Dye image acquiring method, dye image acquiring apparatus, and dye image acquiring program

Abstract

To shorten the whole measuring time.SOLUTION: The dye image acquisition system 1 emits excitation light of C (C is an integer of 1 or more) first wavelength distributions to acquire C first fluorescent images in a first image acquisition period, and emits excitation light of D (D is an integer of 1 or more) second wavelength distributions to acquire D second fluorescent images in a second image acquisition period after the first image acquisition period. Generating K pieces of first dye data partially indicating a distribution of each of K dyes (K is an integer of 2 or more and C + D or less) for the C first fluorescent images in the partial unmixing period, and generating K pieces of second dye data partially indicating a distribution of each of the K dyes for the D second fluorescent images in the unmixing period; K pigment images are generated based on the K first pigment data and the K second pigment data, and at least a part of the partial unmixing processing in the partial unmixing period is executed in parallel with the first image acquisition period or the second image acquisition period.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] One aspect of the embodiment relates to a dye image acquisition method, a dye image acquisition device, and a dye image acquisition program. [Background technology]

[0002] Conventionally, a method has been used in which a substance in a sample such as a biological tissue is stained and a fluorescence image is measured. Patent Document 1 listed below discloses a method of acquiring a plurality of sample images corresponding to different combinations of a wavelength band of a variable illumination light and one or more wavelength bands of emitted light, and performing a decomposition process on the plurality of sample images to obtain an image of the distribution of molecular targets associated with each of the dyes applied to the sample. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] Special Publication No. 2021-526220 Summary of the Invention [Problem to be solved by the invention]

[0004] In the conventional method described above, multiple sample images are acquired by an image acquisition unit including a detector, etc., and then separation processing is performed to obtain images of multiple dye distributions. As a result, the overall measurement time for measuring the distributions of multiple dyes tends to increase.

[0005] Therefore, one aspect of the embodiments has been made in consideration of such problems, and aims to provide a dye image acquisition method, a dye image acquisition device, and a dye image acquisition program that can shorten the overall measurement time for measuring the distribution of multiple dyes. [Means for solving the problem]

[0006] A dye image acquisition method according to a first aspect of the embodiment includes a first image acquisition step of irradiating a sample with each of C (C is an integer of 1 or more) excitation lights having a first wavelength distribution and acquiring C first fluorescence images using an image acquisition unit; a second image acquisition step of, after the first image acquisition step, irradiating the sample with each of D (D is an integer of 1 or more) excitation lights having a second wavelength distribution and acquiring D second fluorescence images using the image acquisition unit; a partial unmixing step of performing a partial unmixing process on the C first fluorescence images to generate K (K is an integer of 2 or more and C+D or less) pieces of first dye data that partially indicate the distribution of each of K dyes; and an unmixing step of performing an unmixing process on the D second fluorescence images to generate K second dye data that partially indicate the distribution of each of the K dyes and to generate K dye images based on the K first dye data and the K second dye data, wherein at least a part of the partial unmixing process in the partial unmixing step is executed in parallel with the first image acquisition step or the second image acquisition step.

[0007] Alternatively, a dye image acquisition device according to a second aspect of the embodiment includes a processing unit that images a sample irradiated with each of C (C is an integer of 1 or more) excitation lights having a first wavelength distribution during a first image acquisition period to acquire C first fluorescence images, images a sample irradiated with each of D (D is an integer of 1 or more) excitation lights having a second wavelength distribution during a second image acquisition period following the first image acquisition period to acquire D second fluorescence images, and performs a partial unmixing process on the C first fluorescence images during a partial unmixing period to generate K (K is an integer of 2 or more and C+D or less) first dye data items that partially indicate the distribution of each of the K dyes, and generates K second dye data items that partially indicate the distribution of each of the K dyes during the unmixing period, and generates K dye images based on the K first dye data items and the K second dye data items, and at least a part of the partial unmixing process during the partial unmixing period is executed in parallel with the first image acquisition period or the second image acquisition period.

[0008] Alternatively, a dye image acquisition program according to a third aspect of the embodiment may include a first image acquisition step of irradiating a sample with each of C (C is an integer of 1 or more) excitation lights having a first wavelength distribution and acquiring C first fluorescence images using an image acquisition unit, a second image acquisition step of irradiating the sample with each of D (D is an integer of 1 or more) excitation lights having a second wavelength distribution and acquiring D second fluorescence images using the image acquisition unit after the first image acquisition step, and a second image acquisition step of partially obtaining K (K is an integer of 2 or more and C+D or less) dye distributions from the C first fluorescence images. and an unmixing step that performs an unmixing process to generate K pieces of second dye data that partially indicate the distribution of each of the K dyes for the D second fluorescence images, and to generate K dye images based on the K pieces of first dye data and the K pieces of second dye data, and at least a part of the partial unmixing process in the partial unmixing step is executed in parallel with the first image acquisition step or the second image acquisition step.

[0009] According to the first, second, or third aspect, C first fluorescence images are acquired, capturing fluorescence images of a sample irradiated with C excitation light beams having first wavelength distributions, respectively. Then, D second fluorescence images are acquired, capturing fluorescence images of the sample irradiated with D excitation light beams having second wavelength distributions, respectively. Additionally, a partial unmixing process is performed on the C first fluorescence images to generate K first dye data pieces partially showing the distributions of the K dyes. K second dye data pieces partially showing the distributions of the K dyes are generated based on the D second fluorescence images. Furthermore, K dye images showing the distributions of the K dyes are generated based on the K first dye data pieces and the K second dye data pieces. According to any of the above aspects, at least a portion of the partial unmixing process is performed in parallel with the acquisition of the C first fluorescence images or the D second fluorescence images. This reduces the overall measurement time from the acquisition of the C first fluorescence images and the D second fluorescence images to the unmixing process.

[0010] In the first or second aspect, it is preferable that the first wavelength distribution and the second wavelength distribution are different. This makes it possible to acquire many fluorescence images under irradiation with excitation light of many wavelength distributions. As a result, it is possible to acquire with high accuracy images showing the distribution of many dyes having different absorption wavelengths.

[0011] In the first aspect, it is also preferable to set the second wavelength distribution to a distribution different from the first wavelength distribution by switching a filter between the excitation light source and the sample between the first image acquisition step and the second image acquisition step. In the second aspect, the processing unit sets the second wavelength distribution to a distribution different from the first wavelength distribution by switching a filter between the excitation light source and the sample between the first image acquisition period and the second image acquisition period. This allows efficient switching of the wavelength of the excitation light, thereby shortening the measurement time for measuring the distributions of multiple dyes.

