Information acquisition method, program, storage medium, image acquisition device, and information acquisition device

Abstract

To acquire information on fluorescence intensity, the reproducibility of which is ensured, in a fluorescence image having morphological information and a fluorescence image having functional information of spots arranged on an array plate.SOLUTION: An information acquisition method includes: a fluorescence image acquisition step; an azimuth correction amount acquisition step; an azimuth-corrected image acquisition step; a template mask information acquisition step; a first translational correction amount acquisition step; a second translational correction amount acquisition step; and a fluorescence intensity acquisition step.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present invention relates to an information acquisition method, a program, a storage medium, an image acquisition device, and an information acquisition device.

Background Art

[0002] Array plates such as protein arrays, peptide arrays, and DNA arrays in which a large number of substances such as proteins, peptides, and nucleic acids are spot-fixed on a substrate such as glass or plastic are known. By using an array plate, the interaction between a large number of fixed substances and substances in a sample can be observed at once. Therefore, the interaction between various liquid samples including biological samples such as blood, cell extracts, saliva, and interstitial fluid and a large number of substances is comprehensively analyzed.

[0003] In the measurement step of the array plate, a method is known in which a spot where an interaction of interest has occurred is selectively fluorescently labeled to obtain optical information. As a measuring device for obtaining optical information, a microarray scanner, a plate reader, etc. are known. Some of these measuring devices have a function for exciting a fluorescent dye at a plurality of wavelengths, and in such a measuring device, a fluorescent image of the array plate at each excitation wavelength is acquired. On the other hand, in the analysis step of the array plate, a method is known in which fluorescence information of each spot is acquired using a template mask in which an analysis area is defined for the fluorescent image of the array plate. In Patent Document 1, the positional deviation between the template mask and the spots of the fluorescent image is automatically adjusted, and information regarding the fluorescence intensity at each spot is acquired.

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0005] However, in Patent Document 1, when the fluorescence intensity of a large number of spots on the array plate is not sufficient, it becomes difficult to adjust the position of the template mask, and thus it also becomes difficult to analyze the fluorescence information of each spot.

[0006] That is, using a measuring device such as a plate reader capable of acquiring two-wavelength fluorescence images, a first fluorescence image captured by irradiating a first primary light having one wavelength is acquired, and this is used as an image for confirming the shape of the spots and adjusting the position of the template mask. A second fluorescence image captured by irradiating a second primary light having a wavelength different from that of the first primary light is acquired, and this can be used as an image for acquiring functional information. However, there may be a case where the fluorescence intensity of a large number of spots on the array plate in the second fluorescence image is not sufficient. In such a case, it is difficult to adjust the position of the template mask. Also, when there is a misalignment between the first fluorescence image and the second fluorescence image, it is difficult to accurately analyze the fluorescence information of each spot with the template mask aligned with the first fluorescence image.

[0007] An object of the present invention is to provide an information acquisition method and an information acquisition device for acquiring information on fluorescence intensity with guaranteed reproducibility in a fluorescence image having morphological information of spots on an array plate and a fluorescence image having functional information.

Means for Solving the Problems

[0008] The present invention is an information acquisition method for acquiring information on the fluorescence intensity of spots arranged on an array plate, comprising: a fluorescence image acquisition step of acquiring a first fluorescence image captured by irradiating a first primary light to an array plate provided with a plurality of arranged spots, and a second fluorescence image captured by irradiating a second primary light having a wavelength different from that of the first primary light to acquire the functional information of the spots; Based on the first fluorescence image, an azimuth correction amount acquisition step of acquiring information regarding the azimuth correction amount within the image plane of the first fluorescence image and the second fluorescence image; An azimuth correction image acquisition step of acquiring a first azimuth correction image obtained by correcting the azimuth of the first fluorescence image and a second azimuth correction image obtained by correcting the azimuth of the second fluorescence image based on the information regarding the azimuth correction amount; A template mask information acquisition step of acquiring information regarding a template mask including a plurality of first regions arranged corresponding to each of the plurality of spots, wherein a reference position and a relative position defining an outer edge with respect to the reference position are determined so that one of the plurality of spots does not overlap with another adjacent spot; A first translation correction amount acquisition step of acquiring information regarding a first translation correction amount for the plurality of first regions included in the first azimuth correction image by adjusting the relative position of the template mask with respect to the first azimuth correction image; A second translation correction amount acquisition step of acquiring a second translation correction amount for the plurality of first regions included in the second azimuth correction image by adjusting the relative position of the template mask with respect to the second azimuth correction image based on the information regarding the first translation correction amount for the plurality of first regions included in the first azimuth correction image; A fluorescence intensity acquisition step of acquiring information regarding the fluorescence intensity corresponding to the plurality of spots in the first fluorescence image based on the information regarding the first translation correction amount and the first azimuth correction image, and acquiring information regarding the fluorescence intensity corresponding to the plurality of spots in the second fluorescence image based on the information regarding the second translation correction amount and the second azimuth correction image; The above problems are solved by providing an information acquisition method including the above steps.

Effect of the Invention

[0009] According to the present invention, it is possible to provide an information method and an information acquisition apparatus for acquiring information regarding fluorescence intensity with ensured reproducibility in each of a fluorescence image having morphological information of spots on an array plate and a fluorescence image having functional information.

Brief Description of the Drawings

[0010]

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BEST MODE FOR CARRYING OUT THE INVENTION

[0011] Hereinafter, the information acquisition method according to the first embodiment of the present invention will be described with reference to the process diagram S10000 shown in FIG. 1. The information acquisition method according to the first embodiment of the present invention is an information acquisition method for a fluorescence image of an array plate provided with a plurality of arranged spots, and includes the following steps.

[0012] A first fluorescence image captured by irradiating with a first primary light and a second fluorescence image captured by irradiating with a second primary light having a wavelength different from that of the first primary light in order to acquire the function information of the spot are acquired (fluorescence image acquisition step S100). Based on the first fluorescence image, information regarding the azimuth correction amount in the image plane of the first fluorescence image and the second fluorescence image is acquired (azimuth correction amount acquisition step S200). Based on the information regarding the azimuth correction amount, a first azimuth correction image obtained by correcting the azimuth of the first fluorescence image and a second azimuth correction image obtained by correcting the azimuth of the second fluorescence image are acquired (azimuth correction image acquisition step S300). A reference position and a relative position defining an outer edge with respect to the reference position are determined so that one of the plurality of spots does not overlap with another adjacent spot, and information regarding a template mask including a plurality of first regions arranged corresponding to each of the plurality of spots is acquired (template mask information acquisition step S400). By adjusting the relative position of the template mask with respect to the first azimuth-corrected image, information regarding the first translation correction amount for a plurality of first regions included in the first azimuth-corrected image is obtained (first translation correction amount acquisition step S500). By adjusting the relative position of the template mask with respect to the second azimuth-corrected image based on the information regarding the first translation correction amount, the second translation correction amount for a plurality of first regions included in the second azimuth-corrected image is obtained (second translation correction amount acquisition step S600). Based on the information regarding the first translation correction amount and the first azimuth-corrected image, information regarding the fluorescence intensity corresponding to a plurality of spots in the first fluorescence image is obtained, and based on the information regarding the second translation correction amount and the second azimuth-corrected image, information regarding the fluorescence intensity corresponding to a plurality of spots in the second fluorescence image is obtained (fluorescence intensity acquisition step S700). Details of each step will be described later.

