JP2013104919A - Virtual slide creating device - Google Patents

Abstract

PROBLEM TO BE SOLVED: To create a virtual slide which is in focus on an entire sample, while reducing computational complexity relating to focus detection.SOLUTION: The VS creating device includes: a moving mechanism 5 for adjusting a relative position between a stage with a sample mounted thereon and an objective lens for condensing light from the sample in an optical direction and a direction crossing the optical direction; an imaging part 7 for acquiring partial images of the sample; an in-focus detection part 93 for detecting an in-focus state of a focus detection area set for a part of a field of view of the imaging part 7; a focus state control part 94 for controlling the moving mechanism 5 to adjust the relative position in the optical direction between the objective lens and the stage, on the basis of the in-focus state detected by the in-focus detection part 93; a contour detection part 91 for detecting contour of the sample; an in-focus detection position control part 92 for controlling the moving mechanism 5 to adjust the relative position between the focus detection area and the partial image, on the basis of the position of the contour detected by the contour detection part 91; and an image processing part 96 for creating a VS from a plurality of partial images acquired by the imaging part 7.

Description

  The present invention relates to a virtual slide creation device.

  Conventionally, a virtual slide obtained by photographing a specimen prepared from a specimen with extremely high resolution is used in pathological diagnosis. The virtual slide is created by repeatedly performing partial high-magnification imaging of an observation target while shifting the imaging range, and arranging a plurality of acquired partial images in two dimensions. As an imaging device for such a virtual slide, partial imaging of a specimen is performed while measuring the in-focus position at a plurality of points on the specimen in advance and performing in-focus adjustment based on the surface to which the measured in-focus position is fitted. There is known an imaging apparatus that performs (see, for example, Patent Document 1). In order to reduce the amount of calculation when detecting the in-focus state of a microscope or the like, the in-focus state is detected only in the in-focus detection area corresponding to a part of the photographing range.

US Patent Application Publication No. 2006/0204072

  However, when the focus state is detected only in the focus detection region that is a part of the sample while moving the imaging range on the sample as in Patent Document 1, one of the focus detection regions is detected at the edge portion of the sample. The sample may be included only in the part. When the in-focus state is detected in this state, the position in the optical axis direction of the surface of the slide glass on which the sample is placed is also added to the detection, and the in-focus state is accurate with respect to the sample to be observed. Will not be detected. As a result, there is a problem that the sample is unclearly displayed in the partial image obtained by photographing the edge portion or the like of the sample.

  The present invention has been made in view of the above-described circumstances, and provides a virtual slide creation device that can create a virtual slide that is accurately focused on a sample as a whole sample while reducing the amount of calculation related to focus detection. The purpose is to provide.

In order to achieve the above object, the present invention provides the following means.
The present invention provides a stage on which a specimen is mounted, an objective lens that collects light from the specimen mounted on the stage, and at least one of the stage or the objective lens in an optical axis direction and a direction that intersects the optical axis. A moving mechanism that moves relative to the imaging unit, an imaging unit that captures the light collected by the objective lens and acquires a partial partial image of the specimen, and a combination corresponding to a part of the field of view of the imaging unit. A focus detection unit that sets a focus detection region and detects a focus state in the focus detection region, and an optical axis of the objective lens and the stage based on the focus state detected by the focus detection unit A focus control unit that adjusts the relative position of the direction by the moving mechanism, a contour detection unit that detects the contour of the sample, and the focus detection region based on the position of the contour detected by the contour detection unit; Above A virtual slide is synthesized by pasting together a plurality of partial images acquired by the imaging unit and an in-focus detection position control unit that adjusts the relative position of the divided image with the imaging range by the moving mechanism or the in-focus detection region. Provided is a virtual slide creation device including an image processing unit.

  According to the present invention, by moving the stage and the objective lens in the direction intersecting the optical axis by the operation of the moving mechanism, the imaging range by the imaging unit is moved with respect to the sample, and a partial partial image of the sample is obtained. Is acquired by the imaging unit, and a virtual slide can be created by combining and synthesizing the acquired plurality of partial images by the image processing unit.

  When acquiring each partial image, the in-focus state of the in-focus detection region corresponding to a part of the shooting range of each partial image is detected by the in-focus detection unit, and the objective lens and the stage are detected at the position where the in-focus state is detected. The moving mechanism is operated by the focusing control unit so that the relative position in the optical axis direction is positioned. Thereby, the amount of calculation related to focus detection can be reduced.

