Image acquisition device, image acquisition method, and image acquisition program

Abstract

An image acquisition device (100) acquires a first fluorescence image, which is an image obtained by imaging a DNA microarray (A) by an imaging device (1) during a first period, and a second fluorescence image, which is an image obtained by imaging the DNA microarray (A) by the imaging device during a second period. Generating a corrected second fluorescence image in which a second fluorescence intensity of a fluorescent spot in the second fluorescence image is corrected on the basis of a first fluorescence intensity of a fluorescent spot in the first fluorescence image, and superimposing the corrected second fluorescence image on the first fluorescence image; a superimposed corrected fluorescence image obtained by correcting the first fluorescence image is generated.

Description

Technical Field

[0001] The present invention relates to an image acquisition device, an image acquisition method, and an image acquisition program. Background Art

[0002] As a measurement, the sample is examined for specific deoxyribonucleic acid (DNA)

[0003] A method for targeting nucleic acid sequences such as those containing d-(4-deoxyribonucleic acid) is known as a DNA microarray method that utilizes a DNA microarray. The DNA microarray method is a method that utilizes the property of detection target molecules modified with fluorescent molecules in a test sample added to the DNA microarray to be captured by the detection probes of the DNA microarray through a hybridization reaction (hybridization) to measure the detection target molecules. In the DNA microarray method, the brightness value or light intensity of the DNA spot portion is calculated by image analysis of the fluorescent image. In addition to determining whether the detection target molecule is contained in the test sample, it is also possible to measure the amount of the detection target molecule contained in the test sample.

[0004] Patent Document 1: Japanese Patent Application Laid-Open No. 2008-203138 Summary of the Invention

[0005] However, the DNA microarray method is difficult to obtain fluorescent images with high detection accuracy. For example, when the measurement time is set to a long time, the estimated amount of the target molecule may be erroneous due to photobleaching of the fluorescent pigment.

[0006] The present invention is proposed in view of the above situation, and its object is to obtain a fluorescence image with high detection accuracy.

[0007] The present invention provides an image acquisition device, wherein the image acquisition device comprises: an acquisition unit that acquires a first fluorescent image and a second fluorescent image, wherein the first fluorescent image is an image obtained by photographing a measurement target sample using a photographing device during a first period, and the second fluorescent image is an image obtained by photographing the measurement target sample using the photographing device during a second period; and a generation unit that generates a corrected fluorescent image by correcting a second fluorescent intensity of a fluorescent spot generating fluorescence in the second fluorescent image based on a first fluorescent intensity of the fluorescent spot generating fluorescence in the first fluorescent image, and superimposes the corrected fluorescent image on the first fluorescent image to generate a superimposed corrected fluorescent image obtained by correcting the first fluorescent image.

[0008] In addition, the present invention provides an image acquisition method, wherein the computer performs the following processing: acquiring a first fluorescent image and a second fluorescent image, wherein the first fluorescent image is an image obtained by photographing a measurement target sample using a photographing device during a first period, and the second fluorescent image is an image obtained by photographing the measurement target sample using the photographing device during a second period, generating a corrected fluorescent image by correcting the second fluorescent intensity of a fluorescent spot generating fluorescence in the second fluorescent image based on the first fluorescent intensity of the fluorescent spot generating fluorescence in the first fluorescent image, and superimposing the corrected fluorescent image on the first fluorescent image to generate a superimposed corrected fluorescent image obtained by correcting the first fluorescent image.

[0009] In addition, the present invention provides an image acquisition program that causes a computer to execute the following processing: acquiring a first fluorescent image and a second fluorescent image, wherein the first fluorescent image is an image obtained by photographing a measurement target sample using a photographing device during a first period, and the second fluorescent image is an image obtained by photographing the measurement target sample using the photographing device during a second period; generating a corrected fluorescent image by correcting the second fluorescent intensity of a fluorescent spot generating fluorescence in the second fluorescent image based on the first fluorescent intensity of the fluorescent spot generating fluorescence in the first fluorescent image; and superimposing the corrected fluorescent image on the first fluorescent image to generate a superimposed corrected fluorescence image obtained by correcting the first fluorescent image.

[0010] Effects of the Invention

[0011] According to the present invention, there is an effect of being able to acquire a fluorescent image with high detection accuracy. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 This is a diagram showing an example of the processing flow of the DNA microarray method.

[0013] Figure 2 FIG. 1 is a diagram for explaining the fluorescence intensity of a fluorescence image according to the reference technique.

[0014] Figure 3 It is a diagram showing a configuration example of an image acquisition system according to an embodiment.

[0015] Figure 4 This is a block diagram showing a configuration example of an image acquisition device and a camera according to an embodiment.

[0016] Figure 5 This is a diagram showing a specific example 1 of the fluorescence intensity of a fluorescence image according to the embodiment.

[0017] Figure 6This is a diagram showing a specific example 2 of the fluorescence intensity of a fluorescence image according to the embodiment.

[0018] Figure 7 This is a diagram showing a specific example 3 of the fluorescence intensity of a fluorescence image according to the embodiment.

[0019] Figure 8 This is a flowchart showing an example of the flow of image acquisition processing according to the embodiment.

[0020] Figure 9 It is a diagram showing a hardware configuration example according to the embodiment. DETAILED DESCRIPTION

[0021] Hereinafter, an image acquisition device, an image acquisition method, and an image acquisition program according to an embodiment of the present invention will be described in detail with reference to the accompanying drawings.

[0022] [Implementation Method]

[0023] Next, the configuration of the image acquisition system 1000 , the configuration of the image acquisition device 100 , and the processing flows according to the embodiment will be described in order, and finally, the effects of the embodiment will be described.

[0024] [1. Structure of Image Acquisition System 1000]

[0025] In use Figures 1 to 3 After explaining the processing flow of the DNA microarray method and the image acquisition process related to the reference technology, the image acquisition system 1000 according to the embodiment will be described in detail. Furthermore, as an example of the image acquisition system 1000 according to the embodiment, the detection process of a biopolymer measurement device that measures specific detection target molecules among biopolymers contained in an inspection sample using the DNA microarray method will be described. However, the scope of application of the image acquisition system 1000 is not particularly limited.

[0026] (1-1. DNA microarray process flow)

[0027] use Figure 1 The processing flow of the DNA microarray method, which is a premise of the image acquisition process according to the reference technique and the image acquisition system 1000 according to the embodiment, will be described. Figure 1 This is a diagram showing an example of the processing flow of the DNA microarray method. Figure 1 As shown, in the DNA microarray method, the following processes (1) to (4) are performed.

[0028] First, the nucleic acid contained in the test sample S, namely the detection target molecule N, is amplified using a nucleic acid amplification technique such as PCR (Polymerase Chain Reaction), and fluorescent molecules F are added to the amplified detection target molecule N (see Figure 1 (1)). Secondly, a solution containing the detection target molecule N to which the fluorescent molecule F is attached is added to the DNA microarray A, and the detection target molecule N fluorescently modified by the fluorescent molecule F is captured by hybridization based on the detection probe P (refer to Figure 1 (2)). At this time, the complementary sequence of the nucleic acid of the detection target molecule N is fixed as a detection probe P on a solid surface such as the base of the DNA microarray A, so that the fluorescently modified detection target molecule N is bound to the detection probe P. Third, in order to remove the fluorescent molecules F from the molecules that are not captured and that are non-specifically bound to the nucleic acid sequence of the detection probe P, the DNA microarray A is washed (refer to Figure 1 (3)). Fourthly, the DNA microarray A is photographed by the imaging device 1 to obtain a fluorescent image of the DNA microarray A in which the fluorescent molecules F are captured in the DNA spots (refer to Figure 1 (4)).

