JP6053327B2 - Microscope system, specimen image generation method and program - Google Patents

Description

  The present invention relates to a microscope system, a specimen image generation method, and a specimen image generation program that connect a plurality of microscope images to generate an image with a wide field of view and a high resolution.

  The range in which the specimen can be observed at a time with a microscope is mainly determined by the magnification of the objective lens. For example, if the magnification of the objective lens is increased, a high-definition image can be obtained, but the observation range is narrowed.

  In recent years, there has been a system that creates a wide-field, high-definition (high-resolution) microscopic image by capturing a plurality of images while moving the field of view using an electric stage or the like, and joining the images. Are known. For example, Patent Documents 1 to 4 divide the range of the sample into a plurality of small sections, and join the images obtained by imaging the portion of the sample corresponding to each small section with a high-resolution objective lens, A microscope system that forms a high-definition and wide-angle microscope image is disclosed. Such a system is called a virtual microscope system and is used in pathological diagnosis and the like. An image obtained by connecting a plurality of images is called a virtual slide image (hereinafter abbreviated as a VS image). Note that the VS image is also referred to as Whole Slide Imaging.

  In this way, the entire specimen is imaged as a VS image with high definition, so that, for example, if there is a personal computer and a network environment, the specimen can be observed regardless of location or time by browsing the VS image. Become. For this reason, the virtual microscope system has begun to be used for medical student pathology training and remote consultation between pathologists.

  By the way, in the pathological diagnosis, various staining techniques and observation methods are used according to the observation object and purpose. For example, hematoxylin-eosin staining (also referred to as HE staining) using two pigments of hematoxylin and eosin is generally used for histopathological diagnosis as a morphological observation stain for observing the morphology of tissues and cells. Bright field observation using an optical microscope is performed on a specimen that has been subjected to such morphological observation staining.

  In pathological diagnosis, in order to complement morphological diagnosis based on morphological information, the specimen is stained to confirm the expression of molecular information, and abnormal functions such as abnormal expression of target molecules (specific genes and proteins) are detected. A molecular pathological examination to diagnose may be done. For example, the sample is fluorescently labeled (stained) by IHC (immunohistochemistry) method, ISH (in situ hybridization) method, or the like, and fluorescence observation is performed, or enzyme field labeling is performed and bright field observation is performed. Hereinafter, staining of a target molecule by the IHC method and the ISH method is referred to as target molecule staining.

  In recent years, treatment using therapeutic agents that act on specific molecules as targets (called molecular target therapy) has been performed as a treatment for cancer and the like, and it is expected to improve the therapeutic effect and reduce side effects. . In the molecular target therapy, a therapeutic drug that targets a molecule (antigen protein) specifically expressed in cancer cells is used. When such molecular target therapy is performed, prior to the treatment, for example, the IHC method or the like is used to observe whether or not an antigen serving as a target molecule of an antibody therapeutic drug is expressed on the cell surface (ie, cell membrane), Indication patient selection is performed.

  For example, in breast cancer treatment, targeted treatment according to the expression state of the target molecule is progressing. In target treatment, disease types (cell subtypes) are classified into four types called “Luminal B”, “Luminal A”, “HER2 disease”, and “Basal like” according to the expression pattern of a plurality of target molecules in the tumor site. Based on this classification, a basic treatment choice is made. Specifically, it is hormone-dependent depending on the presence or absence of expression on the cell nucleus of estrogen receptor (hereinafter abbreviated as “ER”) and progesterone receptor (hereinafter abbreviated as “PgR”), which are hormone receptors. Judgment is made on whether or not the cancer grows, and the applicability of endocrine therapy (hormone therapy) is selected. In addition, whether to apply trastuzumab (Herceptin (registered trademark)), which is an anti-HER2 antibody preparation, is selected depending on whether or not the HER2 receptor (hereinafter abbreviated as “HER2”) is expressed on the cell membrane. In a so-called triple negative breast cancer (TNBC) in which none of ER, PgR, and HER2 is expressed, treatment is performed mainly on chemotherapy. Recently, in Luminal breast cancer, it is also determined whether or not to add chemotherapy to hormone therapy depending on the expression status of Ki-67, a nuclear protein that exhibits cell proliferation ability.

Japanese Patent Laid-Open No. 9-281405 Japanese Patent Laid-Open No. 10-333056 JP 2006-343573 A JP-T-2002-514319

  Thus, expression analysis of target molecules in pathological diagnosis is important in the selection of treatment methods. However, in order to analyze the expression of the target molecule, it is necessary to perform a very laborious operation such as counting the number of cells in which the target molecule expression is positive. For this reason, a technique for automatically counting the number of cells by image analysis has been developed. However, the conventional automatic counting technique has the following problems.

  First, in order to automatically count the number of cells, it is necessary to image a specimen stained with a target molecule with high definition. However, creating a VS image for a wide area of the entire specimen will also image a portion unnecessary for expression analysis, such as a non-tumor part, with high definition. For this reason, an increase in image acquisition time and an increase in image file capacity are incurred.

  In order to prevent an increase in file capacity, the entire specimen stained with the target molecule is imaged at a low magnification, and the pathologist observes the target molecule-stained image and extracts the necessary areas such as the tumor area. It is also conceivable to form a high-definition image only in the region. However, in this case, a pathologist needs to be interposed during image acquisition. For this reason, for pathologists, the work of imaging a sample that is not related to the original work is added, and the workflow of normal pathological diagnosis work is greatly changed, increasing the burden. Such a change is also inefficient as a workflow for pathological diagnosis work when dealing with a large number of specimens. Furthermore, in a low-resolution target molecule-stained image, it may be difficult for a pathologist to appropriately select a tumor site.

  The present invention has been made in view of the above, and can process an image necessary for pathological diagnosis in a short time without greatly changing the workflow of normal pathological diagnosis work, and has an image file capacity. It is an object of the present invention to provide a microscope system, a specimen image generation method, and a specimen image generation program capable of reducing the above.

In order to solve the above-described problems and achieve the object, a microscope system according to the present invention moves a specimen relative to an objective lens included in a microscope in a direction perpendicular to the optical axis of the objective lens, and In a microscope system for acquiring a plurality of microscope images by partially imaging a specimen and generating a virtual slide image obtained by connecting the plurality of microscope images to each other, a first slice sliced from an embedded block A first virtual slide image creating means for creating a first virtual slide image for a first specimen created by staining a section with a form capable of morphological observation in a bright field; An attention area setting means for setting an attention area, and a second slice that is sliced from the embedded block and is different from the first section, Second virtual slide image creating means for creating a second virtual slide image for the observation region corresponding to the region of interest among the second specimen created by applying a label for confirming the expression of the target molecule And a determination unit that determines whether or not focusing is poor with respect to a microscope image acquired by partially imaging the inside of the observation region, and the second virtual slide image creation unit includes: the focus in the observation region is determined to be defective portion, characterized that you reacquire the microscope image.

  In the microscope system, the morphologically observable staining is hematoxylin and eosin staining.

  In the above microscope system, the target molecule expression confirmation label is a detection probe by ISH (In situ hybridization) method, FISH (Fluorescence In situ hybridization) method, or CISH (Chromogenic In situ hybridization) method, or immunostaining. It is characterized by being.

  In the microscope system, the label for confirming the expression of the target molecule is fluorescent staining.

In the microscope system , the determination unit calculates a focus evaluation value of the microscope image, determines whether the focus evaluation value is smaller than a predetermined threshold,
The second virtual slide image creating means re-acquires the microscopic image for a portion determined by the determining means that the focus evaluation value is smaller than a predetermined threshold value .

In the microscope system , the determination unit calculates a focus evaluation value of the microscope image at a position other than a focus position extraction point where the focus position is measured by sampling within the observation region, and the focus evaluation value is a predetermined value. It is characterized by determining whether it is smaller than this threshold value .

  The microscope system further includes expression level calculation means for calculating the expression level of the target molecule in the second virtual slide image.

  In the above microscope system, each of the first and second specimens is provided with specimen information including at least information on an embedding block obtained by slicing a slice and staining type, and specimen information for obtaining the specimen information The apparatus further comprises an acquisition unit and a sample selection unit that selects the second sample based on the sample information acquired from the first sample.

  The microscope system can further include a plurality of specimens, and further includes a slide transport unit that can sequentially transport and transport each of the plurality of specimens to the field of view of the objective lens. The plurality of specimens are continuously processed.

  The microscope system includes a cell number counting unit that counts the number of cells existing in the region of interest set by the region of interest setting unit, and a warning unit that warns a user when the number of cells is less than a predetermined value. And further comprising.

In the specimen image generation method according to the present invention, a plurality of microscopes are obtained by moving a specimen relative to an objective lens included in the microscope in a direction perpendicular to the optical axis of the objective lens and partially imaging the specimen. In a specimen image generation method executed by a microscope system that acquires an image and generates a virtual slide image obtained by connecting the plurality of microscope images to each other, a bright image is obtained with respect to a first section sliced from an embedding block. A first virtual slide image creating step for creating a first virtual slide image and a region of interest in the first virtual slide image are set for the first specimen created by staining with a morphologically observable visual field. A region of interest setting step and a second molecule that is sliced from the embedding block and is different from the first segment; A second virtual slide image creating step for creating a second virtual slide image for an observation region corresponding to the region of interest among the second specimen created by applying a label for confirming expression; unrealized, the second virtual slide image creation step, to a microscope image obtained by partially imaging the observation region, determining whether or not an in-focus is defective, the observation region A re-acquisition step of re-acquiring a microscopic image for a portion determined to be poor in focus .

