JP2007046933A - Fluorescent image-detecting method, fluorescent image-detecting substrate, fluorescent image detector, program and recording medium - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a fluorescent image-detecting method using a gene chip capable of efficiently detecting a fluorescent image with high accuracy, a fluorescent image detecting substrate, a fluorescent image detector, a program and a recording medium. <P>SOLUTION: In the fluorescent image detecting method for making a plurality of the probe substances fixed on the fluorescent image detecting substrate react with a target substance, labeled with a fluorescent substance on the fluorescent image detecting substrate and measuring the fluorescent image of the fluorescence of the reaction product after reaction, to detect the reaction of the probe substances and the target substance, the respective probe substances are fixed on the fluorescent image detecting substrate in a geometrical pattern like state, and the correlation of the geometrical pattern with the fluorescence intensity is detected, with respect to the respective probe substances after reaction. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a fluorescent image detection method using a fluorescent image detection substrate (such as a gene chip) that can be performed efficiently and with high accuracy, a fluorescent image detector using the fluorescent image detection substrate, a program, and a recording medium About.

  In recent years, the progress of gene structure analysis has been remarkable, and many gene structures including human gene structures have been clarified. For analysis of such a gene structure, a DNA chip method using a fluorescent property, a fluorescent antibody method, or the like is often used.

  In the DNA chip method, a plurality of probe materials such as DNA fragments whose sequences are known in advance are arranged in an array on a fluorescent image detection substrate such as a slide glass, and a target material such as an unknown DNA fragment This is a method for analyzing and determining a DNA base sequence by analyzing a hybridization signal obtained by hybridization. The DNA chip method can analyze base sequences simply and quickly, and can monitor gene expression and identify displacements in known base sequences. For example, Patent Document 1 proposes a method for detecting and diagnosing arteriosclerosis using the DNA chip method.

  In order to perform DNA structure analysis by the DNA chip method, a probe substance is prepared, a probe substance is spotted and fixed on a fluorescent image detection substrate, and then a target substance is prepared for hybridization to perform hybridization signal. The process of detecting.

  A fluorescent substance is used to detect a hybridization signal, and an image is analyzed and calculated mainly using analysis software. At this time, in order to fix the probe substance on the slide glass, a method is generally used in which each probe substance is arranged in an array on the fluorescent image detection substrate, and the DNA corresponding to the spot having the maximum fluorescence intensity is detected. It's being used.

  Similar to the DNA chip method, the fluorescent antibody method is a method for examining the presence of virus infection by examining the location of antigens or antibodies in cells using a fluorescent substance. The fluorescent antibody method can be easily and rapidly examined, and includes antinuclear antibody, anti-DNA antibody (Crisidia method), anti-gastric wall cell antibody, anti-smooth muscle antibody, anti-mitochondrial antibody, anti-neutrophil cytoplasmic antibody, etc. Autoimmune disease test, EB virus, rubella virus antibody, herpes virus antibody (antigen), cytomegalovirus antibody, Chlamydia trachomatis antibody (antigen), Kurazimia shitashi antibody (antigen), endotoxin, toxoplasma antibody, FTA-ABS For example, Patent Document 2 proposes a detection and inspection method for subarachnoid hemorrhage using a fluorescent antibody method.

The fluorescent antibody method is classified into a direct fluorescent antibody method and an indirect fluorescent antibody method. In the direct fluorescent antibody method, a cell, tissue, etc. (probe substance) of a subject is immobilized on a fluorescent image detection substrate, and labeled with a fluorescent substance. And reacting the conjugated antibody and observing the fluorescence after the reaction. The indirect fluorescent antibody method includes a step of fixing a subject's cells, tissues, etc. (probe substance) on a fluorescent image detection substrate, a step of reacting an antibody not labeled with a fluorescent substance, and a labeling with a fluorescent substance. After reacting with a non-antibody, the antibody is further reacted with an antibody labeled with a fluorescent substance, and the fluorescence after the reaction is observed.
JP 2005-168498 A JP 2005-151854 A

  However, in the DNA chip method and the fluorescent antibody method, hybridization or reaction in a sample (hereinafter referred to as “reaction” as a general term for hybridization and reaction by an antigen, an antibody, etc.) is not from DNA, antigen, antibody, etc. Fluorescence may cause noise. For this reason, it is usually necessary to clean and remove unreacted DNA, antigens, antibodies, and the like after washing the fluorescent image detection substrate, resulting in a problem of reduced work efficiency. In the above-described DNA chip method and fluorescent antibody method, when the sample concentration is low, the observed fluorescence intensity is weak. Therefore, the fluorescence intensity is buried in the background noise, and there is a problem that erroneous detection occurs. Furthermore, since it is difficult to identify the fluorescent image when the probes overlap, the chip where the probes overlap is treated as a defective product, resulting in a decrease in yield. In addition, when the number of probes increases, the area of the substrate increases, which makes it difficult to ensure reaction uniformity and increases the amount of sample required.

  The present invention has been made in order to solve the above-described problems, and an object of the present invention is to improve the DNA chip method and the fluorescent antibody method, and to perform the method more efficiently and with high accuracy. An object of the present invention is to provide a substrate, an apparatus, a program, and a recording medium to be used.

  The present invention is a fluorescent image detection method capable of preventing erroneous detection and the like even when the sample concentration is low. The object of the present invention is to label a plurality of probe substances fixed on a fluorescent image detection substrate with a fluorescent substance. In the fluorescence image detection method for detecting the reaction between the probe substance and the target substance by reacting the target substance on the fluorescence image detection substrate and measuring the fluorescence fluorescence image of the reaction product after the reaction, This is effectively achieved by fixing the fluorescent image detection substrate so that at least one probe pad made of the plurality of probe substances has a geometric pattern, and measuring the fluorescent image.

  Further, the above-described object of the present invention is to fix the probe pads by fixing the probe pads to the fluorescent image detection substrate in an array when there are two or more probe pads, or when there are two or more probe pads. This is achieved more effectively by fixing it to the fluorescent image detection substrate.

  Furthermore, the object of the present invention is to detect the correlation between the fluorescence distribution and the geometric pattern distribution for each probe substance after the reaction, or the geometric pattern is a pattern to which a pseudo-random code or orthogonal code is applied. Or a geometric pattern is a striped pattern having an equal pitch width, or a geometric pattern is a pattern to which a two-dimensional code is applied, or a pseudo-random pattern. In the code, orthogonal code, two-dimensional code, and striped pattern, there are locations where the probe material is fixed and locations where the probe material is not fixed, or the geometric pattern is a striped pattern. By performing Fourier transform instead of detection, or the probe substance is probe DNA, If the target substance is a target DNA, the reaction and, by a hybridization, it is more effectively achieved.

  An object of the present invention is to provide a fluorescent image detection substrate capable of identifying a fluorescent image even if probe substances overlap, and the object of the present invention is to provide a fluorescent image in which a plurality of probe substances are fixed on at least one surface. In the detection substrate, the plurality of probe substances are effectively achieved by being fixed in a geometric pattern so as to constitute at least one probe pad.