[0013] <Array plate> The array plate has spots containing various types of biological substances on a substrate and is used for comprehensive analysis of a specimen. The array plate may also be referred to as a microchip, microarray, protein chip, DNA chip, etc. As the array plate, commercially available ones can be used. Microarray plates are sold by companies such as Agilent Technologies, Inc. and RayBiotech, Inc. Alternatively, referring to known methods, the array plate is fabricated. That is, the array plate is fabricated by immobilizing a desired biological substance on one surface of a suitable substrate. Here, the immobilization can also be referred to as adsorption and includes immobilization by hydrophobic interaction, electrostatic interaction, van der Waals interaction, hydrogen bond, and covalent bond. The substrate is preferably transparent. Examples of the substrate material include glass, synthetic quartz, quartz, borosilicate glass, etc. Alternatively, examples of the substrate material include resins such as polystyrene, polypropylene, (meth)acrylic resin, polyamide, polyimide, melamine, ABS, polyphenylene oxide urethane, silicone, epoxy, and polydimethylsiloxane.

[0014] <First fluorescence image> The first fluorescence image is an image captured by irradiating the first primary light. The first fluorescence image is the morphological information of the spots on the array plate. For example, when the measurement target is a protein and a plurality of types of proteins are spotted in an array on the array plate, all the spotted proteins are tagged with GST-TAG, and the GST-TAG is labeled with a phosphor that fluoresces with respect to light of the first wavelength. When this is excited with light of the first wavelength, fluorescence from all the proteins can be obtained, and as a result, morphological information can be obtained. Generally, protein spots are transparent to visible light and difficult to observe with a normal optical microscope, so fluorescence labeling is an effective method.

[0015] <Second fluorescence image> The second fluorescence image is an image having the functional information of the spots on the array plate captured by irradiating the first primary light and the second primary light having a different wavelength. By reacting the array plate with a biological sample, a part of the spotted protein is phosphorylated, and the phosphorylated protein is labeled with a phosphor that fluoresces with respect to light of the second wavelength. When this is excited with light of the second wavelength, fluorescence from the phosphorylated protein can be obtained, and as a result, functional information (phosphorylation information) can be obtained.

[0016] In the present specification, the light of the first wavelength may be paraphrased as the first primary light of the first wavelength, the excitation light of the first wavelength, or the first primary light. Similarly, the light of the second wavelength may be paraphrased as the second primary light of the second wavelength, the excitation light of the second wavelength, or the second primary light. To implement the information acquisition method of this embodiment, an image acquisition apparatus is used, which includes an optical system including an irradiation unit that irradiates primary light onto the array plate and a light collection unit that collects secondary light from the spot, a placement unit on which the array plate is placed, a scanning unit that relatively moves the placement unit and the optical system, a control unit that controls the scanning by the scanning unit and the irradiation timing of the optical system, and a computer that executes a program for executing each step of the information acquisition method of this embodiment.

[0017] The above image acquisition apparatus will be described with reference to FIGS. 15 to 17. FIG. 15 is a diagram showing a schematic configuration of an image acquisition apparatus 101 for implementing the information acquisition method according to this embodiment. The image acquisition apparatus 101 includes an apparatus control unit 102, a placement unit 121 on which an array plate 107 is placed, a scanning unit 112-1 that scans the optical system, a scanning unit 112-2 that scans the placement unit 121, an irradiation unit 122 that irradiates primary light onto the array plate 107, a light collection unit 123 that collects secondary light from the array plate 107, and a computer 105. The irradiation unit 122 includes a lens 115-1, a dichroic mirror 114, a half mirror 116, and an objective lens 115-3. The irradiation unit 122 is optically coupled to a light source 110. Note that the light source 110 is composed of an excitation light source. The image acquisition apparatus 101 may include the light source 110. The light collection unit 123 includes a filter 113, a lens 115-2, a dichroic mirror 114, a half mirror 116, and an objective lens 115-3. The optical system 130 includes the irradiation unit 122 and the light collection unit 123. The irradiation unit 122 and the light collection unit 123 share a part of the optical system 130 including a dichroic mirror 114, a half mirror 116, and an objective lens 115-3 as elements in the overlapping region of the two dashed rectangles in FIG. 15. That is, the optical system 130 includes a filter 113, a lens 115-1, a lens 115-2, an objective lens 115-3, a dichroic mirror 114, and a half mirror 116. The irradiation unit 122 is optically coupled to the light source 110 via a lens 115-1 included in the irradiation unit 122. The light collection unit 123 is optically coupled to the photodetection element 111 via a lens 115-2 included in the light collection unit 123. The computer 105 has an information acquisition device 11000 that executes a program for executing each step of S100 to S700 of the information acquisition method S10000 of the present embodiment, and a device control unit 102 that controls the placement unit 121, the irradiation unit 122, and the light collection unit 123 that constitute the optical imaging system. That is, the information acquisition device 11000 is an element that constitutes the image acquisition device 101. The array plate 107 is paraphrased as a target that provides an image to be acquired by the image acquisition device 101.

[0018] FIG. 16 is a diagram showing a schematic configuration of an image acquisition device 101 for implementing the information acquisition method according to the present embodiment in a form different from FIG. 15. As shown in FIG. 16, the image acquisition device 101 includes a placement unit 121 on which the array plate 107 is placed, an irradiation unit 122 that irradiates the array plate with primary light for imaging a first fluorescent image and a second fluorescent image, a light source 110 that is optically coupled to the irradiation unit and emits primary light of two different wavelengths by switching, a light collection unit 123 that receives and detects secondary light including fluorescence, an optical system 130 including the irradiation unit 122 and the light collection unit 123, a placement unit 121, a scanning unit 112-2 that scans the placement unit 121, a device control unit 102 that controls the scanning by the scanning unit and the irradiation timing of the optical system, and an information acquisition device 11000 for executing a program for executing the method of the present embodiment. The pixel pitch of the fluorescent image of the array plate 107 to be acquired is 3 μm to 30 μm.

[0019] The light collection unit 123 may be configured to include a photodetection element 111 such as a digital camera, a fluorescence microscope, a photomultiplier tube (PMT), a photosensor, or the like. The device control unit 102 is composed of an FPGA, a CPU, a memory, embedded software, etc. The first fluorescent image 103 and the second fluorescent image 104 acquired by the image acquisition device 101 are stored in the internal memory of the device control unit 102 in a file format such as binary data or 16-bit grayscale Tiff format. The information acquisition device 11000 is composed of a general-purpose personal computer or the like, reads the first fluorescent image 103 and the second fluorescent image 104 from the device control unit 102, and executes a program for executing the information acquisition method of this embodiment. The first fluorescent image 103, the second fluorescent image 104, and their analysis results may be displayed on the user interface 106.

