JP5471715B2 - Focusing device, focusing method, focusing program, and microscope - Google Patents

Description

  The present invention relates to a focusing device, a focusing method, a focusing program, and a microscope, and is suitable for, for example, observing a tissue section.

  In a pathological diagnosis, a tissue section is fixed to a glass slide and prepared as a preparation through a staining process and an encapsulation process. In general, when the preparation is stored for a long period of time, the visibility of the preparation with a microscope deteriorates due to deterioration or fading of the biological sample. In addition, the preparation may be microscopically examined at facilities other than the facility where the preparation is made, but delivery of the preparation is generally mailed and requires a certain amount of time.

  In view of such circumstances and the like, an apparatus for storing a biological sample as image data has been proposed (see, for example, Patent Document 1). This apparatus employs a focusing technique for focusing based on the contrast of the captured image.

JP 2009-175334 A

  However, in the case of the above-described focusing technique, it is necessary to shift the focus at predetermined intervals in the depth direction (optical axis direction) of the shooting range and to search for an optimum focus position based on the contrast of the captured image at these focus points. Become.

  In general, the search distance in the optical axis direction is approximately 200 [μm] due to factors such as a difference in thickness that occurs during the manufacture of a slide glass and a difference in the amount of encapsulating material that occurs during the preparation of a slide. For example, when the depth of field of an optical lens that focuses on a biological sample is 1 [μm], in order to detect an optimum in-focus position with an accuracy of 0.5 [μm], about 400 captured images Processing to search from is required.

  Since this search process is executed for each imaging range assigned to the biological sample, the time required to detect the optimum focal position is enormous, and as a result, the efficiency of acquiring the entire biological sample as image data is significantly reduced. Will do.

  The present invention has been made in view of the above points, and intends to propose a focusing device, a focusing method, a focusing program, and a microscope that can shorten the time required for focusing.

  In order to solve such a problem, the present invention is a focusing device, in which an image corresponding to an image formed in a recording photographing range smaller than the field of view of the objective lens is formed, and from the field of view of the objective lens A larger focus search imaging range is assigned to the sample, and using the acquisition means for acquiring the image of the sample site within the focus search imaging range at predetermined intervals, and the sample site image, Determining means for determining a focal position in a recording photographing range where an image of a sample portion corresponding to the image is formed; and out of the recording photographing range in the field of view of the objective lens among the images of the sample portion And a predicting means for predicting the focus position in the recording photographing range where the focus position is undetermined.

  Further, the present invention is a focusing method, in which an image corresponding to an image formed in a recording photographing range smaller than the field of view of the objective lens is formed, and the focus search is larger than the field of view of the objective lens. The imaging range is assigned to the sample, and an acquisition step for acquiring an image of the sample part within the focus search imaging range at predetermined intervals and the image of the sample part are used to correspond to the image. A determination step for determining a focal position in an imaging range for recording on which an image of the sample site is formed, and an area outside the imaging range for recording in the field of view of the objective lens in the image of the sample site And a prediction step of predicting the focal position in the recording photographing range in which the focal position is undetermined.

  Further, the present invention is a focusing program, wherein an image corresponding to an image formed in a recording photographing range smaller than the field of view of the objective lens is formed on the computer. An imaging range for the focal point search that is larger than that is assigned to the sample, and an image of the sample part within the imaging range for the focal point search is obtained at predetermined intervals. Determining the focal position in the recording imaging range where the image of the sample region corresponding to the image is formed, and using the region outside the recording imaging range in the field of view of the objective lens in the sample site image Thus, prediction of the focal position in the recording photographing range where the focal position is undetermined is executed.

  Further, the present invention is a microscope having a recording imaging element having a photographing range smaller than the field of view of the objective lens and a photographing range larger than the field of view of the objective lens, and is connected to the photographing range of the recording image sensor. An imaging element for focus search on which an image corresponding to the image to be imaged is formed, and an imaging range of the imaging element for focus search is assigned to the sample, and images of the sample parts within the imaging range are set at predetermined intervals. An acquisition means for acquiring each image, a determination means for determining a focal position in an imaging range of a recording imaging element on which an image of a sample part corresponding to the image is formed, and a sample part Predicting means for predicting the focal position in the imaging range of the recording image sensor for which the focal position is undetermined, using a region outside the recording image sensor in the field of view of the objective lens. Have

  In the present invention, a focus position corresponding to a wider area than the recording imaging range assigned to the sample can be obtained with only images acquired at predetermined intervals from the imaging range for focus searching. The number of shots for the sample to be used is greatly reduced. Thus, a focusing device, a focusing method, a focusing program, and a microscope that can shorten the time required for focusing are realized.