  Further, the above object of the present invention is to fix the probe pads in an array when there are two or more probe pads, or by stacking and fixing the probe pads when there are two or more probe pads, Or, the geometric pattern is a pattern to which a pseudo random code or orthogonal code is applied, or the geometric pattern is a striped pattern having a pitch width of equal intervals, or a geometric pattern The shape is a pattern to which a two-dimensional code is applied, or in a pseudo-random code, orthogonal code, two-dimensional code, and striped pattern, the portion where the probe material is fixed and the probe material are not fixed This is achieved more effectively.

  An object of the present invention is to provide a fluorescence image detector that can detect a fluorescence image without erroneous detection even if the sample concentration is thin, and the object of the present invention is to provide a light source that emits excitation light, A plurality of probe substances in at least one probe pad fixed in a geometric pattern on the fluorescent image detection substrate are reacted with a target substance labeled with a fluorescent substance, and excitation light is applied to the reaction product after the reaction. Irradiate, observe the fluorescence intensity distribution excited by the fluorescent substance, convert the observed fluorescence intensity distribution into an electrical signal, and correlate the electrical signal amplified by the light detection part with the light detection part that amplifies the electrical signal A fluorescence image detector comprising a correlation detection unit for detection and a data input / output unit for inputting and outputting an electrical signal detected by the correlation detection unit, wherein the data input / output unit inputs and stores external information. The input / stored external information is transferred to the correlation detector, and the correlation detector configures the geometric pattern distribution based on the transferred external information and detects the correlation between the electrical signal and the geometric pattern distribution. Is achieved.

  Further, the above object of the present invention is to fix the probe pads to the fluorescent image detection substrate in an array when there are two or more probe pads, or to stack the probe pads when there are two or more probe pads. By fixing to the fluorescent image detection substrate, or the geometric pattern shape is a pattern to which a pseudo random code or orthogonal code is applied, or the geometric pattern shape is a striped pattern having a pitch width of equal intervals. The probe material is fixed by being a pattern, or by being a pattern in which a two-dimensional code is applied, or in a pseudo-random code, orthogonal code, two-dimensional code and striped pattern. Having a portion where the probe material is not fixed, or the geometric pattern is a striped pattern. If the probe substance is a probe DNA and the target substance is a target DNA, the reaction is a hybridization. Achieved more effectively.

  Further, the above object of the present invention is that the external information is the size of the fluorescent image detection substrate, the size of the probe pad, the size of the geometric pattern of each probe substance, the applied geometric pattern, or When the probe substance is fixed to the fluorescence image detection substrate by applying a pseudo-random code, orthogonal code or two-dimensional code, as the external information, the location where the probe substance is fixed and the probe substance are not fixed When the probe substance is fixed to the fluorescent image detection substrate by inputting the total number of points and the size of each probe substance or by applying a striped pattern, the probe substance is further fixed as external information. This is achieved more effectively by inputting the total number of locations where the probe material is not fixed and the pitch width.

  An object of the present invention is to provide a program for causing a computer to execute a fluorescent image detection method, and an object of the present invention is to provide a plurality of probes in at least one probe pad fixed in a geometric pattern on a fluorescent image detection substrate. The probe substance and the target substance labeled with a fluorescent substance are reacted. After the reaction, the reaction material is irradiated with excitation light, and the distribution of the fluorescence intensity excited by the fluorescent substance is observed. A step of converting to an electric signal and amplifying the electric signal, a step of reading external information input in advance from the electric signal amplified in the step, and a geometric pattern distribution based on the external information input in the step A step, a step of detecting a correlation between the electrical signal and the external information, and a step of outputting and displaying the degree of correlation obtained by the step. By executing the fluorescent image detecting method was example to a computer, it is effectively achieved.

  Further, the above object of the present invention is to fix the probe pads to the fluorescent image detection substrate in an array when there are two or more probe pads, or to stack the probe pads when there are two or more probe pads. By fixing to the fluorescent image detection substrate, or the geometric pattern shape is a pattern to which a pseudo random code or orthogonal code is applied, or the geometric pattern shape is a striped pattern having a pitch width of equal intervals. The probe material is fixed by being a pattern, or by being a pattern in which a two-dimensional code is applied, or in a pseudo-random code, orthogonal code, two-dimensional code and striped pattern. Having a portion where the probe material is not fixed, or the geometric pattern is a striped pattern. If the probe substance is a probe DNA and the target substance is a target DNA, the reaction is a hybridization. Achieved more effectively.

  Further, the above object of the present invention is that the external information is the size of the fluorescent image detection substrate, the size of the probe pad, the size of the geometric pattern of each probe substance, the applied geometric pattern, or When the probe substance is fixed to the fluorescence image detection substrate by applying a pseudo-random code, orthogonal code or two-dimensional code, as the external information, the location where the probe substance is fixed and the probe substance are not fixed When the probe substance is fixed to the fluorescent image detection substrate by inputting the total number of points and the size of each probe substance or by applying a striped pattern, the probe substance is further fixed as external information. This is achieved more effectively by inputting the total number of locations where the probe material is not fixed and the pitch width.

  The object of the present invention can also be achieved by providing a computer-readable recording medium characterized by recording the above-described program.

  By the probe substance being probe DNA, RNA, antigen, antibody or protein, or the target substance being target DNA, RNA, antigen, antibody or protein, the above object of the present invention is more effective. Is achieved.

  According to the fluorescence image detection method of the present invention, the probe substance is geometrically patterned and fixed on the fluorescence image detection substrate, and the fluorescence intensity and the geometric pattern distribution of each probe substance after the reaction are detected by correlation to detect the sample concentration. Even when the concentration is low, the S / N ratio between the background noise and the fluorescence signal can be improved, so that erroneous detection can be prevented. Further, by performing correlation detection, even if the fluorescence intensity is weak, it can be detected, and even a low-contrast fluorescence image can be detected, so that the washing step after the reaction is omitted or simplified. As a result, the working efficiency of the DNA chip method and the fluorescent antibody method can be improved, and the measurement can be performed more quickly and without waste.

  Furthermore, according to the fluorescent image detection method of the present invention, the probe substance is fixed in a geometric pattern instead of being fixed in a spot form, so that the probe substance can be identified from the geometric pattern of the fluorescent image after the reaction. Became. As a result, fluorescence can be measured even when probe substances overlap with each other, and the yield can be improved.

  According to the fluorescent image detection substrate used in the fluorescent image detection method of the present invention, a probe chip having a geometric pattern is overlapped and fixed on at least one surface of the fluorescent image detection substrate. It has become possible to provide a fluorescent image detection substrate for use in a fluorescent antibody method or the like.

  According to the fluorescence image detector of the present invention, even if the fluorescence intensity is weak, the distribution of the excited fluorescence intensity of the fluorescent substance of the reaction product is amplified after being converted into an electrical signal, and correlation detection is performed. Now it can be detected. In addition, when performing correlation detection, it is possible to prevent erroneous detection by inputting and storing external information such as the geometric pattern of the probe material in advance in the data input / output unit. You can now do things.

  According to the program of the present invention, the above-described fluorescent image detection method can be performed efficiently and with high accuracy.