[0012] In the first aspect, it is also preferable that, in the first image acquisition step and the second image acquisition step, C first fluorescence images and D second fluorescence images are acquired as images of a region of the sample corresponding to the field of view of an image sensor included in the image acquisition unit. In the second aspect, it is also preferable that, in the first image acquisition period and the second image acquisition period, the processing unit acquires C first fluorescence images and D second fluorescence images as images of a region of the sample corresponding to the field of view of an image sensor included in the processing unit. In this case, it is possible to acquire the distribution of multiple dyes in the region of the sample corresponding to the field of view of the image sensor. Additionally, it is possible to shorten the measurement time for the distribution of multiple dyes in one region of the sample.

[0013] In the first aspect, it is preferable that, in the first image acquisition step and the second image acquisition step, C first fluorescent images and D second fluorescent images are repeatedly acquired for each of a plurality of regions of the sample while moving the field of view of the image acquisition unit relative to the sample. In the second aspect, it is preferable that, in the first image acquisition period and the second image acquisition period, the processing unit repeatedly acquires C first fluorescent images and D second fluorescent images for each of a plurality of regions of the sample while moving the field of view of the image sensor relative to the sample. In this case, it is possible to acquire the distribution of a plurality of dyes in a plurality of regions of the sample corresponding to the field of view of the image sensor that is moved relatively. Additionally, it is possible to shorten the measurement time for the distribution of a plurality of dyes in a plurality of regions of the sample.

[0014] Furthermore, in the first aspect, it is also preferable that the partial unmixing process targeting at least a portion of the multiple regions of the sample in the partial unmixing step is performed in parallel with the second image acquisition step. Furthermore, in the second aspect, it is also preferable that the partial unmixing process targeting at least a portion of the multiple regions of the sample in the partial unmixing period is performed in parallel with the second image acquisition period. In this case, the partial unmixing process targeting a portion of the multiple regions of the sample is performed in parallel with the acquisition of any of the second fluorescence images. This makes it possible to shorten the overall measurement time from the acquisition of C first fluorescence images of the multiple regions and D second fluorescence images of the multiple regions to the unmixing process.

[0015] Furthermore, in the second aspect, it is also preferable that the processing unit includes an image acquisition unit that includes an image sensor and acquires C first fluorescence images and D second fluorescence images, and an image processing unit that performs partial unmixing processing and unmixing processing. With this configuration, at least a part of the partial unmixing processing in the image processing unit is executed in parallel with the acquisition of C first fluorescence images or D second fluorescence images in the image acquisition unit. This makes it possible to shorten the overall measurement time from the acquisition of C first fluorescence images and D second fluorescence images to the unmixing processing.

[0016] Furthermore, in the second aspect, it is also preferable that the processing unit includes an image acquisition unit that includes an image sensor and acquires C first fluorescence images and D second fluorescence images, and a server that is connected to the image acquisition unit via a network and performs the partial unmixing process and the unmixing process. In this case, at least a part of the partial unmixing process in the server is executed in parallel with the acquisition of the C first fluorescence images or the D second fluorescence images by the image acquisition unit. This makes it possible to shorten the overall measurement time from the acquisition of the C first fluorescence images and the D second fluorescence images to the unmixing process.

[0017] The dye image acquisition method of the embodiment includes: [1] a first image acquisition step of irradiating a sample with C (C is an integer of 1 or more) excitation light beams having a first wavelength distribution, and acquiring the C first fluorescence images using an image acquisition unit; a second image acquiring step of irradiating the sample with D (D is an integer of 1 or more) excitation lights having a second wavelength distribution, and acquiring the D second fluorescence images using the image acquiring unit, after the first image acquiring step; a partial unmixing step of performing a partial unmixing process on the C first fluorescence images to generate K pieces of first dye data (K is an integer of 2 or more and C+D or less) partially indicating distributions for each of the K dyes; an unmixing step of generating the K pieces of second dye data partially showing the distribution of each of the K dyes from the D second fluorescence images, and performing an unmixing process of generating the K dye images based on the K pieces of first dye data and the K pieces of second dye data; Equipped with At least a part of the partial unmixing process in the partial unmixing step is executed in parallel with the first image acquisition step or the second image acquisition step. "Method for obtaining dye images."

[0018] In the dye image acquisition method of the embodiment, [2] "the first wavelength distribution and the second wavelength distribution are different, The dye image obtaining method according to the above item [1] may also be used.

[0019] The dye image acquisition method of the embodiment further comprises the steps of: [3] "switching a filter between the excitation light source and the sample between the first image acquisition step and the second image acquisition step, thereby setting the second wavelength distribution to a distribution different from the first wavelength distribution; The dye image obtaining method according to the above item [2] may also be used.

[0020] The dye image acquisition method of the embodiment includes the steps of: [4] "acquiring the C number of first fluorescent images and the D number of second fluorescent images as images of a region of the sample corresponding to a field of view of an image sensor included in the image acquisition unit in the first image acquisition step and the second image acquisition step; The dye image obtaining method may be the method according to any one of [1] to [3] above.

[0021] The dye image acquisition method of the embodiment includes the steps of: [5] "in the first image acquisition step and the second image acquisition step, the C first fluorescent images and the D second fluorescent images are repeatedly acquired for each of a plurality of regions of the sample while moving the field of view of the image acquisition unit relative to the sample; The dye image obtaining method according to the above item [4] may also be used.

[0022] In the dye image acquisition method of the embodiment, [6] "the partial unmixing process for at least a part of the plurality of regions of the sample in the partial unmixing step is performed in parallel with the second image acquisition step, The dye image obtaining method according to the above item [5] may also be used.

[0023] The dye image acquisition device of the embodiment includes: [7] "images of a sample irradiated with C (C is an integer of 1 or more) excitation light beams having a first wavelength distribution during a first image acquisition period to acquire the C first fluorescence images; capturing an image of a sample irradiated with D (D is an integer of 1 or more) excitation light beams having a second wavelength distribution in a second image acquisition period subsequent to the first image acquisition period to obtain the D second fluorescence images; during a partial unmixing period, a partial unmixing process is performed on the C first fluorescence images to generate K pieces of first dye data (K is an integer between 2 and C+D) that partially indicate distributions for each of the K dyes; a processing unit that performs, during an unmixing period, an unmixing process of generating the K pieces of second dye data partially indicating the distribution of each of the K dyes from the D second fluorescence images, and generating the K dye images based on the K pieces of first dye data and the K pieces of second dye data; At least a part of the partial unmixing process in the partial unmixing period is executed in parallel with the first image acquisition period or the second image acquisition period. It may also be a "dye image acquisition device."

[0024] In the dye image acquisition device of the embodiment, [8] "the first wavelength distribution and the second wavelength distribution are different, The dye image obtaining device according to the above item [7] may also be used.

[0025] In the dye image acquisition device of the embodiment, [9] "the processing unit sets the second wavelength distribution to a distribution different from the first wavelength distribution by switching a filter between the excitation light source and the sample between the first image acquisition period and the second image acquisition period. The dye image acquisition device described in [8] above.

