Multi-capillary electrophoresis device

Abstract

To provide a method for discriminating light emission from multiple light emission points and detecting each independently by computationally reducing spatial crosstalk between the multiple light emission points that occurs in an arbitrary optical system that detects the light emission from the multiple light emission points, respectively.SOLUTION: In an analysis method and an analysis system for detecting fluorescences from each of a plurality of light-emitting points in a plurality of wavelength bands in order to identify fluorescence emissions of a plurality of types of fluorophores from the plurality of light-emitting points, spatial crosstalk and spectral crosstalk are present between the plurality of light-emitting points and between signal intensities of the plurality of wavelength bands, and then performance of the identification is reduced. The spatial crosstalk and the spectral crosstalk are eliminated and concentrations of each of the plurality of types of fluorophores at each of the plurality of light-emitting points are derived by inputting all detection signals in the plurality of wavelength bands for the plurality of light-emitting points to a predetermined arithmetic operation expression.SELECTED DRAWING: Figure 30

Description

[Technical Field]

[0001] The present disclosure relates to a method for detecting fluorescence emitted from multiple types of phosphors at multiple light-emitting points, respectively. The present invention relates to an analytical method and an analytical device for detecting while identifying. [Background technology]

[0002] Recently, multiple capillaries have been filled with electrolyte solutions or electrolyte solutions containing polymer gels or polymers. Multi-capillary electrophoresis, in which electrophoretic separation media such as liquids are filled and electrophoretic analysis is performed in parallel The device is widely used. Analytical targets range from small molecules to macromolecules such as proteins and nucleic acids. In addition, the measurement mode involves irradiating the lamp light onto the absorption point of each capillary, This mode detects the absorption of lamp light that occurs when light passes through an absorption point, or it detects laser light. The light is irradiated onto the light-emitting point of each capillary, and the fluorescence or scattering generated when the analyte passes through the light-emitting point is measured. There are many modes available, including one for detecting diffuse light.

[0003] For example, in Patent Document 1, the periphery of A light-emitting points on A capillaries (A is an integer of 2 or more) All capillaries are arranged on the same plane, and a laser beam is introduced from the side of the arrangement plane to measure all capillaries. The light-emitting points of the capillaries are irradiated at once, and the fluorescence generated at each light-emitting point in the direction perpendicular to the array plane is measured. The detector detects the fluorescence emitted from A light-emitting points in one collected light. The light is collimated by an optical lens and passes through a single transmission grating, resulting in the first-order diffraction of each fluorescence. The light is focused onto a single two-dimensional sensor by a single imaging lens. By making the direction of the light spot arrangement and the direction of wavelength dispersion by the diffraction grating perpendicular to each other, Therefore, the wavelength dispersion images of the emitted fluorescence from each capillary do not overlap on the two-dimensional sensor. For each capillary, B arbitrary wavelength dispersion images (B is an integer equal to or greater than 1) are taken. By setting the wavelength band detection area, B color detection is possible. When B=1, monochrome detection is performed, and when B≧2, In the multi-capillary electrophoresis device of Patent Document 1, for example, The capillaries allow for DNA sequencing by the Sanger method on different DNA samples. In the Sanger method, DNA fragments contained in a DNA sample are identified according to the terminal base types A, C, G, and T. The four fluorescent substances are labeled, and the emitted fluorescence of each is identified by multicolor detection.

