizontally to a position where it does not.

In addition, the microscopic image capturing apparatus of the present invention, for example, divides the entire image captured in the field of view by imaging at a low magnification into small sections, and each of these small sections is photographed at a high magnification and bonded together. in the microscope imaging apparatus for forming an overall picture of the resolution, the height coordinate acquisition position for setting a plurality of positions to be obtain the height coordinate from the lattice underlying sampling definitive the lattice image forming small compartments a setting unit, a coordinate reading unit to read the height coordinate is the focal position of the horizontal coordinate of the specimen at high magnification, were read by the coordinate reading unit at a set lattice above the height coordinate acquiring positioning unit A focus correction position calculation unit that calculates the height position at an arbitrary position in the small section using the height coordinate data.

  In addition, the microscopic image capturing apparatus of the present invention, for example, divides the entire image captured in the field of view by imaging at a low magnification into small sections, and each of these small sections is photographed at a high magnification and bonded together. In a microscopic imaging device that forms an overall image of resolution, a sample image segment extraction unit that extracts a small segment in which a sample image exists from a plurality of small segments, and a focal position automatically following the change of the sample image An automatic focusing unit that detects the focal position when moving horizontally to a small section where the sample image extracted by the sample image section extracting unit exists, When moving horizontally to a small section where an image does not exist, the detection of the focal position is stopped to perform image capturing at a high magnification.

In this case, for example, when there is no sample at the center of the first small section, the automatic focusing unit moves to the small section closest to the small section and has the sample at the center, and executes AF. You may comprise.
Further, the microscopic image photographing method of the present invention, for example, takes a wide-field image of the entire observation slide glass with a low-magnification objective lens, extracts a region with a sample image from the wide-field image, and region with the sample image High magnification while correcting the focal position by setting to use real-time AF when the unevenness of the sample is large or the sample is scattered in the field of view based on the presence / absence information of the sample image obtained by the process of extracting Based on the presence / absence information of the sample image obtained by taking the image and extracting the region with the sample image, if the sample is flat and widely present in the entire field of view, it is set to not use real-time AF. It is configured to take a magnification image.

  Further, the focal position correction method of the microscope image photographing apparatus of the present invention extracts, for example, an area where a specimen image exists from an image obtained by photographing the entire specimen, and acquires the height coordinate Z from the extracted specimen image area. Using a coordinate reading unit that sets multiple horizontal positions and reads the height coordinate that is the focal position at the set horizontal position, and the coordinates of the set horizontal position and the height coordinate data read at that horizontal position Thus, the focus correction position at an arbitrary position in the sample image region is calculated, and the height of the sample is moved to the calculated correction focus position when the sample is moved horizontally.

  Further, the focal position correction method of the microscopic image capturing apparatus of the present invention extracts, for example, a region where a sample image exists from an image obtained by photographing the entire sample, and moves horizontally to the position where the extracted sample image exists. An automatic focus position detection operation following the horizontal movement is started, and the automatic focus position detection operation is stopped when the horizontal movement is performed to a position where the sample image does not exist.

  According to the present invention, the correction amount in the height direction of the imaging position is obtained from the reference position for focus correction in a predetermined region of the sample, or the activation is stopped and resumed depending on the state of the sample while real-time autofocus is activated. Therefore, the accuracy of the focus position alignment at the time of capturing the high-resolution entire image is improved, and the time for capturing the entire image at a high magnification can be shortened, so that the operability of the microscope image photographing apparatus is improved.

  Embodiments of the present invention will be described below with reference to the drawings.

FIG. 1 is a diagram showing an overall configuration of a microscope image photographing apparatus as the first embodiment. The microscope image photographing apparatus shown in the figure is roughly composed of a microscope unit 1, a camera unit 2, a computer 3, and a monitor 4.
The microscope unit 1 optically extracts an entire image or a small-section observation slide glass image (microscope image) from the observation slide glass S at a desired magnification. More specifically, the microscope section 1 has a sample stage 5 on which an observation slide glass S to be observed is placed. Below the sample stage 5, a transmission filter unit 6, a transmission field stop 7, and a transmission An aperture stop 8, a condenser optical element unit 9, a condenser top lens unit 11, and a transmission illumination light source 12 made of, for example, a halogen lamp are arranged, and the observation slide glass S on the specimen stage 5 is illuminated from below by these devices. To do.

