WO2020235283A1 - 分析装置および分析方法 - Google Patents

分析装置および分析方法 Download PDF

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WO2020235283A1
WO2020235283A1 PCT/JP2020/017202 JP2020017202W WO2020235283A1 WO 2020235283 A1 WO2020235283 A1 WO 2020235283A1 JP 2020017202 W JP2020017202 W JP 2020017202W WO 2020235283 A1 WO2020235283 A1 WO 2020235283A1
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points
emission
light emitting
fluorescence
time
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French (fr)
Japanese (ja)
Inventor
穴沢　隆
伊名波　良仁
周平 山本
太朗 中澤
満 藤岡
基博 山崎
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Hitachi High Tech Corp
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Hitachi High Tech Corp
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Priority to CN202411307797.XA priority Critical patent/CN119310167A/zh
Priority to CN202411307788.0A priority patent/CN119310164A/zh
Priority to CN202411307791.2A priority patent/CN119310165A/zh
Priority to DE112020002033.9T priority patent/DE112020002033T5/de
Priority to GB2116497.5A priority patent/GB2597202B/en
Priority to CN202080036799.3A priority patent/CN113840902B/zh
Priority to CN202411307793.1A priority patent/CN119310166A/zh
Priority to US17/612,046 priority patent/US12276608B2/en
Application filed by Hitachi High Tech Corp filed Critical Hitachi High Tech Corp
Priority to JP2021520664A priority patent/JP7282880B2/ja
Priority to SG11202112784UA priority patent/SG11202112784UA/en
Publication of WO2020235283A1 publication Critical patent/WO2020235283A1/ja
Anticipated expiration legal-status Critical
Priority to US19/076,669 priority patent/US20250271360A1/en
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C12M1/34—Measuring or testing with condition measuring or sensing means, e.g. colony counters
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6869—Methods for sequencing
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/59—Transmissivity
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/645—Specially adapted constructive features of fluorimeters
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/645—Specially adapted constructive features of fluorimeters
    • G01N21/6456—Spatial resolved fluorescence measurements; Imaging
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/416—Systems
    • G01N27/447—Systems using electrophoresis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/416—Systems
    • G01N27/447—Systems using electrophoresis
    • G01N27/44756—Apparatus specially adapted therefor
    • G01N27/44782—Apparatus specially adapted therefor of a plurality of samples
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N2021/6417—Spectrofluorimetric devices
    • G01N2021/6421—Measuring at two or more wavelengths
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G01N2021/6439—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G01N2021/6439—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks
    • G01N2021/6441—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks with two or more labels
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/416—Systems
    • G01N27/447—Systems using electrophoresis
    • G01N27/44704—Details; Accessories
    • G01N27/44717—Arrangements for investigating the separated zones, e.g. localising zones
    • G01N27/44721—Arrangements for investigating the separated zones, e.g. localising zones by optical means

Definitions

  • the present disclosure relates to an analysis method and an analyzer that detect fluorescence emitted from a plurality of types of phosphors at a plurality of emission points while identifying each of them.
  • the measurement mode is a mode in which the lamp light is irradiated to the absorption point of each capillary to detect the absorption of the lamp light generated when the analysis target passes through the absorption point, or the laser light is applied to the emission point of each capillary.
  • modes such as irradiating and detecting fluorescence or scattered light generated when the analysis target passes through the light emitting point.
  • Patent Document 1 all the capillaries around the A light emitting points on the capillaries of A (A is an integer of 2 or more) are arranged on the same plane, and the laser beam is introduced from the side of the arrangement plane.
  • the emission points of all the capillaries are irradiated at once, and the fluorescence generated at each emission point is wavelength-dispersed from the direction perpendicular to the array plane for batch detection.
  • the fluorescence emitted from the A light emitting points is collectively collimated by one condenser lens, transmitted through one transmission type diffraction grating, and the primary diffracted light of each fluorescence is one.
  • the imaging lens collectively forms an image on one two-dimensional sensor.
  • B color detection is possible by setting B (B is an integer of 1 or more) detection regions of any wavelength band for the wavelength dispersion image of each capillary.
  • B 1 is called single color detection
  • B ⁇ 2 is called multicolor detection.
  • DNA sequencing of different DNA samples by the Sanger method can be performed in each capillary.
  • DNA fragments contained in DNA samples are labeled with four types of phosphors according to the terminal base species A, C, G, and T, and the emission fluorescence of each is identified by multicolor detection. ..
  • Patent Document 2 all the capillaries around the A light emitting points on the capillaries of A (A is an integer of 2 or more) are arranged on the same plane, and the laser beam is introduced from the side of the arrangement plane to all.
  • the emission points of the capillary are collectively irradiated, and the fluorescence generated at each emission point is divided according to the wavelength component from the direction perpendicular to the arrangement plane and collectively detected.
  • the emission fluorescence from A emission points is individually collimated with A condenser lenses to obtain A luminous flux, and B (B is an integer of 1 or more) dichroic mirrors are arranged1.
  • Each luminous flux is incident in parallel on a set of dichroic mirror arrays, each is divided into B luminous fluxes in different wavelength bands, and the generated total A ⁇ B luminous flux is incident in parallel on one two-dimensional sensor. , A ⁇ B divided images are generated on the image.
  • the A ⁇ B divided images overlap each other on the image. It will be possible to set A ⁇ B detection areas. This makes it possible to detect the B color of each capillary. Therefore, in the multi-capillary electrophoresis apparatus of Patent Document 2, for example, as in the case of Patent Document 1, DNA sequencing of different DNA samples by the Sanger method can be performed in each capillary.
  • the emission fluorescence of the C type (C is an integer of 1 or more) phosphor emission and the wavelength band of the B type (B is an integer of 1 or more) are detected.
  • B color detection is performed at each time in the area. However, B ⁇ C.
  • the B color detection result is subjected to color conversion to obtain the concentration of the C type phosphor.
  • the largest element should be 1, and the other elements should be shown as a ratio to the maximum value.
  • the entire row of the matrix Y can be determined by performing the above steps in order for all the C type phosphors D (c).
  • the matrix Y is determined only by the characteristics of the fluorophore D (c) and the detection regions W (b) of different wavelength bands and does not change during the electrophoretic analysis.
  • the matrix Y is kept constant for different electrophoretic analyzes. Therefore, for each emission point, the concentration Z (c) of the phosphor D (c) at each time can be obtained from the signal intensity X (b) of the detection region W (b) at each time by the following equation.
  • Equation 1 is a simultaneous equation showing the relationship between the concentration of the unknown C type phosphor and the known B color fluorescence intensity, and (Equation 6) corresponds to finding the solution. Therefore, in general, the condition B ⁇ C is required as described above. If B ⁇ C, the solution cannot be uniquely obtained (that is, there can be multiple solutions), so the color conversion cannot be performed as in (Equation 6).
  • the emission fluorescence of the phosphor D (c) (c is 1, 2, 3, or 4) is in the wavelength band due to spectral crosstalk.
  • W (b) (b is 1, 2, 3, or 4) represents the intensity ratio detected.
  • the elements Y (b) (c) of the matrix Y are obtained by electrophoretic analysis of a sample in which the phosphor D (c) (c is 1, 2, 3, or 4) fluoresces independently. Can be decided.
  • the four-color fluorescence intensities X (1), X (2), X (3), and X (4) when the phosphor D (1) fluoresces independently are the elements Y (1), respectively.
  • Y (b) (c) is a fixed value determined only by the characteristics of the phosphor D (c) and the wavelength band W (b), and does not change during electrophoresis.
  • the concentrations of the phosphors D (1), D (2), D (3), and D (4) at each time are the four-color fluorescence intensities X (1), X (2) at each time. ), X (3), and X (4), it is obtained by the following equation that embodies (Equation 6).
  • the above color conversion process is performed independently for each of the A capillaries.
