JP4321716B2 - Fluorescence image correction method, apparatus, and program - Google Patents

Description

  The present invention relates to an image correction method and apparatus for correcting fluorescence image data when fluorescence generated from a fluorescently labeled specimen labeled with a fluorescent dye hybridized with a specific binding substance is detected and acquired as fluorescence image data. As well as programs.

  In recent years, rapid progress in technology in the field of genetic engineering has led to the development of a human genome project with the goal of deciphering the base sequence of the human genome, which is thought to be as many as 100,000. On the other hand, research on DNA affecting various gene diseases is also progressing, and microarray technology is attracting attention as one of the methods. In this microarray technology, a large number of different cDNAs (examples of specific binding substances) that have already been decoded have a dot size of 50 to 150 (μm) in the range of 1.8 × 1.8 (cm) on a glass slide as a substrate. )), A so-called microarray in which about 6400 spots are arranged, or a dot size of about 0. 12 (cm), 7 x 5 (cm) or 22 x 22 (cm) on a nylon membrane filter. Dot size within a range of 1.28 x 1.28 (cm) on a silicon substrate, or a 5 to 1 (mm) spot, called a macroarray with 588, 5000, and 27000, respectively. This technique uses what is called a DNA chip in which about 64,000 spots are arranged as 50 (μm) spots. Roarei are collectively microarray and DNA chips is referred to as a "microplate".).

  That is, a fluorescently labeled specimen A of DNA taken out from a healthy person's cells labeled with a fluorescent dye a is dropped onto each cDNA on the microplate with a pipette or the like, and the fluorescently labeled specimen A and the cDNA are hybridized. After this hybridization treatment, the microplate is treated with a predetermined solution, the fluorescently labeled specimen A that has not been hybridized with cDNA is removed from each spot, and the fluorescently labeled specimen A that has been hybridized with cDNA remains in each spot. It is. Then, the spot of each specimen (hereinafter referred to as a spot) on the microplate subjected to the above processing is scanned with the excitation light FL for exciting the fluorescent dye a, and generated by irradiation of the excitation light FL. Fluorescence image data representing the fluorescence detection result is obtained (see, for example, Patent Documents 1 and 2).

  Fluorescence image data representing the detection result of the fluorescence generated by the irradiation of excitation light by performing the same process as described above also on the fluorescence-labeled specimen B of DNA extracted from the cell of a person having a genetic disease labeled with the fluorescent dye b The ratio value of the fluorescence image data obtained from each spot of the fluorescently labeled specimen A and the fluorescence image data obtained from each spot of the fluorescently labeled specimen B is obtained for each spot. Since the ratio of the fluorescence image data increases (or decreases) as the spot where the abnormal DNA is dropped, the fluorescence image data for each spot, for example, in order from the largest, the fluorescence for 50 locations. The ratio value of the image data is read as a table corresponding to the spot from which the signal is obtained, and abnormal DNA is identified based on this table.

  In addition, the optical system that detects fluorescence emitted from each spot on the microplate often uses a confocal optical system with a high spatial resolution and a shallow depth of focus. By detecting the fluorescence emitted from the spot by irradiating the spot on the microplate through the pinhole located at the other focal position of the confocal optical system, Noise such as generated fluorescence can be removed.

Here, the analysis of the fluorescence image data described above requires that the reliability of the data itself is guaranteed. However, due to the experimental environment such as changes in environment such as temperature and humidity, microplate scratches, hybridization reaction efficiency, fluorescence reading accuracy in actual experiments, for example, the same type of specific binding substance Even when fluorescently labeled specimens are hybridized, the obtained fluorescence image data may differ. Therefore, using fluorescence image data acquired from the microplate, a method for visually grasping a defect in the experimental process (see, for example, Patent Document 3), and fluorescence image data acquired from each of a plurality of spots on the microplate are statistically analyzed. A method (for example, see Patent Document 4) for correcting the acquired fluorescent image data by analyzing the data has been proposed.
JP 2001-74656 A JP 2001-74657 A JP 2002-71688 A JP 2003-28862 A

  In the correction of the fluorescence image data for preventing the deterioration of reliability due to the experimental environment as described in Patent Documents 2 and 3, the fluorescence emitted from the fluorescent dye irradiated with the excitation light always has a constant intensity. It is assumed that. That is, it is assumed that the fluorescent dye that labels the specimen always outputs predetermined fluorescence when irradiated with excitation light.

  However, it has been experimentally found that the intensity of the fluorescence emitted from the fluorescent dye itself decreases with time in the combination of the specific fluorescent dye and the specific specimen. That is, when the same kind of fluorescently labeled specimens existing in different spots are irradiated with excitation light at the same time, and the fluorescence emitted from each spot is detected at the same time, the intensity of the fluorescence is the same. Become. However, when the same kind of fluorescently labeled specimens in different spots are irradiated with excitation light at the same time, and the fluorescence emitted from each spot is detected at different times, the intensity of the fluorescence is different. I understood that.

  The fluorescence-labeled specimen that was last irradiated with the excitation light due to this time-dependent change in fluorescence is the first excitation light, even when the first detected fluorescence and the last detected fluorescence emitted at the same intensity. Therefore, there is a problem that it is difficult to perform a highly reliable analysis.

  Therefore, an object of the present invention is to provide a fluorescence image correction method, apparatus, and program for analyzing a fluorescence-labeled specimen whose fluorescence intensity decreases with time.

  The fluorescent image correction method of the present invention detects fluorescence emitted from a fluorescently labeled specimen by irradiating excitation light to the fluorescently labeled specimen hybridized with a specific binding substance present in a spot on a microplate. In the fluorescence image correction method for correcting the main fluorescence image data acquired in this manner, the main fluorescence image data is corrected based on the temporal change characteristic when the intensity of the fluorescence emitted from the fluorescently labeled specimen decreases with time. It is a feature.

  The fluorescence image correction apparatus of the present invention detects the fluorescence emitted from the fluorescently labeled specimen by irradiating the fluorescently labeled specimen hybridized with the specific binding substance present at the spot of the microplate. The image acquisition means for acquiring the main fluorescence image data and the image correction means for correcting the main fluorescence image data acquired by the image acquisition means, and the image correction means is the intensity of the fluorescence emitted from the fluorescence-labeled specimen. Is characterized in that the main fluorescence image data is corrected on the basis of the temporal change characteristic when the value decreases with time.