[0026] In the dye image acquisition device of the embodiment,

[10] "the processing unit acquires the C number of first fluorescent images and the D number of second fluorescent images as images of a region of the sample corresponding to a field of view of an image sensor included in the processing unit during the first image acquisition period and the second image acquisition period; The dye image obtaining device may be the one described in any one of [7] to [9] above.

[0027] The dye image acquisition device of the embodiment is,

[11] "the processing unit, during the first image acquisition period and the second image acquisition period, repeatedly acquires the C first fluorescent images and the D second fluorescent images for each of a plurality of regions of the sample while moving the field of view of the image sensor relative to the sample, The dye image obtaining device according to the above item

[10] may also be used.

[0028] In the dye image acquisition device of the embodiment,

[12] "the partial unmixing process for at least a part of the plurality of regions of the sample during the partial unmixing period is performed in parallel with the second image acquisition period, The dye image obtaining device according to the above item

[11] may also be used.

[0029] In the dye image acquisition device of the embodiment,

[13] "the processing unit includes an image acquisition unit that includes an image sensor and acquires the C number of first fluorescent images and the D number of second fluorescent images, and an image processing unit that performs the partial unmixing process and the unmixing process, The dye image obtaining device may be the one described in any one of [7] to

[12] above.

[0030] In the dye image acquisition device of the embodiment,

[14] "the processing unit includes an image acquisition unit that includes an image sensor and acquires the C first fluorescent images and the D second fluorescent images, and a server that is connected to the image acquisition unit via a network and performs the partial unmixing process and the unmixing process, The dye image obtaining device may be the one described in any one of [7] to

[12] above.

[0031] The dye image acquisition program of the embodiment includes the steps of:

[15] "a computer, a first image acquisition step of irradiating a sample with C (C is an integer of 1 or more) excitation light beams having a first wavelength distribution and acquiring the C first fluorescence images using an image acquisition unit; a second image acquiring step of irradiating the sample with D (D is an integer of 1 or more) excitation lights having a second wavelength distribution, and acquiring the D second fluorescence images using the image acquiring unit, after the first image acquiring step; a partial unmixing step of performing a partial unmixing process on the C first fluorescence images to generate K pieces of first dye data (K is an integer of 2 or more and C+D or less) partially indicating distributions for each of the K dyes; an unmixing step of generating the K pieces of second dye data partially showing the distribution of each of the K dyes from the D second fluorescence images, and performing an unmixing process of generating the K dye images based on the K pieces of first dye data and the K pieces of second dye data; At least a part of the partial unmixing process in the partial unmixing step is executed in parallel with the first image acquisition step or the second image acquisition step. "Dye image acquisition program." [Effects of the Invention]

[0032] According to one aspect of the present invention, the overall measurement time required to measure the distribution of a plurality of dyes can be reduced. [Brief explanation of the drawings]

[0033] [Figure 1] FIG. 1 is a schematic diagram of a dye image acquisition system 1 according to an embodiment. [Figure 2] FIG. 2 is a flowchart showing the flow of the dye image generation process using the dye image acquisition system 1. [Figure 3] FIG. 3 is a diagram showing an image of division of lanes, which are regions of the sample S to be subjected to partial unmixing processing and unmixing processing by the dye image acquisition system 1. [Figure 4] FIG. 4 is a diagram showing an example of a dye image generated by the dye image acquisition system 1 according to the embodiment. [Figure 5] FIG. 5 is a diagram illustrating the execution order of various processes executed by the dye image acquisition system 1 according to the embodiment. [Figure 6] FIG. 6 is a diagram illustrating the order in which various processes are executed by the dye image acquisition system 1 according to the embodiment. [Figure 7] FIG. 7 is a diagram illustrating the order in which each process is executed by the dye image acquisition system 1 according to the modified example. [Figure 8] FIG. 8 is a diagram illustrating the order in which each process is executed by the dye image acquisition system 1 according to the modified example. DETAILED DESCRIPTION OF THE INVENTION

[0034] Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings. In the description, the same elements or elements having the same functions will be denoted by the same reference numerals, and redundant description will be omitted.

[0035] FIG. 1 is a schematic diagram of a dye image acquisition system 1, which is a dye image acquisition device according to an embodiment. The dye image acquisition system 1 is a device for generating dye images for identifying the distribution of dyes in a sample, such as biological tissue, that is the object of observation. Images generated by the dye image acquisition system 1 are used for purposes such as drug development and the study of treatment methods through analysis of the images. For this reason, the dye image acquisition system 1 is required to generate images with high throughput that enable quantitative identification of the distribution of many substances (dyes) contained in a sample.

[0036] The dye image acquisition system 1 includes a sample support section 3 that supports a sample S, an image acquisition device (image acquisition section) 6 that irradiates the sample S with excitation light and acquires an image of the fluorescence generated in response, an excitation light source 7, and an image processing device (image processing section) 8 that processes the image acquired by the image acquisition device 6. The image processing device 8 may be configured to be able to send and receive image data to and from the image acquisition device 6 using wired or wireless communication, or may be configured to be able to input and output image data via a recording medium. The image processing device 8 may also be a server, PC, or the like connected to the image acquisition device 6 via a network.

[0037] In the dye image acquisition system 1, a sample S (e.g., pathological cells stained with multiple fluorescent dyes) is handled while being placed on a glass slide G, and various images of the sample S are acquired. Hereinafter, unless otherwise specified, the "glass slide G on which the sample S is placed" will be simply referred to as the "glass slide G." Furthermore, a predetermined horizontal direction is referred to as the X-axis direction, the horizontal direction perpendicular to the X-axis direction is referred to as the Y-axis direction, and the vertical direction is referred to as the Z-axis direction.

[0038] The excitation light source 7 includes a light output unit 71, a light emitting unit 72, and an optical fiber 73. The light output unit 71 outputs excitation light for each of a plurality of wavelength bands. The light emitting unit 72 emits the excitation light output from the light output unit 71 and guided by the optical fiber 73 along a first optical path P1. The light output unit 71 is a light source that can switch between and irradiate excitation light of a plurality of wavelength bands (wavelength distributions), and is, for example, an LED (Light Emitting Diode) light source, a light source consisting of a plurality of monochromatic laser light sources, or a light source that combines a white light source and a wavelength-selecting optical element.

[0039] The image acquisition device 6 includes a sample support section 3, an objective lens unit 55, a light detection section 61, an optical path switching section 62, a filter switching section 63, a mirror 64, a mirror 65, an imaging lens 66, an optical filter section 67, and a fluorescence filter section 68.

[0040] The sample support unit 3 includes a support unit 31 and a moving stage 33. The support unit 31 is attached to the upper side of the moving stage 33. The support unit 31 supports the slide glass G by holding the slide glass G. In other words, the support unit 31 supports the sample S. The moving stage 33 moves the support unit 31 in each of the X-axis direction and the Y-axis direction in the image acquisition device 6. The moving stage 33 is a device for moving the field of view of the light detection unit 61, which will be described later, relative to the sample S.

