JP2009068996A - Microarray device and microarray analysis method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a microarray device specifying and measuring the position of a spot, even when the position of the spot is deviated from a prescribed position on a microarray substrate. <P>SOLUTION: This device includes: an imaging part 14 for imaging the microarray substrate having a plurality of spots, and outputting digital image data; a spot information storage part 16 for storing information on the spots; a spot position analysis part 15 for calculating a brightness distribution curve of each spot from the digital image data, and analyzing each spot position; a spot position specification part 17 for specifying each spot position from a peak coordinate on the brightness distribution curve of each spot outputted from the spot position analysis part 15, and the information on the spots read from the spot information storage part; and a measuring part 18 for performing measurement on a spot position outputted from the spot position specification part 17. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a microarray apparatus for measuring a microarray substrate having a plurality of spots in which sample specimens are fixed in an array, and more specifically, qualitative and quantitative determination of biomolecules in a sample from the luminescence state of the microarray using a luminescence reaction. The present invention relates to a technique for accurately detecting the position of a spot unit when measuring a spot and preventing a spot measurement error.

  Conventional microarray substrates are used for analysis of biomolecules, and genes are mainly used widely as biomolecules. Hereinafter, when exemplified by genes, the expression of genes in individual cells, tissues or individuals as samples can be comprehensively analyzed by a microarray. As a conventional method for producing a microarray, a microscopic spot is formed on a glass substrate by forming droplets of a gene solution in nanoliter units on a 1 × 3 inch glass substrate. A method for immobilizing fragments has been disclosed (for example, see Patent Document 1).

  The microarray manufactured in this way generates images by labeling the genes on the microarray with a fluorescent dye, and then exciting the fluorescent dye with excitation light and capturing the generated fluorescence with a scanner or a CCD (Charge Coupled Device) camera. However, a microarray measuring apparatus that performs image analysis is disclosed (for example, see Patent Document 2).

In such a conventional microarray apparatus, in order to perform measurement in units of spots formed on the microarray, information stored as microarray analysis parameters (number of spots in the row and column directions, spot radius, position information, etc.) is used. A method has been proposed in which template information for a spot area is generated, the position of the spot area is corrected by image processing so that the spot position on the captured image matches the spot position on the template, and the spot on the template is measured. (For example, refer to Patent Document 3).
Japanese National Patent Publication No. 10-503841 JP 2002-181708 A JP 2005-172840 A

  However, in the above-described conventional configuration, if only a part of the spot on the microarray substrate is misaligned due to spotter dispersion or the like for spotting a droplet of the gene solution on the microarray substrate, the spot position on the template is deviated. However, there was a problem that the position-shifted spot could not be measured.

  An object of the present invention is to solve the above-described conventional problems, and to provide a microarray apparatus capable of specifying and measuring a spot position even when the spot is displaced from a predetermined position where the spot should be originally spotted. To do.

  In order to solve the conventional problems, the microarray apparatus of the present invention is a microarray apparatus for measuring a microarray substrate having a plurality of spots in which a sample specimen is fixed on the substrate. An imaging unit that outputs digital image data that digitally represents the luminance received by the pixels, a spot information storage unit that stores information about the spot, and a luminance distribution curve for each spot calculated from the digital image data A spot position is identified from a spot position analysis unit that analyzes the position of the spot, coordinates that serve as a peak of the luminance distribution curve for each spot output by the spot position analysis unit, and information about the spot read from the spot information storage unit Spot position specifying unit to perform and spot position specifying There are those characterized by comprising a measurement unit for performing measurements in the spot position to be output.

  Further, in the microarray apparatus, the spot position analysis unit performs spot search for a certain range from a predetermined spot position in a noise removal unit that removes a noise component from the digital image data and noise removal image data output by the noise removal unit. A luminance distribution analysis for calculating a luminance distribution curve in the X-axis direction and the Y-axis direction and detecting a peak coordinate of the luminance distribution curve in a predetermined range extraction unit that extracts as a region and a spot search region that is output by the predetermined range extraction unit A spot search area is extracted for each spot from the digital image data output from the imaging unit, and a luminance distribution curve is calculated for each of the X-axis direction and the Y-axis direction. .

  Furthermore, in the microarray apparatus, the spot position specifying unit determines whether the spot is a measurable spot by analyzing the arrangement state of the spot from the coordinates of the peak of the luminance distribution curve output from the spot position analyzing unit. And a spot position calculation unit for calculating the spot position from the spot arrangement information output from the spot position determination unit and the information regarding the spot read from the spot information storage unit, and a luminance distribution curve for each spot search area The positional relationship of the spot is determined from the coordinates of the peak, and the position of the spot is calculated according to the result.

  Furthermore, in the microarray apparatus, the spot position determination unit defines the coordinates of the peak of the luminance distribution curve output by the spot position analysis unit as the coordinates of the center point of the spot, and the spot adjacent to the center point of the spot. It is characterized by determining whether or not the spot is in a measurable position based on the distance from the center point.

  Furthermore, in the microarray apparatus, the spot position calculation unit calculates the spot position only when the spot is in a measurable position in the spot arrangement information output from the spot position determination unit. is there.

  Further, in the microarray analysis method of the present invention, an imaging step for imaging a microarray substrate having a plurality of spots on which a sample specimen is fixed, and a luminance distribution curve for each spot is calculated from the digital image data obtained in the imaging step. A spot position analyzing step for detecting coordinates that become peaks, a spot position determining step for determining a positional relationship of the spots from coordinates that become the peak of the luminance distribution curve for each spot obtained in the spot position analyzing step, It includes a measuring step for measuring the brightness of the spot determined in the spot position determining step.

  According to the microarray apparatus of the present invention, the spot position can be specified and measured even when the spot is displaced from a predetermined position where it should be spotted.

  Embodiments of the microarray device of the present invention will be described below in detail with reference to the drawings.

(Embodiment)
FIG. 1 shows a block diagram of a microarray apparatus according to an embodiment of the present invention. In FIG. 1, the microarray apparatus includes a stage 12 on which a microarray substrate 11 having a plurality of spots on which a sample specimen is fixed can be placed and moved, a stage control unit 13 for controlling the moving direction and amount of movement of the stage 12, and a microarray substrate. An image pickup unit 14 that picks up 11 images and outputs digital image data; a spot information storage unit 16 that stores information (spot arrangement position, spot diameter, etc.) on spots on the microarray substrate; The spot position is determined from the spot position analysis unit 15 that calculates the luminance distribution curve of the spot and analyzes the spot position, the spot position analysis result output from the spot position analysis unit 15, and the information related to the spot read from the spot information storage unit 16. Spot position specifying unit 17 and spot And measurement unit 18 for measuring the spot position output by the location identifying unit 17. Details of each component of the microarray device shown in FIG. 1 in the embodiment of the present invention will be described below.

  First, the stage 12 has a shape in which the microarray substrate 11 can be installed, and the stage control unit 13 outputs a control signal for moving the spot area on the microarray substrate 11 to the imaging field of view of the imaging unit 14.

  The imaging unit 14 images fluorescence or luminescence generated from a plurality of spots on the microarray substrate 11 and outputs digital image data. Imaging is performed by exposure, and the exposure time is controlled so that fluorescence or luminescence can be sufficiently observed. Here, the imaging unit 14 integrates the intensity of light received by each light receiving element (pixel), and outputs data of all the light receiving elements using the value as a luminance value. At this time, the luminance values output from all the light receiving elements are converted (quantized) into discrete digital data by analog-digital conversion. That is, the luminance values of all the light receiving elements of the imaging unit 14 are output as digital image data at every sampling time (ΔT) of analog-digital conversion. In the present invention, the imaging unit 14 is a monochrome CCD (Charge Coupled Device) camera or the like that operates as described above.