It is the schematic which shows the structure of a microscope. It is the schematic where it uses for description of the imaging range in an image sensor. It is a photograph which shows the image of the tissue slice which should be used for a record object, and the phase contrast image of the tissue slice which should be used for a focus search. It is the schematic which shows the structure of a comprehensive control part. It is the schematic which shows the functional structure of CPU based on a focusing program. It is the schematic which shows the example of the biological sample used as the acquisition object of a phase difference image. It is the schematic where it uses for description of the uneven | corrugated state of a biological sample. It is the schematic which shows the distance (parallax) for every pixel of the other image with respect to one image of a phase difference image. It is the schematic where it uses for description of a blank area. It is the schematic where it uses for description of prediction of the depth width of the sample site | part using a blank area | region. It is the schematic where it uses for description of determination of a focus position. It is a flowchart which shows a focusing process procedure. It is a schematic diagram used for explanation of detection of a straight line that most closely approximates the parallax (unevenness distribution) of each pixel in the XZ direction. It is the schematic where it uses for description of the example of a division | segmentation of the depth width in other embodiment.

Hereinafter, modes for carrying out the invention will be described. The description will be in the following order.
<1. Embodiment>
[1-1. Microscope configuration]
[1-2. Functional configuration of overall control unit]
[1-3. Specific contents of focusing process]
[1-4. Focusing procedure]
[1-5. Effect]
<2. Other embodiments>

<1. Embodiment>
[1-1. Microscope configuration]
In FIG. 1, the structure of the microscope 1 which is this one Embodiment is shown. The microscope 1 has a stage 11 on which a preparation PRT can be placed (hereinafter also referred to as a preparation stage) 11.

  The preparation PRT is obtained by fixing a sample (hereinafter also referred to as a biological sample) in a connective tissue such as blood, epithelial tissue, or both tissues to a slide glass by a predetermined fixing method. Is dyed as necessary. Staining includes not only general staining such as HE (hematoxylin and eosin) staining, Giemsa staining or Papanicolaou staining, but also special staining such as FISH (Fluorescence In-Situ Hybridization) and enzyme antibody method. Is included.

  The light source 12 is arranged on the surface side of the preparation stage 11 opposite to the surface on which the preparation PRT is arranged (hereinafter also referred to as a preparation arrangement surface). The light source 12 can irradiate light that illuminates a biological sample that has been subjected to general staining (hereinafter also referred to as bright-field illumination light). A light source (not shown) capable of irradiating light for illuminating a biological sample subjected to special staining (hereinafter also referred to as dark field illumination light) is disposed on the preparation arrangement surface side.

  A condenser lens 13 is disposed between the preparation stage 11 and the light source 12 with the normal line of the reference position on the preparation arrangement surface as the optical axis LX.

  On the preparation arrangement surface side of the preparation stage 11, an objective lens 14 having the normal line of the reference position on the preparation arrangement surface as the optical axis LX is disposed.

  Behind the objective lens 14 is a half mirror 15 that splits the light incident from the objective lens 14 into transmitted light and reflected light.

  An imaging element 16 such as a CCD (Charge Coupled Device) or a CMOS (Complementary Metal Oxide Semiconductor) is disposed behind the transmission side of the half mirror 15. The image sensor 16 is used for capturing an image to be recorded. As shown in FIG. 2, the imaging range FSC1 of the imaging device 16 is a rectangular range that is inscribed in the field of view FOV of the objective lens 14, and is positioned around the optical axis LX of the objective lens 14. Incidentally, the photographing range is a light receiving element portion that is actually used for photographing as a pixel.

  On the other hand, a field lens 17 that relays the subject image of the objective lens 14 projected on the reflection side to the rear (scheduled imaging plane) is disposed behind the reflection side of the half mirror 15. In the field lens 17, the subject light reflected by the half mirror 15 is collected, so that a decrease in brightness around the field of view is suppressed.

  A diaphragm mask 18 is disposed behind the field lens 17. The aperture mask 18 has a pair of openings 18A and 18B at positions symmetrical to each other with respect to the optical axis of the surface orthogonal to the optical axis of the field lens 17. The aperture mask 18 divides the subject luminous flux incident from the field lens 17 through the openings 18A and 18B. The split light beams intersect with each other on the imaging surface of the subject light beam, and become a light beam whose positional relationship is switched before and after the imaging surface.

  Separator lenses 19A and 19B are disposed behind the pair of openings 18A and 18B, respectively. Separator lenses 19A, 19B cause a split light beam divided by corresponding openings 18A, 18B to undergo an oblique image formation (shift), and a pair of images having different viewpoints with respect to a planned image formation surface relayed by field lens 17 (Hereinafter also referred to as a phase difference image).

  When the separator lenses 19A and 19B are subjected to vignetting (vignetting) of the field lens 17, a part of the divided light flux is lost. For this reason, the separator lenses 19A and 19B are arranged close to the center side of the field lens 17 so as not to suffer from vignetting. Further, the depth of field of the separator lenses 19 </ b> A and 19 </ b> B is set wider than the depth of field of the objective lens 14. The setting of the depth of field of the separator lenses 19A and 19B is performed by changing the sizes of the openings 18A and 18B in the aperture mask 18, for example.

  An imaging element 20 for obtaining an image to be used as a focus search target is disposed behind the separator lenses 19A and 19B. As shown in FIG. 2, the imaging range FSC2 of the image sensor 20 is larger than the imaging range FSC1 of the image sensor 16, and is positioned around the optical axis LX of the objective lens 14. In other words, the image sensor 20 is not a line sensor but an area sensor.