  Furthermore, according to the storage medium of the present invention, it is possible to obtain the same effect as the above-described fluorescent image detection method by causing a computer to read and execute a program recorded on the recording medium.

  In the present invention, the probe substance is not spotted and fixed on the fluorescent image detection substrate, but is fixed to the fluorescent image detection substrate in a geometric pattern to react with the target substance, and after the reaction, correlation detection is performed as necessary. It is characterized by that.

  When the probe substance is probe DNA, the probe DNA is not spot-fixed on the fluorescence image detection substrate, but is immobilized on the fluorescence image detection substrate in a geometric pattern, and the target DNA is hybridized. The correlation detection is performed as necessary. As a result, the fluorescence can be measured even if the probe substances or the probe DNAs overlap with each other, and the fluorescence can be detected even if the fluorescence intensity is weak.

  In the present invention, probe DNA, RNA, antigen, antibody, protein or the like can be applied as a probe substance.

  Further, target DNA, RNA, antigen, antibody, protein or the like can be applied as a target substance.

  Hereinafter, the fluorescent image detection method, fluorescent image detection substrate, fluorescent image detector, program, and storage medium of the present invention will be described in detail, but the present invention is not limited to these.

  The fluorescence image detection method of the present invention will be described in detail with reference to the drawings.

  FIG. 1 is a flowchart showing an example of the procedure of the first embodiment of the fluorescent image detection method of the present invention.

  In a normal fluorescent image detection method, the probe substance is spotted and fixed in an array on a fluorescent image detection substrate such as a slide glass. In the present invention, the fixed position is formed in a geometric pattern without spotting (step) 12). FIG. 2 is a diagram showing an example in which the probe substance is fixed in a geometric pattern. In the present invention, the probe substance is not fixed in a spot shape as in the prior art, but a pseudo random code, an orthogonal code, a striped pattern, etc. are applied as shown in (A) to (I) of FIG. Into a geometric pattern. The probe material is fixed to the black portion in FIG. 2 and the probe material is not fixed to the white portion, and the probe material is arranged in a striped pattern, a pseudo random code, and an orthogonal code pattern (shape) as a whole. To fix. That is, the probe substance is fixed to the fluorescent image detection substrate in the form of a barcode. The pattern of the black portion in FIG. 2 where the probe substance is fixed and the entire white portion where the probe substance is not fixed is called a probe pad.

  The striped pattern, pseudo-random code, and orthogonal code are pseudo-random sequences that have a strong autocorrelation and a low possibility of existing in nature, and are code sequences that have a weak cross-correlation with the background component. For this reason, it is widely used in digital spread communication in the field of information communication such as mobile phones where background noise is a problem.

  Various known pseudo-random codes and orthogonal codes can be applied to the pseudo-random code and the orthogonal code applied in the present invention, and examples thereof include an M sequence (Maximum length sequence) and a Golden sequence code.

  Since the target substance labeled with the fluorescent substance is uniformly bonded to the probe substance after the reaction, the fluorescent image detection substrate emits light uniformly. At this time, if there is background light, it cannot be distinguished whether the measured luminescence is background light or fluorescence emitted by the reaction product. In the present invention, when the probe substance is preliminarily geometrically patterned and fixed on the fluorescence image detection substrate, when the fluorescence intensity distribution is observed, light emission in the same form as the patterned and fixed pattern is observed. For example, it can be distinguished that the emitted light is not due to background light or other noise, but is fluorescence emitted by the reaction product. Furthermore, if different geometric patterns are applied to different probe substances, it is possible to distinguish which probe substance corresponds to the observed fluorescence even if different probe substances overlap.

  In addition to the above-described one-dimensional code, a two-dimensional code as shown in FIG. 2 (I) can also be applied for fixing the probe substance according to the present invention by a geometric pattern. In the present invention, as the applicable two-dimensional code, for example, various known two-dimensional codes such as a 2DQR code, a stack type two-dimensional code, and a matrix type two-dimensional code can be applied. Further, an error correction code can be included in the code.

  Whether the probe substance is fixed on the fluorescent image detection substrate by applying any one of the striped pattern, pseudo-random code, orthogonal code, and two-dimensional code can be appropriately changed according to the purpose of use and the method of use. Further, geometric patterns other than those shown in FIG. 2 may be taken, and there is no particular limitation as long as the geometric arrangement has a low correlation with background light such as orthogonal codes and noise.

  Further, by fixing the probe pad in a geometric pattern, the ID of the probe substance can be embedded using a technique such as a barcode or a 2DQR code. Thereby, information embedded in advance at the time of detection is decoded, and it is possible to easily identify which probe corresponds to the probe substance emitting fluorescence after the reaction.

  FIG. 3 is a diagram showing an example in which a probe substance is patterned by applying an M sequence of pseudo-random codes. For example, a location expressed by “0” in the binary system is a location where the probe substance is not fixed (white portion in FIG. 3), and a location expressed by “1” is the location where the probe substance is fixed. (Shown in black in FIG. 3). A probe pad is a group of such portions (unit patterns), and the probe substance fixing method (probe pad fixing method) uses a microlithography technique, uses an ink jet printer, or the like. It can be fixed by various methods.

  The arrangement and setting of the position where the probe substance of the M series and the Golden series orthogonal code is not fixed (the width of the white portion) and the position where the probe substance is fixed are the case where the probe substance is not fixed in the same region (No. 1) 1 embodiment), which is not particularly limited as long as it has a low correlation with background noise components.

  If the pitch width of the portion where the probe substance is not fixed (the white portion in FIG. 3) is p (μm), noise having a spatial frequency other than 1 / p can be removed by correlation detection. Therefore, it is preferable to make the pitch width as small as possible in consideration of the size of the fluorescent image detection substrate and the resolution of the fluorescent image measurement. In addition, when the number of unit patterns (total number) of the portion where the probe substance is fixed (black portion in FIG. 3) is n, the S / N ratio is proportional to the number of unit patterns where the probe substance is fixed. n times and the fluorescence intensity becomes (√n) times. When the size of the fluorescent image detection substrate is L and the resolution at which the probe material can be fixed is d, the probe material having the number of unit patterns of L / d can be fixed on the fluorescent image detection substrate.

In the case of fixing with a striped pattern, a striped pattern with an equal pitch width (width of white portions) is applied. When the pitch width is set to p (μm), noise having a spatial frequency other than 1 / p can be removed by correlation detection. Therefore, the pitch width p is preferably as small as possible in consideration of the size L of the fluorescent image detection substrate. When the number of unit patterns of the striped pattern (total number of black portions in FIG. 2) is n, the S / N ratio is n times in proportion to the number of unit patterns of the striped pattern, and the fluorescence intensity is (√n) times. It becomes. If the resolution at which the probe substance can be fixed is d, the number of unit patterns of L / 2d can be obtained. Even if the number of unit patterns cannot be increased due to the size L of the fluorescent image detection substrate, the S / N ratio can be improved by repeating the number of scans of the fluorescence intensity distribution multiple times. Can be made. In addition, for example, a two-dimensional arrangement such as a 2DQR code can fix (L / d) ² number of unit substance probe substances on a fluorescent image detection substrate.

