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

Abstract

To obtain fluorescence images with high detection accuracy. [Solution] The image acquisition device 100 acquires a first fluorescence image of DNA microarray A captured by imaging device 1 during a first period and a second fluorescence image of DNA microarray A captured by imaging device 1 during a second period. Based on the first fluorescence intensity of the fluorescence spots that emit fluorescence in the first fluorescence image, it corrects the second fluorescence intensity of the fluorescence spots that emit fluorescence in the second fluorescence image to generate a corrected second fluorescence image. The corrected second fluorescence image is added to the first fluorescence image to generate an additively corrected fluorescence image of the corrected first fluorescence image.

Description

Technical Field

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[0005] , , ,

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

Background Art

[0002] As a method for measuring a target having a nucleic acid sequence such as a specific deoxyribonucleic acid (DNA) contained in a test sample, a DNA microarray method using a DNA microarray is known. In the DNA microarray method, a detection target molecule modified with a fluorescent molecule in a test sample added to the DNA microarray is captured by a detection probe of the DNA microarray by a hybridization reaction (hybridization), and the detection target molecule is measured using this property. In the DNA microarray method, by calculating the luminance value or the amount of light of the DNA spot portion by image analysis of a fluorescent image, in addition to determining whether or not the detection target molecule is contained in the test sample, the amount of the detection target molecule contained in the test sample can be measured.

Prior Art Documents

Patent Documents

[0003]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0004] However, in the above DNA microarray method, it is difficult to obtain a fluorescent image with high detection accuracy. For example, in the above DNA microarray method, when the measurement time is set to a long time, an error may occur in the estimation result of the amount of the detection target molecule due to the photobleaching of the fluorescent dye.

[0005] The present invention has been made in view of the above, and an object thereof is to obtain a fluorescent image with high detection accuracy. [Means for solving the problem]

[0006] The present invention provides an image acquisition device comprising: an acquisition unit that acquires a first fluorescence image of a sample to be measured captured by an imaging device during a first period and a second fluorescence image of the sample to be measured captured by the imaging device during a second period; and a generation unit that generates a corrected fluorescence image by correcting the second fluorescence intensity of a fluorescence spot that emits fluorescence in the second fluorescence image based on the first fluorescence intensity of a fluorescence spot that emits fluorescence in the first fluorescence image, and adds the corrected fluorescence image to the first fluorescence image to generate an additively corrected fluorescence image of the first fluorescence image.

[0007] Furthermore, the present invention provides an image acquisition method in which a computer acquires a first fluorescence image of a sample to be measured captured by an imaging device during a first period and a second fluorescence image of the sample to be measured captured by the imaging device during a second period, generates a corrected fluorescence image by correcting the second fluorescence intensity of the fluorescence spots that emit fluorescence in the second fluorescence image based on the first fluorescence intensity of the fluorescence spots that emit fluorescence in the first fluorescence image, and adds the corrected fluorescence image to the first fluorescence image to generate an additively corrected fluorescence image of the corrected first fluorescence image.

[0008] Furthermore, the present invention provides an image acquisition program that causes a computer to acquire a first fluorescence image of a sample to be measured captured by an imaging device during a first period and a second fluorescence image of the sample to be measured captured by the imaging device during a second period, generate a corrected fluorescence image by correcting the second fluorescence intensity of the fluorescence spots that emit fluorescence in the second fluorescence image based on the first fluorescence intensity of the fluorescence spots that emit fluorescence in the first fluorescence image, and add the corrected fluorescence image to the first fluorescence image to generate an additively corrected fluorescence image of the corrected first fluorescence image. [Effects of the Invention]

[0009] According to the present invention, there is an effect that highly accurate fluorescence images can be obtained. [Brief explanation of the drawing]

[0010] [Figure 1] This figure shows an example of the processing flow for DNA microarray technology. [Figure 2] This is a diagram illustrating the fluorescence intensity of fluorescence images related to the reference technology. [Figure 3] This figure shows an example configuration of an image acquisition system according to the embodiment. [Figure 4] This is a block diagram showing an example configuration of an image acquisition device and an imaging device according to an embodiment. [Figure 5] This figure shows a specific example 1 of the fluorescence intensity of a fluorescence image according to the embodiment. [Figure 6] This figure shows a specific example 2 of the fluorescence intensity of a fluorescence image according to the embodiment. [Figure 7] This figure shows a specific example 3 of the fluorescence intensity of a fluorescence image according to the embodiment. [Figure 8] This flowchart shows an example of the image acquisition process according to the embodiment. [Figure 9] This figure shows an example of a hardware configuration according to the embodiment. [Modes for carrying out the invention]

[0011] An image acquisition device, an image acquisition method, and an image acquisition program according to one embodiment of the present invention will be described in detail below with reference to the drawings. However, the present invention is not limited to the embodiments described below.

[0012] [Embodiment] The configuration of the image acquisition system 1000 according to this embodiment, the configuration of the image acquisition device 100, etc., and the flow of each process will be described in order below, and finally the effects of the embodiment will be described.

[0013] [1. Configuration of the image acquisition system 1000] Using FIGS. 1 to 3, after explaining the processing flow of the DNA microarray method and the image acquisition processing according to the related art, the image acquisition system 1000 according to the embodiment will be described in detail. Note that, as the image acquisition system 1000 according to the embodiment, an example regarding the detection processing of a biological polymer measurement device that measures a specific detection target molecule among the biological polymers contained in a test sample by the DNA microarray method will be described, but the application range of the image acquisition system 1000 is not particularly limited.

[0014] (1-1. Processing flow of the DNA microarray method) Using FIG. 1, the processing flow of the DNA microarray method, which is a prerequisite for the image acquisition processing according to the related art and the image acquisition system 1000 according to the embodiment, will be described. FIG. 1 is a diagram showing an example of the processing flow of the DNA microarray method. As shown in FIG. 1, in the DNA microarray method, the following processes (1) to (4) are executed.

[0015] First, a detection target molecule N, which is a nucleic acid contained in a test sample S, is amplified by a nucleic acid amplification technique such as PCR (Polymerase Chain Reaction), and a fluorescent molecule F is added to the amplified detection target molecule N (see FIG. 1(1)). Second, a solution containing the detection target molecule N to which the fluorescent molecule F is added is added to the DNA microarray A, and the detection target molecule N fluorescently modified with the fluorescent molecule F is collected by hybridization with a detection probe P (see FIG. 1(2)). At this time, a complementary sequence of the nucleic acid of the detection target molecule N is fixed as the detection probe P on a solid phase surface such as the base of the DNA microarray A, and the fluorescently modified detection target molecule N binds to the detection probe P. Third, the DNA microarray A is washed to remove the fluorescent molecule F from the uncollected detection target molecule N and the molecules that non-specifically bind to the nucleic acid sequence of the detection probe P (see FIG. 1(3)). Fourth, the DNA microarray A is imaged by the imaging device 1, and a fluorescence image of the DNA microarray A in which the fluorescent molecule F is collected in the DNA spot is obtained (see FIG. 1(4)).