A specimen image generation program according to the present invention moves a specimen relative to an objective lens included in a microscope in a direction perpendicular to the optical axis of the objective lens, and partially images the specimen to thereby obtain a plurality of microscopes. In a specimen image generation program executed by a microscope system that acquires an image and generates a virtual slide image obtained by connecting the plurality of microscope images to each other, the first section sliced from the embedding block is brightened. A first virtual slide image creating step for creating a first virtual slide image and a region of interest in the first virtual slide image are set for the first specimen created by staining with a morphologically observable visual field. A region of interest setting step and a second slice that is sliced from the embedding block and is different from the first slice Second virtual slide image creation for creating a second virtual slide image for an observation region corresponding to the region of interest among the second specimen created by applying a label for confirming the expression of the target molecule And the second virtual slide image creating step determines whether or not focusing is poor with respect to the microscope image acquired by partially imaging the inside of the observation region. And a re-acquisition step of re-acquiring a microscope image for a portion where focusing in the observation region is determined to be poor .

  According to the present invention, after the attention area is set in the virtual slide image of the first specimen, a high-definition virtual slide image for only the area corresponding to the attention area of the second specimen is automatically generated. Therefore, it is possible to execute the image generation processing necessary for pathological diagnosis in a short time without greatly changing the workflow of normal pathological diagnosis work, and to reduce the image file capacity. Is possible.

FIG. 1 is a schematic diagram showing a configuration example of a microscope system according to Embodiment 1 of the present invention. FIG. 2 is a schematic diagram illustrating an example of a slide glass on which a specimen is fixed. FIG. 3 is a block diagram illustrating a configuration example of the host system illustrated in FIG. FIG. 4 is a schematic diagram illustrating a data configuration example of an image file stored in the image data recording unit illustrated in FIG. 3. FIG. 5 is a flowchart showing a specimen image generation method according to Embodiment 1 of the present invention. FIG. 6 is a diagram illustrating an example of specifying a region of interest in a high-resolution image of a HE-stained specimen. FIG. 7 is a flowchart showing processing for creating a VS image of the HE-stained specimen shown in FIG. FIG. 8 is a schematic diagram showing an image corresponding to the specimen existing area shown in FIG. FIG. 9 is a table showing a data configuration example of the focus map. FIG. 10 is a table showing a data configuration example of the VS image list. FIG. 11 is a flowchart showing processing for creating a VS image of an immunostained specimen. FIG. 12 is a schematic diagram showing the scan range determined in the immunostained specimen. FIG. 13 is a schematic diagram illustrating an expression analysis target region set in a region of interest. FIG. 14 is a block diagram showing a configuration of the host system according to Embodiment 2 of the present invention. FIG. 15 is a flowchart showing details of processing for creating a high resolution image in the scan range in the second embodiment. FIG. 16 is a block diagram showing the configuration of the host system according to Embodiment 3 of the present invention.

  Hereinafter, embodiments according to the present invention will be described in detail with reference to the drawings. Note that the present invention is not limited to these embodiments. Moreover, in description of drawing, the same code | symbol is attached | subjected and shown to the same part.

(Embodiment 1)
FIG. 1 is a diagram illustrating a configuration example of the entire microscope system according to the first embodiment.
As shown in FIG. 1, the microscope system 1 according to the first embodiment includes a microscope apparatus 10, a microscope controller 20 that controls each part of the microscope apparatus 10, and a television (TV) camera 30 attached to the microscope apparatus 10. A television (TV) camera controller 40 that controls the operation of the TV camera 30, a host system 50-1 that is connected to each of these units so as to be able to send and receive data, and controls the overall operation of the microscope system 1 and a specimen. A slide transport device 60 capable of accommodating a plurality of fixed slide glasses and transporting one slide glass (hereinafter also referred to as slide glass sample 2) to which the specimen to be observed is fixed to the microscope apparatus 10 one by one; A barcode reader 70 for reading a barcode attached to the glass specimen 2 is provided. The microscope system 1 moves the specimen in a direction orthogonal to the optical axis of the objective lens with respect to the objective lens included in the microscope apparatus 10, images the specimen for each part while shifting the imaging field of view, and obtains a plurality This is a microscope system capable of generating a virtual slide image (hereinafter abbreviated as a VS image) obtained by connecting the images.

  FIG. 2 is a schematic diagram illustrating an example of the slide glass specimen 2. As shown in FIG. 2, the slide glass specimen 2 includes a slide glass S and a slice specimen SP fixed on the slide glass S. Further, on the slide glass S, a label LB on which sample information related to the slice sample SP is described is affixed.

  The slice specimen SP is obtained by performing predetermined staining on a slice obtained by slicing a block-like specimen (also referred to as an embedded block) collected from a living body. The section sample SP is fixed within a sample search range A1 which is a preset region on the slide glass sample 2. The sample search range A1 has, for example, a size of about vertical: 25 mm × width: 50 mm.

  Further, a label LB on which information (hereinafter referred to as specimen information) relating to the slice specimen SP is attached is attached to the slide glass specimen 2. The specimen information includes an examination ID, an embedding block number, a section number, staining information, and the like of the specimen fixed on the slide glass. This staining information includes information such as the type of pigment that stained the section sample SP. The specimen information is printed on the label LB in a form that can be automatically read, such as a two-dimensional barcode.

  The microscope apparatus 10 includes, as a transmission observation optical system 11, a transmission illumination light source 110, a collector lens 111 that collects illumination light of the transmission illumination light source 110, a transmission filter unit 112, a transmission shutter 113, and a transmission A field stop 114, a transmission aperture stop 115, a reflection mirror 116 that bends the optical path of illumination light, a condenser optical element unit 117, and a top lens unit 118 are provided. The microscope apparatus 10 includes an epi-illumination light source 120, a collector lens 121, an epi-illumination filter unit 122, an epi-illumination shutter 123, an epi-illumination field stop 124, and an epi-illumination aperture stop 125 as the epi-illumination optical system 12. Have

  An electric stage 13 on which the slide glass specimen 2 is placed is provided on the observation optical path L that overlaps both the optical path L1 of the transmission observation optical system 11 and the optical path L2 of the incident observation optical system 12. . The electric stage 13 is provided so as to be movable in a plane (XY plane) orthogonal to the optical axis of the objective lens 14a inserted in the observation optical path L and along a direction (Z direction) parallel to the optical axis. Yes. The movement control of the electric stage 13 is performed via the motors 131 and 132 by the stage XY drive control unit 21 and the stage Z drive control unit 22 that operate under the control of the microscope controller 20, respectively. The electric stage 13 has an origin position detection function (not shown) by an origin sensor, and coordinates can be set for each position on the slide glass specimen 2 placed on the electric stage 13. it can.

  Above the electric stage 13, there is provided a revolver 14 capable of holding a plurality of (two in FIG. 1) objective lenses 14a and 14b having different magnifications. As the objective lenses 14a, 14b,..., At least a low magnification (for example, 2 × and 4 ×) objective lens (hereinafter referred to as a low magnification objective lens) having a relatively low magnification and a magnification higher than that of the low magnification objective lens. An objective lens (hereinafter, referred to as a high magnification objective lens) having a high magnification (for example, 10 times, 20 times, or 40 times) may be used. Note that the low magnification and the high magnification are examples, and it is sufficient that at least one magnification is higher than the other magnification. The revolver 14 selectively inserts an objective lens used for observation at that time into the observation optical path L by a rotation operation. FIG. 1 shows a state where the objective lens 14a is inserted into the observation optical path L.

  On the observation optical path L, a plurality (two in FIG. 1) of optical cubes 15a and 15b and an optical cube unit 15 that holds these optical cubes 15a and 15b are provided. The optical cube unit 15 selectively inserts an optical cube corresponding to the microscopic method at that time into the observation optical path L. FIG. 1 shows a state in which the optical cube 15a is inserted into the observation optical path L.

  These revolver 14 and optical cube unit 15 are motorized, and their operations are controlled by the microscope controller 20.

  Further, on the observation light path L, a beam splitter 16 is provided for branching the observation light that has passed through the objective lens 14a to the eyepiece 17 side and the TV camera 30 side.

  The microscope controller 20 has a function of controlling the operation of the entire microscope apparatus 10, and changes the spectroscopic method, the transmitted illumination light source 110, and the epi-illumination light source 120 in accordance with a control signal from the host system 50-1. Each unit is controlled such as dimming, and the current state of each unit is detected and a detection signal is sent to the host system 50-1. Further, the microscope controller 20 controls the movement of the electric stage 13 via the stage XY drive control unit 21 and the stage Z drive control unit 22 in accordance with a control signal from the host system 50-1.

  The TV camera 30 has a color image having, for example, 256 gradation pixel levels (pixel values) for each color component (wavelength component) of R (red), G (green), and B (blue) at each pixel position. It is an imaging device that can be generated. The TV camera 30 is provided with an imaging device such as a CCD that converts received observation light into an electrical signal to generate image data. Image data generated by the TV camera 30 is taken into the host system 50-1 via a video board 51 described later. Various settings such as automatic gain control ON / OFF, gain setting, automatic exposure control ON / OFF, and exposure time setting for the TV camera 30 are controlled by the host system 50-1 via the camera controller 40. Done in

  The host system 50-1 connects a main storage device such as a CPU, a video board, and a main memory, an external storage device such as a hard disk and various storage media, a communication device, an output device such as a display device and a printing device, an input device, and various parts. Alternatively, it is realized by a known hardware configuration (for example, a general-purpose computer such as a workstation or a personal computer) provided with an interface device for connecting an external input.

  FIG. 3 is a block diagram showing the configuration of the host system 50-1. As shown in FIG. 3, the host system 50-1 includes a video board 51 for processing a video signal input from the TV camera 30, an input unit 52, a monitor 53, an image data recording unit 54, and an imaging coordinate recording. A unit 55, a program recording unit 56, and a control unit 57 that controls the operations of these units and the entire microscope system 1. In addition, the host system 50-1 includes a main memory used as a work memory by the control unit 57, an interface unit that manages the exchange of various data with each unit constituting the microscope system shown in FIG. (Not shown).

  The input unit 52 includes input devices such as a mouse and a keyboard that are used when a user inputs various types of information and commands to the host system 50-1, and controls input signals input via these input devices. 57.

