Image acquisition method, image acquisition device, and program

Abstract

To appropriately set a measurement condition sufficiently utilizing a dynamic range of a signal processing system for each measurement sample in fluorescent image photographing of an array plate.SOLUTION: An image acquisition method for acquiring a fluorescence image of an object on a plate, the image acquisition method including a first measurement step of acquiring a fluorescence image by scanning the plate with primary light having a first irradiation light intensity and operating a light detection unit under a first sensitivity condition, a first information acquisition step of acquiring first statistical information relating to fluorescence intensity on the basis of the fluorescence image, a second information acquisition step of acquiring second statistical information obtained by giving a weight relating to the fluorescence intensity to the first statistical information, and a step of, using the second statistical information, the method includes a condition acquisition step of acquiring a multiplication condition regarding at least one of a first irradiation light intensity and a first sensitivity condition, and a second measurement step of performing scanning with primary light having an irradiation light intensity set on the basis of the acquired multiplication condition, operating a light detection unit under the set sensitivity condition, and acquiring a fluorescence image of the plate.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present disclosure relates to an image acquisition method, an image acquisition device, and a program for acquiring a fluorescent image of an array plate. [Background technology]

[0002] Array plates, such as protein arrays, peptide arrays, and DNA arrays, are known, in which numerous substances, such as proteins, peptides, and nucleic acids, are fixed in the form of spots on a substrate such as glass or plastic. Using such array plates, it is possible to simultaneously observe the interactions between numerous fixed substances and substances in a specimen. Therefore, it is also possible to comprehensively analyze interactions between numerous substances, including biological specimens such as blood, cell extracts, saliva, and interstitial fluid.

[0003] In the measurement process of an array plate, a method is known in which spots where an interaction of interest has occurred are selectively fluorescently labeled to obtain optical information. Known measurement devices for obtaining optical information include fluorescence scanners such as microarray scanners and plate readers. Fluorescence scanners use irradiating light to excite fluorophores, and known light sources include laser diodes (LDs), light-emitting diodes (LEDs), mercury lamps, and xenon lamps. Fluorescence scanners are equipped with a photodetector that receives the fluorescence from the fluorophores, and known photodetectors include photodiodes (PDs), photomultiplier tubes (PMTs), and charge-coupled devices (CCD) cameras.

[0004] In a specific array plate measurement, the array plate is irradiated with light, which excites the fluorophores and produces fluorescence. The fluorescence signal obtained by the light detection unit upon receiving the fluorescence is then converted from an analog signal to a digital signal by a signal processing system including an A / D converter. This results in a fluorescence image of the array plate. To ensure that the fluorescence image falls within an appropriate range of brightness values, the intensity of the irradiated light and the sensitivity of the light detection unit must be appropriately set, taking into account the dynamic range of the light detection unit and the signal processing system. Patent Document 1 discloses a technique for simulating the brightness value of each pixel when the PMT control voltage is changed based on the fluorescence image data obtained by pre-scanning, in order to appropriately set the sensitivity of the photodetector when acquiring a fluorescence image. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Japanese Patent Application Laid-Open No. 2002-195948 Summary of the Invention [Problem to be solved by the invention]

[0006] In the technology disclosed in Patent Document 1, the density of the fluorescent image obtained in the pre-scan is shifted to simulate the fluorescent image obtained in the main scan. As a result, some users may focus on the density of the entire fluorescent image, failing to consider saturated pixels in high-brightness spots, which could result in the PMT control voltage being set incorrectly.

[0007] Therefore, one of the objects of the present invention is to appropriately set measurement conditions that fully utilize the dynamic range of the signal processing system for each measurement sample. [Means for solving the problem]

[0008] In order to solve the above problem, an image acquisition method according to one aspect of the present invention includes: 1. An image acquisition method for acquiring a fluorescence image by scanning a plate with primary light to acquire a fluorescence intensity of an object on the plate, comprising: a first measurement step of scanning the plate with primary light having a first irradiation light intensity and operating the light detection unit under a first sensitivity condition to acquire a fluorescent image; a first information acquisition step of acquiring first statistical information related to the fluorescence intensity based on the fluorescence image; a second information acquisition step of acquiring second statistical information by weighting the first statistical information with respect to the fluorescence intensity; a condition acquisition step of acquiring a multiplication condition related to at least one of the first irradiation light intensity and the first sensitivity condition using the second statistical information; and a second measurement step of scanning with primary light of an irradiation light intensity set based on the acquired multiplication conditions, operating the light detection unit under set sensitivity conditions, and acquiring a fluorescent image of the plate. Furthermore, an image acquisition method according to one aspect of the present invention includes: 1. An image acquisition method for acquiring a fluorescence image by scanning a plate with primary light to acquire a fluorescence intensity of an object on the plate, comprising: a first measurement step of scanning the plate with primary light having a first irradiation light intensity and operating the light detection unit under a first sensitivity condition to acquire a fluorescent image; a first information acquisition step of acquiring first statistical information related to the fluorescence intensity based on the fluorescence image; a second information acquisition step of acquiring second statistical information by weighting the first statistical information with respect to the fluorescence intensity; a condition acquisition step of acquiring a multiplication condition related to at least one of the first irradiation light intensity and the first sensitivity condition using the second statistical information; a predicted image acquisition step of simulating a predicted fluorescent image that is predicted to be acquired when a fluorescent image of the plate is captured by scanning with primary light having an irradiation light intensity set based on the acquired multiplication conditions and operating the light detection unit under set sensitivity conditions; and Includes. Furthermore, an image acquisition device according to one aspect of the present invention includes: an image acquisition device that controls a fluorescent scanner and acquires a fluorescent image from the fluorescent scanner, the image acquisition device comprising: a mounting section for mounting a plate on which an object is fixed; an emission section that emits primary light toward the plate to form an irradiation spot on the plate; a light collection section that collects secondary light from the object; a light source optically coupled to the emission section; a light detection section optically coupled to the light collection section; a scanning section that controls the mounting section and the emission section in accordance with scanning conditions to change relative positions of the irradiation spot and the light collection section with respect to the plate; and an image generation section that generates a fluorescent image based on information related to the detection intensity from the light detection section and information related to the relative position, a light intensity control unit that controls the primary light guided from the light source to the emission unit to a predetermined light intensity; a detection sensitivity control unit that controls the light detection unit so that the light detection unit operates at a predetermined detection sensitivity; a scanning instruction unit that instructs the scanning unit on the scanning conditions; a prescan condition acquisition unit that acquires a first light intensity condition and a first sensitivity condition used in the prescan as prescan conditions; a storage unit that stores the fluorescence image acquired under the acquired prescan conditions; a first statistical information acquisition unit that statistically analyzes information about the fluorescence intensity of the fluorescence image to acquire first statistical information; a second statistical information acquiring unit that acquires second statistical information by weighting the first statistical information with respect to the fluorescence intensity; a postscan condition acquisition unit that acquires, as postscan conditions, second light intensity conditions and second sensitivity conditions to be used in a postscan that is performed after the prescan, based on the second statistical information and the prescan conditions; Equipped with. [Effects of the Invention]

[0009] According to an image acquisition method according to one aspect of the present invention, it is possible to appropriately set measurement conditions for each measurement sample, making full use of the dynamic range of the signal processing system. [Brief explanation of the drawings]

[0010] [Figure 1] 3 is a flowchart illustrating steps in an example of an image acquisition method according to an embodiment of the present invention. [Figure 2] 1 is a diagram showing an example of a schematic configuration of a fluorescence image acquisition device according to an embodiment of the present invention. [Figure 3] FIG. 3 is a block diagram showing an example of the configuration of the fluorescence image acquisition device shown in FIG. 2. [Figure 4] FIG. 2 is a schematic diagram of an array plate in the first embodiment. [Figure 5] 10A and 10B are diagrams illustrating an example of a histogram and a probability density function acquired as first statistical information. [Figure 6] FIG. 10 is a diagram showing an example of a probability density function obtained as first statistical information and a weighted probability density function. [Figure 7] FIG. 10 is a diagram showing an example of the relationship between the control voltage and sensitivity of a PMT that has been measured in advance. [Figure 8] 10 is a flowchart showing steps in an example of an image acquisition method according to a second embodiment. [Figure 9] FIG. 10 is a diagram showing an example of the relationship between irradiation light intensity and fluorescent light luminance measured in advance. [Figure 10] 10 is a flowchart showing steps in an example of an image acquisition method according to a third embodiment. [Figure 11] FIG. 10 is a block diagram showing an example of a schematic configuration of a fluorescence image acquisition apparatus according to a fourth embodiment. [Figure 12] 10 is a flowchart showing steps in an example of an image acquisition method according to a fourth embodiment. [Figure 13] 13 is a flowchart showing steps in an example of an image acquisition method according to the fifth embodiment. [Figure 14] 13 is a flowchart showing each step in an example of an image acquisition method according to the sixth embodiment. [Figure 15] FIG. 10 is a diagram showing an example of a relationship between sensitivity and scanning conditions that has been obtained in advance. DETAILED DESCRIPTION OF THE INVENTION

[0011] Exemplary embodiments for carrying out the present invention will be described in detail with reference to the accompanying drawings. However, the materials and relative positions of components described in the following embodiments are arbitrary, and the configuration of an apparatus to which the present invention is applied can be changed according to various conditions. Furthermore, the same reference numerals are used between drawings to indicate identical or functionally similar elements.

