JP2009156715A - Biological substance detector - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a biological substance detector capable of reducing the measuring error by the interference between spots even when a specimen spotted at a minute interval is analyzed. <P>SOLUTION: The biological substance detector is equipped with: a color image acquiring part 102 using a slide 101, on which specimens labeled by a plurality of kinds of labeling enzymes are fixed in an array form so that first and second adjacent sample spots 211 and 212 emit lights in different emission spectra, as a target to acquire the image of the slide 101; an image extraction circuit 103 for extracting the image data corresponding to the kinds of the labeling enzymes from the color image data 111 obtained from the color image acquiring part 102 and outputting a plurality of pieces of the extracted image information 112 and 113; and the first and second image correction circuits 104 and 105 respectively provided corresponding to a plurality of the respective pieces of extracted image information 112 and 113 to correct the respective pieces of extracted image information 112 and 113 so as to enable the quantitative evaluation between the respective pieces of extracted image data 112 and 113 and outputting the pieces of corrected image information 114 and 115. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a biological material detection apparatus, and more particularly to a biological material detection apparatus that acquires and analyzes an examination-based image having a plurality of spots on which a specimen having a luminescent or fluorescent property is fixed.

  In the field of biochemistry and molecular biology, conventionally, images of specimens that have been labeled with a luminescent or fluorescent labeling enzyme and distributed to multiple spot areas such as test bases are acquired, and specimens in each spot area are obtained from the acquired images. There is a biological material detection device to analyze.

  For example, in Patent Document 1, the luminance value of the spot and its surrounding pixels is integrated in a circular area including the spot of the specimen and having the same area from the image photographed by the monochrome CCD camera. A method of making the value of is disclosed.

  However, this method is effective when the light from each spot does not interfere, but when the distance between the spots is short, the light between the spots interferes to cause an error. FIG. 10 shows two-dimensionally the luminance distribution in monochrome image information of the spots of two specimens A and B fixed adjacent to each other. Sample A and sample B are labeled with a labeling enzyme having the same emission spectrum and emit light with the same intensity. In the figure, reference numerals 1005 and 1006 denote spot center positions of the specimen A and the specimen B, respectively. Reference numerals 1001 and 1002 denote ideal luminance distributions of the specimen A and the specimen B that are not subject to interference. Reference numeral 1003 denotes a luminance distribution that is actually acquired, which shows that the two specimens A and B interfere with each other. When the specimen A is actually analyzed, the luminance distribution 1003 is used to integrate the luminance values in the analysis region 1004, so that an error occurs due to the influence of the interference of the luminance distribution 1002. The same applies to the sample B.

In order to reduce such interference between spots, for example, in Patent Document 2, when acquiring images of a plurality of spot regions (wells) of a microtiter plate, an image acquisition means such as a CCD camera, a microtiter plate, An apparatus has been proposed in which a light-shielding plate having an opening corresponding to each well is disposed between the two so as to shield light emitted or fluorescent light from adjacent wells.
International Publication WO2002 / 001477 JP-A-2005-274355

  However, the apparatus of Patent Document 2 uses a light-shielding plate for a microtiter plate as described above, and the opening of the light-shielding plate is used for a test substrate such as a microarray in which the sample spot interval is minute. Miniaturization and positioning of the parts are very difficult and difficult to apply.

  The present invention solves the above-described problem, and an object of the present invention is to provide a biological material detection apparatus capable of reducing measurement errors due to interference between spots when analyzing a sample spotted at a minute interval.

  In order to solve the above problems, the biological material detection device of the present invention acquires a test-based image having a plurality of spots to which a specimen having luminescent or fluorescent properties is fixed, and analyzes the biological material. A color for acquiring an image of the test base as color image information for a test base in which specimens labeled with a plurality of types of labeling enzymes are fixed in an array so that adjacent spots emit light in different emission spectra. An image acquisition unit, an image extraction circuit that extracts image information corresponding to each type of the labeling enzyme from the color image information, outputs a plurality of extracted image information, and corresponds to each of the plurality of extracted image information An image correction circuit that corrects each extracted image information and outputs corrected image information so that quantitative evaluation can be performed between the extracted image information. Characterized by comprising.

