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

Abstract

Information relating to fluorescence intensity, with guaranteed repeatability, is acquired in a fluorescence image including morphological information of spots arranged on an array plate, and a fluorescence image including functional information, by means of the method of the present invention, which includes a fluorescence image acquisition step, an azimuth angle correction amount acquisition step, an azimuth angle corrected image acquisition step, a template mask information acquisition step, a first translation correction amount acquisition step, a second translation correction amount acquisition step, and a fluorescence intensity acquisition step.

Description

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

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

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

[0003] In the process of measuring an array plate, a method is known in which spots where an interaction of interest has occurred are selectively fluorescently labeled to obtain optical information. Known measurement devices for obtaining optical information include microarray scanners and plate readers. Some of these measurement devices are known to have the function of exciting fluorescent dyes at multiple wavelengths, and such measurement devices obtain fluorescent images of the array plate at each excitation wavelength. Meanwhile, in the process of analyzing an array plate, a method is known in which fluorescent information of each spot is obtained using a template mask in which an analysis area is defined for the fluorescent image of the array plate. Patent Document 1 automatically adjusts the positional deviation between the template mask and the spots in the fluorescent image to obtain information about the fluorescent intensity of each spot.

[0004] JP 2011-182705 A

[0005] However, in Patent Document 1, if the fluorescent brightness of many spots on the array plate is insufficient, it becomes difficult to adjust the position of the template mask, which in turn makes it difficult to perform analysis to obtain fluorescent information from each spot.

[0006] That is, a measuring device such as a plate reader capable of acquiring two-wavelength fluorescence images can be used to obtain a first fluorescence image captured by irradiating the plate with a first primary light having one wavelength, which can be 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 the plate with a second primary light having a different wavelength from the first primary light can be obtained, which can be used as an image for acquiring functional information. However, the fluorescence brightness of many spots on the array plate in the second fluorescence image may be insufficient, making it difficult to adjust the position of the template mask. Furthermore, if there is a misalignment between the first and second fluorescence images, it is difficult to accurately analyze the fluorescence information of each spot using the template mask aligned with the first fluorescence image.

[0007] The present invention aims 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.

[0008] The present invention provides an information acquisition method for acquiring information regarding the fluorescence intensity of spots arranged on an array plate, the method comprising: a fluorescence image acquisition step of acquiring 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 about the spots; an azimuth angle correction amount acquisition step of acquiring information regarding an azimuth angle correction amount within the image plane of the first fluorescence image and the second fluorescence image based on the first fluorescence 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 fluorescence image and a second azimuth angle corrected image obtained by correcting the azimuth angle of the second fluorescence image based on the information regarding the azimuth angle correction amount; and 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 as to overlap one of the plurality of spots but not another adjacent spot; The above-mentioned problem is solved by providing an information acquisition method including: a first translational correction amount acquisition step of acquiring information on first translational correction amounts for the plurality of first regions included in the first azimuth-angle-corrected image by adjusting the relative position of the template mask with respect to the first azimuth-angle-corrected image; a second translational correction amount acquisition step of acquiring second translational correction amounts for the plurality of first regions included in the second azimuth-angle-corrected image by adjusting the relative position of the template mask with respect to the second azimuth-angle-corrected image based on information on the first translational correction amounts for the plurality of first regions included in the first azimuth-angle-corrected image; and a fluorescence intensity acquisition step of acquiring information on fluorescence intensity corresponding to the plurality of spots in the first fluorescence image based on the information on the first translational correction amount and the first azimuth-angle-corrected image, and acquiring information on fluorescence intensity corresponding to the plurality of spots in the second fluorescence image based on the information on the second translational correction amount and the second azimuth-angle-corrected image.

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

[0010] 10 is a diagram for explaining an information acquisition method S10000 according to the first embodiment. FIG. 11 is a schematic diagram of an array plate applied to the information acquisition method according to the first embodiment. FIG. 12 is a diagram for explaining an example of a first fluorescent image and a second fluorescent image obtained in a fluorescent image acquisition step S100 according to the first embodiment. FIG. 13 is a diagram for explaining an azimuth angle correction amount acquisition step S200 according to the first embodiment. FIG. 14 is a schematic diagram of a template mask obtained in a template mask information acquisition step S400 according to the first embodiment. FIG. 15 is a diagram for explaining a first translational correction amount acquisition step S500 according to the first embodiment. FIG. 16 is a diagram for explaining a second translational correction amount acquisition step S600 according to the first embodiment. FIG. 17 is a schematic diagram of a second region of a fluorescence intensity acquisition step S700 according to the first embodiment. FIG. 18 is a diagram for explaining a first translational correction amount acquisition step S500 according to the third embodiment. FIG. 19 is a diagram for explaining a second translational correction amount acquisition step S600 according to the third embodiment. FIG. 19 is a diagram for explaining a noise processing step SS220 according to the fifth embodiment. FIG. 19 is a diagram for explaining a spot extraction processing step SS240 (S203) according to the fifth embodiment. FIG. 11 is a diagram showing an example of an image acquired in a region removal processing step S206 in the fifth embodiment. FIG. 12 is a diagram explaining a composite image acquisition step SS260 in the sixth embodiment. FIG. 13 is a diagram showing an example of a schematic configuration of an image acquisition device 101 for carrying out the information acquisition method according to the first embodiment. FIG. 14 is a diagram showing in another form an example of a schematic configuration of an image acquisition device 101 for carrying out the information acquisition method according to the first embodiment. FIG. 15 is a diagram showing a schematic configuration of an information acquisition device 11000 for carrying out the information acquisition method according to the first embodiment.