  • This is based on the premise that the crosstalk between capillaries (referred to as spatial crosstalk in this disclosure) is sufficiently small. In other words, it means that the ratio of the signal intensity obtained by detecting the fluorescence emitted from any one capillary to the signal intensity derived from the fluorescence emitted from the other capillary is sufficiently small. Therefore, the step of determining the matrix Y by sequentially performing the above-mentioned steps of fluorescing one type of phosphor D (c 0 ) independently for the C type phosphor D (c) is a capillary of A capillaries. Are performed substantially at the same time, and the matrix Y of each of the A capillaries is obtained in parallel. This is an inevitable process for deriving the matrix Y in a short time and without any hassle.
  • Spatial crosstalk is basically reduced by devising the optical system, but there are also attempts to reduce it by calculation processing.
  • Patent Document 3 not a plurality of capillaries, but emission fluorescence from a plurality of light emitting points randomly arranged on a plane is collectively imaged on one two-dimensional sensor by one condenser lens. ..
  • the emission fluorescence from each emission point is obtained as the signal intensity of the detection region provided at each imaging position on the two-dimensional sensor.
  • the ratio of spatial crosstalk that the signal intensity of emission fluorescence from any one emission point gives to the signal intensity of emission fluorescence from another emission point is the distance between those two emission points, or the corresponding two detections.
  • the above function is obtained in advance, and in the fluorescence images of a plurality of emission points randomly arranged on a plane, the distance between two arbitrary detection regions and the mutual spatial crosstalk are obtained from the above function, and the original fluorescence image is used. By subtracting the obtained spatial crosstalk, we are trying to reduce the spatial crosstalk.
  • the present inventors tried to reduce spatial crosstalk by the method of Patent Document 3 for the optical system of Patent Document 1 or Patent Document 2, but they did not work well.
  • the signal intensity X ( ⁇ ) ( ⁇ ) of the detection region ⁇ for the emission point ⁇ which is any one emission point of A emission points, is any one emission point ⁇ '( ⁇ ⁇ ) other than the above.
  • the spatial crosstalk given to the signal strength X ( ⁇ ') ( ⁇ ) of the detection region ⁇ for ⁇ ') tends to decrease with the distance between the two detection regions, one of the above distances It became clear that it could not be represented by the function of.
  • the two detection regions are located near the central axis of the optical system and far from the central axis of the optical system.
  • the ratio of spatial crosstalk was different.
  • the ratio of spatial crosstalk from one of the above two detection regions to the other was different from the ratio of spatial crosstalk from the other to one.
  • the method of Patent Document 3 has stopped working.
  • the signal intensity X ( ⁇ ) ( ⁇ ) of the detection region ⁇ , which is any one detection region of the B detection regions, is the signal intensity X ( ⁇ ) ( ⁇ ) of the emission point ⁇ , which is any one emission point of A emission points.
  • any one detection region ⁇ '( ⁇ ⁇ 'or ⁇ ⁇ ⁇ 'of B detection regions.
  • ⁇ ⁇ ⁇ ' spatial crosstalk and spectral crosstalk coexist, so the crosstalk between the above two detection regions is essentially impossible to express as a function of the distance between them. there were. This subject will be described in detail in [Modes for Carrying Out the Invention].
  • the spatial crosstalk between a plurality of light emitting points which occurs in an arbitrary optical system that detects the light emitted from the plurality of light emitting points, is reduced by calculation processing, so that the light emitted from the plurality of light emitting points is emitted.
  • spatial crosstalk between multiple light emitting points which occurs in an arbitrary optical system that detects fluorescence of multiple types of phosphors emitted from multiple light emitting points, and multiple types of each light emitting point.
  • the absorption at multiple absorption points can be identified and each can be identified.
  • spatial crosstalk between multiple absorption points and between multiple types of absorbers for each absorption point occur in an arbitrary optical system that detects the absorption of multiple types of absorbers at multiple absorption points.
  • the concentration of the phosphor D (a, c) at the emission point P (a) at an arbitrary time is Z (a, c), and the signal in the detection region W (a', b) for the emission point P (a').
  • X is an A ⁇ B row and 1 column matrix whose elements are X (a', b)
  • Z is an A ⁇ C row and 1 column matrix whose elements are Z (a, c)
  • Y is Y
  • (Equation 9) is the same formula as (Equation 1), but when comparing (Equation 1) to (Equation 4) and (Equation 9) to (Equation 12), it can be seen that they are completely different.
  • (Equation 9) to (Equation 12) are not only the relational expressions of b and c, but also the relational expression of a, and different emission points P (a) are related to each other. That is, (Equation 9) to (Equation 12) collectively consider spatial crosstalk and spectral crosstalk between different emission points P (a) in addition to spectral crosstalk for the same emission point P (a). It is made possible, and is essentially different from (Equation 1) to (Equation 4).
  • A' ⁇ a that is, the emission fluorescence of phosphor D (a, c) at emission point P (a) is arbitrary detection of P (a') by spatial crosstalk and spectral crosstalk for different emission points.
  • it is generally difficult to control the concentration of the phosphor D (a 0 , c 0 ) it is convenient to standardize the row Y (a, b) (a 0 , c 0 ). ..
  • the largest element should be 1, and the other elements should be shown as a ratio to the maximum value.
  • the entire row of the matrix Y can be determined by sequentially performing the above steps for all combinations of the C-type phosphors D (a, c) at the A light emitting points P (a).
  • the matrix Y is determined only by the characteristics of the emission point P (a), the phosphor D (a, c), and the detection region W (a, b), and does not change during the electrophoretic analysis.
  • the matrix Y is constant for different electrophoretic analyzes. Is kept in. Therefore, for each emission point, the concentration Z (a, c) of the phosphor D (a, c) at each time is determined from the signal intensity X (a, b) of the detection region W (a, b) at each time. It is calculated by the following equation.
  • Equation 14 is the same equation as (Equation 6), but comparing (Equation 2) to (Equation 4) and (Equation 6) with (Equation 10) to (Equation 12) and (Equation 14), It can be seen that the two are completely different.
  • the spectral crosstalk and the spatial crosstalk are Both can be eliminated and the concentration Z (a, c) of A ⁇ C phosphors can be obtained.
  • Y - is a matrix of (A ⁇ C) rows (A ⁇ B) columns.
  • eliminating spectral crosstalk is referred to as color conversion
  • eliminating spatial crosstalk is referred to as spatial correction.
  • the (A ⁇ C) ⁇ (A ⁇ B) elements of Y ⁇ include both the part that executes color conversion and the part that executes spatial correction.
  • the time series data of Z (a, c) obtained by executing (Equation 14) for each time is referred to as color conversion + spatial correction data in the present disclosure.
  • Each element of the matrix X may be a value obtained by subtracting the background light in advance, or may be a value subjected to appropriate noise reduction processing. Similarly, for each element of the matrix Y, the value may be obtained by subtracting the background light in advance, or an appropriate change may be added.
  • Equation 9 and (Equation 14) to (Equation 17) are formally the same as the general formulas used in Patent Document 3, for example, [Equation 7] to [Equation 9] in Patent Document 3.
  • the contents are significantly different (hereinafter, the mathematical formulas used in Patent Document 3 are described by []).
  • the element ⁇ ij of the matrix A of [Equation 7] is a function of the distance dij between the detection regions of the emission points ⁇ (meas) i and ⁇ (meas) j, and is specifically shown in [Equation 10]. As you can see, it is represented by the sum of the exponential functions of dij, and ⁇ ij decays as dij increases.
  • Patent Document 3 different fluorescence images are obtained for different samples, and the positions of the detection regions of a plurality of emission points change randomly in each fluorescence image. Therefore, in Patent Document 3, the above function is obtained in advance, and for each fluorescence image, the distance between the detection regions of any two emission points among the plurality of emission points is the combination of all the two emission points. Is obtained, and ⁇ ij is derived by substituting it into the above function. That is, ⁇ ij, that is, the matrix A changes for each sample or each fluorescence image.