  Further, a revolver 14, an autofocus beam splitter 15, and a plurality of objective lenses 13 (13a to 13f) that are replaceably mounted on the optical axis in the observation optical path above the specimen stage 5; A focus detection light receiving element 16, a zoom lens 17, an observation beam splitter 18 that branches an observation slide glass image, and an eyepiece lens 19 are disposed. Further, the microscope unit 1 is provided with a microscope control unit 20 that controls the entire components.

  In the microscope unit 1 configured as described above, the illumination light generated by the transmission illumination light source 12 is collected by the collector lens, is incident on the transmission filter unit 6, and is adjusted by the transmission filter unit 6. The The adjusted illumination light passes through the transmission field stop 7, the transmission aperture stop 8, the condenser optical element unit 9, and the condenser top lens unit 11, and is illuminated onto the observation slide S from below the illumination opening of the sample stage 5. The

The specimen stage 5 is controlled by the microscope control unit 20 to change the observation part of the observation glass slide S in the optical axis direction for two-dimensional horizontal movement and focusing in a plane orthogonal to the optical axis. At the same time, coordinates can be detected.
The light (observation slide glass image) transmitted through the observation slide glass S and condensed by the objective lens 13 is an autofocus beam splitter 15, a zoom lens 17 that arbitrarily adjusts an observation magnification, and an observation beam splitter 18. And is guided to the camera head 21 of the camera unit 2 disposed above the eyepiece lens 19 of the microscope unit 1.

The autofocus beam splitter 15 is detachable with respect to the optical path, and one of the lights branched by the autofocus beam splitter 15 is guided to the focus detection light receiving element 16 through the imaging lens, and the autofocus. Used for photometry calculation for control.
The observation beam splitter 18 is also detachable from the optical path, and guides the light transmitted through the observation slide glass S to the eyepiece lens 19 or the camera unit 2.

  The camera unit 2 includes a camera head 21 and a camera control unit 22, and the camera head 21 forms an image of a solid-state imaging device made of, for example, CMD (Charge Modulation Device) and light transmitted through the observation slide glass S on the CMD. An image optical system, and converts an observation slide glass image into an image signal.

  The camera control unit 22 controls the camera head 21 and includes an AGC (auto gain contrast) that automatically adjusts the gain of the incident light amount versus the output voltage. The camera control unit 22 transfers analog image data input from the camera head 21 to the A / D converter 23 of the computer 3.

  The computer 3 includes a memory that stores programs and control information for performing various system operations and processes, and stores a plurality of digital image data from the CPU 26 that performs image processing and the frame memory 24, and the height of the sample. A memory 27 capable of recording coordinate data for obtaining a tilt in the vertical direction, an input device 28 such as a mouse and a keyboard, a revolver rotation instruction, a zoom magnification instruction, and an autofocus control instruction to the microscope , A communication device 29 for sending a specimen stage movement instruction and the like, and a capture board unit. The capture board unit includes an A / D converter 23, a frame memory 24, and a D / A converter 25.

The microscope control unit 20 described above receives the above various instructions from the communication device 29 of the computer 3 and controls each component in the corresponding microscope.
The A / D converter 23 of the computer 3 digitizes the image data captured by the camera head 21 and transfers it to the frame memory 24. On the one hand, the digital image data stored in the frame memory 24 is read out by the CPU 26 and processed in various ways, and on the other hand, it is converted into analog data by the D / A converter 25 and displayed on the monitor 4.

  As the autofocus control function in this system, once autofocus execution is started, the sample stage is moved up and down following the change in the sample image until an end command is sent, so that the focus state is always kept ( (Hereinafter referred to as real-time autofocus), control for ending the autofocus operation once the autofocus is executed and in-focus state (hereinafter referred to as one-shot autofocus), and two autofocus modes.

  FIG. 2 is a diagram illustrating an example of a display screen for operating a microscope displayed on the monitor 4. As shown in the figure, an objective lens switching unit 31 is displayed on the left side of the microscope operation display screen 30. In the objective lens switching unit 31, a revolver 32 and six lens mounting portions 33 of an objective lens attached around the revolver 32 are schematically displayed in a button format.