[0004] In Patent Document 2, all capillaries around A light-emitting points on A capillaries (A is an integer of 2 or more) are The capillaries are arranged on the same plane, and a laser beam is introduced from the side of the arrangement plane to The light-emitting points are irradiated at once, and the fluorescence generated at each light-emitting point from the direction perpendicular to the array plane is analyzed according to wavelength components. The detector divides the emitted fluorescence from each of the A emission points into two parts and detects them all at once. The beam is collimated individually by A condenser lenses to form A beams, and then passed through B dichroic lenses (B is an integer greater than or equal to 1). Each beam is incident in parallel onto a set of dichroic mirror arrays, each consisting of an array of dichroic mirrors. Each of these is divided into B beams of different wavelengths, and the total A × B beams are then split into one beam. The light is incident on the two-dimensional sensor in parallel, and A × B divided images are generated on the image. Here, A light emitting points The direction of the arrangement of the dichroic mirrors and the direction of the B splitting of each beam are perpendicular to each other. By making it so that the A × B divided images do not overlap each other on the image. This allows A × B detection areas to be set. This allows B color detection for each capillary. Therefore, in the multi-capillary electrophoresis device of Patent Document 2, for example, As in Reference 1, each capillary was used to sequence different DNA samples using the Sanger method. A can can be performed.

[0005] However, in general, multicolor detection alone is not enough to distinguish between the emitted fluorescence of multiple types of fluorophores. It is not possible to distinguish between them because the fluorescence spectra of each fluorophore overlap with each other. Therefore, the fluorescence of multiple types of phosphors is mixed in any one wavelength band (in this disclosure, This is called spectral crosstalk. This is because the fluorescent material may emit fluorescence at the same time. This technique eliminates spectral crosstalk and makes the above distinction possible.

[0006] For each of A (A is an integer of 2 or more) emission points, C types of fluorescence (C is an integer of 1 or more) are emitted. The fluorescence emitted by the body is detected at each time point in the detection range of B types of wavelength bands (B is an integer greater than or equal to 1). However, B ≥ C. For each light emitting point and at each time, for the B color detection result After color conversion, the concentration of the C-type phosphor is obtained. In this case, the detection region W(b) (b=1, 2, ..., B) of different wavelength bands is detected by the fluorescent substance D(c) (c=1, 2 , ..., C) is detected. At any time, the fluorescent light emitted from the fluorescent substance D(c) at the emission point P(a) is detected. The concentration of P(a) is Z(c), and the signal intensity of the detection area W(b) for the light-emitting point P(a) is X(b). is a B-row, 1-column matrix with signal intensity X(b) as its elements, Z is a C-row, 1-column matrix with concentration Z(c) as its elements, and Y is The following equations hold for a matrix with B rows and C columns, with Y(b)(c) as elements. (Equation 1) to (Equation 4) are The relation between B and c is not the relation between a and b, but it is established independently at each light-emitting point P(a). In the case of detection, since B≧C, C=1, and none of X, Y, and Z are matrices.

[0007]

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[0008] Here, the elements Y(b)(c) of the matrix Y with rows B and columns C are the spectral crosstalk-related components of the phosphor D( c) The emitted fluorescence represents the signal intensity ratio detected in the detection region W(b) of different wavelength bands. By making one type of phosphor D(c0) emit fluorescence alone, one column of the matrix Y, Y(b)(c0) (b=1, 2, ..., B) can be determined. Here, it is generally necessary to control the concentration of the phosphor D(c0). Since this is difficult, it is convenient to normalize one column Y(b)(c0). For example, among the B elements, It is better to set the largest element as 1 and express the other elements as a ratio to the maximum value. It is best to determine the ratio of each element so that the sum of the elements is 1. In other words,

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[0009]

Equation

[0010] , -1 , T , T ,

[0011] , , , , , is the general inverse matrix of Y with C rows and B columns, and Y - =(Y T ×Y)[[ID=二十三]] -1 ×Y T ) is obtained by . When the matrix Y is a square matrix with B = C, Y - is equal to the inverse matrix Y -1 .

[0010] (Equation 1) is a system of simultaneous equations showing the relationship between the concentrations of C types of unknown phosphors and the known B-color fluorescence intensities, and (Equation 6) corresponds to obtaining its solution. Therefore, generally, as described above, the condition B ≥ C is necessary. If B < C, the solution cannot be uniquely obtained (that is, since multiple solutions may exist), color conversion cannot be performed as in (Equation 6).