  In this case, the focus detection position control unit detects the focus so that the focus detection region is arranged inside the contour when the contour of the sample detected by the contour detection unit is included in the imaging range. The position of the focus detection area with respect to the shooting range is adjusted by the unit or the moving mechanism. As a result, only the specimen is included in the in-focus detection area, and the in-focus detection unit accurately detects the in-focus state with respect to the specimen. You can create slides.

In the above invention, the focus detection unit sets the focus detection region at a predetermined position with respect to the field of view of the imaging unit, and the focus detection position control unit includes the contour in the imaging range of the partial image. In this case, it is possible to adjust the photographing range of the partial image by the moving mechanism so that the focus detection area is arranged inside the contour.
By doing in this way, the structure of a focus detection part can be simplified.

Moreover, in the said invention, the said focus detection part can set the said focus detection area to a different position with respect to the visual field of the said imaging part, and the said focus detection position control part image | photographs the said partial image. When the outline is included in the range, the focus detection unit may adjust the position of the focus detection area so that the focus detection area is arranged inside the outline.
In this way, the moving mechanism only needs to shift the photographing range of the partial image at a constant interval, so that the control of the moving mechanism and the processing of positioning the virtual slide composition by the image processing unit are simplified. Can do.

  According to the present invention, it is possible to create a virtual slide that is accurately focused on a sample as a whole while reducing the amount of calculation related to focus detection.

1 is an overall configuration diagram of a virtual slide creation device according to an embodiment of the present invention. It is a figure which shows the relationship between the visual field of the imaging part with which the virtual slide creation apparatus of FIG. 1 is provided, and a focus detection area. It is a functional block diagram of the control unit with which the virtual slide production apparatus of FIG. 1 is provided. It is a figure explaining the movement of the imaging | photography range at the time of the partial image acquisition by the virtual slide creation apparatus of FIG. It is a figure explaining the movement of the focus detection area with respect to the imaging range of a partial image, (a) The imaging range of the partial image arrange | positioned at the edge of a sample, and (b) The imaging area of an image sensor are shown.

A virtual slide (VS) creation device 1 according to an embodiment of the present invention will be described below with reference to the drawings.
As shown in FIG. 1, the VS creation apparatus 1 according to the present embodiment condenses light from the stage 3 on which the slide glass 2 on which the specimen A is attached is placed, and the specimen A on the stage 3. Two objective lenses 41 and 42 having different magnifications, and a motor (movement) that moves the stage 3 in the optical axis direction (Z direction) of the objective lenses 41 and 42 and in two directions (XY directions) perpendicular to the optical axis direction. Mechanism) 5, an imaging lens 6 that forms an image of the specimen A, and an imaging unit such as a CCD that captures an image of the specimen A imaged by the imaging lens 6 and acquires an image of the specimen A 7, a storage unit 8 that stores an image acquired by the imaging unit 7, and a control unit 9 that controls operations of the stage 3 and the imaging unit 7.

  The VS creation apparatus 1 according to this embodiment includes a half mirror 10 that branches the light collected by the imaging lens 6 and an image sensor 11 that captures the light branched by the half mirror 10. Yes.

  The stage 3 is moved in the XY direction and the Z direction by the motor 5 based on movement command signals from an XY control unit 92 and a Z control unit 94 described later. As a result, the imaging range of the specimen A can be sequentially shifted, and the objective lenses 41 and 42 can be adjusted to a position in the Z direction (Z position) at which the specimen A is focused.

  The objective lenses 41 and 42 are attached to a revolver (not shown), and the objective lenses 41 and 42 inserted into the optical path between the specimen A and the imaging unit 7 can be switched by the rotation of the revolver. FIG. 2 is a diagram illustrating a relationship between images captured using the two objective lenses 41 and 42. The objective lens with a lower magnification (hereinafter referred to as a wide-field objective lens) 41 has a sufficiently wide field of view including the entire specimen A. The objective lens with a higher magnification (hereinafter referred to as a high-resolution objective lens) 42 has a sufficiently narrow field of view compared to the wide-field objective lens 41 including only a part of the specimen A.

  When the wide-field objective lens 41 is inserted in the optical path, the imaging unit 7 acquires a wide-field image obtained by capturing the first visual field F1 having a size including the entire image of the specimen A, and the high-resolution objective lens 42 is in the optical path. When the second field of view (field of view) F2 having a size including only a part of the specimen A is photographed, a partial image is obtained. The imaging unit 7 transmits the acquired wide-field image and partial image to the control unit 9.