  The monitor 53 is realized by a display device such as an LCD or an EL display, and includes an operation screen for operating the microscope device 10, a microscope image captured by the microscope device 10, a VS image obtained by connecting a plurality of microscope images, The related information of these images is displayed.

  The image data recording unit 54, the imaging coordinate recording unit 55, and the program recording unit 56 are, for example, various IC memories such as ROM and RAM such as update-recordable flash memory, built-in or external hard disk, or CD-ROM. This is realized by an information recording medium and a reading device thereof. In particular, the image data recording unit 54 is preferably realized by a hard disk, a large capacity memory, or the like.

  The image data recording unit 54 records image data input via the video board 51. The image data recorded in the image data recording unit 54 is read by the control unit 57 at any time (for example, according to an operation on the input unit 52 by the user) and output to the monitor 53, for example. Thereby, a microscope image represented by the image data is displayed on the monitor 53.

The imaging coordinate recording unit 55 records XYZ coordinates (in-focus position coordinates) when imaging the slide glass specimen 2.
The program recording unit 56 records various control programs for controlling the operation of the microscope system 1, data used during the execution of these control programs, and the like.

  The control unit 57 is realized by hardware such as a CPU, and executes various functions such as control of the microscope system 1 and image processing of a microscope image by reading a control program recorded in the program recording unit 56. To do. The control unit 57 includes an in-focus position calculation unit 571, a first VS image creation unit 572, a specimen selection unit 573, a region of interest setting unit 574, a second VS image creation unit 575, and an expression level calculation unit 576. .

  The in-focus position calculation unit 571 calculates the in-focus position coordinates by causing the microscope apparatus 10 to perform an in-focus operation (so-called video AF function) based on the contrast of the image input via the TV camera 30. The calculated in-focus position coordinates are recorded in a focus map (described later) stored in the imaging coordinate recording unit 55.

  The first VS image creation unit 572 creates a VS image of the first slide glass specimen that has been subjected to staining capable of morphological observation in a bright field.

  Based on the specimen information attached to each slide glass specimen, the specimen selection unit 573 generates a VS image from the second slide glass specimen that has been labeled for target molecule expression confirmation. The second glass slide specimen corresponding to the glass slide specimen is selected. The sample information is acquired via the barcode reader 70 as will be described later.

  The attention area setting unit 574 sets the area selected in the VS image of the first glass slide specimen as the attention area according to the signal input by the user operation.

  The second VS image creation unit 575 creates a VS image of a region (observation region) corresponding to the attention region set by the attention region setting unit 574 among the second slide glass samples selected by the specimen selection unit 573. .

  The expression level calculation unit 576 calculates the expression level of the target molecule from the VS image of the second slide glass specimen.

  In addition, the control unit 57 transmits a control signal of the microscope controller 20 when acquiring an image of each glass slide specimen, and controls each part of the microscope apparatus 10 (movement control of the electric stage 13 and the spectroscopic method). Change). In addition, the control unit 57 detects the state of each unit of the microscope apparatus 10 via the microscope controller 20. In the following description, such control and state detection will not be described step by step.

  The host system 50-1 is provided with a general-purpose interface (not shown) such as USB or LAN, and is connected to the slide transport device 60 and the barcode reader 70 via these general-purpose interfaces.

  The slide conveyance device 60 can accommodate a large number of slide glass specimens, for example, several hundred pieces, and the slide glass specimens to be accommodated one by one under the control of the host system 50-1 without human intervention. Automatic conveyance and automatic unloading with the electric stage 13 are performed.

  The barcode reader 70 reads a two-dimensional barcode printed on a label attached to the slide glass specimen and inputs it to the host system 50-1. Thereby, the host system 50-1 can automatically capture the specimen information of the slide glass specimen.

  Next, the file format of the VS image recorded in the image data recording unit 54 will be described. In the first embodiment, the image data generated by the TV camera 30 is stored in units of VS images created for each slide glass specimen. The image data of the VS image may be compressed by a known compression algorithm such as JPEG or JPEG2000 and recorded in the image data recording unit 54.

  FIG. 4 is a schematic diagram illustrating a data configuration example of an image file stored in the image data recording unit 54. As shown in FIG. 4, the image file D includes an incidental information data part D1, a specimen search range image data part D2, a wide-field / high-definition image data part D3, a region-of-interest setting information part D4, and a target molecule. An expression analysis data part D5.

  The incidental information data part D1 includes observation method information (bright field observation, fluorescence observation, etc.) D11 of the slide glass specimen, the above-described specimen information D12, and magnification information (sample search) when an image of the entire specimen search range A1 is captured. Data such as range image data imaging magnification information (D13) is recorded.

  In the sample search range image data portion D2, image data of an image showing the entire sample search range A1 is recorded.

  In the wide-field / high-definition image data section D3, information D30 of the number of a plurality of regions constituting the VS image (that is, the number of connected microscope images) n, and information about each region (region # 1 information D311 to D31) Area #n information D3n) is recorded. Information about each area includes imaging information D321, focus map data D322, and image data D323 of a microscope image acquired by imaging the area. Among these, the imaging information includes information such as imaging magnification, scan start stage X coordinate, scan start stage Y coordinate, number of pixels in the X direction, and number of pixels in the Y direction. At the start of scanning, the XY coordinates of the upper left region (section) are set toward the scanning target region. The focus map data D322 records the focus position coordinates in each area. The focus map will be described in detail later.

  In the attention area setting information section D4, as information regarding the attention area set for the VS image of the slide glass specimen that has been subjected to morphological observation, the attention area number (m) information D40, and each attention area Information (attention area # 1 information D40 to attention area #m information D4m) is recorded.

  In the target molecule expression analysis data part D5, the target molecule expression analysis result detected from the VS image of the slide glass specimen subjected to the target molecule staining is recorded.

Next, the specimen image generation method according to Embodiment 1 will be described. FIG. 5 is a flowchart showing the specimen image generation method according to the first embodiment.
First, in step S <b> 1, an operator such as a laboratory technician accommodates the HE-stained specimen in the slide transport device 60. The HE-stained specimen is a specimen in which a section sliced from the embedding block is subjected to hematoxylin-eosin staining (hereinafter referred to as HE staining) as a staining capable of morphological observation in a bright field, and this is fixed to a slide glass. In normal pathological examination work, slide glass specimens are prepared in a batch process according to the staining method. In this step S1, a plurality of HE stained specimens respectively created from a plurality of embedded blocks are slid. You may accommodate in the conveying apparatus 60. FIG.

  In HE staining, cell nuclei are stained blue-violet with hematoxylin (hereinafter also referred to as H dye), and cytoplasm and connective tissue are stained light red with eosin (hereinafter also referred to as E dye).

  In the subsequent step S2, the microscope system 1 transports the HE-stained specimen accommodated in the slide transport apparatus 60 to the microscope apparatus 10, and obtains a rectangular area (specimen existence area A2) including the section specimen SP from the specimen search range A1. A wide-field and high-definition VS image in which the entire area is captured is created. The VS image is recorded in the image data recording unit 54 as a VS image file in association with the specimen information. When a plurality of HE-stained specimens are accommodated in the slide transport device 60, VS images are sequentially created for these HE-stained specimens, and VS image files are recorded. The process for creating the VS image will be described in detail later.

In subsequent step S3, the microscope system 1 sets a region of interest on the VS image of the HE-stained specimen.
More specifically, the host system 50 sequentially displays the VS images of the HE-stained specimen recorded in the image data recording unit 54 on the monitor 53 according to the operation of the user (for example, a pathologist). FIG. 6 shows a display example of the VS image of the HE-stained specimen displayed on the monitor 53. The pathologist observes the VS image on the monitor 53, creates a diagnosis and a finding based on the morphological information, and reports a pathological diagnosis. Specifically, a diagnosis is made as to whether the tumor seen in the VS image is a benign tumor or a malignant tumor, what is the tissue type, and what is the tumor diameter. Then, for the purpose of selecting a treatment method or confirming the diagnosis, a region is selected that is judged to require confirmation of the expression of the target molecule. This selection is performed, for example, by surrounding the tumor site with a closed figure such as a polygon using an input device such as a mouse for the VS image. Alternatively, the region may be selected with a free curve by a mouse drag operation.

  In response to the pointer operation on the VS image displayed on the monitor 53, the host system 50-1 converts, for example, polygonal vertex coordinates (coordinates on the curve in the case of a free curve) into the attention area setting information section D4 ( (See FIG. 4). Thereby, the attention area A3 is set. As shown in FIG. 6, it is possible to select a plurality of attention areas A3. In this case, a plurality of attention area information (attention area # 1 information D41, attention area # 2 information D42,...) Is recorded. Is done.

  In subsequent step S <b> 4, an operator such as a laboratory technician accommodates the immunostained specimen in the slide transport device 60. The immunostained specimen is a specimen prepared by slicing a section from the same embedding block as that prepared the HE-stained specimen, and immunostaining the section as a label for confirming the expression of the target molecule and fixing it on a slide glass. . At this time, it is preferable to use sections that are continuous or adjacent to each other between the HE-stained specimen and the immunostained specimen. Thereby, it is possible to create an immunostained specimen in which each part in the section (a mode of a component in a tissue or a cell, a manner of appearance of a lesion site, etc.) substantially corresponds to a section of a HE-stained specimen.

  In immunostained specimens, for example, peroxidase is used as a labeling enzyme for immunostaining, and diaminobenzidine (hereinafter referred to as DAB) is used as a chromogenic substrate, and the presence of a target molecule is colored brown, and hematoxylin is used for counterstaining. To stain blue-violet.

  Note that a plurality of immunostained specimens can be accommodated in the slide transport device 60 by batch processing, as in step S1. In this case, an immunostained specimen is created from the same embedded block for each HE-stained specimen in which the region of interest A3 is set.