[0029] (1-2. Image Acquisition Processing of Reference Technology)

[0030] The image acquisition process according to the reference technique will be described below in the order of a processing example of the image acquisition process according to the reference technique, the fluorescence intensity of the fluorescence image according to the reference technique, and the problems of the image acquisition process according to the reference technique.

[0031] (1-2-1. Processing Example of Image Acquisition Processing According to Reference Technology)

[0032] The image acquisition process involved in the reference technology performs the following steps. First, the imaging device irradiates the substrate of DNA microarray A with excitation light of a wavelength corresponding to the fluorochrome of fluorescent molecules F, and uses a light-receiving element to detect the fluorescence generated from DNA microarray A. At this time, the imaging device adjusts the exposure time based on the fluorescence intensity, adjusting the signal-to-noise ratio (S / N) to a range of several seconds to several minutes to capture the signal. Second, the image acquisition device calculates the fluorescence intensity of the detected fluorescent spots on DNA microarray A and estimates the amount of detection target molecules N captured on DNA microarray A.

[0033] (1-2-2. Fluorescence Intensity of Fluorescence Image Related to Reference Technology)

[0034] use Figure 2 The fluorescence intensity of the fluorescence image according to the reference technique will be described. Figure 2FIG is a diagram for explaining the fluorescence intensity of a fluorescence image according to the reference technique. Figure 2 As shown in (1), the fluorescence intensity (arbitrary unit) decreases over time due to photobleaching of the fluorescent pigment. Figure 2 (2) Indicates the basis Figure 2 The equivalent light intensity is calculated by taking the total value of the fluorescence intensity from 0 to 180 seconds of (1) and calculating the average value per unit time.

[0035] (1-2-3. Problems with Image Acquisition Processing Related to Reference Technology)

[0036] In the image acquisition process involved in the reference technique, if the amount of fluorescence generated from DNA microarray A is low, the measurement time needs to be set to a longer time. During this time, the fluorescent pigment is continuously irradiated with excitation light, causing photobleaching of the fluorescent pigment. Consequently, the estimated amount of detection target molecules N captured on the substrate of DNA microarray A will be subject to significant error. Furthermore, the longer the measurement time, the greater the impact of photobleaching, resulting in a lower estimated amount of detection target molecules N.

[0037] Furthermore, in the image acquisition process according to the reference technique, the influence of photobleaching varies depending on the density of fluorescent molecules F, the exposure history of the fluorescent dye, and the like. Therefore, it is difficult to correct the measured signal based on pre-acquired photobleaching data.

[0038] (1-3. Image Acquisition System 1000)

[0039] use Figure 3 The image acquisition system 1000 according to the embodiment will be described below in the order of a configuration example of the image acquisition system 1000 , a processing example of the image acquisition system 1000 , and effects of the image acquisition system 1000 .

[0040] (1-3-1. Configuration Example of Image Acquisition System 1000)

[0041] use Figure 3 A configuration example of the image acquisition system 1000 according to the embodiment will be described. Figure 3 1 is a diagram showing a configuration example of an image acquisition system 1000 according to an embodiment. The image acquisition system 1000 includes a camera 1 and an image acquisition device 100. Figure 3 The image acquisition system 1000 shown may include multiple photographing devices 1 or multiple image acquisition devices 100. In addition, the image acquisition device 100 may be an integrated structure with the photographing device 1.

[0042] The imaging device 1 includes a light source 10, an objective lens 11, a dichroic mirror 12, a filter 13, a lens 14, and a light receiving element 15. The imaging device 1 images a DNA microarray A, which is a set measurement target sample (referred to as "measurement target specimen" as appropriate).

[0043] (1-3-2. Processing Example of Image Acquisition System 1000)

[0044] use Figure 3 An example of processing performed by the image acquisition system 1000 according to the embodiment will be described. The image acquisition process, light intensity calculation process, threshold value determination process, light intensity correction process, and image superposition process of the image acquisition system 1000 according to the embodiment will be described in this order.

[0045] (1-3-2-1. Image acquisition processing)

[0046] The image acquisition system 1000 according to the embodiment executes an image acquisition process. Hereinafter, a first image acquisition process of capturing images by the imaging device 1 during a first period and a second image acquisition process of capturing images by the imaging device 1 during a second period will be described.

[0047] (First Image Acquisition Process)

[0048] First, the image acquisition system 1000 performs the first image acquisition process as the image acquisition process. First, the imaging device 1 sets the DNA microarray A as the measurement target sample. Next, the imaging device 1 irradiates the DNA microarray A with excitation light ( Figure 3 Furthermore, the imaging device 1 uses the light receiving element 15 to detect the fluorescence ( Figure 3 Finally, the image acquisition device 100 acquires the first image I1 of the DNA microarray A captured by the imaging device 1 .

[0049] For example, the first image I1 is an image captured by the imaging device 1 during the initial measurement of the DNA microarray A, the target, when irradiating the DNA microarray A with excitation light for a constant exposure time (e.g., 3 seconds). Alternatively, the first image I1 is an image captured by the imaging device 1 during the initial measurement of the DNA microarray A, the target, when irradiating the DNA microarray A with excitation light for a continuous exposure time (e.g., 300 seconds) at intervals of a constant exposure time (e.g., 3 seconds). The first image I1 is preferably captured before the fluorescence level on the DNA microarray A decreases due to photobleaching of the fluorescent molecules F, but the timing of the capture and the exposure time are not limited.

[0050] (Second Image Acquisition Process)

[0051] Second, the image acquisition system 1000 performs a second image acquisition process as the image acquisition process. First, the imaging device 1 irradiates the DNA microarray A with excitation light ( Figure 3 Furthermore, the imaging device 1 uses the light receiving element 15 to detect the fluorescence ( Figure 3 Finally, the image acquisition device 100 acquires the second image I2 of the DNA microarray A captured by the imaging device 1.

[0052] For example, the second image I2 is an image captured by the imaging device 1 after acquiring the first image I1, capturing fluorescence generated when the imaging device 1 irradiates the sample with excitation light for a constant exposure time (e.g., 3 seconds). Alternatively, the second image I2 is an image captured by the imaging device 1 during the second (3-6 second) capture of fluorescence generated when the imaging device 1 irradiates the sample with excitation light for a continuous exposure time (e.g., 300 seconds) at intervals of a constant exposure time (e.g., 3 seconds) during the initial measurement of the DNA microarray A being measured. Furthermore, the second image I2 may be an image captured by the imaging device 1 before acquiring the first image I1; the timing of capture and exposure time are not limited. Furthermore, the second image I2 may be an image captured by the imaging device 1 under the control of the image acquisition device 100 when the threshold determination process, described later, determines that the fluorescence intensity Q1 of the first image I1 is less than a predetermined signal intensity or that the S / N ratio of the fluorescence intensity Q1 of the first image is less than a predetermined threshold.