  • the elements Y (a') (a) of the matrix Y in (Equation 16) are the light emitting points P ( We have found that it cannot be expressed as a function of the distance between each detection area of a) and P (a'). It has also been found that it is impossible to calculate the elements Y (a') (a) from the configuration of the optical system. Therefore, in the present disclosure, the elements Y (a') and (a) are actually obtained under the condition that the positions of the detection regions of a plurality of light emitting points do not change even for different samples.
  • the concentration of the illuminant D (a, c) at the emission point P (a) is Z (a, c)
  • the emission intensity of W (a', b) for P (a') is X ( Let a', b).
  • (Equation 9) to (Equation 17) are established, and similarly, both spectral crosstalk and spatial crosstalk are eliminated, and the concentration Z (a, c) of A ⁇ C illuminants is determined.
  • the light emission includes fluorescence, phosphorescence, scattered light, and the like.
  • the concentration of the absorber D (a, c) at the absorption point P (a) at an arbitrary time is Z (a, c)
  • the absorbance of W (a', b) at the absorption point P (a') is X.
  • the concentration of the signal generator D (a, c) at the signal generation point P (a) at an arbitrary time is Z (a, c), and the concentration of W (a', b) for the signal generation point P (a').
  • the signal strength be X (a', b).
  • (Equation 9) to (Equation 17) are established, and similarly, both spectral crosstalk and spatial crosstalk are eliminated, and the concentration Z (a, c) of A ⁇ C signal generators is satisfied. Can be sought.
  • spatial crosstalk between a plurality of light emitting points which occurs in an arbitrary optical system for detecting light emission from a plurality of light emitting points, can be eliminated or reduced by calculation processing from a plurality of light emitting points. It is possible to identify the light emission of and detect each of them independently.
  • spatial crosstalk between multiple light emitting points which occurs in an arbitrary optical system that detects the fluorescence of multiple types of phosphors emitted from multiple light emitting points, and multiple types of each light emitting point.
  • Raw data from electrophoretic analysis of unknown samples Color conversion data obtained by performing color conversion on the raw data shown in FIG.
  • Raw data obtained by electrophoretic analysis of samples containing unknown phosphors Color conversion + spatial correction data obtained by performing color conversion + spatial correction on the raw data in Fig. 17.
  • Color conversion data obtained by performing color conversion on the raw data shown in FIG. Color conversion + spatial correction data obtained by performing color conversion + spatial correction on the raw data in Fig. 19.
  • Example 1 In order to investigate the characteristics of spatial crosstalk in detail, we constructed the simple optical system shown in Fig. 1.
  • the optical system in Fig. 1 is a pinhole plate 1-1, a light emitting point side opening plate 1-2, a condenser lens 1-3, a sensor side opening plate 1-4, a colored glass filter 1-5, and a two-dimensional sensor 1-. 6.
  • Halogen lamp A halogen lamp (light source) that irradiates light 1-7 is provided.
  • the optical system shown in FIG. 1 was configured as follows.
  • a light emitting point 1-8 having a diameter of 0.05 mm was formed by irradiating a pinhole plate 1-1 having a pinhole having a diameter of 0.05 mm with halogen lamp light 1-7 from below.
  • 3 was placed at a position 1.54 mm away from the light emitting point 1-8 in the upward direction, and the sensor-side aperture plate 1-4 having an aperture of ⁇ 0.7 mm was placed directly above the condenser lens 1-3.
  • the two-dimensional sensor 1-6 was placed at a position 15 mm above the condenser lens 1-3, and the colored glass filter 1-5 was placed directly under the two-dimensional sensor 1-6.
  • the pinhole plate 1-1, the light emitting point side opening plate 1-2, the sensor side opening plate 1-4, the colored glass filter 1-5, and the two-dimensional sensor 1-6 are arranged in parallel with each other.
  • the light 1-9 emitted from the light emitting points 1-8 is transmitted through the aperture of ⁇ 0.2 mm, condensed by the condenser lens 1-3, transmitted through the aperture of ⁇ 0.7 mm, and the colored glass filter 1-
  • a light emission image 1-10 with a diameter of 0.5 mm was formed on the two-dimensional sensor 1-6 through 5.
  • the light emitting points 1-8 were imaged at an image magnification of 10 times with the two-dimensional sensor 1-6 in focus.
  • Fig. 2 is a light emitting image including images 1-10 obtained by the two-dimensional sensor 1-6 in the simple optical system of Fig. 1.
  • the sensor size of the two-dimensional sensor 1-6 is 13 ⁇ 13 mm, and the signal range of each pixel is 0 to 65536.
  • Fig. 2 (a) and Fig. 2 (b) are the same emission image, but the signal display scale (gradation) of Fig. 2 (a) is set to 0 to 50000, and the signal display scale of Fig. 2 (b) is set to 0 to 5. It is set to 500.
  • the maximum signal value of the luminescent image is about 50,000, and while the signals of all pixels are displayed almost not saturated in FIG. 2 (a), the luminescent image is displayed saturated in FIG. 2 (b).
  • FIGS. 3 (a) to 3 (c) shows the signal intensity distribution in the horizontal direction passing through the center of the luminescent image in the luminescent image of FIG. Fig. 3 (a), Fig. 3 (b), and Fig. 3 (c) use the same data, but the vertical axis is changed.
  • the horizontal axis is common and indicates the distance from the center of the luminescent image.
  • the plus and minus on the horizontal axis indicate the right side and the left side of the luminescent image in the luminescent image, respectively.
  • the vertical axis of FIGS. 3 (a) and 3 (b) shows the absolute signal strength, the vertical axis scale of FIG. 3 (a) is 0 to 60000, and the vertical axis scale of FIG.
  • FIGS. 3 (a) to 3 (c) show the case where the output intensity of the halogen lamp light 1-7 is gradually reduced under the conditions for acquiring the emission image of FIG. Similar to ⁇ , it shows the signal intensity distribution.
  • the maximum signal intensities of the emission images of ⁇ , ⁇ , and ⁇ are about 50,000, about 25,000, and about 10,000, respectively.
  • the signal intensity tends to decrease as the distance from the center of the luminescent image increases, but it is compared with the size of the luminescent image (width of about 0.5 mm) seen in Fig. 3 (a). It can be seen that the hem is much larger. It can also be seen that as the maximum signal strength decreases, so does the strength of the hem. Therefore, looking at Fig. 3 (c), it can be seen that ⁇ , ⁇ , and ⁇ overlap. This is a new discovery and brings some important findings. For example, at a position ⁇ 1 mm away from the center of the luminescent image, that is, at a position outside the luminescent image seen in Fig.
  • Fig. 4 (a) and Fig. 5 (a) are the results derived from Fig. 3.
  • Fig. 4 (a) shows positions -1, -2, -3, -4, -5, -6 mm away from the center of the imaging point
  • Fig. 5 (a) shows +1 and + from the center of the imaging point.
  • the graph is a graph in which the absolute signal intensity at positions 2, +3, +4, +5, +6 mm is on the vertical axis, and the absolute signal intensity at the center of the emission image is on the horizontal axis in each case.
  • the above absolute signal strength is the average value of the absolute signal strength with a width of ⁇ 0.1 mm centered at each position in Fig. 3.
  • the absolute signal intensity of spatial crosstalk at an arbitrary position away from the center of the imaging point is linear with respect to the absolute signal intensity of the center of the imaging point, and the spatial crosstalk ratio is constant. It shows that it becomes. That is, the spatial crosstalk at an arbitrary position is obtained by subtracting the value obtained by multiplying the absolute signal intensity at the center of the imaging point by the spatial crosstalk ratio from the absolute signal intensity at an arbitrary position away from the center of the imaging point. It was newly found that it is possible to eliminate or reduce. This corresponds to subtracting the value of the corresponding approximate straight line at the same position from the absolute signal strength of each plot in FIGS. 4 (a) and 5 (a).
  • Fig. 4 (b) and Fig. 5 (b) are the results of performing the above deduction operation on the results of Fig. 4 (a) and Fig. 5 (a), respectively.
  • the types of plots showing each position and the horizontal axis are common to Fig. 4 (a) and Fig. 4 (b), and Fig. 5 (a) and Fig. 5 (b).