[0011] .

[0011] As an example, the case of C = 4 and B = 4 for 4-color detection by the Sanger method will be described in detail. When making copies of DNA fragments of various lengths for a template DNA by the Sanger reaction, they are labeled with 4 types of phosphors D(1), D(2), D(3), and D(4) according to the terminal base types A, C, G, and T, and while separating them by length by electrophoresis in order, they are irradiated with a laser beam to emit fluorescence ​ Four types of fluorescent materials were prepared according to the maximum emission wavelengths of the fluorescent materials D(1), D(2), D(3), and D(4). Four-color detection of emitted fluorescence was performed in the wavelength ranges W(1), W(2), W(3), and W(4), and the signals were The time series data of intensities X(1), X(2), X(3), and X(4) (referred to as raw data in this disclosure) (This is not limited to the case where B=4 and C=4.) The fluorescent materials D(1), D(2), and D(3) at each time are obtained. , and the concentrations of D(4) are Z(1), Z(2), Z(3), and Z(4), then (Equation 1) becomes the following equation.

[0012]

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[0013] Here, the elements Y(b)(c) of the 4-by-4 matrix Y are the spectral crosstalk-related components of the phosphor D( c) (c is 1, 2, 3, or 4) emitted fluorescence is detected in the wavelength band W(b) (b is 1, 2, 3, or 4) In each capillary, the fluorophore D(c) (c is 1, 2, 3, or 4) is The elements Y(b)(c) of the matrix Y are determined by electrophoretic analysis of independently fluorescent samples. For example, when the fluorescent substance D(1) emits fluorescence alone, the four-color fluorescence intensities X(1), X(2) can be calculated. (2), X(3), and X(4) are the elements Y(1)(1), Y(2)(1), Y(3)(1), and Y(4)(1), respectively. In addition, the four-color fluorescence intensities X(1), X(2), and X(3) when the fluorescent substance D(2) is emitting fluorescence alone are given. , and X(4) give the elements Y(1)(2), Y(2)(2), Y(3)(2), and Y(4)(2), respectively. The same applies to phosphors D(3) and D(4). Y(b)(c) is the characteristic of phosphor D(c) and wavelength band W(b). It is a fixed value determined only by the capacitance of each capillary 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 colors at each time. From the fluorescence intensities X(1), X(2), X(3), and X(4), the following equation, which embodies (6), is used: can be.

[0014]

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[0015] In this way, the inverse matrix Y -1 The spectral crosstalk is calculated by multiplying the four-color fluorescence intensity by The time series of the concentration of the four types of fluorophores, i.e., the concentration of DNA fragments with four types of bases at the end, was calculated by eliminating the block. Column data (in this disclosure, this is called color conversion data. However, this is not limited to the case where B=4, C=4) Get it.

[0016] As already mentioned, the above color conversion process is performed independently for each of the A capillaries. This is because the crosstalk between capillaries (called spatial crosstalk in this disclosure) This is true on the assumption that is sufficiently small. The signal intensity obtained by detecting the fluorescence emitted from the capillary is compared with that derived from the fluorescence emitted from other capillaries. This means that the ratio of mixed signal strengths is sufficiently small. The process of making one type of phosphor D(c0) emit fluorescence independently is carried out for C types of phosphor D(c) in turn. The step of determining the matrix Y by calculating the matrix Y is performed for A capillaries substantially simultaneously, The matrix Y for each of the A capillaries is calculated in parallel. This allows the matrix Y to be derived in a short time. , and is an inevitable process to be carried out without hassle.