[0053] (1-3-2-2. Light Intensity Calculation Processing)

[0054] The image acquisition system 1000 according to the embodiment performs a light quantity calculation process. First, the image acquisition system 1000 performs a first light quantity calculation process as a light quantity calculation process. That is, the image acquisition device 100 determines the fluorescent spot of the acquired first image I1 and calculates the fluorescence intensity (appropriately referred to as "first fluorescence intensity") Q1 of the fluorescent spot as a signal intensity. Second, the image acquisition system 1000 performs a second light quantity calculation process as a light quantity calculation process. That is, the image acquisition device 100 detects the fluorescent spot of the acquired second image I2 and calculates the fluorescence intensity (appropriately referred to as "second fluorescence intensity") Q2 of the fluorescent spot as a signal intensity. Here, the fluorescent spot determined by the above-mentioned first light quantity calculation process is within a specified range on the DNA microarray A where fluorescence generated by irradiating the fluorescent molecule F with excitation light is observed, and its shape, size, etc. are not particularly limited.

[0055] In this case, the image acquisition device 100 can detect fluorescent spots based on linear cross-sections of the first image I1 and the second image I2, and can determine whether the brightness or light intensity of the fluorescent spot portion is significantly higher than that of the background portion. Alternatively, fluorescent spots can be detected through image processing such as contour extraction or feature extraction. Furthermore, if the threshold determination processing described later determines that the fluorescence intensity Q1 of the first image I1 is less than a predetermined signal intensity, or that the S / N ratio of the fluorescence intensity Q1 of the first image is less than a predetermined threshold, the image acquisition device 100 can calculate the fluorescence intensity Q2 of the second image I2.

[0056] (1-3-2-3. Threshold Determination Processing)

[0057] The image acquisition system 1000 according to the embodiment performs threshold determination processing. Specifically, the image acquisition device 100 determines whether the fluorescence intensity Q1 of the first image I1 calculated by the first light intensity calculation processing is greater than or equal to a predetermined signal intensity. Furthermore, the image acquisition device 100 determines whether the signal-to-noise ratio (S / N) of the fluorescence intensity of the first image I1 is greater than or equal to a predetermined threshold (e.g., 0.3). The image acquisition device 100 terminates the processing if the fluorescence intensity Q1 of the first image I1 is greater than or equal to the predetermined signal intensity and the S / N ratio of the fluorescence intensity Q1 of the first image I1 is greater than or equal to the predetermined threshold. On the other hand, if the fluorescence intensity Q1 of the first image I1 is less than the predetermined signal intensity or the S / N ratio of the fluorescence intensity Q1 of the first image I1 is less than the predetermined threshold, the image acquisition device 100 performs the light intensity correction processing and image superposition processing described below.

[0058] Therefore, when the fluorescence intensity Q1 of the first image I1 is greater than or equal to a predetermined signal intensity and the S / N ratio of the fluorescence intensity Q1 of the first image I1 is greater than or equal to a predetermined threshold, it can be determined that the fluorescence intensity Q1 of the first image I1 is sufficient for qualitative analysis, quantitative analysis, etc. On the other hand, when the fluorescence intensity Q1 of the first image I1 is less than the predetermined signal intensity or the S / N ratio of the fluorescence intensity Q1 of the first image I1 is less than a predetermined threshold, it can be determined that the fluorescence intensity Q1 of the first image I1 is insufficient for qualitative analysis, quantitative analysis, etc., and thus correction of the first image I1 is necessary.

[0059] (1-3-2-4. Light Intensity Correction Processing)

[0060] The image acquisition system 1000 according to the embodiment performs light intensity correction processing. Specifically, the image acquisition device 100 corrects the images so that the fluorescence intensity Q1 of the first image I1 and the fluorescence intensity Q2 of the second image I2 are equal. In this process, the image acquisition device 100 maintains a constant background light intensity based on the difference between the background light and the fluorescence intensity of the fluorescent spots. The image acquisition device 100 then generates a corrected second image I2' by correcting the fluorescence intensity Q2 of the fluorescent spots in the second image I2 to account for the decrease in fluorescence intensity due to photobleaching.

[0061] (1-3-2-5. Image overlay processing)

[0062] The image acquisition system 1000 according to the embodiment performs image superposition processing. Specifically, the image acquisition device 100 superimposes a corrected second image I2' on the first image I1 to generate a superimposed corrected fluorescence image I having a signal intensity and S / N ratio sufficient for analysis. This corrected second image I2' is obtained by correcting the fluorescence intensity Q2 using the aforementioned light intensity correction processing. Furthermore, if the fluorescence intensity Q of the superimposed corrected fluorescence image I is less than a predetermined signal intensity or the S / N ratio of the superimposed corrected fluorescence image I is less than a predetermined threshold, the image acquisition device 100 repeatedly performs the image acquisition processing, light intensity correction processing, and image superposition processing.

[0063] (1-4. Effects of Image Acquisition System 1000)

[0064] Next, after describing an overview of the image acquisition system 1000 according to the embodiment, the effects of the image acquisition system 1000 will be described.

[0065] (1-4-1. Summary)

[0066] Regarding the image acquisition system 1000, the image acquisition device 100 acquires a first image I1 obtained by photographing the DNA microarray A using the photographing device 1 and a second image I2 obtained by photographing the DNA microarray A using the photographing device 1, and superimposes the corrected second image I2' on the first image I1, thereby generating a superimposed corrected fluorescent image I that corrects the first image I1. The corrected second image I2' is obtained by correcting the fluorescence intensity Q2 of the fluorescent spot in the second image I2 based on the fluorescence intensity Q1 of the fluorescent spot in the first image I1.

[0067] Specifically, image acquisition system 1000 is a technology that corrects the effects of photobleaching on fluorescent molecules F used to detect target molecules N based on DNA microarray A, thereby performing molecular quantification. Furthermore, image acquisition system 1000 is a technology that acquires multiple images with a short exposure time during shooting that minimizes the effects of photobleaching, and performs light intensity correction based on the initially acquired image data.

[0068] (1-4-2. Effect)

[0069] First, image acquisition system 1000 can reduce quantitative errors in the number of molecules due to photobleaching, thereby improving the accuracy of detection of target molecules N using DNA microarray methods. Second, image acquisition system 1000 can shorten the total measurement time required to achieve the same S / N ratio. Third, image acquisition system 1000 can provide the same quantitative results between images with different exposure times, enabling comparison of the results.

[0070] [2. Configuration of Each Device in Image Acquisition System 1000]

[0071] use Figures 4 to 7 right Figure 3 The functional configuration of each device in the image acquisition system 1000 will be described. Hereinafter, a configuration example of the image acquisition device 100 according to the embodiment, a configuration example of the imaging device 1, and a specific example of the fluorescence intensity of the fluorescence image will be described in detail.