[0016] (1-2. Image acquisition process of the prior art) The image acquisition process according to the prior art will be described. Below, the processing example of the image acquisition process according to the prior art, the fluorescence intensity of the fluorescence image according to the prior art, and the problems of the image acquisition process according to the prior art will be described in this order.

[0017] (1-2-1. Processing example of the image acquisition process according to the prior art) In the image acquisition process according to the prior art, the following processes are executed. First, the imaging device irradiates the substrate of the DNA microarray A with excitation light having a wavelength corresponding to the fluorescent dye of the fluorescent molecule F, and detects the fluorescence generated from the DNA microarray A with a light receiving element. At this time, the imaging device adjusts the exposure time capable of acquiring a signal with a good S / N (appropriately, "signal-to-noise ratio") according to the fluorescence intensity within a range from several seconds to several minutes. Second, the image acquisition device calculates the fluorescence intensity of the detected fluorescence spots of the DNA microarray A, and estimates the amount of the detection target molecule N captured on the DNA microarray A.

[0018] (1-2-2. Fluorescence intensity of the fluorescence image according to the prior art) The fluorescence intensity of the fluorescence image according to the prior art will be described using FIG. 2. FIG. 2 is a diagram for explaining the fluorescence intensity of the fluorescence image according to the prior art. As shown in FIG. 2(1), the fluorescence intensity (arbitrary unit) decreases due to the photobleaching of the fluorescent dye over time. Note that FIG. 2(2) shows the equivalent light amount obtained by calculating the average value per unit time from the total value of the fluorescence intensity from 0 seconds to 180 seconds in FIG. 2(1).

[0019] (1-2-3. Problems of the image acquisition process according to the prior art) In the image acquisition process according to the prior art, when the amount of fluorescence generated from the DNA microarray A is low, it is necessary to set the measurement time to a long time. During this period, since the fluorescent dye is constantly irradiated with excitation light, the fluorescent dye undergoes photobleaching. Therefore, a large error occurs in the estimation result of the amount of the detection target molecule N captured on the substrate of the DNA microarray A. Furthermore, the longer the measurement time, the greater the influence of photobleaching, and the calculated amount of the detection target molecule N will be underestimated.

[0020] Furthermore, in the image acquisition process related to the reference technology, the effect of photobleaching varies depending on the density of fluorescent molecule F and the photoexposure history of the fluorescent dye, making it difficult to correct the signal after measurement using previously acquired photobleaching data.

[0021] (1-3. Image acquisition system 1000) The image acquisition system 1000 according to the embodiment will be described using Figure 3. Below, an example of the configuration of the image acquisition system 1000, an example of the processing of the image acquisition system 1000, and the effects of the image acquisition system 1000 will be described in that order.

[0022] (1-3-1. Example configuration of image acquisition system 1000) An example configuration of the image acquisition system 1000 according to the embodiment will be described using Figure 3. Figure 3 is a diagram showing an example configuration of the image acquisition system 1000 according to the embodiment. The image acquisition system 1000 includes an imaging device 1 and an image acquisition device 100. Note that the image acquisition system 1000 shown in Figure 3 may include multiple imaging devices 1 or multiple image acquisition devices 100. Furthermore, the image acquisition device 100 may be configured to be integrated with the imaging device 1.

[0023] 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 photodetector 15. The imaging device 1 also images the DNA microarray A, which is the sample to be measured (referred to as "the sample to be measured" as appropriate).

[0024] (1-3-2. Example of processing by image acquisition system 1000) Using Figure 3, an example of the processing of the image acquisition system 1000 according to the embodiment will be described. Below, the image acquisition process, light intensity calculation process, threshold determination process, light intensity correction process, and image addition process of the image acquisition system 1000 according to the embodiment will be described in that order.

[0025] (1-3-2-1. Image acquisition process) The image acquisition system 1000 according to this embodiment performs image acquisition processing. Below, the first image acquisition processing performed by the imaging device 1 during a first period and the second image acquisition processing performed by the imaging device 1 during a second period will be described.

[0026] (First image acquisition process) Firstly, the image acquisition system 1000 performs a first image acquisition process as part of the image acquisition process. First, the imaging device 1 places the DNA microarray A, which is the sample to be measured, into the imaging device 1. Next, the imaging device 1 irradiates the DNA microarray A with excitation light (dashed line in Figure 3) from the light source 10. Then, the imaging device 1 captures a first image (referred to as "first fluorescence image") I1 by imaging the fluorescence (single dashed line in Figure 3) generated from the DNA microarray A with the photodetector 15. Finally, the image acquisition device 100 acquires the first image I1 of the DNA microarray A captured by the imaging device 1.

[0027] For example, Image 1 I1 is an image captured by the imaging device 1 of fluorescence generated when excitation light was irradiated for a certain exposure time (e.g., 3 seconds) during the initial measurement of DNA microarray A, which is the target of measurement. Furthermore, Image 1 I1 is the image captured during the first exposure (0-3 seconds) of images taken by the imaging device 1 at regular exposure time intervals (e.g., 3 seconds) when excitation light was irradiated for a continuous exposure time (e.g., 300 seconds) during the initial measurement of DNA microarray A, which is the target of measurement. It is preferable that Image 1 I1 be an image captured before the decrease in fluorescence amount due to photobleaching of the fluorescent molecule F on DNA microarray A progresses, but the timing of imaging and exposure time are not limited.

[0028] (Second image acquisition process) Secondly, the image acquisition system 1000 performs a second image acquisition process as part of the image acquisition process. First, the imaging device 1 irradiates the DNA microarray A with excitation light (dashed line in Figure 3) from the light source 10 at a different timing than when the first image I1 is acquired. Then, the imaging device 1 acquires a second image (referred to as "second fluorescence image") I2 by imaging the fluorescence (single dashed line in Figure 3) generated from the DNA microarray A with the photodetector 15. Finally, the image acquisition device 100 acquires the second image I2 of the DNA microarray A acquired by the imaging device 1.

