JP5953195B2 - Imaging analysis apparatus, control method thereof, and program for imaging analysis apparatus - Google Patents

Description

  The present invention relates to a photographing analysis device, a control method thereof, and a program for the photographing analysis device, and more particularly, an improvement of a photographing analysis device that captures two or more photographed images of monochrome gradation by photographing an observation object. About.

  Fluorescence observation is performed by irradiating an observation object to which a reagent has been applied with excitation light, photographing the observation object using fluorescence emitted by the observation object, and browsing the obtained captured image (for example, Patent Document 1). The reagent is composed of a fluorescent dye that absorbs excitation light and emits fluorescence, and is taken into cells and living tissues, whereby colorless and transparent cells, living tissues, and organs in the cells can be identified by fluorescence. Normally, when observing an observation object that has been multiple-stained using a plurality of reagents, the observation object is irradiated with light containing excitation light of various wavelengths corresponding to the reagent, and the observation object is photographed by a color camera. The

  On the other hand, a monochrome camera having higher sensitivity than a color camera is used for quantitative measurement such as accurately specifying the intensity and position of fluorescence. In addition, when a multiple-stained observation object is photographed with a monochrome camera, the observation object is sequentially irradiated with excitation light having a wavelength corresponding to the reagent. The monochrome camera sequentially captures the observation object every time the excitation light is irradiated, and acquires a monochrome gradation image for each reagent.

  Monochrome grayscale images show the distribution of reagents in cells and living tissues, so it is possible to obtain images in order when confirming how each reagent affects each other on the observation object. A pseudo color image is created from a plurality of monochrome gradation images. This pseudo color image is created by assigning each monochrome gradation image to a different color component. Although the pseudo-color image is different from the original fluorescent color, if the pseudo-color image is viewed, it is easy to identify how the stained parts with different fluorescent colors affect each reagent. Can do.

  Also, when measuring the size of DNA (deoxyribonucleic acid) molecules or chromosomes holding genetic information, the number in the cell, etc., a monochrome gradation image in which the stained site indicates the cell, and the stained site is a DNA molecule or chromosome. A monochrome gradation image indicating such an organ is acquired. Stained parts indicating cells and the like can be extracted as an image area composed of adjacent pixels by binarizing the monochrome gradation image. However, for example, when it is desired to check the genetic information in the cell, the method of simply binarizing the entire monochrome gradation image in which the staining site indicates an organ such as a DNA molecule is not only a staining site in the cell to be confirmed. If a lot of noise is extracted from the extracellular area that is not the target of confirmation, it is difficult not only for the user to see the genetic information on the monitor, but it is also correct when trying to measure the number and area of stained parts. The result may not be obtained.

  In general, a monochrome gradation image in which a stained part indicates a cell and a monochrome gradation image in which a stained part indicates an organ such as a DNA molecule are stored and stored in separate and independent image files. Therefore, even if there is a method for confirming genetic information in a cell using these multiple image files, the user must be familiar with image processing algorithms and image processing programs that handle multiple image files. Therefore, it is difficult for a user with little experience in image processing or programming knowledge to analyze a captured image with a simple operation.

JP 2008-139795 A

  The present invention has been made in view of the above circumstances, and an object thereof is to provide a photographing analysis apparatus capable of analyzing a photographed image with a simple operation even by a user who has little experience in image processing or programming knowledge. To do.

  Another object of the present invention is to provide an imaging analysis apparatus that can correctly extract a stained region indicating an organ such as a DNA molecule or a chromosome without extracting noise from an extracellular region.

  In addition, the present invention is capable of easily identifying a site stained with the first reagent and allowing the user to arbitrarily change a mask threshold for masking a region other than the stained site. For other purposes.

  Further, according to the present invention, the staining site by the second reagent can be easily identified, and the user can arbitrarily specify the extraction threshold value used to binarize the color component image corresponding to the second reagent. Another object of the present invention is to provide a photographing analysis apparatus that can perform the above.

  Another object of the present invention is to provide a method for controlling the above-described imaging analysis apparatus. Furthermore, another object of the present invention is to provide a program that allows a computer to function as the above-described photographing analysis apparatus.

The imaging analysis apparatus according to the first aspect of the present invention includes a stage on which an observation object to which a plurality of fluorescent dyes having different excitation wavelengths are applied is placed, and the plurality of fluorescences with respect to the observation object placed on the stage. Excitation light irradiating means for irradiating a plurality of excitation lights for exciting each of the dyes, and two or more captured images comprising monochrome gradations by photographing the observation object when each of the plurality of excitation lights is irradiated And an overlay image for generating an overlay image by superimposing the different color component images by assigning different color components to each of the captured images and generating a plurality of different color component images The overlay image is configured based on a user operation, a generation unit, a display unit that displays the overlay image generated by the overlay image generation unit, and a user operation. Mask color component selecting means for selecting one of the color components as a mask color component, and mask area specifying means for specifying an area other than the analysis target area as a mask area from the color component image to which the mask color component is assigned And an extracted image obtained by masking a region other than the analysis target region using the mask region from a color component image to which a color component other than the mask color component among the color components constituting the overlay image is assigned. And an extracted image generating means for generating.

The user visually recognizes the overlay image on the display means, and selects a color component to be used as a mask image from a plurality of different color component images constituting the overlay image as a mask color component. Then, the mask area is specified by the mask area specifying means, and the extracted image generation means uses the mask area from the color component image to which the color component other than the mask color component is allocated among the color components constituting the overlay image. Thus, an extracted image in which the area other than the analysis target area is masked is generated. Therefore, even a user who has little image processing experience or programming knowledge can analyze the captured image with a simple operation. For example, when it is desired to check the genetic information in the cell, one of the plurality of different color component images composing the overlay image displayed on the display means is selected as a mask color component. With a simple operation, the noise that appears in the extracellular region can be reduced in the extracted image in which a part of the region is extracted (masked) from the analysis image to be analyzed. In addition, since the observation object is photographed using fluorescence emitted by the fluorescent dye incorporated into the cell or the living tissue, even if it is a colorless and transparent cell or living tissue, it is desired without being affected by noise. Can be extracted. In particular, it is possible to correctly extract a staining site indicating an organ such as a DNA molecule or a chromosome without extracting noise from an extracellular region.

  In addition to the above-described configuration, the imaging analysis apparatus according to the second aspect of the present invention uses the mask area specifying unit to binarize the color component image to which the mask color component is assigned using the mask threshold, and to analyze Binarization means for generating a mask image for masking an area other than the area is provided, and the mask area is specified based on the mask image generated by the binarization means.