  The fluorescent image correction program of the present invention is a program that irradiates a fluorescently labeled specimen hybridized with a specific binding substance present in a spot on a microplate to a computer, thereby emitting fluorescence emitted from the fluorescently labeled specimen. The fluorescent image data is detected and corrected, and the fluorescent image data is corrected based on the temporal change characteristic when the intensity of the fluorescence emitted from the fluorescently labeled specimen decreases with time. To make things happen.

  Here, the “microplate” may be anything as long as it has a plurality of spots and has a plate-like shape in which a specific binding substance is fixed on each spot. A “spot” refers to a unit region to which a specific binding substance in a microplate shape is fixed. “Hybridization” refers to binding of a complementary nucleotide chain to a specific base sequence utilizing the property of nucleic acid forming a complementary duplex. “Fluorescently labeled specimen” means specimens such as hormones labeled with fluorescent dyes, tumor markers, enzymes, antibodies, antigens, abzymes, other proteins, nucleic acids, cDNA, DNA, RNA, etc. "Is hormones, tumor markers, enzymes, antibodies, antigens, abzymes, other proteins, nucleic acids, cDNA, DNA, RNA, mRNA, nucleotide chains, etc., and hormones, tumor markers, enzymes, antibodies, antigens, Abzymes, other proteins, nucleic acids, cDNAs, DNAs, RNAs and the like that can specifically bind to substances derived from living organisms and have known base sequences, base lengths, compositions, and the like.

  “Main fluorescence image data” refers to fluorescence image data for use in analysis of fluorescently labeled specimens, and “auxiliary fluorescence image data” refers to irradiation of excitation light separately from main fluorescence image data. The fluorescence image data for calculating the time-dependent change characteristic of the fluorescently labeled specimen acquired by the above. “Time lapse” refers to the time lapse after the fluorescently labeled specimen is hybridized with the specific binding substance regardless of whether or not the excitation light is irradiated.

  The image correction means has, for example, a time-dependent change characteristic unique to a fluorescently labeled sample hybridized with a specific binding substance when the main fluorescent image data is acquired, and the main fluorescent image data is obtained based on the time-dependent change characteristic. The auxiliary fluorescent image data may be corrected, and when the auxiliary fluorescent image data different from the main fluorescent image data is acquired, the time-dependent change characteristic is calculated from the main fluorescent image data and the main fluorescent image data is corrected. I just need it.

  When acquiring auxiliary fluorescence image data different from the main fluorescence image data, the image acquisition means applies to the fluorescently labeled specimen hybridized with the specific binding substance present on the predetermined spot from which the main fluorescence image data was acquired. On the other hand, the fluorescence emitted from the fluorescently labeled specimen may be detected by irradiating the excitation light to obtain auxiliary fluorescence image data at a predetermined spot. Furthermore, the image correction means includes main fluorescence image data and auxiliary fluorescence image data at a predetermined spot acquired by the image acquisition means, and an excitation light irradiation time interval when acquiring the main fluorescence image data and auxiliary fluorescence image data. There may be provided a change characteristic acquisition means for calculating and acquiring the time-dependent change characteristic based on the above.

  The predetermined spot is a plurality of spots, and the auxiliary fluorescent image data at the predetermined spot is excited in a direction different from the scanning direction of the excitation light with respect to the microplate when acquiring the main fluorescent image data at the predetermined spot. Fluorescence image data acquired by scanning

  Alternatively, the predetermined spot is a plurality of spots, and the auxiliary fluorescent image data at the predetermined spot is a microplate at a speed different from the scanning speed of the excitation light with respect to the microplate when acquiring the main fluorescent image data at the predetermined spot. May be fluorescence image data obtained by scanning with excitation light.

  In addition, the microplate has a plurality of spots for obtaining main fluorescence image data and a plurality of test spots for emitting fluorescence with the same fluorescence intensity, each of which has a fluorescently labeled specimen hybridized with a specific binding substance. It may have.

  The fluorescently labeled specimen hybridized with the specific binding substance present in the test spot has the same time-varying characteristics as the fluorescently labeled specimen hybridized with the specific binding substance of the spot from which the main fluorescence image data is acquired. Each of the inspection spots emits fluorescence with the same fluorescence intensity.

  At this time, the image acquisition means detects the fluorescence emitted from the fluorescently labeled specimen by acquiring the main fluorescent image data and irradiating the fluorescently labeled specimen hybridized at each inspection spot with excitation light. Thus, a plurality of fluorescent image data for examination may be acquired. This “fluorescence image data for examination” refers to the fluorescence-labeled specimen that is obtained by irradiating the examination spot with excitation light at the same time when excitation light irradiation for obtaining main fluorescence image data is performed. Fluorescence image data for calculating the temporal change characteristic.

  Further, the image correction means includes a plurality of inspection fluorescent image data for each inspection spot acquired by the image acquisition means, and an excitation light irradiation time between the inspection spots when the inspection fluorescent image data is acquired. Based on the interval, there may be provided a change characteristic acquisition means for acquiring a time-dependent change characteristic unique to the fluorescently labeled specimen present in the test spot.

  In addition, when the microplate has a plurality of spots arranged in a grid along the row direction and the column direction, the image acquisition means excites the microplate with respect to the fluorescently labeled specimens hybridized at each spot. A plurality of main fluorescence image data may be acquired by detecting the fluorescence emitted from the fluorescently labeled specimen at each spot by moving in the row direction while scanning in the column direction with light.

  At this time, the image correction means has change characteristic acquisition means for acquiring a time-dependent change characteristic based on the average value of the main fluorescence image data for each spot row and the time interval of excitation light irradiation between the respective spot rows. It may be a thing.

  The “spot column” refers to a plurality of spots arranged in the column direction of the microplate. The “time interval of excitation light irradiation between the spot columns” refers to, for example, the same row from among the plurality of spot columns. This is the time interval between the spots existing above.

  According to the fluorescence image correction method and apparatus and program of the present invention, by correcting the main fluorescence image data based on the temporal change characteristic when the intensity of the fluorescence emitted from the fluorescently labeled specimen decreases with time, Even when detecting the fluorescence of a fluorescently labeled specimen that has a characteristic that the fluorescence decreases with time, it can be corrected based on this time-dependent change characteristic, so accurate analysis using the main fluorescence image data It can be performed.