  As a microarray substrate 11 used for imaging, a configuration that can be exemplified in the embodiment of the present invention is shown in FIG. In FIG. 2, the microarray substrate 11 is a colorless and transparent glass substrate having an aspect ratio of 1: 3, and a plurality of spots 22 on which sample specimens are fixed in an array are arranged in the spot region 21. In the spot region 21, the arrangement of the spots 22 as illustrated in FIG. 3 is made by a spotter or the like. FIG. 3 (a) is an enlarged view of the spot region 21 of FIG. 2. In order to explain the embodiment of the present invention, the following description will be given by taking the case where the spots 22 are arranged in 6 rows and 6 columns as an example. Do. In FIG. 3A, a square area surrounding each spot 22 is a data search range for detecting a position in units of spots 22 from the digital image data output from the imaging unit 14, and is referred to as a spot search area 32. And The spot search area 32 is a square area including a predetermined position where the spot 22 should be originally arranged. When the spot 22 is in the predetermined position, the center point 37 coincides with the center point of the spot search area 32. The spot search area reference coordinates 35 and the spot search area width 36 which are the coordinates of the upper left corner of the spot search area 32 are defined. FIG. 3B is an enlarged view of any four adjacent spot search areas 32 in FIG. In FIG. 3B, the spot 22 is defined as a region in the range of the radius 33 of the spot 22 from the center point 37, and the distance between adjacent spots is defined by the distance 34 between the center points 37 of the spots 22. Note that the outer frame of the spot area 21 indicated by the solid line in FIG. 2 and the outer frame of the spot search area 32 indicated by the solid line in FIG. Then it is not necessary to make it visible.

  Information related to the spot 22 on the microarray substrate 11 is stored in the spot information storage unit 16, and in order to detect the position of the spot 22 from the digital image data output by the imaging unit 14, the spot position analysis unit 15. And used by the spot position specifying unit 17. In the embodiment of the present invention, the information included in the spot information storage unit 16 includes the arrangement information 31 of the spots 22 (the number of spots in the row direction and the column direction), the spot radius 33, and the distance 34 between the spots (the center of the spot 22). Distance between points 37), spot search area reference coordinates 35, and spot search area width 36.

  In the case of FIG. 3A, when the spots 22 are arranged in 6 rows and 6 columns in the spot area 21, information of 6 rows and 6 columns is stored in the spot arrangement information 31 of the spot information storage unit 16. To do. In addition, as shown in FIG. 3, when there is no space between the spot search areas 32 and the spot 22 and the spot search area 32 are defined so as to touch each other, the spot search area reference coordinates 35 are set for each spot search area 32. There is no need to specify one spot search area reference coordinate 35 within the spot area 21 (for example, the reference coordinates of the spot search area 32 at the upper left corner in the spot area 21). In the embodiment of the present invention, as shown in FIG. 3, the spot 22 and the spot search area 32 are defined so that there is no space between the spot search areas 32 and contact each other, and the spot search area width 36 has a spot radius 33. The following description will be made on the assumption that the length is four times as long. Note that the arrangement conditions of the spot 22 and the spot search area 32 as described above are an example for explaining the embodiment of the present invention, and can be changed as appropriate.

  The digital image data of fluorescence or light emission of the microarray substrate 11 imaged by the imaging unit 14 is sent to the spot position analysis unit 15. The spot position analysis unit 15 performs a spot search area as preprocessing for specifying the position of the spot 22 from the digital image data acquired by the imaging unit 14 based on the information stored in the spot information storage unit 16 described above. The luminance distribution curve is calculated every 32, and the coordinates that become the peak of the luminance distribution curve are detected. Data processing of the spot position analysis unit 15 will be described with reference to FIGS. FIG. 4 is a block diagram of the spot position analysis unit 15. The spot position analysis unit 15 includes a noise removal unit 41, a predetermined range extraction unit 42, and a luminance distribution analysis unit 43. Hereinafter, details of the processing of the noise removing unit 41, the predetermined range extracting unit 42, and the luminance distribution analyzing unit 43 will be described.

  First, the processing content of the noise removing unit 41 will be described with reference to FIG. As shown in FIG. 5A, the random image 52 is included in the digital image data 51 output by imaging the spot area 21 of the microarray substrate 11 by the imaging unit 14. When the irregular noise 52 is included in the digital image data 51, the irregular noise 52 is included in the data of the spot search area 32 that is input to the luminance distribution analysis unit 43 described later, and is calculated by the luminance distribution analysis unit 43. In the luminance distribution curve, a portion with a sharp change in luminance value appears, which may affect the subsequent processing. Therefore, the noise removal unit 41 needs to remove the irregular noise 52 in advance. In the embodiment of the present invention, the irregular noise 52 is removed from the digital image data 51 by applying a smoothing filter. As a representative smoothing filter, an average value filter that uses the average value of the target pixel and its surrounding eight pixels as the luminance value of the target pixel, or a median filter that uses the central value of the luminance values in the region as the luminance value of the target pixel. However, it is preferable to apply the median filter because the edge portion of the average value filter is easily blurred. When the median filter is applied to the digital image data 51 of FIG. 5A by the noise removal unit 41, noise-removed image data 53 from which the random noise 52 is removed as shown in FIG. 5B is output. The In addition, although two types of smoothing filters were illustrated here, it is applicable also to another smoothing filter.

  Next, the processing content of the predetermined range extraction unit 42 that extracts the spot search region 32 from the noise-removed image data 53 from which the irregular noise 52 has been removed by the noise removal unit 41 will be described with reference to FIG. The predetermined range extracting unit 42 uses the noise-removed image data 53 shown in FIG. 5 and the spot search area reference coordinates 35, the spot search area width 36, and the spot arrangement information 31 stored in the spot information storage unit 16 to determine the spot 22. A fixed range (spot search area 32) is extracted for each spot 22 from a predetermined position to be arranged. For example, as shown in FIG. 6, a plurality of spots 22 are arranged in the spot area 21, and the spot 61 in the m-th row and the n-th column in the spot area 21 is set as a target spot to be extracted, and a search area 62 for the spot 61. The reference coordinate 63 can be calculated by the following equation (1).

  Here, (Xb, Yb) is the spot search area reference coordinate 35 and W is the spot search area width 36. In the case of FIG. 6, since the extraction target spot 61 is m = 2 and n = 3, if (Xb, Yb) = (10, 20) and W = 50, the reference coordinates 63 (X, Y) = ( {10 + 50X (3-1)}, {20 + 50X (2-1)}) = (110, 70).

  Thus, since the search area reference coordinates 63 of the target spot can be specified by the above processing, the range of (spot search area width 36 × spot search area width 36) from the spot search area reference coordinates 63 is set to the spot search area 62 next. And the data in the range of the spot search area 62 is extracted from the noise-removed image data 53.

  Next, the luminance distribution analysis unit 43 calculates a luminance distribution curve in the X-axis direction and the Y-axis direction for every spot search region 32 for all the spot search regions 32 extracted by the predetermined range extraction unit 42, and the luminance distribution Coordinates that are peaks in the curve are detected. The processing of the luminance distribution analysis unit 43 will be described with reference to FIGS. FIG. 7 shows the spots 22 arranged in the spot search area 32, and the luminance distribution analysis unit 43 first scans on the scanning line (thick line arrow in FIG. 7) drawn in the X-axis direction of the spot search area 32. The luminance distribution curve is calculated. The luminance distribution analysis unit 43 sequentially calculates luminance distribution curves on 11 scanning lines whose Y coordinates are Y0, Y1,..., Y9, Y10 in FIG. Hereinafter, an example of the arrangement pattern of the spots 22 in the spot search area 32 will be described, and a luminance distribution curve in the X-axis direction calculated by the luminance distribution analysis unit 43 will be shown.