In addition to the above- described configuration, the imaging analysis apparatus according to the third aspect of the present invention may include any of a plurality of color components other than the mask color component among the plurality of different color components on the display unit on which the overlay image is displayed. or the provided analysis for the color component selecting means for selecting as the analysis for color components, the extracted image generating means with respect to the color component image analysis color component selected is allocated by the color component selecting unit for the analysis, The extracted image is generated by performing a mask process in which a region other than the analysis target region is masked .

According to a fourth aspect of the present invention, in addition to the above- described configuration, the mask color component selection unit includes an analysis target region of the color components constituting the overlay image on the display unit on which the overlay image is displayed. It is configured to accept selection of a color component using a color component in a region other than the color component for masking .

According to a fifth aspect of the present invention, in addition to the above-described configuration, a mask image display unit that displays the mask image, and a mask that changes the mask threshold value based on a user operation on the mask image display unit. Threshold value adjusting means, and the binarizing means binarizes the color component image to which the mask color component is assigned using the changed mask threshold value to generate the mask image, The mask image display means is configured to display the mask image binarized using the changed mask threshold.

  According to such a configuration, the stained region with the first reagent can be easily identified, and the user can arbitrarily change the mask threshold for masking the region other than the stained region.

According to a sixth aspect of the present invention, there is provided an imaging analysis apparatus according to the present invention, wherein the extracted image display means for displaying the extracted image and the extraction threshold value specifying the extraction threshold value based on a user operation on the extracted image display means A threshold adjustment unit, and the extracted image generation unit uses a color component image to which a color component other than the mask color component is allocated among the color components constituting the overlay image using the extraction threshold value. The extracted image is generated by binarizing an image in which an area other than the analysis target area is masked using the mask area.

  According to such a configuration, the staining site by the second reagent can be easily identified, and the user can arbitrarily select an extraction threshold used to binarize the color component image corresponding to the second reagent. Can be changed.

According to a seventh aspect of the present invention, in addition to the above configuration, the extraction threshold adjustment unit accepts a change in the extraction threshold in a state where the extraction image is displayed, and the extraction image generation unit The extracted image is regenerated based on the extraction threshold value changed by the extraction threshold value adjusting means . According to such a configuration, it is possible to adjust the extraction threshold while browsing the extracted image.

  According to an eighth aspect of the present invention, in addition to the above-described configuration, a mask color component, a first analysis color component, and a second analysis color component specified by a user among three different color components are provided. On the other hand, the first extracted image obtained by using the color component image to which the mask color component is assigned, the color component image to which the first analysis color component is assigned, and the mask color component are assigned. And an extracted image synthesizing unit that synthesizes the obtained color component image and the second extracted image obtained by using the color component image to which the second analysis color component is assigned, and generates a synthesized image. Is done.

  According to such a configuration, if the reagent corresponding to the first analytical color component is the second reagent and the reagent corresponding to the second analytical color component is the third reagent, it corresponds to the mask image. Thus, it is possible to easily identify how the respective stained sites by the second and third reagents influence each other on the stained site by the first reagent.

According to a ninth aspect of the present invention, in addition to the above-described configuration, the imaging analysis apparatus is a region of color components other than the mask color component in the analysis target region based on the extracted image generated by the extracted image generation unit. The total number of extraction areas is configured to be displayed on the display means.
According to a tenth aspect of the present invention, there is provided a method for controlling an imaging analysis apparatus , comprising: placing an observation object provided with a plurality of fluorescent dyes having different excitation wavelengths on a stage; and observing the observation object placed on the stage. Irradiating a plurality of excitation lights for exciting each of the plurality of fluorescent dyes, and photographing the observation object when each of the plurality of excitation lights is irradiated, and two or more comprising monochrome gradations A shooting step for acquiring a shot image and assigning different color components to each of the shot images to generate a plurality of different color component images, thereby generating an overlay image in which the plurality of different color component images are superimposed. An overlay image generation step, a display step for displaying the overlay image generated in the overlay image generation step, and a user operation A mask color component selection step for selecting one of the color components constituting the overlay image as a mask color component, and masking an area other than the analysis target area from the color component image to which the mask color component is assigned. A mask area specifying step for specifying as an area, and a color component image other than the analysis target area using the mask area from a color component image to which a color component other than the mask color component among the color components constituting the overlay image is assigned And an extracted image generation step for generating an extracted image in which the region is masked.

A program for an imaging analysis apparatus according to an eleventh aspect of the present invention is directed to a procedure for placing an observation object provided with a plurality of fluorescent dyes having different excitation wavelengths on a stage, and an observation object placed on the stage. , A procedure of irradiating a plurality of excitation lights for exciting each of the plurality of fluorescent dyes, and two or more of monochrome gradations by photographing an observation object when each of the plurality of excitation lights is irradiated An overlay image in which a plurality of different color component images are superimposed is generated by assigning different color components to each of the captured images and generating a plurality of different color component images. Based on the overlay image generation procedure, the display procedure for displaying the overlay image generated in the overlay image generation procedure, and the user operation, the overlay image generation procedure is performed. A mask color component selection procedure for selecting one of the color components constituting the image as a mask color component, and an area other than the analysis target area is specified as a mask area from the color component image to which the mask color component is assigned. From the color component image to which a color component other than the mask color component is assigned among the color components constituting the overlay image, a region other than the analysis target region is masked using the mask region. And an extracted image generation procedure for generating the extracted image.

  With the photographing analysis apparatus according to the present invention, even a user who has little image processing experience or programming knowledge can analyze a photographed image with a simple operation.

  Further, in the imaging analysis apparatus according to the present invention, the observation object is imaged using the fluorescence emitted by the fluorescent dye incorporated into the cell or the living tissue. It is possible to extract a desired stained region without being affected by the above. In particular, it is possible to correctly extract a staining site indicating an organ such as a DNA molecule or a chromosome without extracting noise from an extracellular region.

  Further, in the imaging analysis apparatus according to the present invention, the stained region with the first reagent can be easily identified, and the user can arbitrarily change the mask threshold for masking the region other than the stained region. it can.

  In addition, the imaging analysis apparatus according to the present invention can easily identify the site stained with the second reagent, and sets the extraction threshold value used for binarizing the color component image corresponding to the second reagent to the user. Can be changed arbitrarily.

  Further, according to the present invention, it is possible to provide a control method for the above-described imaging analysis apparatus. Furthermore, according to the present invention, it is possible to provide a program that causes a computer to function as the above-described imaging analysis apparatus.

