Well counting device, well counting method, digital measurement system, and program

Abstract

This well counting device comprises: an image acquisition unit that acquires one or more images obtained by imaging a plurality of wells for capturing a target substance; and a counting unit that counts the number of wells emitting fluorescence among the plurality of wells for each of the one or more images acquired by the image acquisition unit. The counting unit counts the number of wells emitting fluorescence among the plurality of wells, wherein such wells that wells adjacent thereto also emit fluorescence are excluded.

Description

Well counting device, well counting method, digital measurement system, and program

[0001] The present invention relates to a well counting device, a well counting method, a digital measurement system, and a program. This application claims priority to Japanese Patent Application No. 2023-082318, filed on May 18, 2023, the contents of which are incorporated herein by reference.

[0002] In recent years, there has been an increasing need for the accurate detection of target substances in biological samples for purposes such as early detection of diseases. Examples of target substances include, but are not limited to, DNA (Deoxyribonucleic Acid), RNA (Ribonucleic Acid), proteins, and cells. Technologies that perform enzyme reactions in multiple microcompartments have been investigated as a method for accurately detecting target substances in biological samples. These methods are called digital measurements. Examples of digital measurements include digital ELISA (Enzyme-linked Immunosorbent Assay) and digital PCR (Polymerase Chain Reaction).

[0003] In digital measurement, a sample solution is divided into an extremely large number of minute solutions. The signal from each minute solution is then binarized, and the presence or absence of a target substance is determined, and the number of molecules of the target substance is measured. Digital measurement can significantly improve detection sensitivity and quantitativeness compared to conventional methods such as ELISA and real-time PCR.

[0004] In digital measurement using a well array with multiple wells (micropores) for capturing target substances, the captured target substances remain in the wells and do not unintentionally leak out of the wells. Fluorescence is then emitted from the wells that capture the target substances. For example, when the target substance is DNA, fluorescence can be generated by reactions such as ICA or PCR, or by binding a fluorescently labeled probe to the target DNA. Patent Document 1 discloses a technology for detecting target substances when performing digital measurement using a well array with multiple wells (micropores) for capturing target substances. A separately captured image is used as a master, and the difference between the original image and the master image is calculated.

[0005] Japanese Patent No. 6800864

[0006] However, the technique disclosed in Patent Document 1 has the problem that disturbance factors such as foreign matter adhering to the well array or scratches on the well array may be detected as target substances.

[0007] The present invention has been made in view of the above circumstances, and provides a well counting device, a well counting method, a digital measurement system, and a program that can eliminate disturbance factors in digital measurement.

[0008] This invention has been made to solve the above-mentioned problems, and a well counting device according to a first aspect of the present invention comprises an image acquisition unit that acquires one or more images of a plurality of wells that capture a target substance, and a counting unit that counts the number of wells among the plurality of wells that emit fluorescence for each of the one or more images acquired by the image acquisition unit, and the counting unit counts the number of wells among the plurality of wells that emit fluorescence, excluding wells among which adjacent wells also emit fluorescence.

[0009] Furthermore, in the well counting device according to the second aspect of the present invention, in the first aspect described above, each of the one or more images corresponds to a color of fluorescence emitted by the plurality of wells, and the counting unit may count the number of wells emitting the fluorescence by excluding wells emitting fluorescence in an image of a first color of fluorescence corresponding to the target image from wells emitting the fluorescence in an image of a second color of fluorescence different from the first color.

[0010] Furthermore, in the well counting device according to the third aspect of the present invention, in the second aspect described above, the second threshold value used to determine that fluorescence is being emitted even in an image of a fluorescent color different from the fluorescent color corresponding to the target image may be different from the first threshold value used to count the number of wells that are emitting the fluorescence.

[0011] A well counting device according to a fourth aspect of the present invention is any one of the first to third aspects, wherein each of the one or more images corresponds to the color of fluorescence emitted by the plurality of wells, and the well counting device includes a mixing and removing unit that adjusts the brightness of each pixel of the one or more images acquired by the image acquisition unit based on the brightness of corresponding pixels in other images of the one or more images, and the counting unit may use the image whose brightness has been adjusted by the mixing and removing unit when counting the number of wells among the plurality of wells that are emitting the fluorescence.

[0012] Furthermore, a fifth aspect of the present invention provides a well counting device according to the fourth aspect, wherein the mixing removal unit includes a filter that transmits the spectrum of fluorescence of a first color for the target image, and when the transmission spectrum of the filter overlaps with the spectrum of fluorescence of a second color different from the first color, the counting unit may use an image obtained by removing the luminance component due to fluorescence of the second color from the image captured using the filter.