[0020] FIG. 17 is a diagram showing a schematic configuration of an information acquisition device 11000 for use in the information acquisition method according to the first embodiment. As shown in FIG. 17, the information acquisition device 11000 includes a fluorescent image acquisition unit 1051, an azimuth correction amount acquisition unit 1052, an azimuth correction image acquisition unit 1053, a template mask information acquisition unit 1054, a first translation correction amount acquisition unit 1055, a second translation correction amount acquisition unit 1056, and a fluorescence intensity acquisition unit 1057. The fluorescent image acquisition unit 1051, the azimuth correction amount acquisition unit 1052, the azimuth correction image acquisition unit 1053, the template mask information acquisition unit 1054, the first translation correction amount acquisition unit 1055, the second translation correction amount acquisition unit 1056, and the fluorescence intensity acquisition unit 1057 operate on an arithmetic unit (not shown) provided in the computer 105. Each of the elements 1051 to 1057 shown in FIG. 17 constituting the information acquisition device 11000 functionally operates on an arithmetic unit such as a CPU, a GPU, or an ASIC (not shown) provided in the computer 105. That is, it is equivalently said that it is executed as software. The fluorescence image acquisition unit 1051, the azimuth correction amount acquisition unit 1052, the azimuth correction image acquisition unit 1053, the template mask information acquisition unit 1054, the first translation correction amount acquisition unit 1055, the second translation correction amount acquisition unit 1056, and the fluorescence intensity acquisition unit 1057 are implemented to be able to provide and read information from each other via the signal line 1050. The signal line 1050 can be replaced not only by the signal line on the arithmetic processor but also by the system bus on the motherboard, the Internet line, etc. The fluorescence image acquisition unit 1051 acquires a first fluorescence image captured by irradiating an array plate provided with a plurality of arranged spots with first primary light, and a second fluorescence image captured by irradiating the first primary light with second primary light having a different wavelength in order to acquire the function information of the spots. The azimuth correction amount acquisition unit 1052 acquires information regarding the azimuth correction amount within the image plane of the first fluorescence image and the second fluorescence image based on the first fluorescence image. The azimuth correction image acquisition unit 1053 acquires a first azimuth correction image obtained by correcting the azimuth of the first fluorescence image and a second azimuth correction image obtained by correcting the azimuth of the second fluorescence image based on the information regarding the azimuth correction amount. The template mask information acquisition unit 1054 acquires information regarding a template mask that defines a reference position and a relative position that defines an outer edge with respect to the reference position so as not to overlap with other spots that overlap and are adjacent to one of the plurality of spots, and includes a plurality of first regions arranged corresponding to the plurality of spots. The first translation correction amount acquisition unit 1055 acquires information regarding the first translation correction amount for the plurality of first regions included in the first azimuth correction image by adjusting the relative position of the template mask with respect to the first azimuth correction image. The second translation correction amount acquisition unit 1056 acquires the second translation correction amount for the plurality of first regions included in the second azimuth correction image by adjusting the relative position of the template mask with respect to the second azimuth correction image based on the information regarding the first translation correction amount for the plurality of first regions included in the first azimuth correction image. The fluorescence intensity acquisition unit 1057 acquires information on the fluorescence intensity corresponding to the plurality of spots in the first fluorescence image based on the information on the first translation correction amount and the first azimuth angle corrected image, and acquires information on the fluorescence intensity corresponding to the plurality of spots in the second fluorescence image based on the information on the second translation correction amount and the second azimuth angle corrected image.

[0021] According to the present embodiment, it is possible to configure as an information acquisition device that operates a series of processes from fluorescence detection to image analysis and outputs the analyzed data.

[0022] In the present embodiment, it is also possible to further include a step of analyzing another fluorescence image related to the functional information of the array plate corresponding to the second fluorescence image. The present invention also provides a program for causing a computer to execute each of the above-described steps, and a computer-readable recording medium on which the program is recorded.

[0023] <Details of the First Embodiment> With reference to FIG. 1, the information acquisition method S10000 according to the first embodiment will be described in more detail. The first fluorescence image and the second fluorescence image have been imaged in advance by an image acquisition device such as a fluorescence plate reader.

[0024] <Fluorescence Image Acquisition Step S100> In this step, a first fluorescence image captured by irradiating a first primary light on an array plate provided with a plurality of arranged spots, and a second fluorescence image captured by irradiating a second primary light having a wavelength different from that of the first primary light to acquire the functional information of the spots are acquired. The first fluorescence image and the second fluorescence image can be acquired using an image analysis PC. On the array plate, multiple types of proteins are spotted in an array. A schematic diagram of the array plate is shown in Fig. 2. The diameter of one spot is about 100 μm, and the interval between adjacent spots is about 40 μm. The spots are configured in units of 9×9 blocks, and the blocks are arranged in a 3×6 matrix. The spots on the array plate of this embodiment are fluorescently labeled with Alexa Flour 680. Some of the protein spots are phosphorylated, and only the phosphorylated protein spots are fluorescently labeled with Alexa Flour 790. The fluorescence image obtained by exciting this array plate with light of a wavelength of 670 nm is the first fluorescence image. The fluorescence image obtained by exciting this array plate with light of a wavelength of 780 nm is the second fluorescence image.

[0025] It is desirable for the image acquisition device to be equipped with first-wavelength excitation light with a wavelength of 670 nm for exciting protein spots fluorescently labeled with Alexa Flour 680. Also, it is desirable for the image acquisition device to be equipped with second-wavelength excitation light with a wavelength of 780 nm for exciting protein spots fluorescently labeled with Alexa Flour 790.

[0026] The fluorescence image of the array plate obtained in step S100 is shown in Fig. 3. Here, one block is enlarged and displayed. In the first fluorescence image, all spots are measured uniformly brightly in order to obtain morphological information by irradiating the first primary light. In the second fluorescence image, only the phosphorylated protein spots are measured brightly in order to obtain functional information by irradiating the second primary light.

[0027] <Azimuth correction amount acquisition step S200> This step is a step of obtaining information regarding the azimuth correction amount within the image plane of the first fluorescence image and the second fluorescence image obtained in step S100. The method for realizing step S200 will be described with reference to Fig. 4. By averaging the first fluorescence image obtained in Engineering S100 in the x-axis direction (horizontal direction of the image) and the y-axis direction (vertical direction of the image), one-dimensional x-axis averaged data and y-axis averaged data are calculated. Consider the inclination of the array direction of the array plate with respect to the x-axis and y-axis defined from the image. When the array direction of the array plate has a predetermined inclination with respect to the x-axis and y-axis defined from the image, the peak values of the intensities of the x-axis averaged data and the y-axis averaged data are lower compared to the case where the array direction of the array plate coincides with the x-axis and y-axis (Fig. 4(a)). On the other hand, when the array direction of the array plate coincides with the x-axis and y-axis, the peak values of the intensities of the x-axis averaged data and the y-axis averaged data take the maximum value (Fig. 4(b)). From this perspective, a temporary azimuth correction amount is given to the fluorescence image to create an azimuth correction image, and an azimuth correction amount is obtained such that the amplitudes of the x-axis averaged data and the y-axis averaged data are maximized for the created azimuth correction image. For example, the angular misalignment within the image plane of the first fluorescence image or the second fluorescence image depends on the scanning accuracy of the image acquisition device and the manufacturing accuracy of the array plate, but is about ±1 degree. In this embodiment, an azimuth correction image is generated while changing the temporary azimuth correction amount by 0.1 degrees each time, and an azimuth correction amount is obtained such that the amplitudes of the x-axis averaged data and the y-axis averaged data are maximized.