[0072] (2-1. Configuration Example of Image Acquisition Device 100)

[0073] First, use Figure 4 right Figure 3 The configuration example of the image acquisition device 100 shown in FIG. Figure 4 This is a block diagram showing an example configuration of an image acquisition device 100 and an imaging device 1 according to an embodiment. The image acquisition device 100 includes a communication unit 110, a storage unit 120, and a control unit 130. Furthermore, the image acquisition device 100 may include an input unit (e.g., a keyboard, a mouse, etc.) for accepting various operations from an administrator of the image acquisition device 100, and a display unit (e.g., a liquid crystal display, etc.) for displaying various information.

[0074] (2-1-1. Communications Department 110)

[0075] The communication unit 110 is responsible for data communication with other devices. For example, the communication unit 110 communicates data with each communication device via a router, etc. The communication unit 110 can also communicate data with an operator's terminal (not shown).

[0076] (2-1-2. Storage Unit 120)

[0077] The storage unit 120 stores various information that the control unit 130 refers to when performing an action, and various information that the control unit 130 obtains when performing an action. Here, the storage unit 120 can be implemented by, for example, a semiconductor memory element such as RAM (Random Access Memory), a flash memory, or a storage device such as a hard disk or an optical disk. Figure 4 In the example shown in FIG. , the storage unit 120 is provided inside the image acquisition device 100 , but may be provided outside the image acquisition device 100 , or a plurality of storage units may be provided.

[0078] The storage unit 120 stores image data acquired by the acquisition unit 131 of the control unit 130, which will be described later. For example, the storage unit 120 stores image data such as the first fluorescent image (first image) I1, the second fluorescent image (second image) I2, and the third fluorescent image (third image) I3 acquired from the imaging device 1.

[0079] The storage unit 120 also stores image data generated by the generation unit 132 of the control unit 130 described later. For example, the storage unit 120 stores image data such as a second fluorescence image (corrected second image) I2' obtained by correcting the fluorescence intensity Q2 and a superimposed corrected fluorescence image I.

[0080] (2-1-3. Control Unit 130)

[0081] The control unit 130 is responsible for overall control of the image acquisition device 100. The control unit 130 includes an acquisition unit 131 and a generation unit 132. The control unit 130 can be implemented, for example, by an electronic circuit such as a CPU (Central Processing Unit) or an MPU (MicroProcessing Unit), or an integrated circuit such as an ASIC (Application Specific Integrated Circuit) or an FPGA (Field Programmable Gate Array).

[0082] (2-1-3-1. Acquisition Unit 131)

[0083] The acquisition unit 131 acquires various images captured by the imaging device 1. In addition, the acquisition unit 131 may store the acquired various images in the storage unit 120.

[0084] The acquisition unit 131 acquires a first image I1 captured by the imaging device 1 during a first period of time of the DNA microarray A serving as the measurement target sample, and a second image I2 captured by the imaging device 1 during a second period of time of the DNA microarray A. For example, the acquisition unit 131 acquires, as the first period, the first image I1 captured by the imaging device 1 at the first predetermined exposure time (e.g., 3 seconds of excitation light irradiation time) of the DNA microarray A. Furthermore, the acquisition unit 131 acquires, as the second period, the second image I2 captured by the imaging device 1 at each predetermined exposure time (e.g., 3 seconds of excitation light irradiation time) of the DNA microarray A from the second period onward. Similarly, the acquisition unit 131 acquires a third image I3 captured during a third period, a fourth image I4 captured during a fourth period, and so on. In addition, the first period, the second period, the third period, the fourth period, ... may not represent the chronological relationship, but rather the first image I1, the second image I2, the third image I3, the fourth image I4, ... may be selected from multiple images taken during any period.

[0085] To explain using a specific example, the acquisition unit 131 first acquires a fluorescence image obtained by imaging the DNA microarray A, serving as the measurement target sample, using the imaging device 1. This image is a first image I1 captured with a short exposure time of 3 seconds to avoid the effects of photobleaching caused by a long exposure time. Similarly, the acquisition unit 131 acquires a second image I2 captured with the same 3-second exposure time as the first image I1, which is second least affected by photobleaching; a third image I3 captured with the same 3-second exposure time as the first image I1; and so on.

[0086] The acquisition unit 131 acquires a first image I1 obtained by capturing the DNA microarray A using the imaging device 1 with a predetermined exposure time (e.g., 3 seconds of excitation light irradiation time). If the fluorescence intensity Q1 of the first image I1 is less than a predetermined signal intensity or less than a predetermined signal-to-noise ratio, the acquisition unit 131 acquires a second image I2 obtained by capturing the DNA microarray A using the imaging device 1 with a predetermined exposure time (e.g., 3 seconds of excitation light irradiation time). Furthermore, if the fluorescence intensity Q of the corrected first image I1, i.e., the superimposed corrected fluorescence image I, generated by the generation unit 132 (described later), is less than a predetermined signal intensity or less than a predetermined signal-to-noise ratio, the acquisition unit 131 acquires a third image I3, a fourth image I4, and so on.

[0087] To explain using a specific example, the acquisition unit 131 acquires a first image I1 captured by the imaging device 1 with an exposure time of 3 seconds of a DNA microarray A serving as a measurement target sample. If the S / N ratio of the fluorescence intensity Q1 of the first image I1 is less than 0.3, the acquisition unit 131 acquires a second image I2 captured with the same exposure time of 3 seconds as the first image I1. Furthermore, if the S / N ratio of the fluorescence intensity Q of the corrected first image I1, i.e., the superimposed corrected fluorescence image I, is less than 0.3, the acquisition unit 131 acquires a second image I2 captured with the same exposure time of 3 seconds as the first image I1. The acquisition unit 131 acquires a third image I3, a fourth image I4, and so on until the S / N ratio of the fluorescence intensity Q of the superimposed corrected fluorescence image I is greater than or equal to 0.3.

[0088] The acquisition unit 131 acquires a first image I1 and a second image I2 selected from a plurality of fluorescence images captured by the imaging device 1 at different times with a predetermined exposure time (e.g., 3 seconds of excitation light irradiation time) of the DNA microarray A. Similarly, the acquisition unit 131 acquires a third image I3, a fourth image I4, ..., selected from a plurality of fluorescence images captured by the imaging device 1 at different times with a predetermined exposure time (e.g., 3 seconds of excitation light irradiation time).

[0089] To illustrate using a specific example, the acquisition unit 131 acquires 100 fluorescence images (the first image I1, the second image I2, the third image I3, ..., the 100th image I100) obtained by photographing the DNA microarray A, which is the sample to be measured, every 3 seconds using the photographing device 1 with a continuous exposure time of 300 seconds, and selects the fluorescence image acquired during the period of 0 to 3 seconds as the first image I1, the fluorescence image acquired during the period of 3 to 6 seconds as the second image I2, and the fluorescence image acquired during the period of 6 to 9 seconds as the third image I3, ...

[0090] (2-1-3-2. Generation Unit 132)

[0091] The generating unit 132 performs processing related to correction of various images acquired by the acquiring unit 131. Furthermore, the generating unit 132 may store the calculated fluorescence intensity, the image after correction of the fluorescence intensity, and the generated image in the storage unit 120.