[0011] An information acquisition method according to a first embodiment of the present invention will be described below with reference to step S10000 shown in Fig. 1. The information acquisition method according to the first embodiment of the present invention is a method for acquiring information on a fluorescent image of an array plate having a plurality of arranged spots, and comprises the following steps:

[0012] A first fluorescence image captured by irradiating the spot with a first primary light and a second fluorescence image captured by irradiating the spot with a second primary light having a wavelength different from that of the first primary light are acquired (fluorescence image acquisition step S100). Based on the first fluorescence image, information regarding the amount of azimuth angle correction within the image plane of the first fluorescence image and the second fluorescence image is acquired (azimuth angle correction amount acquisition step S200). Based on the information regarding the amount of azimuth angle correction, a first azimuth angle-corrected image in which the azimuth angle of the first fluorescence image is corrected and a second azimuth angle-corrected image in which the azimuth angle of the second fluorescence image is corrected are acquired (azimuth angle-corrected image acquisition step S300). 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), in which a reference position and a relative position defining an outer edge relative to the reference position are determined so as to overlap one of the plurality of spots but not another adjacent spot. The relative position of the template mask with respect to the first azimuth-corrected image is adjusted to acquire information regarding first translational correction amounts for a plurality of first regions included in the first azimuth-corrected image (first translational correction amount acquisition step S500). The relative position of the template mask with respect to the second azimuth-corrected image is adjusted based on the information regarding the first translational correction amounts to acquire second translational correction amounts for a plurality of first regions included in the second azimuth-corrected image (second translational correction amount acquisition step S600). Information regarding fluorescence intensity corresponding to a plurality of spots in the first fluorescence image is acquired based on the information regarding the first translational correction amounts and the first azimuth-corrected image, and information regarding fluorescence intensity corresponding to a plurality of spots in the second fluorescence image is acquired based on the information regarding the second translational correction amounts and the second azimuth-corrected image (fluorescence intensity acquisition step S700). Each step is described in detail below.

[0013] <Array Plate> Array plates have spots containing various types of biological substances on a substrate and are used for comprehensive analysis of samples. Array plates are also called microchips, microarrays, protein chips, DNA chips, etc. Commercially available array plates can be used. Microarray plates are sold by Agilent Technologies Inc., RayBiotech, and other companies. Alternatively, array plates can be prepared using known methods. That is, array plates are prepared by immobilizing desired biological substances on one surface of a suitable substrate. Note that the term "immobilization" here can also be referred to as "adsorption," and includes immobilization via hydrophobic interaction, electrostatic interaction, van der Waals interaction, hydrogen bonding, and covalent bonding. The substrate is preferably transparent. Examples of substrate materials include glass, synthetic quartz, quartz, borosilicate glass, and the like. Alternatively, examples of substrate materials 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 morphological information of spots on an array plate. For example, if the measurement target is a protein and multiple types of proteins are spotted in an array on the array plate, GST-TAGs are attached to all spotted proteins, and the GST-TAGs are labeled with a fluorophore that emits fluorescence in response to light of a first wavelength. When this is excited with light of the first wavelength, fluorescence from all proteins can be obtained, thereby obtaining morphological information. Generally, protein spots are transparent to visible light and are difficult to observe with a normal optical microscope, so fluorescent labeling is an effective method.

[0015] <Second Fluorescence Image> The second fluorescence image is an image containing functional information of spots on an array plate, captured by irradiating the array plate with second primary light having a wavelength different from that of the first primary light. A portion of the spotted proteins is phosphorylated by reacting the array plate with a biological sample, and the phosphorylated proteins are labeled with a fluorophore that emits fluorescence in response to light of the second wavelength. When this is excited with light of the second wavelength, the fluorescence from the phosphorylated proteins can be obtained, thereby obtaining functional information (phosphorylation information).