  First, as shown in FIG. 8A, FIG. 8B shows a luminance distribution curve in the case where the positional deviation of the spot 22 is small and the center point 37 of the spot 22 is in the spot search area 32. FIG. 8B is a luminance distribution curve on the scanning line drawn in the X-axis direction, 82 is Y coordinate Y6, 83 is Y coordinate Y5 and Y7, 84 is Y coordinate Y4 and Y8, 85 is Y The luminance distribution curves on the scanning line when the coordinates are other than the above are shown. In FIG. 8B, the vertical axis represents the luminance value, the horizontal axis represents the X axis coordinate, the left end coordinate is X0, and the right end coordinate is X10.

  Thus, since the random noise 52 is removed by the noise removing unit 41 in the luminance distribution curve in the X-axis direction, there is no sharp change in the luminance value, and the luminance distribution curve is as shown in FIG. The peak of the luminance distribution curve appears only at the center point 37 of the spot 22.

  Next, as shown in FIG. 9A, a luminance distribution curve in the case where the positional deviation of the spot 22 is large and the center point 37 of the spot 22 is outside the spot search area 32 is shown in FIG. 9B. FIG. 9B is a luminance distribution curve on the scanning line drawn in the X-axis direction. 91 is Y coordinate Y6, 92 is Y coordinate Y5 and Y7, 93 is Y coordinate Y4 and Y8, 94 is Y The luminance distribution curves on the scanning line when the coordinates are other than the above are shown. In FIG. 9B, the vertical axis represents the luminance value, the horizontal axis represents the X axis coordinate, the left end coordinate is X0, and the right end coordinate is X10. As shown in FIG. 9A, since the X coordinate of the center point 37 of the spot 22 is outside the X coordinates (X0 to X10) of the spot search area 32, the luminance distribution curve in the X-axis direction of FIG. No peak appears in the luminance distribution curve, and the luminance value increases as the position is shifted (closer to X0).

  Next, the luminance distribution curve when the spots 22 are collectively misaligned in the spot region 21 instead of when the spot 22 alone is misaligned as shown in FIGS. 8 and 9 will be described. FIG. 10 shows a spot search region 32 when the entire spot 22 belonging to the row 101 is shifted to the left side in the spot region 21. The spot search region 32 of the shifted row 101 includes a plurality of spots 22. It is included. In the example of FIG. 10, the spot 22 in the row 101 is entirely shifted to the left side, so that two spots 22 are in the leftmost spot search area 102 and one spot in the rightmost spot search area 103. Contains spots.

  FIG. 11 shows the spot search area 32 when the entire spot area 21 is shifted to the upper left. If the entire spot area 21 is misaligned, a plurality of spots 22 may be included in many spot search areas 32. In the example of FIG. 11, since the entire spot area 21 is shifted to the upper left side, the number of spots 22 in the spot search area 32 may be four, two, or one. It can be seen that in the spot search areas (111, 112, 113, 114) of FIG. Hereinafter, the spot search areas (102, 103) when the entire row 101 of the spot area 21 is displaced as shown in FIG. 10 and the spot search areas (111, 111) when the entire spot area 21 is displaced as shown in FIG. 112, 113, 114) are shown.

  FIG. 12A is an enlarged view of the spot search area 102 at the left end of the row 101 that is displaced in FIG. FIG. 12B is a luminance distribution curve on the scanning line drawn in the X-axis direction, 121 is Y coordinate Y5, 122 is Y coordinate Y4 and Y6, 123 is Y coordinate Y3 and Y7, 124 is Y The luminance distribution curves on the scanning line when the coordinates are other than the above are shown. In FIG. 12B, the vertical axis represents the luminance value, the horizontal axis represents the X axis coordinate, the left end coordinate is X0, and the right end coordinate is X10. As shown in FIG. 12A, since the spots 22 are arranged at both ends of the spot search area 102, two peaks appear at both ends in the luminance distribution curve in the X-axis direction. The left spot 22 has a center point 37 outside the spot search area 102, so no peak appears in the brightness distribution curve. However, the right spot 22 has a center point 37 in the spot search area 102, so Shows a peak.

  Next, FIG. 13A is an enlarged view of the spot search area 103 at the right end of the row 101 that is displaced in FIG. FIG. 13B is a luminance distribution curve on the scanning line drawn in the X-axis direction. 131 is Y coordinate Y5, 132 is Y coordinate Y4 and Y6, 133 is Y coordinate Y3 and Y7, 134 is Y The luminance distribution curves on the scanning line when the coordinates are other than the above are shown. In FIG. 13B, the vertical axis represents the luminance value, the horizontal axis represents the X axis coordinate, the left end coordinate is X0, and the right end coordinate is X10. FIG. 13 shows the spot search area 103 when the entire row is misaligned. However, only one peak appears in the luminance distribution curve in the X-axis direction, and the position misalignment of the spot 22 alone shown in FIG. The luminance distribution curve is the same as in the case.

  Next, FIG. 14A is an enlarged view of the spot search area 111 at the upper left corner when the entire spot area 21 is displaced in FIG. FIG. 14B is a luminance distribution curve on the scanning line drawn in the X-axis direction. 141 is Y coordinate Y9, 142 is Y coordinate Y0 and Y8 and Y10, 143 is Y coordinate Y1 and Y7, 144. Indicates the luminance distribution curve on the scanning line when the Y coordinate is other than the above. In FIG. 14B, the vertical axis represents the luminance value, the horizontal axis represents the X axis coordinate, the left end coordinate is X0, and the right end coordinate is X10. As shown in FIG. 14A, spots 22 are arranged at the four corners of the spot search area 111, and the spots 22 overlap in the X-axis direction and the Y-axis direction, respectively. Two peaks appear in the luminance distribution curve in the X-axis direction. Since the lower right spot 22 of the spot search area 111 includes the center point 37, a peak appears in the luminance distribution curve in the X-axis direction.

  Next, FIG. 15A is an enlarged view of the spot search area 112 at the upper right corner when the entire spot area 21 is displaced in FIG. FIG. 15B is a luminance distribution curve on the scanning line drawn in the X-axis direction, 151 is Y coordinate Y9, 152 is Y coordinate Y0 and Y8 and Y10, 153 is Y coordinate Y1 and Y7, 154. Indicates the luminance distribution curve on the scanning line when the Y coordinate is other than the above. In FIG. 15B, the vertical axis represents the luminance value, the horizontal axis represents the X axis coordinate, the left end coordinate is X0, and the right end coordinate is X10. As shown in FIG. 15A, since the spots 22 are arranged at the upper left and lower left of the spot search area 112, only one mountain appears in the luminance distribution curve in the X-axis direction. This is because there is no positional deviation in the X-axis direction between the two spots 22 in the spot search area 112.

  Next, FIG. 16A is an enlarged view of the spot search area 113 at the lower left corner when the entire spot area 21 is displaced in FIG. FIG. 16B is a luminance distribution curve on the scanning line drawn in the X-axis direction. 161 is the Y coordinate Y0, 162 is the Y coordinate Y1, and 163 is the luminance on the scanning line when the Y coordinate is other than the above. Each distribution curve is shown. In FIG. 16B, the vertical axis represents the luminance value, the horizontal axis represents the X axis coordinate, the left end coordinate is X0, and the right end coordinate is X10. As shown in FIG. 16A, since the spots 22 are arranged on the upper left and upper right of the spot search area 113, two peaks appear in the luminance distribution curve in the X-axis direction. Since the X coordinate of the center point 37 of the upper left spot 22 in the spot search area 113 is not included in the spot search area 113, no peak appears in the luminance distribution curve, but the upper right spot 22 has the X coordinate of the center point 37. Since it is included in the spot search area 113, a peak appears in the luminance distribution curve.

  Next, FIG. 17A is an enlarged view of the spot search area 114 at the lower right corner when the entire spot area 21 is displaced in FIG. FIG. 17B is a luminance distribution curve on the scanning line drawn in the X-axis direction. 171 is the Y coordinate Y0, 172 is the Y coordinate Y1, and 173 is the luminance on the scanning line when the Y coordinate is other than the above. Each distribution curve is shown. In FIG. 17B, the vertical axis represents the luminance value, the horizontal axis represents the X axis coordinate, the left end coordinate is X0, and the right end coordinate is X10. FIG. 17 shows the spot search area 114 when the entire spot area 21 is misaligned. As in the case of the position misalignment of the spot 22 shown in FIG. 9, the peak is 1 in the luminance distribution curve in the X-axis direction. Become one.