[0012] Before describing the embodiments described below, a representative example of an image acquisition method for an array plate according to an embodiment of the present invention will be described with reference to Fig. 1. Fig. 1 is a flowchart illustrating a series of processes executed in the image acquisition method for an array plate.

[0013] The image acquisition method according to the present invention includes, for example, the following steps S101 to S105. <Step S101> In this step, the array plate is scanned with primary light having a first irradiation light intensity, and a fluorescent image is obtained by operating the light detection unit under a first sensitivity condition. <Step S102> In this step, first statistical information regarding the fluorescence intensity is obtained based on the acquired fluorescence image. <Step S103> In this step, second statistical information is obtained by assigning a weight related to the fluorescence intensity to the first statistical information. <Step S104> In this step, the second multiplication condition for the first sensitivity condition is acquired using the second statistical information. Note that although an example of acquiring the second multiplication condition from the second statistical information is shown here, the first multiplication condition for the first irradiation light intensity may be acquired, or both the second multiplication condition for the first sensitivity condition and the first multiplication condition for the first irradiation light intensity may be acquired. <Step S105> In this step, a fluorescent image of the array plate is acquired under the second multiplication condition. Alternatively, a fluorescent image may be acquired under the first multiplication condition, or both of these multiplication conditions. By carrying out the image acquisition method consisting of the above steps, it is possible to appropriately set measurement conditions that fully utilize the dynamic range of the signal processing system for each measurement sample.

[0014] In the above-described embodiment, the object from which the fluorescent image is acquired is an array plate. An example of the array plate used will now be briefly described. The array plate has spots containing various types of biological materials on a substrate and is used for comprehensive analysis of specimens. The array plate is also called a microchip, microarray, protein chip, DNA chip, etc.

[0015] In an embodiment, a commercially available array plate may be used. Examples of commercially available array plates include those sold by Agilent Technologies, RayBiotech, and the like. Alternatively, an array plate can be prepared by referring to a known method. For example, an array plate can be prepared by immobilizing a desired biological material on one surface of a suitable substrate. Note that immobilization can also be referred to as adsorption, and can include immobilization by hydrophobic interaction, electrostatic interaction, van der Waals interaction, hydrogen bonding, and covalent bonding.

[0016] Here, the substrate used in the array plate is preferably transparent. Examples of materials for the substrate include glass, synthetic quartz, quartz, borosilicate glass, etc. Alternatively, examples of materials include resins such as polystyrene, polypropylene, (meth)acrylic resin, polyamide, polyimide, melamine, ABS, polyphenylene oxide urethane, silicone, epoxy, and polydimethylsiloxane.

[0017] Next, an example of a fluorescence image acquisition device for carrying out the fluorescence image acquisition method according to the present invention will be described with reference to Fig. 2. Fig. 2 is a schematic diagram showing the general configuration of an example of a fluorescence image acquisition device 101 according to one embodiment of the present invention.

[0018] The illustrated fluorescence image acquisition device 101 is configured with a confocal optical system and includes an irradiation optical system 116, a light-receiving optical system 117, and a control system. The irradiation optical system 116 includes an irradiation unit 103 including a light source that emits a train of light pulses of a predetermined wavelength, a lens 104, a dichroic mirror 105, an objective mirror 106, and an objective lens 114 that constitutes an emission unit. The objective lens 114 is optically coupled to the irradiation unit 103, which functions as an example of a light source. The light-receiving optical system 117 includes the objective lens 114, objective mirror 106, dichroic mirror 105, a filter 115, a lens 110, and a light detection unit 111 that constitute a light collection unit. The irradiation unit 103, which functions as an example of a light source, is optically coupled to the objective lens 114, which functions as a light collection unit. The control system includes an image acquisition device 102, an image generation unit 112, and a scanning unit 108. The image generation unit 112 generates a fluorescence image based on information about the detection intensity from the light detection unit 111 and information about the relative position of the spot irradiated with light with respect to the plate 107. The scanning unit 108 changes the relative position of the objective lens 114 with respect to the plate 107 according to the scanning conditions so as to two-dimensionally scan the relative position of the spot irradiated with light with respect to the plate 107. The scanning unit 108 has a sub-scanning unit 108s that scans the mounting unit 109 in the Y direction along the longitudinal direction of the plate 107, and a main scanning unit 108p that scans the objective optical system 120 in the X direction along the lateral direction of the plate 107. The components of the fluorescence image acquisition device 101 described here, other than the image acquisition device 102 and the plate 107, constitute a fluorescence scanner 130.

[0019] During actual image acquisition, illumination light from an illumination unit 103 is collimated by a lens 104 and irradiated onto an array plate (plate 107) by an objective mirror 106 that is two-dimensionally scanned by a scanning unit 108. This illumination light excites phosphors in spots on the plate 107, causing them to emit fluorescence. The fluorescence passes through the objective mirror 106, dichroic mirror 105, filter 115, and lens 110 and is received by a photodetector 111. The received fluorescence is converted into an analog electrical signal by the photodetector 111, and is further digitally processed by an image generator 112 that includes an A / D converter, an image format converter, and an image reconstructor (not shown) to convert the signal into a digital signal for 16-bit grayscale TIFF image data. At this time, the image generation unit 112 determines the arrangement of the digital data based on information about the relative position with respect to the plate 107 obtained from the sub-scanning unit 108s that performs sub-scanning on the mounting unit 109 and the main-scanning unit 108p that performs main-scanning on the objective optical system 120. The image acquisition device 102 converts this digital signal into 16-bit grayscale TIFF image data. The image acquisition device 102 also saves the obtained image data.

[0020] Here, an example of the configuration of the above-mentioned fluorescence image acquisition device 101, and in particular the image acquisition device 102, will be described in detail with reference to FIG. 3. FIG. 3 is a block diagram relating to the configuration of the fluorescence image acquisition device 101. As shown in FIG. 3, the image acquisition device 102 in the fluorescence image acquisition device 101 is connected to a scanning unit 108, an irradiating light unit 103, and a light detection unit 111 via a connection unit 16. Although not shown in FIG. 2, the image acquisition device 102 can also be connected to, for example, a display 113 that presents a fluorescence image to the user, or a user interface 118 that functions as a reception unit that receives instructions from the user, as shown in FIG. 3.

[0021] The image acquisition device 102 includes a fluorescence image acquisition unit 121, a first information acquisition unit 122, a second information acquisition unit 123, a pre-scan condition acquisition unit 124, and a postscan condition acquisition unit 125. The image acquisition device 102 also includes a light intensity control unit 126, a detection sensitivity control unit 127, and a scan instruction unit 128. The fluorescence image acquisition unit 121 generates and acquires a fluorescence image based on the fluorescence received by the light detection unit 111. The first information acquisition unit 122 generates and acquires first statistical information related to the fluorescence intensity based on the acquired fluorescence image. The second information acquisition unit 123 assigns a weight, such as a multiplication factor, related to the fluorescence intensity to the first statistical information, and generates and acquires second statistical information. The pre-scan condition acquisition unit 124 acquires scan conditions that are set in advance as defaults or pre-scan conditions specified by the user. The postscan condition acquisition unit 125 evaluates, for example, the saturation tolerance of pixels that become saturated in the light detection unit 111 when receiving fluorescent light based on the second statistical information, and sets and acquires conditions related to acquiring a fluorescent image, such as a new multiplication factor, as postscan conditions based on the second statistical information. The light intensity control unit 126 and the detection sensitivity control unit 127 respectively set the operating conditions of the light irradiation unit 103 and the light detection unit 111 when actually obtaining a fluorescent image based on the set conditions. The scan instruction unit 128 instructs the main scanning unit 108p and the sub scanning unit 108s on the light scanning conditions during prescan and postscan.

[0022] Furthermore, the image acquisition device 102 may have a storage unit 140 that saves the image data described above and stores programs related to the processes executed by the respective acquisition units, etc. Each acquisition unit, etc. functions when, for example, a processor constituting the image acquisition device 102 reads and executes these programs.

[0023] Each of these units may be configured by combining multiple independent processors. Furthermore, each function of each acquisition unit may be realized by a combination of hardware, such as a circuit, and software. While an example in which programs corresponding to each function are stored in a single storage unit 140 has been described here, the embodiment is not limited to this. For example, programs corresponding to the functions of each acquisition unit may be stored in multiple storage circuits, and each acquisition unit may read and execute each program from each storage circuit. Details of the processing performed by each acquisition unit will be described later in detail in the first embodiment with reference to FIG. 1.

[0024] Next, an example of an array plate used in the embodiments described below will be described with reference to FIG. 4. FIG. 4 is a diagram schematically illustrating an array plate (plate 107) viewed from above. On plate 107, multiple types of proteins are spotted in an array. Protein spots 701 have a diameter of approximately 100 μm, and the spots are spaced approximately 40 μm apart. On the illustrated plate 107, three spots are arranged in succession for each type of protein. The protein spots 701 are arranged in blocks 702, with each block consisting of 9 × 9 spots, and the blocks are arranged in a 3 × 6 matrix on plate 107.

[0025] All spots are fluorescently labeled with Alexa Flour (registered trademark) 680. Some protein spots are phosphorylated, and only the phosphorylated protein spots are fluorescently labeled with Alexa Flour (registered trademark) 790. Since the fluorescence intensity increases depending on the degree of phosphorylation, spots with high fluorescence intensity provide more important information in analyzing the degree of phosphorylation.