It is the block diagram which showed the example of 1 structure of the imaging | photography analysis apparatus 100 by embodiment of this invention. FIG. 2 is a diagram showing an example of the operation of the PC 120 in FIG. 1 and shows a fluorescence observation screen 50 displayed on the display unit 121. It is the block diagram which showed the structural example of PC120 of FIG. 3 is a flowchart illustrating an example of an operation during fluorescence observation in the imaging analysis apparatus 100 of FIG. 1. 3 is a flowchart illustrating an example of an operation during fluorescence observation in the imaging analysis apparatus 100 of FIG. 1. FIG. 2 is a diagram showing an example of the operation of the PC 120 in FIG. 1, and shows an extraction setting screen 60 displayed when an analysis button 56 in the fluorescence observation screen 50 is operated. 2 is a diagram illustrating an example of the operation of the PC 120 in FIG. 1, and shows an analysis application screen 70 displayed by operating the start button 62. FIG. 2 is a diagram illustrating an example of the operation of the PC 120 in FIG. 1 and shows an analysis application screen 70 after a mask channel is selected. FIG. 2 is a diagram illustrating an example of the operation of the PC 120 in FIG. 1, and shows an analysis application screen 70 after a mask channel is determined. 2 is a diagram illustrating an example of the operation of the PC 120 in FIG. 1, and shows an analysis application screen 70 in the case of shaping a region of interest determined by binarization of a mask image. FIG. 2 is a diagram showing an example of the operation of the PC 120 in FIG. 1, and shows an analysis application screen 70 after selecting a first analysis channel. It is a figure which showed an example of operation | movement of PC120 of FIG. 1, and the analysis application screen 70 after determining the 1st channel for analysis is shown. It is a figure showing an example of operation of PC120 of Drawing 1, and shows analysis application screen 70 after selecting the 2nd channel for analysis. It is a figure showing an example of operation of PC120 of Drawing 1, and shows analysis application screen 70 after selecting the 2nd channel for analysis. It is the figure which showed an example of operation | movement of PC120 of FIG. 1, and the analysis application screen 70 at the time of confirmation of an extraction result is shown. It is a figure which showed an example of operation | movement of PC120 of FIG. 1, and the measurement result confirmation screen 80 displayed after operating a detailed button is shown.

<Photographing analysis apparatus 100>
FIG. 1 is a block diagram showing a configuration example of a photographing analysis apparatus 100 according to an embodiment of the present invention. This imaging analysis apparatus 100 is a fluorescence microscope system that irradiates an observation object 2 to which a reagent has been applied with excitation light 1 and images the observation object 2 using fluorescence 3 emitted from the observation object 2. 110 and a PC (personal computer) 120.

  For example, the imaging analysis apparatus 100 can be realized by operating a computer based on an imaging analysis program. Such a photographing analysis program is provided by being recorded on a computer-readable recording medium such as a CD-ROM, or provided via a network.

  The imaging unit 110 is a microscope main body that captures the observation object 2 and acquires a monochrome gradation image. The imaging unit 110 performs imaging control by the PC 120 and transmits the acquired monochrome gradation image to the PC 120.

  The PC 120 is an analysis unit that performs quantitative measurement based on a monochrome gradation image, and includes a display unit 121, a keyboard 122, and a mouse 123. The display unit 121 displays a photographed image, a photographing condition setting screen, a result of quantitative measurement, and the like. The keyboard 122 and the mouse 123 are input devices that accept user operations.

  The photographing unit 110 can switch between fluorescence observation and bright field observation using the transmitted light 4, and includes a filter holder 111, an objective lens 112, a movable stage 113, an imaging lens 114, an observation switching unit 115, a color filter. 116, monochrome camera 117, eyepiece lens 118, color camera 119, excitation light source 20, light quantity adjustment unit 21, collector lens 22, transmission illumination light source 30, transmission illumination lens 31, filter switching unit 40, stage drive unit 41, image transfer unit 42 and a control unit 43.

  The excitation light source 20 is a fluorescence observation light source device that generates excitation light 1 for exciting the fluorescent dye contained in the reagent, and the excitation light 1 including a wavelength corresponding to the reagent is emitted. For example, a mercury lamp is used as the excitation light source 20, and ultraviolet light having a shorter wavelength than visible light is emitted as the excitation light 1.

  The light amount adjusting unit 21 adjusts the transmitted light amount of the excitation light 1. The collector lens 22 converts the excitation light 1 incident from the excitation light source 20 via the light amount adjustment unit 21 into a parallel light beam, and emits it toward the excitation filter 12 in the filter holder 111.

  The filter holder 111 holds two or more filter units 10 and moves them in a predetermined direction so that any one of the filter units 10 is arranged on the optical path of the observation light or retracted from the optical path. it can. The filter unit 10 includes an excitation filter 11, a dichroic mirror 12, and an absorption filter 13.

  The excitation filter 11 is a wavelength selection filter that selectively transmits excitation light 1 having a predetermined wavelength. The dichroic mirror 12 reflects the excitation light 1 transmitted through the excitation filter 11 toward the objective lens 112, and transmits the observation light incident from the observation object 2 through the objective lens 112 toward the absorption filter 12.

  The absorption filter 13 is a wavelength selection filter that absorbs the excitation light 1 that is scattered by the observation object 2 and is incident through the objective lens 112 and the dichroic mirror 14 and that selectively transmits the fluorescence 3 having a predetermined wavelength. is there. The excitation light 1 is incident on the observation object 2 from the objective lens 112 side coaxially with the observation light.

  The movable stage 113 is a mounting table for mounting the observation object 2 to which two or more reagents are applied, and can be moved in the x, y, and z directions. The z direction is a direction parallel to the optical axis of the objective lens 112, and the x and y directions are directions perpendicular to the optical axis. The stage drive unit 41 drives the movable stage 113. The captured image can be focused by moving the movable stage 113 in the z direction. Further, the observation object 2 can be moved into the field of view of the monochrome camera 117 by moving the movable stage 113 in the x or y direction.

  Each reagent consists of fluorescent dyes having different excitation wavelengths. The filter holder 111 holds the filter unit 10 for each reagent. The filter switching unit 40 switches the filter unit 10. By changing the filter unit 10 according to the reagent to emit light, the observation object 2 can be sequentially irradiated with the excitation light 1 having the wavelength corresponding to the reagent, and the observation object 2 can be photographed in order. A monochrome gradation image for each reagent can be acquired.