  The signal processing system in the microscope 1 includes a stage drive control unit 31, an illumination control unit 32, an imaging control unit 33, an imaging control unit 34, and an image processing unit 35.

  The stage drive control unit 31 moves the preparation stage 11 in a direction orthogonal to the optical axis (that is, a direction parallel to the preparation arrangement surface). Specifically, as shown in FIG. 2, the preparation stage 11 is scanned so that the imaging range FSC1 of the imaging device 16 is assigned to the biological sample SPL arranged in the preparation PRT.

  Incidentally, in the example illustrated in FIG. 2, the imaging ranges FSC1 assigned to the biological sample SPL are not overlapped. However, the imaging ranges FSC1 may be partially overlapped.

  The stage drive control unit 31 moves the preparation stage 11 in the optical axis direction (that is, the direction orthogonal to the preparation arrangement surface) every time the imaging range FSC1 to be assigned to the biological sample SPL is changed. Specifically, based on the data indicating the amount of deviation of the objective lens 14 in the optical axis direction, the preparation stage 11 is set so that the objective lens 14 is focused on the sample region existing in the imaging range FSC1 assigned to the biological sample SPL. Is adjusted.

  The illumination control unit 32 sets parameters according to a mode for acquiring a bright field image (hereinafter also referred to as a bright field mode) or a mode for acquiring a dark field image (hereinafter also referred to as a dark field mode). The light source 12 or a light source (not shown) is set, and illumination light is irradiated. This parameter is, for example, selection of illumination light intensity or light source type.

  Note that the irradiation light in the bright field mode is generally visible light. On the other hand, the irradiation light in the dark field mode is light including a wavelength that excites a fluorescent marker used in special staining. In the dark field mode, the background portion with respect to the fluorescent marker is cut out.

  When illumination light is irradiated from the light source 12, the light is collected by the condenser lens 13 at the reference position on the preparation arrangement surface of the preparation stage 11. Then, the image of the sample portion assigned as the imaging range FSC1 is enlarged by the objective lens 14 and formed on the image sensor 16. On the other hand, the image of the sample portion assigned as the imaging range FSC2 is enlarged by the objective lens 14, formed as a phase difference image by the separator lenses 19A and 19B, and formed on the image sensor 20.

  Here, a photograph of a tissue section image formed on the image sensor 16 to be used as a recording target and a phase difference image of the tissue section imaged on the image sensor 20 as a focus search target is illustrated. 3 shows. As can be seen from FIG. 3, the image of the sample part assigned as the imaging range FSC1 is formed on the image sensor 16, while the image of the sample part assigned as the imaging range FSC2 is formed on the image sensor 20 as a phase difference image. Is imaged.

  The imaging control unit 33 sets a parameter corresponding to the bright field mode or the dark field mode in the imaging element 16, and acquires subject image data assigned as the imaging range FSC1. This parameter is, for example, the exposure start timing and end timing.

  The imaging control unit 34 sets parameters according to the bright field mode or the dark field mode in the imaging element 20 and acquires phase difference image data allocated as the imaging range FSC2. This parameter is, for example, the exposure start timing and end timing.

  The image processing unit 35 performs various types of image processing on the subject image data output from the image sensor 16.

  In the image processing, for example, the images of the sample parts existing in the imaging range FSC1 assigned to the biological sample SPL are connected using a predetermined connection algorithm, and the biological sample image (enlarged by the magnification of the objective lens 14). And an image of the entire biological sample SPL). As another example, there is a process of generating a magnified image display screen specified by a display command using a predetermined screen generation algorithm.

  In addition, when a biological sample image is generated, the image processing unit 35 stores the biological sample image in a storage medium in units of subjects.

  Incidentally, the stage drive control unit 31, the illumination control unit 32, the imaging control unit 33, the imaging control unit 34, and the image processing unit 35 include a control unit (hereinafter also referred to as a general control unit) 40 that controls these control units. Is connected.

[1-2. Configuration of general control unit]
As shown in FIG. 4, the overall control unit 40 is configured by connecting various hardware to a CPU (Central Processing Unit) 41.

  Specifically, a ROM (Read Only Memory) 42, a RAM (Random Access Memory) 43 serving as a work memory of the CPU 41, an operation input unit 44 for inputting commands according to user operations, an interface 45, a display unit 46, and a storage unit 47 is connected via the bus 48.

  The ROM 42 stores programs for executing various processes. The interface 45 is connected to the stage drive control unit 31, the illumination control unit 32, the imaging control unit 33, the imaging control unit 34, and the image processing unit 35, respectively.

A liquid crystal display, an EL (Electro Luminescence) display, a plasma display, or the like is applied to the display unit 46. For the storage unit 47, a magnetic disk represented by HD (Hard Disk), a semiconductor memory, an optical disk, or the like is applied. USB (Universal Serial Bus) memory and CF (Compact)
A portable memory such as a Flash memory may be applied.