[0026] The light irradiating unit 103 is composed of a semiconductor laser diode with a wavelength of 670 nm that excites Alexa Flour (registered trademark) 680 (trade name) or a semiconductor laser diode with a wavelength of 780 nm that excites Alexa Flour (registered trademark) 790 (trade name). The light intensity of the laser diode is controlled by changing the current value that drives the laser diode. Note that the method for controlling the light intensity irradiated by the light irradiating unit 103 is not limited to this. For example, it is also possible to arrange an ND filter with variable transmittance in the light irradiating unit and control the transmittance of the ND filter instead of the current value of the laser diode to control the light intensity irradiated.

[0027] The photodetector 111 used here is configured as a PMT (Photomultiplier Tube). The light-receiving sensitivity of the photodetector 111 can be changed by the control voltage of the PMT, and the control voltage of the PMT can be controlled by the image acquisition device 102. In the fluorescence image acquisition device 101 illustrated here, the components from the light irradiating unit 103 to the photodetector 111 are configured as a single confocal optical system. However, the optical system is not limited to this, and a configuration in which multiple confocal optical systems are provided for different excitation wavelengths may also be used.

[0028] Regarding the above-described array plate, the process of acquiring a fluorescent image using the above-described fluorescent image acquisition device 101 will be described in detail in the following embodiments. A more detailed configuration and effects of the present invention will become clear from the following embodiments.

[0029] [First embodiment] In the first embodiment, a fluorescent image of the plate 107 is acquired once as a pre-scan (first measurement step). Then, statistical information about the acquired fluorescent image is acquired, and conditions for the main scan (post-scan) are determined based on the statistical information. In the main scan (second measurement step), a fluorescent image is acquired based on these conditions. Below, the fluorescent image acquisition method according to this embodiment will be described in more detail using the flowchart of FIG. 1 referenced above. When an instruction to start acquiring a fluorescent image of the plate 107 is input by the user via, for example, the user interface 118, the image acquisition device 102 shifts the flow of the fluorescent image acquisition process to step S101, and processing begins.

[0030] <Step S101> In step S101, the fluorescence image acquisition unit 121 executes a fluorescence image acquisition process. To perform the first measurement, the image acquisition device 102 sets the illumination light intensity of the illumination light unit 103 to a preset value (first illumination light intensity) via the light intensity control unit 126, and sets the light receiving sensitivity of the light detection unit 111 to a preset value (first sensitivity condition) via the detection sensitivity control unit 127. The preset value is roughly determined based on the concentration of the spotted protein, the degree of phosphorylation of the protein, the reaction conditions of the fluorescent label, the temperature of the array plate, and the like. The image acquisition device 102 operates the scanning unit 108 via the scanning instruction unit 128 to cause the fluorescence image acquisition unit 121 to acquire a fluorescence image of the array plate under the set measurement conditions, and the acquired fluorescence image is stored in the memory unit 140. After acquiring the fluorescence image, the image acquisition device 102 advances the flow to step S102.

[0031] <Step S102> In step S102, the first information acquisition unit 122 executes a process for acquiring first statistical information. Specifically, a region of interest (ROI) is set for all spots in the acquired fluorescent image of the array plate, and a histogram of the average values ​​of each ROI is acquired. At this time, the above-mentioned three identical protein spots may be collectively treated as the same ROI. Then, from the histogram of the average values ​​of each acquired ROI, statistical information such as the mean, variance, and histogram of the ROI with the highest average value is generated and acquired as first statistical information.

[0032] In this case, statistical information may be acquired by extracting not only the region of interest with the highest average value, but also the regions of interest with the highest average values. Furthermore, the region of interest with the highest average value may contain saturated pixels (in the case of 16-bit images, a brightness value of 65535) or pixels with a brightness value of 0 or a brightness value at the noise level of the PMT. In such cases, the measurement conditions may be changed and the processing of steps S101 to S102 may be repeated again. This does not apply if the S / N ratio or reproducibility of the fluorescence image acquisition device is poor. For example, whether the average value of each region of interest is within a threshold value may be used as a criterion for determining whether to change the measurement conditions and repeat the processing of steps S101 to S102 again. After acquiring the first statistical information, the image acquisition device 102 advances the flow to step S103.

[0033] <Step S103> In step S103, the second information acquisition unit 123 executes a process for acquiring second statistical information. Specifically, assuming that the shape of the histogram of the region of interest with the highest average value acquired in step S102 is a normal distribution, a probability density function is created from the mean and variance values. FIG. 5(a) shows an example of a histogram of the region of interest with the highest average value acquired, and FIG. 5(b) shows an example of a probability density function created using the histogram illustrated in FIG. 5(a). The second information acquisition unit 123 executes a process for obtaining a setting value (sensitivity multiplication factor) to be used in the second measurement (step S105, described later) with respect to the value preset in the first measurement (step S101). Specifically, the mean value and variance acquired in step S102 are multiplied by a multiplication factor K to assign weighting to the values, and a new probability density function is created as the second statistical information. The newly created probability density function is the predicted histogram shape of the region of interest with the highest average value acquired in the second measurement. Once the second statistical information is acquired, the image acquisition device 102 moves the flow to step S104.

[0034] <Step S104> In step S104, the postscan condition acquisition unit 125 sets and acquires the setting value used when actually acquiring a fluorescence image (second measurement) by changing the multiplication factor K, which is a weight for the probability density function, relative to the preset value. For example, increasing the multiplication factor K can cause the probability density function to have a distribution greater than the maximum luminance value of the image. This portion of the probability density function corresponds to the probability of saturated pixels in the region of interest with the highest average value obtained in the second measurement. If the saturation tolerance of the probability density function is set to, for example, 3%, the postscan condition acquisition unit 125 calculates and acquires the maximum multiplication factor K' at which the saturation amount does not exceed 3%. FIG. 6(a) shows the original probability density function obtained in step S102, and FIG. 6(b) shows the probability density function obtained by multiplying the multiplication factor K by the saturation tolerance of 3% in the prescan condition acquisition unit 124. Note that the saturation tolerance may be the probability corresponding to one pixel (the reciprocal of the number of pixels in the region of interest). Furthermore, the multiplication factor K' may be a decimal value less than 1 if, for example, more pixels than expected become saturated due to the prescan. Therefore, the multiplication factor K' is a value consisting of a positive real number.

[0035] In this embodiment, the probability density function was created assuming the histogram shape to be a normal distribution for ease of calculation. However, distribution patterns other than a simple normal distribution may be used when creating the probability density function. For example, depending on the accuracy of spot creation on the array plate, a ring structure with low brightness on the inside and high brightness on the outside may be formed, so a probability density function that overlaps two normal distributions may be used. Furthermore, the method for obtaining the multiplication factor K' is not limited to this example. For example, without creating a probability density function, the multiplication factor K' may be calculated so that the maximum brightness value of the region of interest with the highest average value obtained in step S102 is saturated.

[0036] The postscan condition acquisition unit 125 acquires the calculated multiplication factor K' as the PMT sensitivity multiplication factor K2 as the second condition. The set sensitivity multiplication factor K2 is sent to the detection sensitivity control unit 127. The detection sensitivity control unit 127 then calculates a control voltage for the photodetector 111 corresponding to the measured sensitivity of the second measurement, which is calculated by multiplying the sensitivity of the first measurement by the sensitivity multiplication factor K2, based on the relationship between the control voltage and sensitivity of the PMT measured in advance as shown in FIG. 7. In FIG. 7, the vertical axis represents sensitivity and the horizontal axis represents control voltage, and the axes are scaled linearly. Once the sensitivity multiplication factor K2 is calculated and the control voltage of the photodetector 111 is set as the second condition, the image acquisition device 102 proceeds to step S105.

[0037] <Step S105> In step S105, the image acquisition device 102 controls the scanning unit 108, the irradiating light unit 103, and the light detection unit 111 to perform a post-scan fluorescent image acquisition process as the second measurement. Specifically, to perform the second measurement, the image acquisition device 102 sets the control voltage value of the PMT serving as the light detection unit 111 to the value obtained in step S104. Then, a fluorescent image of the array plate is acquired under the set measurement conditions, and the image acquisition device 102 stores the acquired fluorescent image in the storage unit 140. After acquiring the fluorescent image, the image acquisition device 102 advances the flow to end the fluorescent image acquisition process according to this embodiment.

[0038] This embodiment assigns appropriate weights to the bright phosphorylated protein spots of interest, and based on the weights obtained, it is possible to uniquely determine the sensitivity of the light detection unit 111 so that the bright spots fall appropriately within the dynamic range of the signal processing system.

[0039] [Second embodiment] Next, a fluorescence image acquisition method according to a second embodiment of the present invention will be described with reference to Fig. 8. Fig. 8 is a flowchart showing the fluorescence image acquisition process according to this embodiment in the same format as Fig. 1. Note that the same steps as in Fig. 1 are given the same numbers, and their explanation will be omitted here. Furthermore, the fluorescence image acquisition device used to carry out this method is the one exemplified in Figs. 2 and 3, and since it has the same configuration as the first embodiment, its explanation will be omitted here.

[0040] The image acquisition process according to this embodiment differs from the first embodiment in that the sensitivity multiplication factor K2 is obtained in step S104 of the first embodiment, and the irradiated light intensity multiplication factor K1 is obtained in step S201. The process executed in step S201 will be described below.