[0017] Spatial crosstalk can be reduced by improving the optical system, but it can also be reduced by improving the computational processing. Therefore, there are attempts to reduce this. In Patent Document 3, instead of multiple capillaries, The emitted light and fluorescence from multiple light-emitting points arranged on the dam are collected by a single condenser lens and focused onto a single two-dimensional sensor. The fluorescence emitted from each light-emitting point is imaged on the two-dimensional sensor. The signal intensity of the detection area is obtained from any one light-emitting point. The spatial crosstalk between the signal intensity of the emission fluorescence from one point and the signal intensity of the emission fluorescence from another point The ratio of the two points is a function of the distance between the two light sources or the distance between the two corresponding detection areas. It can be expressed as follows, and it has been found that it decreases with the distance. In a fluorescence image of multiple light-emitting points randomly arranged on a plane, any two The mutual spatial crosstalk is calculated from the distance of the detection area and the above function, and the calculated crosstalk is calculated from the original fluorescence image. By subtracting the spatial crosstalk detected by the signal, spatial crosstalk is reduced. [Prior art documents] [Patent documents]

[0018] [Patent Document 1] Patent No. 3897277 [Patent Document 2] Patent No. 6456983 [Patent Document 3] Special Publication No. 2018-529947 Summary of the Invention [Problem to be solved by the invention]

[0019] In both Patent Document 1 and Patent Document 2, the light is emitted from A light-emitting points on A capillaries. The emitted fluorescence is imaged separately on the two-dimensional sensor, so there is no spatial crosstalk. The spatial crosstalk is essentially kept low. The causes of spatial crosstalk are (1) lens aberration, (2) Multi-capillaries, lenses, filters, dichroic mirrors, and 2D sensors Multiple reflections of fluorescent light between elements occurring inside an optical system composed of elements, (3) Two-dimensional sensor Blooming between pixels and other factors can be considered. To reduce spatial crosstalk, the above factors must be met. It is necessary to construct an optical system so that the influence of factors (1) to (3) is minimized. For example, In order to reduce the effect of 2), a coating with a lower reflectivity is applied to the lens. However, it is not possible to completely eliminate spatial crosstalk. Spatial crosstalk occurs when detecting fluorescence emitted from any one capillary. In order to effectively raise the detection limit, the detection sensitivity is reduced and the detection die is also Therefore, it is important to reduce spatial crosstalk even a little. This is extremely important in mitigating or resolving the above issues.

[0020] The present inventors have investigated the optical system of Patent Document 1 or Patent Document 2 by the method of Patent Document 3. We tried to reduce spatial crosstalk by using the The case of monochromatic detection where .gtoreq.1 is explained. The signal intensity X(α)(β) of the detection area β for α is The spatial crosstalk given to the signal intensity X(α')(β) of the detection region β for the above two Although a tendency for the number of detection regions to decrease with the distance between them was observed, It became clear that the distance between the two detection areas could not be expressed as a function. Even if the distance is constant, there are cases where the above two detection areas are located near the central axis of the optical system and cases where the above two detection areas are located near the central axis of the optical system. The spatial crosstalk ratio differed depending on the distance from the central axis of the optical system. The spatial crosstalk ratio from one of the two detection areas to the other and the spatial crosstalk ratio from the other area to the one are In the case of multicolor detection where B≧2, the method in Patent Document 3 is more effective. The number of B detection points for a given emitting point α, which is one of the number of emitting points A, is no longer valid. The signal strength X(α)(β) of the detection area β, which is any one of the detection areas in the detection area, is For one light-emitting point α' (α ≠ α') in the B detection areas, = β' or β≠β', and β=β' is the detection range of the same wavelength band, and β≠β' is the detection range of a different wavelength band. The spatial crosstalk given to the signal strength X(α')(β') of the two detectors mentioned above is In particular, when β ≠ β', the spatial crossover Since crosstalk and spectral crosstalk are mixed, the crosstalk between the two detection areas is small. It was essentially impossible to express the distance between the two points as a function of the distance between them. This will be explained in detail in the Detailed Description of the Invention.