  The image sensor 11 is configured to photograph the focus detection area B corresponding to a partial area of the second visual field F2. In FIG. 2, a rectangular area set at the center of the second visual field F2 is illustrated as the focus detection area B, but the shape and position of the focus detection area B can be changed as appropriate.

  As shown in FIG. 3, the control unit 9 detects the contour of the specimen A from the wide-field image, and moves the motor 5 to the motor 5 based on the contour position detected by the contour detector 91. An XY control unit (focus detection position control unit) 92 for instructing movement, a focus detection unit 93 for detecting the focus state of the image in the focus detection area B input from the image sensor 11, and the focus A Z control unit (focusing control unit) 94 for instructing the motor 5 to move in the Z direction based on a detection signal from the detection unit 93; a main control unit 95 for comprehensively controlling the operation of the VS creating device 1; And an image processing unit 96 that generates a VS from the partial image stored in the storage unit 8.

  The contour detection unit 91 detects the contour of the specimen A from the wide-field image based on, for example, a differential value of luminance, and outputs information on the position of the contour in the wide-field image to the XY control unit 92.

  The XY control unit 92 outputs an XY movement command signal designating the moving direction and moving amount of the stage 3 to the motor 5. At this time, the XY control unit 92 determines the moving direction and moving amount of the stage 3 based on the basic movement pattern set in advance and the information on the contour position input from the contour detecting unit 91.

  Specifically, the XY control unit 92 moves the stage 3 sequentially in the XY direction with a certain amount of movement such that the imaging ranges of adjacent partial images share a predetermined overlap width at the edge portion. A basic movement pattern is set. However, the XY control unit 92 changes each of the basic movement patterns so that the focus detection region B is arranged inside the contour when the contour of the sample A is included in the imaging range of the partial image. The stage 3 is moved by the amount of movement in the direction.

  The focus detection unit 93 calculates the contrast value of the image in the focus detection area B photographed by the image sensor 11, and outputs a detection signal indicating the calculated contrast value to the Z control unit 94. The contrast value is the highest when the high-resolution objective lens 42 is focused on the sample A, and is a value that becomes lower as the focus position of the high-resolution objective lens 42 is shifted from the sample A. Therefore, it can be determined that the high-resolution objective lens 42 is in focus on the focus detection area B of the sample A when the calculated contrast value is the largest.

  The Z control unit 94 moves the stage 3 in the Z direction by the motor 5 while monitoring the change of the detection signal input from the focus detection unit 93, and positions the stage 3 at the Z position where the detection signal becomes the largest.

The main control unit 95 controls a series of operations of other configurations related to the acquisition of the wide-field image and the partial image, and determines the position of the stage 3 in the XY direction of the partial image acquired by the imaging unit 7 at the time of acquisition. The information is stored in the storage unit 8 in association with the information.
The image processing unit 96 reads a plurality of partial images stored in the storage unit 8 and stores them in association with each other after the series of control by the main control unit 95 is completed and partial images are acquired over the entire specimen A. The VS is generated by arranging the partial images according to the position information of the stage 3 in the X and Y directions.

Next, the operation of the VS creation device 1 configured as described above will be described below.
According to the VS creation apparatus 1 according to the present embodiment, first, the main control unit 95 inserts the wide-field objective lens 41 into the optical path by rotating the revolver, and acquires a wide-field image of the specimen A by the imaging unit 7. To do. The main control unit 95 causes the contour detection unit 91 to input the acquired wide-field image. The contour detection unit 91 detects the contour of the specimen A from the input wide-field image.

  Next, the main control unit 95 inserts the high-resolution objective lens 42 in the optical path, and repeats the positioning operation of the stage 3 by the XY control unit 92 and the Z control unit 94 and the partial image acquisition operation by the imaging unit 7. .

  Specifically, the XY control unit 92 moves the stage 3 in the XY directions and positions the photographing range of the partial image with respect to the specimen A. Subsequently, a focusing operation is performed by the focus detection unit 93 and the Z control unit 94. That is, the focus detection unit 93 detects the focus state of the focus detection area B that is a part of the photographing range of the partial image, and the Z control unit 94 adjusts the Z position of the stage 3 based on the detection signal. . As a result, the specimen A is placed at the in-focus position of the high-resolution objective lens 42. Subsequently, the main control unit 95 causes the imaging unit 7 to acquire a partial image. The main control unit 95 stores the acquired partial image in the storage unit 8 together with position information of the stage 3 in the X and Y directions.