[0041] <Step S201> In step S201, as in step S104, the multiplication factor K, which is the weight for the probability density function obtained in step S102, is changed to calculate a setting value to be used when actually acquiring a fluorescent image (second measurement) relative to the preset value. For example, the saturation tolerance of the probability density function is set to 3%, as in step S104, to obtain the maximum multiplication factor K'. However, in step S201, a process is executed to obtain an irradiated light intensity multiplication factor K1 from the calculated multiplication factor K'. Specifically, based on the relationship between the irradiated light intensity and sensitivity (fluorescence brightness value) measured in advance as shown in FIG. 9, the irradiated light intensity for the second measurement is calculated by multiplying the irradiated light intensity preset in the first measurement (step S101) by the multiplication factor K1. Note that in FIG. 9, the vertical axis represents sensitivity and the horizontal axis represents irradiated light intensity, and the axes are scaled linearly. When the processes of steps S104 and S201 are completed, the image acquisition device 102 advances the flow to step S105, and executes the process of acquiring a fluorescent image at the set irradiation light intensity and sensitivity multiplication factor.

[0042] In the above description, the sensitivity multiplication factor K2 is calculated based on the multiplication factor K' in step S104, and the irradiated light intensity multiplication factor K1 is calculated in step S201. However, the method for calculating the irradiated light intensity multiplication factor K1 and the sensitivity multiplication factor K2 is not limited to this example. For example, the calculated multiplication factor K' may be allocated to the irradiated light intensity multiplication factor K1 and the sensitivity multiplication factor K2 obtained in step S104. For example, the square root of the multiplication factor K' may be allocated to the irradiated light intensity multiplication factor K1 and the sensitivity multiplication factor K2, respectively.

[0043] Here, when capturing a fluorescent image of an array plate, for example, excessive light irradiation can cause a phenomenon known as fluorescent browning, in which the coloring efficiency (luminescence efficiency) dynamically decreases. If this fluorescent browning occurs during fluorescent image capture, an appropriate fluorescent image cannot be obtained from the array plate. In this embodiment, taking into account the effect of this fluorescent bleaching in the second measurement, the sensitivity multiplication factor K2 may be set to the maximum that can be achieved by the device, and the irradiated light intensity multiplication factor K1 may be set to K / K2. Alternatively, the irradiated light intensity multiplication factor K1 may be set to the maximum irradiated light intensity in the range where the fluorescent brightness increases linearly with the irradiated light intensity, and the sensitivity multiplication factor K2 may be set to K / K1.

[0044] This embodiment assigns appropriate weights to the bright phosphorylated protein spots of interest, and based on the weights obtained, it is possible to uniquely determine the sensitivity of the photodetector and the irradiated light intensity in the second measurement so that the bright spots fall appropriately within the dynamic range of the signal processing system.

[0045] Furthermore, considering the effect of fluorescence fading due to the prescan (first measurement), it is desirable to keep the illumination light intensity during the prescan as low as possible. In this embodiment, fluorescence fading can be taken into account by setting the illumination light intensity low in the first measurement and high in the second measurement. Generally, increasing the PMT control voltage increases the sensitivity of the PMT, but increasing the sensitivity also increases the variability in signal and noise. Therefore, setting the sensitivity low can improve the S / N ratio and reproducibility in the measurement. According to this embodiment, fluorescence fading can be taken into account by setting the illumination light intensity low in the first measurement and high in the second measurement.

[0046] [Third embodiment] Next, a fluorescence image acquisition method according to a third embodiment of the present invention will be described with reference to Fig. 10. Fig. 10 is a flowchart showing the fluorescence image acquisition process according to this embodiment in the same format as Fig. 1. Note that the same steps as in Fig. 1 are given the same numbers, and their explanation will be omitted here. Furthermore, the fluorescence image acquisition device used to carry out this method is the one exemplified in Figs. 2 and 3, and since it has the same configuration as the first embodiment, its explanation will be omitted here.

[0047] The image acquisition process according to this embodiment differs from the first embodiment in that, instead of step S104 in the first embodiment where the sensitivity multiplication factor K2 is obtained, an irradiation light intensity multiplication factor K1 is obtained in step S304. The process executed in step S304 will be described below.

[0048] <Step S304> In step S304, similar to step S104, the multiplication factor K, which is the weight for the probability density function obtained in step S102, is changed to calculate a setting value to be used when actually acquiring a fluorescence image (second measurement) for the preset value. However, in this embodiment, the sensitivity multiplication factor K2 is not obtained based on the multiplication factor K', but the illumination light intensity multiplication factor K1 is obtained in step S304, which is different. Once the illumination light intensity multiplication factor K1 is obtained in step S304, the image acquisition device 102 transitions the flow to step S105, and processing for acquiring a fluorescence image at the set illumination light intensity is executed.

[0049] This embodiment assigns appropriate weights to the bright phosphorylated protein spots of interest. Based on the weights obtained, the illumination light intensity for the second measurement can be uniquely determined so that the bright spots fall within the dynamic range of the signal processing system. Furthermore, by setting the illumination light intensity low in the first measurement and high in the second measurement, fluorescence bleaching can be taken into account.

[0050] [Fourth embodiment] Next, a fluorescence image acquisition device and a fluorescence image acquisition method according to a fourth embodiment of the present invention will be described with reference to Figures 11 and 12. Figure 11 is a block diagram showing the schematic configuration of a fluorescence image acquisition device according to this embodiment in a format similar to that of Figure 3. Figure 12 is a flowchart showing the fluorescence image acquisition process according to this embodiment in a format similar to that of Figure 1 or Figure 8. Note that the same components as those illustrated in Figure 3 are given the same numbers, and the same steps as those in Figure 1 are given the same numbers, and their explanation will be omitted here.

[0051] The fluorescence image acquisition device 401 according to this embodiment differs from the fluorescence image acquisition device 101 described in the first embodiment in that the image acquisition device 402 includes a predicted image acquisition unit 429, a determination unit 430, and a reception / presentation unit 431. The predicted image acquisition unit 429 generates and acquires a fluorescence image predicted to be obtained using, for example, at least one of the acquired irradiation light intensity multiplication factor K1 and sensitivity multiplication factor K2. Note that if the multiplication factor value includes a decimal and the brightness value of a pixel in the predicted fluorescence image is a decimal, the predicted fluorescence image is converted to an integer by rounding, rounding down, or rounding up, and then image generation is performed. The generated image can be displayed, for example, on the display 113. The determination unit 430 determines whether the input instruction entered via the user interface 118 based on observation of the predicted image is appropriate (suitable), inappropriate (unsuitable), requires correction, or abort, as described in detail below. The reception / presentation unit 431 can display a display format on the display 113 that allows the user to input instructions such as appropriate (suitable), inappropriate (not suitable), correction instructions required, or cancellation. The fluorescence image acquisition process according to this embodiment, which is executed using the fluorescence image acquisition device 401, will be described below.

[0052] The fluorescence image acquisition process according to this embodiment is similar to that of the second embodiment up to step S104 and step S201 (FIG. 8) in which the irradiated light intensity multiplication factor K1 and the sensitivity multiplication factor K2 are acquired. In this embodiment, the processes executed thereafter differ from those of the second embodiment. Below, the processes executed in steps S405 to S409 after the irradiated light intensity multiplication factor K1 and the sensitivity multiplication factor K2 are acquired will be described. Once the irradiated light intensity multiplication factor K1 and the sensitivity multiplication factor K2 have been acquired, the image acquisition device 402 causes the flow to proceed to step S405.

[0053] <Step S405> In step S405, the predicted image acquisition unit 429 generates and acquires a predicted image that will be obtained using the irradiation light intensity and sensitivity multiplication factor set by the irradiation light intensity multiplication factor K1 and sensitivity multiplication factor K2. Specifically, the predicted image is obtained by multiplying the fluorescence image acquired in the first measurement process in step S101 by the irradiation light intensity multiplication factor K1 and the sensitivity multiplication factor K2. Once the predicted image has been acquired, the image acquisition device 402 causes the flow to proceed to step S406.

[0054] <Step S406> In step S406, the image acquisition device 402 instructs the display 113 to display the predicted image acquired in step S405. The user can use the displayed predicted fluorescence image to confirm the presence of saturated pixels when the set irradiation light intensity multiplication factor K1 and sensitivity multiplication factor K2 are used. For example, if the display 113 and the user interface 118 are integrated, the reception / presentation unit 431 can display, on the display 113, buttons, slide bars, dialog boxes, and the like as a reception unit for accepting instructions from the user, along with the predicted fluorescence image. Once the predicted fluorescence image and a display format for reception (not shown) are displayed on the display 113, the image acquisition device 402 causes the flow to proceed to step S407. The display 113 can be used to present the user with images, supplementary information associated with the images, and the like.

[0055] <Step S407> In step S407, the image acquisition device 402 accepts a user instruction regarding the measurement conditions via a display mode for accepting user instructions, such as a button on the display 113. Examples of commands that can be input by the user include stop measurement (stop), appropriate (appropriate), with correction, and inappropriate (inappropriate), and corresponding buttons are displayed on the display 113. The button corresponding to stop measurement can be pressed when the predicted fluorescence image indicates an inappropriate state for acquiring a fluorescence image, such as the possibility of fluorescent browning. The button corresponding to appropriate can be pressed when the predicted fluorescence image indicates that it is appropriate to use the set illumination light intensity multiplication factor K1 and sensitivity multiplication factor K2. When the button corresponding to correction is pressed, it is assumed that the predicted fluorescence image indicates that it is inappropriate to use the set illumination light intensity multiplication factor K1 and sensitivity multiplication factor K2 as they are. However, in this case, it is assumed that correcting either of these factors will enable a second measurement to be performed so that the appropriate high-brightness spot falls appropriately within the dynamic range of the signal processing system. The button corresponding to "No" can be pressed when it is deemed appropriate to use the set irradiation light intensity multiplication factor K1 and sensitivity multiplication factor K2 as they are, and to perform the second measurement using the preset values ​​used in the first measurement.