[0021] Therefore, in this disclosure, in any optical system that detects light emitted from multiple light-emitting points, The spatial crosstalk between multiple light-emitting points is reduced by computational processing. We propose a method to distinguish between the emissions from multiple emission points and detect each one independently. , an arbitrary light that detects the fluorescence of multiple types of phosphors emitted from multiple light-emitting points, Spatial crosstalk between multiple light-emitting points in an optical system and the spatial By reducing the spectral crosstalk between multiple types of phosphors through computational processing, The fluorescent light emitted from multiple types of fluorescent materials from multiple light-emitting points can be identified and detected independently. Alternatively, we propose a method for detecting the absorption at multiple absorption points. The spatial crosstalk between multiple absorption points that occurs in the system is reduced by computational processing. By doing so, we propose a method to distinguish between the absorptions at multiple absorption points and detect each independently. In addition, the absorbance of a plurality of types of light absorbers at a plurality of absorption points can be detected. The spatial crosstalk between multiple absorption points and the By using computational processing to reduce the spectral crosstalk between all the different types of absorbers, This allows the absorption of multiple types of light absorbers at multiple absorption points to be identified and each detected independently. We propose a way to do this. [Means for solving the problem]

[0022] In the optical system of Patent Document 1 or Patent Document 2, etc., there are A (A is an integer of 2 or more) light-emitting points, For each, C types (C is an integer greater than or equal to 1) of wavelength bands are detected in the detection range of B wavelength bands (B is an integer greater than or equal to 1). Here, we will explain the case where the fluorescent light emitted from the fluorescent material is detected. Each of them detects a different wavelength component of the fluorescence. Each emits fluorescence with a different fluorescence spectrum. , 2, ..., A) in the detection area W(a, b) (b = 1, 2, ..., B) of different wavelength bands. The fluorescent light emitted from the phosphor D(a, c) (c = 1, 2, ..., C) present at the light-emitting point P(a) is detected. At any time, the concentration of the phosphor D(a,c) at the luminous point P(a) is Z(a,c), and the concentration of the phosphor D(a,c) at the luminous point P(a') is Z(a,c). The signal strength of the detection area W(a', b) in the above case is assumed to be X(a', b). Here, X is the A with X(a', b) as an element. x B row 1 matrix, Z is an A x C row 1 matrix with Z(a,c) as its element, Y is an A x C row 1 matrix with Y(a',b)(a,c) as its element The inventors have discovered for the first time that the following equation holds true as a matrix of (A × B) rows and (A × C) columns:

[0023]

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[0024] (Equation 9) is the same as (Equation 1), but comparing (Equation 1) to (Equation 4) with (Equation 9) to (Equation 12), It can be seen that the two are completely different. (Equation 9) to (Equation 12) are the relational expressions for b and c. In addition, the relation between the different light-emitting points P(a) is also a relation between the different light-emitting points P(a). Equations 9 to 12 take into account the spectral crosstalk for the same emitting point P(a) as well as the spectral crosstalk for different The spatial crosstalk and spectral crosstalk between the light-emitting points P(a) can be considered together. This is essentially different from (Equation 1) to (Equation 4).

[0025] Here, the elements Y(a',b)(a,c) of the matrix Y with (A × B) rows and (A × C) columns are (1) a' = a, that is, the same The emission of phosphor D(a',c) at P(a') is due to spectral crosstalk about the emission point. In addition to the fluorescence signal intensity ratio W(a', b), (2) a' ≠ a, i.e., spatial crosstalk and spectral crosstalk for different light-emitting points The emitted fluorescence of the fluorescent substance D(a,c) at the emission point P(a) is detected in an arbitrary detection area W(a', b) represents the signal intensity ratio detected at any one of the emission points P(a0). By making the fluorescent substance D(a0,c0) of the same kind emit fluorescence alone, one column Y(a,b)(a0,c0)(a= 1, 2, ..., A, and b = 1, 2, ..., B), where the phosphor D (a0, Since it is generally difficult to control the density of Y(a,b)(a0,c0), it is necessary to normalize one column Y(a,b)(a0,c0). For example, among A × B elements, the largest element is set to 1, and the other elements are set to their maximum values. It is better to show it as a ratio to the total of A × B elements. It is better to decide the ratio.