  The fluorescence 3 that has passed through the filter unit 10 enters the monochrome camera 117 via the imaging lens 114, the observation switching unit 115, and the color filter 116. The observation switching unit 115 transmits the fluorescence 3 incident from the observation object 2 via the imaging lens 114 to the monochrome camera 117 side during fluorescence observation, and from the observation object 2 during bright field observation using the transmitted light 4. The transmitted light 4 incident through the imaging lens 114 is reflected toward the color camera 119.

  The color filter 116 is a filter used when performing bright field observation using the monochrome camera 117, and selectively transmits RGB color components. The monochrome camera 117 is a fluorescence observation imaging device that receives fluorescence 3 and generates a monochrome gradation image, and is configured by an imaging device such as a CCD (Charge Coupled Device).

  The transmitted illumination light source 30 is a light source device that generates visible light for bright field observation. The transmitted illumination lens 31 condenses the visible light incident from the transmitted illumination light source 30 toward the movable stage 113. The eyepiece 118 transmits the observation object 2 and condenses the transmitted light 4 incident through the imaging lens 114 and the observation switching unit 115 toward the color camera 119. The color camera 119 is an imaging device that receives the transmitted light 4 and generates a color image.

  The image transfer unit 42 acquires a monochrome gradation image from the monochrome camera 117 or acquires a color image from the color camera 119 and transmits it to the PC 120 as a captured image. The control unit 43 controls the light amount adjustment unit 21, the filter switching unit 40, the stage drive unit 41, and the image transfer unit 42.

  In the PC 120, a pseudo color image is created using two or more monochrome gradation images acquired by sequentially photographing the observation object 2 every time the excitation light 1 is irradiated during fluorescence observation. The stained part is extracted from the monochrome gradation image.

<Fluorescence observation screen 50>
FIG. 2 is a diagram showing an example of the operation of the PC 120 in FIG. 1, and shows a fluorescence observation screen 50 displayed on the display unit 121. The fluorescence observation screen 50 is an operation screen at the time of fluorescence observation, and can browse monochrome gradation images FIr, FIg, FIb and pseudo color image Pc, and set photographing conditions.

  The fluorescence observation screen 50 includes a captured image display field 51, an observation method selection field 52, an imaging magnification selection field 53, a stage control input field 54, a microscope operation tab 55a, and a camera setting tab 55b. An analysis button 56 is provided. In the display column 51, monochrome gradation images FIr, FIg, FIb and a pseudo color image Pc are displayed.

  The monochrome gradation images FIr, FIg, and FIb are obtained for each reagent obtained by sequentially photographing the observation object 2 every time the observation object 2 is sequentially irradiated with the excitation light 1 having a wavelength corresponding to the reagent. This is a photographed image, and consists of a luminance value of a predetermined gradation held for each pixel, for example, an 8-bit luminance value.

  In this example, three reagents having different excitation wavelengths are applied to the observation object 2, and three monochrome gradation images FIr, FIg, and FIb corresponding to these reagents are acquired. Each monochrome gradation image FIr, FIg, FIb can be displayed by designating an arbitrary color as a display color, and a stained region by a reagent can be identified.

  The pseudo color image Pc is an overlay image created by assigning the monochrome gradation images FIr, FIg, and FIb to different color components. In this example, the pseudo color image Pc is created by assigning the monochrome gradation images FIr, FIg, and FIb to the three color components of RGB and superimposing these monochrome gradation images. Each color component of RGB is called a color channel, green (G) is designated for the color channel “channel 1”, red (R) is designated for the color channel “channel 2”, and the color channel “channel 1” is designated. Blue (B) is designated for “channel 3”.

  The monochrome gradation image FIr is assigned to the color channel “channel 2”, the monochrome gradation image FIg is assigned to the color channel “channel 1”, and the monochrome gradation image FIb is assigned to the color channel “channel 3”. ing. The green stained part in the photographed image is represented by hatching, the red stained part is represented by dots, and the blue stained part is represented by gray gradation. In the display column 51 in the figure, monochrome gradation images FIr, FIg, FIb and a pseudo color image Pc are arranged.

  The selection column 52 is an operation area for designating a display color or monochrome gradation image to be assigned to each color channel or selecting an observation method. The microscope operation tab 55a is an operation item for operating each device in the photographing unit 110. By selecting the microscope operation tab 55a, a selection column 53 and an input column 54 are displayed.

  The selection field 53 is an operation area for selecting a photographing magnification. In this example, any one of 40 times, 20 times, and 10 times can be selected by changing the objective lens 112. The input field 54 is provided with a direction button for moving the movable stage 113 in the xy plane and a direction button for focusing by moving the objective lens 112 in the z direction.

  The camera setting tab 55b is an operation item for setting shooting conditions. The analysis button 56 is an operation icon for performing quantitative measurement using the monochrome gradation images FIr, FIg, and FIb.

<PC120>
FIG. 3 is a block diagram showing a configuration example of the PC 120 of FIG. The PC 120 includes an operation unit 101, a mask color component selection unit 102, an analysis color component selection unit 103, an image storage unit 104, an overlay image generation unit 105, a mask region specification unit 106, an extracted image generation unit 107, and an extracted image composition. The unit 108 and the display unit 121 are configured.

  The operation unit 101 includes a keyboard 122 and a mouse 123, and generates an operation signal corresponding to a user operation. The image storage unit 104 holds two or more monochrome gradation images FI acquired by repeatedly photographing the same observation object 2.

  The overlay image generation unit 105 generates the pseudo color image Pc by assigning two or more monochrome gradation images FI to different color components. Specifically, by assigning two or three monochrome gradation images FI to RGB color channels, a pseudo color image Pc is created as a composite image of these monochrome gradation images FI. The pseudo color image here corresponds to an example of an “overlay image”.

  The “overlay image” will be described in detail. When different color components (for example, RGB) are assigned to two or more monochrome gradation images (for example, in the case of an 8-bit image, each pixel has a luminance value of 0 to 255), A plurality of different color component images are generated. For example, when the red color component (R) is assigned, the luminance value of each pixel is used as the R luminance value, and when the green color component (G) is assigned, the luminance value of each pixel is used as it is. When the blue color component (B) is assigned to the luminance value of G, the luminance value of each pixel can be used as it is as the B luminance value (of course, the luminance value of each pixel is used as the luminance of RGB. Instead of a value, a corrected luminance value subjected to some correction processing may be used as an RGB luminance value).