  • the vertical axes of FIGS. 4 (b) and 5 (b) show the absolute signal strength after the above subtraction calculation, and the scale is enlarged as compared with FIGS. 4 (a) and 5 (a). There is.
  • a straight line with a spatial crosstalk of ⁇ 0.01% is overlaid with a dotted line.
  • Fig. 6 and Fig. 7 are the same experimental results as in Fig. 4 and Fig. 5.
  • the pinhole plate 1-1 was once removed and reattached, and then the same results as in Fig. 2 and Fig. 3 were obtained, and the results in Fig. 6 and Fig. 7 were derived by the same method.
  • the mounting position of the pinhole plate 1-1 is almost the same as the original position, but it is not exactly the same position.
  • the results in FIGS. 6 and 7 are similar to the results in FIGS. 4 and 5, indicating that the method of eliminating or reducing spatial crosstalk at an arbitrary position is highly reproducible by subtraction calculation processing.
  • Patent Document 3 it was found that it is impossible to express the spatial crosstalk ratio that occurs between the center of the imaging point and an arbitrary position as a function of the distance between the two. This means that the method shown in Patent Document 3 does not work effectively.
  • the conditions of the optical system and a plurality of imaging points, that is, a plurality of detection regions are fixed, and the mutual spatial crosstalk ratio is shown in each of FIGS. 4 to 7 regardless of the mutual distance. As described above, it differs from Patent Document 3 in that it is derived experimentally. Whenever the conditions are changed, it is necessary to reacquire the spatial crosstalk ratio.
  • Example 2 A model experiment was conducted on the simplest case of an optical system that detects the fluorescence of multiple types of phosphors emitted from multiple emission points in the detection regions of multiple wavelength bands.
  • A 2 emission points
  • B 2 wavelength bands, respectively.
  • C the same effect can be obtained by the same method when each of A, B, and C has other numerical values. Needless to say.
  • Figure 8 shows a model experimental system.
  • the model experimental system in FIG. 8 is a light source that irradiates a laser beam LB in the arrangement direction of two capillarys Cap (1) and Cap (2) and two capillarys Cap (1) and Cap (2) (not shown). ) And. Emission points P (1) and P (2) are provided at positions where the laser beam LB is irradiated on the two capillary caps (1) and Cap (2), respectively.
  • Two types of phosphors D (1,1) and D (1,2) labeled substances are electrophoresed on the capillary Cap (1), and two types of phosphors D (2,1) are electrophoresed on the capillary Cap (2).
  • D (2,2) labeled substances are electrophoresed.
  • D (1,1) and D (2,1) are the same type of phosphor
  • D (1,2) and D (2,2) are the same type of phosphor.
  • the same type of phosphor means that the fluorescence spectra of the emitted fluorescence are equal.
  • the phosphors D (1,1) and D (1,2) emit fluorescence by irradiation with the laser beam LB at the emission point P (1), and the detection region W (1,1) provided in the sensor S. Detected by and W (1,2).
  • the detection region W (1,1) mainly detects the emission fluorescence of the phosphor D (1,1)
  • the detection region W (1,2) mainly detects the emission fluorescence of the phosphor D (1,2).
  • the detection wavelength band of is designed, mutual spectral crosstalk is significantly present.
  • the phosphors D (2,1) and D (2,2) emit fluorescence by irradiation with the laser beam LB at the emission point P (2), and the detection region W (2) provided in the sensor S is provided. Detected by, 1) and W (2,2).
  • the detection region W (2,1) mainly detects the emission fluorescence of the phosphor D (2,1)
  • the detection region W (2,2) mainly detects the emission fluorescence of the phosphor D (2,2).
  • the detection wavelength band of is designed, mutual spectral crosstalk is significantly present.
  • the fluorescence emission of the phosphors D (1,1) and D (1,2) at the emission point P (1) is also detected in the detection regions W (2,1) and W (2,2), and similarly.
  • Fluorescence emission of phosphors D (2,1) and D (2,2) at the emission point P (2) is also detected in the detection regions W (1,1) and W (1,2), and spatial crosstalk Is significantly present.
  • the signal intensities (fluorescence intensities) in the detection regions W (1,1), W (1,2), W (2,1), and W (2,2) are X (1,1), X (1,2). ), X (2,1), and X (2,2), as shown in the lower part of Fig. 8, electrophoresis for each of the emission point P (1) and emission point P (2).
  • time series data of signal intensities X (1,1) and X (1,2), X (2,1) and X (2,2) are obtained.
  • the detection areas W (1,1), W (1,2), W (2,1), and W (2,2) are provided on one sensor S. It is drawn, but it is not always necessary.
  • the detection areas W (1,1) and W (1,2) may be provided on one sensor, and the detection areas W (2,1) and W (2,2) may be provided on the other sensor. However, the detection areas W (1,1), W (1,2), W (2,1), and W (2,2) may be provided on four different sensors, respectively.
  • some imaging means and spectroscopic means are required between the light emitting points P (1) and P (2) and the sensor S. What kind of imaging means and spectroscopic means are used in this embodiment? In FIG. 8, the imaging means and the spectroscopic means are omitted.
  • FIG. 9 is a schematic diagram showing the relationship between spectral crosstalk and spatial crosstalk in FIG.
  • the fluorescence emission of the phosphor D (1,1) at the emission point P (1) is mainly detected in (i) the detection region W (1,1), and (ii) the detection region W (ii). It is detected as a secondary in 1 and 2).
  • the emission fluorescence was detected in (iii) detection region W (2,1) with much lower intensity than in (i) and (ii), and (iv) detection region W (2,2). However, it is detected with a lower intensity than (iii).
  • (i) and (ii) show spectral crosstalk
  • (iii) and (iv) show spatial crosstalk and spectral crosstalk.
  • FIG. 9 shows the relationship between the strength and weakness of the above detection intensity and the relationship between crosstalk.
  • FIG. 9 (b) shows the fluorescence emission of the phosphor D (1,2) at the emission point P (1)
  • FIG. 9 (c) shows the fluorescence D at the emission point P (2).
  • the fluorescence emission of (2,1) and FIG. 9 (d) are drawn for the fluorescence emission of the phosphor D (2,2) at the emission point P (2).
  • FIGS. 9 (a) to 9 (d) occur all at once at the same time, and their fluorescence intensities are different.
  • Fig. 10 shows a model experimental system similar to Fig. 8.
  • the lamp light LL is applied to the two capillary Caps (1) and Caps (2) from the light source perpendicular to the arrangement direction. Therefore, the emission fluorescence associated with the irradiation of the laser beam LB is not detected, but the absorbance or absorbance associated with the transmission of the lamp light LL is detected.
  • Absorption points P (1) and P (2) are provided on the two capillaries Cap (1) and Cap (2) at positions where the lamp light LL is irradiated.
  • D (1,1) and D (1,2) are electrophoresed on the capillary Cap (1)
  • two types of absorbers D (2,1) and D (2) are electrophoresed on the capillary Cap (2).
  • , 2) are electrophoresed.
  • D (1,1) and D (2,1) are the same type of absorber
  • D (1,2) and D (2,2) are the same type of absorber.
  • the same type of absorber means that the light absorption spectra are equal.
  • the absorbers D (1,1) and D (1,2) absorb the light at the absorption point P (1) by the irradiation of the lamp light LL, and the unabsorbed light is the detection region provided in the sensor S. Detected at W (1,1) and W (1,2).
  • the detection region W (1,1) mainly detects the absorption of the absorber D (1,1)
  • the detection region W (1,2) mainly detects the absorption of the absorber D (1,2).
  • the wavelength band is designed, there is significant mutual spectral crosstalk.
  • the absorbers D (2,1) and D (2,2) absorb the light at the absorption point P (2) by the irradiation of the lamp light LL, and the unabsorbed light is provided in the sensor S. It is detected in the detection areas W (2,1) and W (2,2).
  • the detection region W (2,1) mainly detects the absorption of the absorber D (2,1)
  • the detection region W (2,2) mainly detects the absorption of the absorber D (2,2).