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[0026]

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[0027] (14) is the same as (6), but (2) to (4) and (6) and (10) to Comparing (Equation 12) and (Equation 14), we can see that they are completely different. Then, for each time, the general inverse matrix Y of the matrix Y obtained in advance is - and multiply by A × B signal strengths X(a, b). By combining these, both spectral and spatial crosstalk are eliminated. , the concentration Z(a,c) of A×C phosphors can be calculated. - is a matrix with (A×C) rows and (A×B) columns In this disclosure, the elimination of spectral crosstalk is called color conversion. The process of eliminating spatial crosstalk by adjusting the Y - (A×C)×( The (A × B) elements include both a part that performs color transformation and a part that performs spatial correction. Then, the time series data Z(a, c) obtained by executing (Equation 14) for each time is In this disclosure, this is referred to as color conversion + spatial correction data.

[0028] Each element of the matrix X may be a value obtained by subtracting background light in advance, or may be a value obtained by applying appropriate noise reduction processing. Similarly, for each element of matrix Y, the background light may be subtracted in advance. The value may be the same as the original value, or may be changed appropriately.

[0029] In the case of monochromatic detection with B=1 and C=1, the above is simplified as follows: For each of 1, 2, ..., A), the emitted fluorescence of one type of fluorophore is detected in one detection area. At any time, the concentration of the phosphor at the light-emitting point P(a) is Z(a), and the concentration of the phosphor at the light-emitting point P(a') is Z(a). Let X(a') be the signal strength of the signal. Here, X is a matrix with A rows and 1 column, with X(a') as an element, and Z is a matrix with Z(a) as an element. Let Y be an A-row, 1-column matrix with elements Y(a')(a), then (Equation 10) to (Equation 1) 2) can be simplified as follows: (Equation 9) and (Equation 14) hold as they are.

[0030]

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[0031] (Equation 9) and (Equation 14) to (Equation 17) are general formulas used in Patent Document 3, for example, Although the format is the same as [Number 7] to [Number 9] in Patent Document 3, the content is significantly different (hereafter, The formulas used in Reference 3 are written in brackets. The element αij of the matrix A in [Formula 7] is the luminous point It is a function of the distance dij between the detection areas of φ(meas)i and φ(meas)j, and specifically, As shown in [0], it is expressed as the sum of exponential functions of dij, and αij decays as dij increases. In Patent Document 3, different fluorescent images are obtained for different samples, and in each fluorescent image, Therefore, in Patent Document 3, the positions of the detection areas of the multiple light emitting points change randomly. The above function is calculated in advance, and for each fluorescent image, the function is calculated for any two of the multiple emission points. The distance between the detection areas is calculated for all combinations of two light-emitting points and substituted into the above function. In other words, αij, that is, the row Column A changes. In contrast, in this disclosure, as described above, or As will be described later in the section on the form for the purpose of this analysis, the elements Y(a')(a) of the matrix Y in (Equation 16) are the light-emitting points P(a) and P(a') and It has been found that the distance between the detection areas cannot be expressed as a function of the distance between the detection areas. Therefore, it is impossible to calculate the element Y(a')(a). In the example shown, the position of the detection area of ​​multiple light-emitting points does not change even for different samples. Then, the element Y(a')(a) is measured under these conditions. Specifically, as already mentioned, , by making only one of the light-emitting points P(a0) emit fluorescence alone, one of the matrix Y The sequence Y(a')(a0) (a'=1, 2, ..., A) is determined. Then, the above process is repeated for all A light-emitting points P(a). By performing this process for all the points in order, all columns of the matrix Y are determined. The matrix Y is the characteristic of the light-emitting point P(a). It is determined only by the optical system, the light-emitting point P(a) and its detection area, and does not change during the analysis. As long as the conditions such as It is impossible to carry out the above steps using the optical system or device configuration of Patent Document 3. This is because the positions of the detection areas of multiple light-emitting points cannot be fixed, and even if they could be fixed, Even if it is possible, it is not possible to make each light emitting point emit light independently and in sequence. Therefore, the method disclosed herein cannot be inferred from Patent Document 3.