  When these color component images are superimposed, one color image having RGB luminance values at each pixel, that is, one “overlay image” is generated. This “overlay image” can be color-separated (separated) into each color component image in the same manner as general color separation. In this embodiment, the RGB color components are assigned to generate the overlay image. However, for example, CMY (cyan, magenta, yellow) color components may be assigned. In short, each color component in the color space represented by the orthogonal coordinate space may be assigned.

  The image storage unit 104 holds the pseudo color image Pc generated by the overlay image generation unit 105, and the display unit 121 displays the pseudo color image Pc. The mask color component selection unit 102 selects one of the color components constituting the pseudo color image Pc as a mask color component based on a user operation. The mask color component is a color channel used for masking, as a mask region, a region other than the analysis target region indicating the reagent-stained region or the non-stained region.

  The analysis color component selection unit 103 selects, as an analysis color component, a color component other than the mask color component among a plurality of different color components constituting the pseudo color image Pc based on a user operation. The analysis color component is a color channel for extracting a desired stained site. The display unit 121 displays the monochrome gradation image FI to which the mask color component is assigned and the monochrome gradation image FI to which the analysis color component is assigned as necessary.

  Here, the user arbitrarily designates the mask color component and the analysis color component from among the color components constituting the overlay image Pc, but the mask color component or the analysis color component depends on the combination of the reagents. May be automatically selected.

  The mask area specifying unit 106 specifies an area other than the analysis target area as a mask area from the color component image to which the mask color component is assigned. The mask area specifying unit 106 includes an threshold adjustment unit 61 and a binarization unit 62, and is an image processing unit that binarizes a color component image to which a mask color component is assigned using a mask threshold Thm. .

  The binarization unit 62 compares the luminance value for each pixel with the mask threshold Thm for the color component image to which the mask color component is assigned, and binarizes the luminance value based on the comparison result. Then, a mask image for masking an area other than the analysis target area is generated. The mask area specifying unit 106 specifies a mask area based on the mask image generated by the binarizing unit 62. The mask area specifying unit 106 is not limited to the one that generates a mask image by the binarizing unit 62. For example, in the case of a color component image in which only a region to be analyzed such as a region indicating a cell is colored and the other region (for example, a region other than a region indicating a cell) is hardly colored, a region that is hardly colored (luminance) The region whose value is a certain value or less) may be specified (recognized) as it is as the mask region (mask image information indicating the mask region). In this case, the mask area can be specified without generating the mask image itself.

  The display unit 121 displays a mask image based on a user operation. The analysis target region is displayed so as to be identifiable as a region of interest consisting of a continuous region having an area of a predetermined value or more. This attention area is specified as, for example, an area on the high luminance side in the mask image (binarized image), and the stained region can be identified by visually recognizing the attention area.

  The threshold adjustment unit 61 changes the mask threshold Thm based on a user operation. The binarization unit 62 binarizes the color component image to which the mask color component is assigned using the mask threshold Thm after the change by the threshold adjustment unit 61 to generate a mask image.

  The image storage unit 104 holds a mask image binarized using the changed mask threshold Thm, and the display unit 121 displays the mask image. By browsing such a mask image, a stained site stained with a specific reagent can be easily identified.

  The extracted image generation unit 107 masks a region other than the analysis target region using a mask region from a color component image to which a color component other than the mask color component among the color components constituting the pseudo color image Pc is assigned. Generated extracted images. Specifically, a color component image to which the analysis color component selected by the analysis color component selection unit 103 is assigned is used as an analysis image, and the analysis image is subjected to mask processing to generate an extracted image.

  The extracted image generation unit 107 includes a threshold adjustment unit 71 and a binarization processing unit 72, and masks a part of the analysis image using the mask image and binarizes the analysis image using the extraction threshold Tht. As a result, an extracted image is generated as a binarized image of an unmasked area. Specifically, areas other than the attention area are masked from the analysis image, and the image area corresponding to the attention area in the analysis image is binarized. The attention area is an area that is not masked, that is, a non-mask area.

  The binarization processing of the analysis image is performed by comparing the luminance value for each pixel with the extraction threshold value Tht and binarizing the luminance value based on the comparison result. The image storage unit 104 holds an extracted image generated using the extraction threshold Tht, and the display unit 121 displays the extracted image.

  The threshold adjustment unit 71 specifies an extraction threshold Tht based on a user operation. The binarization processing unit 72 binarizes the analysis image using the extraction threshold Tht designated by the threshold adjustment unit 71, and extracts one or two or more image regions indicating the stained part. The image area extracted by the binarization processing unit 72 is a continuous area composed of adjacent pixels, and is specified as an area on the high luminance side in the binarized image.

  Here, it is assumed that the extracted image is generated by binarizing the entire analysis image and then masking a part of the analysis image using the mask image. However, after masking a part of the analysis image using the mask image, the configuration may be such that only the non-mask area, that is, the attention area is binarized.

  The image storage unit 104 holds an extracted image generated using the changed extraction threshold Tht, and the display unit 121 displays the extracted image. By browsing such an extracted image, it is possible to easily identify a stained region stained with a specific reagent without being affected by noise.

  The extracted image composition unit 108 assigns mask color components to the mask color component, the first analysis color component, and the second analysis color component respectively designated by the user among the three different color components. The first extracted image obtained by using the color component image to which the color component image and the first analysis color component are assigned, the color component image to which the mask color component is assigned, and the second analysis color The second extracted image obtained by using the color component image to which the component is assigned is synthesized to generate a synthesized image. This composite image is created by superimposing two extracted images.

  The image storage unit 104 holds the composite image generated by the extracted image composition unit 108, and the display unit 121 displays the composite image. By browsing such a composite image, it is possible to easily identify how the two reagents influence each other on the observation object 2.

  Here, the extracted image is created using the monochrome gradation image FI held in the image storage unit 104. However, the extracted image and the synthesized image are created using the pseudo color image Pc. It may be.

  In particular, in this embodiment, a plurality of monochrome gradation images (a plurality of color component images) held in the image storage unit 104 are color-separated into one color image having RGB components at each pixel. Can be obtained. That is, if one pseudo color image (overlay image) is held in the image storage unit 104, a color component image can be created as necessary, and an extracted image can be created using these color component images. it can.