  • the wavelength band is designed, there is significant mutual spectral crosstalk.
  • the absorption of the absorbers D (1,1) and D (1,2) at the absorption point P (1) was also detected in the detection regions W (2,1) and W (2,2).
  • the absorption of the absorbers D (2,1) and D (2,2) at the absorption point P (2) was also detected in the detection regions W (1,1) and W (1,2), and the spatial crosstalk was significant. Exists in. This is because a part of the transmitted light of the lamp light LL transmitted through the absorption point P (1) is also detected in the detection regions W (2,1) and W (2,2) and is transmitted through the absorption point P (2).
  • Figure 11 shows the raw data obtained by electrophoresis analysis by injecting known samples into the capillaries Cap (1) and Cap (2) using the model experimental system of FIG. In both cases, the horizontal axis is the electrophoresis time (arbitrary unit) and the vertical axis is the fluorescence intensity (arbitrary unit).
  • Figures 11 (a) and 11 (b) are the same raw data obtained at the emission point P (1), and (b) is an enlargement of the vertical scale of (a).
  • FIGS. 11 (c) and 11 (d) are the same raw data obtained at emission point P (2), and FIG. 11 (d) is an enlargement of the vertical scale of FIG. 11 (c). It is a thing.
  • FIGS. 11 (a) to 11 (d) indicate times 0 to 500
  • the vertical axis scales in FIGS. 11 (a) and 11 (c) are fluorescence intensities 0 to 250
  • the axis scale is fluorescence intensity -0.2 to 0.8
  • X (1,1) and X (2,1) are represented by solid lines
  • X (1,2) and X (2,2) are represented by dotted lines.
  • Z (1,1), Z (1,2), Z (2,1), and Z (2,2) are the phosphors D (1,1), D (1,2) at each time. ), D (2,1), and D (2,2).
  • the 2-by-2 matrix Y and the inverse matrix Y -1 are the same at the emission point P (1) and the emission point P (2). It was. However, in general, the matrix Y and the inverse matrix Y -1 may differ between the light emitting point P (1) and the light emitting point P (2).
  • Each element of the matrix Y for the emission point P (1) is the fluorescence intensity ratio of X (1,1) to X (1,2) by the phosphor D (1,1) at time 100, and the fluorescence at time 300. It was determined by the intensity ratio of fluorescence intensity X (1,1) to X (1,2) by body D (1,2).
  • Each element of the matrix Y for the emission point P (2) is the fluorescence intensity ratio of the fluorescence intensities X (2,1) and X (2,2) by the phosphor D (2,1) at time 200, and the fluorescence at time 400. It was determined by the intensity ratio of the fluorescence intensities X (2,1) and X (2,2) by the body D (2,2).
  • FIG. 12 is color conversion data obtained by performing color conversion of (Equation 18) and (Equation 19) at each time on the raw data of FIG.
  • the notation method is the same as in FIG.
  • the spectral crosstalk of the four large peaks in FIGS. 11 (a) and 11 (c) is resolved by the four large peaks in FIGS. 12 (a) and 12 (c).
  • the spectral crosstalk of the four small peaks in FIGS. 11 (b) and 11 (d) is also shown by the four small peaks in FIGS. 12 (c) and 12 (d) (also indicated by arrows).
  • these peaks themselves still remain, indicating that spatial crosstalk has not been resolved. This is the problem of the conventional method.
  • the matrix Y and the inverse matrix Y -1 are extended from 2 rows and 2 columns of (Equation 18) and (Equation 19) to 4 rows and 4 columns. Further, in (Equation 18) and (Equation 19), the color conversion was performed independently at the emission point P (1) and the emission point P (2), whereas in (Equation 20), the emission point P (1) was performed. ) And the light emitting point P (2) are mixed and color conversion + spatial correction is performed at once.
  • Each element of the matrix Y of (Equation 20) has fluorescence intensities X (1,1), X (1,2), X (2,1) by the phosphor D (1,1) at time 100 in FIG.
  • the upper left 2 rows and 2 columns of the matrix Y of (Equation 20) and the lower right 2 rows and 2 columns correspond to the matrices Y of (Equation 18) and (Equation 19), and the values of each element are almost the same as each other. equal.
  • the upper left 2 rows and 2 columns of the matrix Y -1 of (Equation 20) and the lower right 2 rows and 2 columns correspond to the matrices Y -1 of (Equation 18) and (Equation 19).
  • the values of each element are approximately equal to each other. In other words, these elements are responsible for color conversion that eliminates spectral crosstalk for the same emission point.
  • (Equation 21) Right-hand side 1st term and (Equation 22) Right-hand side 2nd term are responsible for the color conversion of the conventional method, and correspond to (Equation 18) and (Equation 19), respectively.
  • (Equation 21) the second term on the right side and (Equation 22) the first term on the right side are responsible for spatial correction and color conversion, which are not handled by the conventional method.
  • the color conversion, the spatial correction, and the color conversion may be separated and executed individually, or only one of them may be performed.
  • the processing can be changed depending on the light emitting point, for example, the light emitting point P (1) is subjected to spatial correction and color conversion, and the light emitting point P (2) is subjected to only color conversion.
  • FIG. 13 shows the color conversion + spatial correction data obtained by executing the color conversion + spatial correction of (Equation 20) at each time on the raw data of FIG.
  • the notation method is the same as in FIG.
  • the spectral crosstalk of the four large peaks in FIGS. 11 (a) and 11 (c) is resolved by the four large peaks in FIGS. 13 (a) and 13 (c). You can see that it is done.
  • the spatial and spectral crosstalks of the four small peaks in FIGS. 11 (b) and 11 (d) were also eliminated in FIGS. 13 (c) and 13 (d), resulting in peaks. Can be seen to have disappeared (indicated by the arrow).
  • spectral crosstalk for the same emission point and a plurality of emission points generated in an arbitrary optical system for detecting fluorescence of a plurality of types of phosphors emitted from a plurality of emission points. It was shown that spatial crosstalk and spectral crosstalk between them can be eliminated or reduced by computational processing.
  • FIG. 14 shows the raw data obtained as a result of performing electrophoretic analysis by injecting an unknown sample into the capillaries Cap (1) and Cap (2) using the model experimental system of FIG. Since the same model experimental system as in [Example 2] is used, (Equation 18) to (Equation 22) can be used as they are.
  • the notation method is the same as in FIG. However, the vertical scale in Figures 11 (b) and 11 (d) is reduced to fluorescence intensity -0.5 to 2.0. At time 100, 200, 300, and 400, a large peak was observed at emission point P (1), while a small peak (indicated by an arrow) was observed at emission point P (2).
  • FIG. 15 is color conversion data obtained by performing color conversion of (Equation 18) and (Equation 19) at each time on the raw data of FIG.
  • the notation method is the same as in FIG.
  • the emission fluorescence of the phosphor D (1,1) was detected independently at times 100 and 300, and the emission fluorescence of the phosphor D (1) was detected at times 200 and 400.
  • 2) Emission fluorescence was detected alone.
  • the identity of the four small peaks (indicated by arrows) detected at times 100, 200, 300, and 400 at the emission point P (2) is unknown. It was.
  • FIG. 16 shows the color conversion + spatial correction data obtained by executing the color conversion + spatial correction of (Equation 20) at each time on the raw data of FIG.
  • the notation method is the same as in FIG.
  • the emission fluorescence of the phosphor D (1,1) was independently detected at times 100 and 300, as in FIG. 15 (a).
  • Emission fluorescence of phosphor D (1,2) was detected alone at 200 and 400.
  • the fluorescence emission detection regions W (2,1) and W of the phosphors D (1,1) and D (1,2) at the emission point P (1) are shown in FIG. 16 (d).
  • the weak emission fluorescence of phosphor D (2,2) was independently detected at time 100 and 300 at the emission point P (2).
  • the weak emission fluorescence of phosphor D (2,1) was detected alone at times 200 and 400.