[0041] The objective lens unit 55 is composed of multiple objective lenses 55a, a moving stage 55b, and a switching stage 55c. The multiple objective lenses 55a are attached below the moving stage 55b. The moving stage 55b is attached below the switching stage 55c. In the image acquisition device 6, the switching stage 55c rotates to position one of the objective lenses 55a on the optical axis of the light detection unit 61 (the second optical path P2 described below). In the image acquisition device 6, the objective lens 55a arranged on the optical axis of the light detection unit 61 is moved in the Z-axis direction by the moving stage 55b, thereby aligning the focus of the objective lens 55a arranged on the optical axis of the light detection unit 61 with the sample S. The magnification of each objective lens 55a is, for example, 10x, 20x, or 40x. As an example, the switching stage 55c is a rotary stage or a linear stage.

[0042] The light emitting unit 72 of the excitation light source 7 is optically connected to the light path switching unit 62 by a first optical path P1. The support unit 31 is optically connected to the light path switching unit 62 by a second optical path P2. The light detecting unit 61 is optically connected to the light path switching unit 62 by a third optical path P3. In other words, the first optical path P1 extends between the light emitting unit 72 and the light path switching unit 62, the second optical path P2 extends between the support unit 31 and the light path switching unit 62, and the third optical path P3 extends between the light detecting unit 61 and the light path switching unit 62.

[0043] As an example, in the image acquisition device 6, the first optical path P1 extends in the Y-axis direction between the light emitting unit 72 and the optical path switching unit 62. The second optical path P2 extends in the Z-axis direction between the optical path switching unit 62 and the support unit 31. The third optical path P3 extends in the Z-axis direction between the optical path switching unit 62 and the mirror 64, extends in the Y-axis direction between the mirror 64 and the mirror 65, and extends in the Z-axis direction between the mirror 65 and the light detection unit 61.

[0044] The excitation light emitted from the light emitting unit 72 enters the optical path switching unit 62 along the first optical path P1. The excitation light that enters the optical path switching unit 62 is reflected by the optical path switching unit 62 and travels along the second optical path P2, and is irradiated onto the sample S supported by the support unit 31. At this time, the excitation light passes through the objective lens 55a of the objective lens unit 55 arranged on the second optical path P2. Fluorescence emitted from the sample S in response to the irradiation of the excitation light enters the optical path switching unit 62 along the second optical path P2. At this time, the fluorescence passes through the objective lens 55a of the objective lens unit 55 arranged on the second optical path P2. The fluorescence that entered the optical path switching unit 62 passes through the optical path switching unit 62 and travels along the third optical path P3, and is detected by the light detection unit 61.

[0045] The filter switching unit 63 is an optical device disposed on the first optical path P1 between the light emitting unit 72 and the optical path switching unit 62, has two built-in filters 631 and 632, and switches the two filters 631 and 632 so that they can be inserted into or removed from the first optical path P1. For example, the two filters 631 and 632 are multi-bandpass filters that transmit light of a plurality of predetermined wavelength bands different from each other. The filter switching unit 63 configured as described above can switch between two excitation light wavelength bands from the excitation light of a predetermined wavelength band emitted from the light emitting unit 72, transmit the excitation light, and irradiate the sample S with the excitation light via the optical path switching unit 62. In the example of FIG. 1, two filters 631 and 632 are built in, but three or more filters may be built in.

[0046] As described above, the light detection unit 61 detects fluorescence emitted from the sample S when irradiated with excitation light. The light detection unit 61 is, for example, a monochrome area image sensor. The light detection unit 61 may also be a color area image sensor or a color separation sensor (multispectral sensor, hyperspectral sensor, etc.). When a color separation sensor is used as the light detection unit 61, it becomes easier to handle cases where there are a large number of dyes (for example, 10 or more colors). The light detection unit 61 may also be a linear image sensor.

[0047] As described above, the optical path switching unit 62 switches the optical paths of the excitation light and the fluorescence. The optical path switching unit 62 is configured to switch the optical path of the excitation light of a plurality of mutually different first wavelength bands or a plurality of mutually different second wavelength bands from the first optical path P1 to the second optical path P2, and to switch the optical path of the fluorescence of a wavelength band shifted from the plurality of first wavelength bands or the plurality of second wavelength bands from the second optical path P2 to the third optical path P3. The plurality of mutually different first wavelength bands and the plurality of mutually different second wavelength bands refer to a plurality of wavelength bands that are separated from each other. The wavelength band shifted from the plurality of first wavelength bands refers to a wavelength band that does not substantially overlap with the plurality of first wavelength bands. The optical path switching unit 62 has a configuration, for example, in which a pair of triangular prism-shaped light-transmitting members are combined to form a cube, and a dichroic mirror that is a dielectric multilayer film is formed at the interface between the pair of light-transmitting members.

[0048] The image acquisition device 6 is equipped with a plurality of optical path switching units 62 having different specifications (number of wavelength bands, upper limit of wavelength bands, lower limit of wavelength bands, etc.), and is configured to place any one of the optical path switching units 62 at a position where the first optical path P1, the second optical path P2, and the third optical path P3 intersect. The image acquisition device 6 is also configured so that each optical path switching unit 62 can be detached (replaced).

[0049] The mirror 64 is disposed on the third optical path P3. The mirror 65 is disposed on the third optical path P3 between the mirror 64 and the photodetector 61. The mirror 64 reflects the fluorescence of multiple wavelength bands emitted from the optical path switching unit 62 toward the mirror 65. The mirror 65 reflects the fluorescence of multiple wavelength bands reflected by the mirror 64 toward the photodetector 61, causing it to enter the photodetector 61. The imaging lens 66 is disposed on the third optical path P3 between the mirrors 64 and 65. The imaging lens 66 forms an image of the fluorescence of multiple wavelength bands on the photodetector 61. In other words, the imaging lens 66 forms an image of the field of view of the objective lens 55a disposed on the first optical path P1 on the photodetector 61. The imaging lens 66 is, for example, a tube lens.

[0050] The optical filter unit 67 includes an optical filter 67a. The optical filter 67a transmits fluorescence in a plurality of wavelength bands. The optical filter unit 67 is configured to place the optical filter 67a on the third optical path P3 between the optical path switching unit 62 and the imaging lens 66. On the other hand, the optical filter unit 67 is configured to exclude the optical filter 67a from the third optical path P3 between the optical path switching unit 62 and the imaging lens 66.

[0051] The optical filter 67a has transmission characteristics in which the transmittance changes in a fluorescence wavelength band that includes multiple wavelength bands. The optical filter 67a has transmission characteristics in which there is a one-to-one correspondence between wavelength and transmittance in the fluorescence wavelength band. The optical filter 67a has transmission characteristics in which the transmittance changes linearly in the fluorescence wavelength band.