  In addition to the acquisition of the main fluorescence image data, the image acquisition means irradiates the fluorescent labeled sample hybridized with the specific binding substance existing on the predetermined spot from which the main fluorescence image data was acquired. Thus, the fluorescence emitted from the fluorescence-labeled specimen is detected to acquire auxiliary fluorescence image data at a predetermined spot, and the image correction means is the main fluorescence image at the predetermined spot acquired by the image acquisition means. If it has a configuration having change characteristic acquisition means for calculating and acquiring temporal change characteristics based on the data and auxiliary fluorescence image data, and the excitation light irradiation time interval when acquiring the main fluorescence image data and auxiliary fluorescence image data Auxiliary fluorescence image data is acquired when the main fluorescence image data is acquired, and the main fluorescence image data is used as the time-dependent change characteristics using the auxiliary fluorescence image data. The by calculating, it can efficiently calculate the temporal change characteristics to obtain.

  Further, the predetermined spot is a plurality of spots, and the auxiliary fluorescent image data at the predetermined spot is excited in a direction different from the scanning direction of the excitation light with respect to the microplate when acquiring the main fluorescent image data at the predetermined spot. If the fluorescent image data is obtained by scanning the main fluorescent image data obtained from a plurality of predetermined spots, the time interval when the main fluorescent image data and the auxiliary fluorescent image data are acquired is different for each spot. In order to calculate the time-varying characteristics unique to the fluorescently labeled specimen from which the main fluorescence image data was acquired using the data and auxiliary fluorescence image data, the time-varying characteristics are calculated for all spots where the same type of fluorescently labeled specimen is hybridized. It is not necessary to calculate, and the time change characteristic can be calculated efficiently.

  Further, the predetermined spot is a plurality of spots, and the auxiliary fluorescent image data at the predetermined spot is a microplate at a speed different from the scanning speed of the excitation light with respect to the microplate when acquiring the main fluorescent image data at the predetermined spot. If the fluorescence image data obtained by scanning with excitation light, the time interval when obtaining the main fluorescence image data and the auxiliary fluorescence image data is made different for each spot, and obtained from a plurality of predetermined spots. In order to calculate the time-varying characteristics unique to the fluorescently labeled specimen from which the main fluorescent image data was acquired using the main fluorescent image data and the auxiliary fluorescent image data, the time-dependent characteristics of all spots where the same kind of fluorescently labeled specimen was hybridized were calculated. It is not necessary to calculate the change characteristic, and the time change characteristic can be calculated efficiently.

  The microplate has a plurality of spots for obtaining main fluorescence image data, and a plurality of test spots for emitting fluorescence with the same fluorescence intensity, where there are fluorescently labeled specimens hybridized with specific binding substances. And the image acquisition means emits the excitation light to the fluorescently labeled specimens that are respectively hybridized at the inspection spots at different times for each inspection spot, thereby being emitted from the fluorescently labeled specimens at the inspection spots, respectively. And detecting fluorescent image data for each inspection spot, and image correction means includes a plurality of inspection fluorescent image data for each inspection spot acquired by the image acquisition means, and The inspection spot based on the excitation light irradiation time interval between the inspection spots when acquiring the fluorescent image data for inspection If there is a change characteristic acquisition means that acquires the time-dependent change characteristic unique to the fluorescently labeled specimen that exists, it also acquires the fluorescence image data for examination as well as the main fluorescence image data, and irradiates a plurality of examination spots with excitation light. Since the time-dependent change characteristic peculiar to the fluorescently labeled specimen is calculated based on the time interval, the time-dependent change characteristic can be calculated efficiently.

  In addition, when the microplate has a plurality of spots arranged in a grid along the row direction and the column direction, the image acquisition means applies the microplate to the fluorescently labeled specimens respectively hybridized at each spot. Image correction means for detecting a fluorescence emitted from a fluorescently labeled specimen for each spot and acquiring a plurality of main fluorescence image data by moving in a row direction while scanning in a column direction with excitation light, However, if the configuration has a change characteristic acquisition means for acquiring a time-dependent change characteristic based on the average value of the main fluorescence image data for each spot row and the time interval of excitation light irradiation between the spot rows, Since the temporal change characteristic can be calculated using the fluorescence image data, the main fluorescence image data can be efficiently corrected.

  Hereinafter, embodiments of the fluorescence detection system of the present invention will be described in detail with reference to the drawings. FIG. 1 is a configuration diagram showing an example of a fluorescence detection system using the image correction apparatus of the present invention. The fluorescence detection system 1 in FIG. 1 detects fluorescence emitted from a fluorescently labeled specimen by irradiating the fluorescently labeled specimen hybridized with a specific binding substance existing on a microplate, and detects fluorescence image data. The fluorescence detection device 10 that outputs the fluorescence image data and the image correction device 50 that acquires the fluorescence image data from the fluorescence detection device 10 and corrects the fluorescence image data.

  Here, the fluorescence detection apparatus 10 has a plate accommodating portion 10a that opens and closes to accommodate the microplate, and the microplate MP is loaded from the plate accommodating portion 10a. As shown in FIG. 2, the microplate MP has a plurality of lattice-like spots W, and the fluorescently labeled specimen hybridized with a specific binding substance exists in the spots W.

  FIG. 3 is a block diagram showing an example of the fluorescence detection apparatus. The fluorescence detection apparatus 10 will be described with reference to FIGS. 1 to 3. The fluorescence detection apparatus 10 is emitted from the fluorescence-labeled specimen when the excitation-light irradiation means 20 irradiates the microplate MP while scanning the excitation light, and when the excitation-light is irradiated to the fluorescence-labeled specimen by the excitation light irradiation means 20. Fluorescence detection means 30 for detecting fluorescence and data output means 40 for outputting fluorescence detected by the fluorescence detection means 30 as fluorescence image data are provided.

  Here, the excitation light irradiation means 20 includes an excitation light source 21 that emits the excitation light FL, a condensing lens 24 that condenses the emitted excitation light FL onto the fluorescently labeled specimen of the microplate MP, and the excitation light FL as an arrow. Lens moving means 25 for moving the condensing lens 24 for scanning in the Y direction (main scanning direction), and plate moving means 26 for moving the microplate MP in the arrow X direction for scanning the excitation light in the arrow X direction. And.