  In subsequent step S <b> 5, the microscope system 1 transports the immunostained specimen accommodated in the slide transport device 60 to the microscope device 10 to create a VS image. At this time, the microscope system 1 reads the barcode attached to the immunostained specimen to obtain specimen information (examination ID and embedded block number), and the HE-stained specimen of the same embedded block identified by this specimen information VS image file is referred to. Then, a wide-field and high-definition VS image is created for an area on the immunostained specimen corresponding to the attention area A3 recorded in the attention area setting information section D4.

In subsequent step S6, the control unit 57 automatically analyzes the VS image of the immunostained specimen, counts the number of cells with positive expression of the target molecule, and calculates the ratio of positive cells in the total number of cells. Then, the expression level of the target molecule is calculated.
Thereafter, the process is complete.

  Next, step S2 for creating a VS image of a HE-stained specimen will be described in detail. For the VS image creation processing, see also Japanese Patent Application Laid-Open Nos. 9-281405 and 2006-343573.

FIG. 7 is a flowchart showing a process for creating a VS image of a HE-stained specimen.
When the operator performs a process start operation on the operation screen displayed on the monitor 53, the slide transport device 60 transports one HE-stained specimen housed on the electric stage 13 in step S10.

  In subsequent step S11, the barcode reader 70 reads the barcode (for example, a two-dimensional barcode) of the label LB affixed to the transported HE-stained specimen, acquires the specimen information of the slide glass specimen 2, and acquires the host system. 50-1 is transmitted. This sample information is recorded as sample information 612 in the incidental information data portion in the VS image file (see FIG. 4). If the sample information described above is printed as character information on the label LB, the sample information is obtained by separately acquiring image data of the label LB by macro illumination, a macro camera, or the like, and recognizing the image of the label LB. You may get it.

  In subsequent step S12, the host system 50-1 sets the observation method in the microscope apparatus 10 to bright field observation (transmission bright field observation). In response to this, the microscope controller 20 performs control to attach / detach various optical members to / from each optical path of the microscope apparatus 10. Specifically, the microscope apparatus 10 inserts the epi-illumination shutter 123 into the optical path L2, inserts an optical cube for bright field observation into the optical path in the optical cube unit 15, and turns on the light source 110 for transmitted illumination. Thereby, in the microscope apparatus 10, the state which can perform transmission bright field observation is constructed | assembled.

  In subsequent step S13, the microscope controller 20 changes the objective lens used in the microscope apparatus 10 to, for example, a 2 × low magnification objective lens under the control of the host system 50-1. As a result, the revolver 14 rotates and the low-magnification objective lens is inserted into the optical path L.

  In subsequent step S14, the microscope system 1 creates a low-resolution image of the entire HE-stained specimen. More specifically, the host system 50-1 inserts the specimen search range A1 (see FIG. 2) on the HE-stained specimen according to the width of the imaging area projected on the TV camera 30 (in other words, the optical path L). The imaging region is divided into a plurality of sections according to the magnification of the low-magnification objective lens. Then, the microscope controller 20 operates the electric stage 13 in the XY directions to sequentially move the divided sections to the imaging field of view of the microscope apparatus 10. The TV camera 30 sequentially captures the sections in the imaging field of view, generates image data, and inputs the image data to the host system 50-1. Based on the input image data, the host system 50-1 creates a low-resolution image in which the entire specimen search range A1 of the HE-stained specimen is shown by connecting the microscopic images of the sections. The image data of the created low resolution image is recorded in the VS image file 6 as sample search range image data (see FIG. 4).

  In subsequent step S15, the microscope controller 20 replaces the objective lens used in the microscope apparatus 10 with, for example, a 20 × high magnification objective lens under the control of the host system 50-1. As a result, the revolver 14 rotates and the high-magnification objective lens is inserted into the optical path L. As a result, the microscope apparatus 10 can obtain a high-resolution image of the HE-stained specimen.

  In step S16, the control unit 57 automatically extracts the specimen existing area A2 (see FIG. 2) on the HE-stained specimen based on the low resolution image of the entire specimen search range A1 acquired in step S14. A point for measuring the focus position (focus position extraction point) is determined.

More specifically, the control unit 57 first obtains luminance information in each pixel of the low resolution image that is a color (RGB) image. Specifically, the G component may be extracted as luminance information, or the luminance value Y calculated by the following equation (1) may be adopted as luminance information.
Luminance value Y = 0.299 × R + 0.587 × G + 0.114 × B (1)

  Subsequently, the control unit 57 creates a binarized image in which the luminance information of each pixel is binarized depending on whether or not it falls within the range of the predetermined upper limit threshold and lower limit threshold. A pixel whose luminance information falls within the threshold range is a pixel in which an image (specimen image) of the slice specimen SP is captured, and a pixel whose luminance information does not fall within the threshold range is a pixel where the sample image is not captured. Then, the control unit 57 searches the binarized image from each edge toward the center, and obtains a rectangular region circumscribing the sample image. This rectangular area is determined as the specimen existence area A2.

  Note that, in a slide glass specimen such as an HE-stained specimen, an object other than the slice specimen SP such as dust may be attached to the edge portion even in a region where the slice specimen SP does not exist. For this reason, when a pixel having luminance information within the above-mentioned threshold range is found during the search for the specimen existence area A2, it is determined whether the object reflected at the pixel position is a specimen image or a dust image depending on the staining situation. to decide. Here, since dust is not dyed, it is gray in the image, and the RGB values are close to each other. Therefore, for example, if the R / G value and the B / G value, which are color ratios, are within a predetermined range centered at 1.0, it is determined that dust is reflected in the pixel, and the sample image Continue the search. Alternatively, the saturation (S) may be obtained by converting the RGB pixel value into the HSV color space, and if the saturation is lower than a predetermined threshold value, it may be determined that dust is reflected on the pixel.

  In addition, a small isolated region may exist on a slide glass sample such as a HE-stained sample. However, such an isolated region is an unnecessary region for diagnosis even if it is stained, and there is a possibility that accuracy of positional deviation determination with an immunostained specimen described later may be deteriorated. Accordingly, when the pixel area having the luminance information within the threshold range does not have a predetermined area, it is determined that dust is captured, and the pixel area is excluded from the sample image. Note that this exclusion process may be performed after reducing the low-resolution image in which the entire sample search range A1 is captured to an appropriate magnification (resolution) in order to shorten the processing time.

  FIG. 8 is a schematic diagram showing the specimen existence region extracted from the low-resolution image of the HE-stained specimen as described above. A specimen existence area M1 shown in FIG. 8 corresponds to a specimen existence area A2 on the slide glass specimen 2 shown in FIG.

  Subsequently, the control unit 57 divides the sample existing area M1 into a plurality of lattice-shaped small sections C1. The area size of each small section C1 corresponds to the imaging area projected onto the TV camera 30 via the high-magnification objective lens inserted into the optical path L in step S15. Then, the control unit 57 calculates the xy coordinates (specifically, the center coordinates of the small section C1) of each small section C1 in the sample existing area M1 (or the low resolution image corresponding to the sample search range A1). The xy coordinates are converted into the position coordinates (XY coordinates) of the electric stage 13 based on the imaging magnification, image sensor information (number of pixels, pixel size), etc. (see, for example, Japanese Patent Laid-Open No. 9-281405). In the specimen existence area M1 shown in FIG. 8, the small section C1 in the upper left corner of the figure is the origin.

  Further, the control unit 57 automatically extracts a small section C1 (focus position extraction point FP) in which the in-focus position (Z coordinate) is measured by sampling from among the plurality of small sections C1. This is because, when obtaining the in-focus position of each subsection C1, if there are a large number of subsections C1 in the sample existing area M1, it is very difficult to obtain the in-focus position by actual measurement for all the subsections C1. This is because it takes time. The method of extracting the focus position extraction point FP may be random extraction, or may be extracted regularly (for example, every 6 sections). Further, the number of extraction of the focus position extraction points FP may be determined in advance so as to be, for example, a predetermined number or less in accordance with the size of the sample existence area M1. That is, the processing content to be automatically extracted may be arbitrarily determined. It should be noted that the small section C1 that is recognized as a background portion or a cavity portion in which the image (specimen image) SP ′ corresponding to the slice specimen SP does not exist in the specimen existing area M1 is naturally excluded as a focus position extraction point. . In FIG. 8, the small section C1 extracted as the focus position extraction point FP is indicated by hatching.

In step S17, the control unit 57 creates a focus map in which in-focus positions (Z coordinates) at the respective points on the specimen existing area M1 are recorded.
Specifically, the control unit 57 operates the electric stage 13 in the XY directions via the microscope controller 20 to sequentially move the focus position extraction point FP extracted in step S16 to the optical axis position of the objective lens. Subsequently, the control unit 57 inputs the sample image SP ′ via the TV camera 30 while moving the electric stage 13 in the Z-axis direction for each focus position extraction point FP, and performs a focus evaluation calculation (contrast evaluation). ) To obtain a focus position by actual measurement.

  Here, in the focus evaluation calculation, for example, a primary differential filter process (for example, a sobel filter) is used based on the principle that the rise of the edge portion rises sharply when in focus. For this reason, in order to remove the fine edge portion extracted by the filter processing even though the change in luminance is poor, the edge intensity below a predetermined threshold is deleted. The sum of the edge detection data thus obtained is calculated as the focus evaluation value, and the Z coordinate when the focus evaluation value is maximized is determined as the focus position.

  The region for performing the focus evaluation calculation may be the sample image SP ′ input via the TV camera 30, or only the central portion of the sample image SP ′ or a plurality of regions in the central portion or the peripheral portion. There is no particular limitation.

  Next, in step S16, the control unit 57 calculates a focus position by interpolation calculation for the small section C1 that has not been extracted as the focus position extraction point FP, based on the actually measured focus position at the nearby focus position extraction point FP. .