[0016] In this specification, light of a first wavelength may be alternatively referred to as primary light of a first wavelength, excitation light of a first wavelength, or first primary light. Similarly, light of a second wavelength may be alternatively referred to as primary light of a second wavelength, excitation light of a second wavelength, or second primary light. To implement the information acquisition method of this embodiment, an image acquisition device is used that includes: an optical system including an irradiation unit that irradiates the array plate with primary light and a light collection unit that collects secondary light from the spots; a mounting unit on which the array plate is placed; a scanning unit that moves the mounting unit and the optical system relatively; 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 to execute each step of the information acquisition method of this embodiment.

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

[0018] Fig. 16 is a diagram illustrating a schematic configuration of an image acquisition device 101 for implementing the information acquisition method according to this embodiment, in a form different from that of Fig. 15. As shown in Fig. 16, the image acquisition device 101 includes a mounting unit 121 on which an array plate 107 is mounted, an irradiation unit 122 that irradiates the array plate with primary light to capture a first fluorescent image and a second fluorescent image, a light source 110 optically coupled to the irradiation unit and switching between emitting primary light of two different wavelengths, a light collecting unit 123 that receives and detects secondary light including fluorescence, an optical system 130 consisting of the irradiation unit 122 and the light collecting unit 123, the mounting unit 121, a scanning unit 112-2 that scans the mounting 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 that executes a program for carrying out the method according to this embodiment. The pixel pitch of the acquired fluorescent image of the array plate 107 is 3 μm to 30 μm.

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

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

[0021] According to this embodiment, it is possible to configure an information acquisition device that performs a series of operations from fluorescence detection to image analysis and outputs data after analysis.

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

[0023] <Details of First Embodiment> The information acquisition method S10000 according to the first embodiment will be described in more detail with reference to Fig. 1. The first fluorescent image and the second fluorescent image are captured in advance by an image acquisition device such as a fluorescent plate reader.

[0024] <Fluorescence Image Acquisition Process S100> In this process, a first fluorescence image is acquired by irradiating an array plate having a plurality of arranged spots with a first primary light, and a second fluorescence image is acquired by irradiating the spots with a second primary light having a wavelength different from that of the first primary light to acquire functional information of the spots. The first and second fluorescence images can be acquired using an image analysis PC. Multiple types of proteins are spotted in an array on the array plate. A schematic diagram of the array plate is shown in FIG. 2. Each spot has a diameter of approximately 100 μm, and the spacing between adjacent spots is approximately 40 μm. The spots are organized in 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 protein spots are phosphorylated, and only the phosphorylated protein spots are fluorescently labeled with Alexa Flour 790. The first fluorescent image was obtained by exciting this array plate with light having a wavelength of 670 nm, and the second fluorescent image was obtained by exciting this array plate with light having a wavelength of 780 nm.

[0025] The image acquisition device preferably includes excitation light with a first wavelength of 670 nm for exciting protein spots fluorescently labeled with Alexa Flour 680, and excitation light with a second wavelength of 780 nm for exciting protein spots fluorescently labeled with Alexa Flour 790.

[0026] The fluorescence images of the array plate acquired in step S100 are shown in FIG. 3. One block is shown enlarged. In the first fluorescence image, all spots are measured to be uniformly bright in order to obtain morphological information by irradiating with the first primary light. In the second fluorescence image, only phosphorylated protein spots are measured to be bright in order to obtain functional information by irradiating with the second primary light.

[0027] <Azimuth Angle Correction Amount Acquisition Step S200> This step acquires information about the azimuth angle correction amount within the image plane of the first and second fluorescent images obtained in step S100. A method for implementing step S200 will be described with reference to FIG. 4 . The first fluorescent image obtained in step S100 is averaged in the horizontal x-axis direction and vertical y-axis direction of the image to calculate one-dimensional x-axis averaged data and y-axis averaged data. Consider the tilt of the array plate's arrangement direction relative to the x-axis and y-axis defined by the image. When the array plate's arrangement direction has a predetermined tilt relative to the x-axis and y-axis defined by the image, the peak intensity values ​​of the x-axis averaged data and y-axis averaged data are lower than when the array plate's arrangement direction is aligned with the x-axis and y-axis ( FIG. 4( a) ). On the other hand, when the array plate's arrangement direction is aligned with the x-axis and y-axis, the peak intensity values ​​of the x-axis averaged data and y-axis averaged data reach their highest values ​​( FIG. 4( b) ). From this perspective, a provisional azimuth angle correction amount is applied to the fluorescence image to create an azimuth angle-corrected image, and an azimuth angle correction amount is obtained that maximizes the amplitude of the x-axis averaged data and the y-axis averaged data for the created azimuth angle-corrected image. For example, the angular deviation within the image plane of the first fluorescence image or the second fluorescence image is approximately ±1 degree, depending on the scanning accuracy of the image acquisition device and the fabrication accuracy of the array plate. In this embodiment, an azimuth angle-corrected image is generated while changing the provisional azimuth angle correction amount in 0.1 degree increments, and an azimuth angle correction amount is obtained that maximizes the amplitude of the x-axis averaged data and the y-axis averaged data.