[0092] The generating unit 132 generates a corrected second image I2, i.e., a corrected second image I2′, by correcting the fluorescence intensity Q2 (second fluorescence intensity) of the fluorescent spot emitting fluorescence in the second image I2 based on the fluorescence intensity Q1 (first fluorescence intensity) of the fluorescent spot emitting fluorescence in the first image I1, and superimposes the corrected second image I2′ on the first image I1 to generate a superimposed corrected fluorescence image I obtained by correcting the first image I1. For example, the generating unit 132 calculates the fluorescence intensity Q1 and the fluorescence intensity Q2. When the fluorescence intensity Q1 is less than a specified signal intensity or less than a specified signal-to-noise ratio, the difference between the fluorescence intensity Q2 and the intensity of the background light of the second image I2 is used to make the background light of the second image I2 equal to the background light of the first image I1. The fluorescence intensity Q2 is corrected by multiplying it by the inverse of the ratio of the fluorescence intensity reduction between the fluorescence intensity Q1 and the fluorescence intensity Q2. The corrected fluorescence intensity Q2' is superimposed on the fluorescence intensity Q1, thereby generating a superimposed corrected fluorescence image I in which the fluorescence intensity of the fluorescent spot is greater than or equal to the specified signal intensity and greater than or equal to the specified signal-to-noise ratio.

[0093] To illustrate with a specific example, if the fluorescence intensity Q1 of the first image I1 is 110 (arbitrary units) and the background light intensity is 10 (arbitrary units), and the fluorescence intensity Q2 of the second image I2 is 100 (arbitrary units), and the background light intensity is 10 (arbitrary units), the difference between the fluorescence intensity and the background light intensity is 100 (arbitrary units) for the first image I1 and 90 (arbitrary units) for the second image I2. Therefore, the fluorescence intensity Q2 is corrected by multiplying it by 100 / 90, the inverse of the ratio of decrease in the fluorescence intensity between the first image I1 and the second image I2, 90 / 100. The corrected fluorescence intensity Q2' is then superimposed on the fluorescence intensity Q1. While the generation unit 132 preferably performs nonlinear correction to maintain a constant background light intensity when correcting the fluorescence intensity, linear correction may also be performed.

[0094] Furthermore, if the exposure times are the same, the background light in each image can be assumed to be equal. On the other hand, if the exposure times are different, the difference between the fluorescence intensity and the background light in each image is calculated after multiplying by the inverse of the exposure time ratio. The background light is always considered to have a signal intensity of zero, and this difference is used to correct the fluorescence intensity. This allows for comparison of quantitative results between images.

[0095] (2-2. Configuration Example of Image Capturing Device 1)

[0096] use Figure 4 right Figure 3 The imaging device 1 includes a light source 10 , an objective lens 11 , a dichroic mirror 12 , a filter 13 , a lens 14 , and a light receiving element 15 .

[0097] (2-2-1. Light Source 10)

[0098] The light source 10 is a laser light source that irradiates the installed DNA microarray A with laser light. For example, the light source 10 can be implemented by a laser light source that emits single-wavelength laser light or expanded beam light of the laser light, an LED (Light Emitting Diode), a lamp that emits white light, or a light source composed of a combination of an LED and a wavelength filter.

[0099] (2-2-2. Objective lens 11)

[0100] The objective lens 11 transmits or focuses excitation light emitted from the light source 10 during imaging and guides it toward the DNA microarray A. The objective lens 11 also transmits or focuses fluorescence generated from the DNA microarray A and guides it toward the light receiving element 15 via the dichroic mirror 12 , filter 13 , and lens 14 .

[0101] (2-2-3. Dichroic Mirror 12)

[0102] The dichroic mirror 12 reflects the excitation light emitted from the light source 10 during imaging and guides it to the DNA microarray A via the objective lens 11 . The dichroic mirror 12 also transmits the fluorescence generated from the DNA microarray A and guides it to the light receiving element 15 via the filter 13 and the lens 14 .

[0103] (2-2-4. Filter 13)

[0104] The filter 13 transmits the fluorescence generated from the DNA microarray A and guides it to the light receiving element 15 via the lens 14. At this time, the filter 13 removes the reflection of the laser light as the irradiation light and transmits only the fluorescence.

[0105] (2-2-5. Lens 14)

[0106] The lens 14 transmits or converges the fluorescence generated from the DNA microarray A and guides it toward the light receiving element 15 .

[0107] (2-2-6. Light Receiving Element 15)

[0108] The light receiving element 15 converts the fluorescence formed by the lens 14 into an electrical signal. For example, the light receiving element 15 can be realized by an EM (Electron Multiplying)-CCD, a CMOS (Complementary Metal-Oxide-Semiconductor), or the like.

[0109] (2-3. Specific Example of Fluorescence Intensity of Fluorescence Image)

[0110] use Figures 5 to 7A specific example of the fluorescence intensity of the fluorescence image according to the embodiment will be described. Figures 5 to 7 Detailed description is given below of a specific example of fluorescence intensity of a fluorescence image according to an embodiment. Specific example 1 relating to correction of a fluorescence image, specific example 2 and specific example 3 showing effectiveness in comparison with reference techniques, etc. will be described in this order.

[0111] (2-3-1. Correction of Fluorescence Images)

[0112] use Figure 5 A specific example 1 related to correction of a fluorescence image according to the embodiment will be described. Figure 5 As shown in (1), the fluorescence intensity (arbitrary unit) decreases over time due to photobleaching of the fluorochrome, so correction based on the decrease in fluorescence intensity is performed for each image. Figure 5 As shown in (2), it is possible to obtain Figure 5 (1) The equivalent light intensity from 0 seconds to 180 seconds is used as an image of the fluorescence intensity without the influence of photobleaching of the fluorochrome.

[0113] (2-3-2. Effectiveness of Image Acquisition Processing)

[0114] use Figure 6 and Figure 7 Specific Examples 2 and 3 related to the effectiveness of image acquisition processing according to the embodiment will be described. The following describes the experimental process for confirming the effectiveness of image acquisition processing according to the embodiment, followed by a description of the comparison results with reference techniques, photobleaching measurement results, and an examination of the comparison results.

[0115] (2-3-2-1. Experimental process)

[0116] The following describes an experimental procedure for confirming the effectiveness of the image acquisition process according to the embodiment. In the following experimental procedure, a DNA molecule as a detection target molecule N labeled with a Cy3 fluorescent molecule as a fluorescent molecule F is detected.

[0117] First, genomic DNA extracted from Staphylococcus aureus strain (NBRC12732) was used as a template, and PCR amplification was performed using Cy3-modified primers capable of amplifying the 16s ribosomal DNA (16s rDNA) region (Experimental Procedure 1).

[0118] Secondly, as a test sample S containing the detection target molecule N, which is a PCR product diluted to 1 nM, a detection probe P for the Staphylococcus aureus 16s rDNA sequence is provided (supplied) to the immobilized DNA microarray A (experimental flow 2).

[0119] Third, the DNA microarray A was incubated (kept warm) at 66° C. for 1 hour to allow the detection target molecule N to hybridize with the detection probe P (experimental flow 3).

[0120] Fourthly, the DNA microarray A was washed with a 2x concentration SSC (Standard Saline Citrate) solution and a 1x concentration SSC solution, and then dried (experimental process 4).

[0121] Fifth, the 2nd to 100th images were corrected based on the first image, and all images were superimposed (experimental process 5).