[0028] <Azimuth Correction Image Acquisition Step S300> In this step, a first azimuth correction image and a second azimuth correction image are obtained by correcting the first fluorescence image and the second fluorescence image with the azimuth correction amount obtained in the azimuth correction amount acquisition step S200. For the azimuth correction of the fluorescence image, an affine transformation is used. The affine transformation is performed by any interpolation method such as nearest neighbor interpolation, linear interpolation, bilinear interpolation, or bicubic interpolation.

[0029] <Template Mask Information Acquisition Step S400> This step is a step of obtaining information regarding a template mask including a plurality of first regions arranged corresponding to a plurality of spots, where a reference position and a relative position defining an outer edge with respect to the reference position are determined so as not to overlap with other spots adjacent to and overlapping one of the plurality of spots. Information about the template mask is input based on the design information of the array plate using a pre-prepared user interface. Alternatively, a file created in advance based on the design information of the array plate and stored in the memory is read by an analysis PC. The shape of the template mask is shown in FIG. 5. In the template mask, a first region, which is the region of interest, is arranged in a 9×9 matrix. The shape of the first region is a circle with the same radius as the spot in accordance with the shape of the spot on the array plate. Alternatively, the shape of the first region can be set to be slightly larger than the radius of the spot, elliptical in accordance with the shape of the spot, or any other shape that does not overlap with other adjacent first regions. Also, the interval (pitch) between the first regions of the template mask may be determined from the periods of the x-axis averaged data and the y-axis averaged data obtained in the step S200.

[0030] <First translation correction amount acquisition step S500> This step is a step of obtaining information regarding the first translation correction amount for a plurality of first regions included in the first azimuth correction image by adjusting the relative position of the template mask with respect to the first azimuth correction image. The first translation correction amount acquisition step S500 of the present embodiment will be described with reference to FIG. 6. Using the x-axis averaged data and the y-axis averaged data in the first azimuth correction image obtained in the step S300, the coordinates of each block on the first azimuth correction image are set. The relative position of the template mask with respect to a certain block is changed in the x-axis direction and the y-axis direction within a preset scanning range (first scanning range), and the sum of the fluorescence luminance values of the first azimuth correction image that overlaps with a plurality of first regions of the template mask at each relative position is calculated. The relative position at which the sum of the fluorescence luminance values is maximized is obtained as the first translation correction amount in the x-axis direction and the y-axis direction. As a result, before the scanning, the spot positions of the template mask and the block were shifted as shown in FIG. 6(a), but they coincide as shown in FIG. 6(b) by the scanning. For example, when the spot has a diameter of 100 μm, the scanning range of the template mask is preset to 100 μm in each of the x-axis direction and the y-axis direction with respect to the x-axis averaged data and the y-axis averaged data in the first azimuth correction image obtained in step S300. This process is performed for a total of 18 template masks for each block.

[0031] <Second translation correction amount acquisition step S600> This step is a step of obtaining a second translation correction amount for a plurality of first regions included in the second azimuth correction image by adjusting the relative position of the template mask with respect to the second azimuth correction image based on the information regarding the first translation correction amount.

[0032] For a certain block on the second azimuth correction image, using the first translation correction amount obtained in step S500 as the reference position of the relative position of the template mask, the relative position of the template mask is changed within a second scanning range that is more limited than the first scanning range. The sum of the fluorescence luminance values of the second azimuth correction image overlapping with the plurality of first regions of the template mask at each relative position is calculated. The relative position at which the sum of the fluorescence luminance values is maximized is obtained as the second translation correction amount in the x-axis direction and the y-axis direction. As a result, before scanning, the spot positions of the template mask and the block were shifted as shown in Fig. 7(a), but they coincide as shown in Fig. 7(b) by the scanning. For example, when the imaging positions on the array plates of two wavelengths of the image acquisition device are shifted, a relative positional shift occurs between the first fluorescence image and the second fluorescence image. According to the present embodiment, such a shift can be corrected even in an image including such a shift. When there is no positional shift or optimal position shift between the first azimuth correction image and the second azimuth correction image, the second translation correction amount may be set to the same value as the first translation correction amount. Also, when the positional shift or optimal position shift between the first azimuth correction image and the second azimuth correction image takes a fixed value, the second translation correction amount may be a value obtained by adding a fixed value to the first translation correction amount. This process is performed for a total of 18 template masks for each block.

[0033] <Fluorescence intensity acquisition step S700> In this step, based on the information regarding the first translation correction amount and the first azimuth angle corrected image, information regarding the fluorescence intensity corresponding to a plurality of spots in the first fluorescence image is acquired, and based on the information regarding the second translation correction amount and the second azimuth angle corrected image, information regarding the fluorescence intensity corresponding to a plurality of spots in the second fluorescence image is acquired.

[0034] The information regarding the fluorescence intensity includes information (signal) regarding the fluorescence intensity in a plurality of first regions of the template mask, and information (background) regarding the fluorescence intensity in a plurality of second regions defined so as not to overlap with the first regions of other spots adjacent outside the first region. The shape of the second region is shown in FIG. 8 as an example. The second region corresponding to the first region is defined by four circles that do not overlap with a plurality of adjacent first regions and have equal distances from each other. The shape of the second region is not limited, such as a concentric circle with a radius larger than that of the first region, a rectangle, or a shape with adjacent first regions cut out.

[0035] For each spot, statistical quantities such as the average value, median value, and variance value of the signal and the background, and statistical quantities of the net signal obtained by extracting the fluorescence luminance components derived from the spot, such as the difference and ratio between the signal and the background, are calculated and output as a file together with the data related to the position of the spot.

[0036] According to the present embodiment, even when the second fluorescence image is discrete and it is difficult to specify the block coordinates, it is possible to accurately analyze the array plate. Also, even when there is a relative displacement in position between the first fluorescence image and the second fluorescence image, the position of the template mask is automatically adjusted, and the array plate can be analyzed.

[0037] <Second Embodiment> This embodiment is a modified form of the first embodiment, and is different from the first embodiment in that the azimuth correction image acquisition step S300 is performed to calculate the azimuth correction amount by weighting the peak value of the intensity of the x-axis averaged data and the peak value of the intensity of the y-axis averaged data. Due to the shape variation of the spots caused by the manufacturing accuracy of the array plate, the azimuth correction amount at which the amplitudes of the x-axis averaged data and the y-axis averaged data are maximized may be different. In such a case, in step S200, it is effective to calculate the azimuth correction amount by weighting the peak value of the intensity of the x-axis averaged data and the peak value of the intensity of the y-axis averaged data. The shape of the spot tends to be an ellipse that is long in the y-axis direction rather than circular due to the manufacturing accuracy of the array plate. In this case, since it becomes difficult to determine the peak position because the width of the peak indicating the maximum of the x-axis averaged data becomes wide, the azimuth correction amount is calculated by calculating the weighted average of the x-axis averaged data and the y-axis averaged data by weighting the y-axis averaged data with a sharp peak. According to this embodiment, it is possible to obtain the azimuth correction amount even when there is a variation in the spot shape due to the manufacturing accuracy of the array plate.