  Here, the XY control unit 92 moves the stage 3 in the XY direction with a certain amount of movement when only one of the specimen A and the slide glass 2 is included in the entire imaging range of the partial image. On the other hand, the XY control unit 92 determines the amount of movement of the stage 3 in the XY directions so that the specimen A is included in the entire focus detection area B when the imaging range of the partial image crosses the outline of the specimen A. change. As a result, as shown in FIG. 4, the photographing range of the partial image is moved while the end portion overlaps with a constant overlap width at the center portion of the sample A, and the overlap width varies at the edge portion of the sample A. It will be.

  The main control unit 95 performs the positioning operation, the focusing operation, and the partial operation of the stage 3 while sequentially moving the imaging range of the partial image by the XY control unit 92 until a plurality of partial images that cover the entire specimen A are acquired. Repeat the image acquisition operation.

  The main control unit 95 performs the focusing operation only when the sample A is included in the shooting range, and acquires the partial image by omitting the focusing operation when the sample A is not included in the shooting range. It may be done. Whether or not the sample A is included in the shooting range is determined based on the contrast value of the shooting range. That is, the main control unit 95 determines that the sample A is included when the contrast value of the imaging range is equal to or greater than a predetermined threshold, and determines that the sample A is not included when the contrast value is smaller than the predetermined threshold.

  After the series of partial image acquisition control by the main control unit 95 is completed, the image processing unit 96 combines the partial images stored in the storage unit 8 to generate VS.

  As described above, according to the VS creating apparatus 1 according to the present embodiment, when a partial image of the edge portion of the specimen A is captured, the XY direction of the stage 3 so that the specimen A is included in the entire focus detection area B. The position of is adjusted. As a result, the focus detection unit 93 detects an accurate focus state with respect to the specimen A. Therefore, the Z of the stage 3 is arranged so that the specimen A is disposed at the focus position of the high-resolution objective lens 42. The position is determined, which has the advantage that a VS can be created in which the specimen A is clearly imaged throughout the specimen A.

  In the present embodiment, the position of the focus detection area B is fixed with respect to the second visual field F2, and the position of the focus detection area B with respect to the contour of the sample A is moved by the movement of the stage 3 in the XY direction. However, instead of this, the focus detection area B may be moved with respect to the second visual field F2. The movement of the focus detection area B is, for example, that the image sensor 11 has an imaging surface 11c having a size including the second visual field F2, and the focus detection unit 93 is within the imaging surface 11c. This is performed by moving the imaging region C corresponding to.

  For example, as shown in FIG. 5A, when the specimen A is included only in the upper part of the imaging range, as shown in FIG. 5B, the imaging area C is defined in the imaging surface 11a of the image sensor 11. What is necessary is just to move upwards with respect to the imaging range. The focus detection unit 93 may have a plurality of shooting areas set in the imaging surface 11a, and may select one imaging area used for focus detection according to the position of the contour.

  When combining the partial images by the image processing unit 96, in order to prevent the joints between the partial images from becoming conspicuous, a smoothing process is performed on the overlapping end portions. In this case, if the overlapping width of the end portion differs depending on the partial image, it is necessary to determine the area to which the smoothing process is applied for each partial image, and the amount of calculation increases. For such inconvenience, it is possible to move the photographing range by a fixed amount of movement by moving the focus detection area B with respect to the second visual field F2, so that the amount of calculation when generating VS Can be prevented.

  In the present embodiment, a Z position storage unit (not shown) that stores the history of the Z position of the stage 3 positioned by the Z control unit 94 is provided, and the focus detection area B includes the contour of the specimen A. In such a case, the Z position of the stage 3 may be predicted and determined based on the Z position history stored in the Z position storage unit.

  The surface fitting the Z position stored in the Z position storage unit reflects the shape of the surface of the specimen A. Therefore, an appropriate Z position of the stage 3 can be calculated for an imaging range that has not yet been captured based on the gradient of the fitted surface. In this way, even when the specimen A is not included in the entire focus detection area B, the stage 3 is adjusted to the Z position where the specimen A is focused, and the specimen A is photographed sufficiently clearly. Partial images can be acquired. In addition, since the photographing range can be moved with a certain amount of movement according to the basic movement pattern, the overlapping width of the end portions of the partial images can be made constant, and the amount of calculation related to the generation of VS can be reduced.