[0052] The fluorescence filter unit 68 includes a switching mechanism main body 681, multiple single bandpass filters 682, and a light passing unit 683. Each single bandpass filter 682 is disposed in a corresponding one of multiple openings formed in the switching mechanism main body 681. Each single bandpass filter 682 selectively transmits fluorescence for each of multiple wavelength bands. In other words, each single bandpass filter 682 essentially has the function of transmitting only fluorescence of one wavelength band. The light passing unit 683 is an opening formed in the switching mechanism main body 681 and passes fluorescence in the fluorescence wavelength band. The light passing unit 683 may be a light-transmitting member having transmission characteristics that pass fluorescence in the fluorescence wavelength band and disposed in the opening formed in the switching mechanism main body 681. The fluorescence filter unit 68 is configured such that, when the switching mechanism main body 681 is switched, the light passing unit 683 and each of the multiple single bandpass filters 682 are disposed on the third optical path P3 (the third optical path P3 between the optical path switching unit 62 and the mirror 64). As an example, the switching mechanism body 681 is a rotary type switching mechanism (for example, a filter wheel, etc.) or a linear type switching mechanism (for example, a slider, etc.).

[0053] The image processing device 8 includes an image processing unit 81 and a display unit 82. The image processing unit 81 is a computer including a processor, and executes various processes described below. The image processing unit 81 acquires a fluorescence image, which is image data showing the intensity distribution of fluorescence in the sample S, from the light detection unit 61 of the image acquisition device 6, and processes the acquired fluorescence image. The fluorescence image to be processed by the image processing unit 81 is an image of the region of the sample S that corresponds to the field of view of the objective lens 55a (the field of view of the light detection unit 61, which is an image sensor). The display unit 82 is a display, and displays various images of the sample S.

[0054] Below, we will explain the flow of the dye image generation process using dye image acquisition system 1, and also provide a detailed description of the steps of the dye image acquisition method according to this embodiment. Fig. 2 is a flowchart showing the flow of the dye image generation process using dye image acquisition system 1. Each process by dye image acquisition system 1 shown in Fig. 2 is realized by loading a program (dye image acquisition program according to this embodiment) onto hardware such as a processor and memory built into image acquisition device 6 and image processing device 8.

[0055] First, when the process of generating a dye image is started, the filter switching unit 63 is switched so that the filter 631 is placed on the optical path (step S1). Next, the movement of the moving stage 33 is controlled to set the field of view of the light detection unit 61 on the sample S to a predetermined region (step S2). After that, the wavelength of the excitation light output from the excitation light source 7 is switched to a predetermined wavelength band (step S3), and then the excitation light is irradiated from the light output unit 71 (step S4).

[0056] Then, excitation light of one of the plurality of first wavelength bands that has been emitted from the excitation light source 7 and transmitted through the filter switching unit 63 is irradiated onto the sample S, and a first fluorescence image that indicates the intensity distribution of the fluorescence in a predetermined region on the sample S is detected by the light detecting unit 61 (step S5). In response to this, the first fluorescence image is transferred from the light detecting unit 61 to the image processing unit 81 (step S6).

[0057] The processes in steps S3 to S6 are repeated by switching the wavelength of the excitation light output from the excitation light source 7 among C wavelength bands (C is an integer equal to or greater than 1) (step S7). As a result, C first fluorescence images corresponding to the irradiation of the excitation light in the C first wavelength bands are acquired. Thereafter, the image processing unit 81 performs image processing (partial unmixing processing) on ​​the C first fluorescence images (step S8).

[0058] Furthermore, while scanning a plurality of regions on the sample S by moving the moving stage 33, acquisition of C first fluorescent images (first image acquisition step) and image processing of the C first fluorescent images (partial unmixing step) are repeated (step S9). This completes image processing of the C first fluorescent images in all regions, including a plurality of regions, on the sample S.

[0059] Thereafter, if a fluorescence image using the last filter included in the filter switching unit 63 has not been acquired, the processes of steps S1 to S9 are repeated the number of times corresponding to the number of filters included in the filter switching unit 63 (step S10). In this embodiment, the filter switching unit 63 is switched so that filter 632 is positioned on the optical path, and then the processes of steps S2 to S9 are repeated to acquire D second fluorescence images corresponding to the irradiation of each of D (D is an integer equal to or greater than 1) excitation light beams in the second wavelength band (second image acquisition step), and image processing (unmixing processing) is performed on the D second fluorescence images and the processing results of the partial unmixing step (unmixing step), with the execution results being displayed on the display unit 82. This completes the dye image generation process.

[0060] In this embodiment, in the first image acquisition step, two excitation lights in the first wavelength band are repeatedly irradiated onto the sample S, and two first fluorescence images are acquired for each region, and in the second image acquisition step, one excitation light in the second wavelength band is irradiated onto the sample S, and one second fluorescence image is acquired for each region.

[0061] FIG. 3 shows an image of the division of lanes, which are regions of the sample S that are the target of the partial unmixing process and the unmixing process in the partial unmixing step and the unmixing step. As shown in FIG. 3(a), an image of a lane LA obtained by combining multiple (e.g., three) fluorescent images of a region AR on the sample S that corresponds to the field of view of the light detection unit 61, which are continuously scanned by the movement of the moving stage 33, may be the target of the partial unmixing process and the unmixing process. Also, as shown in FIG. 3(b), each of the fluorescent images of a region AR on the sample S that corresponds to the field of view of the light detection unit 61, which are continuously scanned by the movement of the moving stage 33, may be the target of the partial unmixing process and the unmixing process as the image of the lane LA. The arrows shown in FIGS. 3(a) and 3(b) indicate examples of the scanning direction on the sample S. However, the number of divisions of the region AR, the scanning direction, and the number of combined lanes LA on the sample S may be changed arbitrarily.

[0062] Next, the image processing functions executed by the image processing unit 81 will be described in detail.

[0063] The image processing unit 81 pre-stores a mixing matrix A for generating K dye images showing the distribution of each of K dyes (K is an integer of 2 or more and C+D or less) from C first fluorescent images of each lane and D second fluorescent images of each lane. The mixing matrix A is derived and stored in advance by a known method such as that described in International Publication WO2023 / 026742. Let Y be matrix data with (C+D) rows and N columns in which the fluorescence intensity values ​​of N pixels (N is an integer) in each lane constituting each of the C first fluorescent images and the D second fluorescent images are arranged in parallel in a single dimension, and let X be matrix data with K rows and N columns in which the K dye images are arranged in parallel in a single dimension for each pixel in each lane. The relationship between the matrix data Y and the matrix data X is expressed by the following equation using the (C+D) row and K column mixing matrix A: Y=AX Conversely, if the matrix data Y is known, the matrix data X can be expressed by the following formula: X=(A T A) -1 A T Y (This process is called unmixing, which separates a fluorescent image into multiple dye images.)