  The CPU 41 develops, in the RAM 43, a program corresponding to a command given from the operation input unit 44 among the plurality of programs stored in the ROM 42, and appropriately controls the display unit 46 and the storage unit 47 according to the developed program. .

  Further, the CPU 41 performs processing such as designating control conditions in the stage drive control unit 31, the illumination control unit 32, the imaging control unit 33, the imaging control unit 34, or the image processing unit 35 via the interface 45 in accordance with the developed program. It is made to run.

[1-3. Specific contents of focusing process]
When the CPU 41 receives a start command to search for the in-focus position, the CPU 41 develops a program corresponding to the command in the RAM 43. In this case, the CPU 41 functions as a phase difference image acquisition unit 51, a parallax calculation unit 52, a focus position determination unit 53, and a focus position prediction unit 54, as shown in FIG.

  The phase difference image acquisition unit 51 obtains a phase difference image of a sample part in the imaging range FSC2 assigned to the biological sample SPL in the same manner as the imaging range FCS1, for example, as shown in FIG. Opening alternately (portions surrounded by thick broken lines in FIG. 6).

  The parallax calculation unit 52 uses the phase difference image acquired by the phase difference image acquisition unit 51 and uses the other image (hereinafter referred to as “reference image”) for one image (hereinafter also referred to as a reference image) as a reference. (Also called an image) is calculated in pixel units.

  Specifically, for example, each pixel in the reference image in the phase difference image is sequentially selected as a target pixel (hereinafter referred to as a target pixel). Each time a target pixel is selected, a pixel corresponding to the target pixel (hereinafter referred to as a relative pixel) is detected from the reference image in the phase difference image, and the distance of the relative pixel to the target pixel is calculated. Is done.

  As a relative pixel detection method, for example, a block having the highest similarity with the pixel value of the target block centered on the target pixel is searched from the other image of the phase difference image to be referred to by the normalized correlation method. A method is adopted in which the center of the block is a relative pixel.

  The distance calculated by the parallax calculation unit 52 corresponds to parallax. The smaller the distance (parallax) is, the closer the focal point of the objective lens 14 is to the rear, and the larger the distance (parallax), the more forward the position is. Therefore, the distance of the relative pixel of the reference image with respect to each pixel of the standard image corresponds to information indicating the unevenness state of the sample portion reflected in the field of view of the objective lens 14, as shown in FIG.

  Here, the relationship between the position of each pixel of the reference image in the phase difference image of the biological sample shown in FIG. 3 and the distance of the relative pixel of the reference image with respect to the pixel is shown in FIG. 8 as a graph. The thin part of the graph in FIG. 8 indicates the front side, and the dark part indicates the back side. It can be seen from FIG. 8 that the uneven state of the sample portion reflected in the field of view of the objective lens 14 is reflected. Incidentally, the end of the sample region in FIG. 8 is turned up.

  When the parallax (distance of the relative pixel of the reference image with respect to each pixel of the reference image) is calculated by the parallax calculation unit 52, the in-focus position determination unit 53 obtains a phase difference image based on the parallax as a first step. The depth width of the sample region within the imaging range FSC2 that is the acquisition target in the unit 51 is calculated.

  Specifically, the difference between the maximum distance and the minimum distance among the relative pixel distances of the reference image with respect to each pixel of the reference image is calculated as the depth width. That is, the depth width is the distance between the horizontal plane passing through the position closest to one surface side of the preparation PRT and the horizontal plane passing through the position closest to the other surface side.

  As described above, the depth of field of the separator lenses 19 </ b> A and 19 </ b> B is adjusted wider than that of the objective lens 14. For example, the depth of field of the sample region of the imaging range FSC1 imaged on the image sensor 16 is set to about 1 [μm], and the image of the sample region of the imaging range FSC2 imaged on the image sensor 20 is set. It is assumed that the depth of field is set to about 200 [μm]. When searching for a focal position using an image captured by the image sensor 16, it is necessary to acquire a large number of images while moving the sample stage 11 in a range of about ± 100 [μm] in the optical axis direction. On the other hand, when searching for a focal position using an image captured by the image sensor 20, a search range equivalent to a range for searching for a focal position using an image captured by the image sensor 16 is selected using the sample stage 11. It can be acquired by one shooting without moving.

  Therefore, the in-focus position determination unit 53 uses the image (phase difference image) of the image sensor 20 to reduce the number of images to be taken in the field of view of the objective lens 14 compared to the case of using the image of the image sensor 16. The depth of the subject image can be accurately calculated.

  When the focus position determination unit 53 calculates the depth width of the sample part, as a second stage, the focus position of the objective lens 14 to be adjusted in the imaging range FSC1 (hereinafter referred to as the focus position) based on the depth width of the sample part. (Also called the focal position). Incidentally, the imaging range FSC1 is an imaging range corresponding to the imaging range FSC2 that is the acquisition target in the phase difference image acquisition unit 51.

  The specific method for determining the focal position in this embodiment first determines whether the depth width of the sample region is equal to or greater than a threshold set for the depth width. The threshold value is a value corresponding to the depth of field of the objective lens 14, such as an aperture amount set for the aperture of the diaphragm with respect to the objective lens 14.