  FIG. 4 is a conceptual diagram of a fluorescent image detection substrate of the present invention in a fluorescent image detection method such as a DNA chip method. Each probe substance is fixed in a geometric pattern on at least one surface of the fluorescent image detection substrate 1. The probe pad 2 is composed of a part where the probe substance is fixed (black part in FIG. 4) and a part where the probe substance is not fixed (white part in FIG. 4). They are arranged in an array and fixed so as to be in a geometric pattern instead of a spot. In the present invention, the probe pad 2 includes a portion where the probe substance is fixed and a portion where each probe substance is not fixed (each portion is referred to as a unit pattern 3). 4 shows a case where the fluorescent image detection substrate 1 is fixed in the same geometric pattern from A to X, but may be fixed in different geometric patterns. Needless to say, the number of probe pads 2 can be changed as needed, and at least one probe pad 2 is sufficient. Further, for example, each probe pad 2 is fixed so as to form different geometric patterns such as A to H in FIG. 4 as pseudo random codes, I to P as orthogonal codes, and Q to X as stripe patterns. You can also.

  Further, the combination of the location where the probe substance of the unit pattern 3 is fixed and the location where the probe substance is not fixed can be arbitrarily changed according to the purpose of use, the method of use, and the like.

  As the material of the fluorescent image detection substrate 1 of the present invention, various known materials such as a slide glass and a hard glass can be used, and the shape and the like are not particularly limited. It can be changed as appropriate according to the situation.

  Further, although details will be described later, the probe pads 2 may be overlapped and fixed. Note that “fixing the probe pads 2 on top of each other” means that the probe material is not fixed on the probe material, but is fixed in the same region on a macro scale. In a typical scale, it means that each probe substance is fixed to the fluorescent image detection substrate 1.

  Next, the whole is made to react with a target substance on the fluorescence image detection board | substrate 1 shown in FIG. 4 (step 13). When the probe substance is probe DNA and the target substance is target DNA, hybridization is performed on the entire fluorescence image detection substrate 1.

  After the reaction, the distribution of the fluorescence intensity emitted by the reaction product is observed (step 14). When the fluorescence emitted by the reaction product is weak, the correlation of the fluorescence intensity distribution is detected (step 15). When correlation detection is performed on the fluorescence intensity distribution, a geometric pattern distribution p (x) described later is also detected.

  Usually, the fluorescence image detection substrate 1 is washed after the reaction. However, since a noise component can be removed by performing correlation detection described later, in the present invention, the step of washing the fluorescence image detection substrate 1 and removing the floating target material is omitted. Or it can be simplified. It should be noted that correlation detection can be omitted as necessary, such as when the sample concentration is high.

  FIG. 5 is a diagram showing a spatial distribution of fluorescence intensity detected after the reaction between the probe substance and the target substance. For convenience of explanation, a case where the probe substance is fixed in one dimension in the M series will be described.

  As shown in FIG. 5A, the x-axis is taken in the pattern direction. As shown in FIG. 5B, the portion where the probe substance is fixed takes 1, and the portion where the probe substance is not fixed is -1. Let p (x) be a function (pattern signal) that takes the following (hereinafter, p (x) is referred to as a geometric pattern distribution). As shown in FIG. 5C, the spatial distribution of the fluorescence intensity detected after the reaction is defined as s (x), and the correlation I between p (x) and s (x) is calculated by (Equation 1).

最後に相関Ｉが大きい値を示したプローブパッドのプローブに結合する相補的な物質が検出されたと判断する。

Finally, it is determined that a complementary substance that binds to the probe of the probe pad having a large correlation I value has been detected.

  In order for the correlation method to function effectively, p (x) is selected such that the correlation between the background noise distribution and p (x) is small. Since the correlation between the fluorescence intensity spatial distribution from the target substance after the reaction and p (x) is high, the influence of the background can be eliminated. Even in the case of patterning in two dimensions, the two-dimensional distribution p (x, y) can be calculated by (Equation 4). As a result, even if the fluorescence intensity is weak, it can be distinguished from noise, so that the target substance can be detected.

縞状パターンでは、特定の空間周波数をもつため、相関検出のかわりにフーリエ変換を行なうことにより、検出感度を向上させることもできる。すなわち、ｓ（ｘ）をフーリエ変換し、予め設定した縞状パターン固有の空間周波数の強度を相関Ｉの代わりに利用することもできる。

Since the striped pattern has a specific spatial frequency, detection sensitivity can be improved by performing Fourier transform instead of correlation detection. That is, s (x) can be Fourier-transformed, and the intensity of the spatial frequency unique to the stripe pattern can be used instead of the correlation I.

  When the target material includes a plurality of target materials, the concentration per area of the probe material j at the point i on the fluorescence image detection substrate 1 is c (ij). Of the target materials contained in the target material, the concentration of the probe material complementary to the target material j is d (j). The fluorescence intensity distribution I (i) that can be obtained in this case is

のようになる。ここで、αは比例定数である。

become that way. Here, α is a proportionality constant.

  Also, when (Equation 3) is expressed in matrix form,

となる。｛Ｉ｝はＩ（ｉ）をｉ番目成分とするベクトル、｛ｄ｝はｄ（ｊ）をｊ番目成分とするベクトル、［Ｃ］はｃ（ｉｊ）を（ｉｊ）成分とするマトリックスである。

It becomes. {I} is a vector having I (i) as the i-th component, {d} is a vector having d (j) as the j-th component, and [C] is a matrix having c (ij) as the (ij) component. .

  Further, when the fluorescence intensity distribution is obtained, [C] is known at the time of producing the fluorescence image detection substrate 1, and thus the dimensionless concentration α {d} of the target material can be obtained as shown in (Formula 5).

この場合、マトリックスＣが正方でない場合や正則でない場合は適宜最小二乗法、一般逆行列等を利用して解を導出する。また、｛ｄ｝に関する制約や予め得られる情報を制約条件として最適化問題として定式化して、解を求めることもできる。例えば、濃度は正の値であり、上限値が予め得られている場合等これらの情報を制約として｛ｄ｝を決定する。

In this case, when the matrix C is not square or regular, a solution is derived using a least square method, a general inverse matrix, or the like as appropriate. It is also possible to obtain a solution by formulating a constraint on {d} and information obtained in advance as an optimization problem using a constraint condition. For example, if the density is a positive value and the upper limit value is obtained in advance, {d} is determined with these information as constraints.

  After the correlation detecting means (step 15) is finished, the measurement is finished (step 16).

  FIG. 6 is a flowchart showing an example of the procedure of the second embodiment of the fluorescent image detection method according to the present invention. In addition, description of the part which overlaps with 1st Embodiment mentioned above is abbreviate | omitted.

  In the second embodiment, unlike the first embodiment, the probe pads 2 having a geometric pattern are stacked and fixed on the fluorescent image detection substrate 1 (step 22).

  FIG. 7 is a conceptual diagram showing a state in which the probe pad 2 is overlapped and fixed. Conventionally, since the probe pad 2 has the same shape such as a square or a circle for different probe materials, it cannot be distinguished which spot reaction product emits light when the probe pad 2 overlaps. However, in the present invention, since the probe substance is fixed in a geometric pattern, even if the probe pad 2 is overlapped and fixed, it can be easily distinguished from which reaction product the light is emitted. Become.