  FIG. 9 is a diagram illustrating an example of a focus map. As shown in FIG. 9, the focus map M2 includes coordinate numbers (001, 001), (002, 001),... Assigned to each subsection C1 obtained by dividing the specimen existing area M1, and coordinate information of the electric stage 13. (Stage coordinates) and a focus evaluation value. The coordinate information includes coordinates in the X-axis and Y-axis directions of the electric stage 13 when the region on the HE-stained specimen corresponding to each small section C1 is matched with the imaging field of the high-magnification objective lens inserted in the optical path L, and And coordinates in the Z-axis direction representing the in-focus position. Such a focus map M2 is stored in the imaging coordinate recording unit 55.

  In subsequent step S18, the microscope system 1 creates a high-resolution image (VS image) in which the specimen existing area A2 is reflected. Specifically, the microscope controller 20 sequentially moves the electric stage 13 to the stage coordinates (X, Y, Z) registered in the focus map M2. Each time the motorized stage 13 moves, the TV camera 30 captures an area on the HE-stained specimen corresponding to each small section C1 and inputs the image data to the host system 50-1. In the host system 50-1, the control unit 57 performs a connecting process for connecting the microscopic images of the adjacent small sections C1 based on the input image data. By repeating a series of operations such as movement of the motorized stage 13, input of image data, and connection processing of microscope images for all the small sections C1 registered in the focus map M2, the entire specimen existence area A2 is processed. A high-definition VS image with a wide field of view is created. The image data of the VS image created in this way is stored in a VS image file (see FIG. 4).

  At this time, as shown in FIG. 10, the control unit 57 creates a VS image list (file list) representing the correspondence between the information related to the section specimen SP and the VS image file, and stores it in the image data recording unit 54. Store. In the VS image list, information such as an examination ID of the section specimen SP, an embedding block number (No.) from which the section specimen SP is collected, staining information, and a VS image file name are recorded. As will be described later, this VS image list is used when searching for VS images created from the same embedded block.

  Further, in step S <b> 19, the slide conveyance device 60 carries out the slide glass specimen 2 placed on the electric stage 13 and returns it to the slide conveyance device 60. Thereby, the creation process of the VS image of the HE-stained specimen is completed.

  A series of operations of these steps S <b> 10 to S <b> 19 is repeated for all of the HE-stained specimens accommodated in the slide conveyance device 60. Thereafter, the process returns to the main routine.

  In order to prevent the occurrence of discontinuous areas in the VS image, an overlapping area (see Japanese Patent Application Laid-Open No. 9-281405) is provided between the adjacent small sections C1, and the overlapping process is performed so as to overlap each other. (Step S18) may be performed. In this case, in step S16, the sample existing area M1 is divided so that the size of each small section C1 is a size obtained by removing the overlap area from the imaging area projected onto the TV camera 30 via the high-magnification objective lens. Just do it.

Next, step S5 for creating a VS image of an immunostained specimen will be described in detail. FIG. 11 is a flowchart showing a process for creating a VS image of an immunostained specimen.
In step S20 following step S4 (see FIG. 5), the slide transport device 60 transports one immunostained specimen housed on the motorized stage 13.

  Processing in subsequent steps S21 to S25 corresponds to steps S11 to S15 shown in FIG. However, Steps S21 to S25 are different from Steps S11 to S15 in that the observation target is an immunostained specimen.

  In step S26 subsequent to step S25, the control unit 57 sets the amount of positional deviation of the specimen region between the HE-stained specimen prepared from the same embedded block as the immunostained specimen currently being observed and the immunostained specimen. calculate.

  More specifically, the control unit 57 refers to the VS image list (see FIG. 10), and has the same specimen information as the specimen information (test ID and embedded block number) of the immunostained specimen acquired in step S11. The file name of the stained specimen is searched, and the VS image file of the HE stained specimen is acquired. Then, image data (referred to as HE-stained whole specimen data) recorded in the specimen search range image data portion D2 (see FIG. 4) in the acquired VS image file, and an image within the specimen search range of the immunostained specimen being observed. The data (hereinafter referred to as immunostained specimen whole data) is compared, and the deviation amount (parallel movement amount and rotation amount) of the specimen position is calculated.

  The amount of deviation is calculated as follows, for example. First, each of the above two sample whole data is binarized to perform contour extraction, and the entire region is filled as a sample region. Then, the center of gravity of each sample region is obtained, and the difference between these two center of gravity coordinates is obtained. Next, in a state where the center of gravity of the specimen region in the whole immunostained specimen data and the center of gravity of the specimen region in the whole HE stained specimen data are matched, for example, the specimen region of the whole immune specimen data is set in a predetermined angular unit centered on the center of gravity. Rotate clockwise or counterclockwise to obtain the angle (rotation amount) that minimizes the absolute value of the difference from the sample region of the entire HE-stained sample data. The difference between the center-of-gravity coordinates and the amount of rotation (including the direction of rotation) obtained in this way represent the positional misalignment between the immunostained specimen and the HE-stained specimen on the electric stage 13.

  Note that when performing contour extraction on the entire sample data, the luminance value, color ratio (R / G, B / G), or area is outside the predetermined threshold range, as in step S16 described above. A certain area may be judged as dust and removed.

  In subsequent steps S27 to S30, the microscope system 1 follows the region (observation) on the immunostained sample corresponding to each region of interest A3 (see FIG. 6) according to the region-of-interest setting information recorded in the VS image file of the HE-stained sample. A microscopic image with a wide field of view and a high definition is acquired.

  For this purpose, first, in step S27, the control unit 57 performs affine transformation (translation and rotation) on the coordinates of the attention area A3 recorded in the VS image file of the HE-stained specimen based on the positional deviation amount obtained in step S26. The coordinate value of the attention area A3 is corrected. The corrected attention area is set as an observation area in the immunostained specimen, and a rectangular area including the observation area is determined as a scan range.

  In subsequent step S28, the control unit 57 determines a focus position extraction point from the determined scan range. FIG. 12 is a schematic diagram illustrating a scan range including an observation region. The control unit 57 divides the scan range M3 into a plurality of lattice-like small sections C2, and the small section C2 extracted according to the shape of the observation area A4 from these small sections C2 is used as the focus position extraction point FP. decide. The area size of each small section C2 corresponds to the imaging area projected on the TV camera 30 through the high-magnification objective lens inserted into the optical path L in step S25. In FIG. 12, the small section C2 extracted as the focus position extraction point FP is indicated by hatching.

  Here, in general, as an area where a focus failure is likely to occur in the observation area A4, a contour portion of the observation area A4 can be cited. This is because the in-focus position is predicted based on the actually measured value of the in-focus position (Z coordinate) in the area other than the observation area A4 (non-observation area A5). For this reason, in the first embodiment, the focus position extraction point FP is determined according to the shape of the observation region A4.

  Specifically, as shown in FIG. 12, the slice specimen SP is set under the condition that the focus calculation area (for example, the central 30% area of the small section C2 corresponding to the imaging area of the TV camera 30) falls within the observation area A4. The focus position extraction point FP is selected along the contour portion of the. Then, the focus position extraction point FP is added while being extracted toward the inside of the observation area A4. Note that the method of extracting the focus position extraction point FP to be added may be random extraction or regular (for example, every 6 sections). FIG. 12 illustrates the case where the focus position extraction points FP are adjacent along the contour portion of the slice sample SP, but the observation region A4 is large and a large number of focus position extraction points FP are required. Alternatively, the focus position extraction points FP once extracted may be thinned to reduce the number of focus position extraction points to an appropriate number.

  For the non-observation region A5, the focus position extraction point FP is extracted from the non-observation region A5 to increase the focus accuracy, or the processing time is shortened without selecting the focus position extraction point FP. Either of these may be selected. In the former case, the processing time may be shortened by setting the interval between the focus position extraction points FP wider than the interval in the observation region A4.

  In subsequent step S29, the microscope system 1 measures the in-focus position (Z coordinate) at the focus position extraction point FP within the scan range M3. Then, using these actually measured values, a focus map (see FIG. 9) relating to the scan range M3 is created by interpolating the in-focus position in the small section C2. The details of the focus map creation process are the same as in step S2. However, the interpolation calculation for the small section C2 in the attention area M3 is performed using only the actually measured focus position at the close focus position extraction point FP in the observation area A4. The created focus map is stored in the imaging coordinate recording unit 55 (see FIG. 2).

  In subsequent step S30, the microscope system 1 creates a high-resolution image (VS image) in which an area on the immunostained specimen corresponding to the scan range M3 is shown. Specifically, the microscope controller 20 sequentially moves the electric stage 13 to the stage coordinates (X, Y, Z) registered in the focus map created in step S29. Each time the motorized stage 13 moves, the TV camera 30 images an area on the immunostained specimen corresponding to each small section C2, and inputs the image data to the host system 50-1. In the host system 50-1, the control unit 57 performs a connecting process for connecting the microscopic images of the adjacent small sections C2 based on the input image data. A series of operations such as movement of the electric stage 13, input of image data, and connection processing of microscope images is repeated for all the small sections C2 registered in the focus map, thereby corresponding to the scan range M3. A high-definition VS image with a wide field of view is created. The image data of the VS image created in this way is stored in a VS image file (see FIG. 4).

  In the first embodiment, the small section C2 outside the observation area A4 is also imaged by the high-magnification objective lens, but the small section C2 only in the non-observation area A5 is excluded from the imaging target. It is not necessary to create a high resolution image of the observation area A5.

  In subsequent step S31, the control unit 57 determines whether or not the VS image creation processing of the corresponding region on the immunostained specimen has been completed for all the attention areas A3 (see FIG. 6) set on the HE stained specimen. Determine whether. When the observation area A4 where a VS image has not yet been created remains (step S31: No), the operation of the microscope system 1 returns to step S27.

  On the other hand, when the creation of the VS image is completed for all the observation areas A4 (step S31: Yes), the slide transport device 60 unloads the slide glass specimen 2 placed on the electric stage 13, and the slide It returns to the conveying apparatus 60 (step S32). Thereby, the creation process of the VS image for the immunostained specimen is completed.