[0122] Sixth, as comparative data with the reference technique, one image of the DNA microarray A reacted according to the above-mentioned experimental procedures 1 to 4 was captured with an exposure time of 300 seconds (experimental procedure 6).

[0123] (2-3-2-2. Comparison results with reference technologies, etc.)

[0124] use Figure 6 , as a specific example 2 related to the effectiveness of image acquisition processing according to the embodiment, the comparison results with reference technologies etc. are described. Figure 6 (1) As an example of image acquisition processing according to the embodiment, the fluorescence intensity (arbitrary unit 10482) of an image obtained by correcting the fluorescence image acquired with an exposure time of 3 seconds and superimposing 100 images is shown. Figure 6 (2) As an image acquisition process related to the reference technique, the fluorescence intensity (arbitrary unit 8458) of an image acquired with a continuous exposure time of 300 seconds is shown.

[0125] (2-3-2-3. Photobleaching Measurement Results)

[0126] use Figure 7 As a specific example 3 related to the effectiveness of the image acquisition process according to the embodiment, the measurement results of photobleaching are described. Figure 7 As shown, the fluorescence intensity of about 110 (arbitrary unit) gradually decreases within the measurement time of 0 seconds, and becomes a fluorescence intensity of about 80 (arbitrary unit) within the measurement time of 300 seconds.

[0127] (2-3-2-4. Examination of comparison results)

[0128] As described above, the total fluorescence intensity calculated by the image acquisition processing according to the embodiment, that is, the total fluorescence intensity of the detection target molecules N captured on the DNA microarray A, is about 20% higher than the total fluorescence intensity calculated by the image acquisition processing according to the reference technology. Therefore, the image acquisition processing according to the embodiment can Figure 7 The fluorescence intensity of the fluorescent spots shown is corrected for the effects of photobleaching relative to the assay time.

[0129] [3. Processing Flow of Image Acquisition System 1000]

[0130] use Figure 8 The processing flow of the image acquisition system 1000 according to the embodiment will be described. Figure 8 This is a flowchart showing an example of the flow of image acquisition processing according to the embodiment. In addition, the processing of steps S101 to S107 described below may be performed in a different order. In addition, some of the processing of steps S101 to S107 described below may be omitted.

[0131] (3-1. First Image Acquisition Process)

[0132] First, the image acquisition device 100 executes a first image acquisition process (step S101 ). For example, the image acquisition device 100 acquires a first image I1 of the DNA microarray A captured by the imaging device 1 .

[0133] (3-2. First Light Intensity Calculation Process)

[0134] Secondly, the image acquisition device 100 executes a first light intensity calculation process (step S102 ). For example, the image acquisition device 100 identifies a fluorescent spot in the acquired first image I1 and calculates the fluorescence intensity Q1 of the fluorescent spot as the signal intensity.

[0135] (3-3. Threshold Determination Processing)

[0136] Third, the image acquisition device 100 performs threshold determination processing (step S103). For example, the image acquisition device 100 determines whether the fluorescence intensity Q1 of the first image I1 is greater than or equal to a predetermined signal intensity and whether the S / N ratio of the fluorescence intensity Q1 of the first image I1 is greater than or equal to a predetermined threshold. If the fluorescence intensity Q1 is greater than or equal to the predetermined signal intensity and the S / N ratio of the fluorescence intensity Q1 of the first image I1 is greater than or equal to the predetermined threshold (step S103: Yes), the image acquisition device 100 terminates the image acquisition process. On the other hand, if the fluorescence intensity Q1 is less than the predetermined signal intensity or the S / N ratio of the fluorescence intensity Q1 of the first image I1 is less than the predetermined threshold (step S103: No), the image acquisition device 100 proceeds to step S104.

[0137] (3-4. Second Image Acquisition Process)

[0138] Fourthly, the image acquisition device 100 executes a second image acquisition process (step S104 ). For example, the image acquisition device 100 acquires a second image I2 of the DNA microarray A captured by the imaging device 1 .

[0139] (3-5. Second Light Intensity Calculation Process)

[0140] Fifth, the image acquisition device 100 executes a second light intensity calculation process (step S105 ). For example, the image acquisition device 100 identifies a fluorescent spot in the acquired second image I2 and calculates the fluorescence intensity Q2 of the fluorescent spot as the signal intensity.

[0141] (3-6. Light Intensity Correction Processing)

[0142] Sixth, the image acquisition device 200 performs light intensity correction processing (step S106 ). For example, the image acquisition device 200 corrects the image so that the fluorescence intensity Q1 of the first image I1 and the fluorescence intensity Q2 of the second image I2 are equal.

[0143] (3-7. Image Overlay Processing)

[0144] Seventh, the image acquisition device 200 performs image superposition processing (step S107), and then returns to the process of step S103. For example, the image acquisition device 100 superimposes the corrected second image I2', which has been corrected for the fluorescence intensity Q2, with the first image I1 to generate a superimposed corrected fluorescence image I having a sufficient signal intensity and S / N ratio for analysis.

[0145] [4. Effects of Implementation]

[0146] Finally, the effects of the embodiment will be described. Next, effects 1 to 5 corresponding to the processing according to the embodiment will be described.

[0147] (4-1. Effect 1)

[0148] First, in the process according to the above-described embodiment, the image acquisition device 200 acquires a first image I1 obtained by imaging the DNA microarray A during a first period using the imaging device 1, and a second image I2 obtained by imaging the DNA microarray A during a second period using the imaging device 1. The image acquisition device 200 generates a corrected second image I2' by correcting the fluorescence intensity Q2 of the fluorescent spots in the second image I2 based on the fluorescence intensity Q1 of the fluorescent spots in the first image I1. The corrected second image I2' is superimposed on the first image I1 to generate a superimposed corrected fluorescence image I obtained by correcting the first image I1. Therefore, the process according to the embodiment enables the acquisition of fluorescence images with high detection accuracy in the DNA microarray method.

[0149] (4-2. Effect 2)

[0150] Second, in the processing according to the above-described embodiment, the image acquisition device 200 calculates fluorescence intensity Q1 and fluorescence intensity Q2. If fluorescence intensity Q1 is less than a predetermined signal intensity or less than a predetermined signal-to-noise ratio, the background light of the second image I2 is equalized to that of the first image I1 using the difference between fluorescence intensity Q2 and the intensity of the background light of the second image I2. Fluorescence intensity Q2 is corrected by multiplying it by the inverse of the ratio of decrease in fluorescence intensity Q2 and the fluorescence intensity of the second image I2. The corrected fluorescence intensity Q2 is superimposed on fluorescence intensity Q1, thereby generating a superimposed corrected fluorescence image I in which the fluorescence intensity of the fluorescent spot is greater than or equal to a predetermined signal intensity and greater than or equal to a predetermined signal-to-noise ratio. Therefore, the processing according to the embodiment makes it possible to acquire a fluorescence image with high detection accuracy that satisfies a sufficient signal intensity and S / N ratio for analysis in a DNA microarray method.

[0151] (4-3. Effect 3)

[0152] Third, in the process according to the above-described embodiment, the image acquisition device 200 acquires a first image I1 obtained by imaging the DNA microarray A using the imaging device 1 at the first predetermined exposure time during the first period, and acquires a second image I2 obtained by imaging the DNA microarray A using the imaging device 1 at the second and subsequent predetermined exposure times during the second period. Therefore, the process according to the embodiment enables the acquisition of fluorescent images with higher detection accuracy in the DNA microarray method by acquiring multiple fluorescent images captured at the same exposure time.