  The excitation light source 21 has a function of outputting excitation light FL having different wavelengths (for example, 640 nm, 532 nm, and 473 nm) depending on the type of fluorescent dye used for the fluorescently labeled specimen. When the excitation light FL emitted from the excitation light source 21 is incident through the hole of the perforated mirror 23 via the mirrors 22a and 22b, the condenser lens 24 converts the excitation light FL into a fluorescently labeled specimen of the microplate MP. Condensed in

  At this time, the microplate MP is irradiated with the excitation light FL condensed by the condenser lens 24 while scanning in the arrow X direction and the Y direction. Specifically, the condenser lens 24 is provided so as to be able to swing, and is connected to the lens moving means 25. The lens moving means 25 swings the condensing lens 24 so that the condensing position scans in the arrow Y direction of the microplate MP. The microplate MP is provided to be movable in the arrow X direction (sub-scanning direction) and is connected to the plate moving means 26. The plate moving means 26 moves the microplate MP in the arrow X direction. That is, since the excitation light FL scans in the arrow Y direction by the movement of the condenser lens 24 and scans in the arrow X direction by the movement of the microplate MP, a plurality of excitation lights FL arranged in a grid pattern on the microplate MP. It is possible to irradiate the fluorescently labeled specimen with the excitation light FL while scanning.

  The fluorescence detection means 30 is composed of, for example, a PMT (Photomultiplier Tubes), and the fluorescence is detected as an analog signal. The fluorescence emitted from the fluorescently labeled specimen enters the condenser lens 24 to become parallel light, is reflected by the mirror portion of the perforated mirror 23, and enters the color filter 31. Thereafter, the fluorescence emitted from the color filter 31 is incident on the lens 33 through the mirror 32, and is condensed on the fluorescence detection means 30 by the lens 33.

  The data output means 40 has a function of outputting the detected fluorescence as fluorescence image data by returning the analog signal output from the fluorescence detection means 30 to a digital signal and performing signal processing. Here, as described above, each spot W of the microplate MP is irradiated with the excitation light FL while scanning in the arrow X direction and the arrow Y direction. At this time, the fluorescence detection means 30 detects the fluorescence emitted every time the excitation light FL is irradiated to the fluorescently labeled specimen, and the data output means 40 detects the fluorescence emitted from the plurality of fluorescently labeled specimens as fluorescence image data ED. As output.

  An example of the operation of the fluorescence detection apparatus 10 will be described with reference to FIG. First, the excitation light FL is irradiated from the excitation light irradiation means 20 to the spot W of the microplate MP via the mirrors 22 a and 22 b and the condenser lens 24. Then, fluorescence is emitted from the fluorescently labeled specimen existing in the spot W irradiated with the excitation light FL. The emitted fluorescence is detected by the fluorescence detection means 30 and output from the data output means 40 as fluorescence image data. At this time, since the excitation light FL is irradiated while scanning the microplate MP by the lens moving means 25 and the plate moving means 26, the fluorescence image data ED is output for each spot W existing on the microplate MP. It will be done.

  FIG. 4 is a block diagram showing an embodiment of the image correction apparatus of the present invention. The image correction apparatus 50 will be described with reference to FIGS. The image correction device 50 irradiates the fluorescence-labeled specimen hybridized with the specific binding substance present in the spot W of the microplate MP with excitation light, thereby causing the fluorescence emitted from the fluorescent-labeled specimen to be the main fluorescence image. The image acquisition means 51 which acquires as data ED1 and the image correction means 60 which correct | amends the main fluorescence image data ED1 acquired by the image acquisition means 51 are provided.

  Here, the image acquisition means 51 separates the main fluorescence image data ED1 used for analyzing the fluorescence-labeled specimen and the main fluorescence image data ED1 on the predetermined spot W from which the main fluorescence image data ED1 has been acquired. By irradiating the fluorescently labeled specimen hybridized with the specific binding substance with the excitation light FL, the fluorescence emitted from the fluorescently labeled specimen is detected, and the auxiliary fluorescence image data ED2 at the predetermined spot W is obtained. It has a function to acquire.

  Specifically, as shown in FIG. 5A, the image acquisition unit 51 is acquired when scanning a plurality of spots W arranged on the microplate MP while irradiating the excitation light FL. Acquire a plurality of auxiliary fluorescence image data ED2 and main fluorescence image data ED1 acquired when the excitation light FL is irradiated in a scanning direction different from the scanning direction of the excitation light FL when the auxiliary fluorescence image data ED2 is acquired. It is supposed to be. That is, in FIG. 5A, scanning is performed so that the excitation light FL is first irradiated in the order of the spots Wa, Wb, and Wc, and auxiliary fluorescence image data ED2a, ED2b, and ED2c are acquired. Next, when the microplate MP is mounted on the fluorescence detection device 10 with its orientation changed, the microplate MP is scanned so that the excitation light FL is irradiated in the order of the spots Wc, Wb, Wa, and auxiliary fluorescence image data. ED2c, ED2b, and ED2a are acquired.

  In acquiring two fluorescent image data ED1 and ED2 having different scanning directions, the two fluorescent image data ED1 and ED2 are changed by changing the direction of the microplate MP without changing the scanning direction of the excitation light by the excitation light irradiation means 20. Or by reversing the scanning direction of the excitation light of the excitation light irradiation means 20 and the moving direction of the microplate MP without changing the direction of the microplate MP. You may make it acquire. Such scanning control of excitation light is performed by the controller 70.

  Next, the image correction means 60 will be described with reference to FIGS. The image correction unit 60 in FIG. 4 corrects the main fluorescence image data ED1 based on the temporal change characteristic when the intensity of the fluorescence emitted from the fluorescently labeled specimen decreases with time. Based on the main fluorescence image data ED1 and auxiliary fluorescence image data ED2 at the predetermined spot W acquired by 51, and the time interval of excitation light irradiation when acquiring the main fluorescence image data ED1 and auxiliary fluorescence image data ED2 It has a change characteristic acquisition unit 61 that calculates and acquires a change characteristic, and a correction unit 62 that corrects the main fluorescence image data ED1 using the acquired temporal change characteristic.