  FIG. 7A is a diagram showing a case where a probe substance is overlapped and fixed by a conventional method, and FIG. 7B shows a fluorescence intensity after reacting with a target substance by the method. It is the figure which showed the mode at the time of observing distribution of. FIG. 7C is a diagram showing a case where the probe pad 2 is overlapped and fixed using different geometric patterns, and FIG. 7D shows the fluorescence intensity after reacting with the target substance by the method. It is the figure which showed the mode at the time of observing distribution.

  When 26 types of probe pads 2 from A to Z are stacked and fixed as shown in FIG. 7A, the reaction products detected as shown in FIG. Even if the fluorescent substance emits fluorescence, since the conventional method discriminates at the position of the spot, it cannot be distinguished which reaction product reacted with which probe substance emits fluorescence.

  On the other hand, as shown in FIG. 7C, when the probe pad 2 is fixed in a different geometric pattern with respect to different probes, A to Z can be identified from the geometric pattern of the probe pad 2. Even if the probe pads 2 are overlapped, it is possible to distinguish which reaction product reacts with which probe substance emits fluorescence. In addition, since the area of the substrate can be reduced by stacking and fixing the probe pads, there are effects such as ensuring the uniformity of the reaction and reducing the required sample amount.

  Examples of a method for fixing the probe pad 2 on the fluorescent image detection substrate 1 in an overlapping manner include, for example, a method in which a solution containing a probe material is sequentially stacked and spotted as shown in FIG. There are various known methods such as a method of mixing and spotting a solution containing.

  For example, patterning is performed by assigning ID numbers representing different probe substances in three-digit binary numbers like Sample A to Sample G in Table 1. That is, the probe pad has three unit patterns. In Table 1, “1” in the binary system indicates a position where the probe substance is fixed, and “0” in the binary system indicates a position where the probe substance is not fixed.

蛍光画像検出基板１上に、表１中の３桁目が「１」であるプローブ物質（試料Ａ、試料Ｂ、試料Ｃ、試料Ｄ）を重ねて固定する（以下、「プローブ物質３」という）。また、プローブ物質３を固定した蛍光画像検出基板１上に、プローブ物質３と重ならないように、表１中の２桁目が「１」であるプローブ物質（試料Ａ、試料Ｂ、試料Ｅ、試料Ｆ）を重ねて固定する（以下、「プローブ物質２」という）。さらに、プローブ物質２およびプローブ物質３を固定した蛍光画像検出基板１上に、プローブ物質２およびプローブ物質３と重ならないように、表１中の１桁目が「１」であるプローブ物質（試料Ａ、試料Ｃ、試料Ｅ、試料Ｇ）を重ねて固定する（以下、「プローブ物質１」という）。

A probe substance (sample A, sample B, sample C, sample D) whose third digit in Table 1 is “1” is superimposed and fixed on the fluorescence image detection substrate 1 (hereinafter referred to as “probe substance 3”). ). In addition, on the fluorescent image detection substrate 1 to which the probe substance 3 is fixed, the probe substance (sample A, sample B, sample E, second digit in Table 1 is “1” so as not to overlap with the probe substance 3. Sample F) is stacked and fixed (hereinafter referred to as “probe substance 2”). Further, on the fluorescent image detection substrate 1 on which the probe substance 2 and the probe substance 3 are fixed, the probe substance (sample) whose first digit is “1” in Table 1 so as not to overlap the probe substance 2 and the probe substance 3. A, Sample C, Sample E, and Sample G) are stacked and fixed (hereinafter referred to as “probe substance 1”).

  As shown in FIG. 9, after the probe substance 1, the probe substance 2 and the probe substance 3 are all fixed on the same fluorescent image detection substrate 1, they are reacted with the target substance on the fluorescent image detection substrate 1 (step 23).

  After the reaction, the distribution of the fluorescence intensity emitted by the reaction product is observed (step 24). For example, if it is assumed that the probe substance of the sample C and the target substance have reacted, the fluorescence emitted by the reaction product will be emitted from the probe substance 1 and the probe substance 3 containing the sample C. From Table 1, it can be determined that the sample C has reacted. After observation of the fluorescence intensity distribution (step 24), correlation detection is performed on the fluorescence intensity distribution as necessary (step 25).

In the above, an example in which there are seven types of probe substances of sample A to sample G has been described. For example, in the case of 1000 types of probe substances, 2 ¹⁰ becomes 1024, so that patterning is performed by applying a binary ID number. In this case, a probe pad having a unit pattern of 10 digits can be used. As a result, a substrate having 1000 arrays is required in the conventional method, but in the present invention, the density can be increased to a substrate composed of 10 unit patterns.

  Next, the fluorescence image detector of the present invention will be described in detail with reference to the drawings. In addition, description is abbreviate | omitted about the location which overlaps with the content mentioned above.

  FIG. 14 is a schematic view of a fluorescence image detector according to the present invention. Inside the fluorescent image detector main body 4 of the present invention, there are a light source 5 for irradiating excitation light, an excitation filter 7, and a dichroic mirror for reflecting the fluorescence so as to be perpendicular to the incident direction to change the optical path. 8 and an objective lens 10 for collecting excitation light through an XY scanner (galvano scanner) 9 is housed.

  In addition, a fluorescent filter 11 is installed on the opposite side of the XY scanner 9 through the dichroic mirror 8, and the distribution of the fluorescence intensity transmitted through the fluorescence filter 11 is observed, and the distribution of the observed fluorescence intensity is converted into an electrical signal. The photo detector 13 for amplifying the signal and the correlation detector 14 for detecting the correlation of the measurement data are contained in the main body 4.

  Excitation light is irradiated from the light source 5 in the main body 4, passes through the collimator lens 6, and light having a wavelength suitable for excitation is transmitted through the excitation filter 7. The excitation light is reflected perpendicularly to the incident direction of the excitation light by the dichroic mirror 8 and passes through the XY scanner 9. The excitation light transmitted through the XY scanner 9 is collected by the objective lens 10 and irradiates the fluorescence image detection substrate 1 with the excitation light.

  When excitation light is irradiated to the reaction product resulting from the reaction between the probe substance and the target substance labeled with the fluorescent substance, the fluorescent substance is excited and emits fluorescence. The fluorescence emitted by the reaction product passes through the objective lens 10, the XY scanner 9 and the dichroic mirror 8 again. The fluorescence emitted by the reaction product passes through the fluorescent filter 11 installed on the opposite side via the dichroic mirror 8 of the XY scanner 9, is collected by the condenser lens 12, and enters the light detection unit 13. .

  The light detection unit 13 observes the distribution of the fluorescence intensity emitted by the reaction product, and converts the observed fluorescence intensity distribution into an electrical signal. This electric signal is amplified by the light detector 13 and the correlation detector 14 detects the correlation. The correlation detection unit 14 has a data input / output unit (not shown), and previously inputs external information such as the size of the probe pad of the fluorescent image detection substrate 1 and the shape of the geometric pattern to the data input / output unit. Store the external information stored in the data input / output unit to the correlation detection unit 14. The correlation detection unit 14 forms a geometric pattern distribution based on the transferred external information, and performs correlation detection together with the electrical signal. The correlation-detected electrical signal is output by the data input / output unit.