[0038] <The Third Embodiment> This embodiment is a modified form of the first embodiment, and is different from the first embodiment in that the second translation correction amount acquisition step S600 is performed to determine the second translation correction amount based on the first translation correction amount. The step S600 of this embodiment is also effective when there are different curvatures in the fluorescence images of two wavelengths of the plate reader. For example, since Alexa Flour 790 is excited even by a slight excitation light source of wavelength 670 nm, the second fluorescence image is affected by the excitation light of wavelength 670 nm. In order to avoid this influence, the plate reader separates the excitation wavelengths in the forward and backward paths during the scanning in the short side direction of the array plate to acquire the fluorescence image. Depending on the scanning accuracy of the scanner, there may be a misalignment of the relative positions of the scanning lines and a curvature in the reverse direction of the scanning lines between the forward and backward paths, and an image with a curvature in the reverse direction may be acquired for the fluorescence image of wavelength 670 nm and the fluorescence image of wavelength 780 nm. For example, assuming that in step S500, the first fluorescence image is convexly curved as shown in FIG. 9, the position of the template mask is adjusted and the first translation correction amount is obtained. In step S600, when the second fluorescence image is concavely curved, at the relative position of the same template mask, the first region of the template mask will be displaced with respect to the spot. As shown in FIG. 10, by determining the second translation correction amount based on the first translation correction amount, it becomes possible to correctly adjust the position of the template mask also for the second fluorescence image. According to this method, since the positional deviation of the spot caused by the curvature of the image in step S600 can be adjusted by the second translation correction amount, it is effective also for an image including such a curvature.

[0039] <Fourth Embodiment> This embodiment is a modified form of the first embodiment, and is different from the first embodiment in that the second translation correction amount acquisition step S600 is performed so as to adjust the spot positional deviation derived from the array plate also by the second translation correction amount. Even when spots are generated at different positions in the first fluorescence image for acquiring morphological information by irradiating the first primary light and the second fluorescence image for acquiring functional information by irradiating the second primary light in the process of manufacturing the array plate, the step S600 of this embodiment is effective. For example, assuming that the positions where Alexa Flour 680 and Alexa Flour 790 are fluorescently labeled are different. According to this method, since the spot positional deviation derived from this array plate in step S600 can also be adjusted by the second translation correction amount, it is effective also for an array plate including such a positional deviation.

[0040] <Fifth Embodiment> This embodiment is a modified form of the first embodiment, and is different from the first embodiment in that a noise processing step SS220 is performed after the fluorescence image acquisition step and before the azimuth angle correction amount acquisition step. In the first embodiment, it was assumed that the first fluorescence image did not contain a noise signal. However, when a noise signal is included, it may be difficult to calculate the azimuth correction amount. Unwanted signals may include the signal-to-background ratio (SN) of the scanner, dust, fluorescent dyes, etc. that accidentally adhere to or mix into the array plate. In that case, the noise removal processing step shown in FIG. 11 is effective. The method will be described in this embodiment. The noise processing step SS220 shown in FIG. 11 can be performed after the fluorescence image acquisition step and before the azimuth correction amount acquisition step. The noise removal processing step includes a filter processing step (S201), a threshold processing step (S202) for reducing the number of gradations related to pixel values, and a spot extraction processing step (S203) for extracting spots. The details of each step will be described below.

[0041] <Filter processing step S201> By performing arbitrary filter processing such as a median filter or a Gaussian filter on the first fluorescence image obtained by step S100, the influence of system noise applied to the entire fluorescence image is reduced. The filter mask size of the filter processing is set to an arbitrary size so as not to affect the general shape of the spots. For example, when the SN of the scanner is poor, noise with a high-luminance pixel size may be included in areas other than the spots. Also, low-luminance pixels may occur in the high-luminance areas inside the spots. Furthermore, the fluorescence image may include high-luminance points with a size of several pixels generated by fluorescent labeling of proteins non-specifically adsorbed outside the spot area. To remove these, filter processing is performed using a 5×5 pixel median filter.

[0042] <Threshold processing step S202> By performing threshold processing on the first fluorescence image after filter processing obtained by step S201, the number of gradations is reduced. For example, a threshold is determined to distinguish between the spot area and the area outside the spots, and the first fluorescence image is converted into a binary image. The threshold may be determined in advance by input or by an algorithm such as Otsu's binarization.

[0043] <Spot extraction processing step S203> <For the first fluorescence image after threshold processing obtained in step S202, by performing spot extraction processing, unnecessary signals are removed. The spot extraction processing step SS240 includes the region shape calculation processing step (S204), the spot determination processing step (S205), and the region removal processing step (S206) shown in FIG. 12. Details of each step will be described later.>

[0044] <Region shape calculation processing step S204> <For the first fluorescence image after threshold processing obtained in step S202, for a high-luminance region where pixels of the same luminance are adjacent, information regarding the shape of the region is acquired. For example, in a binary image, the luminance value is 0 or 1, and the region of adjacent pixels with a luminance value of 1 becomes a plurality of high-luminance regions including spots. As information regarding the shape of this high-luminance region, the length of the outer periphery of the region, the area of the region, the length in the longitudinal direction and the length in the short direction of the region, etc. are acquired.>

[0045] <Spot determination processing step S205> <Based on the information regarding the shape of the high-luminance region obtained in step S204, it is determined whether the high-luminance region is a spot or derived from other unnecessary signals. For example, if the length of the outer periphery of the region is larger than the circumference length calculated from the spot diameter in the design of the array plate, it is determined that it is not a spot. Also, whether the high-luminance region is a spot or not is judged by a determination formula such as when the circularity calculated from the length of the outer periphery and the area of the region is larger than a preset circularity, or when the ratio of the length in the longitudinal direction to the length in the short direction is larger than a preset value.>

[0046] <Region removal processing step S206> <For the high-luminance region determined not to be a spot in step S205, pixel values of a low-luminance region are given. For example, when fibrous dust as shown in FIG. 13(a) is included in the first fluorescence image binarized as an unnecessary high-luminance signal, as shown in FIG. 13(b), the high-luminance region is converted from a luminance value of 1 to a luminance value of 0.>

[0047] The noise removal processing step according to this embodiment is executed after S100 and before S200 of the first embodiment. A noise removal image with noise removed from the first fluorescence image is obtained through the noise removal processing step. The subsequent steps are the same as those of the first embodiment. In step S200, information regarding the azimuth correction amount is obtained by using the noise removal image instead of the first fluorescence image. According to this embodiment, it is possible to calculate the azimuth correction amount independent of the SN of the scanner and the state of the array plate.

[0048] <Sixth Embodiment> This embodiment is a modified form of the first embodiment. In the first embodiment, based on the first fluorescence image, information regarding the azimuth correction amount within the image plane of the first fluorescence image and the second fluorescence image is obtained. However, this embodiment is different from the first embodiment in that a composite image acquisition step of obtaining a composite image of the first fluorescence image and the second fluorescence image is performed to calculate the azimuth correction amount.