  On these six lens mounting portions 33, the magnifications of the mounted objective lenses are displayed as 40 times, 20 times, 10 times, 4 times, and 1.25 times in the counterclockwise direction from the upper right. In the example shown in the figure, five types of objective lenses having different magnifications are attached, and no objective lens is attached to the sixth lens attachment portion 33 (button labeled “NONE”). It is empty.

  When instructing the switching of the objective lens, an input operation of the switching instruction can be performed by clicking the lens mounting portion 33 having the objective lens having a desired magnification with the mouse of the input device 28. In the lens mounting portion 33 that has been input, the display of the button changes to a shape in which the button is pushed in, so that the magnification of the objective lens currently in use can be seen at a glance.

  Also, on the right side of the microscope operation display screen 30, an instruction setting unit 34 for performing instructions and settings relating to photographing is displayed. The instruction setting unit 34 displays a “macro image capture” button 35, a “high resolution image capture” button 36, and a check input window 37 from the top. Real-time AF (autofocus) is displayed on the right side of the check input window 37.

  When instructing to shoot a macro image, the “macro image shooting” button 35 is clicked with the mouse to perform an input operation. When an instruction for capturing a high resolution image is given, the “high resolution image capturing” button 36 is input. In either case, the input button changes the display of the button to the pressed shape.

  As described above, in this example, in the case of “macro image shooting” for shooting the entire specimen, the “whole image” to be shot may be shot using a low-magnification objective lens, or shot using a macro device. It is also possible to specify any shooting method. Further, when a check (x) is displayed in the check input window 37 as shown in the figure, real-time AF is instructed. To cancel the real-time AF instruction, click on the check input window 37 with the mouse, the check (x) display disappears, and the real-time AF instruction is cancelled. Further, when instructing real-time AF from the state in which the real-time AF instruction is canceled, similarly, when the check input window 37 is clicked with the mouse, a check (x) is displayed again and real-time AF is instructed. It can be confirmed that there is.

  FIG. 3 is a flowchart for explaining the processing operation in the microscope image photographing apparatus having the above-described configuration. In the first embodiment, the undulation state of the focal position on the specimen is obtained in advance as Z position data, and thereby the focal position is corrected during horizontal movement in high magnification image shooting.

In FIG. 3, first, a wide-field image of the entire observation slide glass S is taken (S01).
In this process, on the microscope operation display screen 30 in FIG. 2, the button of the lens mounting unit 33 to which the desired low-magnification objective lens of the objective lens switching unit 31 is mounted is clicked (clicked with the mouse, hereinafter Similarly, the revolver 14 is rotated to switch to a desired low-magnification objective lens. Subsequently, in response to pressing of the macro image capturing button 35, wide field image capturing of the entire observation slide glass S is performed.

Subsequently, an area with a sample is extracted on the observation slide glass S using the captured wide-field image (S02).
This region extraction process can be performed by, for example, a method proposed in Japanese Patent Laid-Open No. 2000-295462.

  In this example, in parallel with this, the captured wide-field image is divided into small sections. This is for photographing a high-definition image. In order to photograph a high-definition image, it is necessary to photograph with a high-magnification objective lens. For this reason, it is necessary to determine the minimum visual field size that can be taken with a high-magnification objective lens.

FIG. 4 is a diagram for explaining the correspondence between the field size of the smallest unit for photographing and the observation slide glass and the basic principle of the photographing control method in this example.
The small section 39 obtained by dividing the captured image region other than the region where the label 38 is attached to the observation slide glass S shown in the same figure vertically and horizontally in a grid pattern is the minimum unit visual field size for capturing the above-described high-definition image. is there. The minimum visual field size is determined by the CCD size of the objective lens 13, zoom lens 17, and camera head 21 set at the time of shooting.

  In the example shown in the figure, m × n small sections 39 from the small section 39 at the position indicated by the coordinates (1, 1) in the lower right corner to the small sections 39 at the position indicated by the coordinates (m, n) in the upper left corner. It is divided into In this way, the photographing positions of the m × n small sections 39 at the positions indicated by the coordinates (1, 1) to the coordinates (m, n) are determined on the specimen slide glass S. Is done. These small sections 39 are sequentially photographed with a high-magnification objective lens as indicated by arrows in the figure.