According to the above configuration, image information corresponding to each type of labeling enzyme is extracted and corrected, so that interference due to light emission between adjacent spots can be reduced.
“Emission spectrum” means both emission and fluorescence spectra. “Specimen labeled with a labeling enzyme” refers to a specimen, that is, a test object containing a plurality of biological substances, which is subjected to labeling treatment of luminescence with an enzyme. An example of the specimen is human blood. Biological substances refer to biological macromolecules (nucleic acids, proteins, polysaccharides) that are the basic materials that make up living organisms, and nucleotides, nucleosides, amino acids, various sugars, etc., and lipids, vitamins, hormones, etc. .

An image combining circuit that combines a plurality of pieces of corrected image information into one and outputs combined image information can be further provided.
The image correction circuit can be configured to calculate a correction coefficient from the emission spectrum of the labeling enzyme and the visibility characteristic of the color image acquisition unit, and to correct each extracted image information using the calculated correction coefficient.

  When a reference spot for quantifying the amount of luminescence for each type of labeled enzyme is provided on the inspection platform, the image correction circuit extracts the reference spot from the extracted image information for each type of labeled enzyme, and the amount of luminescence And calculating a correction coefficient from the light emission amount value, and using the calculated correction coefficient, the extracted image information for each type of the labeling enzyme so that the light emission amount of the reference spot for each type of the labeling enzyme becomes equal. Can be configured to correct.

  According to the biological material detection apparatus of the present invention, when analyzing each specimen spotted on the examination base, interference due to light emission between adjacent spots can be reduced, and measurement errors can be reduced.

Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings.
(Embodiment 1)
A biological substance detection apparatus according to Embodiment 1 of the present invention and an analysis method using the same will be described with reference to FIGS.

  FIG. 1 shows a system configuration of a biological material detection apparatus. Reference numeral 102 denotes a color image acquisition unit, 103 denotes an image extraction circuit, 104 denotes a first image correction circuit, and 105 denotes a second image correction circuit. Reference numeral 101 denotes a slide to be inspected.

  The slide 101 is one in which specimens that emit light by a biochemical reaction are fixed in an array on a small substrate, and is generally called a microarray or a DNA chip. The specimens are labeled with a plurality of types of labeling enzymes and fixed (spotted) in an array so that one adjacent specimen and the other specimen emit light with different emission spectra. Here, two kinds of specimens, which are labeled with labeling enzymes having different emission spectra, are used. One specimen forms the first sample spot 211 and the other specimen is the first specimen. Two sample spots 212 are formed. The first sample spot 211 and the second sample spot 212 are alternately arranged in the vertical direction and the horizontal direction so as not to be adjacent to each other.

  The color image acquisition unit 102 captures the slide 101, acquires color image information 111, and outputs it. The image extraction circuit 103 extracts, from the color image information 111, first extracted image information 112 that is image information in a specific wavelength region and second extracted image information 113 that is image information in a different wavelength region. And output. The first image correction circuit 104 performs correction to quantitatively evaluate the first extracted image information 112 and the second extracted image information 113, and outputs first corrected image information 114. The second image correction circuit 105 performs correction in the same manner as the first image correction circuit 104, and outputs second corrected image information 115.

  This biological material detection apparatus has a system configuration corresponding to the above-described slide 101 with two types of emission spectra of the specimen, but when there are three or more types of emission spectra, the number of extracted images corresponding to the number of types in the wavelength region. Information is output, and the system configuration includes an image correction circuit for each extracted image information.

Hereinafter, an analysis method using the biological material detection apparatus of FIG. 1 will be described.
First, a slide 101 to be inspected shown in an enlarged manner in FIG. 2 is prepared. In other words, on a small substrate made of glass (which may be made of silicon), the first sample spot 211 and the second sample spot 212 adjacent to each other are labeled with a plurality of types of labeling enzymes so that they emit light with different emission spectra. Fix specimens in an array.

  Here, a specimen labeled with a labeling enzyme is preliminarily bound to a biological substance (antigen) to be analyzed, a specific reactive substance (antibody) that specifically binds to it, and either the biological substance or the specific reactive substance. Labeled with the amount of luminescence corresponding to the amount of the complex of the biological substance, the specific reactive substance and the labeling enzyme, that is, the amount of luminescence corresponding to the amount of the biological substance to be analyzed. The enzyme emits light. As the labeling enzyme, two types of luciferases having different emission colors are used, the first sample spot 211 is labeled with a luciferase that emits green light, and the second sample spot 212 is labeled with a luciferase that emits red light.