[0056] In step S407, the user presses one of these buttons to input a command regarding the measurement conditions into the image acquisition device 402. Note that the type, number, and display mode of the push buttons exemplified here are merely examples and can be changed as appropriate depending on the actual device configuration, the configuration of the array plate from which the fluorescent image is to be acquired, the display format of the fluorescent image, etc. Furthermore, the processing corresponding to the push button is not limited to the example described here and can be changed as appropriate depending on the user's options. Furthermore, for example, if no command is input within a predetermined period after the start of display of a predicted fluorescent image, etc., it can be determined by default that "OK" or "NO" has been pressed. When a command is input from the user, the image acquisition device 402 transitions the flow to step S408.

[0057] <Step S408> In step S408, the determination unit 430 determines whether the input command was abort, pass, correction required, or no. If the input command is abort, the image acquisition device 402 skips the subsequent step S409 and aborts and terminates the fluorescence image acquisition process. If the input command is anything other than abort, the image acquisition device 402 transitions the flow to step S409.

[0058] <Step S409> In step S409, measurement conditions are set based on the multiplication conditions according to the input command, and a second measurement step is performed as a postscan. More specifically, if appropriate, the second measurement is performed using the set irradiation light intensity multiplication factor K1 and sensitivity multiplication factor K2 as they are. If corrections are required, the multiplication factor K' obtained based on the first statistical information is changed again, and more appropriate irradiation light intensity multiplication factors K1' and sensitivity multiplication factors K2' are set. In this case, a fluorescence image may be predicted using these again, displayed, and presented to the user. If not appropriate, the second measurement is performed using the preset values ​​used in the first measurement, as described above. After the second measurement is completed, the image acquisition device 402 moves the flow and terminates the fluorescence image capture process according to this embodiment.

[0059] Note that the above description deals with an embodiment in which the predicted fluorescence image is actually displayed on the display 113, and the user observes it and selects from options displayed on the display 113 to proceed with the processing. However, the embodiments of the present invention are not limited to this example. For example, by focusing on saturated pixels (saturated pixels) in high-brightness spots, thresholds corresponding to each of the above options may be set for the presence state of saturated pixels in the predicted fluorescence image, such as the number and distribution, and the determination unit 430 may evaluate these evaluation items. Then, processing corresponding to each option may be executed depending on the evaluation results.

[0060] In this embodiment, the user can confirm and appropriately weight the highly bright phosphorylated protein spots of interest, and based on the weights obtained, the sensitivity of the photodetector and the intensity of the irradiated light in the second measurement can be appropriately determined so that the highly bright spots fall within the dynamic range of the signal processing system.

[0061] [Fifth embodiment] Next, a fluorescence image acquisition method according to a fifth embodiment of the present invention will be described with reference to Fig. 13. Fig. 13 is a flowchart showing the fluorescence image acquisition process according to this embodiment in the same format as Figs. 10 and 12. Note that the same steps as in Figs. 10 and 12 are given the same numbers, and their explanation will be omitted here. Furthermore, the fluorescence image acquisition device used to carry out this embodiment is the one exemplified in Figs. 2 and 11, and since it has the same configuration as the fourth embodiment, its explanation will be omitted here.

[0062] This embodiment differs from the fourth embodiment in that only the irradiated light intensity multiplication factor K1 is acquired, and the sensitivity multiplication factor K2 is not acquired. That is, in this embodiment, only the irradiated light intensity multiplication factor K1 is acquired in step S304 based on the multiplication factor K', and the image acquisition device 402 proceeds to step S405, where the acquisition process for the predicted fluorescence image at the set irradiated light intensity is executed.

[0063] In steps S406 to S409, the predicted fluorescence image is displayed, commands related to measurement conditions are received, the commands are judged, and a second measurement is performed based on the judgment results, as in the fourth embodiment. After the second measurement is completed, the image acquisition device 402 moves the flow and ends the fluorescence image acquisition process according to this embodiment.

[0064] This embodiment allows the user to confirm and appropriately weight the highly bright phosphorylated protein spots of interest. Based on the weights obtained, the illumination light intensity for the second measurement can be appropriately determined so that the highly bright spots fall within the dynamic range of the signal processing system. Furthermore, by setting the illumination light intensity low in the first measurement and high in the second measurement, fluorescence fading can be taken into account.

[0065] [Sixth embodiment] Next, a fluorescence image acquisition method according to a sixth embodiment of the present invention will be described with reference to Fig. 14. Fig. 14 is a flowchart showing the fluorescence image acquisition process according to this embodiment in the same format as Fig. 1 or Fig. 8. Note that the same steps as those in Fig. 1 or Fig. 8 are given the same numbers, and their explanation will be omitted here. Furthermore, the fluorescence image acquisition device used to carry out this method is the one exemplified in Figs. 2 and 3, and since it has the same configuration as that of the first embodiment, its explanation will be omitted here.

[0066] In the image acquisition process according to the second embodiment, the sensitivity multiplication factor K2 is obtained in step S104 of the first embodiment, and the irradiation light intensity multiplication factor K1 is obtained in step S201. In contrast, this embodiment differs from the first or second embodiment in that a multiplication factor related to the scanning conditions is obtained in step S601 in addition to the multiplication factors K1 and K2. The process executed in step S601 will be described below.

[0067] The conditions for acquiring a fluorescence image also include scanning conditions for scanning the plate 107 with an illumination spot formed by the primary light emitted onto the plate 107. When acquiring fluorescence with the light detection unit 111, one of the factors that can cause, for example, brown fluorescence is the effective irradiation dose of the primary light. This effective irradiation dose is affected not only by the illumination intensity of the primary light but also by the illumination time of the primary light. For example, when scanning the plate 107 with the illumination spot, a slow scanning speed results in a long illumination time. Therefore, in order to reduce the influence of brown fluorescence, it is preferable to consider control of the scanning conditions in addition to control of the light intensity and detection sensitivity of the primary light described in the second embodiment. In this embodiment, the multiplication conditions are determined taking these scanning conditions into account.

[0068] In the description of step S101 in the first embodiment, only the first irradiation light intensity value and the first sensitivity condition value are mentioned as preset values, but there are also preset values ​​that are determined in advance for the scanning conditions. The preset values ​​for the scanning conditions are acquired in step S101 like the other preset values, and the first measurement step is performed using these preset values. Therefore, only the preset values ​​for the scanning conditions will be described here. The actual fluorescence acquisition time is determined by the time (the reciprocal of the sampling frequency) for which the light detection unit 111 samples the fluorescence and the duty ratio of the time the light detection unit 111 detects the fluorescence to the scanning time. For example, the scanning conditions when the first measurement step is performed in step S101 are defined as first scanning conditions, and these are expressed as the value obtained by dividing the first sampling time, which corresponds to the time per unit time that the light detection unit 111 detects the fluorescence, by the first scanning speed, which corresponds to the movement speed of the irradiation spot in the pre-scan.

[0069] <Step S601> In this embodiment, step S601 is added as a process executed after the second statistical information is acquired in step S103. In step S601, a process is executed to obtain a scan multiplication factor K3 from the calculated multiplication factor K'. Specifically, based on the relationship between the previously measured fluorescence sampling time and detection sensitivity shown in FIG. 15, the scanning conditions for the second measurement are calculated by multiplying the scanning conditions used as preset values ​​in the first measurement (step S101) by the scan multiplication factor K3. In FIG. 15, the vertical axis represents sensitivity and the horizontal axis represents sampling time, and the axes are scaled linearly. Furthermore, the scan multiplication factor K3 may be calculated as K3 relative to K1, for example, using a method similar to the method used to calculate K2 from K' in the second embodiment. Alternatively, after K1 is determined, the remaining values ​​may be allocated to K2 and K3. For example, the allocation method can use the method used to allocate K' to K1 and K2. When the process of determining and acquiring the multiplication factors K1 to K3 is completed, the image acquisition device 102 moves the flow to step S105, and executes the process of acquiring a fluorescent image under the scanning conditions set using the scanning multiplication factor K3, as well as the conditions set by the irradiation light intensity and sensitivity multiplication factor.

[0070] For example, it may be difficult to directly adopt the optimum value for the multiplication factor obtained in step S104 or step S201 due to the conditions shown in Fig. 7 or 9. By appropriately determining the scan multiplication factor K3 in step S601, it becomes possible to appropriately determine the multiplication factors K1 and K2 even when their setting ranges are narrow.

[0071] According to this embodiment, appropriate weighting is performed on the bright phosphorylated protein spots of interest. Based on the obtained weighting, the sensitivity of the photodetector and the irradiated light intensity for the second measurement can be uniquely determined so that the bright spots fall appropriately within the dynamic range of the signal processing system. Furthermore, according to this embodiment, by also adjusting the scanning conditions, the second measurement can be performed even with a narrower range of change in the sensitivity of the photodetector and the irradiated light intensity, which is expected to reduce the possibility of generating brown fluorescence. While the first and second embodiments have been described above with step S601 and a configuration for implementing this step, this embodiment may also be combined with the predicted image acquisition process and presentation process in the third to fifth embodiments, for example.