Of course, the photographing position on the specimen slide glass S may be set by providing an overlap area in the small section 39. The setting of the small section 39 and the setting when the overlap area is provided are proposed in Japanese Patent Laid-Open No. 9-281405.
Further, the photographing order may be moved in the line-sequential direction while reciprocating in the horizontal direction instead of moving in the line-sequential direction while reciprocating in the vertical direction as indicated by the arrows in FIG.

Subsequently, in FIG. 3, a reference point for focus correction is determined in order to obtain a focus position for correction in the height direction (Z direction) from a region of the sample extracted in the process of step S02. (Step S03).
FIG. 5 is a diagram for explaining a method for determining a focus correction reference point for obtaining the correction focus position. In general, as a reference point for focus correction for obtaining the focus position for correction in the height direction of the sample, the position of the reference point and the reference point should be separated by an appropriate distance so that the inclination of the sample can be corrected. is there. In order to cope with this, in this example, as shown in FIG. 5, with respect to the specimen 40 extracted on the specimen slide glass S, first, as a region where the specimen 40 exists, a quadrilateral circumscribing the specimen 40 is formed. A sample area 41 is set, and then the sample area 41 is divided into sections 42 having a predetermined interval L.

This section 42 is a section set for determining a focus correction reference point, and is not directly related to the small section 39 for determining the minimum photographing unit and the photographing order in FIG.
After the section 42 is set in this way, the intersections (a, b, c,..., G) of the lattice in the region where the sample 40 is located are used as the focus correction reference points. The coordinates (X, Y) of the position of the reference point are obtained. In this case, if the division interval L is reduced, the number of reference points increases and the accuracy of height direction correction (focus correction), which will be described later, improves, but it takes time as the number of reference points increases. become. Therefore, an appropriate value is set as the division interval L.

  In the above example, the grid point of the small section on the region where the sample exists is set as the acquisition position as the reference point of the acquisition position of the height spot. The center point of the section may be set, or may be set regardless of the position of the small section. In any case, it is a matter of course that the setting is made on the area where the sample exists.

Subsequently, in FIG. 3, the objective lens is replaced with a high-magnification objective lens (step S04).
In this process, the lens mounting portion 33 on which a desired high-magnification objective lens is mounted is pressed from among the lens mounting portions 33 displayed in the button format on the microscope operation display screen 30 in FIG. The revolver 14 in FIG. 1 is driven to rotate, and the desired high-magnification objective lens is set on the observation slide glass S.

Subsequently, in FIG. 3, the focal position of the reference point for focus correction is acquired for correction in the height direction (step S05).
In this process, the height of the focus correction reference point set (determined) at the grid intersection in the process of step S03 is obtained in order to correct the height position of the image to be taken for each small section 39. Therefore, the sample stage 5 shown in FIG. 1 is moved so that the focus correction reference point comes to the objective lens position, and the height coordinate (Z) as the focus position of the focus correction reference point (X, Y) is set. Desired. The acquisition of the coordinate (Z) can be easily performed by autofocusing.

In FIG. 3, it is determined whether or not it is the last focus correction reference point (step S06). If it is not yet the last focus correction reference point (S06 is No), the process returns to step S05. The process of obtaining the coordinates (Z) of the next focus correction reference point is repeated.
Thereby, the height coordinate (Z) of each focus correction reference point (X, Y) is sequentially obtained, and the three-dimensional position coordinates (X, Y, X) including the height of each focus correction reference point are obtained. Z) is determined, and data of the determined position coordinates (X, Y, Z) of each focus correction reference point is recorded in the memory 27.

  FIG. 6 is a diagram showing a data structure of the position coordinates (X, Y, Z) of the focus correction reference point recorded in the memory 27. As shown in FIG. As shown in FIG. 5, the focus correction reference points set on the intersections of the grid with the interval L as shown in FIG. 5 are sequentially stored in the memory 27 from the first to the nth.

  Here, a method for correcting the height (focus) of the imaging position of the specimen 40 using the focus correction reference point will be described. In FIG. 5, for example, in order to know (correct) the correct height (focus) of the position 43, a focus correction reference point in the range of “L × 2” centered on the position 43 is searched. At this time, if there are three or more focus correction reference points, the nearest three focus correction reference points are searched.