  FIG. 3 shows the relative emission spectrum of the labeling enzyme (luciferase) used. 301 is an emission spectrum of luciferase emitting green light (referred to as a first emission spectrum). Reference numeral 302 denotes an emission spectrum of luciferase that emits red light (referred to as a second emission spectrum). The first emission spectrum 301 is defined as a function L1 (λ) with respect to the wavelength λ, and the second emission spectrum 302 is also defined as a function L2 (λ). In the figure, the peak of L2 (λ) is taken as 1, and the intensity (relative intensity) is shown. The first emission spectrum 301 and the second emission spectrum 302 are measured in advance with a spectrometer or the like. An example of a luciferase having such an emission spectrum is Multicolor Look (registered trademark) Ultra-HTS luminescence reagent manufactured by Toyo B-Net Co., Ltd.

  When the prepared slide 101 is set at a predetermined position and the system is started, the color image acquisition unit 102 captures the slide 101 to acquire the color image information 111 and outputs it.

  Although a general color CCD camera can be used as the color image acquisition unit 102, a color CCD having RGB visibility characteristics as shown in FIG. 4 is used here. In the figure, 401 is a blue sensitivity curve indicating the blue sensitivity with respect to the wavelength of the object to be imaged. Similarly, 402 is a green sensitivity curve indicating the green sensitivity, and 403 is a red sensitivity curve indicating the red sensitivity.

  The blue sensitivity curve 401 is defined as a function Fb (λ) with respect to the wavelength λ, the green sensitivity curve 402 is defined as a function Fg (λ), and the red sensitivity curve 403 is defined as a function Fr (λ). In the figure, the peak of Fr (λ) is set to 1, and the sensitivity (relative sensitivity) is shown. For the blue sensitivity curve 401, the green sensitivity curve 402, and the red sensitivity curve 403, the spectral distribution is measured in advance by a spectrometer or the like.

  Also, the image information acquired with blue sensitivity is IB, the luminance value is IB (m, n), the image information acquired with green sensitivity is IG, the luminance value is acquired with IG (m, n), and the red sensitivity is acquired. The brightness value of the image information thus obtained is defined as IR, and the brightness value is defined as IR (m, n). m is a pixel position in the horizontal axis direction, and n is a pixel position in the vertical axis direction. The size of the color image information 111 is 480 pixels vertically and 640 pixels horizontally, m is a pixel position from 0 to 639, and n is a pixel position from 0 to 479.

FIGS. 5A, 5B and 5C show the luminance distribution of the specimen in the image information acquired as described above.
To simplify the explanation, assuming that there are two specimen spots and the same amount of luciferase is bound to both spots, the luminance distribution is two-dimensional (that is, the luminance value on the line passing through the center position of both spots). ). Both spots are referred to as a first sample spot 211 and a second sample spot 212, as before.

  Reference numeral 501 denotes a luminance distribution of the image information IR, 502 denotes a luminance distribution of the image information IG, and 503 denotes a luminance distribution of the image information IB. Reference numeral 504 denotes the center position of the first sample spot 211, and reference numeral 502 denotes the center position of the first sample spot 212. Even if the binding amount of luciferase in both spots is equal, the emission spectrum distribution of luciferase and the sensitivity curve of each color of the color CCD camera are different as described above, so that the emission intensity is not equivalent.

  In response to the output from the color image acquisition unit 102, the image extraction circuit 103 separates and extracts the light emission of the first sample spot 211 and the light emission of the second sample spot 212 from the color image information 111.

  Assuming that the luminance value of the image information extracted for the first sample spot 211 is I1 (m, n) and the luminance value of the image information extracted for the second sample spot 212 is I2 (m, n), respectively, It is calculated by the following calculation formulas (1) and (2).