  Specifically, as shown in FIG. 5A, the change characteristic acquisition unit 61 includes three auxiliary fluorescent image data ED2a and ED2b acquired from fluorescently labeled specimens existing in a plurality of predetermined spots Wa, Wb, and Wc. , ED2c is acquired. Further, the change characteristic acquisition unit 61 acquires three main fluorescence image data ED1a, ED1bv, and ED1c acquired from fluorescently labeled specimens existing in a plurality of predetermined spots Wa, Wb, and Wc. Then, for the spots Wa, Wb, and Wc, it is calculated how much the fluorescence intensity has decreased in the time interval from the acquisition of the main fluorescence image data ED1 to the acquisition of the auxiliary fluorescence image data ED2. Specifically, the fluorescence intensity of the auxiliary fluorescence image data ED2 is divided by the fluorescence intensity of the main fluorescence image data ED1, and the decrease rate of the fluorescence intensity is calculated.

  At this time, the time interval from the acquisition of the main fluorescence image data ED1 to the acquisition of the auxiliary fluorescence image data ED2 is different for each of the spots Wa, Wb, and Wc. When scanning of the excitation light as shown in FIG. 5A is performed, the time interval from the first excitation light irradiation to the second excitation light irradiation is the shortest in the spot Wc, and then the spots Wb and Wa. It becomes shorter in order.

  Therefore, as shown in FIG. 5B, the change characteristic acquisition unit 61 acquires three reduction rates having different time intervals from the acquisition of the main fluorescence image data ED1 to the acquisition of the auxiliary fluorescence image data ED2. be able to. Therefore, the change characteristic acquisition means 61 calculates and acquires a time-dependent change characteristic in which the fluorescence intensity decreases with time by connecting the three reduction rates with lines. Thereafter, from the time when the correction unit 62 acquires the main fluorescence image data ED1, the decrease rate used for the main fluorescence image data ED1 is detected from the temporal change characteristic of FIG. 5B, and the main fluorescence image data ED1 is detected at the decrease rate. It corrects by dividing.

  Thereby, even when the fluorescence intensity decreases with time, the decrease can be corrected, and the fluorescence analysis can be performed with high accuracy and high reliability. That is, in the fluorescence detection system 1 as described above, since the fluorescent dye emits fluorescence when irradiated with excitation light, it has been considered that the fluorescence does not decay over time. That is, in FIG. 5B, it was considered that the main fluorescence image data ED1 and the auxiliary fluorescence image data ED2 are equal. However, in experiments, it was observed that the fluorescence decayed with time. This is considered to be caused by the fact that the fluorescent dye and the specimen to be examined cause some chemical reaction, and the fluorescence by the fluorescent dye becomes weaker with time. Therefore, even when detecting fluorescence of a fluorescently labeled specimen having a characteristic that the fluorescence decreases with time, correction can be made based on the calculated temporal change characteristic, so the main fluorescence image data ED1 is used. Accurate analysis.

  Furthermore, a single microplate MP is irradiated with excitation light a plurality of times while changing the scanning direction, and the time interval from the acquisition of the auxiliary fluorescence image data ED2 to the acquisition of the main fluorescence image data ED1 is the spot Wa of each spot. , Wb, and Wc can be made different from each other, so that the time-varying characteristics can be calculated more efficiently than when the time-varying characteristics are calculated for all the spots W.

  FIG. 6 is a flowchart showing a preferred embodiment of the image correction method of the present invention. The image correction method will be described with reference to FIGS. First, the fluorescence detection apparatus 10 irradiates the fluorescently labeled specimen existing in the spot W with the excitation light FL (step ST1). Then, the fluorescence emitted from the fluorescently labeled specimen is acquired by the image acquisition means 51 as auxiliary fluorescent image data ED2 (step ST2). The excitation light FL is irradiated while scanning the plurality of spots W of the microplate MP, and the auxiliary fluorescence image data ED2 corresponding to the plurality of spots is acquired by the image acquisition means 51 (step ST1 to step ST3).

  Next, the excitation light FL is scanned in the direction opposite to the scanning direction when the auxiliary fluorescence image data ED2 is acquired with respect to the microplate MP (see FIG. 5A) (step ST4). The excitation light FL is irradiated while scanning a plurality of spots W of the microplate MP, and main fluorescence image data ED1 corresponding to the plurality of spots is acquired by the image acquisition means 51 (step ST4 to step ST6).

  Thereafter, the image correction device 50 calculates the time-varying characteristics when the intensity of the fluorescence emitted from the fluorescently labeled specimen decreases with time using the main fluorescence image data ED1 and the auxiliary fluorescence image data ED2 (step ST7). ), The main fluorescence image data ED1 is corrected based on the temporal change characteristic (step ST8). Specifically, the change characteristic acquisition unit 61 calculates a time change characteristic as shown in FIG. 5B, and the correction unit 62 corrects the main fluorescence image data ED1 based on the time change characteristic.

  As a result, even when the fluorescence intensity decreases with time, the time-varying characteristics can be calculated, and the decrease in fluorescence intensity can be corrected based on the time-varying characteristics. Highly sensitive fluorescence analysis can be performed.

  By the way, as a method for calculating the temporal change characteristic, in addition to the method for changing the scanning direction of the excitation light described above, auxiliary fluorescence image data indicating fluorescence when the excitation light is irradiated at a scanning speed different from the main fluorescence image data. A method for acquiring time-varying characteristics using a fluorescence detection method, detecting fluorescence of fluorescently labeled specimens hybridized at a plurality of test spots that emit fluorescence with the same fluorescence intensity, and measuring a plurality of time-lapse values from the obtained plurality of test fluorescence image data. Method for obtaining change characteristics, detecting fluorescence of fluorescently labeled specimens hybridized at a plurality of spots arranged in a lattice form where a specific binding substance exists, and obtaining time-dependent change characteristics from the obtained fluorescence image data There are methods. Each method will be described below.