  In the example shown in FIG. 5, seven focus correction reference points a to g exist within a range of “L × 2” centered on the position 43. In such a case, among the seven focus correction reference points a to g, the three closest focus correction reference points a, b, and c are adopted as the position 43, and their position coordinates (X , Y, Z) is obtained. Then, the height coordinate (Z) of the position 43 is obtained by substituting the horizontal position coordinates (X, Y) of the position 43 into this plane expression.

  Further, when there are only two focus correction reference points a and b within the range of “L × 2” centering on the position 44 as in the position 44 shown in FIG. 5, in other words, two. In the following cases, the Z coordinate value of the closest focus correction reference point is set as the height coordinate (Z) of the position 44. In the example shown in FIG. 5, the focus correction reference point that is closest to the position 44 is the focus correction reference point a. Therefore, the Z coordinate value of the focus correction reference point a is the height coordinate of the position 44. (Z).

When all the focal positions (Z coordinates) of the focus correction reference points for height direction correction have been acquired in this way, in FIG. 3, first, the sample stage 5 is moved horizontally to the first section 39 to be imaged. (Step S07).
The first small section 39 to be photographed is the small section 39 at the position indicated by the coordinates (1, 1) in the lower right corner shown in FIG. Further, in the processing after the horizontal movement, the check input window 37 of real-time AF (autofocus) is unchecked on the microscope operation display screen 30 in FIG. In response to the input operation of 36, the computer 2 in FIG.

Following the above, photographing of the small section 39 is performed while performing Z correction (step S08).
In this process, the sample 40 does not exist in the small section 39 at the position indicated by the first coordinate (1, 1) in the example shown in FIG. In such a case, the Z correction is substantially “0”.
Subsequently, it is determined whether or not it is the last small section 39 at the position indicated by the coordinates (m, n) (step S09).

  If it is not the last small section 39 (S09 is No), there is a small section 39 to be photographed next, so that it moves horizontally to the next small section 39 (step S10) and returns to the processing of step S8. The photographing of the horizontally moved small section 39 while performing Z correction and the determination in step S9 are repeated until the photographing of the last small section 39 is completed (Yes in S09).

  As a result, imaging from the small section 39 at the position indicated by the first coordinates (1, 1) to the last small section 39 at the position indicated by the coordinates (m, n) is sequentially performed, and the small section where the sample 40 is located. In 39, the sample stage 5 moves horizontally and the height direction also moves to the position corrected by the focus correction reference point, and the focus of the sample 40 is corrected, and a high-definition image that is correctly focused is photographed. .

  As described above, according to the first embodiment, the sample image region on the sample slide glass is extracted, and several points in the sample image region are selected as reference points for focus correction, and the reference points at the reference points are selected. A series of processing for detecting the focal position and obtaining the inclination of the specimen can be automatically performed, and this improves the operability of the microscope apparatus when taking a microscope image.

  By the way, when the sample 40 has fine irregularities, or the sample is not scattered in one place as shown in FIG. 5, it is scattered sparsely as described above. In some cases, the focal position cannot be obtained with high accuracy only by correction in the height direction using. Even in such a case, the present invention performs focusing by real-time autofocus following the change of the imaging surface due to the movement of each small section, and reproduces the sample image in accordance with reality. Hereinafter, this will be described as a second embodiment.

  FIG. 7 is a flowchart for explaining the processing operation of the microscope image photographing apparatus according to the second embodiment. The hardware configuration of the microscope image capturing apparatus and the configuration of the display screen for microscope operation of the monitor in this example are the same as the hardware configuration shown in FIG. 1 and the display screen shown in FIG.

FIG. 8 is a diagram specifically showing an example of the operation in the above processing.
In FIG. 7, the process of capturing a wide-field image of the entire observation slide S with a low-magnification objective lens (step S31) and the process of extracting a region with a specimen image (step S32) are the steps shown in FIG. It is the same as the process of S01 and the process of step S02. In FIG. 7, the process of converting to a high-magnification objective lens (step S33) is the same as the process of step S04 shown in FIG.

