JP2006300795A - Biological information measuring device and method, program, and recording medium - Google Patents

Abstract

An intensity of excitation light used for measuring a state of hybridization generated in a reaction region can be set without depending on an input by a user.
A scan for measuring the amount of hybridization generated at each spot 12 of a DNA chip 11 is performed separately for a pre-scan and a main scan. The pre-scan is a low-accuracy scan for detecting the fluorescence intensity (hybridization amount) of each spot 12 necessary for determining the intensity of excitation light used in the main scan. This is a highly accurate scan performed using excitation light having an intensity determined from the result. In the main scan, for example, excitation light having different intensity is used for each spot to be measured. The present invention can be applied to an apparatus for measuring the fluorescence intensity of a DNA chip.
[Selection] Figure 1

Description

  The present invention relates to a biological information measuring apparatus and method, a program, and a recording medium, and in particular, sets the intensity of excitation light used for measuring the state of hybridization that has occurred in a reaction region, regardless of user input. The present invention relates to a biological information measuring apparatus and method, a program, and a recording medium.

  In recent years, a DNA (deoxyribonucleic acid) chip or a DNA microarray (hereinafter simply referred to as a DNA chip when it is not necessary to distinguish between them) has been put into practical use. A DNA chip is a DNA chip in which various and many DNA oligo chains are integrated and immobilized on a substrate surface as nucleic acids for detection. Comprehensive analysis of gene expression in collected cells by detecting hybridization between probes immobilized on spots on the substrate surface and targets in samples collected from cells using a DNA chip Can do.

  With the improvement of hybridization detection technology in gene expression analysis using a DNA chip, it is becoming possible not only to detect the presence or absence of gene expression but also to quantitatively measure the gene expression level. For example, a technique for obtaining a quantitative numerical value indicating a gene expression level by quantitatively measuring fluorescence intensity at the time of hybridization detection has been partially put into practical use.

For example, Patent Documents 1 to 3 are prior art documents related to a method for analyzing and correcting a gene expression level obtained with a DNA chip or the like.
JP 2002-71688 A JP 2002-267668 Gazette JP 2003-28862 A

  By the way, the intensity of the excitation light used to obtain an expression profile image (an image obtained by irradiating the DNA chip with excitation light and scanning the generated fluorescence) used to quantitatively measure the fluorescence intensity is determined by the observer ( This causes a problem that the reliability of the hybridization detection result is lowered and the rate-determining site when the entire analysis of the expression profile image is performed. It was.

  In other words, since the observer sets the excitation light intensity, human noise is included in the expression profile image, and thus the measurement result using the expression profile image, and it takes time to set an appropriate intensity. It will take.

  The present invention has been made in view of such a situation, and sets the intensity of excitation light used for measuring the state of hybridization generated in a spot (reaction region) regardless of input by the user. Is to be able to.

  According to an aspect of the present invention, the first biological material and the second biological material in the reaction region are obtained from the fluorescence intensity obtained when the portion where the first biological material and the second biological material are hybridized is irradiated with excitation light. Is obtained from storage means for storing conversion information that is uniquely obtained for each intensity of excitation light, and fluorescence intensity obtained by the first measurement using the excitation light of the first intensity. Excitation light used for the second measurement of the state of hybridization of the first biological material and the second biological material based on the amount of hybridization between the first biological material and the second biological material and the conversion information A determination means for determining a second intensity higher than the first intensity, and a measurement control means for performing the second measurement using the excitation light having the second intensity determined by the determination means. Biological information measuring device characterized by It is.

  In the biological information measuring device of the present invention, the first biological material and the first biological material in the reaction region are obtained from the fluorescence intensity obtained when the portion where the first biological material and the second biological material are hybridized is irradiated with excitation light. Conversion information, which is information for which the amount of hybridization of the two biological substances is uniquely determined for each intensity of excitation light, is stored, and from the fluorescence intensity obtained by the first measurement using the excitation light of the first intensity Excitation used for the second measurement of the hybridized state of the first biological material and the second biological material based on the obtained amount of hybridization between the first biological material and the second biological material and the conversion information As the light intensity, a second intensity higher than the first intensity is determined. Further, the second measurement is performed using the excitation light having the determined second intensity.

  The measurement control means can cause the second measurement to be performed with the excitation light having the second intensity and having an irradiation range narrower than the irradiation range of the excitation light used for the first measurement.

  The determining means is configured to obtain the fluorescence intensity obtained by the second measurement based on the amount of hybridization between the first biological material and the second biological material obtained from the fluorescence intensity obtained by the first measurement, and the conversion information. However, the intensity of the excitation light having an intensity not exceeding the upper limit value of the fluorescence intensity set in the conversion information can be determined as the second intensity.

  The determining unit determines the second intensity for each reaction region provided on the substrate, and the measurement control unit targets each reaction region using the excitation light having the second intensity determined by the determining unit. As a result, the second measurement can be performed.

  According to the present invention, the intensity of excitation light used for measuring the state of hybridization that has occurred in the reaction region can be set regardless of input by the user.

  Embodiments of the present invention will be described below. The correspondence relationship between the invention described in this specification and the embodiments of the invention is exemplified as follows. This description is intended to assure that embodiments supporting the claimed invention are described in this specification. Therefore, although there is an embodiment which is described in the embodiment of the invention but is not described here as corresponding to the invention, it means that the embodiment is not It does not mean that it does not correspond to the invention. Conversely, even if an embodiment is described herein as corresponding to an invention, that means that the embodiment does not correspond to an invention other than the invention. Absent.

  Further, this description does not mean all the inventions described in this specification. In other words, this description is for the invention described in the present specification and not claimed in this application, i.e., the existence of an invention that will be filed in the future or added by amendment. There is no denial.

  The biological information measuring apparatus according to claim 1, wherein a second biological material (for example, a probe) fixed to a reaction region provided on a substrate and a second biological material that hybridizes to the first biological material. A biological information measuring device for measuring the state of hybridization with a biological material (for example, a target) when the portion where the first biological material and the second biological material are hybridized is irradiated with excitation light Conversion information (for example, fluorescence intensity), which is information that is uniquely determined for each intensity of excitation light, from the fluorescence intensity obtained in the reaction region, the amount of hybridization between the first biological material and the second biological material in the reaction region. -In the first measurement (prescan) using the storage means (for example, the fluorescence intensity-hybridization amount conversion formula part 30 in FIG. 1) for storing the hybridization amount conversion formula and the excitation light having the first intensity. Based on the amount of hybridization between the first biological material and the second biological material determined from the obtained fluorescence intensity, and the conversion information, the first biological material and the second biological material Determining means for determining a second intensity higher than the first intensity as the intensity of the excitation light used for the second measurement (main scan) of the hybridized state (for example, the excitation light intensity calculation unit 23 in FIG. 1) And a measurement control means (for example, the control unit 43 in FIG. 1) that performs the second measurement using the excitation light having the second intensity determined by the determination means.