  A series of operations of these steps S20 to S32 is repeated for all of the immunostained specimens accommodated in the slide conveyance device 60. Thereafter, the process returns to the main routine.

  Next, the expression analysis step S6 for calculating the expression level of the target molecule in the immunostained specimen will be described in detail. For specific processing for calculating the expression level of the target molecule, see Japanese Patent Application Laid-Open No. 2011-179924.

  Further, in the following description, a case where the VS image of the immunostained specimen is composed of a normal color (RGB) image will be described. A multiband image may be captured by switching the optical cube (see Japanese Patent Application Laid-Open No. 2011-179924), and expression analysis may be performed on a VS image formed of the multiband image.

  First, the expression level calculation unit 576 creates an HSV image obtained by converting the RGB value of each pixel constituting the VS image of the immunostained specimen into an HSV value. Then, the hue area near blue-violet is extracted as an area dyed with H dye, and the dye area near brown is extracted as an area dyed with DAB dye.

  In addition, the expression level calculation unit 576 acquires staining information among the sample information of the immunostained specimen to be processed. Here, the staining information includes, for example, H + DAB (ER) and H + DAB (HER2), the dye used for staining the immunostained specimen, and information on the target molecule that the dye stains. . Therefore, the expression level calculation unit 576 identifies the type of pigment and the site of expression of the target molecule (cell nucleus, cytoplasm, cell membrane) from the staining information. And the positive cell rate according to the expression site | part of a target molecule is calculated.

For example, when calculating the expression level of intracellular proteins such as estrogen receptor (ER), progesterone receptor (PgR), and Ki-67, the expression level calculation unit 576 targets areas stained with H dye and DAB dye. Perform cell nucleus extraction. Specifically, cell nuclei stained only with H dye are counted as cells that are negative in expression of the target molecule, while cell nuclei stained only with DAB dye or both DAB dye and H dye are expressed as target molecules. Count as positive cells. Then, the ratio P of positive cells is calculated by the following equation (2).
P = N _P ÷ (N _N + N _P ) (2)
In the formula (1), code N _P represents the number of positive cells, code N _N denotes the number of negative cells.

  In addition, when obtaining the expression level of a protein such as HER2 on the cell membrane, the expression level calculation unit 576 performs cell nucleus extraction on a region stained with H dye, and extracts a region stained with DAB dye as a cell membrane. Then, HER2-positive cells are determined based on the positional relationship between the cell nucleus and the cell membrane, the expression status of DAB (presence / absence of expression), and the positive cell rate is calculated (reference: JP 2011-179924 A).

  The expression analysis result (positive cell rate) acquired in this way is recorded in the target molecule expression analysis result data part D5 (see FIG. 4) in the VS image file, and the VS image of the immunostained specimen is viewed. Sometimes, it is displayed on the monitor 53 together with the VS image.

  It should be noted that when the attention area A3 that is a target for generating a high-resolution image is set on the HE-stained specimen (step S3 in FIG. 5), when the tumor is large and the attention area A3 covers a wide area, or within the attention area A3 There may be a non-tumor area. In such a case, for example, as shown in FIG. 13, by further specifying (limiting) the expression analysis target region A6 that is the target of the expression analysis within the attention region A3, May be limited to only a necessary region such as a tumor part. In this case, when the pathologist selects the attention area A3, the host system 50-1 may display the predetermined message on the monitor 53 and specify the expression analysis target area A6 together. In this case, the time required for expression analysis can be shortened.

  Further, when a non-tumor region such as a stromal cell exists in the attention region A3, the stromal cell is excluded from the calculation target of the expression analysis by using the characteristic that the nuclei of the nuclei are low in the stromal cell. Also good.

  As described above, according to the first embodiment, a target region (region of interest) for expression analysis of a target molecule such as a tumor site is designated to a pathologist at the time of morphological diagnosis using a HE-stained specimen that is the basis of histopathological diagnosis. After that, a high-resolution image (VS image) of a specified region in the target molecule stained specimen can be created without the intervention of a pathologist. Therefore, it is possible to create a VS image necessary for pathological diagnosis efficiently and in a short time in accordance with a normal pathological diagnosis workflow.

  Further, according to the first embodiment, since the pathologist designates the attention area based on the high-resolution image of the HE-stained specimen, the pathologist observes the target molecule-stained image with low resolution. In contrast, it is possible to select a tumor part or the like as a region of interest appropriately and easily.

  Further, according to the first embodiment, since a high-resolution image is created only for a necessary region of the target molecule stained specimen based on the attention region set by the pathologist's judgment, the image file capacity is reduced. be able to. Therefore, the storage capacity of the storage device that stores the image data can be reduced as compared with the conventional case.

  Further, according to the first embodiment, when creating a high-resolution image of a target molecule-stained specimen, only the necessary part of the target molecule-stained specimen is focused on the basis of image information of the HE-stained specimen. Since the accuracy is improved, a high-definition image can be acquired efficiently and in a short time.

  Further, according to the first embodiment, it is possible to automatically analyze the expression state of the target molecule in a region (for example, a tumor part) designated in advance by a pathologist. Therefore, the processing time can be shortened, unlike the case where the expression analysis of the target molecule is performed by designating the region at the time of browsing the VS image as in the prior art. Thereby, it is also possible to execute expression analysis on a wide range of tumor parts and the like. Furthermore, although the expression analysis may be hindered when the focusing accuracy of the tumor part or the like is poor, as described above, in the first embodiment, the VS image of the target molecule-stained specimen is focused with high accuracy. Therefore, highly reliable expression analysis can be performed.

  In addition, according to the first embodiment, the expression analysis of the target molecule staining can be executed in a continuous batch process on a large number of slide glass specimens, so that the efficiency of molecular pathological examination can be improved. It becomes.

(Modification)
Next, a modification of the first embodiment will be described.
In the first embodiment, DAB staining, which is immunostaining, is used as a label for confirming the expression of a target molecule, and hematoxylin staining is used as a counterstaining. However, fluorescence staining may be performed as a label for confirming the expression of the target molecule.

  Here, DAPI which is a fluorescent dye is generally used as counterstaining (for example, staining of cell nuclei) when fluorescently labeling a target molecule, but in this modification, bright field observation is used instead of DAPI. A non-fluorescent or low quantum yield dye capable of observing the morphology is used. For example, cell nuclei are stained with H dye as counterstain, anti-ER antibody recognizing estrogen receptor (ER) is fluorescently labeled with Alexa555, and anti-HER2 antibody recognizing HER2 receptor (HER2) is fluorescently labeled with Cy5 Make multiple stained specimens.

  Note that the reason why DAPI is not used as counterstaining is that, when correction of the positional deviation of the specimen region and determination of the scanning range are performed, if the oblique illumination (dark field illumination) is performed, the specimen region position detection accuracy decreases. Because.

  When creating a VS image of such multiple stained specimen (target molecule stained specimen), a low resolution image of the entire multiple stained specimen is created by bright field observation (step S24 in FIG. 11), and the corresponding HE staining is performed. After correcting the positional deviation of the specimen area with the low-resolution image of the specimen (step S16), a scan range including an observation area corresponding to the attention area set on the HE-stained specimen is determined (step S27). . Then, a focus map of the scan range is created (step S29), the observation method in the microscope apparatus 10 is switched to fluorescence observation, and a high resolution image of the scan range is created (step S30).

  According to the modification described above, even in a specimen in which the target molecule is fluorescently labeled, the specimen region on the slide glass can be extracted using the nuclear staining information with the H dye. At this time, since the staining information is used, it is possible to improve the extraction accuracy of the specimen region by eliminating the influence of dust and the like existing on the slide glass. As a result, it is possible to accurately correct the positional deviation of the specimen region between the HE-stained specimen and the target molecule-stained specimen.

  Heretofore, as a method for visualizing the expression of a plurality of target molecules such as estrogen receptor (ER), progesterone receptor (PgR), HER2 receptor (HER2), Ki-67 in breast cancer, for example, as a VS image, Multiple types of target molecules are labeled with different fluorescent dyes, and multiple stained specimens are prepared at the same time for morphological observation. Attention areas such as tumors are designated by bright field observation, and labeled by fluorescence observation. A technique for acquiring molecular expression information has been proposed (see, for example, JP-A-2009-14939). In this case, the number of necessary sections of the pathological specimen (that is, the number of slides to be created) can be reduced from 4 to 1, for example, and the expression information of a plurality of target molecules can be confirmed at the cell level. There are benefits. However, the above technique requires the intervention of a pathologist in the process of image generation. On the other hand, according to the above-described modified example, after the pathologist designates the attention area at the time of morphological diagnosis using the HE-stained specimen, the inside of the multiple staining specimen corresponding to the attention area without intervention of the pathologist. It is possible to automatically create a high-resolution image (VS image) in which the above area is reflected.

  In general, the tumor site is determined by morphological observation by HE staining. However, if HE staining and fluorescent dye are labeled simultaneously, autofluorescence of eosin becomes a problem, and the expression of the target molecule can be detected correctly. There wasn't. In addition, in specimens labeled with hematoxylin and fluorescent dye at the same time, the tumor site is selected only from the nuclear information stained with hematoxylin, and the selection of the tumor site is inappropriate when the nuclear or structural variant is weak. And it was difficult to determine the infiltrated site. On the other hand, in this modified example, only hematoxylin is used as a counterstaining dye, so that autofluorescence does not become a problem in fluorescence observation of multiple stained specimens. Moreover, according to this modification, since a tumor site is selected on the image of the HE-stained specimen, it is possible to appropriately set the attention area.