  • the peak intensity of these weak emission fluorescence was less than 1% of the peak intensity of the emission fluorescence observed at the emission point P (1).
  • the spatial crosstalk ratio generated in the model experimental system in Fig. 8 is also less than 1%. Therefore, in Fig. 15, the true weak emission fluorescence and the false weak emission fluorescence due to spatial crosstalk coexist, making it impossible to distinguish between the two.
  • the above results show that spatial crosstalk can push up the lower limit of detection in detecting luminescence from each emission point.
  • the spatial crosstalk ratio is 1%, even if the detection limit is 0.1% when there is one light emitting point, is the signal of 1% or less a true signal? Or, because it is not possible to distinguish whether it is a fake signal due to spatial crosstalk, the effective lower limit of detection rises to 1%.
  • the detection sensitivity and dynamic range are both orders of magnitude lower in the emission detection of multiple emission points than in the emission detection of one emission point. The present disclosure can solve such a problem and avoid a decrease in detection sensitivity and dynamic range in light emission detection of a plurality of light emitting points.
  • Fluorescent material D (1,1) at emission point P (1) at time 100, phosphor D (1,2) at emission point P (1) at time 200, and phosphor at emission point P (1) at time 300 Samples are prepared so that the phosphor D (1,3) fluoresces independently at the emission point P (1) at D (1,1) and time 400. Then, the sample is prepared so that the fluorescence other than these is not emitted. As shown in Fig. 17 (a), only four large peaks corresponding to the above fluorescence emission were observed. It was found that the spectral crosstalk ratio of fluorophore D (1,3) was similar to that of fluorophore D (1,2), but slightly different. On the other hand, the four small peaks shown by the arrows shown in Fig. 17 (d) are the results of the spatial crosstalk and spectral crosstalk of the emission fluorescence that are the sources of the four large peaks shown in Fig. 17 (a), respectively. It can be judged that.
  • FIG. 18 shows the color conversion + spatial correction data obtained by executing the color conversion + spatial correction of (Equation 20) at each time on the raw data of FIG.
  • the notation method is the same as in FIG.
  • the emission fluorescence of the phosphor D (1,1) was detected independently at time 100 and 300, and the emission fluorescence of the phosphor D (1,2) was detected at time 200.
  • Emission fluorescence was detected alone.
  • the emission fluorescence of phosphor D (1,3) was detected independently at time 400, but the spectral crosstalk ratios were between phosphor D (1,1) and phosphor D (1,2). ), So the spectral crosstalk was not resolved.
  • the phosphor D (1,1) fluoresces independently at the emission point P (1), and the concentration and fluorescence intensity of the phosphor D (1,1) are stepwise. Samples have been prepared to rise to. Then, the sample is prepared so that the fluorescence other than these is not emitted. As shown in FIG. 19 (a), four large peaks corresponding to the above fluorescence emission were observed, but the sensor S used in the model experimental system of FIG. 8 is saturated at the fluorescence intensity of 200. , Times 200, 300, and 400 were detected saturated.
  • FIG. 20 is color conversion data obtained by performing color conversion of (Equation 18) and (Equation 19) at each time on the raw data of FIG.
  • the notation method is the same as in FIG.
  • the emission fluorescence of the phosphor D (1,1) at time 100 at the emission point P (1) was detected independently after the spectral crosstalk was eliminated.
  • the spectral crosstalk was not resolved because the emission fluorescence of the phosphor D (1,1) at times 200, 300, and 400 was saturated. This is because, as is clear from Fig. 19 (a), when the fluorescence intensity is saturated, the spectral crosstalk ratio derived from the intensity ratio of X (1,1) and X (1,2) changes (number).
  • FIG. 21 is color conversion + spatial correction data obtained by executing (Equation 20) color conversion + spatial correction on the raw data of FIG. 19 at each time.
  • the notation method is the same as in FIG. In Fig. 21 (a), equivalent results are obtained for the same reason as in Fig. 20 (a).
  • Fig. 21 (d) both spatial crosstalk and spectral crosstalk were eliminated for the emission fluorescence of phosphor D (2,1) at time 100.
  • spatial crosstalk and spectral crosstalk were not resolved for the emission fluorescence of phosphor D (2,1) at times 200, 300, and 400. This is because the fluorescence intensity of X (1,1) or X (1,2) shown in FIG.
  • Example 6 Here, a method for determining the matrix Y of (Equation 20) will be described by a method different from [Example 2].
  • the phosphors D (1,1), D (2,1), D (1,2), and D (2,2) are used in this order.
  • the matrix Y was determined by fluorescing each of them independently, that is, by fluorescing them alternately at the emission point P (1) and the emission point P (2).
  • the electrophoresis speed of either Capillary Cap (1) or Cap (2) deviates from the assumption, fluorescence may be emitted simultaneously at the emission point P (1) and the emission point P (2). Can no longer determine the matrix Y.
  • Figure 22 shows a more realistic way to avoid the above problems.
  • the sample was injected only into the capillary Cap (1), electrophoretic analysis was performed with the capillaries Cap (1) and Cap (2), and the fluorophore D (1,1) was obtained. Fluorescence is emitted from each of D (1,2) independently, and fluorescence is detected in the detection regions W (1,1), W (1,2), W (2,1), and W (2,2).
  • the sample was injected only into the capillary Cap (2), and the electrophoretic analysis was performed with the capillaries Cap (1) and Cap (2) to obtain the phosphor D (2,1).
  • Fluorescence is emitted from each of D (2,2) independently, and fluorescence is detected in the detection regions W (1,1), W (1,2), W (2,1), and W (2,2).
  • the matrix Y can be determined as in [Example 2].
  • the matrix Y can be determined more easily and reliably than in [Example 2].
  • 2 and 2) are drawn so as to emit fluorescence multiple times, it is sufficient to emit fluorescence once for each.
  • the next electrophoresis analysis is performed after the first electrophoresis analysis is completed, but it is not necessary to extend the interval of the electrophoresis analysis to that extent.
  • the same effect as in FIG. 22 can be obtained.
  • the electrophoresis may be performed for a short time, then the sample may be injected into the capillary Cap (2), and then the electrophoresis may be restarted. This can reduce the time required to determine the matrix Y.
  • the sample injected into the capillary Cap (1) and the sample injected into the capillary Cap (2) can have the same composition or the same composition. This simplifies the sample to be prepared and reduces the cost for it.
  • FIG. 23 shows a method for further simplifying the determination of the matrix Y.
  • the phosphor D (1,1) in the capillary Cap (1) was similar to that in FIG. ) And D (1,2), and the phosphors D (2,1) and D (2,2) in the capillary Cap (2) are prepared so that they fluoresce at different times.
  • the composition of the sample injected into the capillary Cap (1) and the sample injected into the capillary Cap (2) may be changed so that the electrophoresis rate of the substance labeled with each phosphor is different.
  • a sample injected into Capillary Cap (1) has a 50-base-long DNA fragment labeled with fluorescent substance D (1,1) and a 60-base-long DNA fragment labeled with fluorescent substance D (1,2).
  • Samples containing DNA fragments and injected into Capillary Cap (2) were labeled with a 70-base-long DNA fragment labeled Fluorescent D (2,1) and Fluorescent D (2,2). It may be prepared so as to contain a DNA fragment having a length of 80 bases.
  • FIG. 23 shows that even if samples of the same composition are injected into the capillaries Cap (1) and Cap (2) at the same timing, different electrophoresis conditions are set for the capillaries Cap (1) and Cap (2). Can also be realized. For example, during electrophoresis, the applied voltage of Capi (2) is temporarily lowered, the temperature of Capi (2) during electrophoresis is lowered, and so on.
  • FIG. 24 is a block diagram of a multi-capillary electrophoresis device, which is an example of an analyzer.
  • the multi-capillary electrophoresis device is widely used as an analyzer for analyzing DNA sequences and DNA fragments.
  • the multi-capillary electrophoresis equipment includes capillary 24-1, cathode 24-4, anode 24-5, cathode side buffer solution 24-6, anode side buffer solution 24-7, and pump block 24-9. , Syringe 24-11, laser light source 24-12.