  The biological information processing method according to claim 5, wherein a first biological material (for example, a probe) fixed to a reaction region provided on a substrate is hybridized with the first biological material. Fluorescence intensity obtained by a first measurement (prescan) using excitation light having a first intensity, which is a biological information measurement method for measuring the state of hybridization with two biological substances (for example, a target) Obtained when the amount of hybridization between the first biological material and the second biological material determined from the above and the portion where the first biological material and the second biological material are hybridized are irradiated with excitation light. From the fluorescence intensity, conversion information (for example, fluorescence intensity−high) is obtained by uniquely determining the amount of hybridization between the first biological substance and the second biological substance in the reaction region for each intensity of excitation light. The first intensity as the intensity of the excitation light used for the second measurement (main scan) of the hybridized state of the first biological substance and the second biological substance based on the conversion amount conversion formula) A determination step (for example, step S23 in FIG. 6) for determining a higher second intensity, and the second measurement is performed using the excitation light having the second intensity determined by the processing of the determination step. And a measurement control step (for example, step S24 in FIG. 6).

  In the program according to claim 6 and the program recorded on the recording medium according to claim 7, the embodiment (however, an example) to which each step corresponds corresponds to the biological information measuring method according to claim 5. It is the same.

  Here, the meanings of terms used in this specification will be described.

  A probe refers to a biological substance fixed on a bioassay substrate such as a DNA chip, which reacts biologically with a target.

  The target refers to a biological material that bioreacts with a biological material fixed on a bioassay substrate such as a DNA chip.

  Biological substances include genes generated in vivo such as proteins, nucleic acids, and sugars, as well as genes having mutually complementary base sequences or substances derived therefrom.

  Biological reaction means that two or more biological substances react biochemically. A typical example is hybridization.

  Hybridization refers to a complementary strand (double strand) forming reaction between nucleic acids having a complementary base sequence structure.

  Hereinafter, embodiments of the present invention will be described with reference to the drawings.

  FIG. 1 illustrates a configuration example of a biological information processing apparatus according to an embodiment of the present invention. The biological information processing apparatus 1 includes a DNA chip 11, a pickup unit 21, a fluorescence intensity acquisition unit 22, an excitation light intensity calculation unit 23, a hybridization amount estimation unit 24, an expression amount calculation unit 25, a standardization unit 26, an output unit 27, It comprises an expression profile data storage unit 28, a user interface (UI) unit 29 having a display unit 29A, a fluorescence intensity-hybridization amount conversion type storage unit 30, and a mechanical learning unit 31.

  The DNA chip 11 has a spot 12 and a guide 13. FIG. 2 shows a more detailed configuration example of the DNA chip 11.

  The DNA chip 11 has an expression analysis reaction vessel 101 and a cell number counting reaction vessel 102 on the substrate 11A. A linear start position guide 13A is provided at the lower end of the substrate 11A in the drawing, and an end position guide 13B is provided at the upper end of the substrate 11A. Specifically, the guide 13 shown in FIG. 1 includes a start position guide 13A and an end position guide 13B.

  The expression analysis reaction tank 101 and the cell number counting reaction tank 102 are arranged between the start position guide 13A and the end position guide 13B.

  In the expression analysis reaction tank 101, a plurality of spots 12 as reaction regions are formed, and in each spot 12, a hybridization verification probe 111 as a biological material (first biological material), an expression analysis A probe 112 and an expression standardization control probe 113 are fixed. When a sample is dropped into the expression analysis reaction tank 101, a target 111A as a biological material (second biological material) having a base having a complementary structure to the base is hybridized to the hybridization verification probe 111. To do. Similarly, the target 112A as a biological material (second biological material) having a base having a complementary structure to the base is hybridized to the expression analysis probe 112. In addition, the expression standardization control probe 113 is hybridized with a target 113A as a biological material (second biological material) having a base complementary to the base.

  In the cell number counting reaction tank 102, a hybridization verification probe 114 as a biological material (first biological material) and a cell number counting control probe 115 are respectively attached to a spot 12 as a reaction region. When a sample is dropped into the cell number counting reaction tank 102, a target 114A as a biological material (second biological material) having a base complementary to the base is hybridized to the hybridization verification probe 114. The target 115A as a biological material (second biological material) having a base complementary to the base hybridizes with the control probe 115 for counting the number of cells.

  An intercalator 116 is bonded to a probe and a target as a hybridized (bioreacted) biological material. The intercalator 116 generates fluorescence when irradiated with excitation light.

  FIG. 2 shows a state in which the target is hybridized to each probe as described above. In FIG. 2, for convenience, only one probe is shown for one spot 12, but actually, a plurality of probes of the same type are fixed to one spot 12. Further, in each reaction tank, an arbitrary number of spots to which the same type of probe is fixed are arranged at predetermined positions.

  The pickup unit 21 shown in FIG. 1 includes a fluorescence intensity acquisition pickup 41, a guide signal acquisition pickup 42, a control unit 43, an objective coordinate calculation unit 44, and a convolution expansion unit 45.

  The fluorescence intensity acquisition pickup 41 is a pickup that acquires images of the expression analysis reaction tank 101 and the cell number counting reaction tank 102 of the DNA chip 11 of FIG. On the other hand, the guide signal acquisition pickup 42 is a pickup for reading the start position guide 13A and the end position guide 13B.

  The fluorescence intensity acquisition pickup 41 includes an objective lens 51, a prism 52, a semiconductor laser 53, and a photodiode 54. Laser light (excitation light) emitted from the semiconductor laser 53 is incident on the objective lens 51 via the prism 52, and the objective lens 51 irradiates the incident laser light on the substrate 11A (spot 12). The objective lens 51 also makes the light from the spot 12 incident on the photodiode 54 via the prism 52. A plurality of probes are fixed to each spot 12, and when the probe and the target are hybridized, an intercalator 116 is further coupled to both. That is, when the probe and the target are not hybridized, there is no intercalator 116 between them, and only when they are hybridized, there is an intercalator 116 between them. The intercalator 116 generates fluorescence when irradiated with excitation light. The fluorescence condensed by the objective lens 51 is separated from the excitation light by the prism 52 and is incident on the photodiode 54.