[0028] <Azimuth angle-corrected image acquisition step S300> In this step, a first azimuth angle-corrected image and a second azimuth angle-corrected image are acquired by correcting the first fluorescence image and the second fluorescence image using the azimuth angle correction amount obtained in the azimuth angle correction amount acquisition step S200. Affine transformation is used to correct the azimuth angles of the fluorescence images. The affine transformation is performed using any interpolation method, such as nearest neighbor interpolation, linear interpolation, bilinear interpolation, or bicubic interpolation.

[0029] <Template Mask Information Acquisition Process S400> This process acquires information about a template mask including a plurality of first regions arranged corresponding to each of the plurality of spots, each of which has a reference position and a relative position defining its outer edge relative to the reference position so that the first region overlaps with one of the plurality of spots but not with adjacent spots. The template mask information is input using a pre-prepared user interface based on the design information of the array plate. Alternatively, a file created in advance based on the design information of the array plate and stored in memory is read by the analysis PC. The shape of the template mask is shown in FIG. 5. The template mask has first regions of interest arranged in a 9x9 matrix. The shape of the first region is circular with the same radius as the spot to match the shape of the spot on the array plate. Alternatively, the shape of the first region can be set slightly larger than the radius of the spot, elliptical to match the spot shape, or any other shape that does not overlap with adjacent first regions. The spacing (pitch) of the first regions of the template mask may be determined from the period of the x-axis averaged data and y-axis averaged data obtained in process S200.

[0030] <First Translational Correction Amount Acquisition Step S500> This step acquires information regarding first translational correction amounts for multiple first regions included in the first azimuth-corrected image by adjusting the relative position of the template mask with respect to the first azimuth-corrected image. The first translational correction amount acquisition step S500 of this embodiment will be described with reference to FIG. 6 . The coordinates of each block on the first azimuth-corrected image are set using the x-axis averaged data and y-axis averaged data in the first azimuth-corrected image obtained in step S300. The relative position of the template mask with respect to a certain block is changed in the x-axis and y-axis directions within a predetermined scanning range (first scanning range), and the sum of the fluorescence brightness values ​​of the first azimuth-corrected image that overlaps with the multiple first regions of the template mask at each relative position is calculated. The relative position at which the sum of the fluorescence brightness values ​​is maximized is acquired as the first translational correction amount in the x-axis and y-axis directions. As a result, the spot positions of the template mask and the block, which were misaligned before scanning as shown in FIG. 6( a), become aligned as shown in FIG. 6( b) after scanning. For example, when the spot diameter is 100 μm, the scanning range of the template mask is preset to 100 μm in each of the x-axis and y-axis directions for the x-axis averaged data and y-axis averaged data in the first azimuth angle corrected image obtained in step S300. This process is performed for a total of 18 template masks for each block.

[0031] <Second translational correction amount acquisition process S600> This process is a process of acquiring second translational correction amounts for a plurality of first regions included in the second azimuth-corrected image by adjusting the relative position of the template mask with respect to the second azimuth-corrected image based on information related to the first translational correction amount.

[0032] For a certain block on the second azimuth-corrected image, the first translational correction amount obtained in step S500 is used as the reference position for the relative position of the template mask, and 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 brightness values ​​of the second azimuth-corrected image that overlaps with the multiple first regions of the template mask at each relative position is calculated. The relative position at which the sum of the fluorescence brightness values ​​is maximized is acquired as the second translational correction amount in the x-axis and y-axis directions. As a result, the spot positions of the template mask and the block, which were misaligned before scanning as shown in FIG. 7(a), are aligned after scanning as shown in FIG. 7(b). For example, if the imaging positions of the two wavelengths on the array plate of the image acquisition device are misaligned, a relative positional deviation occurs between the first and second fluorescent images. According to this embodiment, the positional deviation can be corrected even for images containing such a deviation. If there is no positional deviation or deviation from the optimal position between the first and second azimuth-corrected images, the second translational correction amount may be set to the same value as the first translational correction amount. Furthermore, if the positional deviation or the deviation of the optimal position between the first azimuth angle-corrected image and the second azimuth angle-corrected image is a fixed value, the second translational correction amount may be a value obtained by adding a constant value to the first translational correction amount. This process is performed for a total of 18 template masks for each block.

[0033] <Fluorescence intensity acquisition process S700> This process is a process of acquiring information about fluorescence intensity corresponding to multiple spots in the first fluorescence image based on information about the first translational correction amount and the first azimuth-corrected image, and acquiring information about fluorescence intensity corresponding to multiple spots in the second fluorescence image based on information about the second translational correction amount and the second azimuth-corrected image.