式（１）（２）は、重み付けをした画像情報ＩＢ、ＩＧ、ＩＲの輝度値を加算・減算することを表している。第１のサンプルスポット２１１にかかるＩ１（ｍ，ｎ）は、上述のように緑色で発光するルシフェラーゼで標識化しているため、つまりＩＧ（ｍ，ｎ）のみの成分と仮定しているため、ａ１＝０、ｂ１＝１、ｃ１＝０となり、式（１）′で表されることとなり、図５（ｂ）と同じ輝度分布となる。同様に、第２のサンプルスポット２１２にかかるＩ２（ｍ，ｎ）は、ＩＲ（ｍ，ｎ）のみの成分と仮定しているため、ａ２＝０、ｂ２＝０、ｃ２＝１となり、式（２）′で表されることとなり、図５（ａ）と同じ輝度分布となる。

Expressions (1) and (2) represent adding and subtracting the luminance values of the weighted image information IB, IG, and IR. Since I1 (m, n) applied to the first sample spot 211 is labeled with luciferase that emits green light as described above, that is, it is assumed that only IG (m, n) is present, a1 = 0, b1 = 1, and c1 = 0, which are expressed by Expression (1) ′, and have the same luminance distribution as that in FIG. 5B. Similarly, since I2 (m, n) applied to the second sample spot 212 is assumed to be a component of only IR (m, n), a2 = 0, b2 = 0, c2 = 1, and the expression ( 2) ′, and the luminance distribution is the same as that in FIG.

  The image extraction circuit 103 uses the image information having the luminance value of I1 (m, n) as the first extracted image information 112 and the image information having the luminance value of I2 (m, n) as the second extracted image information. As 113, it outputs.

(Step 4)
The first image correction circuit 104 and the second image correction circuit 105 that have received the output from the image extraction circuit 103 can quantitatively evaluate the first extracted image information 112 and the second extracted image information 113. Correction is performed, and first corrected image information 114 and second corrected image information 115 are output.

  In the correction, the above-described emission spectrum distribution (L1 (λ), L2 (λ)) and color sensitivity curves (Fb (λ), Fg (λ), Fr () corresponding to the emission spectrum of the color CCD camera are used. λ)) to calculate the correction coefficient, and use the calculated correction coefficient.

  Specifically, first, the correction coefficient R1 of the first extracted image information 112 and the correction coefficient R2 of the second extracted image information 113 are calculated by the following equations (3) and (4).

式（３）（４）において、Ｆｂ（λ）、Ｆｇ（λ）、及びＦｒ（λ）への重み付けａ１，ｂ１，ｃ１，ａ２，ｂ２，ｃ２は、上述の式（１）（２）で各色の画像情報に付した重み付けと同じものを用いる。

In the equations (3) and (4), the weights a1, b1, c1, a2, b2, and c2 to Fb (λ), Fg (λ), and Fr (λ) are expressed by the above equations (1) and (2). The same weighting is used for the image information of each color.

  In equation (3), {a1 × Fb (λ) + b1 × Fg (λ) + c1 × Fr (λ)} gives the same weighting as that used in equation (1) to the color sensitivity curve of each color of the CCD camera. The luminance value obtained is shown. Therefore, by multiplying this luminance value by the emission spectrum L1 (λ) and integrating in the entire wavelength region, a fixed amount of luciferase image that emits green light is acquired by the color CCD camera, and calculated by the equation (1). It is possible to obtain the brightness value when

  That is, Equation (3) is obtained by calculating the ideal luminance value obtained when a luciferase having an emission spectrum of L1 (λ) is photographed with a monochrome camera. L1 (λ) × Fb (λ) is a term for converting a blue component into a luminance value, L1 (λ) × Fg (λ) is a term for converting a green component into a luminance value, and L1 (λ) × Fr (λ) is This is a term for converting a red component into a luminance value. In Equation (1), each color is weighted when the luminance values of blue, green, and red are combined. Therefore, the same weight is also given in Equation (3). As a result, the luminance value obtained by calculating the emission spectrum of L1 (λ) with the same weighting as in equation (1) can be obtained by calculation. This is the correction coefficient R1 of the first extracted image information 112.

Equation (4) is to calculate an ideal luminance value obtained when a luciferase having an emission spectrum of L2 (λ) is photographed with a monochrome camera. In the same manner as described above, the luminance value obtained by obtaining the emission spectrum of L2 (λ) with the same weighting as that of the expression (2) can be obtained by calculation. This is the correction coefficient R2 of the second extracted image information 113.
λ_max indicates the upper limit of integration. Since the integration is performed in the entire wavelength range, it is theoretically infinite, but 1000 nm is adopted as a finite value in the present embodiment. Therefore, R1 is represented and calculated by Expression (3) ′ using a1 = 0, b1 = 1, c1 = 0, and λ_max = 1000. R2 is expressed and calculated by the equation (4) ′ using a2 = 0, b2 = 0, c2 = 1, and λ_max = 1000.