  The greater the amount of hybridization, the greater the amount of intercalator 116, and hence the greater the amount of fluorescence generated therefrom. Therefore, it is possible to measure the state of hybridization (obtain information on hybridization) based on the intensity of fluorescence.

  The control unit 43 controls the current of the semiconductor laser 53 and adjusts the intensity of the excitation light. Further, the control unit 43 reads the output (current amount change) of the photodiode 54.

  The convolution developing unit 45 receives a signal based on the change in the amount of current output from the photodiode 54 from the control unit 43, and generates image data in units of pixels.

  The guide signal acquisition pickup 42 includes an objective lens 61, a prism 62, a semiconductor laser 63, and a photodiode 64. The semiconductor laser 63 generates laser light based on control from the control unit 43 (this laser light functions as guide detection light). The prism 62 makes the laser light from the semiconductor laser 63 incident on the objective lens 61, and the objective lens 61 irradiates the substrate 11A with this laser light. The objective lens 61 receives the reflected light from the substrate 11A, and the prism 62 separates the reflected light from the irradiation light and emits it to the photodiode 64. The photodiode 64 photoelectrically converts the reflected light incident from the prism 62 and outputs it as a guide signal to the control unit 43. The control unit 43 outputs the guide signal input from the photodiode 64 to the objective coordinate calculation unit 44. The guides 13 (the start position guide 13A and the end position guide 13B) are formed so that the reflectance is higher (or lower) than other areas of the substrate 11A. The objective coordinate calculation unit 44 determines the positions of the start position guide 13A and the end position guide 13B and the start position guide 13A based on the level of the guide signal supplied from the guide signal acquisition pickup 42 via the control unit 43. The position (coordinates) of the guide signal acquisition pickup 42 moved at a constant speed toward the end position guide 13B is calculated.

  The control unit 43 controls the position of the fluorescence intensity acquisition pickup 41 (objective lens 51) based on the position of the guide signal acquisition pickup 42 calculated by the objective coordinate calculation unit 44. The guide signal acquisition pickup 42 and the fluorescence intensity acquisition pickup 41 are fixed to each other in a predetermined positional relationship, and the fluorescence intensity acquisition pickup 41 is arranged between the start position guide 13A and the end position guide 13B in FIG. The guide signal acquisition pickup 42 is disposed at a predetermined position between the start position guide 13A and the end position guide 13B.

The fluorescence intensity acquisition unit 22 receives the input of the fluorescence intensity (pf _{x, y} ) from each spot 12 (its coordinates (x, y)) output from the photodiode 54 of the fluorescence intensity acquisition pickup 41, and receives this fluorescence intensity. Is output to the creation unit 81 of the hybridization amount estimation unit 24. The fluorescence intensity acquisition unit 22 also receives control signals for controlling the objective coordinates (x, y), the objective area radius (r), and the excitation light intensity of the objective lens 51 of the fluorescence intensity acquisition pickup 41 on the substrate 11A. Output to 43. The control unit 43 controls the objective lens 51 based on this control signal. Thereby, the objective lens 51 is arranged at predetermined coordinates (x, y) on the substrate 11A, and the radius (objective area radius) (r) of the irradiation range of the laser light emitted from the objective lens 51 becomes a predetermined value. The laser light intensity (excitation light intensity) is adjusted to a predetermined value.

  The fluorescence intensity acquisition unit 22 outputs the fluorescence intensity supplied from the control unit 43 to the excitation light intensity calculation unit 23. The excitation light intensity calculation unit 23 is based on the fluorescence intensity-hybridization amount conversion equation stored in the fluorescence intensity-hybridization amount conversion equation storage unit 30 and the fluorescence intensity input from the fluorescence intensity acquisition unit 22 during pre-scanning. Then, the optimum excitation light intensity is calculated, and the excitation light intensity obtained by the calculation is output to the fluorescence intensity acquisition unit 22. During the main scan, the fluorescence intensity acquisition unit 22 controls the current of the semiconductor laser 53 based on the excitation light intensity from the excitation light intensity calculation unit 23, and causes the semiconductor laser 53 to emit excitation light having a predetermined intensity.

  The hybridization amount estimation unit 24 includes a creation unit 81, an image processing unit 82, a verification unit 83, and a hybridization amount calculation unit 84.

  The creation unit 81 creates an expression hybridize (pf) that uniquely determines the amount of hybridization from the fluorescence intensity based on the data from the fluorescence intensity acquisition unit 22. The image processing unit 82 processes the image data input from the creation unit 81 and outputs the processed image data to the verification unit 83 and the user interface unit 29. The user interface unit 29 displays the image input from the image processing unit 82 on the display unit 29A. The image processing unit 82 removes components of debris (substance that is an obstacle to observation) from the image of the DNA chip 11 based on an input instructed by the user via the user interface unit 29, and the spot 12 A process of disassembling each image is performed.

  The verification unit 83 verifies that the hybridization is correctly performed based on the amount of hybridization in the spot 12 of the hybridization verification probes 111 and 114 in the image data input from the image processing unit 82.

  The hybridization amount calculation unit 84 calculates the hybridization value and reliability, and outputs the hybridization value and reliability in spot units.

  The expression level calculation unit 25 estimates the expression level corresponding to the fluorescence intensity by obtaining the binding strength of the target to the probe based on the output from the hybridization level calculation unit 84. The standardization unit 26 performs a standardization process using the expression standardization control probe 113 and the cell number counting control probe 115. The output unit 27 supplies the standardized data to the expression profile data storage unit 28. The expression profile data storage unit 28 stores the data supplied from the output unit 27 as expression profile data. The data stored in the expression profile data storage unit 28 is supplied to the user interface unit 29 as necessary and displayed on the display unit 29A. Data output from the expression level calculation unit 25 is also displayed on the display unit 29A as necessary.

  The fluorescence intensity-hybridization amount conversion equation storage unit 30 is a conversion equation (information for conversion without necessarily forming an equation) that is information for uniquely determining the relationship between the fluorescence intensity and the corresponding hybridization amount. May be stored in advance.

Figure 3 is a fluorescence intensity - is a diagram showing an example of a formula hybridize _e (pf) which defines the hybridized amount conversion equation fluorescence intensity stored in the storage unit 30 and the hybridized amount relational.