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

[0035] For each spot, statistical quantities such as the mean, median, and variance of the signal and background, as well as statistical quantities of the net signal extracted from the fluorescent brightness components originating from the spot, such as the difference and ratio between the signal and background, are calculated and output as a file together with data related to the spot position.

[0036] According to this embodiment, even if the second fluorescent image is discrete and it is difficult to identify block coordinates, the array plate can be accurately analyzed. Furthermore, even if there is a relative positional deviation between the first and second fluorescent images, the position of the template mask can be automatically adjusted, enabling analysis of the array plate.

[0037] Second Embodiment This embodiment is a modification of the first embodiment and differs from the first embodiment in that the azimuth angle correction image acquisition step S300 is performed to calculate the azimuth angle correction amount by weighting the peak intensity values ​​of the x-axis averaged data and the y-axis averaged data. Due to variations in spot shape caused by the fabrication precision of the array plate, the azimuth angle correction amount at which the amplitudes of the x-axis averaged data and the y-axis averaged data are maximized may differ. In such cases, it is effective to calculate the azimuth angle correction amount by weighting the peak intensity values ​​of the x-axis averaged data and the y-axis averaged data in step S200. Depending on the fabrication precision of the array plate, the spot shape tends to be an ellipse elongated along the y-axis rather than a circle. In this case, the peak width indicating the maximum of the x-axis averaged data becomes wider, making it difficult to determine the peak position. Therefore, the azimuth angle correction amount is calculated by weighting the y-axis averaged data with a steeper peak and calculating the weighted average of the x-axis averaged data and the y-axis averaged data. According to this embodiment, it is possible to obtain the amount of azimuth angle correction even when there is variation in the spot shape due to the manufacturing precision of the array plate.

[0038] Third Embodiment This embodiment is a variation of the first embodiment and differs from the first embodiment in that a second translational correction amount acquisition step S600 is performed to determine a second translational correction amount based on a first translational correction amount. Step S600 of this embodiment is also effective when two fluorescence images of a plate reader have different curvatures. For example, Alexa Flour 790 is only slightly excited by an excitation light source with a wavelength of 670 nm, so the second fluorescence image is affected by the excitation light with a wavelength of 670 nm. To avoid this effect, the plate reader acquires fluorescence images by separating the excitation wavelengths during forward and backward scanning of the array plate in the short direction. Depending on the scanning accuracy of the scanner, misalignment of the relative positions of the scanning lines and curvature of the scanning lines in opposite directions may occur between the forward and backward scanning passes, resulting in images acquired with curvatures in opposite directions between the 670 nm and 780 nm fluorescence images. For example, suppose that in step S500, the first fluorescence image is convexly curved as shown in FIG. 9 , and the position of the template mask is adjusted to obtain a first translational correction amount. In step S600, if the second fluorescence image is concavely curved, the first region of the template mask will be misaligned with respect to the spot when the same template mask is used in its relative position. As shown in FIG. 10 , by determining the second translational correction amount based on the first translational correction amount, the position of the template mask can be accurately adjusted for the second fluorescence image as well. According to this method, the misalignment of the spot caused by the image curvature in step S600 can be adjusted using the second translational correction amount, and therefore this method is effective even for images that include such curvature.

[0039] Fourth Embodiment This embodiment is a modified version of the first embodiment and differs from the first embodiment in that a second translational correction amount acquisition step S600 is performed to adjust spot position deviations resulting from the array plate using the second translational correction amount. Step S600 of this embodiment is effective even when, due to a process performed during fabrication of the array plate, spots are generated at different positions in a first fluorescence image in which morphological information is acquired by irradiating the array plate with the first primary light and a second fluorescence image in which functional information is acquired by irradiating the array plate with the second primary light. For example, assume that the fluorescently labeled positions of Alexa Flour 680 and Alexa Flour 790 are different. According to this method, the spot position deviation resulting from the array plate can also be adjusted using the second translational correction amount in step S600, and therefore this method is effective even for array plates containing such position deviations.

[0040] Fifth Embodiment This embodiment is a modification of the first embodiment and differs 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 noise signals. However, the presence of noise signals can make it difficult to calculate the azimuth angle correction amount. Possible unwanted signals include the scanner's signal-to-background ratio (SN), dust or fluorescent dye accidentally attached to or mixed into the array plate, etc. In such cases, the noise removal processing step shown in FIG. 11 is effective. This embodiment describes a method for this. The noise processing step SS220 shown in FIG. 11 can be performed after the fluorescence image acquisition step and before the azimuth angle correction amount acquisition step. The noise removal processing step includes a filtering processing step (S201), a threshold processing step (S202) that reduces the number of gradations related to pixel values, and a spot extraction processing step (S203) that extracts spots. Details of each step are described below.