[0029] For example, the second image I2 is an image captured by the imaging device 1 of fluorescence generated when excitation light was irradiated for a certain exposure time (e.g., 3 seconds) after the acquisition of the first image I1. Alternatively, the second image I2 is the image captured at the second exposure (3-6 seconds) of images taken by the imaging device 1 at regular exposure times (e.g., 3 seconds) when excitation light was irradiated for a continuous exposure time (e.g., 300 seconds) during the initial measurement of the DNA microarray A, which is the target of measurement. Note that the second image I2 may also be an image captured by the imaging device 1 before the acquisition of the first image I1, and the timing of imaging 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 fluorescence intensity Q1 of the first image I1 is less than a predetermined signal intensity, or when the S / N ratio of the fluorescence intensity Q1 of the first image is less than a predetermined threshold, as determined by the threshold determination process described later.

[0030] (1-3-2-2. Light intensity calculation process) The image acquisition system 1000 according to the embodiment performs a light intensity calculation process. Firstly, the image acquisition system 1000 performs a first light intensity calculation process as a light intensity calculation process. That is, the image acquisition device 100 identifies a fluorescent spot in the acquired first image I1 and calculates the fluorescence intensity of the fluorescent spot (appropriately, "first fluorescence intensity") Q1 as the signal intensity. Secondly, the image acquisition system 1000 performs a second light intensity calculation process as a light intensity calculation process. That is, the image acquisition device 100 detects a fluorescent spot in the acquired second image I2 and calculates the fluorescence intensity of the fluorescent spot (appropriately, "second fluorescence intensity") Q2 as the signal intensity. Here, the fluorescent spot identified by the first light intensity calculation process is a predetermined area on the DNA microarray A where fluorescence generated by the irradiation of a fluorescent molecule F with excitation light is observed, and its shape, size, etc., are not particularly limited.

[0031] In this case, the image acquisition device 100 may determine the detection of a fluorescent spot from the line profiles of the first image I1 and the second image I2 by checking if the brightness value or light intensity of the fluorescent spot is significantly higher than that of the background, or it may detect the fluorescent spot by image processing such as contour extraction or feature extraction. Alternatively, the image acquisition device 100 may calculate the fluorescence intensity Q2 of the second image I2 by a threshold determination process described later, if the fluorescence intensity Q1 of the first image I1 is less than a predetermined signal intensity, or if the S / N ratio of the fluorescence intensity Q1 of the first image is less than a predetermined threshold.

[0032] (1-3-2-3. Threshold determination process) The image acquisition system 1000 according to the embodiment performs a threshold determination process. That is, the image acquisition device 100 determines whether the fluorescence intensity Q1 of the first image I1 calculated by the first light intensity calculation process described above is equal to or greater than a predetermined signal intensity. The image acquisition device 100 also determines whether the signal-to-noise ratio (S / N) of the fluorescence intensity of the first image I1 is equal to or greater than a predetermined threshold (e.g., 0.3). If the fluorescence intensity Q1 of the first image I1 is equal to or greater than a predetermined signal intensity, and the S / N of the fluorescence intensity Q1 of the first image I1 is equal to or greater than a predetermined threshold, the image acquisition device 100 terminates the process. On the other hand, if the fluorescence intensity Q1 of the first image I1 is less than a predetermined signal intensity, or if the S / N of the fluorescence intensity Q1 of the first image I1 is less than a predetermined threshold, the image acquisition device 100 performs a light intensity correction process and an image addition process, which will be described later.

[0033] Therefore, if the fluorescence intensity Q1 of the first image I1 is above a predetermined signal intensity, and the signal-to-noise ratio (S / N) of the fluorescence intensity Q1 of the first image I1 is above a predetermined threshold, it can be seen that the fluorescence intensity Q1 of the first image I1 is sufficient for qualitative analysis, quantitative analysis, and other analyses. On the other hand, if the fluorescence intensity Q1 of the first image I1 is below a predetermined signal intensity, or if the S / N of the fluorescence intensity Q1 of the first image I1 is below a predetermined threshold, it can be seen that the fluorescence intensity Q1 of the first image I1 is insufficient for qualitative analysis, quantitative analysis, and other analyses, and therefore correction of the first image I1 is necessary.

[0034] (1-3-2-4. Light intensity correction processing) The image acquisition system 1000 according to the embodiment performs light intensity correction processing. That is, 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. At this time, the image acquisition device 100 makes the background light constant by taking into account the difference between the background light and the fluorescence intensity of the fluorescence spot, and then corrects the fluorescence intensity Q2 of the fluorescence spot in the second image I2 to account for the decrease in fluorescence intensity due to the effect of photobleaching, thereby generating a corrected second image I2'.

[0035] (1-3-2-5. Image addition processing) The image acquisition system 1000 according to the embodiment performs image addition processing. That is, the image acquisition device 100 adds the corrected second image I2', in which the fluorescence intensity Q2 has been corrected by the light intensity correction processing described above, to the first image I1 to generate an additively corrected fluorescence image I with sufficient signal intensity and S / N ratio for analysis. If the fluorescence intensity Q of the additively corrected fluorescence image I is less than a predetermined signal intensity, or if the S / N ratio of the additively corrected fluorescence image I is less than a predetermined threshold, the image acquisition device 100 repeatedly performs image acquisition processing, light intensity correction processing, and image addition processing.

[0036] (1-4. Effects of Image Acquisition System 1000) The following describes an overview of the image acquisition system 1000 according to the embodiment, followed by a description of the effects of the image acquisition system 1000.

[0037] (1-4-1. Overview) In the image acquisition system 1000, the image acquisition device 100 acquires a first image I1 of DNA microarray A captured by the imaging device 1 and a second image I2 of DNA microarray A captured by the imaging device 1. It then adds a corrected second image I2', which is obtained by correcting the fluorescence intensity Q2 of the fluorescence spots in the second image I2 based on the fluorescence intensity Q1 of the fluorescence spots in the first image I1, to the first image I1 to generate an additively corrected fluorescence image I.

[0038] In other words, the image acquisition system 1000 is a technology that corrects for the effect of photobleaching of the fluorescent molecule F used to detect the target molecule N by the DNA microarray A and performs molecular quantification. Furthermore, the image acquisition system 1000 is a technology that acquires multiple images with a short exposure time during shooting, which minimizes the effect of photobleaching, and performs light intensity correction based on the image data acquired initially.

[0039] (1-4-2. Effects) Firstly, the image acquisition system 1000 can reduce quantitative errors in the number of molecules due to the effects of photobleaching, thereby improving the accuracy of detection of the target molecule N using the DNA microarray method. Secondly, the image acquisition system 1000 can shorten the total measurement time required to obtain the same signal-to-noise ratio (S / N). Thirdly, the image acquisition system 1000 can compare the results of images taken at different exposure times, as the quantitative results from both images are equivalent.