  • four capillaries 24-1 were used, and DNA sequences of different samples were performed in each capillary 24-1. Samples of DNA sequences consist of DNA fragments labeled with four fluorophore.
  • each capillary 24-1 near the light emitting point 24-14 is removed in advance, each capillary 24-1 near the light emitting point 24-14 is arranged on the same plane, and the laser beams 24-13 are collected. After illuminating, it was introduced along the array plane from the side of the above array plane. (5) Then, DNA fragments labeled with four types of phosphors are electrophoresed inside each capillary 24-1, and are excited by irradiation with a laser beam 24-13 as they pass through emission points 24-14. , It emitted fluorescence. In other words, four types of phosphors emitted fluorescence from the four emission points, and the fluorescence intensity of each changed from moment to moment with electrophoresis.
  • Figure 25 shows Raman emitting light from four emission points P (1) to P (4) due to laser beam irradiation when four capillary Caps (1) to Cap (4) are filled with a standard solution. This is the acquisition result of a two-dimensional sensor image including a wavelength dispersion image of scattered light.
  • the horizontal axis direction is the arrangement direction of the four capillaries, and the vertical axis direction is the wavelength direction.
  • the four vertically elongated streaky images indicated by the arrows are the wavelength dispersion images of the Raman scattered light of the capillaries Cap (1) to Cap (4), respectively.
  • the wavelength of the wavelength dispersion image on the two-dimensional sensor image was calibrated for each light emitting point, and the relationship between the pixel position and the wavelength was obtained.
  • B 20 different wavelength band detection regions are set for each of the emission points P (1) to P (4) on the two-dimensional sensor image. did.
  • Each of the 20 detection regions was set to detect light emission of 500 to 700 nm in an evenly divided wavelength band at 10 nm intervals.
  • the detection regions W (1,1), W (1,2), ..., W (1,20) set for the emission point P (1) are 500 to 510 nm, 510 to 520 nm, ..., respectively.
  • 690-700 nm wavelength band emission was set to be detected, and the signal intensities were set to X (1,1), X (1,2), ..., X (1,20). The same applies to the light emitting points P (2) to P (4).
  • the detection area set in 26 is also valid for multiple different emission detections or for multiple different analysis sessions.
  • the emission fluorescence of dR110, dR6G, dTAMRA, and dROX at the emission point P (1) is in the detection regions W (1,5), W (1,7), W (1,10), and W (1), respectively. , 12), it is mainly detected.
  • each emission fluorescence is also detected in the other detection regions of emission points P (1) by spectral crosstalk, and in the detection regions of emission points P (2) to P (3) by spatial crosstalk and spectral crosstalk. Is also detected with weak intensity.
  • dR110, dR6G, dTAMRA, and dROX at the emission point P (1) are set to D (1,1), D (1,2), D (1,3), and D (1,4), respectively.
  • Their concentrations were Z (1,1), Z (1,2), Z (1,3), and Z (1,4).
  • X is a matrix of 80 rows and 1 column
  • Y is a matrix of 80 rows and 16 columns
  • Z is a matrix of 16 rows and 1 column.
  • the method for determining the matrix Y is as described above. However, it is not always necessary to set all 80 x 16 elements. For example, an element whose absolute value is sufficiently small compared to other elements may be replaced with zero to simplify the related calculation. If the range covered by spatial crosstalk is limited, (Equation 9) to (Equation 12) may be defined within the limited range. In this case, a wider range of analysis can be performed by sequentially sliding the limited range, if necessary. For example, if the range of spatial crosstalk can be limited to adjacent capillaries, spatial crosstalk and spectral crosstalk for two or more distant capillaries may be ignored and related calculations may be omitted.
  • FIG. 27 shows the configuration of this optical system and a quadrant image of four light emitting points acquired by this optical system.
  • the optical system shown in Fig. 27 includes a condenser lens array 27-1, a long-pass filter 27-3, a dichroic mirror 27-4, 27-5, 27-6, and 27-7, and a two-dimensional sensor 27-8. As shown in FIGS.
  • each luminous flux 27-9 is incidentally incident on a set of dichroic mirror arrays consisting of four dichroic mirrors 27-4, 27-5, 27-6, and 27-7.
  • the divided luminous fluxes 27-10, 27-11, 27-12, and 27-13 are 520 to 550 nm, 550 to 580 nm, and 580 to 610 nm, respectively.
  • the arrangement directions of the four capillarys Cap (1) to Cap (4) that is, the arrangement directions of the light emitting points P (1) to P (4) and the division direction by the dichroic mirror array, that is, the wavelength directions are mutual. Since it is vertical, as shown in Fig. 27 (c), the divided images W (1,1) to W (4,4) are aligned in the two-dimensional sensor image 27-14 without overlapping each other. Was detected in. For example, the fluorescence emitted from the emission point P (1) is detected as a divided image of W (1,1), W (1,2), W (1,3), and W (1,4).
  • the divided images W (1,1) to W (4,4) in the two-dimensional sensor image 27-14 were set in the detection area, respectively.
  • the signal intensities detected in each detection area were set to X (1,1) to X (4,4), respectively.
  • the detection area set in Fig. 27 (c) is also valid for multiple different emission detections or for multiple different analysis sessions.
  • the DNA fragments of A and G were labeled. Since the maximum fluorescence emission wavelengths of dR110, dR6G, dTAMRA, and dROX are 541 nm, 568 nm, 595 nm, and 618 nm, respectively, the emission fluorescence is 520 to 550 nm, 550 to 580 nm, and 580 to, respectively.
  • the emission fluorescence of dR110, dR6G, dTAMRA, and dROX at the emission point P (1) is the detection regions W (1,1), W (1,2), W (1,3), and W (1), respectively. , 4) are mainly detected.
  • each emission fluorescence is also detected in the other detection regions of emission points P (1) by spectral crosstalk, and in the detection regions of emission points P (2) to P (3) by spatial crosstalk and spectral crosstalk. Is also detected.
  • dR110, dR6G, dTAMRA, and dROX at the emission point P (1) are the phosphors D (1,1), D (1,2), D (1,3), and D (1,4), respectively.
  • concentrations were Z (1,1), Z (1,2), Z (1,3), and Z (1,4). The same applies to the light emitting points P (2) to P (4).
  • X is a 16-by-1 matrix
  • Y is a 16-by-16 matrix
  • Z is a 16-by-1 matrix.
  • Figure 28 shows four types of multi-channel multi-channel reaction tanks arranged on a plane, in which a complementary strand extension reaction using a different DNA fragment in each channel as a template is performed in units of one base and incorporated into the complementary strand in the extension reaction.
  • a complementary strand extension reaction using a different DNA fragment in each channel as a template is performed in units of one base and incorporated into the complementary strand in the extension reaction.
  • the multi-channel extension reactor includes a laser light source 28-1, a dichroic mirror 28-3, a lens 28-4, a dichroic mirror 28-8, 28-9 and 28-10, a lens 28-11, and a first two-dimensional sensor 28. -12, lens 28-13, second 2D sensor 28-14, lens 28-15, third 2D sensor 28-16, lens 28-17 and 4th 2D sensor 28-18.
  • the maximum fluorescence emission wavelengths of the phosphors labeled with the base types T, C, A, and G were 535 nm, 565 nm, 595 nm, and 625 nm, respectively.
  • the laser beam 28-2 oscillated from the laser light source 28-1 was transmitted through the dichroic mirror 28-3, then focused by the lens 28-4 and irradiated to the multi-channel sample 28-5.
  • the fluorescence emission 28-6 of each phosphor excited by the laser beam irradiation was collectively collimated with the lens 28-4, and then the obtained luminous flux 28-7 was transferred to the dichroic mirror 28-3. It was reflected by.
  • the dichroic mirror 28-3 has a spectral characteristic that transmits laser beam light and reflects emission fluorescence.