  Here, when the same amount of luciferase is coupled to the first sample spot 211 and the second sample spot 212 to emit light, the calculated ratio of R1 and R2 is the first extracted image. The ratio between the luminance value I1 (m, n) acquired from the information 112 and I2 (m, n) acquired from the second extracted image information 113 is shown. Therefore, the luminance value of the image information that can be directly compared with I2 (m, n) can be obtained by correcting I1 (m, n) by the following calculation formula (5).

  Detailed description. As described above, L1 (λ) and L2 (λ) are emission spectra obtained when the same amount of luciferase is emitted, and these L1 (λ) and L2 (λ) show relative characteristics. Equation (3) is a calculated value of the luminance value when a luciferase having the emission spectrum of L1 (λ) is emitted and photographed with a monochrome camera. Equation (4) is the emission spectrum of L2 (λ). This is a calculated value of the brightness value when the same amount of luciferase is emitted and photographed with a monochrome camera.

  Therefore, when the ratio between the value R1 in the expression (3) and the value R2 in the expression (4) is taken, the ratio of the luminance values acquired by the camera when the same amount of luciferase is emitted. In order to compare the amount of luciferase, it is necessary to normalize using the value of formula (3) or formula (4).

Here, I1 (m, n) and I2 (m, n) are images obtained by extracting the component of L1 (λ) and the component of L2 (λ), respectively. Therefore, even if the same amount of luciferase is photographed, the same luminance value is not obtained as shown in equations (3) and (4). In order to compare the amount of luciferase of the L1 (λ) component in I1 (m, n) and the amount of luciferase of the L2 (λ) component in I2 (m, n) in terms of luminance values, it is ideal. It is necessary to normalize with a ratio (that is, Expression (3) and Expression (4)). The following formula (5) performs calculation for normalizing formula (3), that is, R2 is 1. Since Expression (6) is normalized by R2, it remains as it is.

  C1 (m, n) shown in Expression (5) is a luminance value of the first corrected image information 114 that can be corrected by correcting the first extracted image information 112. C2 (m, n) shown in Expression (6) is a luminance value of the second corrected image information 115 that can be corrected by correcting the second extracted image information 113. In C2 (m, n), I2 (m, n) is used as it is, and correction using a correction coefficient is not performed. This is because I1 (m, n) is corrected so that it can be compared with I2 (m, n) as described above. Expressions (5) ′ (6) ′ are obtained from Expressions (5) and (6). Note that I2 (m, n) may be corrected so that it can be compared with I1 (m, n), or both I2 (m, n) and I1 (m, n) may be corrected.

図６は、上述の式（５）′（６）′に基づいて、第１の抽出画像情報１１２（図５（ｂ））と第２の抽出画像情報１１３（図５（ａ））を補正して得られた、第１の補正画像情報１１４と第２の補正画像情報１１５の輝度分布である。図６（ａ）において、６０１は第１の補正画像情報１１４の輝度分布であり、６０３は第２のサンプルスポット２１２の干渉を受けていない場合の、理想となる第１のサンプルスポット２１１の輝度分布を示す。図６（ｂ）において、６０２は第２の補正画像情報１１５の輝度分布であり、６０４は第１のサンプルスポット２１１の干渉を受けていない場合の、理想となる第２のサンプルスポット２１２の輝度分布を示す。

FIG. 6 corrects the first extracted image information 112 (FIG. 5 (b)) and the second extracted image information 113 (FIG. 5 (a)) based on the above formula (5) ′ (6) ′. The brightness distribution of the first corrected image information 114 and the second corrected image information 115 obtained as described above. In FIG. 6A, reference numeral 601 denotes a luminance distribution of the first corrected image information 114, and reference numeral 603 denotes an ideal luminance of the first sample spot 211 when not receiving interference from the second sample spot 212. Show the distribution. In FIG. 6B, reference numeral 602 denotes a luminance distribution of the second corrected image information 115, and reference numeral 604 denotes an ideal luminance of the second sample spot 212 when not receiving interference from the first sample spot 211. Show the distribution.