  As shown in the figure, when the fluorescence intensity is given, the corresponding hybridization amount is uniquely determined based on the function (curves 121 to 124). However, as shown in the figure, the curve 121 shown on the uppermost side in the figure represents the curve when the level of the excitation light intensity is the weakest. Hereinafter, the lower curve 122, the curve 123, and the excitation are sequentially shown. The light intensity level becomes strong, and the lowermost curve 124 represents the curve when the excitation light intensity level is strongest. That is, the fluorescence intensity-hybridization amount conversion formula storage unit 30 is provided with a fluorescence intensity-hybridization amount conversion formula that can uniquely determine the hybridization amount from the fluorescence intensity for each excitation light intensity.

  Each of the curves 121 to 124 shows a slight change in the fluorescence intensity in the portion 121A to 124A on the left end portion in the drawing and the portion 121B to 124B on the right end portion in the drawing. Thus, since the amount of hybridization has changed significantly, it is difficult to uniquely determine the amount of hybridization corresponding to the fluorescence intensity in these regions.

  Therefore, since the hybridization amount obtained by inputting the fluorescence intensity from these portions 121A to 124A and the portions 121B to 124B can be said to be low in reliability, for example, in the center except for such a low reliability section. Only the solid line section is used for the calculation of the amount of hybridization corresponding to the fluorescence intensity.

  Returning to the description of FIG. 1, the mechanical learning unit 31 includes an SVM (Support Vector Machine) 91 and a spot removal pattern database 92 as means for mechanical learning. In the learning mode, the SVM 91 performs learning based on data from the user interface unit 29 and the expression profile data storage unit 28, and stores the learning result in the spot removal pattern database 92. In the determination mode, the SVM 91 also determines the data from the expression profile data storage unit 28 based on the pattern stored in the spot removal pattern database 92 and outputs the determination result to the hybridization amount calculation unit 84.

  The quantitative measurement of the gene expression level is performed by the experimental process processor 131 shown in FIG. The biological information processing apparatus 1 in FIG. 1 constitutes a part of the experimental process processing apparatus 131 in FIG.

  That is, the experimental process processing device 131 includes an adjustment unit 141, a hybridization unit 142, an acquisition unit 143, an expression level estimation unit 144, a standardization unit 145, an output unit 146, and a storage unit 147. Among these, the acquisition unit 143, the expression level estimation unit 144, the standardization unit 145, the output unit 146, and the storage unit 147 are configured by the biological information processing apparatus 1. Specifically, the acquisition unit 143 includes a pickup unit 21, a fluorescence intensity acquisition unit 22, an excitation light intensity calculation unit 23, and a fluorescence intensity-hybridization amount conversion expression storage unit 30, and the expression level estimation unit 144 includes: The hybridizing amount estimating unit 24, the expression amount calculating unit 25, and the mechanical learning unit 31 are configured, the standardizing unit 145 is configured by the standardizing unit 26, the output unit 146 is configured by the output unit 27, and the storage unit 147 is expressed. The profile data storage unit 28 is used.

  The adjustment unit 141 adjusts the target. The hybridizing unit 142 performs hybridization between the probe and the target. The acquisition unit 143 acquires the fluorescence intensity. The expression level estimation unit 144 performs expression level estimation processing. The standardization unit 145 standardizes data. The output unit 146 outputs expression profile data. The storage unit 147 stores expression profile data.

  Next, the process of the experimental process processor 131 of FIG. 4 will be described with reference to the flowchart of FIG.

  First, in step S11, the adjustment unit 141 adjusts the target. Specifically, a sample containing cells is taken out, the protein is denatured and removed from the sample, RNA (ribonucleic acid) extraction, fragmentation, DNA (deoxyribonucleic acid) extraction, fragment The target (target 112A for the expression analysis probe 112) is generated.

  In step S12, the hybridizing unit 142 executes a hybridizing process. Specifically, in the solution containing the target generated in step S11, the targets 111A and 114A for the hybridization verification probes 111 and 114, the target 113A for the expression standardization control probe 113, and the cell count counter A target 115A for the control probe 115 is added, and this solution is dropped into the expression analysis reaction tank 101 and the cell number counting reaction tank 102, whereby the target and the probe are hybridized. Then, the intercalator 116 is introduced and combined with the hybridized target and probe to obtain the DNA chip 11 as shown in FIG. As shown in the figure, in the spot 12 of the expression analysis reaction tank 101, the target 112A is hybridized to the expression analysis probe 112, and the target 113A is hybridized to the expression standardization control probe 113. The target 111A is hybridized with the probe 111 for hybridization verification. And the intercalator 116 has couple | bonded between the probe and target which couple | bonded those double strands.

  Similarly, also in the spot 12 of the cell number counting reaction tank 102, the target 114A is hybridized to the hybridization verification probe 114, and the target 115A is hybridized to the cell number counting control probe 115. ing. An intercalator 116 is also coupled between these hybridized probe and target.

  In step S13, the acquisition unit 143 acquires the fluorescence intensity. The fluorescence intensity acquisition process performed by the acquisition unit 143 will be described later with reference to the flowchart of FIG. 6. In this process, the fluorescence intensity acquisition unit 22 that constitutes the acquisition unit 143 receives the fluorescence intensity via the control unit 43. The acquisition pickup 41 is driven to cause the semiconductor laser 53 to emit laser light as excitation light. The excitation light is incident on the objective lens 51 via the prism 52, and the objective lens 51 irradiates the expression analysis reaction tank 101 on the substrate 11A.

  The intercalator 116 generates fluorescence when irradiated with excitation light. This fluorescence is collected by the objective lens 51 and is incident on the photodiode 54 via the prism 52. The photodiode 54 outputs a current corresponding to the fluorescence. The control unit 43 causes the convolutional expansion unit 45 to convert a signal corresponding to the current into an image signal, and outputs a signal corresponding to the fluorescence intensity generated by the conversion to the fluorescence intensity acquisition unit 22.

  The control unit 43 moves the position of the objective lens 51 from the start position guide 13A toward the end position guide 13B. At this time, laser light as guide detection light emitted from the semiconductor laser 63 of the guide signal acquisition pickup 42 is incident on the objective lens 61 via the prism 62, and the objective lens 61 irradiates the substrate 11A with this guide detection light. To do. The intensity of the reflected light of the guide detection light increases when it is applied to the start position guide 13A and the end position guide 13B. This reflected light is incident on the prism 62 via the objective lens 61, and is incident on the photodiode 64 from the prism 62. The objective coordinate calculation unit 44 acquires a guide signal from the photodiode 64 via the control unit 43, and on the basis of this signal, the guide signal acquisition pickup 42 (therefore, the fluorescence intensity acquisition pickup 41 integrated therewith). Is located between the start position guide 13A and the end position guide 13B of the substrate 11A. Based on the coordinates, the control unit 43 moves (scans) the guide signal acquisition pickup 42 (fluorescence intensity acquisition pickup 41) from the start position guide 13A to the end position guide 13B at a constant speed.