  • the luminous flux 28-7 was divided into four luminous fluxes having four different wavelength components by three types of dichroic mirrors 28-8, 28-9, and 28-10.
  • the first divided light beam is imaged on the first two-dimensional sensor 28-12 by the lens 28-11, and the second divided light beam is formed on the second two-dimensional sensor 28-14 by the lens 28-13.
  • An image is formed, the third divided light beam is formed on the third two-dimensional sensor 28-16 by the lens 28-15, and the fourth divided light beam is formed on the fourth two-dimensional sensor 28-18 by the lens 28-17. The image was formed on the top.
  • Sample 28-19 is a schematic diagram of sample 28-5 observed from the front.
  • A 5 channels each formed emission points P (1) to P (5).
  • the emission point P (3) and the emission point P (3) of interest Is directly affected by spatial crosstalk, and only the emission points P (1), P (2), P (4), and P (5) existing around the emission point P (3) are analyzed. Therefore, only the emission points P (1) to P (5) are simply drawn on the sample 28-19.
  • the same analysis may be performed for the light emitting point and the light emitting points around it.
  • the first two-dimensional sensor image 28-20 is a divided image of the sample 28-19 acquired by the first two-dimensional sensor 28-12, and each of the light emitting points P (1) to P (5) is imaged.
  • the detection areas W (1,1) to W (5,1) were set at the points, and the respective signal intensities were set to X (1,1) to X (5,1).
  • fluorescent components in the wavelength band of 520 to 550 nm were detected.
  • the second two-dimensional sensor image 28-21 is a divided image of the sample 28-19 acquired by the second two-dimensional sensor 28-14, and each of the light emitting points P (1) to P (5) is imaged.
  • the detection areas W (1,2) to W (5,2) were set at the points.
  • the third two-dimensional sensor image 28-22 is a divided image of the sample 28-19 acquired by the third two-dimensional sensor 28-16, and each of the light emitting points P (1) to P (5) is imaged.
  • the detection areas W (1,3) to W (5,3) were set at the points.
  • fluorescent components in the wavelength band of 580 to 610 nm were detected.
  • the fourth two-dimensional sensor image 28-23 is a divided image of the sample 28-19 acquired by the fourth two-dimensional sensor 28-18, and each of the light emitting points P (1) to P (5) is imaged.
  • the detection areas W (1,4) to W (5,4) were set at the points.
  • the emission fluorescence of the four types of phosphors at the emission point P (3) is in the detection regions W (3,1), W (3,2), W (3,3), and W (3, 3, respectively, in order of wavelength. It is mainly detected in 4).
  • each emission fluorescence is also detected in the other detection regions of emission point P (3) by spectral crosstalk, and emission points P (1), P (2), P (4) by spatial crosstalk and spectral crosstalk.
  • P (5) is also detected in the detection area.
  • the four types of phosphors at the emission point P (3) are divided into D (3,1), D (3,2), D (3,3), and D (3,4), respectively, in order of wavelength. Their concentrations were Z (3,1), Z (3,2), Z (3,3), and Z (3,4). The same applies to the emission points P (1), P (2), P (4), and P (5).
  • X is a matrix of 20 rows and 1 column
  • Y is a matrix of 20 rows and 20 columns
  • Z is a matrix of 20 rows and 1 column.
  • FIG. 29 is a flowchart showing one analysis session of the conventional method.
  • the traditional analysis session is performed by an analytical system that has an analyzer, computer, display, and database that analyzes sample 29-1.
  • the analyzer has a sensor (not shown) that receives light from sample 29-1.
  • the sample 29-1 of type A (A is an integer of 2 or more) labeled with the phosphor of type C (C is an integer of 1 or more) is input to the analyzer.
  • each sample was analyzed in parallel by step 29-2, and the emission fluorescence was detected for each analysis and at each time in the wavelength band of type B (B is an integer of 1 or more).
  • a ⁇ B fluorescence intensity X (a, b) time-series raw data are acquired, and these time-series raw data are sent to the computer.
  • step 29-3 analyzed for (a 0), the matrix Y of C rows B string stored in the database - spectral crosstalk by color conversion using (a 0) (a 0) Is solved, and the concentration Z (a 0 , c) of the C type phosphor is obtained from the fluorescence intensity X (a 0 , b) at each time.
  • the time series color conversion data of Z (a 0 , c) is output in step 29-4. Steps 29-3 and 29-4 are performed for all a 0 . As shown in Fig.
  • the conventional method is characterized in that A analyzes and analyzes are performed independently.
  • the fluorescence intensity X (a, b) is expressed as X (b)
  • the concentration Z (a 0 , c) is expressed as Z (c).
  • FIG. 30 is a flowchart showing one analysis session of this method. Similar to FIG. 29, the analysis session of this example shown in FIG. 30 is performed by an analysis system having an analyzer, a computer, a display device, and a database for analyzing sample 29-1.
  • the analyzer has a sensor (not shown) that receives light from sample 29-1.
  • the sample 30-1 of type A (A is an integer of 2 or more) labeled with the phosphor of type C (C is an integer of 1 or more) is input to the analyzer.
  • each sample was analyzed in parallel by step 30-2, and emission fluorescence was detected for each analysis and at each time in the wavelength band of type B (B is an integer of 1 or more).
  • a ⁇ B fluorescence intensity X (a, b) time-series raw data are acquired, and these time-series raw data are sent to the computer.
  • Fig. 29 as shown by the dotted arrow in step 30-2, there is a significant spatial crosstalk of luminescence fluorescence between different analyzes.
  • the step 30-3, all analyzed (a), the matrix of which is stored in the database (A ⁇ C) line (A ⁇ B) column Y - color conversion + space correction using Spectral crosstalk and spatial crosstalk are eliminated by, and at each time, the fluorescence intensity X (a, b) is combined with the concentration Z (a, c) of the C type phosphor for the A type sample or analysis.
  • the time-series color conversion + spatial correction data of Z (a, c) is output for each analysis (a) in step 30-4.
  • this method is characterized in that the color conversion + spatial correction in step 30-3 is performed collectively for all analyzes.
  • common matrix Y color conversion + space correction to all analyzes - is also characteristic points used.
  • Figure 31 is performed prior to FIG. 30, the matrix Y stored in the database - is a flowchart illustrating a method for obtaining the.
  • the above is realized by the sample 31-1 for determining the matrix Y, but the above may be realized by the setting of the analyzer.
  • the analyzer in step 31-2, the above A ⁇ C single fluorescent emission was detected in the wavelength band of B type (B is an integer of 1 or more), and A ⁇ B fluorescence intensity X.
  • the time-series raw data of (a, b) is acquired, and these time-series raw data are sent to the computer.
  • a matrix of (A ⁇ B) rows (A ⁇ C) columns is derived from the (A ⁇ B) ⁇ (A ⁇ C) data obtained in step 31-3.
  • FIG. 32 is a flowchart in which the analysis sessions of steps 30-2 to 30-4 for the A type sample 30-1 shown in FIG. 30 are repeated a plurality of times while utilizing the matrix Y ⁇ obtained in FIG. 31. is there.
  • the A-type samples 30-1 to be analyzed in each analysis session are different from each other.
  • the same matrix Y stored in the database - that are utilizing a is a feature of the present method.
  • Figure 33 shows an example of a computer configuration.
  • the calculator is connected to the analyzer.
  • the computer not only analyzes the data but also controls the analyzer.
  • the database and display device are drawn outside the computer, but in Fig. 33, they are drawn inside the computer.
  • Data analysis conditions and analyzer control conditions are set through the keyboard, which is the input unit.
  • the time-series raw data of the fluorescence intensity X (a, b) output from the analyzer is sequentially stored in the memory. Further, it stored in the database internal to the HDD (A ⁇ C) line (A ⁇ B) column of matrix Y - is stored in the memory.
  • the CPU the fluorescence intensity X (a, b) stored in the memory and Y - the product calculated in the phosphor concentration Z (a, c) deriving a time-series color conversion + space correction data, sequentially memory
  • the analysis results can be collated with the information on the network through the network interface NIF.

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