[0032] The above can be interpreted as follows: A number of emissions (A is an integer of 2 or more) For each point, C types (C is 1) are detected in the detection area of ​​B arbitrary wavelength bands (B is an integer of 1 or more). For each light-emitting point P(a) (a=1, 2, ..., A), In the detection area W(a, b) (b=1, 2, ..., B) of different wavelength bands, the light emitting point P(a) Detect the light emitted by the light emitter D(a,c) (c=1,2,...,C). At any time, the light emitting point P(a) Let Z(a,c) be the concentration of the luminous substance D(a,c) in the luminous medium, and X(a',b) be the luminous intensity of W(a',b) for P(a'). In this case, equations (9) to (17) hold, and spectral crosstalk and spatial By eliminating both crosstalk and luminous flux, the concentration Z(a, c) of A × C luminous bodies can be obtained. Here, luminescence includes fluorescence, phosphorescence, scattered light, etc.

[0033] The above can also be read as follows: A number of absorbances (A is an integer of 2 or more) For each point, C types (C is 1) are detected in the detection area of ​​B arbitrary wavelength bands (B is an integer of 1 or more). For each absorption point P(a) (a=1, 2, ..., A), In the detection region W(a, b) (b=1, 2, ..., B) of different wavelength bands, the absorption point P(a) exists. Detect the absorption of the light absorber D(a,c) (c=1,2,...,C). At any time, the absorption point P(a) The concentration of the absorber D(a,c) at the absorption point P(a') is Z(a,c), and the absorbance of W(a',b) at the absorption point P(a') is X(a',b). In this case, (Equation 9) to (Equation 17) hold, and similarly, the spectral crosstalk and By eliminating both spatial crosstalk and quantization, the concentration Z(a, c) of A × C absorbers can be calculated. .

[0034] Alternatively, the above can be interpreted as multi-point detection other than optical measurement. For each of the signal generating points (B is an integer greater than or equal to 2), B arbitrary frequencies (B is an integer greater than or equal to 1) In the detection area of ​​the band, signals from C types of signal generators (C is an integer equal to or greater than 1) are detected. (a) (a=1,2,...,A) for each, the detection area W(a,b) (b=1,2, ..., B), the signal of the signal generator D(a, c) (c = 1, 2, ..., C) existing at the signal generating point P(a) is The concentration of the signal generator D(a, c) at the signal generating point P(a) at any time is calculated as Let Z(a,c) and the signal strength of W(a',b) for the signal generation point P(a') be X(a',b). Similarly, (Equation 9) to (Equation 17) hold, and both spectral crosstalk and spatial crosstalk By solving this problem, the concentrations Z(a, c) of A × C signal generators can be obtained.

[0035] The above explanation mainly uses mathematical formulas, but this is to make it easier to understand the contents of this disclosure. When implementing the technology of this disclosure, a method based on the contents of this disclosure is required. It is not necessary to follow the formula exactly, you can transform the formula and use the formula. It is not necessary to use the formula. In addition, in this disclosure, the expression "concentration of the luminous substance" means "luminous The term "fluorescence intensity of the fluorescent substance" can be read as "luminescence intensity of the fluorescent substance." The term "concentration of absorber" can be read as "absorbance of absorber." It can be done.

[0036] Further features related to the present disclosure will become apparent from the description of this specification and the accompanying drawings. Aspects of the disclosure also relate to the elements and combinations of various elements and the accompanying detailed description that follows. This is achieved and realized by the aspects of the appended claims. The descriptions in this specification are exemplary and illustrative only and are not intended to limit the scope or application of the present disclosure in any way. There is no limitation on the taste.