  In this way, the fluorescence intensity acquisition pickup 41 is moved from the start position guide 13A to the end position guide 13B in FIG. 2, and the scanning position is further changed to the start position guide 13A (end position guide 13B). ) In the direction parallel to () in the figure (x-coordinate direction) by one pitch, and similarly, from the start position guide 13A to the end position guide 13B at the new movement position. In this manner, the entire expression analysis reaction tank 101 and cell number counting reaction tank 102 are scanned, and image signals at respective coordinates are output from the fluorescence intensity acquisition pickup 41.

  As described above, prescan and main scan are executed at least twice as scans (image acquisition) for the entire expression analysis reaction tank 101 and cell number counting reaction tank 102. The pre-scan is a low-accuracy scan for detecting the fluorescence intensity (hybridization amount) of each spot 12 necessary for determining the intensity of excitation light used in the main scan. This is a highly accurate scan performed using excitation light having an intensity determined from the result. In the main scan, for example, the excitation light intensity is switched for each spot to be measured, and the scan is performed.

  In step S14, the expression level estimation unit 144 executes an expression level estimation process. The expression level estimation process will be described later with reference to the flowchart of FIG. 15, and the calculation of the hybridization level and the reliability is performed by this process, and the expression level is calculated.

  In step S15, the standardization unit 145 (standardization unit 26) performs processing for standardizing data. As the standardization, standardization by the expression standardization control probe 113 and standardization by the cell number counting control probe 115 are performed. Standardization by the expression standardization control probe 113 is performed as follows.

  That is, in FIG. 2, the expression standardization control probe 113 is shown only in one place, but actually, this expression standardization control probe 113 is a predetermined predetermined in the expression analysis reaction tank 101. It is distributed and arranged at a plurality of positions (for example, four corners of the expression analysis reaction tank 101 and approximately five at the center). Then, based on the fluorescence value of the expression standardization control probe 113 arranged at each position, a correction curved surface is calculated based on, for example, a B-spline curved surface, and each pixel is calculated based on the fluorescence value obtained by the correction curved surface. Normalization is performed by dividing the fluorescence value. By this normalization, variation in hybridization due to the position of the spot 12 in the reaction tank 101 for expression analysis is corrected.

  Further, the standardization by the cell number counting control probe 115 is performed based on the value of the amount of hybridization to the cell number counting control probe 115 (fluorescence value based on the cell number counting control 115). This is done by dividing the fluorescence value of the pixel on the spot 12. As the control probe 115 for calculating the number of cells, a repetitive sequence (for example, Alu sequence for humans) in the genome of the living body from which the expression analysis probe 112 is extracted is used. By this processing, the expression level of the acquired gene can be converted into a value per certain number of cells.

  Furthermore, in step S16, the output unit 146 (output unit 27) outputs the expression profile data. Specifically, the image data obtained as described above is supplied to the storage unit 147 (expression profile data storage unit 28) and recorded.

  Next, the fluorescence intensity acquisition process performed in step S13 of FIG. 5 will be described with reference to the flowchart of FIG.

  In step S <b> 21, the control unit 43 controls the current of the semiconductor laser 53 to perform pre-scanning with low excitation light intensity and low resolution. That is, control signals for controlling the objective coordinates (x, y), the objective area radius (r), and the excitation light intensity are supplied from the fluorescence intensity acquisition unit 22 to the control unit 43.

  Since the pre-scan is performed at a low resolution, the objective area radius (r) controlled here is larger than the objective area radius (r) controlled during the main scan. In addition, since the pre-scan is performed with low-intensity excitation light, the excitation light intensity controlled here is lower than the excitation light intensity controlled during the main scan.

  FIG. 7 is a diagram showing how the DNA chip 11 is pre-scanned. In FIG. 7, the DNA chip 11 of FIG. 2 is shown from above.

As indicated by the white arrow A ₁ , the pre-scan is performed in the y-axis direction of FIG. This is performed by sequentially changing the irradiation position of the excitation light toward the upper side in the figure of the DNA chip 11 corresponding to the end on the side where the light is provided. A circle PS in the figure indicates an irradiation range of excitation light used in the pre-scan. As shown by being surrounded by a dotted line in FIG. 7, the pre-scan is performed with a plurality of (three in the case of FIG. 7) spots 12 arranged in the y-axis direction as one unit.

When the scanning of one unit of the spot 12 is completed, the unit of the target spot 12 is changed to one shifted by one pitch as shown by an arrow A ₃ , and scanning is performed sequentially. For example, the left end of FIG. When the scanning of one unit of spot 12 is finished, the pre-scan is finished. A solid arrow A ₂ shown in the y-axis direction in FIG. 7 indicates the scan position and direction of the guide signal acquisition pickup 42.

  FIG. 8 is a diagram for explaining the irradiation range of the excitation light at the time of pre-scanning. In FIG. 8, the spot 12 and its vicinity are shown enlarged.

As shown in FIG. 8, in this example, as shown by dotted circles PS _{1 to} PS ₅ , spot neighboring regions are defined so as to cover the entire spots 12 _{1 to} 12 ₅ , and excitation is performed in this region. Light is irradiated. The radius of the spot vicinity region is, for example, ½ of the distance from the center of one spot 12 to the center of the adjacent spot 12 so that the same position is not overlapped and scanned. That is, the ratio of the distance (radius) R ₁ to the distance R _{2 in} the figure is 2: 1.

  FIG. 9 is a diagram illustrating an example of signal (signal indicating fluorescence intensity and guide signal) intensity obtained by pre-scanning.

In FIG. 9, the horizontal axis represents time and the vertical axis represents signal intensity. The signal S ₁ represents, for example, a guide signal output from the photodiode 64 to the control unit 43, and the signal S ₂ represents, for example, the amount of current (fluorescence intensity) output from the photodiode 54 to the control unit 43.

As shown in FIG. 9, it is detected from the change in the signal intensity of the guide signal S ₁ surrounded by a dotted line on the left side in the figure that the start position guide 13A is at the scan position at that time, and similarly, on the right side. From the change in the signal intensity of the guide signal S ₁ indicated by being surrounded by a dotted line, it is detected that the end position guide 13B is present at the scan position at that time. As described above, the start position guide 13A and the end position guide 13B are formed so that the reflectance is higher than that of the other regions of the substrate 11A, for example.