[0013] Furthermore, the well counting device according to the sixth aspect of the present invention may be any one of the first to fifth aspects, in which one target substance is introduced into one well out of the plurality of wells.

[0014] Another aspect of the present invention is a well counting method comprising a first step of acquiring one or more images of a plurality of wells that capture a target substance, and a second step of counting the number of wells that emit fluorescence among the plurality of wells for each of the one or more images acquired in the first step, wherein in the second step, the number of wells that emit fluorescence is counted, excluding wells among the plurality of wells that also emit fluorescence from adjacent wells.

[0015] Another aspect of the present invention is a digital measurement system comprising a microscope and a well counting device, wherein the microscope generates one or more images of a plurality of wells that capture a target substance, and the well counting device comprises an image acquisition unit that acquires the one or more images acquired by the microscope, and a counting unit that counts the number of wells among the plurality of wells that emit fluorescence for each of the one or more images acquired by the image acquisition unit, and the counting unit counts the number of wells among the plurality of wells that emit fluorescence, excluding wells among which adjacent wells also emit fluorescence.

[0016] Another aspect of the present invention is a program for causing a computer to function as an image acquisition unit that acquires one or more images of multiple wells that capture a target substance, and a counting unit that counts the number of wells among the multiple wells that emit fluorescence for each of the one or more images acquired by the image acquisition unit, wherein the counting unit counts the number of wells among the multiple wells that emit fluorescence, excluding wells among which adjacent wells also emit fluorescence.

[0017] According to the present invention, the well counting device, well counting method, digital measurement system, or program can eliminate external disturbance factors.

[0018] FIG. 1 is a schematic diagram showing the configuration of a digital measurement system 10 according to a first embodiment of the present invention. FIG. 2 is a diagram showing an example of an image generated by a microscope 200 in the first embodiment. FIG. 3 is a schematic block diagram showing the configuration of a well counting device 300 in the first embodiment. FIG. 4 is a spectrum diagram for explaining the operation of a mixing and removing unit 324 in the first embodiment. FIG. 5 is a schematic diagram for explaining the operation of the mixing and removing unit 324 in the first embodiment. FIG. 6 is a diagram showing examples of images before and after mixing and removing processing by the mixing and removing unit 324 in the first embodiment. FIG. 7 is a flowchart for explaining the operation of the well counting device 300 in the first embodiment. FIG. 8 is a flowchart for explaining the operation of a counting unit 330 in the first embodiment. FIG. 9 is a schematic diagram showing a first example of an arrangement of adjacent wells in the first embodiment. FIG. 10 is a schematic diagram showing a second example of an arrangement of adjacent wells in the first embodiment. FIG. 11 is a schematic diagram showing a third example of an arrangement of adjacent wells in the first embodiment. FIG. 12 is a schematic diagram showing the configuration of a digital measurement system 10 according to a second embodiment of the present invention.

[0019] <First Embodiment> A first embodiment of the present invention will now be described with reference to the drawings. Fig. 1 is a schematic diagram showing the configuration of a digital measurement system 10 according to a first embodiment of the present invention. Digital measurement system 10 is a system that counts the number of wells that capture each type of target substance among a plurality of wells provided in a well array 100, and includes a microscope 200 and a well counting device 300.

[0020] Each of the multiple wells captures one or more types of target substances. When irradiated with excitation light while capturing a target substance, the well emits fluorescence of a color corresponding to the type of target substance captured. The shape, dimensions, and arrangement of the wells are not particularly limited. In this embodiment, there are cases where one target substance is introduced into one well, and cases where no target substance is introduced into one well. The wells are preferably microwells with small volumes. For example, the volume of one well may be approximately 10 fL to 100 pL. The well array 100 includes multiple wells of the same shape and size. The term "same shape and size" means that the wells have the same shape and capacity to the extent required for digital measurement, and variations within the range of manufacturing errors are acceptable.

[0021] When the wells are cylindrical, the maximum diameter of the wells in plan view is preferably, for example, 10 nm to 100 μm, more preferably 100 nm to 50 μm, and even more preferably 1 μm to 20 μm. The depth of the wells is preferably, for example, 10 nm to 100 μm, more preferably 100 nm to 50 μm, and even more preferably 1 μm to 20 μm. The volume of the wells is preferably, for example, 1 fL to 6 nL, more preferably 1 fL to 5 pL, even more preferably 1 fL to 2 pL, and particularly preferably 1 fL to 300 fL. When the volume of each well is within this range, enzymatic reactions performed in microspaces such as digital PCR and invader reactions can be suitably carried out. Digital PCR can be used to detect gene mutations, for example. The arrangement of the wells is not particularly limited; for example, they may be arranged in a triangular lattice pattern, a square lattice pattern, or randomly arranged.