[0153] (4-4. Effect 4)

[0154] Fourth, in the process according to the above embodiment, the image acquisition device 200 acquires a first image I1 of the DNA microarray A captured by the imaging device 1 with a predetermined exposure time. When the fluorescence intensity Q1 is less than a predetermined signal intensity or less than a predetermined signal-to-noise ratio, the image acquisition device 200 acquires a second image I2 of the DNA microarray A captured by the imaging device 1 with a predetermined exposure time. Therefore, the process according to the embodiment enables the acquisition of fluorescence images with high detection accuracy in the DNA microarray method by acquiring multiple fluorescence images in real time.

[0155] (4-5. Effect 5)

[0156] Fifth, in the process according to the above embodiment, the image acquisition device 200 acquires a first image I1 and a second image I2 selected from a plurality of fluorescence images obtained by imaging device 1 at a plurality of different times and at predetermined exposure intervals of the DNA microarray A. Therefore, the process according to the embodiment enables the acquisition of fluorescence images with higher detection accuracy in the DNA microarray method by batch-acquiring a plurality of fluorescence images.

[0157] [Application Examples of Embodiments]

[0158] Hereinafter, application examples of the embodiment will be described. However, the present invention is not limited to the application examples of the embodiment described below.

[0159] The embodiment can be applied to biopolymers such as nucleic acids, sugar chains, and proteins as specific target molecules.

[0160] The embodiment can be applied to image acquisition and analysis based on fluorescence, chemiluminescence, and colorimetry using DNA microarrays.

[0161] The embodiment can be applied to nucleic acid sequence measurement equipment in which signal sources such as fluorescent molecules are modified by probes fixed in a solid state on a substrate in a DNA microarray, etc., non-modified target detection based on signal array probes, and microarray detection of nucleic acid sequence measurement equipment that does not require cleaning.

[0162] The embodiment can be applied to detection in a nucleic acid sequence measurement device that captures and observes a target molecule in which a signal generator such as a fluorescent molecule is modified by a probe solid-state immobilized on a substrate in a DNA microarray or the like.

[0163] The embodiment can be applied to the detection of nucleic acid sequence measurement equipment that captures and observes target molecules in DNA microarrays, etc., wherein the target molecules are combined with labeled molecules that are modified signal generators such as fluorescent molecules that specifically bind to the target molecules in a probe fixed in a solid state on a substrate.

[0164] When nucleic acid is used as a target, the embodiment can be applied to gene expression analysis, genotyping, and base sequence analysis.

[0165] The embodiment can be applied to sugar chain profile analysis when sugar chains are used as targets.

[0166] When proteins are used as targets, the embodiments can be applied to antibody profiling and protein-protein interaction analysis.

[0167] As industrial applications, the implementation methods can be applied to drug development, biomarker detection, disease diagnosis, disease prevention / prognosis management, biomarker exploration, development of diagnostic methods, antibody antigen exploration, plant variety discrimination, genetic identification detection of agricultural and livestock products, genetic recombination tests of foods, allergen display tests, microbial detection / microbial identification in environmental sanitation inspections, microbial detection in product quality management, monitoring of fermentation processes, strain management in fermentation processes, exploration of useful bacteria in microbial materials, and evaluation of the properties of harmful bacteria in pharmaceutical / food manufacturing processes.

[0168] The embodiment can be applied to a technique for acquiring a fluorescence image based on absolute light quantity detection.

[0169] [system]

[0170] Information including the processing flow, control flow, specific names, various data, and parameters described above and shown in the drawings can be arbitrarily changed unless otherwise specified.

[0171] Furthermore, the components of the devices shown in the diagrams are functional concepts and do not necessarily need to be physically configured as shown. That is, the specific distribution and integration of the devices is not limited to that shown. In other words, all or part of the devices can be functionally or physically distributed / integrated in arbitrary units based on various loads, usage conditions, and the like.

[0172] Furthermore, all or any part of each processing function performed by each device may be realized by a CPU and a program analyzed and executed by the CPU, or may be realized as hardware based on wired logic.

[0173] [hardware]

[0174] Next, a hardware configuration example of the image acquisition device 100 will be described. Figure 9 FIG is a diagram showing an example of a hardware configuration according to an embodiment of the present invention. Figure 9 As shown, the image acquisition device 100 includes a communication device 100a, a HDD (Hard Disk Drive) 100b, a memory 100c, and a processor 100d. Figure 9The components shown are connected to each other by a bus or the like.

[0175] The communication device 100a is a network interface card, etc., and performs communication with other server devices. Figure 4 The program that executes the functions shown is stored in the DB.

[0176] The processor 100d will execute the Figure 4 The program for the same processing as that of each processing unit shown is read from the HDD 100b or the like and expanded in the memory 100c, thereby executing the same processing as that of the processing unit shown in FIG. Figure 4 The processes for the functions described in [ 131 ] and [ 132 ] execute actions. For example, this process performs the same functions as the processing units included in the image acquisition device 100. Specifically, the processor 100d reads a program having the same functions as the acquisition unit 131, the generation unit 132, etc. from the HDD 100b, etc. The processor 100d then executes a process that performs the same processing as the acquisition unit 131, the generation unit 132, etc.

[0177] In this manner, the image acquisition device 100 operates as a device that performs various processing methods by reading and executing programs. Furthermore, the image acquisition device 100 can also read the programs from a recording medium using a media reader and execute the read programs, thereby achieving the same functions as the above-described embodiments. Furthermore, the programs described in the other embodiments are not limited to being executed by the image acquisition device 100. For example, the present invention can also be applied to other computers or servers executing the programs, or to other computers or servers operating in concert to execute the programs.

[0178] The program can be distributed via a network such as the Internet. In addition, the program can be recorded on a computer-readable recording medium such as a hard disk, a floppy disk (FD), a CD-ROM, an MO (Magneto-Optical Disk), or a DVD (Digital Versatile Disc), and executed by the computer after reading from the recording medium.

[0179] [other]

[0180] Several examples of combinations of disclosed technical features are described below.

[0181] (1) An image acquisition device, wherein the image acquisition device comprises: an acquisition unit that acquires a first fluorescent image and a second fluorescent image, the first fluorescent image being an image obtained by photographing a measurement target sample using a photographing device during a first period, and the second fluorescent image being an image obtained by photographing the measurement target sample using the photographing device during a second period; and a generation unit that generates a corrected fluorescent image by correcting a second fluorescent intensity of a fluorescent spot generating fluorescence in the second fluorescent image based on a first fluorescent intensity of a fluorescent spot generating fluorescence in the first fluorescent image, and superimposes the corrected fluorescent image on the first fluorescent image to generate a superimposed corrected fluorescent image obtained by correcting the first fluorescent image.