[0064] Using the above principle, the image processing unit 81 has the function of performing partial unmixing based only on the C first fluorescent images in the partial unmixing step. That is, the image processing unit 81 generates matrix data Y1 with (C+D) rows and N columns in which the fluorescence intensity values ​​of N pixels in each lane constituting each of the C first fluorescent images are arranged in parallel in one dimension, and the fluorescence intensity values ​​of N pixels in each lane constituting each of the D second fluorescent images are set to zero. Then, as the partial unmixing process, the following equation is used: X1=(A T A) -1 A T Y1 By calculating the above, matrix data X1 with K rows and N columns is calculated in which K first dye data partially showing the distribution of each of the K dyes are arranged in parallel one-dimensionally for each pixel of each lane.

[0065] For example, in this embodiment, the image processing unit 81 calculates three pieces of first dye data from two first fluorescent images, and if the values ​​of specific pixels in the two first fluorescent images are Ex1 and Ex2, it calculates the distributions of the specific pixels for each of the three dyes, Dye1_1, Dye1_2, and Dye1_3, using the following formulas.

number

[0066] Furthermore, the image processing unit 81 has a function of performing partial unmixing based on D second fluorescent images in the unmixing step, and simultaneously performing unmixing by adding the partial unmixing result from the partial unmixing step to the result. That is, the image processing unit 81 sets the fluorescence intensity values ​​of N pixels in each lane constituting each of C first fluorescent images to zero, and generates matrix data Y2 with (C+D) rows and N columns in which the fluorescence intensity values ​​of N pixels in each lane constituting each of D second fluorescent images are arranged in parallel in one dimension. Then, as the unmixing process, it calculates the following equation: X3=(A T A) -1 A T Y2+X1=X2+X1 By calculating the above, matrix data X2 of K rows and N columns is calculated in which K pieces of second dye data, which partially indicate the distribution of each of the K dyes, are arranged in parallel in one dimension for each pixel of each lane, and by adding together the elements of the K pieces of first dye data and the K pieces of second dye data, matrix data X3 of K rows and N columns is calculated in which K dye images, which indicate the distribution of each of the K dyes, are arranged in parallel in one dimension for each pixel of each lane.

[0067] For example, in this embodiment, the image processing unit 81 calculates three pieces of second dye data based on one second fluorescence image, and adds the distributions Dye1_1, Dye1_2, and Dye1_3 of the specific pixel for each of the three dyes calculated in the partial unmixing step to the three pieces of second dye data. For example, if the value of a specific pixel in one second fluorescence image is Ex3, the image processing unit 81 calculates the distributions Dye1, Dye2, and Dye3 of the specific pixel for each of the three dyes using the following equations.

number

[0068] Furthermore, the image processing unit 81 calculates matrix data X3 for all lanes to be measured, and generates, from the calculation results, K dye images that indicate the distribution of the K dyes in a range that includes all lanes on the sample S. The image processing unit 81 then displays the generated K dye images on the display unit 82.

[0069] 4 shows an example of a dye image generated by the dye image acquisition system 1 according to this embodiment. As described above, according to this embodiment, the distribution of rhodamine, which is a dye contained in the sample S, can be obtained with high accuracy.

[0070] Next, the execution order of each processing step executed by the dye image acquisition system 1 will be described. FIG. 5 is a diagram illustrating the execution order of each processing executed by the dye image acquisition system 1. In FIG. 5, the period of each processing on the time axis is indicated by an arrow, the execution order of the processing according to this embodiment is shown at the bottom, and the execution order of the processing according to the first comparative example is shown at the top. Here, it is assumed that there are a total of 40 lanes to be observed in the sample S.

[0071] In the dye image acquisition system 1 according to this embodiment, first, a first image acquisition step is performed for the first lane of the sample S, and two first fluorescent images are acquired (during a period P S1_L1 ), and then the first image acquisition step is performed successively for the second lane to the fortieth lane (period P S1_L2 ~Period P S1_L40 Immediately after that, the filter switching unit 63 is switched, and then the second image acquisition step is executed for the first lane of the sample S, and one second fluorescent image is acquired (period P S3_L1 ), and then the second image acquisition step is performed successively for the second lane to the 40th lane (period P S3_L2 ~Period P S3_L40 ).

[0072] In parallel with the above process, for each period P S1_L1 ~P S1_L40 Immediately after this, partial unmixing processing is performed on the two first fluorescent images acquired in each period (period P S2_L1 ~Period P S2_L40 ), each period P S3_L1 ~P S3_L40 Immediately after this, an unmixing process is performed on one second fluorescent image acquired in each period and the partial unmixing results of each lane (period P S4_L1~Period P S4_L40 ).

[0073] As described above, in this embodiment, the partial unmixing process for at least a part of all the lanes to be observed is executed in parallel with the second image acquisition step. In detail, during the partial unmixing period P S2_L40 is the period P of the second image acquisition step S3_L1 In addition, the partial unmixing process for some lanes is executed in parallel with the first image acquisition step. In detail, the partial unmixing period P S2_L1 is overlapped with the period of the first image acquisition step for the next second lane. In contrast, in the first comparative example, the entire period P S1_L1 ~P S1_L40 ,P S3_L1 ~P S3_L40 After this, an unmixing process is performed using the first and second fluorescent images for each lane together (period P S5_L1 ~P S5_L40 ) As a result, according to this embodiment, the measurement time of the dye image is set to the period P S5_L1 ~P S5_L40 The total time and period P S4_L40 For example, the difference between the period P S5_L1 ~P S5_L40 The total time is 150 seconds, and the period P S4_L40 becomes 0.1 seconds, and the measurement time is shortened by approximately 149.9 seconds.

[0074] 6 is a diagram illustrating the execution order of the processing steps executed by the dye image acquisition system 1 in comparison with a second comparative example. The upper part of FIG. 6 shows the execution order of the processing according to the second comparative example.

[0075] In the second comparative example, the sample S is scanned only once, and the first and second fluorescent images are acquired for each lane in one scan, and unmixing processing is performed for each lane in parallel. That is, during the first image acquisition step P S6_L1 In parallel with this, the filter switching unit 63 switches and acquires one second fluorescent image for each region of the first lane during a period P S7_L1 is set. After that, the period P S6_L2 ~P S6_L40 and the period P of the second image acquisition step S7_L2 ~P S7_L40 This is continuously repeated for each of the 2nd to 40th lanes while the sample S is scanned. In parallel with this, immediately after the second image acquisition step for each lane, a period P S8_L1 ~P S8_L40 is set.