[0022] The microscope 200 generates one or more images by capturing images of multiple wells. The microscope 200 generates images by irradiating the multiple wells with excitation light and capturing images of the wells capturing target substances emitting fluorescence. The microscope 200 may use a filter corresponding to the color of the fluorescence when capturing images of the multiple wells. That is, each of the one or more images generated by the microscope 200 may correspond to the color of the fluorescence emitted by the multiple wells. Capture using a filter corresponding to the color of the fluorescence is called fluorescence capture. The microscope 200 may also generate bright-field images by capturing images of the multiple wells without using a filter.

[0023] The well counting device 300 counts the number of wells emitting fluorescence using the image generated by the microscope 200. This allows the well counting device 300 to quantitatively measure the number of target substances. The well counting device 300 may be realized by one or more computers reading and executing a program.

[0024] 2 is a diagram showing an example of an image generated by the microscope 200 in this embodiment. In the example image shown in FIG. 2, multiple wells are arranged in a triangular lattice pattern, and some wells among them have high brightness. These wells with high brightness are wells that emit fluorescence and are counted by the well counting device 300.

[0025] 3 is a schematic block diagram showing the configuration of a well counting device 300 according to this embodiment. The well counting device 300 includes an image acquisition unit 310, a preprocessing unit 320, and a counting unit 330. The image acquisition unit 310 acquires one or more images generated by the microscope 200. The image acquisition unit 310 may acquire images from the microscope 200 via an IP (Internet Protocol) network or the like, or may acquire images from the microscope 200 via a recording medium such as a USB (Universal Serial Bus) memory.

[0026] The preprocessing unit 320 performs processes such as brightness value correction, well position identification, image duplication removal, and mixed removal on the images acquired by the image acquisition unit 310, and generates images for counting in the counting unit 330. The preprocessing unit 320 includes a brightness value correction unit 321, a well position identification unit 322, a duplication removal unit 323, and a mixed removal unit 324. Note that the preprocessing unit 320 may not include at least some of the brightness value correction unit 321, the well position identification unit 322, the duplication removal unit 323, and the mixed removal unit 324, or may not perform at least some of the brightness value correction, well position identification, image duplication removal, and mixed removal.

[0027] The luminance value correction unit 321 corrects the luminance values ​​of the central and peripheral portions of the image acquired by the image acquisition unit 310. Generally, luminance values ​​are greater near the center of the image than at the edges. Therefore, the luminance value correction unit 321 assumes that luminance values ​​change depending on the distance from the center of the lens, creates a function of the average luminance value change from the center of the screen to the periphery, and corrects the image so as to compensate for the change in this function. This function is created using a reference image obtained by fluorescent imaging without a sample in the image. Note that instead of creating a function of the average luminance value change, a pre-set function may be input and the image may be corrected using this pre-set function.

[0028] The well position identifying unit 322 creates a mesh with nodes corresponding to the positions of the wells for the image acquired by the image acquiring unit 310, and identifies the positions of the wells. The image used by the well position identifying unit 322 may be an image obtained by fluorescent photography or a bright field image. The well position identifying unit 322 may set multiple positions stored in advance in the image as the positions of multiple wells, respectively.

[0029] If the images acquired by image acquisition unit 310 are images of well array 100 divided into multiple regions and photographed, overlapping deletion unit 323 deletes overlapping regions between the images. Overlap deletion unit 323 may combine the images from which the overlapping regions have been deleted so that a single image includes the entire region of well array 100 where wells are provided.

[0030] The mixing and removal unit 324 performs a mixing and removal process in which the brightness of each pixel in one or more images acquired by the image acquisition unit 310 is adjusted based on the brightness of corresponding pixels in other images of the one or more images. This mixing and removal process is performed, for example, when the transmission spectrum of a first filter corresponding to a first color of fluorescence overlaps with the spectrum of a second color of fluorescence, in order to remove the brightness component of the second color of fluorescence from an image captured using the first filter. Note that the mixing and removal process may not be performed if the spectra of the fluorescence of each color are sufficiently different such that none of the fluorescence is transmitted through the filters of the other colors. Furthermore, the mixing and removal process may not be performed when a single wavelength is used to capture images of target particles introduced into the well array 100 (when capturing images of a single color).