In the example of FIG. 9, the start position guide 13A is detected from time t ₁ to t ₂ and the end position from time t ₁₅ to t ₁₆ from the change in signal intensity of the guide signal S _1. The guide 13B is detected.

Further, as described above, the relative positions of the two pickups of the fluorescence intensity acquisition pickup 41 and the guide signal acquisition pickup 42 do not change, and they move at a constant speed, so the time when the start position guide 13A is detected. From the time until the end position guide 13B is detected and the moving speed of the pickup, the position of each spot of one unit arranged in the y-axis direction of the DNA chip 11 is specified, The fluorescence intensity of each spot is acquired from the fluorescence intensity (signal S ₂ ) detected at the position.

In the example of FIG. 9, from the change in the signal intensity of the signal S ₂ , the spot closest to the start position guide 13A is detected from the spots constituting one unit of a certain spot from time t ₄ to t ₅ . Has been. Similarly, in the t ₉ from time t _8, it is detected spots from time t ₄ to the detected neighboring spot (end position guide 13B side) at t _5, at t ₁₃ from the time t _12, from the time t ₈ spot next to the detected spots have been detected in t _9.

The arrows shown between time t ₃ to t ₆ , time t ₇ to t ₁₀ , and time t ₁₁ to t ₁₄ are those when the center of each spot 12 is the center of the irradiation range of the excitation light. The irradiation range is shown. That is, for example, between time t ₃ and time t ₄ , a region near each spot 12 (region indicated by a dotted circle in FIG. 8) is represented.

  Returning to the description of FIG. 6, in step S <b> 22, the control unit 43 obtains the average value of the fluorescence intensity at each spot 12 from the fluorescence intensity detected as described above, and uses the obtained average value for the fluorescence intensity acquisition unit 22. Output to. The average value of the fluorescence intensity obtained by the control unit 43 is supplied from the fluorescence intensity acquisition unit 22 to the excitation light intensity calculation unit 23.

  In step S <b> 23, the excitation light intensity calculation unit 23 calculates the fluorescence intensity supplied from the fluorescence intensity acquisition unit 22 and the fluorescence intensity-hybridization amount conversion formula stored in the fluorescence intensity-hybridization amount conversion formula storage unit 30. Based on the above, the intensity of the excitation light used for scanning each spot 12 during the main scan is calculated.

  FIG. 10 is a diagram illustrating an example of calculation of excitation light intensity. 10, the same parts as those in FIG. 3 are denoted by the same reference numerals.

  First, the excitation light intensity calculation unit 23 includes the fluorescence intensity-hybridization at the excitation light intensity used in the prescan among the fluorescence intensity-hybridization amount conversion expressions read from the fluorescence intensity-hybridization amount conversion expression storage unit 30. Pay attention to the quantity conversion formula. Information indicating the intensity of the excitation light used in the pre-scan is also output from the fluorescence intensity acquisition unit 22 to the excitation light intensity calculation unit 23.

The excitation light intensity calculation unit 23 corresponds to the fluorescence intensity MF _{1 of} a certain spot 12 (spot S ₁ ) supplied from the fluorescence intensity acquisition unit 22 using the focused fluorescence intensity-hybridization amount conversion formula. Determine the amount of hybridization MH ₁ . In the example of FIG. 10, the pre-scan is performed using the excitation light intensity whose relationship between the fluorescence intensity and the amount of hybridization is represented by a curve 121.

Next, the excitation light intensity calculation unit 23 takes the hybridization amount α ₁ as a margin, and the excitation light intensity such that the hybridization amount of MH ₁ + α ₁ becomes the hybridization amount at the boundary point with the low reliability interval. The fluorescence intensity-hybridization amount conversion formula is determined. In the example of FIG. 10, the fluorescence intensity-hybridization amount conversion formula at the excitation light intensity such that the hybridizing amount of MH ₁ + α ₁ becomes the hybridizing amount at the boundary point with the low confidence interval is represented by a curve 123. It is supposed to be done.

As will be described later, the excitation light intensity of the curve 123 is used in the main scan for the spot S ₁ to which the fluorescence intensity has been supplied from the fluorescence intensity acquisition unit 22.

  FIG. 11 is a diagram for explaining another example of calculation of excitation light intensity.

When the fluorescence intensity MF _{2 of a} certain spot 12 (spot S ₂ ) has been supplied, the excitation light intensity calculation unit 23 converts the fluorescence intensity-hybridization amount at the excitation light intensity used in the prescan as described above. Paying attention to the equation, the amount of hybridization MH ₂ corresponding to the fluorescence intensity MF ₂ supplied from the fluorescence intensity acquisition unit 22 is obtained.

Further, the excitation light intensity calculation unit 23 takes the hybridization amount α ₂ as a margin, and the excitation light intensity is such that the hybridization amount of MH ₂ + α ₂ becomes the hybridization amount at the boundary point with the low reliability interval. the fluorescence intensity - seeking those represented as hybridized weight conversion equation in the curve 124 shown in FIG. 11, the excitation light intensity of the curve 124 is selected as the excitation light intensity used during the main scanning spot S _2.

  The excitation light intensity of each spot 12 obtained by the excitation light intensity calculation unit 23 as described above is output to the control unit 43 via the fluorescence intensity acquisition unit 22 and used during the main scan.

  As described above, since the intensity of the excitation light used in the main scan is automatically set by the apparatus without being operated by the user, human noise that may occur when the user himself sets the noise. Is not included in the measurement result. Moreover, it prevents that the reliability of a measurement result falls.

  Furthermore, since it is not necessary for the user to adjust the intensity of the excitation light, a series of analysis processes of measurement results can be quickly completed by the time required for the adjustment.

  In addition, when trying to obtain the fluorescence intensity of one spot 12, normally, the higher the intensity of the excitation light that irradiates the spot 12, it is preferable in terms of ensuring a dynamic range of measurable fluorescence intensity, This can be achieved by selecting the intensity of the excitation light during the main scan as described above from the fluorescence intensity obtained by the pre-scan. For example, it is better to use excitation light having an intensity whose relationship between the fluorescence intensity and the hybridization amount is represented by the curve 123 than excitation light having an intensity whose relationship between the fluorescence intensity and the hybridization amount is represented by the curve 121 in FIG. Wide range of measurable fluorescence intensity (high reliability interval).