Subsequently, in FIG. 7, it is determined whether or not there is a sample at the center of the first subsection (for example, the subsection 39 at the position indicated by the first coordinates (1, 1) shown in FIG. 4) (step S34). ).
If there is a sample (S34 is Yes), the sample stage 5 is moved horizontally to the first small section 39, and real-time autofocus is activated (step S35).

  On the other hand, when there is no sample (No in S34), based on a region of the sample extracted in step S32, the small section 39 closest to the first small section 39 and having the sample at the center thereof. The sample stage 5 is moved to the obtained small section 39, one-shot autofocus is executed to determine the focal position (see step S36, arrow S36 in FIG. 8), and then moved to the first small section 39. (See step S37, arrow S37 in FIG. 8).

  Since a series of processes of these steps S36 and S37 is performed for the center point of the photographing field of view, if there is no sample at the center of the first small section 39a in FIG. When started, an error occurs and the operation of the entire apparatus stops.

  Therefore, in order to avoid this problem, a temporary Z coordinate is set in the first small section 39a. The provisional Z coordinate is obtained from the small section 39b that is closest to the first small section 39a and has a sample at the center thereof. As a result, even if the first small section 39a having no sample is photographed, the photographing is performed based on the set temporary Z coordinate, so that no error is derived.

Thus, in the first photographing of the small section 39a, real-time autofocus is activated when there is a sample, and a temporary Z coordinate is set when there is no sample.
Then, high resolution imaging of the small section 39 is performed (step S38).
Subsequently, it is determined whether or not the subsection 39 just photographed is the last subsection 39 (step S39).

  In other words, this is processing for checking whether or not there is a small section 39 to be photographed next. If it is not the last small section 39, that is, if there is a small section 39 to be photographed next (S39 is No), then it is determined whether or not there is a sample at the center of the small section 39 (step S40). ).

  If there is a sample at the center of the small section 39 (S40 is Yes), in this case, after starting real-time AF (autofocus) (step S41), on the other hand, when there is no sample at the center of the small section 39 (S40 is No). In that case, after starting the real-time AF is stopped (step S42), it moves to the next sub-section (step S43). And it returns to the process of step S38 and repeats the process of step S38-S43. In this manner, the high-resolution shooting of the small section 39 is continued until it is determined in the process of step S39 that the small section 39 just shot is the last small section 39.

  Here, the relationship between the absence of a sample at the center of the small section 39 and the start and stop of real-time AF (autofocus) will be described with reference to FIG. In FIG. 8, it is assumed that high-resolution imaging of the small section 39 has progressed to the small section 39p as indicated by the arrow A as described above. Since there is no sample at the center of the small section from the upper small section to the small section 39p, the real-time AF is stopped.

When moving to the small section 39q, since there is a sample at the center of the small section 39q, the activation of the real-time AF is resumed, and the same state continues to the small section 39r.
Then, when moving to the next small section 39s, since there is no sample at the center of the small section 39s, the real-time AF is stopped. This state is continued until the imaging surface moves to the position of the small section having the sample at the center.

In this way, high-resolution imaging of the small section 39 is repeated from the first small section 39a to the last small section 39 at the position indicated by the coordinates (m, n) while the real-time AF is restarted and stopped repeatedly.
As described above, according to the second embodiment, even when specimens or specimens with many irregularities are scattered, the stop and restart can be freely switched according to the presence or absence of the specimen while the real-time autofocus is activated. The time until the in-focus state is shortened, and the time until the state in which the image can be captured from the movement of the small section can be shortened.

  In addition, after setting the autofocus or temporary focus position in the first small section and performing shooting, the target range of autofocus is narrowed, and the center position of the small section obtained by this autofocus is set in the height direction. Since the correction position is set, the focus of autofocus is accelerated, and even when an autofocus error occurs due to insufficient contrast or the like, there is no significant shift from the actual focus position, thereby preventing a problem of a defocused image.

  As described above, in the microscope image capturing apparatus of the present invention, the horizontal movement position and the height position of the specimen are obtained in advance and the correction in the height direction is performed simultaneously with the horizontal movement. There is no need to manually perform the operation for obtaining, and the work efficiency of sample photographing is improved.