[0040] [2. Configuration of each device in the image acquisition system 1000] Using Figures 4 to 7, the functional configuration of each device in the image acquisition system 1000 shown in Figure 3 will be explained. Below, a detailed explanation will be given in the following order: an example of the configuration of the image acquisition device 100 according to the embodiment, an example of the configuration of the imaging device 1, and a specific example of the fluorescence intensity of the fluorescence image.

[0041] (2-1. Example of the configuration of the image acquisition device 100) First, an example of the configuration of the image acquisition device 100 shown in Figure 3 will be described using Figure 4. Figure 4 is a block diagram showing an example of the configuration of the image acquisition device 100 and imaging device 1 according to this embodiment. The image acquisition device 100 has a communication unit 110, a storage unit 120, and a control unit 130. The image acquisition device 100 may also have an input unit (e.g., a keyboard or mouse) for receiving various operations from the administrator of the image acquisition device 100, and a display unit (e.g., a liquid crystal display) for displaying various information.

[0042] (2-1-1. Communications Section 110) 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 or the like. The communication unit 110 can also communicate data with an operator's terminal (not shown).

[0043] (2-1-2. Storage section 120) The storage unit 120 stores various information that the control unit 130 references when it operates, and various information acquired when the control unit 130 operates. Here, the storage unit 120 can be implemented as, for example, a semiconductor memory element such as RAM (Random Access Memory) or flash memory, or a storage device such as a hard disk or optical disc. In the example shown in Figure 4, the storage unit 120 is installed inside the image acquisition device 100, but it may be installed outside the image acquisition device 100, or multiple storage units may be installed.

[0044] 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 acquired from the imaging device 1, such as the first fluorescence image (first image) I1, the second fluorescence image (second image) I2, and the third fluorescence image (third image) I3.

[0045] Furthermore, the storage unit 120 stores image data generated by the generation unit 132 of the control unit 130, which will be described later. For example, the storage unit 120 stores image data such as a second fluorescence image (corrected second image) I2' with corrected fluorescence intensity Q2, and an additively corrected fluorescence image I.

[0046] (2-1-3. Control Unit 130) The control unit 130 is responsible for controlling the entire image acquisition device 100. The control unit 130 has an acquisition unit 131 and a generation unit 132. Here, the control unit 130 can be implemented by electronic circuits such as a CPU (Central Processing Unit) or an MPU (Micro Processing Unit), or by integrated circuits such as an ASIC (Application Specific Integrated Circuit) or an FPGA (Field Programmable Gate Array).

[0047] (2-1-3-1. Acquisition Department 131) The acquisition unit 131 acquires various images captured by the imaging device 1. The acquisition unit 131 may also store the acquired images in the storage unit 120.

[0048] The acquisition unit 131 acquires a first image I1, in which the DNA microarray A, which is the sample to be measured, is imaged by the imaging device 1 during a first period, and a second image I2, in which the DNA microarray A is imaged by the imaging device 1 during a second period. For example, as the first period, the acquisition unit 131 acquires the first image I1, in which the DNA microarray A is imaged by the imaging device 1 during the first predetermined exposure time (e.g., 3 seconds of excitation light irradiation). The acquisition unit 131 also acquires the second image I2, in which the DNA microarray A is imaged by the imaging device 1 during the second and subsequent predetermined exposure times (e.g., 3 seconds of excitation light irradiation). Furthermore, the acquisition unit 131 similarly acquires a third image I3, in which the DNA microarray A is imaged during a third period, a fourth image I4, in which the DNA microarray A is imaged during a fourth period, and so on. Note that the first period, second period, third period, fourth period, etc., do not indicate a temporal sequence; rather, images can be selected and acquired as the first image I1, second image I2, third image I3, fourth image I4, etc., from multiple images captured during any given period.

[0049] To explain using a specific example, the acquisition unit 131 acquires the first image I1, which is the fluorescence image with the least effect of photobleaching, of the DNA microarray A, the sample to be measured, as captured for the first time by the imaging device 1, and is captured with a short exposure time of 3 seconds to avoid the effect of photobleaching due to long exposure times. Similarly, the acquisition unit 131 acquires the second image I2, which has the second least effect of photobleaching and is captured with the same 3-second exposure time as the first image I1, the third image I3, which has the third least effect of photobleaching and is captured with the same 3-second exposure time as the first image I1, and so on.

[0050] The acquisition unit 131 acquires a first image I1, which is an image of the DNA microarray A taken by the imaging device 1 for a predetermined exposure time (e.g., 3 seconds of excitation light irradiation). If the fluorescence intensity Q1 of the first image I1 is less than a predetermined signal intensity or a predetermined signal-to-noise ratio, the acquisition unit 131 acquires a second image I2, which is an image of the DNA microarray A taken by the imaging device 1 for a predetermined exposure time (e.g., 3 seconds of excitation light irradiation). Furthermore, if the fluorescence intensity Q of the additively corrected fluorescence image I, which is the corrected first image I1 generated by the generation unit 132 (described later), is less than a predetermined signal intensity or a predetermined signal-to-noise ratio, the acquisition unit 131 acquires a third image I3, a fourth image I4, ...

[0051] To explain using a specific example, the acquisition unit 131 acquires a first image I1, in which the DNA microarray A, which is the sample to be measured, is imaged by the imaging device 1 with an exposure time of 3 seconds. If the signal-to-noise ratio (S / N) of the fluorescence intensity Q1 of the first image I1 is less than 0.3, the acquisition unit 131 acquires a second image I2, which is in the same 3-second exposure time as the first image I1. Furthermore, if the S / N of the fluorescence intensity Q of the corrected additive-corrected fluorescence image I is less than 0.3, the acquisition unit 131 acquires a second image I2, which is in the same 3-second exposure time as the first image I1, and continues to acquire third images I3, fourth images I4, and so on, until the S / N of the fluorescence intensity Q of the additive-corrected fluorescence image I becomes 0.3 or greater.

[0052] The acquisition unit 131 acquires a first image I1 and a second image I2 selected from multiple fluorescence images captured by the imaging device 1 at predetermined exposure times (e.g., 3 seconds of excitation light irradiation) at multiple different times on the DNA microarray A. Furthermore, the acquisition unit 131 similarly acquires a third image I3, a fourth image I4, ... selected from multiple fluorescence images captured by the imaging device 1 at predetermined exposure times (e.g., 3 seconds of excitation light irradiation) at multiple different times on the

[0053] To explain using a specific example, the acquisition unit 131 acquires 100 fluorescence images (first image I1, second image I2, third image I3, ..., 100th image I100) of the DNA microarray A, which is the sample to be measured, captured by the imaging device 1 every 3 seconds over a continuous exposure time of 300 seconds. The acquisition unit selects the fluorescence images acquired between 0 and 3 seconds as the first image I1, the fluorescence images acquired between 3 and 6 seconds as the second image I2, the fluorescence images acquired between 6 and 9 seconds as the third image I3, ...