[0041] <Filtering Step S201> The first fluorescent image obtained in step S100 is subjected to any filtering process, such as a median filter or Gaussian filter, to reduce the influence of system noise present in the entire fluorescent image. The filter mask size for the filtering process is set to any size that does not affect the overall shape of the spot. For example, if the scanner's signal-to-noise ratio is poor, noise with high-brightness pixel size may be included in areas other than the spot. Also, low-brightness pixels may occur in high-brightness areas within the spot. Furthermore, the fluorescent image may contain high-brightness spots of several pixels in size that are caused by fluorescent labeling of nonspecifically adsorbed proteins outside the spot area. To remove these, filtering is performed using a 5 x 5 pixel median filter.

[0042] <Threshold Processing Step S202> The filtered first fluorescence image obtained in step S201 is subjected to threshold processing to reduce the number of gradations. For example, a threshold is determined to distinguish between spot regions and non-spot regions, and the first fluorescence image is converted into a binarized image. The threshold may be input in advance or determined from an algorithm such as Otsu's binarization.

[0043] <Spot extraction processing step S203> The first fluorescence image obtained in step S202 after threshold processing is subjected to spot extraction processing to remove unnecessary signals. The spot extraction processing step S240 includes a region shape calculation processing step (S204), a spot determination processing step (S205), and a region removal processing step (S206) shown in Fig. 12. Each step will be described in detail below.

[0044] <Region shape calculation process step S204> For the first fluorescence image after threshold processing obtained in step S202, high-brightness regions where adjacent pixels have the same brightness are targeted and information about the shape of the regions is obtained. For example, in a binary image, the brightness value is 0 or 1, and regions of adjacent pixels with a brightness value of 1 form multiple high-brightness regions including spots. Information about the shape of these high-brightness regions, such as the perimeter of the region, the area of ​​the region, and the longitudinal and lateral lengths of the region, is obtained.

[0045] <Spot Determination Processing Step S205> Based on the information about the shape of the high-brightness region obtained in step S204, it is determined whether the high-brightness region is a spot or is derived from other unwanted signals. For example, if the outer perimeter of the region is longer than the circumferential length calculated from the designed spot diameter of the array plate, it is determined not to be a spot. In addition, whether the high-brightness region is a spot is determined based on a determination formula such as if the circularity calculated from the outer perimeter and area of ​​the region is greater than a preset circularity, or if the ratio of the longitudinal to lateral lengths is greater than a preset value.

[0046] <Region Removal Processing Step S206> High-brightness regions determined not to be spots in step S205 are assigned the pixel values ​​of low-brightness regions. For example, if fibrous dust as shown in Fig. 13(a) is included in the binarized first fluorescence image as an unnecessary high-brightness signal, the high-brightness region is converted from a brightness value of 1 to a brightness value of 0, as shown in Fig. 13(b).

[0047] The noise removal processing step according to this embodiment is executed after S100 and before S200 in the first embodiment. The noise removal processing step removes noise from the first fluorescent image to obtain a noise-removed image. The subsequent steps are the same as those in the first embodiment. In step S200, information regarding the amount of azimuth angle correction is obtained by using the noise-removed image instead of the first fluorescent image. This embodiment makes it possible to calculate the amount of azimuth angle correction independent of the signal-to-noise ratio of the scanner or the state of the array plate.

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

[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 angle correction amount is calculated from the first fluorescent image. However, depending on the measurement parameters used when capturing the fluorescent image, the contrast between the spots and areas outside the spots in the first fluorescent image may be low, making it difficult to calculate the azimuth angle correction amount. In this case, it is effective to calculate the azimuth angle correction amount from the composite image of the first and second fluorescent images obtained in the composite image acquisition step. The composite image acquisition step can be performed after the fluorescent image acquisition step and before the azimuth angle correction amount acquisition step. FIG. 14 shows the composite image acquisition step SS260. The composite image acquisition step includes a step of acquiring weightings for the first fluorescent image (S211), a step of acquiring weightings for the second fluorescent image (S212), and an image synthesis processing step (S213). By reading preset weighting values ​​in steps S211 and S212, the synthesis ratio for image synthesis is acquired. For example, a threshold is determined by Otsu's binarization process, assigning pixel values ​​on the high-brightness side as 1 and pixel values ​​on the low-brightness side as 0. In this case, a high-contrast fluorescent image has a pixel value of 1 in the spot area and a pixel value of 0 outside the spot area. On the other hand, a low-contrast fluorescent image has a pixel value of 1 regardless of whether it is inside or outside the spot area, resulting in large variation in the in-plane distribution of pixel values ​​1. Using this binarized image characteristic, it is effective to increase the weight when the in-plane distribution of pixel values ​​1 is small. In step S213, a compositing method such as arithmetic average compositing or comparatively bright compositing is set based on the obtained compositing ratio, thereby increasing the contrast between the spot and outside the spot. The noise removal process shown in FIG. 11 may be performed on the composite image or fluorescent image before or after the composite image acquisition process. Information regarding the amount of azimuth angle correction within the image plane of the first fluorescent image and the second fluorescent image is acquired using the composite image of the first and second fluorescent images obtained in the composite image acquisition process. According to this embodiment, even if the contrast between the spot and the outside of the spot in the first fluorescent image is low, it is possible to calculate the amount of azimuth angle correction.