[0072] [Other embodiments] Although the embodiments of the present invention have been described above in detail, the present invention can be embodied as a system, device, method, program, recording medium (storage medium), etc. Specifically, the present invention may be applied to a system made up of multiple devices (for example, a host computer, an interface device, an imaging device, a Web application, etc.), or may be applied to an apparatus made up of a single device.

[0073] The object of the present invention can also be achieved by: providing a recording medium (or storage medium) on which is recorded software program code (computer program) that realizes the functions of the above-described embodiments to a system or device; the storage medium is computer-readable; and a computer (or CPU or MPU) of the system or device reads and executes the program code stored on the recording medium. In this case, the program code itself read from the recording medium realizes the functions of the above-described embodiments, and the recording medium on which the program code is recorded constitutes the present disclosure.

[0074] As described above, the present invention relates to an image acquisition method for acquiring a fluorescence image by scanning a plate with primary light to acquire the fluorescence intensity of an object on the plate. The image acquisition method can include a first measurement step (step S101), a first information acquisition step (step S102), and a second information acquisition step (step S103). This image acquisition method can further include a condition acquisition step (steps S104, S201, S304) and a second measurement step (step S105). In the first measurement step, the plate is scanned with first primary light having a preset first irradiation light intensity, and the light detection unit 111 is operated under a preset first sensitivity condition to acquire a fluorescence image. In the first information acquisition step, first statistical information related to fluorescence intensity can be acquired based on the fluorescence image acquired in the first measurement step. In the second information acquisition step, second statistical information can be acquired by weighting the first statistical information with respect to fluorescence intensity. In the condition acquisition step, the second statistical information can be used to acquire multiplication conditions (multiplication factors K1, K2) for at least one of the first irradiation light intensity and the first sensitivity condition. In the second measurement step, a fluorescent image of the plate can be acquired using the irradiation light intensity and sensitivity conditions set based on the acquired multiplication conditions.

[0075] The image acquisition methods according to the first to third embodiments may include cases where a related multiplication condition is not acquired from either the first irradiation light intensity or the first sensitivity condition. In such cases, for one of the first irradiation light intensity and the first sensitivity condition for which a multiplication condition is not acquired, the second measurement step can be performed using one of the first irradiation light intensity and the first sensitivity condition (preset value). Furthermore, cases where a related multiplication condition is not acquired from either the first irradiation light intensity or the first sensitivity condition may also occur depending on the allocation of the multiplication factor K'. In such cases, for example, for one of the first irradiation light intensity and the first sensitivity condition for which a multiplication condition is not acquired, the multiplication factor (multiplication condition) can be set to 1. Furthermore, for example, if there are more saturated pixels than expected due to the pre-scan, the multiplication factor K' may be a decimal value less than 1. In such cases, the multiplication factor (multiplication condition) can be defined as a positive real number depending on the multiplication factor K'.

[0076] Furthermore, the present invention can include a predicted image acquisition step (step S405) in addition to the above-described first measurement step, first information acquisition step, second information acquisition step, and condition acquisition step. The predicted image acquisition step can simulate a predicted fluorescence image that is predicted to be acquired when a fluorescence image of the plate is captured using irradiation light intensity and sensitivity conditions set based on the multiplication conditions (multiplication factors K1, K2) acquired in the condition acquisition step. Note that such an image acquisition method can further include a presentation step (step S406) of presenting this predicted fluorescence image to a user on the display 113. Furthermore, this image acquisition method can further include a step (step S407) of accepting instructions regarding the suitability or correction of the multiplication conditions acquired in the condition acquisition step, based on the predicted fluorescence image.

[0077] This image acquisition method can further include a second measurement step (step S105) of acquiring a fluorescent image of the plate using irradiation light intensity and sensitivity conditions set based on the multiplication conditions acquired based on the received instruction. The multiplication conditions acquired based on the received instruction can include, for example, the following four multiplication conditions: multiplication conditions corrected based on the received modification instruction, multiplication conditions acquired in the condition acquisition step based on the received appropriate instruction, and predetermined multiplication conditions acquired based on the received unacceptable instruction. The predetermined multiplication conditions include, for example, a multiplication factor that sets the irradiation light intensity and sensitivity conditions to preset values.

[0078] Furthermore, the present invention can configure an image acquisition device 102 that acquires a fluorescent image from a fluorescent scanner 130 in a fluorescent image acquisition device 101 that acquires a fluorescent image by scanning primary light across a plate to acquire the fluorescent intensity of an object on the plate. The fluorescent scanner 130 can include, for example, a mounting unit 109, an emission unit (114), a light collecting unit (114), a light source (103), a light detection unit 111, a scanning unit 108, and an image generation unit 112. A plate (107) on which an object is fixed is mounted on the mounting unit 109. In the above-described embodiment, the function of the emission unit is performed by, for example, the objective lens 114, which emits primary light toward the plate 107 and forms an illumination spot on the plate. The function of the light collecting unit is performed by, for example, the objective lens 114, and which collects secondary light from the object. In the above-described embodiment, the light source can be configured by the light irradiation unit 103, for example, and is optically coupled to the emission unit (114). The light detection unit 111 can be optically coupled to the light collection unit (114). The scanning unit 108 controls the mounting unit 109 and the emission unit (114) according to the scanning conditions, and changes the relative positions of the irradiation spot and the light collection unit (114) with respect to the plate 107. The image generation unit 112 can generate a fluorescence image based on information about the detection intensity from the light detection unit 111 and information about the relative position, which is the scanning condition of the scanning unit 108.

[0079] An image acquisition device 102 according to one aspect of the present invention is capable of controlling a fluorescent scanner 130 and acquiring a fluorescent image from the fluorescent scanner 130. The image acquisition device 102 includes a light intensity control unit 126, a detection sensitivity control unit 127, a scanning instruction unit 128, a pre-scan condition acquisition unit 124, a storage unit 140, a first statistical information acquisition unit (122), a second statistical information acquisition unit (123), and a second statistical information acquisition unit (125). The light intensity control unit 126 can control, for example, the irradiating unit 103 to control the primary light guided from the light source to the emission unit (114) to a predetermined light intensity. The detection sensitivity control unit 127 can control the light detection unit 111 to operate at a predetermined detection sensitivity. The scanning instruction unit 128 can instruct the scanning unit 108 on scanning conditions. The prescan condition acquisition unit 124 can acquire, as prescan conditions, first light intensity conditions and first sensitivity conditions to be used in a prescan. The storage unit 140 can store, for example, fluorescent images acquired under the acquired prescan conditions. The first information acquisition unit 122, which is an example of a first statistical information acquisition unit, can acquire first statistical information by statistically analyzing information related to the fluorescent intensity of the fluorescent image. The second information acquisition unit 123, which is an example of a second statistical information acquisition unit, can acquire second statistical information in which the first statistical information is weighted with respect to the fluorescent intensity. The postscan condition acquisition unit 125 can acquire, as postscan conditions, second light intensity conditions and second sensitivity conditions to be used in a postscan performed after the prescan, based on the second statistical information and the prescan conditions.

[0080] The image acquisition device 102 (402) described above may further include a presentation unit, such as the display 113 in the above-described embodiment. The display 113 may present to the user a fluorescence image acquired under the acquired prescan conditions and a second fluorescence image acquired under the acquired postscan conditions. In this case, the image acquisition device 102 (402) may further include a predicted image acquisition unit 429 that can simulate a predicted fluorescence image that is predicted to be acquired when a fluorescence image of the plate 107 is captured under the postscan conditions. In this configuration, the presentation unit (display 113) may be configured to present the predicted image to the user. The image acquisition device 402 may also include a display mode that accepts instructions regarding the suitability of the acquired postscan conditions or instructions to correct them. For example, the image acquisition device 402 may further include a reception / presentation unit 431 that causes the presentation unit (display 113) to present the above-described "suitable" or similar.

[0081] The prescan conditions described above can include a first scanning condition, as described in the sixth embodiment. The first scanning condition can be obtained, for example, by dividing a first sampling time corresponding to the time it takes the light detection unit 111 to detect a fluorescent image per unit time by a first scanning speed corresponding to the moving speed of the irradiation spot in the prescan. The first scanning condition described here is just one example, and is not limited to this, as long as the scanning condition can specify the effective dose of primary light in the prescan. The postscan conditions can include a second scanning condition. The second scanning condition can be obtained, for example, by dividing a second sampling time corresponding to the time it takes the light detection unit 111 to detect a fluorescent image per unit time by a second scanning speed corresponding to the moving speed of the irradiation spot in the postscan. The second scanning condition described here is just one example, and is not limited to this, as long as the scanning condition can specify the effective dose of primary light in the postscan. In the sixth embodiment described above, the coefficient used when determining the second scanning condition with respect to the first scanning condition can be, for example, the scanning multiplication factor K3, which is an example of the third multiplication condition.