  In addition, the intensity of excitation light used for the main scan is selected by taking a margin for the amount of hybridization determined based on the fluorescence intensity obtained by the pre-scan and the fluorescence intensity-hybridization amount conversion formula. Therefore, the hybridization amount obtained based on the fluorescence intensity and the fluorescence intensity-hybridization amount conversion formula obtained by the main scan is prevented from being within the low reliability interval. That is, there may be an error in the amount of hybridization detected in the pre-scan (it may be detected less than the actual amount of hybridization), and the intensity of the excitation light used in the main scan should be considered in consideration of this error. By being determined, it is possible to prevent the amount of hybridization detected in the main scan from being saturated (ie, entering the low reliability interval beyond the high reliability interval).

  In step S24 of FIG. 6, the control unit 43 performs the main scan with excitation light having the excitation light intensity obtained as described above and high resolution.

  FIG. 12 is a diagram illustrating a state in which the DNA chip 11 is actually scanned. The same parts as those in FIG.

As indicated by the arrow A _1, the scan or in the y-axis direction in FIG. 12, sequentially carried out by changing the scanning position. A circle DS in the figure indicates an irradiation range of excitation light used in the main scan. As indicated by the circle DS, the irradiation range of the excitation light during the main scan is narrower than the irradiation range of the excitation light during the pre-scan, thereby obtaining a higher resolution measurement result. For example, when the scanning of one unit spot 12 at the left end in FIG. 12 is finished, the main scanning is finished.

  FIG. 13 is a diagram illustrating an example of signal (signal indicating fluorescence intensity and guide signal) intensity obtained by the main scan. The same parts as those in FIG. 9 are denoted by the same reference numerals.

As shown in FIG. 13, by performing scanning at a higher resolution than in pre-scanning, the position of the spot 12 can be specified from the signal S ₂ with high accuracy, and the fluorescence intensity can be determined. Can be measured.

Further, since the intensity of the excitation light used in the main scan is determined for each spot 12 according to the amount of hybridization in each spot 12 determined at the time of pre-scanning, variation in fluorescence intensity obtained by the main scan is determined. Can be suppressed. As shown in the signal S ₂ in FIG. 13, the fluorescence intensity of the three spots 12, (less vertical variation) less variation compared to that of FIG.

  In step S25 of FIG. 6, the control unit 43 quantizes the fluorescence intensity of each spot 12 obtained in this way by the moving distance of the pickup, and develops the convolution by the convolution expansion unit 45 to cause the fluorescence intensity in units of pixels. After that, the obtained fluorescence intensity information for each pixel is output as image data to the fluorescence intensity acquisition unit 22. After the image data is output from the control unit 43 to the fluorescence intensity acquisition unit 22, the processing returns to step S13 in FIG. 5 and the subsequent processing is performed.

  FIG. 14 is a diagram illustrating an example of a format of image data obtained by the fluorescence intensity acquisition unit 22.

  For each spot, the image data is derived from the excitation light intensity at the time of the main scan, the index that specifies the probe gene sequence, the number of vertical and horizontal pixels of the image of the entire spot, and the fluorescence image (image taken by irradiating excitation light). Composed. Image data having such a format is output from the fluorescence intensity acquisition unit 22 to the hybridization amount estimation unit 24, and various processes are performed based thereon.

  Next, the expression level estimation process in step S14 of FIG. 5 will be described with reference to the flowchart of FIG. In step S31, the creation unit 81 inputs image information (image data). Specifically, image information is input from the fluorescence intensity acquisition unit 22.

In step S <b> 32, the creation unit 81 determines whether the input image information is image information of an image captured with a plurality of excitation light intensities. In the case of image information of images captured with a plurality of excitation light intensities, in step S33, the creation unit 81 determines the amount of hybridization from the fluorescence intensity based on the following expressions (1) to (3). Create hybridize (pf).

The expressions hybridize _s (pf _s ) and hybridize _w (pf _w ) in the expression (3) are respectively the expression hybridize _e (pf) having higher excitation light intensity and the weaker one of the obtained data. Represents the hybridize _e (pf).

  If it is determined in step S32 that the input image data is not image data of an image captured with a plurality of excitation light intensities, the process of step S33 cannot be executed and is skipped.

  In step S34, the image processing unit 83 performs image processing. By this process, a debris region straddling the spot boundary is removed from the image of the DNA chip 11, and the image is decomposed into an image for each spot.

  In step S35, the verification unit 83 executes a process for verifying hybridization. Specifically, as shown in FIG. 2, a hybridization verification probe 111 is provided in the expression analysis reaction tank 101, and a hybridization verification probe 114 is provided in the cell number counting reaction tank 102, respectively. It is fixed to. As the hybridization verification probes 111 and 114, gene sequences that do not exist in the biological species to be experimented are used. For example, when the experiment target is an animal (when the expression analysis probe 112 is an animal gene), a plant chlorophyll gene is used as the hybridization verification probes 111 and 114, and the targets 111A and 114A are as follows: Its complementary sequence is used.

  That is, as the hybridization verification probes 111 and 114 and the targets 111A and 114A, those that reliably cause hybridization regardless of the hybridization of the expression analysis probe 112 and the target 112A are used. In addition, since a completely different species from that of the experiment target is used, when the hybridization verification probes 111 and 114 are sufficiently hybridized, in this experiment (in the measurement), the hybridization surely occurred. Can be verified. On the contrary, when the hybridization verification probes 111 and 114 are not sufficiently hybridized, there is a possibility that this measurement is an environment in which hybridization is difficult to occur for some reason. Therefore, by measuring the fluorescence value of the hybridization verification probes 111 and 114, if the fluorescence value is equal to or higher than a preset reference value, it is verified that correct hybridization processing is being performed. be able to.

  In step S36, the hybridization amount calculation unit 84 obtains a hybridization amount based on, for example, the fluorescence intensity included in the image data and the expression hybridize (pf) obtained in step S33, and determines the obtained hybridization amount. The reliability is obtained by a predetermined method.

  In step S <b> 37, the expression level calculation unit 25 executes a process of calculating the expression level based on the hybridization value and the reliability calculated by the hybridization level calculation unit 84. Based on this processing, the expression level corresponding to the calculated (acquired) fluorescence value is calculated. Thereafter, the process returns to step S14 in FIG. 5, and the subsequent processes are executed.

  As described above, the embodiment in the case of measuring the hybridization of the DNA chip has been described. However, the present invention is not limited to the DNA chip. In the case of measuring whether various biological materials are biologically bound to other predetermined biological materials. It is possible to apply to.

  The series of processes described above can be executed by hardware or can be executed by software. In this case, for example, the biological information processing apparatus 1 is configured by a personal computer as shown in FIG.

  In FIG. 16, a CPU (Central Processing Unit) 201 executes various processes according to a program stored in a ROM (Read Only Memory) 202 or a program loaded from a storage unit 208 to a RAM (Random Access Memory) 203. To do. The RAM 203 also appropriately stores data necessary for the CPU 201 to execute various processes.