  In this example, the number of scanning lines in the X-axis direction when calculating the luminance distribution curve is exemplified as 11. However, when the number of scanning lines is increased, the accuracy of the calculated luminance distribution curve increases, and spot search is performed. Since it becomes easy to grasp the luminance distribution of the entire region 32, it is better that the number of scanning lines is larger. In addition, when it can be predicted that there is little spotter variation and the amount of positional deviation of the spot is small in advance, the luminance distribution analysis unit 43 is reduced by reducing the search region width 36 of the spot search region 32 extracted by the predetermined range extraction unit 42. It is possible to reduce the amount of data to be processed and improve the processing speed. The luminance distribution analysis unit 43 can calculate a luminance distribution curve in the X-axis direction for each spot search region 32 by performing the processing as described above.

  Next, the luminance distribution analysis unit 43 adds the calculated luminance distribution curve in the X-axis direction. The scanning line for calculating the luminance distribution curve does not necessarily pass on the spot 22. Since the luminance distribution curve on the scanning line that does not pass on the spot 22 does not have a peak or inclination, the coordinates at which the luminance distribution curve reaches the peak may not be detected using only the luminance distribution curve on one scanning line. In the embodiment of the present invention, since the luminance distribution analysis unit 43 selects a scanning line so that the luminance distribution of the entire spot search region 32 can be grasped, the luminance distribution curve calculated by the luminance distribution analysis unit 43 is used. Always includes a plurality of luminance distribution curves on the scanning line passing on the spot 22, and by adding the luminance distribution curves, the luminance distribution curve in which the peak of the luminance distribution curve is emphasized can be extracted. The coordinate detection becomes easier.

  Next, in the added luminance distribution curve in the X-axis direction, a coordinate that becomes a peak is detected. Depending on how the spot 22 is misaligned, in the spot search area 32, the coordinates of the peak of the luminance distribution curve may not be detected. In this case, in the spot search area 32, the coordinate with the maximum luminance value is detected in the inclination of the luminance distribution curve. Next, the luminance distribution curve in the Y-axis direction is calculated for the X coordinate that is the peak of the luminance distribution curve in the X-axis direction or the X coordinate that has the maximum luminance value in the inclination. Regarding the calculation of the luminance distribution curve in the Y-axis direction, a plurality of luminance distribution curves are not calculated as in the case of the luminance distribution curve in the X-axis direction. This is because, in the luminance distribution curve in the X-axis direction, the coordinates at which the peak is already reached or the coordinates at which the luminance value is maximum at the inclination are already known. Next, in the luminance distribution curve in the Y-axis direction, coordinates that have a peak are detected as in the luminance distribution curve in the X-axis direction. Here, in the case where a peak coordinate cannot be detected in the luminance distribution curve in the X-axis direction or the Y-axis direction, the positional deviation direction of the spot 22 is estimated from the inclination of the luminance distribution curve, and is adjacent to the positional deviation direction. The peak detection is performed again for the spot search area 32.

  Hereinafter, addition of the luminance distribution curve and peak detection in the luminance distribution analysis unit 43 will be described with reference to FIGS.

  First, as shown in FIG. 8A, when the center point 37 of the spot 22 is in the spot search area 32, the luminance distribution curve in the X-axis direction is 82, 83, 84, 85 in FIG. It is the curve shown by. When these luminance distribution curves in the X-axis direction are added, a luminance distribution curve 181 is obtained. In FIG. 18A, the X coordinate that is the peak of the luminance distribution curve 181 is X4, and the X coordinate of the center point 37 of the spot 22 is determined to be X4. Next, a luminance distribution curve in the Y-axis direction at the X coordinate where the luminance distribution curve in the X-axis direction reaches a peak is calculated. In FIG. 18A, since the peak X coordinate is X4, when the luminance distribution curve in the Y-axis direction on X coordinate X4 in FIG. 8A is calculated, 182 in FIG. It becomes like this. In the luminance distribution curve 182, the peak Y coordinate is Y6. Here, as shown in FIG. 8A, when the center point 37 of the spot 22 is on the scanning line, the coordinates of the peak of the luminance distribution curve coincide with the coordinates of the center point 37 of the spot 22, but the spot is not necessarily spotted. The 22 center points 37 are not necessarily on the scanning line. Therefore, when the number of scanning lines is small, in the luminance distribution curve calculated by the luminance distribution analysis unit 43, an approximate curve is calculated using the local maximum point and surrounding points, and the peak coordinates of the approximate curve are calculated. Thus, the peak coordinates of the luminance distribution curve may be calculated.

  Next, as shown in FIG. 9A, when the center point 37 of the spot 22 is outside the spot search region 32, the luminance distribution curves in the X-axis direction are 91, 92, 93, 94, a luminance distribution curve 191 is obtained by adding these. In the added luminance distribution curve 191 in the X-axis direction, as shown in FIG. 19A, the coordinates that become the peak of the luminance distribution curve cannot be detected. As described above, when the coordinate that becomes the peak of the luminance distribution curve cannot be detected, the coordinate having the maximum luminance value in the inclination of the luminance distribution curve is detected, and the positional deviation direction of the spot 22 is determined. In the case of FIG. 19A, in the added luminance distribution curve 191, the X coordinate at which the luminance value is maximum is X0, and it can be determined that the spot 22 is displaced in the decreasing direction of the X axis. Next, a luminance distribution curve in the Y-axis direction at the X coordinate at which the luminance value becomes maximum at the inclination of the luminance distribution curve in the X-axis direction is calculated. In FIG. 19A, the X coordinate at which the luminance value becomes maximum at the inclination of the luminance distribution curve is X0. Therefore, in FIG. 9A, when the luminance distribution curve in the Y-axis direction on the X coordinate X0 is calculated. 192 of FIG. 19B. In the luminance distribution curve 192, the peak Y coordinate is Y6. As described above, in the X-axis direction or Y-axis direction luminance distribution curve, when the peak coordinates cannot be detected, the peak coordinates are detected for the spot search region 32 adjacent to the spot 22 in the position shift direction. As shown in FIG. 19A, when the peak in the luminance distribution curve in the Y-axis direction can be detected and the peak coordinate in the luminance distribution curve in the X-axis direction cannot be detected, the spot search area 32 adjacent in the decreasing direction of the X-axis. , The luminance distribution curve on the Y coordinate that is the peak in the luminance distribution curve in the Y-axis direction is calculated, and the peak coordinate is detected.

  Taking the case of FIG. 9A as an example, peak detection of the luminance distribution curve in the adjacent spot search region 32 will be described with reference to FIG. FIG. 20A shows the situation of the spot search area 32 on the left side (X axis decreasing direction) of FIG. In FIG. 20A, the X coordinate of the search area 32 of the adjacent spot 22 is represented by (X0 ', X1', ..., X9 ', X10'). Since the spot search area 32 of the detection target spot 201 and the adjacent spot 22 is in contact with each other, X10 'and X0 have the same coordinates, and are indicated as X0 in the drawing.

  In the case of FIG. 9A, the Y coordinate that is the peak of the luminance distribution curve in the Y-axis direction is Y6. Therefore, when the luminance distribution curve in the X-axis direction on Y6 is calculated, as indicated by 203 in FIG. 20B. become. In this example, the detection target spot 201 and the adjacent spot 22 emit the same light.