[0082] When determining second scanning conditions for the postscan, the prescan condition acquisition unit 124 can acquire the first light intensity condition, the first sensitivity condition, and the first scanning condition as prescan conditions. Correspondingly, the postscan condition acquisition unit 125 can acquire the second light intensity condition (K1) and the second sensitivity condition (K2) based on the second statistical information, the prescan conditions, and the second scanning conditions. In this case, for example, the ratio of the second light intensity condition to the first light intensity condition can be used as the first multiplication condition (K1). The ratio of the second sensitivity condition to the first sensitivity condition can be used as the second multiplication condition (K2). The ratio of the second scanning condition to the first scanning condition can be used as the third multiplication condition (K3). When the above multiplication conditions (K1 to K3) are used, the postscan condition acquisition unit 125 can acquire the multiplication factor K1, which is an example of the first multiplication condition, the multiplication factor K2, which is an example of the second multiplication condition, and the multiplication factor K3, which is an example of the third multiplication condition, as positive real numbers. Furthermore, the emission unit (114) can irradiate the primary light so as to focus on the object.

[0083] As described above, the present invention allows the user to confirm and appropriately weight the highly bright phosphorylated protein spots of interest. Based on the weights obtained, the irradiated light intensity for the second measurement can be appropriately determined so that the highly bright spots fall within the dynamic range of the signal processing system. Furthermore, by setting the irradiated light intensity low in the first measurement and high in the second measurement, fluorescence fading can be taken into account. Furthermore, by including scanning conditions as variables, the second measurement can be performed even with a narrower range of change in the sensitivity of the light detection unit and the irradiated light intensity, further reducing the possibility of brown fluorescence.

[0084] The above-mentioned invention includes the following methods, programs, and configurations. (Method 1) 1. An image acquisition method for acquiring a fluorescence image by scanning a plate with primary light to acquire a fluorescence intensity of an object on the plate, comprising: a first measurement step of scanning the plate with primary light having a first irradiation light intensity and operating the light detection unit under a first sensitivity condition to acquire a fluorescent image; a first information acquisition step of acquiring first statistical information related to the fluorescence intensity based on the fluorescence image; a second information acquisition step of acquiring second statistical information by weighting the first statistical information with respect to the fluorescence intensity; a condition acquisition step of acquiring a multiplication condition related to at least one of the first irradiation light intensity and the first sensitivity condition using the second statistical information; a second measurement step of scanning with primary light having an irradiation light intensity set based on the acquired multiplication conditions and operating the light detection unit under set sensitivity conditions to acquire a fluorescent image of the plate; An image acquisition method comprising: (Method 2) An image acquisition method as described in Method 1, wherein, if the associated multiplication condition in the first irradiation light intensity and the first sensitivity condition is not obtained from either one of the first irradiation light intensity and the first sensitivity condition for which the multiplication condition was not obtained, a fluorescent image of the plate is acquired in the second measurement step using one of the first irradiation light intensity and the first sensitivity condition. (Method 3) An image acquisition method according to Method 1, wherein, if the associated multiplication condition in either the first irradiation light intensity or the first sensitivity condition is not acquired from either one of the first irradiation light intensity and the first sensitivity condition for which the multiplication condition was not acquired, a fluorescent image of the plate is acquired in the second measurement step using the irradiation light intensity or sensitivity condition set by regarding the multiplication condition as 1 for either the first irradiation light intensity or the first sensitivity condition. (Method 4) 4. An image acquisition method according to any one of methods 1 to 3, wherein the multiplication condition is a positive real number. (Method 5) 1. An image acquisition method for acquiring a fluorescence image by scanning a plate with primary light to acquire a fluorescence intensity of an object on the plate, comprising: a first measurement step of scanning the plate with primary light having a first irradiation light intensity and operating the light detection unit under a first sensitivity condition to acquire a fluorescent image; a first information acquisition step of acquiring first statistical information related to the fluorescence intensity based on the fluorescence image; a second information acquisition step of acquiring second statistical information by weighting the first statistical information with respect to the fluorescence intensity; a condition acquisition step of acquiring a multiplication condition related to at least one of the first irradiation light intensity and the first sensitivity condition using the second statistical information; a predicted image acquisition step of simulating a predicted fluorescent image that is predicted to be acquired when a fluorescent image of the plate is captured by scanning with primary light having an irradiation light intensity set based on the acquired multiplication conditions and operating the light detection unit under set sensitivity conditions; and An image acquisition method comprising: (Method 6) The image acquisition method of Method 5, further comprising a presentation step of presenting the predicted fluorescence image to a user. (Method 7) 7. The image acquisition method according to Method 5 or 6, further comprising the step of receiving an instruction as to whether the acquired multiplication conditions are appropriate or to correct them. (Method 8) The image acquisition method described in Method 7 further includes a second measurement step of acquiring a fluorescent image of the plate using irradiation light intensity and sensitivity conditions set based on the multiplication conditions acquired based on the received instructions. (Method 9) The image acquisition method described in Method 7 or 8, wherein the multiplication conditions acquired based on the received instructions include at least one of the multiplication conditions modified based on the received modification instructions, the multiplication conditions acquired in the condition acquisition process based on the received appropriate instructions, and predetermined multiplication conditions acquired based on the accepted or unaccepted instructions. (Method 10) An image acquisition method described in Method 8, wherein, if the associated multiplication condition in the first irradiation light intensity and the first sensitivity condition is not obtained from either one of the first irradiation light intensity and the first sensitivity condition for which the multiplication condition was not obtained, a fluorescent image of the plate is acquired in the second measurement step using one of the first irradiation light intensity and the first sensitivity condition. (Method 11) An image acquisition method according to Method 8, wherein, if the associated multiplication condition in either the first irradiation light intensity or the first sensitivity condition is not acquired from either one of the first irradiation light intensity and the first sensitivity condition for which the multiplication condition was not acquired, a fluorescent image of the plate is acquired in the second measurement step using the irradiation light intensity or sensitivity condition set by regarding the multiplication condition as 1 for either the first irradiation light intensity or the first sensitivity condition. (Method 12) 12. The image acquisition method according to any one of methods 5 to 11, wherein the multiplication condition is a positive real number. (program) A program for causing a computer to execute the image acquisition method according to any one of Methods 1 to 12. (Configuration 1) an image acquisition device that controls a fluorescent scanner and acquires a fluorescent image from the fluorescent scanner, the image acquisition device comprising: a mounting section for mounting a plate on which an object is fixed; an emission section that emits primary light toward the plate to form an irradiation spot on the plate; a light collection section that collects secondary light from the object; a light source optically coupled to the emission section; a light detection section optically coupled to the light collection section; a scanning section that controls the mounting section and the emission section in accordance with scanning conditions to change relative positions of the irradiation spot and the light collection section with respect to the plate; and an image generation section that generates a fluorescent image based on information related to the detection intensity from the light detection section and information related to the relative position, a light intensity control unit that controls the primary light guided from the light source to the emission unit to a predetermined light intensity; a detection sensitivity control unit that controls the light detection unit so that the light detection unit operates at a predetermined detection sensitivity; a scanning instruction unit that instructs the scanning unit on the scanning conditions; a prescan condition acquisition unit that acquires a first light intensity condition and a first sensitivity condition used in the prescan as prescan conditions; a storage unit that stores the fluorescence image acquired under the acquired prescan conditions; a first statistical information acquisition unit that statistically analyzes information about the fluorescence intensity of the fluorescence image to acquire first statistical information; a second statistical information acquiring unit that acquires second statistical information by weighting the first statistical information with respect to the fluorescence intensity; a postscan condition acquisition unit that acquires, as postscan conditions, second light intensity conditions and second sensitivity conditions to be used in a postscan that is performed after the prescan, based on the second statistical information and the prescan conditions; An image acquisition device comprising: (Configuration 2) 2. The image acquisition device according to claim 1, further comprising a presentation unit that presents to a user a fluorescence image acquired under the acquired prescan conditions and a second fluorescence image acquired under the acquired postscan conditions. (Configuration 3) 3. The image acquisition device according to configuration 2, further comprising a predicted image acquisition unit that simulates a predicted fluorescent image that is predicted to be acquired when a fluorescent image of the plate is captured under the postscan conditions. (Configuration 4) 4. The image acquisition device of claim 3, wherein the presentation unit is configured to present the predicted fluorescence image to a user. (Configuration 5) 5. The image acquisition device according to configuration 4, further comprising a reception / presentation unit that presents on the presentation unit a display format for accepting an instruction to correct or not correct the acquired post-scan conditions. (Configuration 6) the pre-scanning conditions include a first scanning condition obtained by dividing a first sampling time corresponding to a time taken for detection by the light detection unit per unit time by a first scanning speed corresponding to a moving speed of the irradiation spot in the pre-scanning; The image acquisition device according to any one of configurations 1 to 5, wherein the post-scan condition includes a second scanning condition obtained by dividing a second sampling time corresponding to the time taken for the light detection unit to detect per unit time by a second scanning speed corresponding to the moving speed of the irradiation spot in the post-scan. (Configuration 7) 7. The image acquisition device according to configuration 6, wherein the pre-scan condition acquisition unit acquires the first light intensity condition, the first sensitivity condition, and the first scanning condition as pre-scan conditions. (Configuration 8) The image acquisition device according to configuration 6 or 7, wherein the postscan condition acquisition unit acquires the second light intensity condition and the second sensitivity condition based on the second statistical information, the prescan condition, and the second scanning condition. (Configuration 9) a ratio of the second light intensity condition to the first light intensity condition is set as a first multiplication condition; a ratio of the second sensitivity condition to the first sensitivity condition is set as a second multiplication condition; When the ratio of the second scanning condition to the first scanning condition is defined as a third multiplication condition, 9. The image acquisition device according to any one of configurations 6 to 8, wherein the postscan condition acquisition unit acquires the first multiplication condition, the second multiplication condition, and the third multiplication condition as positive real numbers. (Configuration 10) 10. The image acquisition device according to any one of configurations 1 to 9, wherein the emission unit irradiates the primary light so as to focus on the object.