[0054] (2-1-3-2. Generation unit 132) The generation unit 132 performs processing related to the correction of various images acquired by the acquisition unit 131. The generation unit 132 may also store the calculated fluorescence intensity, the image with corrected fluorescence intensity, and the generated image in the storage unit 120.

[0055] The generation unit 132 generates a corrected second image I2', which is a corrected second image I2, by correcting the fluorescence intensity Q2 (second fluorescence intensity) of the fluorescence spots emitting fluorescence in the second image I2 based on the fluorescence intensity Q1 (first fluorescence intensity) of the fluorescence spots emitting fluorescence in the first image I1. The corrected second image I2' is then added to the first image I1 to generate an additively corrected fluorescence image I, which is the corrected first image I1. For example, the generation unit 132 calculates fluorescence intensity Q1 and fluorescence intensity Q2. If fluorescence intensity Q1 is less than a predetermined signal intensity or a predetermined signal-to-noise ratio, it uses the difference between fluorescence intensity Q2 and the background light intensity of the second image I2 to make the background light of the second image I2 equal to the background light of the first image I1. It then corrects fluorescence intensity Q2 by multiplying it by the reciprocal of the ratio of the decrease in fluorescence intensity between fluorescence intensity Q1 and fluorescence intensity Q2. Finally, it adds the corrected fluorescence intensity Q2' to fluorescence intensity Q1 to generate an additively corrected fluorescence image I in which the fluorescence intensity of the fluorescent spot is equal to or greater than a predetermined signal intensity and a predetermined signal-to-noise ratio.

[0056] To explain using a specific example, if the fluorescence intensity Q1 of the first image I1 is 110 (arbitrary unit) and the background light intensity is 10 (arbitrary unit), and the fluorescence intensity Q2 of the second image I2 is 100 (arbitrary unit) and the background light intensity is 10 (arbitrary unit), the generation unit 132 corrects the difference between the fluorescence intensity and the background light intensity by multiplying the fluorescence intensity Q2 by 100 / 90, which is the reciprocal of the decrease ratio of fluorescence intensity between the first image I1 and the second image I2 (90 / 100). The corrected fluorescence intensity Q2' is then added to the fluorescence intensity Q1. Here, when correcting the fluorescence intensity, the generation unit 132 prefers a nonlinear correction such that the background light remains constant, but a linear correction may also be performed.

[0057] If the exposure times are the same, the background light for each image can be considered equal. On the other hand, if the exposure times are different, the quantitative results between each image can be compared by multiplying by the reciprocal of the ratio of the exposure times, calculating the difference between the fluorescence intensity of each image and the background light, always considering the background light as having a signal intensity of 0, and correcting the fluorescence intensity using this difference.

[0058] (2-2. Example of configuration of imaging device 1) Using Figure 4, an example of the configuration of the imaging device 1 shown in Figure 3 will be explained. 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.

[0059] (2-2-1.Light source 10) Light source 10 is a laser light source that irradiates the installed DNA microarray A with laser light. For example, light source 10 can be realized by a laser light source that emits single-wavelength laser light or expanded light of said laser light, an LED (Light Emitting Diode), a lamp that emits white light, or a light source consisting of an LED and a wavelength filter.

[0060] (2-2-2. Objective lens 11) The objective lens 11 transmits or focuses the excitation light emitted from the light source 10 during imaging and guides it to the DNA microarray A. The objective lens 11 also transmits or focuses the fluorescence generated from the DNA microarray A and guides it to the photodetector 15 via the dichroic mirror 12, filter 13, and lens 14.

[0061] (2-2-3. Dichroic mirror 12) 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 photodetector 15 via the filter 13 and lens 14.

[0062] (2-2-4. Filter 13) Filter 13 transmits the fluorescence generated from DNA microarray A and guides it to the photodetector 15 via lens 14. At this time, filter 13 removes the reflection of the laser light, which is the irradiating light, and transmits only the fluorescence.

[0063] (2-2-5. Lens 14) Lens 14 transmits or collects fluorescence generated from DNA microarray A and guides it to photodetector 15.

[0064] (2-2-6. Photodetector 15) The photodetector 15 converts the fluorescence imaged by the lens 14 into an electrical signal. For example, the photodetector 15 can be realized using an EM (Electron Multiplying)-CCD, a CMOS (Complementary Metal-Oxide-Semiconductor), or the like.

[0065] (2-3. Specific examples of fluorescence intensity in fluorescence images) Specific examples of fluorescence intensity in fluorescence images according to the embodiment will be explained using Figures 5 to 7. Figures 5 to 7 are diagrams showing specific examples of fluorescence intensity in fluorescence images according to the embodiment. Below, specific example 1 regarding fluorescence image correction, specific example 2 and specific example 3 regarding effectiveness compared with reference technologies, etc. will be explained in that order.

[0066] (2-3-1. Correction of fluorescence images) Using Figure 5, a specific example 1 of the correction of fluorescence images according to the embodiment will be explained. As shown in Figure 5(1), the fluorescence intensity (in arbitrary units) decreases over time due to photobleaching of the fluorescent dye, so a correction is performed for each image based on the amount of decrease in fluorescence intensity. As a result, as shown in Figure 5(2), the equivalent light amount from 0 seconds to 180 seconds in Figure 5(1) can be obtained as an image with fluorescence intensity unaffected by photobleaching of the fluorescent dye.

[0067] (2-3-2. Effectiveness of Image Acquisition Processing) Using Figures 6 and 7, specific examples 2 and 3 regarding the effectiveness of the image acquisition process according to the embodiment will be described. Below, the experimental procedure for confirming the effectiveness of the image acquisition process according to the embodiment will be described, followed by a comparison with reference technologies, measurement results of photobleaching, and a discussion of the comparison results.

[0068] (2-3-2-1. Experimental Procedure) The following describes an experimental procedure to confirm the effectiveness of the image acquisition process according to the embodiment. In the following experimental procedure, a DNA molecule, which is the target molecule N, is detected, which is labeled with a fluorescent molecule F, which is a Cy3 fluorescent molecule.

[0069] First, using genomic DNA extracted from Staphylococcus aureus strain (NBRC12732) as a template, PCR amplification is performed using Cy3-modified primers capable of amplifying the 16s ribosomal DNA (16s rDNA) region (Experimental Procedure 1).