  First, a method for obtaining the temporal change characteristic using two fluorescent image data having different scanning speeds will be described. FIG. 7 is a schematic diagram illustrating an example of a method for acquiring the main fluorescence image data ED1 and the auxiliary fluorescence image data ED2, and an aging characteristic. In FIG. 7A, the image acquisition means 51 includes main fluorescence image data ED1 acquired when scanning a plurality of fluorescently labeled specimens on the microplate MP while irradiating the excitation light FL, and the main fluorescence image. The auxiliary fluorescence image data ED2 acquired when the excitation light FL is irradiated at a scanning speed different from the scanning speed when the data ED1 is acquired is acquired.

  Specifically, the plurality of main fluorescence image data ED1 is obtained by using the excitation light FL at a main scanning speed SV1 such that the excitation light is emitted to each spot only for the time required for performing the fluorescence detection. Acquired when scanning while scanning (main scan). On the other hand, the plurality of auxiliary fluorescence image data ED2 is acquired when the excitation light FL is irradiated while being scanned at an auxiliary scanning speed SV2 higher than the main scanning speed SV1, which is performed before acquisition of the main fluorescence image data ED1. (Pre-scan). By using the main fluorescence image data ED1 and the auxiliary fluorescence image data ED2, the decrease rate of the fluorescence intensity can be calculated as described above, and the temporal change characteristic can be acquired. At this time, the time interval from the acquisition of the main fluorescence image data ED1 to the acquisition of the auxiliary fluorescence image data ED2 is different for the spots Wa, Wb and Wc of each spot W. When scanning with the excitation light as shown in FIG. 7A, the time interval from the first excitation light irradiation to the second excitation light irradiation is the shortest in the spot Wc, and then the spots Wb and Wa. It becomes shorter in order.

  Therefore, as shown in FIG. 7B, the change characteristic acquisition unit 61 acquires three reduction rates having different time intervals from the acquisition of the main fluorescence image data ED1 to the acquisition of the auxiliary fluorescence image data ED2. be able to. Therefore, the change characteristic acquisition means 61 calculates and acquires a time-dependent change characteristic in which the fluorescence intensity decreases with time by connecting the three reduction rates with lines. Thereafter, from the time when the correction unit 62 acquires the main fluorescence image data ED1, the decrease rate used for the main fluorescence image data ED1 is detected from the temporal change characteristic of FIG. 7B, and the main fluorescence image data ED1 is detected at the decrease rate. It corrects by dividing.

  Even with the method as shown in FIG. 7, it is possible to calculate the temporal change characteristic when the fluorescence intensity decreases with time, and to correct the decrease in the fluorescent intensity based on the temporal change characteristic. Highly accurate and reliable fluorescence analysis can be performed. Furthermore, since the decrease rate of the fluorescence intensity of the fluorescence-labeled specimen can be calculated and acquired using the pre-scan and the main scan that are normally performed in the fluorescence detection device 10, no special control for the fluorescence detection device 10 is necessary, The fluorescent image data can be corrected efficiently.

  Next, a description will be given of a method for acquiring the temporal change characteristic using the inspection fluorescence image data EXD acquired from a plurality of inspection spots that emit fluorescence with the same fluorescence intensity. FIG. 8 is a schematic diagram showing an example of a microplate MP having a plurality of spots for acquiring the main fluorescence image data ED1 and inspection spots for acquiring the inspection fluorescence image data EXD. Specifically, in FIG. 8 (a), each of the microplates MP has a hybridized fluorescently labeled specimen that emits fluorescence with the same fluorescence intensity to each of the inspection spots Wa to Wf. ing. FIG. 8A illustrates a case where inspection spots Wa to Wf are arranged one by one in each row of the microplate MP. The arrangement positions of the inspection spots Wa to Wf only need to be such that the excitation light FL is irradiated to the inspection spots Wa to Wf at different times, as will be described later.

  Then, the excitation light irradiation means 20 irradiates each spot of the microplate MP including the inspection spots Wa to Wf while scanning the excitation light FL. Then, each of the inspection spots Wa to Wf is irradiated with the excitation light FL at different times. Then, the image acquisition means 51 acquires the main fluorescence image data ED1 from each spot on the microplate MP, and acquires the inspection fluorescence image data EXDa to EXDf from each inspection spot Wa to Wf. Thereafter, the change characteristic acquisition unit 61 uses the fluorescence intensity indicated by the plurality of inspection fluorescence image data EXDa to EXDf and the time when the excitation spots FL are irradiated with the excitation light FL, as shown in FIG. A time-dependent change characteristic as shown is calculated. Then, the correction means 62 corrects the intensity of the fluorescence that has decreased over time by correcting the main fluorescence image data ED1 using the calculated decrease rate in accordance with the time when the main fluorescence image data ED1 was acquired.

  Even with the method shown in FIG. 8, it is possible to calculate the time-varying characteristic when the fluorescence intensity decreases with time, and to correct the decrease in the fluorescence intensity based on the time-varying characteristic. Highly accurate and reliable fluorescence analysis can be performed.

  Next, a method for acquiring time-varying characteristics using main fluorescence image data emitted from a plurality of fluorescently labeled specimens hybridized with the same specific binding substance will be described. FIG. 9 is a schematic diagram showing an example of a microplate MP in which a plurality of fluorescently labeled specimens hybridized with the same specific binding substance are arranged in a lattice pattern.

  In FIG. 9A, the image acquisition means 51 has a plurality of main fluorescence image data when the microplate MP is moved in the row direction (arrow X1 direction) while scanning in the column direction (arrow Y1 direction) with excitation light. ED1 is acquired. Then, as shown in FIG. 9B, the change characteristic acquisition unit 61 calculates the average value of the plurality of main fluorescence image data ED1 emitted from the plurality of spot rows W31 to W36 arranged in the scanning direction of the excitation light, and the excitation A time-dependent change characteristic is calculated and acquired based on the time when the light FL is scanned. Then, the correction means 62 corrects the acquired main fluorescence image data ED1 using the calculated temporal change characteristic and the time when each fluorescently labeled specimen is irradiated with the excitation light.

  As described above, when calculating the temporal change characteristic, by using the main fluorescence image data ED1 that is an actual image acquisition target, the temporal change characteristic can be efficiently calculated and the image can be corrected.

  In the method of FIG. 9, the temporal change characteristic is obtained using the main fluorescence image data ED1 obtained from the measurement object. That is, the temporal change characteristic is calculated on the assumption that the average value of the main fluorescent image data ED1 of the spot rows is substantially the same in each spot row if the temporal change property is not considered.