  The light source 5 is not particularly limited as long as it emits excitation light. For example, various known light sources such as a laser, a tungsten lamp, and an LED can be used.

  The collimator lens 6 is mainly for making the light emitted from the light source 5 into parallel luminous fluxes, and various known collimator lenses can be used in the present invention.

  The excitation filter 7 passes light having a wavelength suitable for excitation from the excitation light emitted from the light source 5. Before passing the excitation light through the excitation filter 7, it may be transmitted through the collimator lens 6 and then through the excitation filter 7, or directly through the excitation filter 7 without passing through the collimator lens 6. You may make it make it.

  The dichroic mirror 8 is installed so as to reflect perpendicularly to the incident direction of the excitation light. In FIG. 14, the optical system of the excitation filter 7, the dichroic mirror 8, and the XY scanner (galvano scanner) 9 shows an example of a confocal scanner, but a non-confocal optical system or the like can also be applied. . Further, a normal half mirror or the like may be used instead of the dichroic mirror 8, and a prism or the like may be used instead of the XY scanner 9.

  The excitation light that has passed through the dichroic mirror 8 and the XY scanner 9 is collected by the objective lens 10 and irradiates the fluorescence image detection substrate 1 with the excitation light.

  The fluorescent filter 11 transmits only fluorescence having a specific wavelength. The fluorescent filter 11 can be appropriately changed, such as an LP filter, an SP filter, and a BP filter, depending on the fluorescence wavelength emitted by the reaction product.

  The condensing lens 12 condenses the fluorescence emitted by the reaction product, and the collected fluorescence enters the light detection unit 13. As the condensing lens 12 used in the present invention, various known condensing lenses can be used.

  When the light detector 13 observes the distribution of the fluorescence intensity emitted by the reaction product, it converts it into an electrical signal and amplifies it. For the light detection unit 13, various known amplifiers such as an array type detector such as a CCD, a photomultiplier tube (PMT), and a photodiode can be used. If a CCD is used for the light detection unit 13, the installation of the XY scanner 9 can be omitted.

  Even when the probe pad 2 is fixed on the fluorescent image detection substrate 1 in a geometric pattern, the distribution of the fluorescence intensity emitted by the reaction product is converted and amplified into an electrical signal.

  The electrical signal amplified by the light detection unit 13 is subjected to correlation detection by the correlation detection unit 14.

  The correlation detection unit 14 includes a data input / output unit (not shown), and inputs and stores external information in advance in the data input / output unit before measurement. The external information stored in the data input / output unit is transferred to the correlation detection unit 14, and a geometric pattern distribution is constructed based on the external information, and the correlation between the geometric pattern distribution and the electrical signal is detected. The correlation-detected electrical signal is output and displayed as a fluorescence image by the data input / output unit.

  As external information to be input to the data input / output unit in advance, for example, the size of each probe pad 2 of the fluorescent image detection substrate 1 and what geometric pattern is fixed in an array as shown in FIG. It is input whether the probe substance is fixed or the probe pad 2 is overlapped and fixed.

  When the probe pad 2 is fixed to the fluorescent image detection substrate 1 with a pseudo random code or orthogonal code, the code or code, the size of each unit pattern 3 of the probe pad 2, the total number of unit patterns 3, etc. are also combined. Enter.

  Further, when the probe pad 2 is fixed to the fluorescent image detection substrate 1 in a striped pattern, the total number of the positions where the probe substance is fixed and the positions where the probe material is not fixed, the pitch width, and the like are also input. It should be noted that these external information can be stored in a program or a storage medium described later.

  FIG. 15 is a circuit configuration diagram of the correlation detection unit 14, and the fluorescence generated from the reaction product when the geometric pattern of the fluorescence image detection substrate 1 is a striped pattern and the spatial angular frequency is ω. The scanned state is shown. Integration in correlation detection is performed by a low-pass filter (LPF) 15. In the correlation detection, a signal having a high correlation with the geometric pattern distribution p (x) is detected. Of the various signals included in the measurement signal s (x), only the component having a high correlation with the geometric pattern distribution p (x) becomes a direct current and can pass through the low-pass filter 15. A component having a low correlation with the pattern signal p (x) is removed by the low-pass filter 15 because it is converted into an AC signal having a frequency other than 0 Hz.

  Further, as shown in FIG. 16, the phase circuit 16 that adjusts the phase difference between the geometric pattern distribution p (x) and the measurement signal s (x) to 0 ° and inputs the geometric pattern distribution p (x) to the PSD 17 is used. You can also The correlation detection may be performed using an analog circuit, a digital circuit, or numerical correlation analysis using a computer and software.

  Furthermore, when the geometric pattern of the probe material is a striped pattern, a spatial frequency of a known striped pattern may be extracted in advance by performing Fourier transform instead of the correlation detection.

  Next, the program of the present invention will be described in detail with reference to the drawings. In addition, description is abbreviate | omitted about the location which overlaps with the content mentioned above.

  FIG. 16 is a flowchart showing the flow of processing of the program of the present invention. Steps 32 to 36 correspond to the program of the present invention, and the degree of correlation is analyzed by a computer based on the digital image data such as jpeg and the information such as the geometric pattern of the probe substance by the fluorescence image detector of the present invention.

  In the operation of the program of the present invention, first, the distribution of the fluorescence intensity emitted by the reaction product of the probe substance and the target substance is observed, converted into an electric signal by the light detection unit 13, and amplified (step 32).

  The distribution of the fluorescence intensity emitted by the reaction product is observed, converted into an electrical signal, amplified, and then external information relating to the geometric pattern of the probe substance input and stored in advance by the data input / output unit is read (step 33). Specifically, it is determined in what geometric pattern the probe material is fixed to the fluorescent image detection substrate 1 and whether the probe material is fixed to the fluorescent image detection substrate 1 in an overlapping manner. When the probe substance is stacked and fixed, it is read after confirming which unit pattern 3 emits fluorescence.

  When the probe substance is fixed to the fluorescent image detection substrate 1 with a pseudo-random code or orthogonal code, the size of each unit pattern 3 of the probe pad 2, the total number of unit patterns 3, etc. are also confirmed and read. .

  Further, when the probe substance is fixed to the fluorescent image detection substrate 1 in a striped pattern, the total number of the places where the probe substance is fixed and the places where the probe substance is not fixed, the pitch width, etc. are also confirmed and read.

  After the end of step 33, a geometric pattern distribution of the probe substance is constructed based on the read external information (step 34).

  After constructing the geometric pattern distribution of the probe substance, correlation detection is performed (step 35), and the correlation degree is output and displayed (step 36).

  The series of processes described above can be executed by hardware, but can also be executed by software. When a series of processing is executed by software, a computer in which a program constituting the software is incorporated in dedicated hardware, or a computer capable of executing the function of the program by installing the program It can also be installed from a recording medium or the like.