[0049] The composite image acquisition step SS260 according to the sixth embodiment will be described with reference to FIG. 14. In the first embodiment, the azimuth correction amount is calculated from the first fluorescence image. However, depending on the measurement parameters during the imaging of the fluorescence image, the contrast between the spots and outside the spots in the first fluorescence image may be low, making it difficult to calculate the azimuth correction amount. At this time, it is effective to calculate the azimuth correction amount from the composite image of the first fluorescence image and the second fluorescence image obtained in the composite image acquisition step. The composite image acquisition step can be performed after the fluorescence image acquisition step and before the azimuth correction amount acquisition step. The steps of the composite image acquisition step SS260 are shown in FIG. 14. The composite image acquisition step includes a step of obtaining the weighting of the first fluorescence image (S211), a step of obtaining the weighting of the second fluorescence image (S212), and an image composite processing step (S213). By reading the pre-set weighting values in step S211 and step S212, the synthesis ratio at the time of image synthesis is obtained. For example, the threshold value is determined by Otsu's binarization process, and the pixel value on the high luminance side is given as 1 and the pixel value on the low luminance side is given as 0. At this time, in the fluorescence image with high contrast, the spot area has a pixel value of 1 and the area outside the spot has a pixel value of 0. On the other hand, in the fluorescence image with low contrast, the pixel value is 1 regardless of whether it is inside or outside the spot area, and the variation in the in-plane distribution of the pixel value of 1 becomes large. Using the characteristics of this binarized image, it is effective to increase the weight when the variation in the in-plane distribution of the pixel value of 1 is small. In step S213, based on the obtained synthesis ratio, a synthesis method such as addition average synthesis or comparative brightness synthesis is set so that the contrast between the spot and outside the spot becomes high. The noise removal processing step in FIG. 11 may be performed on the synthesized image or the fluorescence image before and after the synthesized image acquisition step. Using the synthesized image of the first fluorescence image and the second fluorescence image obtained by the synthesized image acquisition step, information regarding the azimuth correction amount in the image plane of the first fluorescence image and the second fluorescence image is obtained. According to the present embodiment, even when the contrast between the spot and outside the spot of the first fluorescence image is low, it is possible to calculate the azimuth correction amount.

[0050] <The Seventh Embodiment> This embodiment is a modified form of the first embodiment. In the first embodiment, the azimuth correction amount was calculated from the peak values of the intensities of the x-axis averaged data and the y-axis averaged data. However, in this embodiment, it is different from the first embodiment in that the azimuth correction amount is calculated from the positions of the local peaks appearing in a matrix form in the two-dimensional frequency image. A method different from the first embodiment for realizing the step of step S200 in FIG. 1 in the seventh embodiment will be described. In the first embodiment, the azimuth correction amount was calculated from the peak values of the intensities of the x-axis averaged data and the y-axis averaged data. However, in the azimuth correction amount acquisition step, information regarding the azimuth correction amount can be obtained by performing two-dimensional frequency analysis of the first fluorescence image. In the present embodiment, the azimuth correction amount is calculated from the two-dimensional frequency image obtained by the two-dimensional Fourier transform of the first fluorescence image. The two-dimensional frequency image represents the absolute value of the complex amplitude in the frequency space after the two-dimensional Fourier transform. Specifically, the azimuth correction amount is calculated from the positions of local peaks that appear in a matrix form in the two-dimensional frequency image. In the first embodiment, it was necessary to create the azimuth correction image a plurality of times with a provisional azimuth correction amount, whereas in this embodiment, there is an advantage that it is only necessary to generate the two-dimensional frequency image once.

[0051] <Eighth Embodiment> This embodiment is a modified form of the first embodiment. In the first embodiment, the relative position of the template mask at which the fluorescence luminance value of the azimuth correction image in the region overlapping the plurality of first regions of the template mask is maximized is obtained as the translation correction amount. However, in this embodiment, it differs from the first embodiment in that the relative position of the template mask at which the fluorescence luminance value of the azimuth correction image in the region overlapping the second region in step S700 of the first embodiment is minimized is calculated as the translation correction amount. In the eighth embodiment, a method for realizing the steps corresponding to steps S500 and S600 in FIG. 1 will be described. In the first embodiment, the relative position of the template mask at which the fluorescence luminance value of the azimuth correction image in the region overlapping the plurality of first regions of the template mask is maximized is obtained as the translation correction amount. In contrast, in this embodiment, the relative position of the template mask at which the fluorescence luminance value of the azimuth correction image in the region overlapping the second region in step S700 of the first embodiment is minimized is calculated as the translation correction amount. According to this embodiment, even when the first region of the template mask is larger than the spot, it is possible to calculate the translation correction amount.

[0052] <Ninth Embodiment> This embodiment is a modified form of the first embodiment. In the first embodiment, the relative position of the template mask at which the fluorescence luminance value of the azimuth-corrected image in the region overlapping with the plurality of first regions of the template mask is maximized is obtained as the translational correction amount. However, in this embodiment, a mask image is created from the array information of the template mask, and the relative position at which the maximum value of the two-dimensional cross-correlation between the azimuth-corrected image and the mask image with the relative position shifted is calculated as the translational correction amount, which is different from the first embodiment in this regard.

[0053] A method for realizing steps S500 and S600 in FIG. 1 in the ninth embodiment will be described. In the first embodiment, the relative position of the template mask at which the fluorescence luminance value of the azimuth-corrected image in the region overlapping with the plurality of first regions of the template mask is maximized is obtained as the translational correction amount. Depending on the fluorescence image, high-luminance signals other than the spots may be included in the vicinity of the spots, and the maximum value search is affected by this. In this embodiment, a mask image is created from the array information of the template mask, and the relative position at which the maximum value of the two-dimensional cross-correlation between the azimuth-corrected image and the mask image with the relative position shifted is calculated as the translational correction amount. According to this embodiment, it is possible to reduce the influence of unnecessary high-luminance signals included in the fluorescence image and calculate the translational correction amount.

[0054] <The Tenth Embodiment> This embodiment is a modified form of the first embodiment. In the first embodiment, the translational correction amount is calculated for each template mask, but in this embodiment, after calculating the translational correction amount, a fine-tuning translational correction amount for fine-tuning each spot is calculated, which is different from the first embodiment in this regard.

[0055] A method for realizing steps S500 and S600 in FIG. 1 in the tenth embodiment will be described. In the first embodiment, the translation correction amount was calculated for each template mask. However, due to variations in the spot shape caused by the manufacturing accuracy of the array plate and image curvature caused by the scanning accuracy of the scanner, spots that do not fit correctly into the first region of the template mask occur. In this embodiment, after calculating the translation correction amount in steps S500 and S600, a fine translation correction amount for fine adjustment is calculated for each spot. The fine translation correction amount is obtained by scanning the first region so that the luminance value inside the first region of the template mask for each spot becomes maximum. According to this embodiment, even if there are variations in the spot shape or image curvature due to the scanning accuracy of the scanner, it is possible to correctly set the first region of the template mask.

[0056] <The 11th Embodiment> This embodiment is a modified form of the first to tenth embodiments. In the first to tenth embodiments, only one fluorescence image captured by irradiating the second primary light for acquiring the functional information of the spot was obtained. However, this embodiment differs from the first to tenth embodiments in that a plurality of fluorescence images corresponding to the second fluorescence image are obtained.