[0076] Compared to Comparative Example 2, this embodiment can reduce the measurement time of the dye image even more than Comparative Example 2. Specifically, assuming that the imaging time of each region, including the exposure time, excitation light switching time, and image transfer time, is 50 msec, the switching time of the filter switching unit 63 is 500 msec, the number of regions in one lane is 25, the movement time of the moving stage 33 is 50 msec, the number of lanes is 40, and the unmixing process time is 100 msec, the measurement time in Comparative Example 2 is (3*0.05+2*0.5+0.05)*25*40+0.1 = 1200.1sec In contrast to this, in this embodiment, (2*0.05+0.05)*25*40+(1*0.05+0.05)*25*40+0.5+0.1 = 250.6sec The calculation shows that the measurement time is reduced by approximately 949.5 seconds compared to the second comparative example.

[0077] According to the dye image acquisition system 1 described above, C first fluorescence images are acquired, capturing fluorescence images of the sample S when irradiated with excitation light beams of C first wavelength bands, respectively. Then, D second fluorescence images are acquired, capturing fluorescence images of the sample S when irradiated with excitation light beams of D second wavelength bands, respectively. Additionally, a partial unmixing process is performed on the C first fluorescence images to generate K first dye data pieces partially showing the distributions of the K dyes. K second dye data pieces partially showing the distributions of the K dyes are generated based on the D second fluorescence images. Furthermore, K dye images showing the distributions of the K dyes are generated based on the K first dye data pieces and the K second dye data pieces. According to this embodiment, at least a portion of the partial unmixing process is performed in parallel with the acquisition of any of the C first fluorescence images or any of the D second fluorescence images. This shortens the overall measurement time from the acquisition of the C first fluorescence images and the D second fluorescence images to the unmixing process.

[0078] In the dye image acquisition system 1, the first waveband and the second waveband are set to be different. This makes it possible to acquire many fluorescence images under irradiation with excitation light of many wavelength bands. As a result of the unmixing process using many fluorescence images, it is possible to acquire with high precision an image showing the distribution of many dyes with different absorption wavelengths.

[0079] Furthermore, in the dye image acquisition system 1, the second wavelength band is set to a distribution different from that of the first wavelength band by switching the filter switching unit 63 between the first image acquisition step and the second image acquisition step. This allows efficient switching of the wavelength of the excitation light, and shortens the measurement time required to measure the distributions of multiple dyes.

[0080] Furthermore, in the dye image acquisition system 1, in the first image acquisition step and the second image acquisition step, C first fluorescent images and D second fluorescent images are acquired as images of the region of the sample S that corresponds to the field of view of the light detection unit 61. In this case, it is possible to acquire the distribution of multiple dyes in the region of the sample S that corresponds to the field of view of the light detection unit 61. In addition, it is possible to shorten the measurement time for the distribution of multiple dyes in one region of the sample S.

[0081] Furthermore, in the dye image acquisition system 1, in the first image acquisition step and the second image acquisition step, C first fluorescent images and D second fluorescent images are repeatedly acquired for each of multiple regions of the sample S while the field of view of the light detection unit 61 is moved relatively to the sample S. In this case, it is possible to acquire the distribution of multiple dyes in multiple regions of the sample S that correspond to the field of view of the light detection unit 61 that is moving relatively. In addition, it is possible to shorten the measurement time for the distribution of multiple dyes in multiple regions of the sample S.

[0082] Furthermore, in the dye image acquisition system 1, the partial unmixing process for at least some of the multiple regions of the sample S in the partial unmixing step is executed in parallel with the second image acquisition step, thereby shortening the overall measurement time from acquisition of C first fluorescent images in the multiple regions and D second fluorescent images in the multiple regions to the unmixing process.

[0083] Various embodiments of the present invention have been described above, but the present invention is not limited to the above embodiments, and may be modified or applied to other things within the scope that does not change the gist of the claims.

[0084] For example, in the dye image acquisition system 1 of this embodiment, two first fluorescent images are acquired in the first image acquisition step, one second fluorescent image is acquired in the second image acquisition step, and the partial unmixing process and the unmixing process are performed using these images in the partial unmixing step and the unmixing step. As a modified example, any number of first fluorescent images may be acquired in the first image acquisition step, any number of second fluorescent images may be acquired in the second image acquisition step, and the partial unmixing process and the unmixing process may be performed using these images in the partial unmixing step and the unmixing step.

[0085] For example, as a modified example, three first fluorescence images may be acquired in the first image acquisition step, and three second fluorescence images may be acquired in the second image acquisition step, and the partial unmixing process and unmixing process may be performed using these images in the partial unmixing step and unmixing step to generate five dye images showing the distribution of the five dyes. In such a modified example, the partial unmixing process and unmixing process are performed as follows.

[0086] That is, the image processing unit 81 calculates five pieces of first dye data from three first fluorescent images using a 6-row, 5-column mixing matrix A, and if the values ​​of specific pixels in the three first fluorescent images are Ex1, Ex2, and Ex3, it calculates the distributions of the specific pixels for each of the five dyes, Dye1_1, Dye2_1, Dye3_1, Dye4_1, and Dye5_1, using the following formula.

number

[0087] Furthermore, the image processing unit 81 calculates five pieces of second dye data based on the three second fluorescence images, and adds the distributions Dye1_1, Dye2_1, Dye3_1, Dye4_1, and Dye5_1 of the specific pixels for each of the five dyes calculated in the partial unmixing step to the five pieces of second dye data. For example, if the values ​​of the specific pixels of the three second fluorescence images are Ex4, Ex5, and Ex6, the image processing unit 81 calculates the distributions Dye1, Dye2, Dye3, Dye4, and Dye5 of the specific pixels for each of the five dyes using the following formulas:

number

[0088] Furthermore, in the dye image acquisition system 1 of the present embodiment, the partial unmixing step and the partial unmixing process in the unmixing step are performed for each lane. In a modified example, the partial unmixing step and the partial unmixing process in the unmixing step may be performed collectively for all lanes to be measured.

[0089] 7 is a diagram illustrating the execution order of each process executed by the dye image acquisition system 1 according to this modified example, in comparison with the first comparative example. As described above, in this modified example, the period P S1_L40 Immediately after this, a partial unmixing step for each lane is performed with a duration of P S2_L1 ~P S2_L40 is set consecutively, and the duration of the second image acquisition step, P, is set for the last lane. S3_L40 Immediately after this, there is an unmixing step for each lane, with a duration of P S4_L1 ~P S4_L40 In other words, in this modification, the period P S2_L1 ~P S2_L40 is the period P of the second image acquisition step. S3_L1 ~P S3_L40 As a result, in this modification, the measurement time of the dye image is set to be parallel to a part of the period P S5_L1 ~PS5_L40 The total time and period P S4_L1 ~P S4_L40 For example, the time period P S5_L1 ~P S5_L40 The total time is 150 seconds, and the period P S4_L1 ~P S4_L40 The total time is 50 seconds, reducing the measurement time by about 100 seconds.