  When analysis is performed using the obtained first corrected image information 114 and second corrected image information 115, the integrated value of the luminance values of a predetermined analysis region 1004 of the luminance distribution 601 is calculated for the first sample spot 211. Use to analyze. Similarly, the second sample spot 212 is analyzed using an integrated value of luminance values in a predetermined analysis region of the luminance distribution 602.

  The analysis region 1004 is provided between adjacent spots. If the area is increased, the amount of light taken from the target spot increases, while the influence of interference from the adjacent spot increases, but the former signal component (S) and the latter noise component ( N), the region where the S / N is the maximum is the analysis region 1004. Actually, slides on which the respective luciferases are spotted are prepared in advance, images (IA, IB) are acquired without causing interference with each other, and the two images are synthesized to form a composite image (IC). Create When synthesizing, the same interval as that for measuring each spot is set. The circular area is expanded around the luciferase spot in IA, the total luminance value in the circular area is S, the total luminance value in the same area of IC is S + N, and S / N is obtained. An area where the S / N is maximum is set as an analysis area 1004.

  When the specimen of the first sample spot 211 is analyzed using the integrated value of the analysis region 1004 of the luminance distribution 601 as described above, the difference from the integrated value of the luminance distribution 603 is the total integrated value (the hatched portion in the figure). ) Of 3% or less. Compared to the case where the integrated value of the analysis region 1004 (using the label enzyme having the same emission spectrum) shown in FIG. 10 is used, the error is about one third. This is because the first corrected image information 114 is affected by noise due to light emission from outside the spot area, that is, light emission at the second sample spot 212. Similarly, when the specimen of the second sample spot 212 is analyzed using the integrated value of the predetermined analysis region of the luminance distribution 602, the second corrected image information 115 is caused by light emission at the first sample spot 211. Since the influence is reduced, the error is reduced.

  The above describes the case where the same amount of luciferase is bound to two spots, the first sample spot 211 and the second sample spot 212, and emits light. The same method can also be used when providing.

As described above, according to the biological material detection apparatus of the first embodiment, from the captured image of the slide 101 in which two types of specimens labeled with labeling enzymes having different emission spectra are spotted in an array, the first By extracting the extracted image information 112 and the second extracted image information 113 and correcting each using the emission spectrum and the sensitivity curve of the imaging means, measurement caused by interference caused by light emission between adjacent spots The error can be reduced.
(Embodiment 2)
FIG. 7 shows a system configuration of the biological material detection apparatus according to the second embodiment of the present invention.

  The difference between the biological material detection device of the second embodiment and that of the first embodiment is that the slide 701 provided with the first and second reference spots 803 and 804 is the inspection object, and correspondingly, The first image correction circuit 702 and the second image correction circuit 703 are configured to perform correction based on the light emission amounts of the first and second reference spots 803 and 804 of the slide 701, and the corrected image thereof. An image synthesizing circuit 704 that synthesizes grayscale image information from the information is provided.

  In the figure, reference numeral 711 denotes first corrected image information corrected by the first image correction circuit 702, 712 denotes second corrected image information corrected by the second image correction circuit 703, and 713 denotes an image composition circuit 704. This is combined image information (grayscale image information). Components having the same configuration or operation as those of the first embodiment are denoted by the same reference numerals, and detailed description thereof is omitted.

  FIG. 8 shows an enlarged view of the slide 701 to be inspected. A first sample spot 211 and a second sample spot 212 described in Embodiment 1 are formed on the slide 701. Each emits light with a light emission amount corresponding to the amount of the biological material to be analyzed. The first reference spot 803 serves as a reference for correction in the first image correction circuit 702 and has the same emission spectrum as that of the first sample spot 211. Similarly, the second reference spot 804 serves as a reference for correction in the second image correction circuit 703 and has the same emission spectrum as the second sample spot 212.

  The first reference spot 803 and the second reference spot 804 may be formed in a reference spot region 801 that is separate from the region where the first sample spot 211 and the second sample spot 212 are fixed as shown in the figure. However, they may be provided in the same region. When provided in the same region, the first reference spot 803 and the first sample spot 211 are not adjacent to each other, and the second reference spot 804 and the second sample spot 212 are not adjacent to each other. To do.