  Next, in the luminance distribution curve 203 of FIG. 20B, two X coordinates (X5 ′, X9 ′) that are the peaks of the luminance distribution can be detected in the range of the adjacent spot 22 (X0 ′ to X10 ′). Therefore, the peak coordinate in the luminance distribution curve in the X-axis direction of the detection target spot 201 is determined as X9 ′ close to the search area 32 of the detection target spot 201. Here, the case where the peak cannot be detected for the luminance distribution curve in the X-axis direction in the spot search region 32 due to the positional deviation of the spot 22 is illustrated, but the peak cannot be detected also for the luminance distribution curve in the Y-axis direction. By processing in the same way, it is possible to detect the peak of the luminance distribution in the Y-axis direction.

  As in the embodiment of the present invention, when there is no space between the spot search areas 32 and the spot search areas 32 are defined so as to be in contact with each other, even if the coordinates at the peak of the luminance distribution curve cannot be detected, the above As described above, the peak position can be detected by expanding the peak search range of the luminance distribution to the search area 32 of the adjacent spot 22. However, if the amount of positional deviation of the detection target spot 201 is large and the distance between the center point 202 of the detection target spot 201 and the center point 37 of the adjacent spot 22 becomes short, the luminance distributions interfere with each other, and the peak position of the luminance distribution is There is a possibility that it cannot be separated. In such a case, the spot position specifying unit 17 described later determines that the spot is not a measurement target.

  Next, the luminance distribution curve when the entire row 101 of the spot area 21 is displaced as shown in FIG. 10 (FIGS. 12 and 13) and the luminance distribution curve when the entire spot area 21 is displaced as shown in FIG. The addition of the luminance distribution curve and the peak detection in the luminance distribution analysis unit 43 in (FIGS. 14, 15, 16, and 17) will be described.

  First, as shown in FIG. 12A, the processing of the luminance distribution analysis unit 43 in the spot search region 102 at the left end of the misaligned row 101 will be described with reference to FIG. In the case of FIG. 12A, the luminance distribution curves in the X-axis direction are curves indicated by 121, 122, 123, and 124 in FIG. 21A, and when these are added, a luminance distribution curve 211 is obtained. In the added luminance distribution curve 211 in the X-axis direction, the peak X coordinate X9.5 (an intermediate point between X9 and X10) can be detected, but the inclination of the luminance distribution is also seen on the left side. As described above, in the luminance distribution curve in the X-axis direction, when the slope of the luminance distribution is different from that of one peak, the spot search area 102 has two positions shifted in the X-axis direction. It can be determined that there is a spot 22. In the gradient of the luminance distribution on the left side of the added luminance distribution curve 211, the X coordinate at which the luminance value is maximum is X0, and it can be determined that the spot 22 is displaced in the decreasing direction of the X axis.

  Next, a luminance distribution curve in the Y-axis direction is calculated at the X coordinate that is the peak of the luminance distribution curve in the X-axis direction and the X coordinate that has the maximum luminance value in the inclination. In FIG. 21A, the X coordinate that is the peak of the luminance distribution curve is X9.5, and the X coordinate that maximizes the luminance value in the inclination is X0. Therefore, in FIG. When the luminance distribution curve in the Y-axis direction on X9.5 is calculated, both are as indicated by 212 in FIG. In the luminance distribution curve 212, the peak Y coordinate is Y5. Here, with respect to the slope on the left side in the luminance distribution curve 211 in the X-axis direction, the peak coordinates could not be detected, so the spot search region 32 adjacent in the X-axis decreasing direction is Detect peak coordinates. As shown in FIG. 21A, when the peak in the luminance distribution curve in the Y-axis direction can be detected and the peak coordinate in the luminance distribution curve in the X-axis direction cannot be detected, the spot search area 32 adjacent in the decreasing direction of the X-axis. , The luminance distribution curve on the Y coordinate that is the peak in the luminance distribution curve in the Y-axis direction is calculated, and the peak coordinate is detected.

  Next, as shown in FIG. 13A, processing of the luminance distribution analysis unit 43 in the spot search region 103 at the right end of the misaligned row 101 will be described with reference to FIG. In the case of FIG. 13A, the luminance distribution curves in the X-axis direction are curves indicated by 131, 132, 133, 134 in FIG. 22A, and when these are added, a luminance distribution curve 221 is obtained. In the added luminance distribution curve 221 in the X-axis direction, as shown in FIG. Thus, in the luminance distribution curve in the X-axis direction, when the inclination of the luminance distribution is detected on the left side, it can be determined that one spot 22 is arranged in the spot search area 103. In the luminance distribution curve in the X-axis direction, it can be determined that the X coordinate at which the luminance value is maximum is X0, and the spot 22 is displaced in the X-axis decreasing direction. Next, a luminance distribution curve in the Y-axis direction at the X coordinate at which the luminance value becomes maximum at the inclination of the luminance distribution curve in the X-axis direction is calculated. In FIG. 22A, the X coordinate at which the luminance value becomes maximum at the inclination of the luminance distribution curve is X0. Therefore, in FIG. 13A, when the luminance distribution curve in the Y-axis direction on the X coordinate X0 is calculated. 22 in FIG. 22B. In the luminance distribution curve 222, the peak Y coordinate is Y5. Here, in the luminance distribution curve in the X-axis direction, since the coordinate that becomes the peak could not be detected, the peak coordinate in the luminance distribution curve in the X-axis direction is detected for the spot search region 32 adjacent in the decreasing direction of the X-axis. As shown in FIG. 22A, when the peak in the luminance distribution curve in the Y-axis direction can be detected and the peak coordinate in the luminance distribution curve in the X-axis direction cannot be detected, the spot search region 32 adjacent in the decreasing direction of the X-axis. , The luminance distribution curve on the Y coordinate that is the peak in the luminance distribution curve in the Y-axis direction is calculated, and the peak coordinate is detected.

  Next, as shown in FIG. 14A, the luminance distribution curve addition and peak detection in the luminance distribution analysis unit 43 of the spot search region 111 in the upper left corner when the entire spot region 21 is displaced are shown in FIG. Will be described. In the case of FIG. 14A, the X-axis direction luminance distribution curves output by the luminance distribution calculation unit 15 are curves indicated by 141, 142, 143, and 144 in FIG. The distribution curve 231 is obtained. In the added luminance distribution curve 231 in the X-axis direction, a peak X coordinate X9.5 (an intermediate point between X9 and X10) can be detected, but a slope of the luminance distribution is also seen on the left side. As described above, in the luminance distribution curve in the X-axis direction, when the inclination of the luminance distribution is different from one peak coordinate, two spot search areas 111 are located at positions shifted in the X-axis direction. It can be determined that there is a spot 22. In the gradient of the luminance distribution detected by the added luminance distribution curve 231, the coordinate at which the luminance value becomes maximum is X0, and it can be determined that the spot 22 is displaced in the decreasing direction of the X axis.

  Next, a luminance distribution curve in the Y-axis direction is calculated at the X coordinate that is the peak of the luminance distribution curve in the X-axis direction and the X coordinate that has the maximum luminance value in the inclination. In FIG. 23 (a), the X coordinate that is the peak of the luminance distribution curve is X9.5, and the X coordinate that maximizes the luminance value in the inclination is X0. Therefore, in FIG. 14 (a), the X coordinate is on X0. When the luminance distribution curve in the Y-axis direction on X9.5 is calculated, both are as indicated by 232 in FIG. In the luminance distribution curve 232, the peak Y coordinate Y9 can be detected, but the inclination of the luminance distribution is also seen on the left side. As described above, in the luminance distribution curve in the Y-axis direction, when the inclination of the luminance distribution is different from that at one peak coordinate, the spot search area 111 has 2 in the position shifted in the Y-axis direction. It can be determined that there are two spots 22. In the inclination of the luminance distribution curve detected by the luminance distribution curve 232, the coordinate at which the luminance value becomes maximum is Y0, and it can be determined that the spot 22 is misaligned in the decreasing direction of the Y axis. As shown in FIG. 23, when two peaks and inclinations are detected in the luminance distribution curves in the X-axis direction and the Y-axis direction, it can be determined that there are one spot 22 at each of the four corners of the spot search area 111. . Since it can be determined that the spot 22 in the upper left corner is displaced in the decreasing direction of the X axis and the Y axis, the peak coordinates in the luminance distribution curve are detected for the spot search region 32 adjacent in the diagonally upper left direction. Similarly, for the spot 22 at the upper right corner and the lower left corner, the peak coordinates in the luminance distribution curve are detected for the adjacent spot search areas 32.