  By the way, since the state of the sample varies from time to time, it is convenient to be able to select whether to correct with the reference point for focus correction or to use real-time autofocus when moving to a small section depending on the state of the sample. It is. This will be described below as a third embodiment.

  FIG. 9 is a flowchart for explaining the processing operation of the microscope image photographing apparatus according to the third embodiment. The hardware configuration of the microscope image capturing apparatus and the configuration of the display screen for microscope operation of the monitor in this example are the same as the hardware configuration shown in FIG. 1 and the display screen shown in FIG.

In FIG. 9, the process of capturing a wide-field image of the entire observation slide S with a low-magnification objective lens (step S60) and the process of extracting a region with a specimen image (step S61) are the steps shown in FIG. It is the same as the process of S01 and the process of step S02.
Next, the observer of the microscope image determines the state of the specimen from the wide-field image of the entire observation slide glass S displayed on the monitor 4, and selects whether or not to perform real-time autofocus (step S62).

In this selection, for example, when the unevenness of the specimen is large or the specimen is scattered in the field of view, it is better to use real-time AF. If the sample is flat and widely present in the entire field of view, it is selected to not use real-time AF.
In this case, whether the specimens are scattered or spread over the entire field of view can be determined using the presence / absence information of the specimen image extracted by the specimen area extraction process. Therefore, the process of S62 is the result of the duplicate area extraction process. You may make it carry out automatically using.

  Here, when the real-time autofocus is not used (S62 is No), the check (x) in the check input window 37 displayed on the left of the “real-time AF” display on the microscope operation display screen 30 in FIG. With “) removed, the“ Get high resolution image ”button 36 is pressed.

Thereby, the process which image | photographs a high magnification image by focus position correction | amendment is performed (step S63).
The high-magnification image capturing process without using the real-time autofocus is the same as the process from steps S04 to S09 in FIG.

  On the other hand, when using real-time autofocus (S62 is Yes), check (x) in the check input window 37 displayed on the left side of the “real-time AF” display on the microscope operation display screen 30 in FIG. While the mark is on, the “Get high resolution image” button 36 is pressed.

Thereby, a process of taking a high-magnification image while using real-time autofocus is executed (step S64).
The process of taking a high-magnification image while using this real-time autofocus is the same as the process from steps S33 to S41 in FIG.

As described above, according to the third embodiment, before capturing a high-magnification image, it is possible to obtain a room for selecting a high-magnification image capturing method by viewing a low-magnification wide-field image. In this case, the operability of the microscope apparatus is improved.
In addition, since the case where auto focus is used and the case where it is not used can be selected or automatically set, there is no trouble that an auto focus error occurs in a place where there is no sample and operation becomes difficult.

In addition, since the horizontal position and its height position are calculated in advance and the height position is also moved to the focal position at the same time as the horizontal movement, auto-focusing is performed until the horizontal movement and the correction movement in the height direction as seen in the past are completed. This eliminates the time wasted of not being able to execute and improves the work efficiency of sample photography.
(Appendix 1)
A sample image region extraction unit that extracts a region where a sample image exists from an image of the entire sample;
A height coordinate acquisition position setting unit for automatically setting a plurality of XY direction positions for acquiring the height coordinate Z from the sample image area extracted by the sample image area extraction unit;
A coordinate reading unit that reads the height coordinate that is the focal position at the position in the XY direction set by the height coordinate acquisition position setting unit;
Focus correction position calculation for calculating a focus correction position at an arbitrary position in the sample image area using the height coordinate data read by the coordinate reading unit at the position set by the height coordinate acquisition position setting unit Unit,
A sample moving unit that moves the height of the sample to the corrected focus position calculated by the focus correction position calculating unit when moving the sample horizontally;
A microscope image photographing device characterized by comprising:
(Appendix 2)
The coordinate reading unit executes AF processing in a state where the sample is horizontally moved to a set position, and reads the height position of the sample moving unit after completion of AF as height coordinates. Microscope image photographing device.
(Appendix 3)
The height coordinate acquisition position setting unit sets the position of the lattice point where the sample image is present among the lattice points of the section obtained by dividing the sample image area into the lattice-shaped sections at predetermined intervals as the position for acquiring the height coordinate. The microscope image photographing device according to appendix 1.
(Appendix 4)
A sample image region extraction unit that extracts a region where a sample image exists from an image of the entire sample;
An automatic focusing unit that automatically detects the focal position following the horizontal movement of the specimen;
With
The automatic focusing unit starts detecting the focal position when moving horizontally to the position where the sample image extracted by the sample image area extracting unit exists, and when moving horizontally to the position where the sample image does not exist, A microscope image photographing device characterized by stopping detection.
(Appendix 5)
In a microscopic image capturing apparatus that forms a high-resolution overall image by dividing an overall image captured at a low magnification and obtained in a field of view into small sections, and shooting and pasting each of these small sections at a high magnification. ,
A sample image section extracting unit that extracts a small section in which a sample image exists from a plurality of small sections, an automatic focusing unit that automatically detects a focal position following changes in the sample image, and
With
The automatic focusing unit starts detection of the focal position when moving horizontally to a small section where the sample image extracted by the sample image section extracting unit exists, and the focal point when moving horizontally to a small section where the sample image does not exist. When the position detection is stopped and the image is taken at a high magnification, and there is no sample at the center of the first small section, it moves to the small section that is closest to the center and has the sample at the center. A microscope image photographing apparatus characterized by executing AF.