[0031] Fig. 4 is a spectrum diagram for explaining the operation of the mixing removal unit 324 in this embodiment. In Fig. 4, the horizontal axis represents wavelength. Spectrum S1 is the spectrum of fluorescence of the first color. Spectrum S2 is the spectrum of fluorescence of the second color. Spectrum S3 is the spectrum of fluorescence of the third color. Region F1 is the transmission region of the filter corresponding to fluorescence of the first color. Region F2 is the transmission region of the filter corresponding to fluorescence of the second color. Region F3 is the transmission region of the filter corresponding to fluorescence of the third color.

[0032] For example, the region F1, which is the transmission region of the filter corresponding to the first color of fluorescence, includes not only the spectrum S1, which is the spectrum of the first color of fluorescence, but also the spectrum S2, which is the spectrum of the second color of fluorescence, and the spectrum S3, which is the spectrum of the third color of fluorescence. Note that, although the spectrums of the first to third colors of fluorescence are shown in this embodiment, the present invention is not limited to three-color fluorescence spectra. Fluorescence spectra of four or more colors are also possible. For example, six-color fluorescence spectra are also possible.

[0033] 5 is a schematic diagram illustrating the operation of the mixing and removing unit 324 in this embodiment. The mixing and removing unit 324 calculates a luminance value L by subtracting a luminance value corresponding to the spectrum S2-1 included in the region F1 of the spectrum S2 of the second color fluorescence and a luminance value corresponding to the spectrum S3-1 included in the region F1 of the spectrum S3 of the third color fluorescence from a luminance value corresponding to the spectrum S1-1 included in the region F1 in an image captured using a filter corresponding to the fluorescence of the first color. １ Let's say.

[0034] In this case, the luminance value corresponding to spectrum S2-1 is calculated by multiplying the area ratio of the portion of spectrum S2 included in region F2 to the portion included in region F1 by the pixel value in the image captured using a filter corresponding to the second color of fluorescence. The same is true for the luminance value corresponding to spectrum S3-1. Note that while FIG. 5 describes an image captured using a filter corresponding to the first color of fluorescence, the same applies to images captured using filters corresponding to the other colors of fluorescence.

[0035] Specifically, the mixed removal unit 324 calculates the luminance value L using the following equation (1): ｉ (x, y) is adjusted. In equation (1), the luminance value L ｉ (x, y) is the brightness value at the coordinates (x, y) of the image corresponding to the fluorescence of the i-th color, and C ｉｊ is a coefficient for adjustment, which represents the influence of the jth color fluorescence on the luminance of the image corresponding to the ith color fluorescence. ｉｊ is 1 when i=j, and is less than or equal to 0 and greater than -1 when i≠j. ｉｊ may be a value input by the operator of the well counting device 300, or may be stored in advance in the well counting device 300.

[0036]

[0037] 6 is a diagram showing example images before and after the mixing and removal process by the mixing and removal unit 324 in this embodiment. In Fig. 6, image G1 is an example image before the mixing and removal process by the mixing and removal unit 324, and image G2 is an example image after the mixing and removal process by the mixing and removal unit 324. Some of the wells that had brightness in image G1 have almost no brightness in image G2, which indicates that this was due to the influence of fluorescence of other colors.

[0038] Returning to FIG. 3 , the counting unit 330 counts the number of fluorescent wells among the multiple wells for each of one or more images acquired by the image acquisition unit 310 and preprocessed by the preprocessing unit 320. In this case, the counting unit 330 counts the number of fluorescent wells, excluding wells among the multiple wells that also emit fluorescence from adjacent wells. Foreign matter adhering to the well array 100 or scratches on the well array 100 often do not fit into a single well but extend across adjacent wells. In contrast, in digital measurement, it can be assumed that the rate at which adjacent wells capture the same target substance is not high. Therefore, by counting while excluding wells that also emit fluorescence from adjacent wells, the counting unit 330 can eliminate the effects of disturbances such as foreign matter or scratches.

[0039] Furthermore, the counting unit 330 may count the number of fluorescent wells by excluding wells that emit fluorescence in an image of a second fluorescent color different from the first fluorescent color from wells that emit fluorescence in an image of a first fluorescent color corresponding to the target image. Foreign matter attached to the well array 100 or scratches on the well array 100 often emit fluorescence not only in an image of one fluorescent color but also in an image of a second fluorescent color. In contrast, in digital measurement, wells that capture target substances emit fluorescence of only one color. Therefore, by counting wells while excluding wells that also emit fluorescence in an image of the second fluorescent color, the counting unit 330 can eliminate the influence of external disturbances such as foreign matter or scratches.