  The CPU 201, the ROM 202, and the RAM 203 are connected to each other via the bus 204. An input / output interface 205 is also connected to the bus 204.

  The input / output interface 205 includes an input unit 206 including a keyboard and a mouse, a display including a CRT (Cathode Ray Tube) and an LCD (Liquid Crystal display), an output unit 207 including a speaker, and a hard disk. A communication unit 209 including a storage unit 208 and a modem is connected. The communication unit 209 performs communication processing via a network including the Internet.

  A drive 210 is connected to the input / output interface 205 as necessary, and a removable medium 211 such as a magnetic disk, an optical disk, a magneto-optical disk, or a semiconductor memory is appropriately attached, and a computer program read from them is It is installed in the storage unit 208 as necessary.

  When a series of processing is executed by software, a program constituting the software executes various functions by installing a computer incorporated in dedicated hardware or various programs. For example, a general-purpose personal computer is installed from a network or a recording medium.

  As shown in FIG. 16, the recording medium is distributed to provide a program to the user separately from the apparatus main body, and includes a magnetic disk (including a floppy disk) on which the program is recorded, an optical disk (CD- It is not only composed of removable media 211 consisting of ROM (compact disk-read only memory), DVD (digital versatile disk), magneto-optical disk (including MD (mini-disk)), or semiconductor memory. The program is provided with a ROM 202 in which a program is recorded and a hard disk included in the storage unit 208, which is provided to the user in a state of being incorporated in the apparatus main body in advance.

  In the present specification, the step of describing the program recorded on the recording medium is not limited to the processing performed in chronological order according to the described order, but is not necessarily performed in chronological order. It also includes processes that are executed individually.

It is a block diagram showing the example of a structure of the biometric information processing apparatus as embodiment of this invention. It is a perspective view showing the structural example of a DNA chip. It is a figure showing the relationship between fluorescence intensity and the amount of hybridization. It is a block diagram showing the structural example of an experimental process processing apparatus. It is a flowchart explaining the process of an experiment process. It is a flowchart explaining a fluorescence intensity acquisition process. It is a figure which shows a mode that a DNA chip is pre-scanned. It is a figure explaining the irradiation range of the excitation light at the time of a pre scan. It is a figure which shows the example of the signal strength obtained by prescan. It is a figure explaining the example of calculation of excitation light intensity | strength. It is a figure explaining other examples of calculation of excitation light intensity. It is a figure which shows a mode that a DNA chip is actually scanned. It is a figure which shows the example of the signal intensity | strength obtained by this scanning. It is a figure which shows the example of the format of image data. It is a flowchart explaining an expression level estimation process. And FIG. 11 is a block diagram illustrating a configuration example of a personal computer.

Explanation of symbols

  DESCRIPTION OF SYMBOLS 1 Biological information processing apparatus, 11 DNA chip, 21 Pickup part, 22 Fluorescence intensity acquisition part, 23 Excitation light intensity calculation part, 24 Hybridization amount estimation part, 25 Expression amount calculation part, 28 Expression profile data storage part, 29 User interface , 30 fluorescence intensity-hybridization amount conversion type storage unit, 81 creation unit, 82 image processing unit, 83 verification unit, 84 hybridization amount calculation unit

Claims (7)

In a biological information measuring apparatus for measuring the state of hybridization between a first biological material fixed on a reaction region provided on a substrate and a second biological material that hybridizes to the first biological material ,
From the fluorescence intensity obtained when the portion where the first biological material and the second biological material are hybridized is irradiated with excitation light, the first biological material and the second biological material in the reaction region Storage means for storing conversion information, which is information for which the amount of hybridization is uniquely determined for each intensity of excitation light;
Based on the amount of hybridization between the first biological material and the second biological material determined from the fluorescence intensity obtained by the first measurement using the excitation light of the first intensity, and the conversion information, Determining means for determining a second intensity higher than the first intensity as the intensity of the excitation light used for the second measurement of the hybridized state of the first biological substance and the second biological substance;
And a measurement control unit that performs the second measurement using the excitation light having the second intensity determined by the determination unit. The measurement control means causes the second measurement to be performed with excitation light having the second intensity and having an irradiation range narrower than an irradiation range of excitation light used for the first measurement. The biological information measuring device according to claim 1. The determining means is configured to determine whether the second biological material is hybridized based on the amount of hybridization between the first biological material and the second biological material obtained from the fluorescence intensity obtained by the first measurement, and the conversion information. The intensity of excitation light that takes an intensity that does not exceed the upper limit value of the fluorescence intensity set in the conversion information is determined as the second intensity. The biological information measuring device described. The determining means determines the second intensity for each reaction region provided on the substrate,
The measurement control unit causes the second measurement to be performed for each reaction region using the excitation light having the second intensity determined by the determination unit. Biological information measuring device. In a biological information measurement method for measuring a hybridization state between a first biological material fixed in a reaction region provided on a substrate and a second biological material that hybridizes to the first biological material. ,
The amount of hybridization between the first biological material and the second biological material determined from the fluorescence intensity obtained by the first measurement using the excitation light of the first intensity, the first biological material, and the From the fluorescence intensity obtained when the portion where the second biological material is hybridized is irradiated with excitation light, the amount of hybridization between the first biological material and the second biological material in the reaction region is the intensity of the excitation light. The intensity of excitation light used for the second measurement of the hybridized state of the first biological material and the second biological material based on the conversion information that is uniquely obtained for each time, A determining step of determining a second intensity higher than the intensity of 1;
And a measurement control step of performing the second measurement using the excitation light having the second intensity determined by the processing of the determination step. A computer executes a process for measuring the state of hybridization between the first biological material fixed in the reaction region provided on the substrate and the second biological material that hybridizes to the first biological material. In the program to let
The amount of hybridization between the first biological material and the second biological material determined from the fluorescence intensity obtained by the first measurement using the excitation light of the first intensity, the first biological material, and the From the fluorescence intensity obtained when the portion where the second biological material is hybridized is irradiated with excitation light, the amount of hybridization between the first biological material and the second biological material in the reaction region is the intensity of the excitation light. The intensity of excitation light used for the second measurement of the hybridized state of the first biological material and the second biological material based on the conversion information that is uniquely obtained for each time, A determining step of determining a second intensity higher than the intensity of 1;
And a measurement control step for performing the second measurement using the excitation light having the second intensity determined by the processing of the determination step.   A recording medium on which the program according to claim 6 is recorded.

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