  Next, as shown in FIG. 15A, the processing of the luminance distribution analysis unit 43 in the spot search area 112 in the upper right corner when the entire spot area 21 is displaced will be described with reference to FIG. In the case of FIG. 15A, the luminance distribution curves in the X-axis direction output from the luminance distribution calculation unit 15 are curves indicated by 151, 152, 153, and 154 in FIG. A luminance distribution curve 241 is obtained. In the added luminance distribution curve 241 in the X-axis direction, as shown in FIG. 24A, the coordinates that are the peak of the luminance distribution curve cannot be detected. In the gradient of the luminance distribution on the left side of the added luminance distribution curve 241, it can be determined that the X coordinate at which the luminance value is maximum is X0, and the spot 22 is displaced in the decreasing direction of the X axis. Next, a luminance distribution curve in the Y-axis direction at the X coordinate at which the luminance value becomes maximum at the inclination of the luminance distribution curve in the X-axis direction is calculated. In FIG. 24A, since the X coordinate at which the luminance value becomes maximum at the inclination of the luminance distribution curve is X0, in FIG. 15A, the luminance distribution curve in the Y-axis direction on the X coordinate on X0 is calculated. Then, it becomes like 242 in FIG. In the luminance distribution curve 232, the peak Y coordinate Y9 can be detected, but the inclination of the luminance distribution is also seen on the left side. As described above, in the luminance distribution curve in the Y-axis direction, when the inclination of the luminance distribution is different from one peak, the spot search area 112 has two positions shifted in the Y-axis direction. It can be determined that there are spots 22 and these spots 22 overlap in the X-axis direction. In FIG. 24, it can be determined that there are one spot 22 at each of the upper left corner and the lower left corner of the spot search area 112. Since it can be determined that the spot 22 in the upper left corner is displaced in the decreasing direction of the X axis and the Y axis, the peak coordinates in the luminance distribution curve are detected for the spot search region 32 adjacent in the diagonally upper left direction. Similarly, for the spot 22 in the lower left corner, the peak coordinates in the luminance distribution curve are detected for the adjacent spot search area 32.

  Next, as shown in FIG. 16A, the processing of the luminance distribution analysis unit 43 in the spot search area 113 in the lower left corner when the entire spot area 21 is displaced will be described with reference to FIG. In the case of FIG. 16A, the luminance distribution curves in the X-axis direction are curves indicated by 161, 162, and 163 in FIG. 25A, and when these are added, a luminance distribution curve 251 is obtained. In the added luminance distribution curve 251 in the X-axis direction, the peak X coordinate X9.5 (an intermediate point between X9 and X10) can be detected, but the inclination of the luminance distribution is also seen on the left side. As described above, in the luminance distribution curve in the X-axis direction, when the inclination of the luminance distribution is different from one peak, the spot search area 113 has two positions shifted in the X-axis direction. It can be determined that there is a spot 22. In the gradient of the luminance distribution on the left side of the added luminance distribution curve 251, it can be determined that the X coordinate at which the luminance value is maximum is X0, and the spot 22 is displaced in the decreasing direction of the X axis. Next, a luminance distribution curve in the Y-axis direction is calculated at the X coordinate that is the peak of the luminance distribution curve in the X-axis direction and the X coordinate that has the maximum luminance value in the inclination.

  In FIG. 25A, the X coordinate that is the peak of the luminance distribution curve is X9.5, and the X coordinate that maximizes the luminance value in the inclination is X0. Therefore, in FIG. When the luminance distribution curve in the Y-axis direction on X9.5 is calculated, both are as indicated by 252 in FIG. In the luminance distribution curve 242, the coordinates at the peak cannot be detected, but the inclination of the luminance distribution is observed. As described above, when the slope of the luminance distribution is detected in the luminance distribution curve in the Y-axis direction, the spot search area 113 has two spots 22 at positions shifted in the X-axis direction. Can be determined to overlap in the Y-axis direction. In FIG. 25, it can be determined that there are one spot 22 at each of the upper left corner and the upper right corner of the spot search area 32. Since it can be determined that the spot 22 in the upper left corner is displaced in the decreasing direction of the X axis and the Y axis, the peak coordinates in the luminance distribution curve are detected for the spot search region 32 adjacent in the diagonally upper left direction. Similarly, for the spot 22 in the upper right corner, the peak coordinates in the luminance distribution curve are detected for the adjacent spot search area 32.

  Next, as shown in FIG. 17A, the processing of the luminance distribution analysis unit 43 in the spot search area 114 in the lower right corner when the entire spot area 21 is displaced will be described with reference to FIG. In the case of FIG. 17A, the luminance distribution curves in the X-axis direction output from the luminance distribution calculation unit 15 are curves indicated by 171, 172, and 173 in FIG. A curve 251 is obtained. In the added luminance distribution curve 261 in the X-axis direction, as shown in FIG. 26A, the coordinates that are the peak of the luminance distribution curve cannot be detected. In the inclination of the luminance distribution of the added luminance distribution curve 261, it can be determined that the X coordinate at which the luminance value is maximum is X0, and the spot 22 is displaced in the decreasing direction of the X axis. Next, a luminance distribution curve in the Y-axis direction at the X coordinate at which the luminance value becomes maximum at the inclination of the luminance distribution curve in the X-axis direction is calculated. In FIG. 26 (a), the X coordinate at which the luminance value becomes maximum at the inclination of the luminance distribution curve is X0. Therefore, in FIG. 17 (a), when calculating the luminance distribution curve in the Y-axis direction on the X coordinate X0. Both of them are as indicated by 262 in FIG. In the luminance distribution curve 262, the peak coordinates cannot be detected, but the inclination of the luminance distribution is observed. As described above, when the inclination of the luminance distribution is detected in the luminance distribution curve in the Y-axis direction, it can be determined that the spot 22 in the spot search region 114 is displaced in the decreasing direction of the Y-axis. In FIG. 26, since it can be determined that the spot 22 is displaced in the decreasing direction of the X axis and the Y axis, the peak coordinates in the luminance distribution curve are detected for the spot search region 32 adjacent in the diagonally upper left direction.

  Next, the spot position specifying unit 17 first defines the coordinates of the peak of the luminance distribution curve output from the spot position analyzing unit 15 as the center point 37 of the spot 22, and determines the distance between the center points 37 of the adjacent spots 22. By calculating, it is determined whether or not the spot 22 is in a measurable position. Thereafter, the position of each spot 22 is specified by referring to the center point 37 of each spot 22 and the information in the spot information storage unit 16. The spot position specifying unit 17 will be described with reference to FIGS. FIG. 28 is a block diagram of the spot position specifying unit 17, and the spot position specifying unit 17 includes a spot position determining unit 271 and a spot position calculating unit 272. Details of each component of the spot position specifying unit 17 will be described.

  First, the spot position determination unit 271 defines the peak coordinates of the luminance distribution curve in the X-axis direction and the Y-axis direction in the spot search region 32 output from the spot position analysis unit 15 as the center point 37 of the spot 22. Whether the spot is measurable from the distance between the center points 37 is determined.

  This is because when the spot 201 is displaced from the predetermined position 81 as shown in FIG. 20A, the detection target spot 201 calculated by the spot position analysis unit 15 and the adjacent spot 22 on the left side in the X-axis direction. The luminance distribution curve is like the curve shown in FIG. 20B, and the luminance distribution curves are in the form of interference with each other. Therefore, not only the misaligned spot 201 but also the measurement result of the spot 22 at a predetermined position adjacent to the misaligned spot is affected. Therefore, the spot position determination unit 271 grasps the positional relationship between the spot 22 and the adjacent spot 22 and determines whether the measurement result has an influence of light emission from the adjacent spot 22.