[0040] In addition, the second threshold value used to determine whether fluorescence is being emitted in the image of the second fluorescent color may be different from the first threshold value used to count the number of wells emitting fluorescence.

[0041] FIG. 7 is a flowchart illustrating the operation of the well counting device 300 according to this embodiment. The image acquisition unit 310 acquires one or more images of multiple wells from the microscope 200 (step Sa1). Next, the preprocessing unit 320 performs preprocessing (e.g., brightness correction, well position identification, image duplication removal, and mixed removal) on the one or more images acquired in step Sa1 (step Sa2). Next, the counting unit 330 counts the number of fluorescent wells for each of the one or more images preprocessed in step Sa2 (step Sa3). The counting unit 330 then outputs the counting results. The output method may be displaying the results on a display, writing them to a recording medium such as a USB memory stick, or transmitting them to another device.

[0042] Fig. 8 is a flowchart illustrating the operation of the counting unit 330 in this embodiment. The flowchart shown in Fig. 7 corresponds to step Sa3 in Fig. 6. The counting unit 330 performs steps Sb2 to Sb7 on one or more preprocessed images, i.e., one or more images each corresponding to a fluorescent color (step Sb1). Here, each of the one or more images corresponds to a fluorescent color, i.e., a type of target substance.

[0043] The counting unit 330 performs the processes of steps Sb3 to Sb6 for each of the multiple wells included in the target image (step Sb2). In step Sb3, the counting unit 330 determines whether the target well satisfies a fluorescence condition. The fluorescence condition is a condition for determining whether the target well emits fluorescence, as will be described in detail later. For example, the fluorescence condition may include whether the representative value of the luminance of the target well exceeds a first threshold. The representative value of the luminance of the target well may be any value that represents the luminance of the target well, such as the average, mode, median, or integrated value of the luminance values ​​in the target well.

[0044] If it is determined in step Sb3 that the target well does not satisfy the fluorescence condition (step Sb3-No), the counting unit 330 skips steps Sb4 to Sb6 and proceeds to step Sb7. On the other hand, if it is determined in step Sb3 that the target well satisfies the fluorescence condition (step Sb3-Yes), the counting unit 330 determines whether any wells adjacent to the target well are emitting light (step Sb4).

[0045] The condition for determining whether an adjacent well emits light may be that the same condition as the fluorescence condition in step Sb3 is satisfied, or may be that some of the conditions in the fluorescence condition are satisfied. For example, this determination condition may include whether the representative value of the luminance of the adjacent well exceeds a third threshold. The third threshold may be different from or the same as the first threshold in the fluorescence condition. The representative value of the luminance may be the average, mode, median, or integrated value of the luminance values ​​in the well. The arrangement of the wells adjacent to the target well is pre-stored in the counting unit 330.

[0046] If it is determined in step Sb4 that there is a luminescent well among the adjacent wells (step Sb4—Yes), the counting unit 330 skips steps Sb5 and Sb6 and proceeds to step Sb7. On the other hand, if it is determined in step Sb4 that there is no luminescent well among the adjacent wells (step Sb4—No), the counting unit 330 determines whether the target well also luminesces in any of the other images (step Sb5). Here, the other images are images other than the target image, and are included in one or more preprocessed images, i.e., one or more images each corresponding to a fluorescent color.

[0047] The condition for determining whether the target well emits light in the other image may be that the same condition as the fluorescence condition in step Sb3 is satisfied, or may be that some of the fluorescence conditions are satisfied. For example, this condition may include whether the representative brightness value of the well in the other image exceeds a second threshold. The second threshold may be different from or the same as the first threshold in the fluorescence condition and the third threshold in step Sb4. The representative brightness value may be the average, mode, median, or integrated value of the brightness values ​​in the well.

[0048] In step Sb5, if it is determined that the target well also emits light in any of the other images (step Sb5—Yes), the counting unit 330 skips step Sb6 and proceeds to step Sb7. On the other hand, in step Sb5, if it is determined that the target well does not emit light in any of the other images (step Sb5—No), the counting unit 330 counts up the number of fluorescent wells in the target image (step Sb6).

[0049] Step Sb7 is the end of the loop for each well performed in step Sb2. That is, in step Sb7, if the counting unit 330 has not yet completed processing for all of the multiple wells in the target image, it selects the next well as the target well and returns to step Sb3, but if the counting unit 330 has completed processing for all of the multiple wells in the target image, it proceeds to step Sb8.

[0050] Step Sb8 is the end of the loop for each image performed in step Sb1. That is, in step Sb8, if the counting unit 330 has not yet completed processing for all of the preprocessed image or images, it selects the next image as the target image and returns to step Sb2; if the counting unit 330 has completed processing for all of the preprocessed image or images, it ends the processing.