  When forming the first reference spot 803 and the second reference spot 804, the same amount of luciferase is bound to each. Since the amount of luciferase binding in the first and second sample spots 211 and 212 and the first and second reference spots 803 and 804 depends on the amount of the biological material to be analyzed, the first and second reference spots The spots 803 and 804 have a binding ability equivalent to that of the biological substance to be analyzed and are produced by a housekeeping gene whose content is known (for example, GAPDH (glyceraldehyde-3-phosphate dehydrogenase), β -Actin, β2-microglobulin) may be supplied, or may be supplied after being collected from a sample whose biological material content is known in advance.

Hereinafter, an analysis method using the biological material detection apparatus of FIG. 7 will be described.
As in the first embodiment, the color image acquisition unit 102 acquires and outputs the color image information 111 of the slide 701, and the image extraction circuit 103 extracts the first extracted image information 112 and the second extracted image information from the color image information 111. The extracted image information 113 is extracted. The luminance values of the first extracted image information 112 and the second extracted image information 113 are I1 (m, n) and I2 (m, n).

Next, correction is performed so that the first image correction circuit 702 and the second image correction circuit 703 can quantitatively evaluate each other's image information.
For this purpose, first, the amount of light emitted from the reference spot for each type of labeling enzyme (luciferase) is determined. That is, the first image correction circuit 702 extracts the first reference spot 803 from the first corrected image information 112 and calculates the light emission amount. This light emission amount may be a peak value of the first reference spot 803 or may be a value obtained by integrating luminance values in the first reference spot 803. The calculated value is set as a correction coefficient R1. Similarly, the second image correction circuit 703 extracts the second reference spot 804 from the second corrected image information 113 and calculates the light emission amount. The calculated value is set as a correction coefficient R2.

  Next, the extracted image information for each type of labeling enzyme is corrected so that the amount of light emitted from the reference spot for each type of labeling enzyme becomes equal. That is, the first image correction circuit 702 applies the correction coefficients R1 and R2 to the equation (5) described in the first embodiment, thereby correcting the first corrected image information 711 (C1 (m, n)). Similarly, the second image correction circuit 703 applies the correction coefficients R1 and R2 to the equation (6) described in the first embodiment, thereby correcting the second corrected image information 712 (C2 (m , N)). The first and second image correction circuits 702 and 703 output first corrected image information 711 and second corrected image information 712, respectively.

  Thereafter, the image composition circuit 704 composes the first corrected image information 711 and the second corrected image information 712 to create composite image information 713. That is, when obtaining the luminance value of each coordinate, the luminance value of that coordinate in the first corrected image information 711 is compared with the luminance value of that coordinate in the second corrected image information 712, and a larger luminance value is obtained. The brightness value of the composite image information 713 is used.

  In FIG. 9, reference numeral 901 denotes the luminance distribution of the composite image information 713. Reference numeral 603 denotes an ideal luminance distribution of the first sample spot 211 in the case where the second sample spot 212 is not affected by the interference described in the first embodiment. Similarly, reference numeral 604 denotes an ideal luminance distribution of the second sample spot 212 when not receiving the interference of the first sample spot 211. The composite image information 713 is grayscale image information in which the amount of interference between the first sample spot 211 and the second sample spot 212 is reduced.

  In the conventional method of spotting a specimen that emits light with one color luciferase and acquiring it with a normal monochrome camera, an image in which the spots interfere with each other is produced. In this method, two corrected images are created. Each corrected image has reduced interference between adjacent spots, and since the two corrected images are combined, all the spots are contained in one image as in the prior art, and between the spots. This is a gray scale image with reduced interference.

  For this reason, it is possible to perform analysis using this single grayscale image information. The first sample spot 211 is analyzed using the integrated value of the luminance value in the analysis region 1004 of the luminance distribution 603, and the integrated value of the luminance value in the predetermined analysis region 1004 of the luminance distribution 604 is related to the second sample spot 212. It can be analyzed using.

  As described above, according to the biological material detection apparatus of the second embodiment, the first reference spot 802 and the second reference spot 803 are provided on the slide 701, so that the emission spectrum of the labeling enzyme can be measured in advance. Even if detailed information and detailed sensitivity characteristics of the color image acquisition unit 102 are not obtained, interference due to light emission of the adjacent first and second sample spots 211 and 212 and measurement errors caused thereby can be reduced. .