  Next, the determination process of whether or not the spot 22 is a measurable position will be described with reference to FIG. It is determined whether or not the adjacent spot 22 is affected by the distance between the center point 37 of the spot 22 to be determined and the center point 37 of the adjacent spot 22. FIG. 28A shows a state of a spot 281 shifted from a predetermined position 81 to be originally arranged and eight spots 282 adjacent thereto. First, the distance between the coordinates of the center point 283 of the spot 22 output from the spot position analysis unit 15 and the center point 37 of the adjacent spot 282 is calculated. If the coordinates of the center point 283 of the target spot 281 are (Xm, Ym) and the coordinates of the center point 37 of the adjacent spot 282 are (Xn, Yn), the distance d between the center points can be calculated by the following equation (2).

  As in the embodiment of the present invention, when there is no space between the spot search areas 32 and the spot search areas 32 are defined so as to touch each other, and the spot search area width 36 is four times the spot radius 33, When the spot radius 33 is r, the spot search area width 36 is 4r. Here, when each spot 22 is at a predetermined position, the distance d between the center points of the spot 22 is 4r for a spot in the vertical and horizontal directions and 4√2r for a spot in the oblique direction, as shown in FIG. When the position is shifted from the predetermined position 81, the distance between the center points 37 of the spot 22 changes from 4r and 4√2r.

  For example, as shown in FIG. 28 (b), when the spot 281 is displaced from the predetermined position 81 by the length r of the spot radius 33 in the decreasing direction of the X axis, the positional relationship comes into contact with the adjacent spot 282. The distance between the center points is 2r. At this time, the mutual luminance distributions interfere with each other in contact with each other, and even if each spot is measured as it is, the influence of each spot is included in the result. Therefore, when the distance d between the center points of the spots 22 is 2r or less, that is, when the distance between the center points is closer than the position where the spots 22 are in contact, it is determined as a spot that is not a measurement target. Judge as. Thus, by grasping the positional relationship of the spots 22 based on the distance between the center points 37 of the spots 22, it is possible to determine whether or not the spot 22 is a measurement target, eliminating the need to measure a spot 22 that is not a measurement target. To do.

  Next, the spot position calculation unit 272 reads the coordinates of the center point 37 of the spot 22 and the spot information storage unit 16 only when the spot position determination result in the spot position determination unit 271 determines that the spot 22 is measurable. The position of the spot 22 is calculated from the information regarding the read spot. The process of the spot position calculation part 272 is demonstrated using FIG. First, referring to the spot position determination result, it is checked whether or not the spot 22 can be measured. In the case of the measurable spot 22, first, the coordinates (Xp, Yp) of the center point 37 of the spot 22 output from the spot position analyzing unit 15 are defined as shown in FIG. Next, as shown in FIG. 29B, the range 291 (within the dotted line in FIG. 29B) of the spot radius 33 from the center point 37 of the spot 22 is specified as the target spot region. On the other hand, in the case of the spot 22 that is not the measurement target, the area of the spot 22 is not specified because it is affected by the light emission of the adjacent spot 22.

  The spot position specifying unit 17 determines whether or not each spot 22 can be measured from the luminance distribution of the spot search area 32 by performing the processing as described above, and can specify the position of only the spot 22 to be measured. It becomes.

  Next, the measuring unit 18 uses the spot position information for each spot search area 32 output from the spot position specifying unit 17 to measure the light amount of the spot from the digital image data.

  By configuring the microarray device as described above, the position of the spot 22 can be specified even when the spot 22 is misaligned from a predetermined position where it should be spotted, and further adjacent to the spot 22 where the misalignment occurred. It is possible to identify the spot 22 to be measured and prevent measurement errors.

  1 to 29, the components of the microarray apparatus of the present invention have been described. Next, the measurement procedure of the microarray apparatus according to the embodiment of the present invention will be described with reference to the flowchart shown in FIG.

  First, the microarray substrate 11 is set on the stage 12, and the stage controller 13 instructs the stage 12 to move to a position where the spot area 21 of the microarray substrate 11 enters the imaging field of the imaging unit 14. When the movement of the stage 12 is completed, the imaging unit 14 images the spot area 21 of the microarray substrate 11 (step S101).

  Next, as shown in FIG. 5A, the digital image data 51 output from the image pickup unit 14 is input to the spot position analysis unit 15, and the spot position analysis unit 15 uses the noise removal unit 41, and FIG. As shown in FIG. 5, the smoothing filter is applied to remove the irregular noise 52 (step S102).

  Next, the noise-removed image data 53 output by the noise removing unit 41 is input to the predetermined range extracting unit 42, and the predetermined range extracting unit 42 searches the search area for each spot 22 from within the spot area 21 as shown in FIG. 32 is defined and extracted. Next, the spot search region 32 output by the predetermined range extraction unit 42 is input to the luminance distribution analysis unit 43. First, as shown in FIG. 8, a luminance distribution curve in the X-axis direction is calculated for the spot search region 32, and the peak Next, the Y coordinate in the Y-axis direction is calculated, and the peak coordinate is detected (step S103).

  Next, the peak coordinates of the luminance distribution curve in the X-axis direction and the Y-axis direction output from the spot position analysis unit 15 are input to the spot position specifying unit 17. In the spot position specifying unit 17, first, the spot position determining unit 271 defines the peak coordinates of the luminance distribution curve output from the spot position analyzing unit 15 as the coordinates of the center point 37 of the spot 22, and as shown in FIG. The distance between the center point 37 of the spot 281 and the center point 37 of the adjacent spot 282 is calculated. If the distance is equal to or less than the length of the diameter of the spot 22, the spot position is determined to be NG and determined as a spot not to be measured. On the other hand, when the distance between the center points of the spot 22 is larger than the length of the diameter of the spot 22, the spot position is determined to be OK and set as a measurement target spot (step S104).

  Next, the spot position determination result output by the spot position determination unit 271 is input to the spot position calculation unit 272. When the spot 22 is determined to be a measurement target spot in step S104, the spot position analysis unit 15 outputs the spot. The center point 37 of the spot 22 is specified from the coordinates of the center point 37 of 22 and the information about the spot stored in the spot information storage unit 16 as shown in FIG. 29, and the range 291 of the spot radius 33 is determined from there. The measurement unit 18 performs measurement. On the other hand, when the spot 22 is determined to be a spot not to be measured in step S104, measurement by the measurement unit 18 is not performed (step S105).

  As described above, in the embodiment, after a spot search area is set for each spot on the microarray substrate and a luminance distribution curve is generated, the center point of the spot is detected and the spot position is specified. Even when the spot is misaligned from the predetermined position, the spot position can be specified, and a spot adjacent to the misaligned spot can be specified to prevent measurement errors.

  In the microarray device of the present invention, a monochrome CCD is exemplified as an element of the imaging unit. However, the color CCD, the complementary metal oxide semiconductor (CMOS), the charge injection element (CID), and the horizontal embedded are not limited thereto. An amplification type imaging sensor (LBCAST) having a charge storage unit, a photodiode, a photomultiplier tube, or the like is also applicable. It is also possible to introduce an image intensifier into the imaging unit.

  In the microarray apparatus of the present invention, a colorless and transparent glass substrate is exemplified as the microarray substrate. However, the present invention is not limited to this, and a silicon substrate, a colored and opaque substrate, or a plastic substrate is also applicable. Furthermore, the size of the microarray substrate and the size of the spot area shown in the embodiment of the present invention are examples, and can be changed as appropriate.

  The microarray apparatus according to the present invention can identify the position of a spot even when the spot is displaced from a predetermined position to be originally spotted on the microarray substrate, and further identifies a spot adjacent to the displaced spot. It is possible to prevent measurement errors and is useful as an imaging device for imaging a sample fixed on a substrate.