[0051] 9 is a schematic diagram showing a first example of the arrangement of adjacent wells in this embodiment. The first example shown in FIG. 9 is an example in which the wells are arranged in a triangular lattice. In the example of FIG. 9, six wells W2 surrounding the target well W1 are adjacent wells.

[0052] 10 is a schematic diagram showing a second example of the arrangement of adjacent wells in this embodiment. The second example shown in FIG. 10 is an example in which the wells are arranged in a square lattice pattern. In the example of FIG. 10, the eight wells W4 surrounding the target well W3 are adjacent wells. Note that when the wells are arranged in a square lattice pattern, the four wells above, below, left, and right of the target well may also be considered adjacent wells.

[0053] 11 is a schematic diagram showing a third example of the arrangement of adjacent wells in this embodiment. The third example shown in FIG. 11 is an example in which the wells are arranged randomly. In the example of FIG. 11, four wells W6 within a predetermined distance range C from the target well W5 are adjacent wells.

[0054] Next, the fluorescence conditions in step Sb3 of Fig. 8 will be described. The fluorescence conditions may include some or all of the following conditions, or may include other conditions: 1) circularity, 2) area, 3) correlation coefficient, 4) brightness of the peripheral area, and 5) average brightness.

[0055] The circularity of the above 1) is based on the condition that the shape of a region (hereinafter referred to as a light-emitting region) whose brightness value is equal to or greater than a threshold value in a certain region (hereinafter referred to as an extracted region) from the center of the well is close to a circle. Specifically, the area of ​​the light-emitting region is S, the perimeter is L, and the circularity is 4πS / L. ２ The condition is that the circularity 4πS / L is equal to or greater than a threshold value. ２ The closer the shape is to a circle, the larger the value becomes, and when the shape is a circle, the value becomes 1. The threshold value may be a value input by the operator of the well counting device 300, or may be a value stored in advance in the well counting device 300.

[0056] The area of ​​2) above is determined on the condition that the area of ​​the light-emitting region is equal to or greater than a threshold value, or is between an upper limit and a lower limit value. The threshold value, upper limit value, and lower limit value may be values ​​input by an operator of well counting device 300, or may be stored in advance in well counting device 300.

[0057] The correlation coefficient in 3) above is determined on the condition that the correlation coefficient between the image of the extracted region and a standard image prepared in advance is equal to or greater than a threshold value. The standard image is an image that serves as a model of when the well is emitting fluorescence. A normalized correlation coefficient may also be used as the correlation coefficient. The threshold value may be a value input by the operator of the well counting device 300, or may be stored in advance in the well counting device 300.

[0058] The brightness of the peripheral portion in 4) above is determined based on the condition that the brightness of the peripheral portion of the well in the extracted region is equal to or less than a threshold value. The brightness of the peripheral portion of the well is, for example, the average brightness of the region extending from the center of the well to the well radius multiplied by the coefficient rate_min and equal to or less than the well radius multiplied by the coefficient rate_max. The threshold value, well radius, coefficient rate_min, and coefficient rate_max may be values ​​input by the operator of the well counting device 300, or may be stored in advance in the well counting device 300.

[0059] The average brightness in 5) above is determined based on the condition that the average brightness value within the well is equal to or greater than a first threshold. The first threshold is calculated by multiplying the mode in a histogram of the average brightness values ​​of the wells by a multiplier and adding an offset. This histogram may be a histogram of wells among multiple wells whose correlation coefficient in 3) above is equal to or greater than a threshold. Furthermore, if brightness varies depending on the region of the well array, the first threshold may be calculated for each region. For example, when calculating the first threshold for the target region, the offset may be multiplied by the offset obtained by dividing the mode of brightness of the entire well array by the mode of brightness of the target region, thereby increasing the offset in regions with low brightness and decreasing the offset in regions with high brightness. The threshold, multiplying factor, and offset may be values ​​input by the operator of the well counting device 300 or may be stored in advance in the well counting device 300. The second and third thresholds may be calculated in the same manner as the first threshold, or may be calculated in the same manner using a multiplying factor and offset different from those used for the first threshold.

[0060] It is preferable that one target substance be introduced into one well among the multiple wells, since it is easier to count the number of luminescent wells when one target substance is introduced into one well than when multiple target substances are introduced into one well.