  In this embodiment, one pseudo color image (overlay image) is stored in a single file. In the present invention, since orthogonal color components such as RGB are used for pseudo color display, it is not necessary to introduce a dedicated file format or browsing software, and a general-purpose file format such as Jpeg or tiff can be used. Accordingly, the user can browse and edit the pseudo color image using general image browsing and editing software, and versatility is improved. The user can confirm the pseudo color image in advance by opening the file using general image processing software. Then, a pseudo color image to be analyzed is selected, and the imaging analysis apparatus 100 according to the present embodiment may be used as necessary. Thereby, the usability and versatility of the imaging analysis apparatus 100 can be improved.

  For example, a plurality of pseudo color images Pc are held as different image files of the observation object 2, and an extracted image is created using the pseudo color images Pc arbitrarily designated by the user. Specifically, the pseudo color image Pc is separated into monochrome gradation images for each color channel, and an extracted image is created by selecting a mask image and an analysis image from these monochrome gradation images.

  Steps S101 to S114 in FIG. 4 and FIG. 5 are flowcharts showing an example of the operation at the time of fluorescence observation in the imaging analysis apparatus 100 in FIG. First, the imaging unit 110 images the observation object 2 based on an instruction from the PC 120, and acquires a monochrome gradation image FI for each reagent (step S101).

  Next, the overlay image generation unit 105 of the PC 120 assigns three monochrome gradation images FI obtained by sequentially photographing the same observation object 2 to each of the RGB color channels, and displays a pseudo color image as a three-color composite image. Pc is generated (step S102). The display unit 121 displays the pseudo color image Pc (step S103).

  Next, when the mask channel is selected, the mask region specifying unit 106 executes a process of binarizing the mask image designated as the mask channel based on the mask threshold Thm, and determines the region of interest. (Steps S104 and S105). The display unit 121 displays the binarized mask image (step S106). If the mask threshold Thm is changed, the processing procedure of step S105 and step S106 is repeated (step S107).

  Next, if the analysis channel is selected as a color channel different from the mask channel, the extracted image generation unit 107 binarizes the entire analysis image specified as the analysis channel based on the extraction threshold Tht. Processing is executed, and an extracted image is generated by masking a region other than the region corresponding to the region of interest in the analysis image (steps S108 and S109). The display unit 121 displays this extracted image (step S110). If the extraction threshold Tht is changed, the processing procedure of step S109 and step S110 is repeated (step S111).

  The processing procedure from step S104 to step S111 is repeated until one color channel is selected as a mask channel and two different color channels are selected as analysis channels (step S112). The extracted image composition unit 108 creates two extracted images and, if there is a merge instruction from the user, synthesizes these extracted images to generate a synthesized image (step S113). The display unit 121 displays this composite image (step S114).

<Extraction setting screen 60>
FIG. 6 is a diagram illustrating an example of the operation of the PC 120 in FIG. 1, and shows an extraction setting screen 60 displayed when the analysis button 56 in the fluorescence observation screen 50 is operated. The extraction setting screen 60 is an operation screen for setting extraction conditions, and is displayed based on the operation of the analysis button 56.

  The extraction setting screen 60 is provided with three selection buttons 61 and an activation button 62. By operating the selection button 61, it is possible to select one of creating one extracted image, creating two extracted images, or not creating an extracted image. When the start button 62 is operated, an analysis application for performing quantitative measurement using the monochrome gradation images FIr, FIg, and FIb can be started.

<Analysis application screen 70>
FIG. 7 is a diagram illustrating an example of the operation of the PC 120 in FIG. 1, and shows an analysis application screen 70 displayed by operating the activation button 62. The analysis application screen 70 is an operation screen for performing quantitative measurement, and is displayed based on the operation of the start button 62.

  The analysis application screen 70 is an operation screen at the time of selection of a mask image, and includes an analysis flow display field 71, a display field 72 for displaying a photographed image and a processed image, a mask threshold value input field 74, A display setting field 75 is provided. The display column 71 displays a processing procedure from selection of a mask image to confirmation of a measurement result. The display column 72 displays a pseudo color image Pc, a mask image before and after binarization, an analysis image, an extracted image, a composite image, and the like.

  The input field 74 is an operation area for adjusting the value of the mask threshold Thm, and any integer between 0 and 255 can be specified as the mask threshold Thm. The display setting column 75 selects whether or not to display the extraction result in the display column 72, or designates a display color to be assigned to an image area indicating a stained region as an extraction color or an image filled with the extraction color This is an operation area for designating the transmittance of the area. In this example, it is selected that the extraction result is displayed in the display field 72, and that red is set as the extraction color.

  In the display column 72 in the figure, the pseudo color image Pc of FIG. 2 is enlarged and a dialog screen 73 for selecting a mask channel is displayed. The dialog screen 73 is provided with a selection button for selecting each color channel of RGB and a confirmation button.

  FIG. 8 is a diagram showing an example of the operation of the PC 120 of FIG. 1, and shows the analysis application screen 70 after the mask channel is selected. The analysis application screen 70 is an operation screen when a blue color channel is selected as a mask channel, and a monochrome gradation image assigned to the color channel is displayed in the display field 72.

  When the confirm button in the dialog screen 73 is operated, the mask channel is confirmed, the mask image is binarized using the mask threshold Thm, and the setting content of the display setting column 75 is applied to the mask image. .

  FIG. 9 is a diagram illustrating an example of the operation of the PC 120 in FIG. 1, and shows the analysis application screen 70 after the mask channel is determined. In the display column 72 of the analysis application screen 70, a mask image binarized using the mask threshold Thm is displayed. The attention area indicating the stained region is filled with the display color designated as the extraction color.

  FIG. 10 is a diagram showing an example of the operation of the PC 120 in FIG. 1, and shows an analysis application screen 70 in the case of shaping a region of interest determined by binarization of a mask image. The analysis application screen 70 is an operation screen at the time of shaping the attention area, and a selection column 76 for selecting image processing for shaping the attention area is arranged.

  In the selection column 76, an area removal button, a hole filling button, an area correction button, and an area separation button are arranged. The area removal button is an operation icon for selecting image processing for removing an unnecessary area. This image processing removes a region in which a feature amount such as an area, a perimeter length, and a long diameter does not satisfy a predetermined condition from a continuous region on the high luminance side in the binarized image.

  The hole filling button is an operation icon for selecting image processing for filling a hole in the region. In this image processing, a low-brightness region that is entirely overlapped is removed from the continuous region on the high-luminance side in the binarized image by expansion or contraction of the region.

  The area correction button is an operation icon for selecting image processing for correcting a missing area. This image processing removes the dent of the boundary of the region from the continuous region on the high luminance side in the binarized image by the expansion or contraction of the region.