[0050] Seventh Embodiment This embodiment is a modification of the first embodiment. In the first embodiment, the azimuth angle correction amount is calculated from the peak values ​​of the intensities of the x-axis averaged data and the y-axis averaged data. However, in this embodiment, the azimuth angle correction amount is calculated from the positions of local peaks that appear in a matrix in the two-dimensional frequency image. The seventh embodiment differs from the first embodiment in that a method for implementing step S200 in FIG. 1 that differs from the first embodiment will be described. In the first embodiment, the azimuth angle correction amount is calculated from the peak values ​​of the intensities of the x-axis averaged data and the y-axis averaged data. However, in the azimuth angle correction amount acquisition step, information regarding the azimuth angle correction amount can be acquired by performing two-dimensional frequency analysis of the first fluorescence image. In this embodiment, the azimuth angle correction amount is calculated from a two-dimensional frequency image obtained by two-dimensional Fourier transform of the first fluorescence image. The two-dimensional frequency image represents the complex amplitude in frequency space after the two-dimensional Fourier transform as an absolute value. Specifically, the azimuth angle correction amount is calculated from the positions of local peaks that appear in a matrix in the two-dimensional frequency image. In the first embodiment, it was necessary to generate an azimuth angle corrected image multiple times using a provisional azimuth angle correction amount, whereas in this embodiment, there is an advantage that it is only necessary to generate a two-dimensional frequency image once.

[0051] Eighth Embodiment This embodiment is a modification of the first embodiment. In the first embodiment, the relative position of the template mask at which the fluorescence intensity value of the azimuth angle-corrected image in the region overlapping with the plurality of first regions of the template mask is maximized is acquired as the translation correction amount. However, in this embodiment, the translation correction amount is calculated as the relative position of the template mask at which the fluorescence intensity value of the azimuth angle-corrected image in the region overlapping with the second region in step S700 of the first embodiment. This embodiment differs from the first embodiment in that the translation correction amount is calculated as the relative position of the template mask at which the fluorescence intensity value of the azimuth angle-corrected image in the region overlapping with the plurality of first regions of the template mask is minimized in step S700 of the first embodiment. In the eighth embodiment, a method for implementing steps corresponding to steps S500 and S600 of FIG. 1 will be described. In the first embodiment, the translation correction amount is calculated as the relative position of the template mask at which the fluorescence intensity value of the azimuth angle-corrected image in the region overlapping with the plurality of first regions of the template mask is maximized. In contrast, in this embodiment, the translation correction amount is calculated as the relative position of the template mask at which the fluorescence intensity value of the azimuth angle-corrected image in the region overlapping with the second region in step S700 of the first embodiment. 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 version of the first embodiment, and in the first embodiment, the relative position of the template mask at which the fluorescence luminance value of the azimuth angle-corrected image is maximized in an area overlapping with a plurality of first areas of the template mask is acquired as the translation 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 two-dimensional cross-correlation between the azimuth angle-corrected image and the mask image whose relative position has been moved is maximized is calculated as the translation correction amount. This differs from the first embodiment in that

[0053] A method for implementing steps S500 and S600 in FIG. 1 will be described in the ninth embodiment. In the first embodiment, the relative position of the template mask at which the fluorescence intensity value of the azimuth-corrected image in the region overlapping with the plurality of first regions of the template mask is maximized was acquired as the translation correction amount. Depending on the fluorescence image, high-intensity signals other than spots may be included near the spots, and this may affect maximum value search. In this embodiment, a mask image is created from the array information of the template mask, and the relative position at which the two-dimensional cross-correlation between the azimuth-corrected image and the mask image whose relative position has been shifted is maximized is calculated as the translation correction amount. This embodiment makes it possible to calculate the translation correction amount while minimizing the influence of unnecessary high-intensity signals included in the fluorescence image.

[0054] Tenth Embodiment This embodiment is a modified version of the first embodiment, and differs from the first embodiment in that, while in the first embodiment, the translational correction amount is calculated for each template mask, in this embodiment, after the translational correction amount is calculated, a fine adjustment translational correction amount for fine adjustment is calculated for each spot.