[0061] Furthermore, after determining in step Sb4 that there are no adjacent wells that emit light, the counting unit 330 determines whether the target well also emits light in any of the other images (step Sb5), but this is not limited to this. For example, the counting unit 330 may determine whether the target well also emits light in any of the other images (step Sb5) and then determine whether there are adjacent wells that emit light (step Sb4). Furthermore, the determination of whether there are adjacent wells that emit light (step Sb4) and the determination of whether there are adjacent wells that emit light in any of the other images (step Sb5) may be performed in parallel. In this case, if either step Sb4 or step Sb5 applies, i.e., if the adjacent well emits light or emits light in the other images, proceed to step Sb6.

[0062] Second Embodiment Fig. 12 is a schematic diagram showing the configuration of a digital measurement system 10 according to a second embodiment of the present invention. In the first embodiment, the microscope 200 and the well counting device 300 were separate devices, but these functions may be provided by a single device. That is, as shown in Fig. 12, the microscope 200 and the well counting device 300 may be included in a single housing of the digital measurement system 10.

[0063] The following embodiments may also be used: (1) One embodiment is a well counting device that includes an image acquisition unit that acquires one or more images of a plurality of wells that capture a target substance, and a counting unit that counts the number of wells that emit fluorescence among the plurality of wells for each of the one or more images acquired by the image acquisition unit, and the counting unit counts the number of wells that emit fluorescence among the plurality of wells, excluding wells whose adjacent wells also emit fluorescence.

[0064] (2) Another embodiment is the well counting device described in (1), in which each of the one or more images corresponds to the color of fluorescence emitted by the multiple wells, and the counting unit counts the number of wells emitting the fluorescence, excluding wells among the multiple wells that emit fluorescence even in images with a fluorescent color different from the fluorescent color corresponding to the target image.

[0065] (3) Another embodiment is the well counting device described in (2), in which the second threshold value used to determine that an image of a fluorescent color different from the fluorescent color corresponding to the target image is emitting fluorescence is different from the first threshold value used to count the number of wells emitting the fluorescence.

[0066] (4) Another embodiment is a well counting device described in any one of (1) to (3), wherein each of the one or more images corresponds to the color of fluorescence emitted by the plurality of wells, and the well counting device includes a mixing and removing unit that adjusts the brightness of each pixel of the one or more images acquired by the image acquisition unit based on the brightness of corresponding pixels in other images of the one or more images, and the counting unit uses the image whose brightness has been adjusted by the mixing and removing unit when counting the number of wells among the plurality of wells that are emitting the fluorescence.

[0067] (5) Another embodiment is the well counting device of (1), wherein the mixed removal unit includes a filter that transmits the spectrum of a first color of fluorescence for the target image, and when the transmission spectrum of the filter overlaps with the spectrum of a second color of fluorescence that is different from the first color, the counting unit uses an image obtained by removing the brightness component due to the second color of fluorescence from the image captured using the filter.

[0068] (6) Another embodiment is the well counting device according to any one of (1) to (5), wherein one of the target substances is introduced into one of the multiple wells.

[0069] (7) Another embodiment is a well counting method having a first step of acquiring one or more images of a plurality of wells that capture a target substance, and a second step of counting the number of wells that emit fluorescence among the plurality of wells for each of the one or more images acquired in the first step, wherein in the second step, the number of wells that emit fluorescence is counted, excluding wells among the plurality of wells that also emit fluorescence from adjacent wells.

[0070] (8) Another embodiment is a digital measurement system including a microscope and a well counting device, wherein the microscope generates one or more images of a plurality of wells that capture a target substance, and the well counting device includes an image acquisition unit that acquires the one or more images acquired by the microscope, and a counting unit that counts the number of wells among the plurality of wells that emit fluorescence for each of the one or more images acquired by the image acquisition unit, and the counting unit counts the number of wells among the plurality of wells that emit fluorescence, excluding wells among the plurality of wells that also emit fluorescence from adjacent wells.

[0071] (9) Another embodiment is a program for causing a computer to function as an image acquisition unit that acquires one or more images of multiple wells that capture a target substance, and a counting unit that counts the number of wells among the multiple wells that emit fluorescence for each of the one or more images acquired by the image acquisition unit, wherein the counting unit counts the number of wells among the multiple wells that emit fluorescence, excluding wells among the multiple wells that also emit fluorescence from adjacent wells.

[0072] 1 may be recorded on a computer-readable recording medium, and the program recorded on the recording medium may be read into a computer system and executed to realize the well counting device 300. Note that the term "computer system" as used herein includes hardware such as an OS and peripheral devices.