  Here, for example, as shown in FIG. 9A, when the depth width of the sample region is less than the threshold value, the middle of the depth width is determined as the in-focus position. On the other hand, when the depth width of the sample region is equal to or larger than the threshold, for example, as shown in FIG. 9B, a plurality of layers (hereinafter referred to as imaging layers) whose depth width falls within the depth of field of the objective lens 14 are used. Also, the middle of the depth direction in each of the photographing layers is determined as the focus position.

  As described above, when the depth width of the sample part is less than the depth of field of the objective lens 14, the in-focus position determining unit 53 determines the in-focus position according to the depth width of the sample part 14, and the depth is more than the depth of field. In this case, the in-focus position is determined for each imaging layer that falls within the depth of field. That is, the focus position determination unit 53 determines the focus position and the number thereof according to the depth of field of the objective lens 14.

  Therefore, the in-focus position determination unit 53 can avoid an imaging state in which a part of the sample part is missing or blurred even when the sample part exists in an inclined state or a turned-up state.

  When determining the in-focus position, the in-focus position determining unit 53 calculates the amount of movement of the sample stage 11 to the in-focus position (hereinafter referred to as the defocus amount) as a third stage, and drives the stage. This is given to the control unit 31.

  By the way, as shown in FIG. 6, the phase difference image acquisition unit 51 does not acquire the phase difference images of all the sample parts to which the imaging range FSC2 is assigned, but alternately acquires it by opening one imaging range FSC2. The

  For this reason, in the imaging range FSC1 corresponding to the imaging range FSC2 that is excluded as a phase difference image acquisition target, the focus position (focus position) to be adjusted is not determined.

  However, in the case of this embodiment, the imaging range FSC2 of the imaging device 20 for acquiring an image to be used as a focus search target is an image pickup for acquiring an image to be used as a recording target, as shown in FIG. The range is larger than the imaging range FSC1 of the element 16. Therefore, as shown in FIG. 10, there are areas (hereinafter also referred to as blank areas) MAR1 to MAR4 in the field of view FOV of the objective lens 14 and outside the field of view FSC1 in the field of view FSC2.

  In this blank area MAR, as shown in FIG. 11, a part of the sample part P2 to be excluded as a phase difference image acquisition target is adjacent to the sample part P2 in the longitudinal direction of the preparation PRT. Alternatively, it is included in the phase difference image of P3. Although not shown, it is also included in the phase difference image of the sample part adjacent to the sample part P2 in the short direction of the preparation PRT.

  The in-focus position predicting unit 54 predicts the in-focus position in the imaging range FSC1 in which the focal position has not been determined, based on the blank areas MAR1 to MAR4 adjacent to the sample part P2.

  Specifically, in the blank areas MAR1 to MAR4, the difference between the average value of the relative distances of the relative pixels of the reference image with respect to the respective pixels of the reference image is the photographing range in which the focal position is undetermined. Predicted as the depth width of the sample site in FSC1. Then, in accordance with the depth width, the in-focus positions and the number thereof are predicted as in the in-focus position determination unit 53.

  When the in-focus position predicting unit 54 predicts the in-focus position of the sample part, the in-focus position calculating unit 54 calculates the movement amount (defocus amount) of the sample stage 11 with respect to the in-focus position, and supplies this to the stage drive control unit 31.

[1-4. Focusing procedure]
Next, the focusing process procedure in the CPU 41 will be described with reference to the flowchart shown in FIG. However, in FIG. 12, as shown in FIG. 6, the imaging range FSC2 whose focal position should be determined using the phase difference image and the imaging range FSC2 whose focal position should be predicted using the margin area MAR are alternately displayed. The setting content to be specified in is shown in the case where the overall control unit 40 and the stage drive control unit 31 are set.

  When the CPU 41 receives a start command to search for the in-focus position, the CPU 41 starts the in-focus processing procedure and proceeds to the first step SP1. In the first step SP1, the CPU 41 waits for data indicating that the recording imaging range FSC1 has been assigned to the sample region for which the in-focus position is to be searched.

  When the data to be received in the first step SP1 is given from the stage drive control unit 31, for example, the CPU 41 proceeds to the second step SP2. In the second step SP2, the CPU 41 determines whether or not a phase difference image should be acquired from the focus search shooting range FSC2 corresponding to the recording shooting range FSC1.

  If it is determined that a phase difference image should be acquired, the CPU 41 proceeds to the third step SP3 and uses the phase difference image formed in the focus search photographing range FSC2 to perform parallax (reference image). Relative pixel distance of the reference image with respect to each of the pixels. Then, the CPU 41 proceeds to the fourth step SP4, and uses the parallax calculated in the third step SP3 to determine the in-focus position and the number thereof with respect to the recording shooting range FSC1. Further, the CPU 41 proceeds to the fifth step SP5, calculates the defocus amount based on the in-focus position calculated in the fourth step SP4, and gives this to the stage drive control unit 31. Further, the CPU 41 proceeds to the sixth step SP6, and determines whether or not data indicating that the allocation of the imaging range FSC1 for recording has been completed for the biological sample SPL has been received.