  That is, in general, DNA is randomly arranged at each spot on a microplate (DNA chip). On the other hand, tens of thousands of spots are provided on the microplate (DNA chip), and there are 100 or more spots in one row. Note that the detection level of the fluorescence detection device 10 is also a finite integer of 0 digit to 65535 digit. If a sample is sampled from a finite population of tens of thousands of spots in which DNA is randomly arranged and spot sequences containing several hundred spots are averaged, the average of each spot sequence is statistically abbreviated. Be the same. Therefore, the temporal change characteristic can be efficiently calculated using the main fluorescence image data ED1.

  According to each of the embodiments described above, by correcting the main fluorescence image data ED1 based on the time-varying characteristics when the intensity of the fluorescence emitted from the fluorescently labeled specimen decreases with time, the fluorescence increases with time. Even when detecting fluorescence of a fluorescently labeled specimen having a decreasing characteristic, correction can be made based on this time-varying characteristic, so that accurate analysis can be performed using the main fluorescent image data ED1. it can.

  In addition to the acquisition of the main fluorescence image data ED1, the image acquisition means 51 excites the fluorescently labeled specimen hybridized with the specific binding substance existing on the predetermined spot from which the main fluorescence image data ED1 was acquired. By irradiating the light FL, the fluorescence emitted from the fluorescently labeled specimen is detected and the auxiliary fluorescence image data ED2 at a predetermined spot is acquired. The image correcting unit 60 is acquired by the image acquiring unit 51. A time-varying characteristic is calculated and acquired based on main fluorescence image data ED1 and auxiliary fluorescence image data ED2 at a predetermined spot, and excitation light irradiation time intervals when acquiring the main fluorescence image data ED1 and auxiliary fluorescence image data ED2. If the configuration having the change characteristic acquisition means 61 is provided, the auxiliary fluorescent image data is acquired when the main fluorescent image data ED1 is acquired. Get the ED2, by calculating the time-change characteristic of the main fluorescence image data ED1 by using the auxiliary fluorescent image data ED2, it can efficiently calculate the temporal change characteristics to obtain.

  Further, as shown in FIG. 5, the predetermined spot is a plurality of spots Wa to Wc, and the auxiliary fluorescent image data ED2 at the predetermined spot is the excitation light when the main fluorescent image data ED1 at the predetermined spot is acquired. If the fluorescence image data is obtained by scanning the excitation light in a direction different from the scanning direction with respect to the microplate MP, the time interval when the main fluorescence image data ED1 and the auxiliary fluorescence image data ED2 are obtained is different for each spot. In order to calculate the time-varying characteristics peculiar to the fluorescence-labeled specimen from which the main fluorescence image data ED1 is obtained using the main fluorescence image data ED1 and the auxiliary fluorescence image data ED2 obtained from a plurality of predetermined spots Wa to Wc. Calculate the time course characteristics for all spots where the same type of fluorescently labeled specimen is hybridized. Becomes unnecessary, it is possible to efficiently calculate the aging characteristics.

  Further, as shown in FIG. 7, the predetermined spot is a plurality of spots Wa to Wc, and the auxiliary fluorescent image data ED2 at the predetermined spot is used when acquiring the main fluorescent image data ED1 at the predetermined spots Wa to Wc. If fluorescence image data acquired by scanning the microplate MP with the excitation light FL at a speed different from the scanning speed of the excitation light with respect to the microplate MP, the main fluorescence image data ED1 and the auxiliary fluorescence image data ED2 are acquired. In which the main fluorescent image data ED1 is acquired using the main fluorescent image data ED1 and the auxiliary fluorescent image data ED2 acquired from a plurality of predetermined spots Wa to Wc. The same type of fluorescently labeled sample is hybridized in order to calculate the time-varying characteristics unique to the labeled sample. It becomes unnecessary to calculate the temporal change characteristics for each of all spots can be efficiently calculated aging characteristics.

  Further, as shown in FIG. 8, the image acquisition means 51 irradiates the fluorescently labeled specimens hybridized in the plurality of test spots Wa to Wf with excitation light at different times for each test spot Wa to Wf. Thus, the fluorescence emitted from the fluorescently labeled specimens of the inspection spots Wa to Wf is detected to obtain the inspection fluorescent image data EXD for each of the inspection spots Wa to Wf. Excitation between the plurality of inspection fluorescent image data EXDa to EXDf for each inspection spot Wa to Wf acquired by the image acquisition means 51 and the inspection spots Wa to Wf when acquiring the inspection fluorescent image data EXD Based on the time interval of light irradiation, a change characteristic for acquiring a time-dependent change characteristic specific to the fluorescently labeled specimen existing in the inspection spots Wa to Wf. By having an acquisition unit 61, for calculating the temporal change characteristics to obtain a test fluorescence image data EXD with the acquisition of the main fluorescence image data ED1, can be calculated efficiently aging characteristics.

  Furthermore, as shown in FIG. 9, when the microplate MP has a plurality of spots arranged in a grid along the row and column directions, the image acquisition means 51 has the same number of specific binding substances. By moving the microplate MP in the row direction while scanning in the column direction with the excitation light FL, the fluorescently labeled samples respectively hybridized in the plurality of spots are emitted from the fluorescently labeled samples for each spot. A plurality of main fluorescence image data ED1 is acquired by detecting fluorescence, and the image correction means 60 is adapted to irradiate excitation light between the average value of the fluorescence image data ED1 for each of the spot rows W31 to W36 and between the spot rows. The main fluorescence image data ED1 can be obtained by providing a change characteristic acquisition means for acquiring a change characteristic with time based on the time interval of It is possible to calculate the temporal change characteristics have, it is possible to perform efficiently the fluorescence image data correction.