  The region separation button is an operation icon for selecting image processing for separating regions. In this image processing, a continuous area on the high luminance side in the binarized image is separated based on a feature amount such as a squeezing degree of the area. For example, the regions are separated by a circular separation process using a water-shed method.

  In the display column 72 in the figure, a plurality of attention regions 5 extracted by binarization of the mask image and a separation line 6 indicating that the regions have been separated by the region separation processing are displayed. By performing such a region shaping process, it is possible to correctly identify the stained site indicating the cell or the organ in the cell.

  FIG. 11 is a diagram illustrating an example of the operation of the PC 120 in FIG. 1, and shows the analysis application screen 70 after the first analysis channel is selected. The analysis application screen 70 is an operation screen when the green color channel is selected as the first analysis channel, and the display column 72 displays a monochrome gradation image assigned to the color channel. Has been.

  When the confirm button in the dialog screen 73 is operated, the analysis channel is confirmed, the analysis image is binarized using the extraction threshold Tht1, the extracted image is created, and the setting contents of the display setting column 75 are changed. Applied to the extracted image.

  FIG. 12 is a diagram showing an example of the operation of the PC 120 in FIG. 1, and shows the analysis application screen 70 after the first analysis channel is determined. In the display column 72 of the analysis application screen 70, an extracted image that is binarized using the extraction threshold value Tht1 and masks an area other than the attention area 5 is displayed. In addition, the extraction region indicating the stained region on the extracted image is filled with the display color designated as the extraction color. In addition, the extraction region can be subjected to the same shaping process as that performed on the attention region of the mask image.

  FIG. 13 is a diagram illustrating an example of the operation of the PC 120 in FIG. 1, and shows the analysis application screen 70 after the second analysis channel is selected. The analysis application screen 70 is an operation screen when the red color channel is selected as the second analysis channel, and the display column 72 displays a monochrome gradation image assigned to the color channel. Has been.

  When the confirm button in the dialog screen 73 is operated, the analysis channel is confirmed, the analysis image is binarized using the extraction threshold Tht2, an extracted image is created, and the setting contents of the display setting column 75 are changed. Applied to the extracted image.

  FIG. 14 is a diagram showing an example of the operation of the PC 120 of FIG. 1, and shows the analysis application screen 70 after the second analysis channel is selected. In the display column 72 of the analysis application screen 70, an extracted image that is binarized using the extraction threshold Tht2 and masks an area other than the attention area 5 is displayed. In addition, the extraction region indicating the stained region on the extracted image is filled with the display color designated as the extraction color. In addition, the extraction region can be subjected to the same shaping process as that performed on the attention region of the mask image.

  FIG. 15 is a diagram showing an example of the operation of the PC 120 of FIG. 1, and shows an analysis application screen 70 when the extraction result is confirmed. The analysis application screen 70 is an operation screen after the second extracted image is confirmed, a measurement result display field 77 is arranged, and the display field 72 is a composite image obtained from the two extracted images. Is displayed.

  In the display column 77, the total number of extracted areas extracted from the attention area 5 is displayed as a count value, and the total area of the extracted areas is displayed. If the detail button in the display column 77 is operated, the detailed information of the measurement result can be confirmed.

  FIG. 16 is a diagram showing an example of the operation of the PC 120 in FIG. 1, and shows a measurement result confirmation screen 80 displayed after operating the detail button in the analysis application screen 70. (A) in the drawing shows the case where the standard tab is selected, and (b) shows the case where the mask tab is selected.

  The measurement result confirmation screen 80 is an operation screen displayed based on the operation of the detail button, and includes a standard tab 81 and a mask tab 82. When the standard tab 81 is selected, the measurement results for each extraction region indicating the stained site are displayed in a tabular format. A serial number is automatically assigned to each of the extraction area and the attention area 5.

  In the standard tab 81, the area of the extraction region, the perimeter, the major axis, the minor axis, and the luminance are displayed as measurement results. The luminance is an integrated value of luminance values for each pixel in the original monochrome gradation image. The serial number of the attention area 5 to which the extraction area belongs is displayed as a mask number. Moreover, the result of having performed various statistical processing is displayed with respect to each measured value of area, perimeter, major axis, minor axis, and luminance. Specifically, an average value, standard deviation, maximum value, minimum value, and total are displayed as statistical processing results.

  When the mask tab 82 is selected, the measurement results for each region of interest 5 are displayed in a table format. In the mask tab 82, the total number and total area of the extraction regions and the area of the attention region 5 are displayed as measurement results.

  The total number and total area of the extraction regions obtained from the first extraction image are displayed as “count 1” and “area 1”, and the total number and total area of the extraction regions obtained from the second extraction image are , “Count 2” and “Area 2”. Moreover, the result of having performed various statistical processing with respect to each measured value of the total number and total area of the extraction regions and the area of the attention region 5 is displayed.

  According to the present embodiment, when the reagent corresponding to the mask image is the first reagent and the reagent corresponding to the analysis image is the second reagent, the first portion is extracted when the stained region by the second reagent is extracted. Regions other than the stained region with one reagent can be masked in the analysis image. For this reason, it is possible to correctly extract the site stained with the second reagent without extracting noise from the region other than the site stained with the first reagent.

  In particular, since the observation object 2 is photographed using the fluorescence 3 emitted by the reagent taken into the cells or the living tissue, even if it is a colorless and transparent cell or living tissue, it is not affected by noise, A desired staining site can be extracted. For example, it is possible to correctly extract a staining site indicating an organ such as a DNA molecule or a chromosome without extracting noise from an extracellular region.

  In addition, the stained region by the first reagent can be easily identified by the binarized mask image, and the user can arbitrarily change the mask threshold Thm for masking the region other than the stained region. Can do. Further, the stained region with the second reagent can be easily identified by the extracted image, and the user arbitrarily changes the extraction threshold Tht used to binarize the analysis image corresponding to the second reagent. be able to.

  Further, if the reagent corresponding to the first analysis image is the second reagent and the reagent corresponding to the second analysis image is the third reagent, the first reagent corresponding to the mask image is stained on the stained region. It can be easily identified from the synthesized image how the stained sites of the second and third reagents influence each other.

  In the present embodiment, an example in which the present invention is applied to a fluorescence microscope system that photographs the observation object 2 using the fluorescence 3 when the excitation light 1 is irradiated has been described. However, the present invention can be applied not only to such a fluorescence microscope system but also to an optical microscope system that photographs an observation object using visible light. The present invention can also be applied to a confocal microscope, particularly a confocal microscope system that scans laser light.