  On the other hand, when determining that the phase difference image should not be acquired in the second step SP2, the CPU 41 proceeds to the sixth step SP6 without going through the respective processes from the third step SP3 to the fifth step SP5.

  As described above, the CPU 41 performs the processing from the first step SP1 to the sixth step SP6 until it receives data indicating that the allocation of the recording range FSC1 for recording to the biological sample SPL in the sixth step SP6. repeat.

  On the other hand, if the CPU 41 receives data indicating that the recording range FSC1 for recording has been assigned to the biological sample SPL in the sixth step SP6, the CPU 41 proceeds to the seventh step SP7.

  In the seventh step SP7, the CPU 41 sets the in-focus position and the number of the image-capturing range FSC1 in which the in-focus position is not determined in the image-recording range FSC1 to the image-capturing range FSC1 located around the image-capturing range FSC1. Prediction is performed using the margin area MAR of the corresponding focus search imaging range FSC2, and the process proceeds to the eighth step SP8. In the eighth step SP8, the CPU 41 calculates a defocus amount based on the in-focus position calculated in the seventh step SP7, gives this to the stage drive control unit 31, and then ends this focusing processing procedure.

[1-5. Effect]
In the above configuration, the microscope 1 alternately obtains phase difference images from the focus search imaging range FSC2 corresponding to the recording imaging range FSC1 (see FIGS. 2 and 6), and uses the phase difference image. Thus, the focal position in the recording range FSC1 for recording is determined (see FIG. 9).

  Further, the microscope 1 uses a blank area MAR outside the recording imaging range FSC1 within the field of view of the objective lens 14 in the phase difference image, and uses the blank area MAR within the recording imaging range FSC1 where the focal position is undetermined. The focal position is predicted (see FIGS. 10 and 11).

  Therefore, in this microscope 1, since the focal position is obtained in all the recording imaging ranges FSC1 assigned to the biological sample SPL only by the phase difference images obtained alternately from the imaging range FSC2 for focus search, the focus search is performed. The number of images taken with respect to the biological sample SPL to be used is greatly reduced.

  When searching for a focal position based on the contrast of an image, generally, an imaging range in one image sensor is assigned to a biological sample, and an image within these imaging ranges is used for focus search and recording. . In this case, in order to search for the focal position, it is essential to obtain an image of the entire imaging range assigned to the biological sample.

  Therefore, compared with the case where the focal position is searched based on the contrast of the image, the number of images taken for the biological sample SPL to be used for the focal point search is greatly reduced.

  By the way, when searching for a focal position based on the contrast of an image, the time required to detect the optimum focal position is determined by selecting a representative part of the imaging range assigned to the biological sample as a search target. It may be possible to shorten the time. However, in this case, as the portion other than the search target increases, the detection accuracy of the focal position becomes worse. In other words, when searching for the focal position based on the contrast of the image, the reduction in time until the optimal focal position is detected and the detection accuracy of the focal position are in a trade-off relationship, and the effect of the other is not affected unless one is sacrificed. Will not be expected.

  On the other hand, the images acquired alternately from the focus search shooting range FSC2 are not only the entire recording shooting range FSC1 corresponding to the shooting range FSC2 to be acquired but also the shooting range FSC2 that is not to be acquired. A part of the corresponding recording photographing range FSC1 is included. That is, the ratio of the observation range used for determining or predicting the focal position is larger than when searching for the focal position based on the contrast of the image. Therefore, compared with the case where the focal position is searched based on the contrast of the image, the detection accuracy of the focal position is improved, but the number of images taken for the biological sample SPL to be used for the focal search is greatly reduced.

  In the case of this embodiment, the depth of field with respect to the shooting range FSC2 for focus search is set to be larger than the shooting range FSC1 for recording. Therefore, the uneven state in the shooting range FSC2 for focus search is an image reflected in more detail than the shooting range FSC1 for recording (see FIG. 8). In the microscope 1, the focal position is based on the uneven state. Determined or predicted. Therefore, it is possible to determine or predict the in-focus position more accurately than in the case where the focus position is searched based on the contrast of the imaging range FSC1.

  Further, the subject depth of the lens that forms the phase difference image in the focus search photographing range FSC2 is adjusted to be wider than the subject depth of the lens that forms an image in the recording photographing range FSC1. For this reason, compared with the case where the focus position is searched based on the contrast, the number of shots per shooting range to be used for the focus search is significantly reduced.

  According to the above configuration, the microscope 1 that can reduce the time required for focusing can be realized by greatly reducing the number of images taken for the biological sample SPL to be used for the focus search.

<2. Other embodiments>
In the above embodiment, the tissue section is the biological sample SPL. However, the biological sample SPL is not limited to this embodiment. For example, smear cells and chromosomes can be applied as the biological sample SPL. Further, for example, a sample other than a living body (organism) such as a semiconductor element may be used.