  Further, since the composite image information 713 is created by the image composition circuit 704, analysis can be performed using only this single composite image information 713, that is, not using two or more pieces of image information. This means that the analysis means can be used with one type of enzyme labeling method so far. Since it can be combined into a single gray scale image of composite image information 713 and interference between spots is reduced, a conventional analysis means for analyzing one image can be used. In Embodiment 1 described above, images are created as many as the number of colors of luciferase used, so actual analysis needs to be performed for each image, compared to the need to provide an analysis means for each color, It is advantageous.

  Since the biological material detection apparatus according to the present invention can reduce interference of light emission between spots, it is useful as an analysis apparatus for a specimen spotted at a high density on a test substrate.

1 is a system configuration diagram of a biological material detection device according to Embodiment 1 of the present invention. Enlarged view of the slide to be examined Relative emission spectrum of the labeling enzyme used for the slide Relative sensitivity characteristic diagram of color image acquisition unit in biological material detection apparatus of FIG. The figure which shows the luminance distribution of the image information acquired in the color image acquisition part with the relative sensitivity characteristic shown in FIG. The figure which shows the luminance distribution of the correction | amendment image information in the biological material detection apparatus of FIG. System configuration diagram of the biological material detection apparatus in Embodiment 2 of the present invention Enlarged view of the slide to be examined The figure which shows the luminance distribution of the composite image information in the biological material detection apparatus of FIG. The figure which shows the luminance distribution with the labeling enzyme which has been used for the detection of biological material conventionally

Explanation of symbols

101 Slide 111 Color image information 112 First extracted image information 113 Second extracted image information 114 First corrected image information 115 Second corrected image information 211 First sample spot 212 Second sample spot 301 First Emission spectrum 302 Second emission spectrum 401 Blue sensitivity curve 402 Green sensitivity curve 403 Red sensitivity curve 501 Brightness distribution of image information IR 502 Brightness distribution of image information IG 503 Brightness distribution of image information IB 504 Spot center position of specimen C 505 specimen D spot center position 601 Luminance distribution of first corrected image information 602 Luminance distribution of second corrected image information 603 Luminance distribution 604 Luminance distribution 701 Slide 702 First image correction circuit 703 Second image correction circuit 704 Image composition Circuit 711 First correction image Information 712 Second corrected image information 713 Composite image information 801 Reference spot area 803 First reference spot 804 Second reference spot 901 Brightness distribution of composite image information 1001 Brightness distribution 1002 Brightness distribution 1003 Acquisition brightness distribution 1004 Analysis area 1005 Sample Spot center position of A 1006 Spot center position of specimen B

Claims (4)

A biological substance detection apparatus for acquiring and analyzing an image of a test base having a plurality of spots to which a specimen having a luminescent or fluorescent property is fixed,
A color image acquisition unit for acquiring an image of the test base as color image information for a test base in which specimens labeled with a plurality of types of labeling enzymes are fixed in an array so that adjacent spots emit light in different emission spectra; ,
An image extraction circuit that extracts image information corresponding to each type of the labeling enzyme from the color image information, and outputs a plurality of extracted image information;
An image correction circuit that is provided corresponding to each of the plurality of extracted image information, corrects each extracted image information so that quantitative evaluation can be performed between the extracted image information, and outputs corrected image information A biological material detection device comprising:   The biological material detection apparatus according to claim 1, further comprising an image synthesis circuit that synthesizes a plurality of corrected image information into one and outputs the synthesized image information.   The image correction circuit calculates a correction coefficient from the emission spectrum of the labeling enzyme and the visibility characteristic of the color image acquisition unit, and corrects each extracted image information using the calculated correction coefficient. The biological substance detection apparatus described.   A reference spot for quantifying the amount of luminescence for each type of labeling enzyme is provided on the inspection platform, and the image correction circuit extracts the reference spot from the extracted image information for each type of the labeling enzyme, and the light emission thereof. The amount is calculated, the correction coefficient is calculated from the light emission amount value, and the extracted image for each type of labeling enzyme is equalized using the calculated correction coefficient so that the light emission amount of the reference spot for each type of the labeling enzyme becomes equal. The biological substance detection device according to claim 1, wherein the information is corrected.

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