It is a figure which shows the whole structure of the microscope image imaging device as 1st Embodiment. It is a figure which shows the example of the display screen for microscope operation displayed on a monitor. It is a flowchart explaining the processing operation in the microscope image photographing device of the first embodiment. It is a figure explaining the basic principle of the imaging | photography control method in a corresponding relationship between the visual field size of the minimum unit for imaging | photography, and an observation slide glass, and this example. It is a figure explaining the determination method of the reference point for focus correction | amendment for calculating | requiring the focus position for correction | amendment. It is a figure which shows the data structure of the position coordinate (X, Y, Z) of the reference point for focus correction | amendment recorded on a memory. It is a flowchart explaining the processing operation of the microscope image photographing device in the second embodiment. It is a figure which shows concretely the operation | movement in the process of the microscope image imaging device of 2nd Embodiment as an example. It is a flowchart explaining the processing operation of the microscope image photographing device in the third embodiment.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Microscope part 2 Camera part 3 Computer 4 Monitor S Observation slide 5 Specimen stage 6 Transmission filter unit 7 Transmission field stop 8 Transmission aperture stop 9 Condenser optical element unit 11 Condenser top lens unit 12 Light source 13 for transmission illumination 13 (13a-13f) Objective lens 14 Revolver 15 Autofocus beam splitter 16 Focus detection light receiving element 17 Zoom lens 18 Observation beam splitter 19 Eyepiece 20 Microscope control unit 21 Camera head 22 Camera control unit 23 A / D converter 24 Frame memory 25 D / A converter 26 CPU
24 Frame memory 27 Memory 28 Input device 29 Communication device 30 Microscope operation display screen 31 Objective lens switching unit 32 Revolver 33 Lens mounting unit 34 Instruction setting unit 35 Macro image capture button 36 High resolution image capture button 37 Check input window 38 Label 39 39a, 39b, 39p, 39q, 39r, 39s Small section 40, 40 'Sample 41 Sample area 42 Section 43, 44 Position on the sample

Claims (1)

In a microscopic image capturing apparatus that forms a high-resolution overall image by dividing an overall image captured at a low magnification and obtained in a field of view into small sections, and shooting and pasting each of these small sections at a high magnification. ,
A height coordinate acquisition position setting unit for setting a plurality of positions from which a height coordinate is to be acquired from within a grid having a sample image in a grid forming a small section;
A coordinate reading unit that reads the height coordinate, which is the focal position of the sample in the horizontal coordinate at high magnification,
A focus correction position calculation unit for calculating a height position at an arbitrary position in the small section using the height coordinate data read by the coordinate reading unit in the lattice set by the height coordinate acquisition position setting unit; ,
With
When the small section where the sample image exists is extracted from the whole image and moved horizontally to the small section where the extracted sample image exists, the automatic focus position detection operation following the horizontal movement of the sample is started, A microscope image photographing apparatus characterized by stopping an automatic focus position detecting operation when horizontally moving to a small section where no image exists .

Images