[0090] Furthermore, the dye image acquisition system 1 according to the above embodiment repeatedly scans the sample S about twice, and performs the first image acquisition step and the partial unmixing step on multiple regions scanned once. In a modified example, the first image acquisition step and the partial unmixing step may be performed on multiple regions scanned multiple times. Then, by switching the filter switching unit 63 between the two scans, it is possible to acquire many fluorescence images in a state where excitation light of many wavelength bands is irradiated. As a result of the unmixing process using many fluorescence images, it is possible to accurately acquire an image showing the distribution of many dyes with different absorption wavelengths.

[0091] Furthermore, in the dye image acquisition system 1 of the above embodiment, the filter switching unit 63 may be switched and the second image acquisition step may be executed after the partial unmixing process for C first fluorescent images is completed, as shown in Fig. 8. Fig. 8 is a diagram illustrating the execution order of each process executed by the dye image acquisition system 1 according to a modified example. Even with this modified example, the partial unmixing process for some lanes is executed in parallel with the first image acquisition step, and the overall measurement time from the acquisition of C first fluorescent images and D second fluorescent images to the unmixing process can be shortened. [Explanation of symbols]

[0092] 1...dye image acquisition system, 6...image acquisition device (image acquisition section), 8...image processing device, 81...image processing section, 631, 632...filters, AR...area, S...sample.

Claims

1. a first image acquisition step of irradiating a sample with C (C is an integer of 1 or more) excitation light beams having a first wavelength distribution, and acquiring the C first fluorescence images using an image acquisition unit; a second image acquiring step of irradiating a sample with D (D is an integer of 1 or more) excitation light beams having a second wavelength distribution, and acquiring the D second fluorescence images using the image acquiring unit, after the first image acquiring step; a partial unmixing step of performing a partial unmixing process on the C first fluorescence images to generate K pieces of first dye data (K is an integer of 2 or more and C+D or less) partially indicating distributions for each of the K dyes; an unmixing step of generating the K pieces of second dye data partially showing the distributions of the K dyes from the D second fluorescence images, and performing an unmixing process of generating the K dye images based on the K pieces of first dye data and the K pieces of second dye data; Equipped with At least a part of the partial unmixing process in the partial unmixing step is executed in parallel with the first image acquisition step or the second image acquisition step. Dye image acquisition method.

2. the first wavelength distribution and the second wavelength distribution are different; The method for obtaining a dye image according to claim 1 .

3. and setting the second wavelength distribution to a distribution different from the first wavelength distribution by switching a filter between an excitation light source and the sample between the first image acquisition step and the second image acquisition step. The method for obtaining a dye image according to claim 2 .

4. In the first image acquisition step and the second image acquisition step, the C first fluorescent images and the D second fluorescent images are acquired as images of a region of the sample corresponding to a field of view of an image sensor included in the image acquisition unit. The method for obtaining a dye image according to claim 1 or 2.

5. In the first image acquiring step and the second image acquiring step, the C first fluorescent images and the D second fluorescent images are repeatedly acquired for each of a plurality of regions of the sample while moving the field of view of the image acquiring unit relative to the sample. The method for obtaining a dye image according to claim 4 .

6. the partial unmixing process for at least a part of the plurality of regions of the sample in the partial unmixing step is performed in parallel with the second image acquisition step. The method for obtaining a dye image according to claim 5 .

7. capturing an image of a sample irradiated with C (C is an integer of 1 or more) excitation light beams having a first wavelength distribution during a first image acquisition period to acquire the C first fluorescence images; capturing an image of a sample irradiated with D (D is an integer of 1 or more) excitation light beams having a second wavelength distribution in a second image acquisition period subsequent to the first image acquisition period to acquire the D second fluorescence images; during a partial unmixing period, a partial unmixing process is performed on the C first fluorescence images to generate K pieces of first dye data (K is an integer equal to or greater than 2 and equal to or less than C+D) that partially indicate distributions for each of the K dyes; a processing unit that performs, during an unmixing period, an unmixing process of generating the K pieces of second dye data partially indicating the distribution of each of the K dyes from the D second fluorescence images, and generating the K dye images based on the K pieces of first dye data and the K pieces of second dye data, At least a part of the partial unmixing process in the partial unmixing period is executed in parallel with the first image acquisition period or the second image acquisition period. Dye image acquisition device.

8. the first wavelength distribution and the second wavelength distribution are different; The dye image acquisition device according to claim 7 .

9. the processing unit sets the second wavelength distribution to a distribution different from the first wavelength distribution by switching a filter between an excitation light source and the sample between the first image acquisition period and the second image acquisition period. The dye image acquisition device according to claim 8 .

10. the processing unit acquires, during the first image acquisition period and the second image acquisition period, the C first fluorescent images and the D second fluorescent images as images of a region of the sample corresponding to a field of view of an image sensor included in the processing unit. The dye image obtaining apparatus according to claim 7 or 8.

11. the processing unit, during the first image acquisition period and the second image acquisition period, repeatedly acquires the C first fluorescent images and the D second fluorescent images for each of a plurality of regions of the sample while moving the field of view of the image sensor relative to the sample. The dye image acquisition device according to claim 10.

12. the partial unmixing process for at least a part of the plurality of regions of the sample during the partial unmixing period is performed in parallel with the second image acquisition period. The dye image acquisition device according to claim 11.

13. the processing unit includes an image acquisition unit that includes an image sensor and acquires the C first fluorescent images and the D second fluorescent images, and an image processing unit that performs the partial unmixing process and the unmixing process. The dye image obtaining apparatus according to claim 7 or 8.

14. the processing unit includes an image acquisition unit that includes an image sensor and acquires the C first fluorescent images and the D second fluorescent images, and a server that is connected to the image acquisition unit via a network and performs the partial unmixing process and the unmixing process. The dye image obtaining apparatus according to claim 7 or 8.

15. Computer, a first image acquisition step of irradiating a sample with C (C is an integer of 1 or more) excitation light beams having a first wavelength distribution, and acquiring the C first fluorescence images using an image acquisition unit; a second image acquiring step of irradiating a sample with D (D is an integer of 1 or more) excitation light beams having a second wavelength distribution, and acquiring the D second fluorescence images using the image acquiring unit, after the first image acquiring step; a partial unmixing step of performing a partial unmixing process on the C first fluorescence images to generate K pieces of first dye data (K is an integer of 2 or more and C+D or less) partially indicating distributions for each of the K dyes; an unmixing step of generating the K pieces of second dye data partially showing the distribution of each of the K dyes from the D second fluorescence images, and performing an unmixing process of generating the K dye images based on the K pieces of first dye data and the K pieces of second dye data; At least a part of the partial unmixing process in the partial unmixing step is executed in parallel with the first image acquisition step or the second image acquisition step. Dye image acquisition program.

Images