[0070] Secondly, a test sample S containing the target molecule N, prepared by diluting the PCR product concentration to 1 nM, is applied (supplied) to DNA microarray A on which the detection probe P for the Staphylococcus aureus 16s rDNA sequence is immobilized (Experimental Procedure 2).

[0071] Thirdly, DNA microarray A is incubated at 66°C for 1 hour to allow the target molecule N to hybridize with the detection probe P (Experimental Procedure 3).

[0072] Fourth, DNA microarray A is washed with 2x concentration SSC (Standard Saline Citrate) solution and then with 1x concentration SSC solution, and then dried (experimental procedure 4).

[0073] Fifth, the images acquired from the 2nd to the 100th are corrected based on the first image acquired, and all images are added together (Experimental Procedure 5).

[0074] Sixth, as comparative data corresponding to the reference techniques, one image is captured of DNA microarray A reacted according to the procedures in experimental steps 1 to 4 above, with an exposure time of 300 seconds (experimental step 6).

[0075] (2-3-2-2. Comparison results with reference technologies, etc.) Using Figure 6, we will explain the results of a comparison with reference technologies, etc., as a specific example 2 of the effectiveness of the image acquisition process according to the embodiment. Here, Figure 6(1) shows the fluorescence intensity (arbitrary unit 10482) of an image obtained by correcting and adding 100 fluorescence images acquired with an exposure time of 3 seconds as the image acquisition process according to the embodiment. Also, Figure 6(2) shows the fluorescence intensity (arbitrary unit 8458) of an image obtained with a continuous exposure time of 300 seconds as the image acquisition process according to reference technologies, etc.

[0076] (2-3-2-3. Measurement results of photobleaching) Using Figure 7, we will explain the measurement results of photobleaching as a specific example 3 of the effectiveness of the image acquisition process according to the embodiment. As shown in Figure 7, the fluorescence intensity, which is about 110 (arbitrary unit) at a measurement time of 0 seconds, gradually decreases, and at a measurement time of 300 seconds, the fluorescence intensity becomes about 80 (arbitrary unit).

[0077] (2-3-2-4. Discussion of the comparison results) Based on the above, the total fluorescence intensity calculated by the image acquisition process according to the embodiment, that is, the detection target molecule N collected on DNA microarray A, is calculated to be approximately 20% higher than the total fluorescence intensity calculated by the image acquisition process according to the reference technology, etc. Therefore, the image acquisition process according to the embodiment can correct for the effect of photobleaching on the measurement time of the fluorescence intensity of the fluorescence spot shown in Figure 7.

[0078] [3. Processing flow of image acquisition system 1000] The processing flow of the image acquisition system 1000 according to the embodiment will be explained using Figure 8. Figure 8 is a flowchart of an example of the image acquisition processing flow according to the embodiment. Note that the processes in steps S101 to S107 below can be executed in a different order. Also, some of the processes in steps S101 to S107 below may be omitted.

[0079] (3-1. First image acquisition process) Firstly, the image acquisition device 100 performs 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.

[0080] (3-2. First light intensity calculation process) Secondly, the image acquisition device 100 performs a first light intensity calculation process (step S102). For example, the image acquisition device 100 identifies the fluorescent spot in the acquired first image I1 and calculates the fluorescence intensity Q1 of the fluorescent spot as the signal intensity.

[0081] (3-3. Threshold determination process) Thirdly, the image acquisition device 100 performs a threshold determination process (step S103). For example, the image acquisition device 100 determines whether the fluorescence intensity Q1 of the first image I1 is equal to or greater than a predetermined signal intensity, and whether the signal-to-noise ratio (S / N) of the fluorescence intensity Q1 of the first image I1 is equal to or greater than a predetermined threshold. If the fluorescence intensity Q1 is equal to or greater than a predetermined signal intensity, and the S / N of the fluorescence intensity Q1 of the first image I1 is equal to or greater than a 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 a predetermined signal intensity, or the S / N of the fluorescence intensity Q1 of the first image I1 is less than a predetermined threshold (step S103: No), the image acquisition device 100 proceeds to the process in step S104.

[0082] (3-4. Second image acquisition process) Fourth, the image acquisition device 100 performs 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.

[0083] (3-5. Second light intensity calculation process) Fifth, the image acquisition device 100 performs a second light intensity calculation process (step S105). For example, the image acquisition device 100 identifies the fluorescent spot in the acquired second image I2 and calculates the fluorescence intensity Q2 of the fluorescent spot as the signal intensity.

[0084] (3-6. Light intensity correction processing) Sixth, the image acquisition device 200 performs light intensity correction processing (step S106). For example, 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.

[0085] (3-7. Image Addition Processing) Seventh, the image acquisition device 200 performs image addition processing (step S107) and returns to the processing in step S103. For example, the image acquisition device 100 generates an additively corrected fluorescence image I with sufficient signal intensity and S / N ratio for analysis by adding the corrected second image I2', which has been corrected for fluorescence intensity Q2, to the first image I1.

[0086] [4. Effects of the Embodiment] Finally, the effects of the embodiment will be described. Below, effects 1 to 5 corresponding to the processing according to the embodiment will be described.

[0087] (4-1. Effect 1) Firstly, in the process according to the embodiment described above, the image acquisition device 200 acquires a first image I1 of the DNA microarray A captured by the imaging device 1 during a first period and a second image I2 of the DNA microarray A captured by the imaging device 1 during a second period. It then generates a corrected second image I2' by correcting the fluorescence intensity Q2 of the fluorescence spots in the second image I2 based on the fluorescence intensity Q1 of the fluorescence spots in the first image I1, and adds the corrected second image I2' to the first image I1 to generate an additively corrected fluorescence image I. Therefore, in the process according to the embodiment, fluorescence images with high detection accuracy can be obtained in the DNA microarray method.

[0088] (4-2. Effect 2) Secondly, in the processing according to the embodiment described above, 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 a predetermined signal-to-noise ratio, the difference between fluorescence intensity Q2 and the background light intensity 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. Fluorescence intensity Q2 is corrected by multiplying it by the reciprocal of the ratio of the decrease in fluorescence intensity between fluorescence intensity Q2 and the second image I2. The corrected fluorescence intensity Q2 is then added to fluorescence intensity Q1 to generate an additively corrected fluorescence image I in which the fluorescence intensity of the fluorescence spot is equal to or greater than a predetermined signal intensity and a predetermined signal-to-noise ratio. Therefore, in the processing according to the embodiment, in the DNA microarray method, a fluorescence image with high detection accuracy that satisfies sufficient signal intensity and S / N for analysis can be obtained.