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

[0056] Eleventh Embodiment This embodiment is a modified version of the first to tenth embodiments, and differs from the first to tenth embodiments in that, while in the first to tenth embodiments, one fluorescence image is captured by irradiating the spot with second primary light to obtain functional information of the spot, this embodiment acquires multiple fluorescence images corresponding to the second fluorescence image.

[0057] In the first to tenth embodiments, one fluorescent image was captured by irradiating the spot with the second primary light to obtain functional information about the spot. In this embodiment, multiple fluorescent images corresponding to the second fluorescent image captured by irradiating the spot with the second primary light, which is a fluorescent image for obtaining functional information about the spot, are acquired. This embodiment makes it possible to simultaneously obtain functional information other than phosphorylation information.

[0058] The present invention is not limited to the above-described embodiments, and various modifications and variations can be made without departing from the spirit and scope of the present invention. Therefore, the following claims are appended to apprise the public of the scope of the present invention.

[0059] This application claims priority based on Japanese Patent Application No. 2023-213063 filed on December 18, 2023 and Japanese Patent Application No. 2024-188835 filed on October 28, 2024, the entire contents of which are incorporated herein by reference.

[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 angle correction amount acquisition unit 1053: Azimuth angle corrected 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: Light detection element 112: Scanning unit 113: Filter 114: Dichroic mirror 115: Lens 116: Half mirror 121: Placement unit 122: Irradiation unit 123: Light collection unit 130: Optical system

Claims

1. An information acquisition method for acquiring information regarding 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 an array plate having a plurality of arranged spots with a first primary light, 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 the image plane of the first fluorescence image and the second fluorescence image based on the first fluorescence image; an azimuth angle corrected image acquisition step of acquiring a first azimuth angle corrected image in which the azimuth angle of the first fluorescence image is corrected, and a second azimuth angle corrected image in which the azimuth angle of the second fluorescence image is corrected based on the information regarding the azimuth angle correction amount; and 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 as to overlap one of the plurality of spots but not 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 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 regarding the first translation correction amount for the plurality of first regions included in the first azimuth angle-corrected image; and a fluorescence intensity acquisition step of acquiring information regarding fluorescence intensity corresponding to the plurality of spots in the first fluorescent image based on the information regarding the first translation correction amount and the first azimuth angle-corrected image, and acquiring information regarding fluorescence intensity corresponding to the plurality of spots in the second fluorescent image based on the information regarding the second translation correction amount and the second azimuth angle-corrected image.

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

3. The information acquisition method according to claim 1 or 2, 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. An information acquisition method according to any one of claims 1 to 3, wherein in the azimuth angle correction amount acquisition step, information regarding the azimuth angle correction amount is acquired by performing two-dimensional frequency analysis of the first fluorescent image.

5. An information acquisition method according to any one of claims 1 to 4, comprising, in the first translational correction amount acquisition step, a step of acquiring information relating to 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 with the first areas of other adjacent spots.

6. A method for acquiring information relating to a plurality of first fine translation correction amounts for the first region by adjusting a relative position of a 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 angle corrected image to acquire information relating to a plurality of first fine translation correction amounts for the first region, and a plurality of second fine translation correction amounts for the first region by adjusting the relative positions of the plurality of first regions included in the second azimuth angle corrected image, the method including a step replacing the first translation correction amount acquisition step and the second translation correction amount acquisition step.

7. An information acquisition method described in any one of claims 1 to 6, 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 having the program according to claim 8 recorded thereon.

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 relatively, 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 said illumination portion.

12. The image acquisition device according to claim 11, wherein the light source switches between emitting the first primary light and 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 on which a plurality of arranged spots are provided 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 in the image plane of the first fluorescence image and the second fluorescence image based on the first fluorescence image; an azimuth angle corrected image acquisition unit that acquires a first azimuth angle corrected image in which the azimuth angle is corrected for the first fluorescence image and a second azimuth angle corrected image in which the azimuth angle is corrected for the second fluorescence image based on the information regarding the azimuth angle correction amount; and 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 that defines an outer edge with respect to the reference position are determined so as to overlap one of the plurality of spots but not another adjacent spot; an information acquiring device including: a first translation correction amount acquiring unit that acquires information regarding a first translation 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 acquiring unit that acquires 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 regarding the first translation correction amount for the plurality of first regions included in the first azimuth angle-corrected image; and a fluorescence intensity acquiring unit that acquires information regarding fluorescence intensity corresponding to the plurality of spots in the first fluorescence image based on information regarding the first translation correction amount and the first azimuth angle-corrected image, and acquires information regarding fluorescence intensity corresponding to the plurality of spots in the second fluorescence image based on information regarding the second translation correction amount and the second azimuth angle-corrected image.

Images