  Further, in the present embodiment, for all partial images, the focus detection unit 93 detects the focus state and the Z control unit 94 positions the Z position of the stage 3, but instead, For some partial images, the Z position of the stage 3 may be determined by the user. In this case, in the VS creating apparatus 1, the operation of the stage 3 and the imaging unit 7 is controlled by the control unit 9 as described above, and the operation of the stage 3 and the imaging unit 7 is controlled by the user. Manual focus (MF) mode.

  According to the VS creation device 1 configured in this way, first, preprocessing is performed by the user in the MF mode. That is, the user observes the whole image of the specimen A in the wide-field image, and selects a part in the specimen A for which the Z position of the stage 3 is to be determined, for example, a part containing dust or a part containing a specific tissue. To do. In the case where the specimen A is a thick slice, for example, when the cell nucleus is an observation object, if the sample A is focused on a portion other than the nucleus, the nucleus is unclearly photographed.

  In this way, the user determines the Z position of the stage 3 for the portion that may not be accurately focused on the observation target of the specimen A in the AF mode. Information on the determined Z position is held in the Z control unit 94 in association with the position in the XY direction. Thereafter, the user switches to the AF mode to repeatedly perform the positioning operation of the stage 3 and the partial image acquisition operation as described above.

At this time, the Z control unit 94 omits the focusing operation for the shooting range including the position in the XY direction in which the Z position of the stage 3 is determined by the MF mode, and moves the stage 3 to the Z position determined by the user. And the Z position of the stage 3 is adjusted based on the in-focus state detected by the in-focus detection unit 93 for other imaging ranges.
In this way, the observation object can be clearly photographed even in a portion where it is difficult to accurately focus the observation object of the specimen A only in the AF mode.

  In the present embodiment, the motor 5 that moves the stage 3 in the three-axis directions has been described as the moving mechanism. However, the configuration of the moving mechanism is not limited to this. For example, the moving mechanism may be configured by a motor that moves the revolver in three axial directions, and the objective lenses 41 and 42 may be moved in the three axial directions with respect to the stage 3 whose position is fixed. Alternatively, the moving mechanism may include a first motor that moves the stage 3 in the XY direction and a second motor that moves the revolver in the Z direction.

1 virtual slide creation device 2 slide glass 3 stage 5 motor (movement mechanism, focus adjustment mechanism)
6 Imaging Lens 7 Imaging Unit 8 Storage Unit 9 Control Unit 10 Half Mirror 11 Image Sensor 11a Imaging Surface 41 Wide Field Objective Lens 42 High Resolution Objective Lens (Objective Lens)
91 Contour detection unit 92 XY control unit (focus detection position control unit)
93 Focus detection unit 94 Z control unit (focus control unit)
95 Main Control Unit 96 Image Processing Unit A Specimen B Focus Detection Area C Imaging Area F1 First Field of View F2 Second Field of View

Claims (3)

A stage with a specimen,
An objective lens that collects light from the specimen mounted on the stage;
A moving mechanism for relatively moving at least one of the stage or the objective lens in an optical axis direction and a direction crossing the optical axis;
An imaging unit that captures light collected by the objective lens and acquires a partial partial image of the sample;
A focus detection unit that sets a focus detection region corresponding to a part of the field of view of the imaging unit and detects a focus state in the focus detection region; and
A focus control unit that adjusts the relative position of the objective lens and the stage in the optical axis direction by the moving mechanism based on the focus state detected by the focus detection unit;
A contour detector for detecting the contour of the specimen;
A focus detection position control unit that adjusts the relative position between the focus detection area and the imaging range of the partial image by the moving mechanism or the focus detection area based on the position of the contour detected by the contour detection unit. When,
An image processing unit comprising: an image processing unit that combines a plurality of partial images acquired by the imaging unit to synthesize a virtual slide. The focus detection unit sets the focus detection region at a predetermined position with respect to the field of view of the imaging unit;
The focus detection position control unit, when the outline is included in the imaging range of the partial image, the imaging range of the partial image by the moving mechanism so that the focus detection area is arranged inside the outline. The virtual slide creating apparatus according to claim 1, wherein the virtual slide creating apparatus adjusts the angle. The focus detection unit can set the focus detection region at a different position with respect to the field of view of the imaging unit,
The focus detection position control unit adjusts the position of the focus detection region so that the focus detection region is arranged inside the contour when the contour is included in the imaging range of the partial image. The virtual slide creation apparatus according to claim 1.

Images