[0073] Furthermore, "computer system" also includes the homepage provision environment (or display environment) if the WWW system is used. Furthermore, "computer-readable recording medium" refers to portable media such as flexible disks, optical magnetic disks, ROMs, and CD-ROMs, as well as storage devices such as hard disks built into computer systems. Furthermore, "computer-readable recording medium" also includes devices that dynamically store programs for a short period of time, such as communication lines used when transmitting programs over networks like the Internet or communication lines like telephone lines, and devices that store programs for a fixed period of time, such as volatile memory within the computer systems that serve as servers or clients in such cases. Furthermore, the above-mentioned programs may be those that implement some of the aforementioned functions, or may be those that can realize the aforementioned functions in combination with programs already stored in the computer system.

[0074] The above-described embodiment can be expressed as follows: A computer-readable non-transitory storage medium storing a program that causes a computer to acquire one or more images of a plurality of wells that capture a target substance, count the number of wells that emit fluorescence among the plurality of wells for each of the one or more images acquired by the image acquisition unit, and count the number of wells that emit fluorescence while excluding wells that also emit fluorescence from adjacent wells among the plurality of wells.

[0075] Although an embodiment of the present invention has been described in detail above with reference to the drawings, the specific configuration is not limited to this embodiment, and design changes and the like are also included within the scope that does not deviate from the gist of the present invention.

[0076] According to the present invention, the well counting device, well counting method, digital measurement system, or program can eliminate external disturbance factors.

[0077] 10 Digital measurement system 100 Well array 200 Microscope 300 Well counting device 310 Image acquisition unit 320 Preprocessing unit 321 Brightness value correction unit 322 Well position identification unit 323 Duplicate removal unit 324 Mixing removal unit 330 Counting unit

Claims

1. an image acquisition unit that acquires one or more images of a plurality of wells that capture target substances; a counting unit that counts the number of wells that emit fluorescence among the plurality of wells for each of the one or more images acquired by the image acquisition unit; Equipped with The counting unit counts the number of wells emitting fluorescence, excluding adjacent wells emitting fluorescence, among the plurality of wells. Well counting device.

2. each of the one or more images corresponds to a color of fluorescence emitted by the plurality of wells; the counting unit counts the number of wells emitting fluorescence by excluding wells emitting fluorescence in an image of a second color of fluorescence different from the first color from wells emitting fluorescence in an image of a first color of fluorescence corresponding to a target image, among the plurality of wells; The well counting device of claim 1.

3. a second threshold value used when determining that an image having a different fluorescent color from the fluorescent color corresponding to the target image is emitting fluorescence is different from a first threshold value used when counting the number of wells emitting fluorescence; The well counting device of claim 2.

4. each of the one or more images corresponds to a color of fluorescence emitted by the plurality of wells; The well counting device comprises: a mixture removal unit that adjusts the brightness of each pixel of the one or more images acquired by the image acquisition unit based on the brightness of a corresponding pixel in another image of the one or more images; Equipped with the counting unit uses the image whose brightness has been adjusted by the mixing and removing unit when counting the number of wells emitting the fluorescence among the plurality of wells. The well counting device according to any one of claims 1 to 3.

5. the mixed removal unit includes a filter that transmits a spectrum of fluorescence of a first color for a target image; When the transmission spectrum of the filter overlaps with the spectrum of fluorescence of a second color different from the first color, the counting unit uses an image obtained by removing a luminance component due to fluorescence of the second color from the image captured using the filter. The well counting device of claim 4.

6. One of the target substances is introduced into one of the wells. The well counting device according to any one of claims 1 to 3.

7. a first step of acquiring one or more images of a plurality of wells capturing target material; a second step of counting the number of wells emitting fluorescence among the plurality of wells for each of the one or more images acquired in the first step; and In the second step, the number of wells emitting fluorescence is counted, excluding wells in which adjacent wells also emit fluorescence. Well counting method.

8. A digital measurement system comprising a microscope and a well counting device, the microscope generates one or more images of the plurality of wells capturing the target material; The well counting device comprises: an image acquisition unit that acquires one or more images captured by the microscope; a counting unit that counts the number of wells that emit fluorescence among the plurality of wells for each of the one or more images acquired by the image acquisition unit; Equipped with the counting unit counts the number of wells emitting fluorescence, excluding wells in which adjacent wells also emit fluorescence, among the plurality of wells; Digital measurement system.

9. Computer, an image acquisition unit that acquires one or more images of a plurality of wells that capture the target substance; a counting unit that counts the number of wells that emit fluorescence among the plurality of wells for each of the one or more images acquired by the image acquisition unit; A program for functioning as the counting unit counts the number of wells emitting fluorescence, excluding wells in which adjacent wells also emit fluorescence, among the plurality of wells; program.