  In the above-described embodiment, the phase difference image of the biological sample SPL is acquired from the image sensor 20. However, the acquisition source is not limited to the image sensor 20. For example, it may be acquired from a storage medium connected to the overall control unit 40. Further, it may be acquired from outside the microscope 1 through a wired or wireless communication medium such as a local area network or the Internet.

  In the above-described embodiment, the image to be acquired is a phase difference image. However, the acquired image is not limited to the phase difference image. For example, the imaging element 20 may not pass through the field lens 17, the openings 18A and 18B, and the separator lenses 19A and 19B. In this case, an image corresponding to the image sensor 16 is formed on the image sensor 20 via one or a plurality of lenses or openings arranged behind the reflection side of the half mirror 15.

  Further, as a method for determining the in-focus position using the image, for example, the depth position in the imaging range FSC2 is changed at predetermined intervals, and the depth position having the largest image contrast at the depth position is adjusted. A method of determining the focal position can be employed. Furthermore, as a method for predicting the in-focus position using the margin area MAR of the image, for example, a technique for predicting, as the in-focus position, the average of the depth positions with the largest contrast in the margin areas MAR1 to MAR4 can be employed. .

  In the above-described embodiment, the in-focus position is predicted based on the blank areas MAR1 to MAR4 adjacent to the sample site P2. However, the margin area MAR to be used for prediction of the in-focus position is not limited to all areas. For example, only a part of the margin areas MAR1 and MAR2, the margin areas MAR3 and MAR4, or the margin areas MAR1 and MAR3 may be used. However, the blank area MAR3 or MAR4 in which the degree of distortion of the image to be formed is smaller than that of the blank area MAR1 or MAR2 is preferable from the viewpoint of prediction accuracy.

  In the above-described embodiment, the depth width of the sample portion in the imaging range FSC1 in which the focal position is undetermined is in the blank area MAR of the phase difference image acquired from the imaging range FSC2 near the imaging range FSC1. The difference (distance) between the average of the maximum parallax and the average of the minimum was predicted. However, the depth width prediction method is not limited to this.

  For example, the prediction can be performed using the inclination angle of the sample portion in the blank area MAR. For example, as shown in FIG. 13, the inclination angle is calculated using a straight line SL that most closely approximates the parallax distribution for each pixel in the XZ direction, for each column Y0, Y1,. Detect by least squares method. Then, the plane that passes through the most straight lines in each Y row is determined by, for example, averaging the angles of the respective straight lines with respect to the horizontal plane. That is, the sample region is approximated to a plane closest to the uneven state when viewed from the XZ direction. The angle formed by this plane and the horizontal plane is calculated as the inclination angle of the sample region.

  The depth width prediction method based on the inclination angle acquires, for example, the depth width assigned to the inclination angle using a predetermined function or a database stored in a storage medium. The depth width is predicted by using both the inclination angle and the difference between the average of the maximum value and the average of the minimum values of the relative pixels of the reference image relative to the pixels of the reference image in the blank area MAR. Also good.

  In the above-described embodiment, the depth of the sample part in the blank area MAR of the phase difference image acquired from the imaging range FSC2 in the vicinity of the imaging range FSC1 is the in-focus position in the imaging range FSC1 in which the focal position is undetermined. Predicted based on width.

  However, the parameter to be predicted for the in-focus position is not limited to the depth width of the sample part. For example, the in-focus position may be predicted based on the inclination angle of the sample part. Specifically, the in-focus position assigned to the tilt angle is acquired using a predetermined function or a database stored in a storage medium.

  Note that the depth width or the inclination angle predicted from the sample region in the margin area MAR may be a parameter for determining whether the in-focus position should be predicted or whether the in-focus position should be determined by acquiring a phase difference image. Can be used.

  Further, in the above-described embodiment, the acquisition target of the phase difference image is alternated with one shooting range FSC2 as shown in FIG. However, two or more photographing ranges FSC2 may be opened and alternated or may not be alternated. In short, it is only necessary to acquire images at predetermined intervals to the extent that the sample region in the blank area MAR can be used. However, it is more desirable in the viewpoint of prediction accuracy because the number of blank areas MAR that can be used for prediction is larger than in the case of not alternating.

  In the above-described embodiment, the movement amount (defocus amount) of the focal point of the objective lens 14 with respect to the focal position is calculated by the overall control unit 40. However, the defocus amount calculation place can be the stage drive control unit 31 instead of this embodiment.

  In the above-described embodiment, the depth of field in the objective lens 14 is fixed, but may be variable. Although the number of objective lenses 14 is one in the above-described embodiment, the objective lens 14 may be electrically or manually selected from a plurality of objective lenses having different magnifications by a lens switching mechanism.

  Further, in the above-described embodiment, when the difference between the maximum value and the minimum value in the distance (parallax) of the relative pixel of the reference image with respect to each pixel of the reference image is equal to or greater than the threshold value, the depth width is the object image of the objective lens 14. Divided into layers that fit within the depth of field (photographing layer). However, the division mode is not limited to this embodiment.