100 imaging analysis apparatus 110 imaging unit 111 filter holder 112 objective lens 113 movable stage 114 imaging lens 117 monochrome camera 10 filter unit 11 excitation filter 12 dichroic mirror 13 absorption filter 20 excitation light source 21 light quantity adjustment unit 40 filter switching unit 41 stage drive unit 42 Image transfer unit 43 Control unit 50 Fluorescence observation screen 70 Analysis application screen 120 PC
121 Display unit 122 Keyboard 123 Mouse 101 Operation unit 102 Mask color component selection unit 103 Analysis color component selection unit 104 Image storage unit 105 Overlay image generation unit 106 Mask area identification unit 107 Extracted image generation unit 108 Extracted image composition unit 1 Excitation Light 2 Observation object 3 Fluorescence

Claims (11)

A stage for placing an observation object to which a plurality of fluorescent dyes having different excitation wavelengths are attached;
Excitation light irradiating means for irradiating a plurality of excitation lights for exciting each of the plurality of fluorescent dyes to the observation object placed on the stage;
Photographing means for photographing an observation object when each of the plurality of excitation lights is irradiated to obtain two or more photographed images having monochrome gradation;
An overlay image generation unit that generates an overlay image by superimposing the plurality of different color component images by assigning different color components to each of the captured images and generating a plurality of different color component images;
Display means for displaying the overlay image generated by the overlay image generating means;
Mask color component selection means for selecting any one of the color components constituting the overlay image as a mask color component based on a user operation;
A mask area specifying means for specifying an area other than the analysis target area as a mask area from the color component image to which the mask color component is assigned;
From the color component image to which the color component other than the mask color component among the color components constituting the overlay image is assigned, an extracted image in which an area other than the analysis target area is masked using the mask area is generated. And an extracted image generating means.   The mask area specifying means binarizes a color component image to which the mask color component is assigned using a mask threshold value, and generates a mask image for masking an area other than the analysis target area. The imaging analysis apparatus according to claim 1, further comprising: a converting unit configured to identify the mask region based on the mask image generated by the binarizing unit. On the display means the overlay image is displayed, among the plurality of different color components, the analysis color component selecting means for selecting one of a plurality of color components other than the color component for the mask as the analysis for color components Prepared,
The extracted image generation means performs a mask process in which a region other than the analysis target area is masked on the color component image to which the analysis color component selected by the analysis color component selection means is assigned. The imaging analysis apparatus according to claim 1, wherein an image is generated. The mask color component selection unit selects a color component on the display unit on which the overlay image is displayed, using a color component other than the analysis target region as a mask color component among the color components constituting the overlay image. imaging analysis apparatus according to claim 1, characterized in that accepts. Mask image display means for displaying the mask image;
A mask threshold adjusting means for changing the mask threshold based on a user operation on the mask image display means;
The binarization means binarizes the color component image to which the mask color component is assigned, using the mask threshold after the change, and generates the mask image,
The imaging analysis apparatus according to claim 2, wherein the mask image display unit displays the mask image binarized using the mask threshold value after the change. Extracted image display means for displaying the extracted image;
An extraction threshold value adjusting means for specifying an extraction threshold value based on a user operation on the extracted image display means;
The extracted image generation means uses the mask region from a color component image to which a color component other than the mask color component among the color components constituting the overlay image is assigned using the extraction threshold value. The imaging analysis apparatus according to claim 1, wherein an extracted image is generated by binarizing an image in which a region other than the analysis target region is masked. The extraction threshold adjustment means accepts a change in the extraction threshold in a state where the extraction image is displayed ,
The imaging analysis apparatus according to claim 6, wherein the extraction image generation unit regenerates the extraction image based on the extraction threshold value changed by the extraction threshold value adjustment unit .   Among the three different color components, a color component image in which the mask color component is assigned to the mask color component, the first analysis color component, and the second analysis color component respectively designated by the user, and the above A first extracted image obtained using a color component image to which a first analysis color component is assigned, a color component image to which the mask color component is assigned, and a second analysis color component are assigned. 4. The photographing analysis apparatus according to claim 3, further comprising an extracted image synthesizing unit that synthesizes the second extracted image obtained using the obtained color component image and generates a synthesized image.   Based on the extracted image generated by the extracted image generating means, the total number of extracted areas that are areas of color components other than the mask color component in the analysis target area are displayed on the display means. The imaging | photography analysis apparatus in any one of Claims 1-8. Placing an observation object provided with a plurality of fluorescent dyes having different excitation wavelengths on a stage;
Irradiating the observation object placed on the stage with a plurality of excitation lights for exciting each of the plurality of fluorescent dyes;
An imaging step of capturing an observation object when each of the plurality of excitation lights is irradiated and acquiring two or more captured images composed of monochrome gradations;
An overlay image generation step of generating an overlay image by superimposing the plurality of different color component images by assigning different color components to each of the photographed images and generating a plurality of different color component images;
A display step for displaying the overlay image generated in the overlay image generation step;
A mask color component selection step of selecting any one of the color components constituting the overlay image as a mask color component based on a user operation;
A mask area specifying step for specifying an area other than the analysis target area as a mask area from the color component image to which the mask color component is assigned;
From the color component image to which the color component other than the mask color component among the color components constituting the overlay image is assigned, an extracted image in which an area other than the analysis target area is masked using the mask area is generated. A method for controlling an imaging analysis apparatus, comprising: A procedure for placing an observation object to which a plurality of fluorescent dyes having different excitation wavelengths are attached on a stage;
A procedure of irradiating a plurality of excitation lights for exciting each of the plurality of fluorescent dyes on the observation object placed on the stage,
An imaging procedure for capturing an observation object when each of the plurality of excitation lights is irradiated and acquiring two or more captured images of monochrome gradation;
An overlay image generation procedure for generating an overlay image by superimposing the plurality of different color component images by assigning different color components to each of the captured images and generating a plurality of different color component images;
A display procedure for displaying the overlay image generated in the overlay image generation procedure;
A mask color component selection procedure for selecting, as a mask color component, one of the color components constituting the overlay image based on a user operation;
A mask region specifying procedure for specifying a region other than the analysis target region as a mask region from the color component image to which the mask color component is assigned;
From the color component image to which the color component other than the mask color component among the color components constituting the overlay image is assigned, an extracted image in which an area other than the analysis target area is masked using the mask area is generated. A program for an imaging analysis apparatus that causes a computer to execute an extracted image generation procedure.