1 is an external view showing a preferred embodiment of an image correction apparatus of the present invention. The schematic diagram which shows an example of the microplate used when acquiring the fluorescence image data correct | amended in the image correction apparatus of this invention The block diagram which shows an example of the fluorescence detection apparatus for acquiring the fluorescence image data correct | amended in the image correction apparatus of this invention 1 is a block diagram showing a preferred embodiment of an image correction apparatus of the present invention. 4 is a schematic diagram illustrating an example of a scanning method of excitation light when the image acquisition unit acquires two pieces of fluorescence image data in the image correction apparatus of FIG. The flowchart which shows preferable embodiment of the image correction method of this invention The schematic diagram which shows another example of the scanning method of the excitation light when an image acquisition means acquires two fluorescence image data in the image correction apparatus of this invention. The schematic diagram which shows an example of the microplate at the time of acquiring the fluorescence image data used for the image acquisition means to acquire a time-dependent change characteristic in the image correction apparatus of the present invention. The schematic diagram which shows an example of the microplate at the time of acquiring the fluorescence image data used for the image acquisition means to acquire a time-dependent change characteristic in the image correction apparatus of the present invention.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Fluorescence detection system 10 Fluorescence detection apparatus 20 Excitation light irradiation means 21 Excitation light source 30 Fluorescence detection means 50 Image correction apparatus 51 Image acquisition means 60 Image correction means 61 Change characteristic acquisition means 62 Correction means ED1 Main fluorescence image data ED2 Auxiliary fluorescence image data EXD Fluorescence image data for inspection FL Excitation light MP Microplate W Spot

Claims (7)

Main fluorescence image data obtained by detecting fluorescence emitted from the fluorescently labeled specimen by irradiating the fluorescently labeled specimen hybridized with the specific binding substance present in the spot of the microplate with excitation light; and Irradiating excitation light to the fluorescently labeled specimen hybridized with the specific binding substance existing on the predetermined spot from which the main fluorescence image data was acquired separately from the acquisition of the main fluorescence image data Detecting fluorescence emitted from the fluorescently labeled specimen to obtain auxiliary fluorescence image data at the predetermined spot;
Based on the acquired main fluorescent image data and auxiliary fluorescent image data at the acquired predetermined spot, and the time interval of excitation light irradiation when acquiring the main fluorescent image data and the auxiliary fluorescent image data, the fluorescent label A fluorescence image correction method, comprising : calculating a temporal change characteristic when the intensity of the fluorescence emitted from the specimen decreases with time, and correcting the main fluorescent image data based on the temporal change characteristic. Present in microplates spot, by irradiating the excitation light to the specific binding substance and hybridized fluorescently labeled analyte, wherein the fluorescent labeled analytes main fluorescent image data that has detected the emitted fluorescence from the main By irradiating the fluorescence-labeled specimen hybridized with the specific binding substance existing on the predetermined spot from which the main fluorescence image data was acquired separately from the acquisition of the fluorescence image data, Image acquisition means for detecting fluorescence emitted from the labeled specimen and acquiring auxiliary fluorescence image data at the predetermined spot;
Image correction means for correcting the main fluorescence image data acquired by the image acquisition means,
The image correction means includes the main fluorescence image data and the auxiliary fluorescence image data at the predetermined spot acquired by the image acquisition means, and excitation light used when acquiring the main fluorescence image data and the auxiliary fluorescence image data. Based on the time interval of irradiation, a temporal change characteristic is calculated when the intensity of the fluorescence emitted from the fluorescently labeled specimen decreases with time, and the main fluorescent image data is corrected based on the temporal change characteristic A fluorescent image correction apparatus characterized by comprising: The predetermined spot is a plurality of spots;
The auxiliary fluorescent image data at the predetermined spot is acquired by scanning the excitation light in a direction different from the scanning direction of the excitation light with respect to the microplate when acquiring the main fluorescent image data at the predetermined spot. The fluorescence image correction apparatus according to claim 2 , wherein the fluorescence image data is obtained. The predetermined spot is a plurality of spots;
The auxiliary fluorescent image data at the predetermined spot is used to drive the microplate with the excitation light at a speed different from the scanning speed of the excitation light with respect to the microplate when acquiring the main fluorescent image data at the predetermined spot The fluorescence image correction apparatus according to claim 2 , wherein the fluorescence image data is acquired by scanning. A plurality of examinations in which the microplate emits fluorescence at the same fluorescence intensity, wherein there are a plurality of spots from which the main fluorescence image data is acquired and the fluorescently labeled specimen hybridized with the specific binding substance. Have a spot for
The image acquisition means acquires the main fluorescence image data and irradiates excitation light to the fluorescently labeled specimens that are respectively hybridized at the inspection spots, thereby obtaining fluorescence emitted from the fluorescently labeled specimens. Detect and acquire a plurality of fluorescent image data for examination,
Excitation light irradiation between the plurality of inspection fluorescent image data for each inspection spot acquired by the image acquisition unit and the inspection spot at the time of acquisition of the inspection fluorescent image data. 3. The fluorescence image according to claim 2, further comprising a change characteristic acquisition unit that acquires the time-dependent change characteristic unique to the fluorescently labeled specimen existing in the test spot based on the time interval of Correction device. The microplate has the plurality of spots arranged in a grid along the row direction and the column direction,
The image acquisition means moves the microplate in the row direction while scanning the microplate in the column direction with the excitation light with respect to the fluorescence-labeled specimens that are respectively hybridized in the spots. A plurality of the main fluorescence image data is obtained by detecting fluorescence emitted from each fluorescently labeled specimen,
The characteristic correction means for acquiring the time-varying characteristic based on the average value of the main fluorescence image data for each spot row and the time interval of excitation light irradiation between the spot rows. The fluorescence image correction apparatus according to claim 2, wherein On the computer,
Main fluorescence image data obtained by detecting fluorescence emitted from the fluorescently labeled specimen by irradiating the fluorescently labeled specimen hybridized with the specific binding substance present in the spot of the microplate with excitation light; and Irradiating excitation light to the fluorescently labeled specimen hybridized with the specific binding substance existing on the predetermined spot from which the main fluorescence image data was acquired separately from the acquisition of the main fluorescence image data Detecting fluorescence emitted from the fluorescently labeled specimen to obtain auxiliary fluorescence image data at the predetermined spot;
Based on the acquired main fluorescent image data and auxiliary fluorescent image data at the acquired predetermined spot, and the time interval of excitation light irradiation when acquiring the main fluorescent image data and the auxiliary fluorescent image data, the fluorescent label A fluorescence image correction program for calculating a temporal change characteristic when the intensity of the fluorescence emitted from the specimen decreases with time and correcting the main fluorescent image data based on the temporal change characteristic.

Images