[0089] (4-3. Effect 3) Thirdly, in the process according to the embodiment described above, the image acquisition device 200 acquires a first image I1, in which the DNA microarray A is imaged by the imaging device 1 for the first predetermined exposure time, as a first period, and second images I2, in which the DNA microarray A is imaged by the imaging device 1 for the second and subsequent predetermined exposure times, as a second period. Therefore, in the process according to the embodiment, by acquiring multiple fluorescence images imaged with the same exposure time in the DNA microarray method, fluorescence images with high detection accuracy can be obtained.

[0090] (4-4. Effect 4) Fourth, in the process according to the embodiment described above, the image acquisition device 200 acquires a first image I1 of the DNA microarray A captured by the imaging device 1 for a predetermined exposure time, and if the fluorescence intensity Q1 is less than a predetermined signal intensity or less than a predetermined signal-to-noise ratio, it acquires a second image I2 of the DNA microarray A captured by the imaging device 1 for a predetermined exposure time. Therefore, in the process according to the embodiment, by acquiring multiple fluorescence images in real time in the DNA microarray method, fluorescence images with high detection accuracy can be obtained.

[0091] (4-5. Effect 5) Fifth, in the process according to the embodiment described above, the image acquisition device 200 acquires a first image I1 and a second image I2 selected from a plurality of fluorescence images captured by the imaging device 1 at predetermined exposure times at multiple different times for the DNA microarray A. Therefore, in the process according to the embodiment, by acquiring multiple fluorescence images in batches in the DNA microarray method, fluorescence images with high detection accuracy can be obtained.

[0092] [Examples of application of embodiments] The following describes examples of applications of the embodiments. However, the present invention is not limited to the examples of applications of the embodiments described below.

[0093] The embodiments can be applied to specific target molecules, such as nucleic acids, glycans, and proteins, which are biomacromolecules.

[0094] The embodiments can be applied to imaging and analysis of DNA microarrays using fluorescence, chemiluminescence, and colorimetric methods.

[0095] The embodiment can be applied to nucleic acid sequence measurement devices in which a signal source such as a fluorescent molecule is modified on a probe immobilized on a substrate in a DNA microarray, to detection of unmodified targets using a signaling array probe, and to microarray detection of nucleic acid sequence measurement devices that do not require washing.

[0096] The embodiment can be applied to the detection of nucleic acid sequence measurement devices, such as DNA microarrays, in which a target molecule modified with a signal source such as a fluorescent molecule is attached to a probe immobilized on a substrate, and then observed.

[0097] The embodiment can be applied to the detection of nucleic acid sequence measurement devices, such as DNA microarrays, in which a target molecule is captured and observed by a labeling molecule to which a signal source, such as a fluorescent molecule that specifically binds to the target molecule, is attached to a probe immobilized on a substrate.

[0098] The embodiment can be applied to gene expression analysis, genotyping, and base sequence analysis when targeting nucleic acids.

[0099] The embodiment can be applied to glycan profiling when targeting glycans.

[0100] The embodiment can be applied to antibody profiling and protein-protein interaction analysis when targeting proteins.

[0101] The embodiments can be applied to industrial applications such as drug discovery and research and development, biomarker detection, disease diagnosis, disease prevention and prognosis management, biomarker exploration, development of diagnostic methods, antibody antigen exploration, plant variety identification, genetic identification testing of livestock and agricultural products, genetic modification testing of food products, allergy labeling testing, microbial detection and identification in environmental hygiene inspections, microbial testing in product quality control, monitoring of fermentation processes, inoculum management in fermentation processes, exploration of beneficial bacteria in microbial materials, and characterization of harmful bacteria in pharmaceutical and food manufacturing processes.

[0102] The embodiment can be applied to image acquisition technology for fluorescence images using absolute light intensity detection.

[0103] 〔system〕 Unless otherwise specified, the processing procedures, control procedures, specific names, and various data and parameters shown in the above documents and drawings may be changed at will.

[0104] Furthermore, the components of each illustrated device are functionally conceptual and do not necessarily need to be physically configured as shown. In other words, the specific forms of distribution and integration of each device are not limited to those shown. That is, all or part of them can be functionally or physically distributed and integrated in any unit according to various loads and usage conditions.

[0105] Furthermore, each processing function performed by each device may be implemented, in whole or in part, by a CPU and a program executed for analysis by that CPU, or by hardware using wired logic.

[0106] [Hardware] Next, an example of the hardware configuration of the image acquisition device 100 will be described. Figure 9 is a diagram showing an example of the hardware configuration according to the embodiment. As shown in Figure 9, the image acquisition device 100 has a communication device 100a, an HDD (Hard Disk Drive) 100b, memory 100c, and a processor 100d. Furthermore, each of the parts shown in Figure 9 is interconnected by a bus or the like.

[0107] The communication device 100a is a network interface card or the like, and communicates with other servers. The HDD 100b stores programs and databases that operate the functions shown in Figure 4.

[0108] The processor 100d operates a process that performs the functions described in Figure 4 by reading a program that performs the same processing as each processing unit shown in Figure 4 from the HDD 100b or the like and loading it into memory 100c. For example, this process performs the same functions as each processing unit of the image acquisition device 100. Specifically, the processor 100d reads a program that has the same functions as the acquisition unit 131, the generation unit 132, etc. from the HDD 100b or the like. Then, the processor 100d executes a process that performs the same processing as the acquisition unit 131, the generation unit 132, etc.

[0109] Thus, the image acquisition device 100 operates as a device that executes various processing methods by reading and executing a program. Furthermore, the image acquisition device 100 can also achieve the same functionality as the embodiment described above by reading the program from the recording medium using a media reader and executing the read program. It should be noted that the program referred to in this other embodiment is not limited to being executed by the image acquisition device 100. For example, the present invention can be similarly applied when another computer or server executes the program, or when they collaborate to execute the program.

[0110] This program can be distributed via networks such as the Internet. Furthermore, this program can be recorded on computer-readable storage media such as hard disks, flexible disks (FDs), CD-ROMs, MO (Magneto-Optical disks), and DVDs (Digital Versatile Discs), and executed by reading the program from these media using a computer.

[0111] 〔others〕 Some examples of the combinations of technical features that will be disclosed are listed below.

[0112] (1) An image acquisition device comprising: an acquisition unit that acquires a first fluorescence image of a sample to be measured captured by an imaging device during a first period and a second fluorescence image of the sample to be measured captured by the imaging device during a second period; and a generation unit that generates a corrected fluorescence image by correcting the second fluorescence intensity of a fluorescence spot that emits fluorescence in the second fluorescence image based on the first fluorescence intensity of a fluorescence spot that emits fluorescence in the first fluorescence image, and adds the corrected fluorescence image to the first fluorescence image to generate an additively corrected fluorescence image of the first fluorescence image.