  The program of the present invention can also be stored in a computer-readable recording medium. The recording medium is, for example, a portable medium such as a flexible disk, a magneto-optical disk, a CD-ROM, an MO, or a DVD, a fixed medium such as a ROM, RAM, or HD, or a program via a network such as a LAN or the Internet. Various known communication media for holding a program such as a communication line or the like when transmitting a message are included.

  By executing the program read by the computer, not only the above-described series of processing is executed, but also the OS (operating system) running on the computer is a part of the actual processing or based on the instruction of the program. It is also possible to perform all of the above and realize the above-described series of processes.

  Furthermore, after the program read from the storage medium is written to the memory provided in the function expansion board inserted into the computer or the function expansion unit connected to the computer, the function expansion is performed based on the instructions of the program. The CPU or the like provided in the board or the function expansion unit may perform part or all of the actual processing, and the above-described series of processing may be executed by the processing.

  EXAMPLES Hereinafter, although this invention is demonstrated in detail using an Example, this invention is not limited to these.

  A fluorescent dye (fluorescein solution) was printed on the fluorescent image detection substrate 1 in a striped pattern as shown in FIG. The pitch width p of the striped pattern was 0.2 mm, and the number of unit patterns was 30 (hereinafter referred to as Example 1). Further, as shown in FIG. 10B, as a comparative example, a fluorescent dye was uniformly printed in a rectangular region under the same conditions as in Example 1. Example 1 and the comparative example were measured for fluorescence intensity with a microscope under poor conditions with strong background light and weak excitation light. In the measurement, excitation light was blocked with a filter having a cutoff wavelength of 500 nm.

  The measurement results are shown in FIG. The gradation of the image is 256 gradations. White indicates fluorescence intensity 0, and black indicates that fluorescence intensity is high.

  The distribution of fluorescence intensity is shown in FIG. FIG. 12 shows the data scan position. A-A ′ represents the distribution on the pattern. BB ′ represents a uniform distribution on the pad in the conventional method. CC ′ represents the distribution of detection light in a region where the fluorescent dye is not fixed.

  Table 2 shows signal and noise powers and S / N ratios obtained in the examples together with comparative examples.

従来法ではＳ／Ｎ比が０．１３程度であるのに対し、本発明では、７．１３となり、約５５倍Ｓ／Ｎ比が向上することが確認された。このことにより、本発明は、バックグラウンドのノイズと蛍光信号のＳ／Ｎ比を向上させることができるので、誤検出を防止することができることがわかる。また、相関検出を行なうことにより、蛍光強度が微弱であってもシグナルを誤検出することなく検出することができることがわかる。

While the S / N ratio is about 0.13 in the conventional method, it is 7.13 in the present invention, and it has been confirmed that the S / N ratio is improved by about 55 times. Thus, it can be seen that the present invention can improve the S / N ratio between the background noise and the fluorescence signal, thereby preventing erroneous detection. It can also be seen that by performing correlation detection, a signal can be detected without erroneous detection even if the fluorescence intensity is weak.

It is a flowchart which shows the example of the procedure of 1st Embodiment of this invention. It is the figure which showed an example which arrange | positions a probe substance in a pattern. It is the figure which extracted a part of fluorescence image detection board | substrate at the time of applying a M series and patterning a probe substance. It is a conceptual diagram of the DNA chip fluorescence image detection board | substrate of this invention. (A): It is the figure which showed the probe substance patterned geometrically. (B): It is the figure which used what was geometrically patterned as a function. (C): Spatial distribution of fluorescence intensity. It is a flowchart which shows the example of the procedure of 2nd Embodiment of this invention. (A): It is the figure which showed the case where the probe substance was piled up and fixed using the conventional method. (B): It is the figure which showed the mode at the time of performing fluorescence detection by the method of (a). (C): It is the figure which showed the case where the geometrically patterned probe substance was piled up and fixed. (D): It is the figure which showed the mode at the time of performing fluorescence detection by the method of (c). It is the figure which showed the method of overlapping and spotting the solution containing a probe substance sequentially. It is the figure which showed the case where a probe substance is overlapped and hybridization is performed. (A): It is the figure which showed the pattern of the printed striped pattern used in the Example. (B) It is the figure which showed the pattern of the comparative example. It is the figure which showed the fluorescence image obtained in the Example. It is the figure which showed the scanning position which scanned the data of fluorescence intensity distribution. It is a graph which shows fluorescence intensity distribution. It is the schematic of a fluorescence image detector. It is the figure which showed the example of a circuit structure at the time of scanning the fluorescence of a correlation detection part. FIG. 5 is a diagram illustrating a phase circuit of a pattern signal (x) and a measurement signal s (x) in a correlation detection unit. It is the flowchart which showed the process of the program of this invention.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Fluorescence image detection board | substrate 2 Probe pad 3 Unit pattern 4 Main body 5 Light source 6 Collimator lens 7 Excitation filter 8 Dichroic mirror 9 XY scanner (galvano scanner)
10 Objective lens 11 Fluorescence filter (absorption filter)
12 Condensing Lens 13 Light Detection Unit 14 Correlation Detection Unit 15 Low Pass Filter (LPF)
16 Phase circuit 17 PSD

Claims (49)