[0057] In the first to tenth embodiments, only one fluorescence image captured by irradiating the second primary light for acquiring the functional information of the spot was obtained. This embodiment acquires a plurality of fluorescence images corresponding to the second fluorescence image captured by irradiating the second primary light, which is a fluorescence image for acquiring the functional information of the spot. According to this embodiment, functional information other than phosphorylation information can be acquired simultaneously.

[0058] The present invention is not limited to the above embodiments, and various changes and modifications can be made without departing from the spirit and scope of the present invention. Therefore, the following claims are appended to disclose the scope of the present invention. This application claims priority based on Japanese Patent Application No. 2023-213063 filed on December 18, 2023, and incorporates herein by reference all of the descriptions thereof.

[0059] Embodiments of the present invention include the following configurations. (Configuration 1) An information acquisition method for acquiring information regarding the fluorescence intensity of spots arranged on an array plate, a fluorescence image acquisition step of irradiating an array plate provided with a plurality of arranged spots with a first primary light to obtain a first fluorescence image captured thereby, and a second fluorescence image captured by irradiating the array plate with a second primary light having a wavelength different from that of the first primary light in order to acquire the functional information of the spots; an azimuth correction amount acquisition step of acquiring information regarding the azimuth correction amount within the image plane of the first fluorescence image and the second fluorescence image based on the first fluorescence image; an azimuth correction image acquisition step of acquiring a first azimuth correction image obtained by correcting the azimuth of the first fluorescence image and a second azimuth correction image obtained by correcting the azimuth of the second fluorescence image based on the information regarding the azimuth correction amount; a template mask information acquisition step of acquiring information regarding a template mask including a plurality of first regions arranged corresponding to each of the plurality of spots, wherein a reference position and a relative position defining an outer edge with respect to the reference position are determined so that one of the plurality of spots does not overlap with another adjacent spot; a first translation correction amount acquisition step of acquiring information regarding a first translation correction amount for the plurality of first regions included in the first azimuth correction image by adjusting the relative position of the template mask with respect to the first azimuth correction image; a second translation correction amount acquisition step of acquiring a second translation correction amount for the plurality of first regions included in the second azimuth correction image by adjusting the relative position of the template mask with respect to the second azimuth correction image based on the information regarding the first translation correction amount for the plurality of first regions included in the first azimuth correction image; Based on the information regarding the first translation correction amount and the first azimuth angle correction image, information regarding the fluorescence intensity corresponding to the plurality of spots in the first fluorescence image is acquired, and based on the information regarding the second translation correction amount and the second azimuth angle correction image, information regarding the fluorescence intensity corresponding to the plurality of spots in the second fluorescence image is acquired in a fluorescence intensity acquisition step. An information acquisition method including this. (Configuration 2) After the fluorescence image acquisition step and before the azimuth angle correction amount acquisition step, a filter process, a threshold process for reducing the number of gradations related to pixel values, a spot extraction process for extracting spots, or a noise removal process for acquiring a noise removal image is further performed on the first fluorescence image in the information acquisition method of Configuration 1. (Configuration 3) After the fluorescence image acquisition step and before the azimuth angle correction amount acquisition step, a composite image acquisition step for acquiring a composite image of the first fluorescence image and the second fluorescence image is further included in the information acquisition method of Configuration 1 or 2. (Configuration 4) In the azimuth angle correction amount acquisition step, the information acquisition method according to any one of Configurations 1 to 3, wherein the information regarding the azimuth angle correction amount is acquired by performing two-dimensional frequency analysis of the first fluorescence image. (Configuration 5) In the first translation correction amount acquisition step, the step of acquiring information regarding the first translation correction amount based on the fluorescence intensity in a plurality of second regions defined so as not to overlap the first region of other spots adjacent to the outside of the first region in the template mask is included in the information acquisition method according to any one of Configurations 1 to 4. (Configuration 6) Adjusting the relative position of the template mask related to the first translation correction amount and the second translation correction amount, and adjusting the relative positions of the plurality of first regions included in the first azimuth correction image to obtain information on a plurality of first fine translation correction amounts for the first region, and adjusting the relative positions of the plurality of first regions included in the second azimuth correction image to obtain information on a plurality of second fine translation correction amounts for the first region, the method for acquiring information according to any one of Configurations 1 to 5 including a process replacing the first translation correction amount acquisition process and the second translation correction amount acquisition process. (Configuration 7) The method for acquiring information according to any one of Configurations 1 to 6, wherein the first fluorescence image includes the morphological information of the spot, and the second fluorescence image includes the functional information of the spot. (Configuration 8) A program for causing a computer to execute each process of the method for acquiring information according to any one of Configurations 1 to 7. (Configuration 9) A computer-readable recording medium on which the program of Configuration 8 is recorded. (Configuration 10) An image acquisition device including a computer that executes the program of Configuration 8, an irradiation unit that irradiates the array plate with primary light, an optical system including a light collection unit that collects secondary light from the spot, a placement unit on which the array plate is placed, a scanning unit that relatively moves the placement unit and the optical system, and a control unit that controls the scanning by the scanning unit and the irradiation timing of the optical system. (Configuration 11) The image acquisition device according to Configuration 10, further including a light source optically coupled to the irradiation unit. (Configuration 12) The image acquisition device according to Configuration 11, wherein the light source emits light by switching between the first primary light and the second primary light. (Configuration 13) An information acquisition device for acquiring information on the fluorescence intensity of spots arranged on an array plate, For an array plate provided with a plurality of arranged spots, a first fluorescence image captured by irradiating a first primary light, and a fluorescence image acquisition unit that acquires a second fluorescence image captured by irradiating a second primary light having a wavelength different from that of the first primary light in order to acquire functional information of the spots, An azimuth correction amount acquisition unit that acquires information regarding an azimuth correction amount within the image plane of the first fluorescence image and the second fluorescence image based on the first fluorescence image, An azimuth correction image acquisition unit that acquires a first azimuth correction image obtained by correcting the azimuth of the first fluorescence image and a second azimuth correction image obtained by correcting the azimuth of the second fluorescence image based on the information regarding the azimuth correction amount, A template mask information acquisition unit that acquires information regarding a template mask including a plurality of first regions arranged corresponding to each of the plurality of spots, wherein a reference position and a relative position defining an outer edge with respect to the reference position are determined so that one of the plurality of spots does not overlap with another adjacent spot, A first translation correction amount acquisition unit that acquires information regarding a first translation correction amount for the plurality of first regions included in the first azimuth correction image by adjusting the relative position of the template mask with respect to the first azimuth correction image, A second translation correction amount acquisition unit that acquires a second translation correction amount for the plurality of first regions included in the second azimuth correction image by adjusting the relative position of the template mask with respect to the second azimuth correction image based on the information regarding the first translation correction amount for the plurality of first regions included in the first azimuth correction image, A fluorescence intensity acquisition unit that acquires information regarding the fluorescence intensity corresponding to the plurality of spots in the first fluorescence image based on the information regarding the first translation correction amount and the first azimuth correction image, and acquires information regarding the fluorescence intensity corresponding to the plurality of spots in the second fluorescence image based on the information regarding the second translation correction amount and the second azimuth correction image, An information acquisition device including the above.