Block diagram of a microarray device according to the present invention Microarray substrate configuration diagram Configuration of spot area Block diagram of spot position analysis unit in the present invention The figure explaining the effect of a noise removal part Diagram explaining the spot search area The figure explaining the scanning method of the spot search area in a luminance distribution analysis part 1st figure explaining calculation of a luminance distribution curve The 2nd figure explaining calculation of a luminance distribution curve Diagram explaining spot area shifted in line units The figure explaining the case where the whole spot area has shifted. 3rd figure explaining calculation of a luminance distribution curve FIG. 4 is a diagram for explaining calculation of a luminance distribution curve. 5th figure explaining calculation of a luminance distribution curve FIG. 6 is a diagram for explaining calculation of a luminance distribution curve. FIG. 7 illustrates the calculation of the luminance distribution curve. FIG. 8 illustrates the calculation of the luminance distribution curve. 1st figure explaining the analysis method of a luminance distribution curve The 2nd figure explaining the analysis method of a luminance distribution curve The figure explaining the brightness distribution curve of the spot which shifted 3rd figure explaining the analysis method of a luminance distribution curve 4th figure explaining the analysis method of a luminance distribution curve The 5th figure explaining the analysis method of a luminance distribution curve FIG. 6 is a diagram for explaining a luminance distribution curve analysis method; FIG. 7 is a diagram for explaining a luminance distribution curve analysis method; FIG. 8 is a diagram for explaining a luminance distribution curve analysis method; Block diagram of spot position specifying unit in the present invention The figure explaining the method of determining the position shift of a spot Diagram explaining how to identify the spot position Flow chart of microarray measurement process

Explanation of symbols

DESCRIPTION OF SYMBOLS 11 Microarray substrate 12 Stage 13 Stage control part 14 Imaging part 15 Spot position analysis part 16 Spot information memory | storage part 17 Spot position specific | specification part 18 Measurement part 21 Spot area 22 Spot 31 Spot arrangement | positioning information 32 Spot search area 33 Spot radius 34 Between spots Distance 35 Spot search area reference coordinates 36 Spot search area width 37 Spot center point 41 Noise removal section 42 Predetermined range extraction section 43 Luminance distribution analysis section 51 Digital image data 52 Irregular noise 53 Noise removal image data 61 Target spot 62 Target spot 62 Search area 63 Search area reference coordinates of the target spot 81 Predetermined spot positions 82, 91 Luminance distribution on the scanning line with Y coordinate Y6 83, 92 Luminance distribution on the scanning line with Y coordinate Y5 and Y7 84, 93 Y position Luminance distribution on Y4 and Y8 scanning lines 85, 94 Luminance distribution on Y0, Y1, Y2, Y3, Y9, and Y10 scanning lines 101 Misaligned rows 102 First state of spot search area 103 Spot search The second state of the area 111 The third state of the spot search area 112 The fourth state of the spot search area 113 The fifth state of the spot search area 114 The sixth state of the spot search area 121, 131 The Y coordinate is Y5 Luminance distribution on the scanning line 122, 132 Luminance distribution on the scanning line whose Y coordinate is Y4 and Y6 123, 133 Luminance distribution on the scanning line whose Y coordinate is Y3 and Y7 124, 134 Y coordinate is Y0, Y1, Y2, Y8, Y9 , Y10 luminance distribution on the scanning line 141, 151 Y coordinate luminance distribution on the Y9 scanning line 142, 152 Y coordinate Y0 and Y8 Luminance distribution on Y10 scanning line 143, 153 Y-coordinate distribution on Y1 and Y7 scanning line 144, 154 Y-coordinate luminance distribution on Y2, Y3, Y4, Y5, Y6 scanning line 161, 171 Y-coordinate Y0 Luminance distribution on the scanning line 162, 172 Luminance distribution on the scanning line with Y coordinate Y1 163, 173 Luminance distribution on the scanning line with Y coordinate Y2, Y3, Y4, Y5, Y6, Y7, Y8, Y9, Y10 181 X Addition curve 1 of axial luminance distribution
182 Luminance distribution curve 1 in the Y-axis direction
191 Addition curve 2 of luminance distribution in the X-axis direction
192 Y-axis direction luminance distribution curve 2
201 Target spot shifted in position 202 Center point of target spot shifted in position 203 Luminance distribution curve on scanning line whose Y coordinate is Y6 211 Addition curve 3 of luminance distribution in X axis direction
212 Y-axis direction luminance distribution curve 3
221 Addition curve 4 of luminance distribution in the X-axis direction
222 Luminance distribution curve 4 in the Y-axis direction
231 Addition curve 5 of luminance distribution in the X-axis direction
232 Y-axis direction luminance distribution curve 5
241 Addition curve 6 of luminance distribution in the X-axis direction
242 Luminance distribution curve 6 in the Y-axis direction
251 X-axis direction luminance distribution addition curve 7
252 Y-axis luminance distribution curve 7
261 Addition curve 8 of luminance distribution in the X-axis direction
262 Y-axis luminance distribution curve 8
271 Spot position determining unit 272 Spot position calculating unit 281 Misaligned spot 282 Adjacent spot of misaligned spot 283 Center point of misaligned spot 291 Spot calculated by spot position calculating unit

Claims (6)

In a microarray apparatus for measuring a microarray substrate having a plurality of spots in which a sample specimen is fixed on the substrate,
An imaging unit that outputs the digital image data that digitally represents the luminance received by each pixel of the light receiving element by imaging the microarray substrate;
A spot information storage unit storing information about the spot;
A spot position analysis unit for calculating a luminance distribution curve for each spot from the digital image data and analyzing the position of the spot;
A spot position specifying unit for specifying a spot position from coordinates corresponding to the peak of the luminance distribution curve for each spot output by the spot position analyzing unit and information about the spot read from the spot information storage unit;
Including a measurement unit that performs measurement at the spot position output by the spot position specifying unit,
A microarray apparatus characterized by that. The spot position analysis unit
A noise removing unit for removing a noise component from the digital image data;
In the noise-removed image data output by the noise removing unit, a predetermined range extracting unit that extracts a certain range as a spot search area from a predetermined position of the spot;
In the spot search area that is output by the predetermined range extraction unit, a luminance distribution analysis unit that calculates a luminance distribution curve in the X-axis direction and the Y-axis direction and detects peak coordinates of the luminance distribution curve,
Extracting the spot search area for each spot from the digital image data output by the imaging unit, and calculating a luminance distribution curve for each of the X-axis direction and the Y-axis direction;
The microarray apparatus according to claim 1, wherein: The spot position specifying unit
A spot position determination unit that determines whether or not the spot is measurable by analyzing the arrangement status of the spot from the coordinates of the peak of the luminance distribution curve output by the spot position analysis unit;
A spot position calculation unit that calculates the spot position from the spot arrangement information output by the spot position determination unit and information about the spot read from the spot information storage unit;
Determining the positional relationship of the spots from the coordinates of the peak of the luminance distribution curve for each spot search area, and calculating the position of the spot according to the result,
The microarray apparatus according to claim 2, wherein: The spot position determination unit
The coordinates of the peak of the luminance distribution curve output by the spot position analysis unit are defined as the coordinates of the center point of the spot, and the spot can be measured by the distance between the center point of the spot and the center point of the adjacent spot Determine if it is in position,
The microarray apparatus according to claim 3. The spot position calculation unit
In the spot arrangement information output by the spot position determination unit, the spot position is calculated only when the spot is in a measurable position.
The microarray apparatus according to claim 3. An imaging step of imaging a microarray substrate having a plurality of spots in which a sample specimen is fixed on the substrate;
A spot position analyzing step of calculating a luminance distribution curve for each spot from the digital image data obtained in the imaging step and detecting coordinates that are peaks; and
A spot position determination step for determining the positional relationship of the spots from the coordinates of the peak of the luminance distribution curve for each spot obtained in the spot position analysis step;
Including a measurement step of measuring the brightness of the spot determined in the spot position determination step,
And a microarray analysis method.

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