[0113] (2) The image acquisition apparatus according to (1), wherein the generation unit calculates the first fluorescence intensity and the second fluorescence intensity, and if the first fluorescence intensity is less than a predetermined signal intensity or less than a predetermined signal-to-noise ratio, it uses the difference between the second fluorescence intensity and the background light intensity 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 reciprocal of the ratio of the decrease in fluorescence intensity between the first fluorescence intensity and the second fluorescence intensity, and adds the corrected second fluorescence intensity to the first fluorescence intensity to generate the additively corrected fluorescence image in which the fluorescence intensity of the fluorescence spot is equal to or greater than the predetermined signal intensity and the predetermined signal-to-noise ratio.

[0114] (3) The image acquisition apparatus according to (1) or (2), wherein the first period is the first predetermined exposure time during which the sample to be measured is imaged by the imaging device, and the second period is each predetermined exposure time from the second onward during which the sample to be measured is imaged by the imaging device.

[0115] (4) The image acquisition apparatus according to any one of (1) to (3), wherein the acquisition unit acquires the first fluorescence image of the sample to be measured captured by the imaging device for a predetermined exposure time, and if the first fluorescence intensity is less than a predetermined signal intensity or less than a predetermined signal-to-noise ratio, acquires the second fluorescence image of the sample to be measured captured by the imaging device for the predetermined exposure time.

[0116] (5) The image acquisition apparatus according to any one of (1) to (4), wherein the acquisition unit acquires a first fluorescence image and a second fluorescence image selected from a plurality of fluorescence images taken by the imaging device at predetermined exposure times at multiple different times of the sample to be measured.

[0117] (6) The sample to be measured is a DNA microarray, and the image acquisition device is one of the items in (1) to (5).

[0118] (7) An image acquisition method in which a computer acquires a first fluorescence image of the sample to be measured captured by an imaging device during a first period and a second fluorescence image of the sample to be measured captured by the imaging device during a second period, generates a corrected fluorescence image by correcting the second fluorescence intensity of the fluorescence spot in the second fluorescence image based on the first fluorescence intensity of the fluorescence spot in the first fluorescence image, and adds the corrected fluorescence image to the first fluorescence image to generate an additively corrected fluorescence image of the first fluorescence image.

[0119] (8) An image acquisition program that causes a computer to perform the following processes: acquire a first fluorescence image of the sample to be measured captured by an imaging device during a first period, and a second fluorescence image of the sample to be measured captured by the imaging device during a second period; generate a corrected fluorescence image by correcting the second fluorescence intensity of the fluorescence spot in the second fluorescence image based on the first fluorescence intensity of the fluorescence spot in the first fluorescence image; and add the corrected fluorescence image to the first fluorescence image to generate an additively corrected fluorescence image of the first fluorescence image. [Explanation of Symbols]

[0120] 1. Imaging device 10 light source 11 Objective lens 12 Dichroic Mirrors 13 Filters 14 lenses 15. Photodetector 100 Image acquisition device 110 Communications Department 120 Storage section 130 Control Unit 131 Acquisition Department 132 Generation part 1000 Image Acquisition System

Claims

1. An acquisition unit that acquires a first fluorescence image of the sample to be measured captured by an imaging device during a first period, and a second fluorescence image of the sample to be measured captured by the imaging device during a second period. A corrected fluorescence image is generated by correcting the second fluorescence intensity of the fluorescence spots that generate fluorescence in the second fluorescence image based on the first fluorescence intensity of the fluorescence spots that generate fluorescence in the first fluorescence image. A generation unit that adds the corrected fluorescence image to the first fluorescence image to generate an additively corrected fluorescence image in which the first fluorescence image has been corrected, An image acquisition device equipped with the following features.

2. The generating unit is The first fluorescence intensity and the second fluorescence intensity are calculated, and if the first fluorescence intensity is less than a predetermined signal intensity or less than a predetermined signal-to-noise ratio, the difference between the second fluorescence intensity and the background light intensity of the second fluorescence image is used to make the background light of the second fluorescence image equal to the background light of the first fluorescence image, and the second fluorescence intensity is corrected by multiplying it by the reciprocal of the ratio of the decrease in fluorescence intensity between the first fluorescence intensity and the second fluorescence intensity. By adding the corrected second fluorescence intensity to the first fluorescence intensity, an additively corrected fluorescence image is generated in which the fluorescence intensity of the fluorescence spot is equal to or greater than the predetermined signal intensity and the predetermined signal-to-noise ratio. The image acquisition device according to claim 1.

3. The first period is the first predetermined exposure time during which the sample to be measured is imaged by the imaging device. The second period is the predetermined exposure time for each subsequent image of the sample to be measured by the imaging device, starting from the second image. The image acquisition device according to claim 1.

4. The acquisition unit is, The first fluorescence image of the sample to be measured is captured by the imaging device for a predetermined exposure time, If the first fluorescence intensity is less than a predetermined signal intensity or less than a predetermined signal-to-noise ratio, the second fluorescence image of the sample to be measured is acquired by the imaging device for a predetermined exposure time. The image acquisition device according to claim 1.

5. The acquisition unit is, The first fluorescence image and the second fluorescence image are obtained from a plurality of fluorescence images taken by the imaging device at predetermined exposure times at multiple different times from the sample to be measured. The image acquisition device according to claim 1.

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

7. Computers A first fluorescence image of the sample to be measured, captured by the imaging device during a first period, and a second fluorescence image of the sample to be measured, captured by the imaging device during a second period are acquired. A corrected fluorescence image is generated by correcting the second fluorescence intensity of the fluorescence spots that generate fluorescence in the second fluorescence image based on the first fluorescence intensity of the fluorescence spots that generate fluorescence in the first fluorescence image. The corrected fluorescence image is added to the first fluorescence image to generate an additively corrected fluorescence image in which the first fluorescence image has been corrected. The method for acquiring images to be processed.

8. On the computer, A first fluorescence image of the sample to be measured, captured by the imaging device during a first period, and a second fluorescence image of the sample to be measured, captured by the imaging device during a second period are acquired. A corrected fluorescence image is generated by correcting the second fluorescence intensity of the fluorescence spots that generate fluorescence in the second fluorescence image based on the first fluorescence intensity of the fluorescence spots that generate fluorescence in the first fluorescence image. The corrected fluorescence image is added to the first fluorescence image to generate an additively corrected fluorescence image in which the first fluorescence image has been corrected. An image acquisition program that executes processing.

Images