Explanation of Reference Numerals

[0060] S10000: Information acquisition method 11000: Information acquisition device 101: Image acquisition device 102: Device control unit 103: First fluorescence image 104: Second fluorescence image 105: Computer 1051: Fluorescence image acquisition unit 1052: Azimuth correction amount acquisition unit 1053: Azimuth correction image acquisition unit 1054: Template mask information acquisition unit 1055: First translation correction amount acquisition unit 1056: Second translation correction amount acquisition unit 1057: Fluorescence intensity acquisition unit 106: User interface 107: Array plate 110: Light source 111: Photodetector element 112: Scanning unit 113: Filter 114: Dichroic mirror 115: Lens 116: Half mirror 121: Mounting unit 122: Irradiation unit 123: Light collection unit 130: Optical system

Claims

1. 1. A method for acquiring information regarding fluorescence intensity of spots arranged on an array plate, comprising: a fluorescence image acquiring step of acquiring a first fluorescence image captured by irradiating a first primary light onto an array plate having a plurality of arranged spots, and a second fluorescence image captured by irradiating a second primary light having a wavelength different from that of the first primary light in order to acquire functional information of the spots; an azimuth angle correction amount acquisition step of acquiring information regarding an azimuth angle correction amount within an image plane of the first fluorescent image and the second fluorescent image based on the first fluorescent image; an azimuth angle-corrected image acquisition step of acquiring a first azimuth angle-corrected image obtained by correcting the azimuth angle of the first fluorescent image based on information about the amount of azimuth angle correction, and a second azimuth angle-corrected image obtained by correcting the azimuth angle of the second fluorescent image; a template mask information acquiring step of acquiring information about a template mask including a reference position and a relative position that defines an outer edge with respect to the reference position so as to overlap one of the plurality of spots and not overlap another adjacent spot, the template mask including a plurality of first regions that are arranged corresponding to each of the plurality of spots; a first translational correction amount acquisition step of acquiring information regarding a first translational correction amount for the plurality of first regions included in the first azimuth angle corrected image by adjusting a relative position of the template mask with respect to the first azimuth angle corrected image; a second translation correction amount acquisition step of acquiring second translation correction amounts for the plurality of first regions included in the second azimuth angle corrected image by adjusting a relative position of the template mask with respect to the second azimuth angle corrected image based on information on first translation correction amounts for the plurality of first regions included in the first azimuth angle corrected image; a fluorescence intensity acquiring step of acquiring information about fluorescence intensity corresponding to the plurality of spots of the first fluorescence image based on information about the first translational correction amount and the first azimuth angle-corrected image, and acquiring information about fluorescence intensity corresponding to the plurality of spots of the second fluorescence image based on information about the second translational correction amount and the second azimuth angle-corrected image; Methods for obtaining information, including:

2. 2. The information acquiring method according to claim 1, further comprising, after the fluorescence image acquiring step and before the azimuth angle correction amount acquiring step, a noise removal processing step of performing a filter process, a threshold process for reducing a number of gradations related to pixel values, or a spot extraction process for extracting spots on the first fluorescence image, or a noise removal processing step of acquiring a noise-removed image.

3. 2. The information acquisition method according to claim 1, further comprising a composite image acquisition step of acquiring a composite image of the first fluorescent image and the second fluorescent image after the fluorescent image acquisition step and before the azimuth angle correction amount acquisition step.

4. The information acquiring method according to claim 1 , wherein in the azimuth angle correction amount acquiring step, information regarding the azimuth angle correction amount is acquired by performing a two-dimensional frequency analysis of the first fluorescent image.

5. 2. The information acquiring method according to claim 1, wherein the first translational correction amount acquiring step comprises a step of acquiring information about the first translational correction amount based on fluorescence intensity in a plurality of second areas defined outside the first area in the template mask so as not to overlap the first area of ​​another adjacent spot.

6. 2. The information acquisition method according to claim 1, further comprising: a step of adjusting a relative position of a template mask related to the first translational correction amount and the second translational correction amount, and adjusting the relative positions of the plurality of first regions included in the first azimuth-corrected image to acquire information related to a plurality of first fine translational correction amounts for the first region, and adjusting the relative positions of the plurality of first regions included in the second azimuth-corrected image to acquire information related to a plurality of second fine translational correction amounts for the first region, the information acquisition method including a step of replacing the first translational correction amount acquisition step and the second translational correction amount acquisition step.

7. The information acquiring method according to claim 1 , wherein the first fluorescent image includes morphological information of the spots, and the second fluorescent image includes functional information of the spots.

8. A program for causing a computer to execute each step of the information acquisition method according to any one of claims 1 to 7.

9. A computer-readable recording medium on which the program according to claim 8 is recorded.

10. An image acquisition device having a computer that executes the program described in claim 8, an optical system including an irradiation unit that irradiates primary light onto the array plate and a light collection unit that collects secondary light from the spot, a mounting unit on which the array plate is placed, a scanning unit that moves the mounting unit and the optical system relative to each other, and a control unit that controls scanning by the scanning unit and the irradiation timing of the optical system.

11. The image capture device of claim 10 , further comprising a light source optically coupled to the illumination portion.

12. The image acquisition device according to claim 11 , wherein the light source switches between emitting the first primary light and emitting the second primary light.

13. An information acquisition device for acquiring information regarding the fluorescence intensity of spots arranged on an array plate, comprising: a fluorescence image acquisition unit that acquires a first fluorescence image captured by irradiating an array plate having a plurality of arranged spots with a first primary light, and a second fluorescence image captured by irradiating the array plate with a second primary light having a wavelength different from that of the first primary light in order to acquire functional information of the spots; an azimuth angle correction amount acquisition unit that acquires information regarding an azimuth angle correction amount within an image plane of the first fluorescent light image and the second fluorescent light image based on the first fluorescent light image; an azimuth angle-corrected image acquisition unit that acquires a first azimuth angle-corrected image obtained by correcting the azimuth angle of the first fluorescent image based on information about the azimuth angle correction amount, and a second azimuth angle-corrected image obtained by correcting the azimuth angle of the second fluorescent image based on information about the azimuth angle correction amount; a template mask information acquisition unit that acquires information about a template mask including a reference position and a relative position that defines an outer edge with respect to the reference position so as to overlap one of the plurality of spots and not overlap another adjacent spot, the template mask including a plurality of first regions that are arranged corresponding to each of the plurality of spots; a first translational correction amount acquisition unit that acquires information regarding a first translational correction amount for the plurality of first regions included in the first azimuth angle corrected image by adjusting a relative position of the template mask with respect to the first azimuth angle corrected image; a second translational correction amount acquisition unit that acquires second translational correction amounts for the plurality of first regions included in the second azimuth angle corrected image by adjusting a relative position of the template mask with respect to the second azimuth angle corrected image based on information on first translational correction amounts for the plurality of first regions included in the first azimuth angle corrected image; a fluorescence intensity acquisition unit that acquires information about fluorescence intensity corresponding to the plurality of spots of the first fluorescence image based on information about the first translational correction amount and the first azimuth angle-corrected image, and acquires information about fluorescence intensity corresponding to the plurality of spots of the second fluorescence image based on information about the second translational correction amount and the second azimuth angle-corrected image; An information acquisition device comprising:

Images