[0085] Although the present invention has been described above with reference to the embodiments, the present invention is not limited to the above-described embodiments. Inventions modified within the scope of the present invention and inventions equivalent to the present invention are also included in the present disclosure. Furthermore, the above-described embodiments can be combined as appropriate within the scope of the present invention. [Explanation of symbols]

[0086] 101: Fluorescence image acquisition device 102: Device control unit 103: Irradiation unit 104: Lens 105:Dichroic mirror 106: Scanning unit 107: Array plate 108: Scanning unit 109: Placement section 110: Lens 111: Light detection unit 112: Signal processing unit 113: User Interface 115: Filter 118: Display 121: Fluorescence image acquisition unit 122: First information acquisition unit 123: Second information acquisition unit 124: Pre-scan condition acquisition unit 125: Post-scan condition acquisition unit 126: Light intensity control unit 127: Detection sensitivity control section 128: Scanning instruction section 429: Prediction image generation unit 430: Judgment section 431: Reception and presentation department

Claims

1. 1. An image acquisition method for acquiring a fluorescence image by scanning a plate with primary light to acquire a fluorescence intensity of an object on the plate, comprising: a first measurement step of scanning the plate with primary light having a first irradiation light intensity and operating the light detection unit under a first sensitivity condition to acquire a fluorescent image; a first information acquisition step of acquiring first statistical information related to the fluorescence intensity based on the fluorescence image; a second information acquisition step of acquiring second statistical information by weighting the first statistical information with respect to the fluorescence intensity; a condition acquisition step of acquiring a multiplication condition related to at least one of the first irradiation light intensity and the first sensitivity condition using the second statistical information; a second measurement step of scanning with primary light having an irradiation light intensity set based on the acquired multiplication conditions and operating the light detection unit under set sensitivity conditions to acquire a fluorescent image of the plate; An image acquisition method comprising:

2. 2. The image acquisition method according to claim 1, wherein, when the multiplication condition associated with either the first irradiation light intensity or the first sensitivity condition is not acquired from either one of the first irradiation light intensity and the first sensitivity condition for which the multiplication condition is not acquired, a fluorescence image of the plate is acquired in the second measurement step using either the first irradiation light intensity or the first sensitivity condition.

3. 2. The image acquisition method according to claim 1, wherein, when the multiplication condition associated with either the first irradiation light intensity or the first sensitivity condition is not acquired from either one of the first irradiation light intensity and the first sensitivity condition for which the multiplication condition is not acquired, a fluorescence image of the plate is acquired in the second measurement step using an irradiation light intensity or a sensitivity condition that is set by regarding the multiplication condition as 1 for one of the first irradiation light intensity and the first sensitivity condition.

4. The image acquisition method according to claim 1 , wherein the multiplication condition is a positive real number.

5. 1. An image acquisition method for acquiring a fluorescence image by scanning a plate with primary light to acquire a fluorescence intensity of an object on the plate, comprising: a first measurement step of scanning the plate with primary light having a first irradiation light intensity and operating the light detection unit under a first sensitivity condition to acquire a fluorescent image; a first information acquisition step of acquiring first statistical information related to the fluorescence intensity based on the fluorescence image; a second information acquisition step of acquiring second statistical information by weighting the first statistical information with respect to the fluorescence intensity; a condition acquisition step of acquiring a multiplication condition related to at least one of the first irradiation light intensity and the first sensitivity condition using the second statistical information; a predicted image acquisition step of simulating a predicted fluorescent image that is predicted to be acquired when a fluorescent image of the plate is captured by scanning with primary light having an irradiation light intensity set based on the acquired multiplication conditions and operating the light detection unit under set sensitivity conditions; and An image acquisition method comprising:

6. The image acquisition method according to claim 5 , further comprising the step of presenting the predicted fluorescence image to a user.

7. The image acquisition method according to claim 6 , further comprising the step of receiving an instruction as to whether the acquired multiplication conditions are appropriate or to correct them.

8. The image acquisition method according to claim 7, further comprising a second measurement step of acquiring a fluorescent image of the plate using irradiation light intensity and sensitivity conditions set based on the multiplication conditions acquired based on the received instruction.

9. The image acquisition method described in claim 8, wherein the multiplication conditions acquired based on the received instruction include at least one of the multiplication conditions modified based on the received instruction to modify, the multiplication conditions acquired in the condition acquisition process based on the received instruction to suit, and predetermined multiplication conditions acquired based on the instruction to not accept.

10. 9. The image acquisition method according to claim 8, wherein, when the multiplication condition associated with either the first irradiation light intensity or the first sensitivity condition is not acquired from either one of the first irradiation light intensity and the first sensitivity condition for which the multiplication condition is not acquired, a fluorescence image of the plate is acquired in the second measurement step using either the first irradiation light intensity or the first sensitivity condition.

11. 9. The image acquisition method according to claim 8, wherein, when the multiplication condition associated with either the first irradiation light intensity or the first sensitivity condition is not acquired from either one of the first irradiation light intensity and the first sensitivity condition for which the multiplication condition is not acquired, a fluorescence image of the plate is acquired in the second measurement step using the irradiation light intensity or sensitivity condition that is set by regarding the multiplication condition as 1 for one of the first irradiation light intensity and the first sensitivity condition.

12. The image acquisition method according to claim 5 , wherein the multiplication condition is a positive real number.

13. A program for causing a computer to execute the image acquisition method according to claim 1 or 5.

14. an image acquisition device that controls a fluorescent scanner and acquires a fluorescent image from the fluorescent scanner, the image acquisition device comprising: a mounting section for mounting a plate on which an object is fixed; an emission section that emits primary light toward the plate to form an irradiation spot on the plate; a light collection section that collects secondary light from the object; a light source optically coupled to the emission section; a light detection section optically coupled to the light collection section; a scanning section that controls the mounting section and the emission section in accordance with scanning conditions to change relative positions of the irradiation spot and the light collection section with respect to the plate; and an image generation section that generates a fluorescent image based on information related to the detection intensity from the light detection section and information related to the relative position, a light intensity control unit that controls the primary light guided from the light source to the emission unit to a predetermined light intensity; a detection sensitivity control unit that controls the light detection unit so that the light detection unit operates at a predetermined detection sensitivity; a scanning instruction unit that instructs the scanning unit on the scanning conditions; a prescan condition acquisition unit that acquires a first light intensity condition and a first sensitivity condition used in the prescan as prescan conditions; a storage unit that stores the fluorescence image acquired under the acquired prescan conditions; a first statistical information acquisition unit that statistically analyzes information about the fluorescence intensity of the fluorescence image to acquire first statistical information; a second statistical information acquiring unit that acquires second statistical information by weighting the first statistical information with respect to the fluorescence intensity; a postscan condition acquisition unit that acquires, as postscan conditions, second light intensity conditions and second sensitivity conditions to be used in a postscan that is performed after the prescan, based on the second statistical information and the prescan conditions; An image acquisition device comprising:

15. 15. The image acquisition device according to claim 14, further comprising a presentation unit that presents to a user the fluorescence image acquired under the acquired prescan conditions and the second fluorescence image acquired under the acquired postscan conditions.

16. The image acquisition device according to claim 15 , further comprising a predicted image acquisition unit that simulates a predicted fluorescent image that is predicted to be acquired when a fluorescent image of the plate is captured under the postscan condition.

17. The image acquisition device of claim 16 , wherein the presentation unit is configured to present the predicted fluorescence image to a user.

18. The image acquisition device according to claim 17 , further comprising a reception / presentation unit that presents, on the presentation unit, a display format for accepting an instruction to correct or not correct the acquired post-scan conditions.

19. the pre-scanning conditions include a first scanning condition obtained by dividing a first sampling time corresponding to a time taken for detection by the light detection unit per unit time by a first scanning speed corresponding to a moving speed of the irradiation spot in the pre-scanning; 16. The image acquisition device according to claim 14 or 15, wherein the post-scan condition includes a second scanning condition obtained by dividing a second sampling time corresponding to a time taken for the light detection unit to detect the light per unit time by a second scanning speed corresponding to a moving speed of the irradiation spot in the post-scan.

20. The image acquisition device according to claim 19 , wherein the prescan condition acquisition unit acquires the first light intensity condition, the first sensitivity condition, and the first scanning condition as prescan conditions.

21. 21. The image acquisition device according to claim 20, wherein the postscan condition acquisition unit acquires the second light intensity condition and the second sensitivity condition based on the second statistical information, the prescan condition, and the second scanning condition.

22. a ratio of the second light intensity condition to the first light intensity condition is defined as a first multiplication condition; a ratio of the second sensitivity condition to the first sensitivity condition is set as a second multiplication condition; When a ratio of the second scanning condition to the first scanning condition is defined as a third multiplication condition, The image acquisition device according to claim 20 , wherein the postscan condition acquisition unit acquires the first multiplication condition, the second multiplication condition, and the third multiplication condition as positive real numbers.

23. The image acquisition device according to claim 14 , wherein the emission unit irradiates the primary light so as to focus the primary light on the object.

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