  A plurality of probe substances fixed on the fluorescence image detection substrate and a target material labeled with the fluorescence substance are reacted on the fluorescence image detection substrate, and a fluorescence fluorescence image of the reaction product after the reaction is obtained. In the fluorescent image detection method for detecting a reaction between the probe substance and the target substance by measuring, the fluorescent image detection substrate is arranged such that at least one probe pad made of the plurality of probe substances has a geometric pattern. A fluorescence image detection method comprising fixing and measuring the fluorescence image.   The fluorescent image detection method according to claim 1, wherein when there are two or more probe pads, the probe pads are fixed to the fluorescent image detection substrate in an array.   The fluorescent image detection method according to claim 1, wherein when there are two or more probe pads, the probe pads are overlapped and fixed to the fluorescent image detection substrate.   4. The fluorescence image detection method according to claim 1, wherein a correlation between a fluorescence intensity distribution and a geometric pattern distribution is detected for each probe substance after the reaction.   The fluorescent image detection method according to claim 1, wherein the geometric pattern is a pattern to which a pseudo random code or an orthogonal code is applied.   The fluorescent image detection method according to claim 1, wherein the geometric pattern is a striped pattern having a pitch width of equal intervals.   The fluorescent image detection method according to claim 1, wherein the geometric pattern is a pattern to which a two-dimensional code is applied.   8. The device according to claim 1, wherein the pseudo random code, the orthogonal code, the two-dimensional code, and the striped pattern have a portion where the probe material is fixed and a portion where the probe material is not fixed. 9. The fluorescent image detection method as described in 2.   The fluorescent image detection method according to claim 1, 6 or 8, wherein when the geometric pattern is the striped pattern, Fourier transform is performed instead of the correlation detection.   The fluorescent image detection method according to claim 1, wherein the probe substance is probe DNA, RNA, an antigen, an antibody, or a protein.   The fluorescent image detection method according to claim 1, wherein the target substance is target DNA, RNA, an antigen, an antibody, or a protein.   The fluorescence image detection method according to claim 1, wherein when the probe substance is the probe DNA and the target substance is the target DNA, the reaction is hybridization.   A fluorescent image detection substrate having a plurality of probe substances fixed on at least one surface, wherein at least one probe pad made of the plurality of probe substances is fixed in a geometric pattern. .   The fluorescent image detection substrate according to claim 13, wherein when there are two or more probe pads, the probe pads are fixed in an array.   The fluorescent image detection substrate according to claim 13, wherein when there are two or more probe pads, the probe pads are overlapped and fixed.   The fluorescent image detection substrate according to claim 13, wherein the geometric pattern is a pattern to which a pseudo random code or an orthogonal code is applied.   The fluorescent image detection substrate according to claim 13 or 16, wherein the geometric pattern is a striped pattern having a pitch width of equal intervals.   The fluorescent image detection substrate according to claim 13, wherein the geometric pattern is a pattern to which a two-dimensional code is applied.   19. The pseudo random code, the orthogonal code, the two-dimensional code, and the striped pattern, each having a location where the probe material is fixed and a location where the probe material is not fixed. The fluorescent image detection substrate according to 1.   The fluorescent image detection substrate according to claim 13, wherein the probe substance is probe DNA, RNA, an antigen, an antibody, or a protein.   Reacting a light source for irradiating excitation light, a plurality of probe substances in at least one probe pad fixed in a geometric pattern on the fluorescent image detection substrate, and a target substance labeled with the fluorescent substance, A light detection unit that irradiates the reaction product with the excitation light after the reaction, observes the distribution of the fluorescence intensity excited by the fluorescent material, converts the observed fluorescence intensity distribution into an electrical signal, and amplifies the electrical signal A fluorescence image detector comprising: a correlation detection unit that detects correlation of the electrical signal amplified by the light detection unit; and a data input / output unit that inputs and outputs the electrical signal detected by the correlation detection unit The data input / output unit inputs / stores external information, transfers the input / stored external information to the correlation detection unit, and the correlation detection unit relies on the transferred external information. In What constitutes a pattern distribution, the fluorescence image detector and detects the correlation of the electrical signal and the geometric pattern distribution.   The fluorescence image detector according to claim 21, wherein when there are two or more probe pads, the probe pads are fixed to the fluorescence image detection substrate in an array.   The fluorescence image detector according to claim 21, wherein when there are two or more probe pads, the probe pads are overlapped and fixed to the fluorescence image detection substrate.   The fluorescent image detector according to any one of claims 21 to 23, wherein the geometric pattern is a pattern to which a pseudo random code or an orthogonal code is applied.   The fluorescent image detector according to any one of claims 21 to 24, wherein the geometric pattern is a striped pattern having a pitch width of equal intervals.   The fluorescence image detector according to any one of claims 21 to 25, wherein the geometric pattern is a pattern to which a two-dimensional code is applied.   27. The device according to claim 21, wherein in the pseudo-random code, the orthogonal code, the two-dimensional code, and the striped pattern, the probe material is fixed and the probe material is not fixed. The fluorescent image detector according to 1.   28. The fluorescence image detector according to claim 21, wherein when the geometric pattern is the striped pattern, Fourier transform is performed instead of the correlation detection.   The fluorescent image detector according to any one of claims 21 to 28, wherein the probe substance is probe DNA, RNA, an antigen, an antibody, or a protein.   30. The fluorescent image detector according to any one of claims 21 to 29, wherein the target substance is target DNA, RNA, an antigen, an antibody, or a protein.   31. The fluorescence image detector according to claim 21, wherein when the probe substance is the probe DNA and the target substance is the target DNA, the reaction is hybridization.   The external information is the size of the fluorescent image detection substrate, the size of the probe pad, the size of the geometric pattern of each probe substance, and the applied geometric pattern. The fluorescent image detector as described.   When the probe substance is fixed to the fluorescent image detection substrate by applying the pseudo-random code, the orthogonal code or the two-dimensional code, the external information further includes a place where the probe substance is fixed The fluorescence image detector according to any one of claims 21 to 24, 26, 27, and 29 to 32, wherein a total number of portions where the probe substance is not fixed and a size of each probe substance are input.   When the probe material is fixed to the fluorescence image detection substrate by applying the striped pattern, the external information includes a location where the probe material is fixed and a location where the probe material is not fixed. The fluorescence image detector according to any one of claims 21 to 23, 25, and 28 to 32, wherein a total number and the pitch width are input.   A plurality of probe substances in at least one probe pad fixed in a geometric pattern on a fluorescent image detection substrate are reacted with a target substance labeled with a fluorescent substance, and after the reaction, the reaction product is subjected to the excitation light. , Observing the fluorescence intensity distribution excited by the fluorescent substance, converting the observed fluorescence intensity distribution into an electrical signal, amplifying the electrical signal, and the electrical signal amplified by the step Reading external information input in advance; configuring a geometric pattern distribution based on the external information input in the step; detecting a correlation between the electrical signal and the external information; Outputting and displaying the degree of correlation obtained in this step, and causing a computer to execute a fluorescence image detection method comprising: Program.   36. The program according to claim 35, wherein when there are two or more probe pads, the probe pads are fixed to the fluorescent image detection substrate in an array.   The program according to claim 35, wherein when there are two or more probe pads, the probe pads are overlapped and fixed to the fluorescent image detection substrate.   The program according to any one of claims 35 to 37, wherein the geometric pattern is a pattern to which a pseudo random code or an orthogonal code is applied.   The program according to any one of claims 35 to 38, wherein the geometric pattern is a striped pattern having a pitch width of equal intervals.   40. The program according to claim 35, wherein the geometric pattern is a pattern to which a two-dimensional code is applied.   41. The device according to claim 35, wherein the pseudo random code, the orthogonal code, the two-dimensional code, and the striped pattern have a portion where the probe material is fixed and a portion where the probe material is not fixed. The program described in.   42. The program according to claim 35, wherein when the geometric pattern is the striped pattern, Fourier transform is performed instead of the correlation detection.   The program according to any one of claims 35 to 42, wherein the probe substance is probe DNA, RNA, an antigen, an antibody, or a protein.   44. The program according to any one of claims 35 to 43, wherein the target substance is target DNA, RNA, an antigen, an antibody, or a protein.   45. The program according to claim 35, wherein when the probe substance is the probe DNA and the target substance is the target DNA, the reaction is hybridization.   46. The program according to claim 35, wherein the external information is a size of the fluorescent image detection substrate, a size of the probe pad, and a size of the geometric pattern of each probe substance.   When the probe substance is fixed to the fluorescent image detection substrate by applying the pseudo-random code, the orthogonal code or the two-dimensional code, the external information further includes a place where the probe substance is fixed 45. The program according to any one of claims 35 to 38, 40, 41, and 43 to 44, wherein the total number of locations where the probe substance is not fixed and the size of each probe substance are input.   When the probe material is fixed to the fluorescence image detection substrate by applying the striped pattern, the external information includes a location where the probe material is fixed and a location where the probe material is not fixed. The program according to any one of claims 35 to 37, 39, and 42 to 44, wherein the total number and the pitch width are input.   49. A computer-readable recording medium on which the program according to any one of claims 35 to 48 is recorded.

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