  Also, conventionally, in order to perform morphological observation and fluorescence observation on the same section specimen, a technique of imaging a specimen stained with HE, and then destaining and fluorescently labeling is known (for example, Japanese Patent Laid-Open No. 2009-2009). No. 14939). However, in this case, work such as HE staining and destaining is troublesome. In addition, on the fluorescently labeled specimen, the specimen area is judged based only on contrast information such as phase difference, so it is impossible to distinguish between dust on the slide glass and the specimen, and the specimen area is accurately detected. It was difficult to do. For this reason, it is not easy to determine a region of interest in which a high-resolution VS image is to be created. On the other hand, according to the present modification, a sample obtained by performing multiple staining including fluorescent staining on another section collected from the same embedding block is prepared for a certain HE-stained sample. The dyeing work can be omitted. Further, according to the present modification, the attention area is designated on the image of the HE-stained specimen, the positional deviation of the specimen area on the multiple-stained specimen with respect to the HE-stained specimen is corrected, and then the designated attention area is supported. Since a high-definition image of a region is created, a high-resolution image of a necessary region can be acquired efficiently in a short time.

  Conventionally, an HE-stained specimen and a fluorescently-labeled specimen are created from the same specimen block, a region of interest is determined based on the HE-stained sample, and a sample region is extracted and determined from the fluorescently-labeled sample based on this region of interest. A technique to be performed has also been proposed (see, for example, JP 2009-14939 A). However, in this case as well, since the extraction and determination of the sample area is performed only with the contrast information, there is a possibility that a problem may occur in the determination of the attention area. On the other hand, according to this modification, the sample region is detected by binarizing the image data and extracting the contour, so that it is less affected by dust on the slide glass, and the sample region extraction accuracy is improved. It becomes possible to improve. In addition, according to the present modification, the positional deviation of the specimen region of the multiple stained specimen with respect to the HE stained specimen is corrected using the barycentric coordinates of the specimen area, so that multiple staining corresponding to the region of interest designated in the HE stained specimen is performed. It becomes possible to specify the region on the specimen more accurately.

(Embodiment 2)
Next, a second embodiment of the present invention will be described.
FIG. 14 is a block diagram showing a configuration of a host system provided in the microscope system according to Embodiment 2 of the present invention. The overall configuration of the microscope system is the same as that in the first embodiment.

  As illustrated in FIG. 14, the host system 50-2 according to the second embodiment includes a control unit 58 instead of the control unit 57 illustrated in FIG. The control unit 58 further includes a focus evaluation unit 581 with respect to the control unit 57. The configuration of the host system 50-2 other than the control unit 58 is the same as that of the first embodiment.

  In the second embodiment, when a high-resolution image in the scan range is created (see step S30 in FIG. 11) in the VS image creation process (see step S5 in FIG. 5) of the immunostained specimen (see step S30 in FIG. 11). In-focus evaluation is performed, and if the focus is poor, a microscope image is re-acquired.

FIG. 15 is a flowchart showing details of processing for creating a high-resolution image in the scan range.
In step S201 following step S29 (see FIG. 11), the control unit 58 acquires the minimum value of the focus evaluation value at the focus position extraction point registered in the focus map of the scan range. At this time, only the focus evaluation value in the small section C2 in the observation area A4 (see FIG. 12) is set as the determination target of the minimum value, and the focus evaluation value in the non-observation area A5 is excluded from the determination target. In addition, when the minimum value of the focus evaluation value in the focus map is larger than a predetermined threshold value, the predetermined threshold value may be set as the minimum value.

In subsequent steps S202 to S210, the microscope system 1 performs the following processing on all the small sections C2 registered in the focus map.
In step S <b> 202, the host system 50-2 acquires from the TV camera 30 a microscope image obtained by capturing the processing target small section C <b> 2 using the high-magnification objective lens replaced in step S <b> 25 (see FIG. 11).

  In subsequent step S203, the focus evaluation unit 581 determines whether or not the processing target small section C2 is included in the observation region A4. When the small section C2 is not included in the observation area A4 (step S203: No), the second VS image creation unit 575 performs a connection process for joining the microscope image acquired in step S202 with the microscope image of the adjacent small section C2. (Step S210).

  On the other hand, when the processing target small section C2 is included in the observation area A4 (step S203: Yes), the focus evaluation unit 581 determines whether the small section C2 is the focus position extraction point FP. (Step S204). That is, it is determined whether or not the microscope image is taken at the actually measured focus position. When the small section C2 is a focus position extraction point (step S204: Yes), the process proceeds to step S210.

  On the other hand, when the small section C2 to be processed is not the focus position extraction point (step S204: No), the focus evaluation unit 581 determines whether or not the sample image SP ′ is present in the small section C2 (whether it is a cavity). (Step S205). This determination is made as to whether or not the pixels in which the specimen image SP ′ appears in a focus evaluation calculation area (for example, the center 30% area of the microscope image) of the microscope image acquired in step S202 at a ratio equal to or higher than a predetermined value. This is done by judging whether or not. For example, each of the pixel values (R, G, B) is greater than or equal to a predetermined value (for example, 218), and the absolute values | R−G | and | B−G | For example, in the case of 2) or less, it is determined that the sample image SP ′ is a pixel.

  Note that it is also conceivable that the sample image SP ′ is not detected because the microscope image acquired in step S202 is poorly focused. In this case, the presence / absence of the sample image SP ′ may be determined by evaluating the corresponding area in the low-resolution image of the entire slide acquired in step S24 shown in FIG.

When the sample image SP ′ does not exist in the processing target small section C2 (step S205: No), the process proceeds to step S210.
On the other hand, when the sample image SP ′ is present in the processing-target small section C2 (step S205: Yes), the focus evaluation unit 581 calculates the focus evaluation value by executing the focus evaluation calculation on the acquired microscope image. (Step S206).

  In step S207, the focus evaluation unit 581 determines whether or not the focus evaluation value calculated by the focus evaluation calculation is smaller than the minimum value of the focus evaluation value recorded in the focus map. If the calculated focus evaluation value is greater than or equal to the recorded minimum value (step S207: No), the process proceeds to step S210.

  On the other hand, when the calculated focus evaluation value is smaller than the recorded minimum value (step S207: Yes), the focus evaluation unit 581 determines that the processing target small section C2 is poorly focused. In response to this, the microscope system 1 searches for the in-focus position by performing Z-axis movement control, and re-acquires a microscope image at the in-focus position (step S208).

In subsequent step S209, the focus evaluation unit 581 updates the minimum value of the focus evaluation value acquired from the focus map.
Furthermore, in step S210, the second VS image creation unit 575 executes a connection process for joining the re-acquired microscope image with the microscope image of the adjacent small section C2.

  In step S211, the control unit 58 determines whether or not the processing has been completed for all the small sections C2 within the scan range. When the small section C2 that has not yet been processed remains (step S211: No), the process proceeds to step S202. On the other hand, when the processing for all the small sections C2 is completed (step S211: No), the processing returns to the main routine.

  As described above, according to the second embodiment, the input image (microscopic image) of the small section C2 in which the focus position is calculated by the interpolation calculation with reference to the focus evaluation value actually measured in the observation region A4. ) Is determined. That is, according to the second embodiment, since the focus evaluation is performed without performing the Z-axis movement control, the processing time can be shortened. Further, when it is determined that the focus is poor, a microscope image at the actually measured focus position is re-acquired, so that a VS image with high image quality can be created.

  In addition, as described above, when the focusing accuracy of the region where the tumor part or the like is shown is poor, the expression analysis of the target molecule may be hindered. When this occurs, the microscope image is re-acquired and a VS image focused with high accuracy is created, so that the reliability in the expression analysis can be further increased.

(Embodiment 3)
Next, a third embodiment of the present invention will be described.
FIG. 16 is a block diagram showing a configuration of a host system included in the microscope system according to Embodiment 3 of the present invention. The overall configuration of the microscope system is the same as that in the first embodiment.

  As shown in FIG. 16, the host system 50-3 according to the third embodiment includes a control unit 59 instead of the control unit 57 shown in FIG. The control unit 59 further includes a condition determination unit 591 and a warning control unit 592 with respect to the control unit 57. The configuration of the host system 50-3 other than the control unit 59 is the same as that of the first embodiment. Further, a condition determination unit 591 and a warning control unit 592 may be added to the control unit 58 shown in FIG.

The condition determination unit 591 determines whether or not the attention area A3 selected by the user for the calculation of the expression level of the target molecule by the expression level calculation unit 576 satisfies a predetermined condition.
The warning control unit 592 performs control for warning the user, for example, on the monitor 53 when the condition determination unit 591 determines that the attention area A3 selected by the user does not satisfy the predetermined condition. Do it. In this case, the warning control unit 592 and the monitor 53 constitute warning means.

  For example, when detecting the expression of Ki-67 in a tumor site, it is desirable to calculate the positive cell rate by examining the expression status of at least 500 cells. For this reason, a predetermined number of cells is required as a premise for calculating the positive cell rate of the target molecule.

  Therefore, when the user selects a region of interest A3 on the VS image of the HE-stained specimen (see step S3 in FIG. 5), the condition determination unit 591 stains the hue region near the blue-violet color in the VS image with H dye. The extracted regions are extracted, and cell nuclei are extracted from the region, and the number is counted.

  Extraction of cell nuclei is performed, for example, as follows. First, a contour of a candidate region of a cell nucleus is extracted by a known method such as contour tracking. Then, based on the extracted contour, a morphological feature amount representing the morphological feature is calculated. Examples of morphological feature quantities of cell nuclei include circumscribed rectangle, center of gravity, area, perimeter, circularity, major axis, minor axis, aspect ratio, and the like. A contour region in which one or more of these morphological features are included in a predetermined range is extracted as a cell nucleus (see JP 2011-179924 A).

  The condition determining unit 591 further determines that the counted cell nucleus number is equal to the cell number, and determines that the calculation condition is not satisfied when the cell number does not reach a predetermined number (for example, 500).