  For example, as shown in FIG. 14, the portion other than the portion that is interrupted in the depth direction in the sample portion in the depth width may be divided into imaging layers. In this way, since the process of focusing and shooting for the discontinuous portion is eliminated, it is possible to further improve the subject image acquisition efficiency. Incidentally, the discontinuous portion can be easily detected from the distance (parallax) of the relative pixel of the reference image with respect to each pixel of the reference image.

  In the above-described embodiment, two separator lenses 44A and 44B are used. However, the number of separator lenses 44 is not limited to this embodiment. A plurality of separator lenses 44 can be used with one pair of separator lenses 44A and 44B as a unit (set). In this case, it is necessary to provide openings corresponding to each set of separator lenses 44 in the aperture mask 43.

  In the above-described embodiment, the phase difference image is formed by the separator lenses 44A and 44B. However, the forming method is not necessarily limited to this embodiment, and other known ones may be adopted. Good.

  In the above-described embodiment, the light source that illuminates the bright field illumination light and the light source that illuminates the dark field illumination light are separately provided. However, one light source capable of switching between bright field illumination light or dark field illumination light may be provided. The light source may be realized by a single light source element having different wavelengths, or may be realized by a plurality of light source elements having different center wavelengths.

  It should be noted that various forms can be widely adopted without departing from the spirit of the present invention, even if other than the matters described as other embodiments.

  The present invention can be used in the bio-industry such as genetic experiments, creation of medicines, or patient follow-up.

  DESCRIPTION OF SYMBOLS 1 ... Microscope, 11 ... Preparation stage, 12 ... Light source, 13 ... Condenser lens, 14 ... Objective lens 14 ... Half mirror, 16, 20 ... Image sensor, 17 ... Field lens, 18 ... Aperture mask, 18A, 18B ... Aperture, 19A, 19B ... Separator lens, 31 ... Stage drive control unit, 32 ... Illumination control unit, 33,34 ... Imaging control unit, 35 ... Image processing unit, 40 ...... General control unit, 41 ... CPU, 42 ... ROM, 43 ... RAM, 44 ... operation input unit, 45 ... interface, 46 ... display unit, 47 storage unit, 51 ... retardation , 52... Parallax calculation unit, 53... In-focus position determination unit, 54... In-focus position prediction unit, FSC 1, FSC 2 .. Shooting range, FOV ... Field of view, PRT ... Preparation slide, SPL. Sample.

Claims (6)

An image corresponding to an image formed in a recording shooting range smaller than the field of view of the objective lens is formed, and a shooting range for focus search larger than the field of view of the objective lens is assigned to the sample, An acquisition means for acquiring an image of a sample part within the imaging range for focus search at predetermined intervals;
Determining means for determining a focal position in an imaging range for recording in which an image of the sample part corresponding to the image is formed using the image of the sample part;
Prediction for predicting a focal position in a recording imaging range in which the focal position is undetermined using an area outside the recording imaging range in the field of view of the objective lens in the image of the sample part And a focusing device. The shooting range for focus search is
As an image corresponding to the image formed in the photographing range for recording, a set of images having different viewpoints is formed,
The determination means is
The focusing apparatus according to claim 1, wherein a distance between one image and the other image in the set of images having different viewpoints is calculated in units of pixels, and the focal position and the number thereof are determined using the distance. The prediction means is
The focusing device according to claim 2, wherein the focal position in the recording photographing range in which the focal position is undetermined is predicted using the distance in the region. An image corresponding to an image formed in a recording shooting range smaller than the field of view of the objective lens is formed, and a shooting range for focus search larger than the field of view of the objective lens is assigned to the sample, An acquisition step of acquiring an image of a sample part within the imaging range for the focus search at predetermined intervals;
A determination step of determining a focal position in an imaging range for recording on which an image of the sample portion corresponding to the image is formed using the image of the sample portion;
Prediction for predicting a focal position in a recording imaging range in which the focal position is undetermined using an area outside the recording imaging range in the field of view of the objective lens in the image of the sample part A focusing method having steps. Against the computer,
An image corresponding to an image formed in a recording shooting range smaller than the field of view of the objective lens is formed, and a shooting range for focus search larger than the field of view of the objective lens is assigned to the sample, Obtaining images of sample parts within the imaging range for focus search at predetermined intervals;
Using the image of the sample part to determine a focal position in a recording photographing range where an image of the sample part corresponding to the image is formed;
Predicting a focal position in a recording imaging range in which the focal position is undetermined using an area outside the recording imaging range within the field of view of the objective lens in the image of the sample part Focusing program to execute. An image sensor for recording having a photographing range smaller than the field of view of the objective lens;
An imaging device for focus search having an imaging range larger than the field of view of the objective lens, and an image corresponding to an image formed in the imaging range of the recording imaging device;
An acquisition unit that assigns an imaging range of the imaging device for focus search to the sample and acquires an image of a sample part within the imaging range at predetermined intervals;
Determining means for determining a focal position in an imaging range of a recording image sensor on which an image of a sample part corresponding to the image is formed using the image of the sample part;
Of the image of the sample part, using the region outside the recording image sensor within the field of view of the objective lens, the focal position in the imaging range of the recording image sensor for which the focal position is undetermined is determined. A microscope having a predicting means for predicting.