[0182] (2) An image acquisition device according to (1), wherein the generating unit calculates the first fluorescence intensity and the second fluorescence intensity, and when the first fluorescence intensity is less than a specified signal intensity or less than a specified signal-to-noise ratio, uses the difference between the second fluorescence intensity and the intensity of the background light of the second fluorescence image to make the background light of the second fluorescence image equal to the background light of the first fluorescence image, corrects the second fluorescence intensity by multiplying it by the inverse of the ratio of the fluorescence intensity reduction between the first fluorescence intensity and the second fluorescence intensity, and superimposes the corrected second fluorescence intensity on the first fluorescence intensity, thereby generating the superimposed corrected fluorescence image in which the fluorescence intensity of the fluorescent spot is greater than or equal to the specified signal intensity and greater than or equal to the specified signal-to-noise ratio.

[0183] (3) An image acquisition device according to (1) or (2), wherein the first period is a prescribed exposure time for the first time when the image capturing device is used to capture the sample to be measured, and the second period is each prescribed exposure time thereafter when the image capturing device is used to capture the sample to be measured.

[0184] (4) An image acquisition device according to any one of (1) to (3), wherein the acquisition unit acquires the first fluorescence image obtained by photographing the measurement object sample using the photographing device with a prescribed exposure time, and when the first fluorescence intensity is less than a prescribed signal intensity or less than a prescribed signal-to-noise ratio, acquires the second fluorescence image obtained by photographing the measurement object sample using the photographing device with the prescribed exposure time.

[0185] (5) An image acquisition device according to any one of (1) to (4), wherein the acquisition unit acquires the first fluorescent image and the second fluorescent image selected from a plurality of fluorescent images obtained by photographing the measurement object sample at a plurality of different times at predetermined exposure times using the photographing device.

[0186] (6) The image acquisition device according to any one of (1) to (5), wherein the measurement target sample is a DNA microarray.

[0187] (7) An image acquisition method, wherein the image acquisition method is performed by a computer to perform the following processing: acquiring a first fluorescent image and a second fluorescent image, wherein the first fluorescent image is an image obtained by photographing a measurement target sample using a photographing device during a first period, and the second fluorescent image is an image obtained by photographing the measurement target sample using the photographing device during a second period, generating a corrected fluorescent image obtained by correcting a second fluorescent intensity of a fluorescent spot in the second fluorescent image based on a first fluorescent intensity of a fluorescent spot in the first fluorescent image, and superimposing the corrected fluorescent image on the first fluorescent image to generate a superimposed corrected fluorescent image obtained by correcting the first fluorescent image.

[0188] (8) An image acquisition program, wherein the image acquisition program causes a computer to perform the following processing: acquiring a first fluorescent image and a second fluorescent image, wherein the first fluorescent image is an image obtained by photographing a measurement target sample during a first period using a photographing device, and the second fluorescent image is an image obtained by photographing the measurement target sample during a second period using the photographing device, generating a corrected fluorescent image obtained by correcting a second fluorescent intensity of a fluorescent spot in the second fluorescent image based on a first fluorescent intensity of a fluorescent spot in the first fluorescent image, and superimposing the corrected fluorescent image on the first fluorescent image to generate a superimposed corrected fluorescent image obtained by correcting the first fluorescent image.

[0189] Description of the label

[0190] 1. Camera

[0191] 10 Light Source

[0192] 11 Objective lens

[0193] 12 dichroic mirrors

[0194] 13 Filter

[0195] 14 Lens

[0196] 15 Light receiving element

[0197] 100 Image acquisition device

[0198] 110 Communications Department

[0199] 120 Storage Department

[0200] 130 Control Department

[0201] 131 Acquisition Department

[0202] 132 Generation Department

[0203] 1000 Image Acquisition System

Claims

1. An image acquisition device, wherein: The image acquisition device comprises: an acquisition unit configured to acquire a first fluorescent image obtained by imaging the sample to be measured using an imaging device during a first period, and a second fluorescent image obtained by imaging the sample to be measured using the imaging device during a second period; as well as a generating unit that generates a corrected fluorescence image by correcting the second fluorescence intensity of the fluorescent spot generating fluorescence in the second fluorescence image based on the first fluorescence intensity of the fluorescent spot generating fluorescence in the first fluorescence image, and superimposes the corrected fluorescence image on the first fluorescence image to generate a superimposed corrected fluorescence image obtained by correcting the first fluorescence image.

2. The image acquisition device according to claim 1, wherein: The generating unit calculates the first fluorescence intensity and the second fluorescence intensity, and when the first fluorescence intensity is less than a predetermined signal intensity or less than a predetermined signal-to-noise ratio, uses a difference between the second fluorescence intensity and the intensity of the background light of the second fluorescent image to make the background light of the second fluorescent image equal to the background light of the first fluorescent image, and corrects the second fluorescence intensity by multiplying it by the inverse of a ratio of fluorescence intensity reduction between the first fluorescence intensity and the second fluorescence intensity. The corrected second fluorescence intensity is superimposed on the first fluorescence intensity, thereby generating the superimposed corrected fluorescence image in which the fluorescence intensity of the fluorescent spot is greater than or equal to the predetermined signal intensity and greater than or equal to the predetermined signal-to-noise ratio.

3. The image acquisition device according to claim 1, wherein: The first period is a predetermined exposure time for the first time when the imaging device is used to image the measurement target sample. The second period is each predetermined exposure time for the second and subsequent times when the imaging device captures the measurement target sample.

4. The image acquisition device according to claim 1, wherein: The acquisition unit acquires the first fluorescent image obtained by imaging the measurement target sample with a predetermined exposure time using the imaging device, When the first fluorescence intensity is lower than a predetermined signal intensity or lower than a predetermined signal-to-noise ratio, the second fluorescence image is acquired by imaging the measurement target sample with the imaging device at the predetermined exposure time.

5. The image acquisition device according to claim 1, wherein: The acquisition unit acquires the first fluorescent image and the second fluorescent image selected from a plurality of fluorescent images obtained by imaging the measurement target sample at a plurality of different times at predetermined exposure times using the imaging device.

6. The image acquisition device according to any one of claims 1 to 5, wherein: The sample to be measured is a DNA microarray.

7. A method for acquiring an image, wherein: The image acquisition method is performed by a computer as follows: acquiring a first fluorescent image and a second fluorescent image, wherein the first fluorescent image is an image captured by an imaging device during a first period of time and the second fluorescent image is an image captured by the imaging device during a second period of time; generating a corrected fluorescence image by correcting a second fluorescence intensity of a fluorescent spot generating fluorescence in the second fluorescence image based on the first fluorescence intensity of the fluorescent spot generating fluorescence in the first fluorescence image; The corrected fluorescence image is superimposed on the first fluorescence image to generate a superimposed corrected fluorescence image obtained by correcting the first fluorescence image.

8. An image acquisition program, wherein: The image acquisition program causes the computer to execute the following processing: acquiring a first fluorescent image and a second fluorescent image, wherein the first fluorescent image is an image captured by an imaging device during a first period of time and the second fluorescent image is an image captured by the imaging device during a second period of time; generating a corrected fluorescence image by correcting a second fluorescence intensity of a fluorescent spot generating fluorescence in the second fluorescence image based on the first fluorescence intensity of the fluorescent spot generating fluorescence in the first fluorescence image; The corrected fluorescence image is superimposed on the first fluorescence image to generate a superimposed corrected fluorescence image obtained by correcting the first fluorescence image.

Images