  In response to this, the warning control unit 592 displays the number of cells extracted by the condition determination unit 591 and a warning display on the monitor 53, and prompts a user such as a pathologist to change the attention area. The content of the warning display may be, for example, a text display of a message such as “Insufficient number of cells. Please select the attention area again.” Or a warning display (flashing display) ). Alternatively, the warning control unit 592 may warn the user by reading a message aloud or warning sound.

  As described above, according to the third embodiment, the stage before the actual preparation of the immunostained specimen and the generation process of the VS image, that is, the stage where the pathologist is observing the form of the specimen. The pathologist can recognize the validity of the selection of the region of interest. Therefore, by causing the pathologist to redo the selection of the attention area as necessary, it is possible to avoid a wasteful work such as re-acquisition of the VS image due to the insufficient number of cells in the attention area.

(Embodiment 4)
Next, a fourth embodiment of the present invention will be described.
In Embodiments 1 to 3 described above, immunostaining was performed as a label for confirming the expression of the target molecule. In addition to this, ISH (In situ hybridization) method, FISH (Fluorescence In situ hybridization) method, or You may process a sample with the detection probe by CISH (Chromogenic In situ hybridization) method. In this Embodiment 4, the case where the label | marker for the expression confirmation of a target molecule is performed by methods other than immuno-staining is demonstrated. The overall configuration of the microscope system according to the fourth embodiment is the same as that of the first embodiment.

  First, the case where a detection probe by FISH (fluorescence in situ hybridization) method is used as a label for confirming the expression of a target molecule will be described. The detection probe is not particularly limited. For example, a known FISH detection probe such as HER2 FISH pharmDx Kit, which is a product of Dako, can be used.

  Here, the FISH method is a method for detecting a gene abnormality in a cell nucleus by a fluorescence observation method. In order to visualize a gene abnormality, a high magnification objective lens of about 40 to 100 times is used in a microscope system. Must be used to observe the expression of the target molecule. However, in such a high-magnification objective lens, since the depth of field is usually shallow, it is possible to detect a signal from a labeled probe that is unevenly distributed in the cell depth direction (i.e., the focal depth direction of the objective lens). There is a risk that the observation accuracy may deteriorate due to incompleteness. In such a case, in order to improve the observation accuracy, it is conceivable to generate a plurality of high-resolution images that are respectively focused at a plurality of different positions in the depth of focus direction. However, if a plurality of high-resolution images are acquired by changing the focus position for all of the attention area A3, it takes a long time to acquire the images.

  Therefore, in the fourth embodiment, the pathologist is further set in the expression analysis target area A6 (see FIG. 13) in the attention area A3 that is the generation target of the high resolution image. For the expression analysis target area A6, a plurality of high-resolution images are acquired by changing the focus position. For the area other than the expression analysis target area A6 in the attention area A3, the user Only one high-resolution image at a desired in-focus position is acquired. This makes it possible to acquire a high resolution image necessary for observation by a pathologist in a short time for the entire expression analysis target area A6.

  As described above, the timing and method of setting the expression analysis target area A6 are as follows. When the pathologist selects the attention area A3, the pathologist displays a predetermined message on the monitor 53, and the expression analysis is performed on the pathologist. The target area A6 may be designated.

  The image data of a plurality of high resolution images acquired for the expression analysis target area A6 may be stored separately as image data having different in-focus positions, or a plurality of images having different in-focus positions may be stored. An omnifocal image may be generated by synthesis by image processing, and stored as image data representing one image with an expanded depth of field.

  In addition, when the ISH method or the CISH method is used as a label for confirming the expression of the target molecule, when the pathologist selects the attention area A3, the expression analysis target area is also included in the attention area A3. Specify A6. Even in these cases, it is possible to acquire a high resolution image necessary for observation by a pathologist in a short time by acquiring a plurality of images while changing the focus position with respect to the expression analysis target region A6. Become.

  The present invention is not limited to the above-described first to fourth embodiments and modification examples, but various combinations of a plurality of constituent elements disclosed in the first to fourth embodiments and modification examples are various. Can be formed. For example, some components may be excluded from all the components shown in the embodiment. Or you may form combining the component shown in different embodiment suitably.

DESCRIPTION OF SYMBOLS 1 Microscope system 2 Slide glass specimen 10 Microscope apparatus 11 Transmission observation optical system 110 Transmission illumination light source 111 Collector lens 112 Transmission filter unit 113 Transmission shutter 116 Reflection mirror 117 Condenser optical element unit 118 Top lens unit 12 Episcopic observation optical system 120 Light source for epi-illumination 121 Collector lens 122 Epi-illumination filter unit 123 Epi-illumination shutter 13 Electric stage 131, 132 Motor 14 Revolver 14a, 14b Objective lens 15 Optical cube unit 15a, 15b Optical cube 16 Beam splitter 17 Eyepiece 20 Microscope controller 21 Stage XY drive control unit 22 Stage Z drive control unit 30 TV camera 40 Camera controller 50-1, 50-2, 50- 3 Host system 51 Video board 52 Input unit 53 Monitor 54 Image data recording unit 55 Imaging coordinate recording unit 56 Program recording unit 57, 58, 59 Control unit 571 Focus position calculation unit 572 First VS image creation unit 573 Specimen selection unit 574 Attention Area setting unit 575 Second VS image creation unit 576 Expression amount calculation unit 581 Focus evaluation unit 591 Condition determination unit 592 Warning control unit 60 Slide transport device 70 Bar code reader

Claims (12)

A plurality of microscope images are obtained by moving the specimen relative to the objective lens included in the microscope in a direction perpendicular to the optical axis of the objective lens, and partially imaging the specimen. In a microscope system that generates a virtual slide image by connecting
First virtual slide image creation for creating a first virtual slide image for a first specimen created by staining the first section sliced from the embedding block with a bright field capable of morphological observation Means,
Attention area setting means for setting an attention area in the first virtual slide image;
Among the second specimen prepared by applying a label for confirming the expression of the target molecule to a second section that is sliced from the embedded block and is different from the first section, A second virtual slide image creating means for creating a second virtual slide image for the corresponding observation region;
Determination means for determining whether or not focusing is poor for a microscopic image obtained by partially imaging the inside of the observation area;
Equipped with a,
The microscope system according to claim 2, wherein the second virtual slide image creating means re-acquires a microscope image for a portion determined to have poor focus in the observation region .   The microscope system according to claim 1, wherein the staining capable of morphological observation is hematoxylin / eosin staining.   The label for confirming the expression of the target molecule is a detection probe by ISH (In situ hybridization), FISH (Fluorescence In situ hybridization), or CISH (Chromogenic In situ hybridization), or immunostaining. The microscope system according to claim 1.   The microscope system according to claim 1, wherein the label for confirming the expression of the target molecule is fluorescent staining. The determination means calculates a focus evaluation value of the microscope image, determines whether the focus evaluation value is smaller than a predetermined threshold,
5. The second virtual slide image creation unit re-acquires the microscope image for a portion where the determination unit determines that the focus evaluation value is smaller than a predetermined threshold. The microscope system according to any one of the above. The determination means calculates a focus evaluation value of the microscope image at a position other than a focus position extraction point where the focus position is measured by sampling within the observation region, and the focus evaluation value is smaller than a predetermined threshold value. 6. The microscope system according to claim 5 , wherein it is determined whether or not .   The microscope system according to claim 1, further comprising an expression level calculation unit that calculates an expression level of the target molecule in the second virtual slide image. Each of the first and second specimens is provided with specimen information including at least information on an embedding block obtained by slicing a slice and the type of staining,
Specimen information acquisition means for acquiring the specimen information;
Sample selecting means for selecting the second sample based on the sample information acquired from the first sample;
The microscope system according to claim 1, further comprising: A slide transporting means capable of accommodating a plurality of specimens and capable of sequentially transporting and transporting each of the plurality of specimens to the visual field range of the objective lens;
The microscope system according to claim 8, wherein the plurality of specimens conveyed by the slide conveying unit are continuously processed. Cell number counting means for counting the number of cells present in the attention area set by the attention area setting means;
Warning means for warning the user when the number of cells is less than a predetermined value;
The microscope system according to claim 1, further comprising: A plurality of microscope images are obtained by moving the specimen relative to the objective lens included in the microscope in a direction perpendicular to the optical axis of the objective lens, and partially imaging the specimen. In a specimen image generation method executed by a microscope system that generates a virtual slide image formed by mutually connecting
First virtual slide image creation for creating a first virtual slide image for a first specimen created by staining the first section sliced from the embedding block with a bright field capable of morphological observation Steps,
An attention area setting step for setting an attention area in the first virtual slide image;
Among the second specimen prepared by applying a label for confirming the expression of the target molecule to a second section that is sliced from the embedded block and is different from the first section, A second virtual slide image creating step for creating a second virtual slide image for the corresponding observation area;
Only including,
The second virtual slide image creation step includes:
A determination step for determining whether or not focusing is poor for a microscope image acquired by partially imaging the inside of the observation region;
Re-acquisition step for re-acquiring a microscope image for the portion determined to be poor in focus in the observation area;
Sample image generation method characterized by having a. A plurality of microscope images are obtained by moving the specimen relative to the objective lens included in the microscope in a direction perpendicular to the optical axis of the objective lens, and partially imaging the specimen. In the sample image generation program executed by the microscope system that generates a virtual slide image by connecting
First virtual slide image creation for creating a first virtual slide image for a first specimen created by staining the first section sliced from the embedding block with a bright field capable of morphological observation Steps,
An attention area setting step for setting an attention area in the first virtual slide image;
Among the second specimen prepared by applying a label for confirming the expression of the target molecule to a second section that is sliced from the embedded block and is different from the first section, A second virtual slide image creating step for creating a second virtual slide image for the corresponding observation area;
To the microscope system ,
The second virtual slide image creation step includes:
A determination step for determining whether or not focusing is poor for a microscope image acquired by partially imaging the inside of the observation region;
Re-acquisition step for re-acquiring a microscope image for the portion determined to be poor in focus in the observation area;
Sample image generation program characterized by having.