[0037] Further features related to the present disclosure will become apparent from the description of this specification and the accompanying drawings. Aspects of the disclosure also relate to the elements and combinations of various elements and the accompanying detailed description that follows. This is achieved and realized by the aspects of the appended claims. The descriptions in this specification are exemplary and illustrative only and are not intended to limit the scope or application of the present disclosure in any way. There is no limitation on the taste. [Effects of the Invention]

[0038] According to the present disclosure, an optical system that detects light emitted from a plurality of light-emitting points can be used. Eliminate or reduce spatial crosstalk between multiple light-emitting points through computational processing. This makes it possible to distinguish between the emissions from multiple emission points and detect each one independently. In addition, the fluorescence of multiple types of phosphors emitted from multiple light-emitting points is detected. Spatial crosstalk between multiple light-emitting points and the spatial crosstalk between each light-emitting point that occurs in any optical system. The spectral crosstalk between multiple types of phosphors is eliminated or reduced by computational processing. By reducing the number of fluorescent light sources, it is possible to distinguish between the fluorescent light emitted by multiple types of phosphors from multiple emission points and It is possible to detect each independently.

[0039] Furthermore, by eliminating or reducing spatial and spectral crosstalk, Therefore, the decrease in detection sensitivity caused by spatial crosstalk and spectral crosstalk is This makes it possible to avoid a decrease in the detection dynamic range. Problems, configurations, and effects other than those described above will become clear from the following description of the embodiments. . [Brief explanation of the drawings]

[0040] [Figure 1] Schematic diagram of a simple optical system [Figure 2] Luminescence image captured by a 2D sensor using a simple optical system [Figure 3] Signal intensity distribution of luminescence image in luminescence image [Figure 4] The relationship between the absolute signal intensity at the center of the luminescence image and the absolute signal intensity at a position far to the left of the center of the luminescence image [Figure 5] The relationship between the absolute signal intensity at the center of the luminescence image and the absolute signal intensity at a position far to the right of the center of the luminescence image [Figure 6] The relationship between the absolute signal intensity at the center of the luminescence image and the absolute signal intensity at a position far to the left of the center of the luminescence image [Figure 7] The relationship between the absolute signal intensity at the center of the luminescence image and the absolute signal intensity at a position far to the right of the center of the luminescence image [Figure 8] Schematic diagram of the model experiment system [Figure 9] Schematic diagram of spectral crosstalk and spatial crosstalk in a model experimental system [Figure 10] Schematic diagram of the model experiment system [Figure 11] Raw data obtained from electrophoretic analysis of known samples [Figure 12] Color transformation data obtained by performing color transformation on the raw data in Figure 11 [Figure 13] Color conversion + spatial correction data obtained by performing color conversion + spatial correction on the raw data in Figure 11 [Figure 14] Raw data obtained from electrophoretic analysis of unknown samples [Figure 15]Color-transformed data obtained by performing color transformation on the raw data in Figure 14 [Figure 16] Color conversion + spatial correction data obtained by performing color conversion + spatial correction on the raw data in Figure 14 [Figure 17] Raw data from electrophoretic analysis of a sample containing an unknown fluorophore [Figure 18] Color conversion + spatial correction data obtained by performing color conversion + spatial correction on the raw data in Figure 17 [Figure 19] Raw data from electrophoretic analysis of samples containing increasing concentrations of known fluorophores [Figure 20] Color-transformed data obtained by performing color transformation on the raw data in Figure 19 [Figure 21] Color conversion + spatial correction data obtained by performing color conversion + spatial correction on the raw data in Figure 19 [Figure 22] Schematic diagram of a method for injecting samples into multiple capillaries at different times [Figure 23] Schematic diagram of a method for obtaining fluorescence emission by staggering the timing using multiple capillaries [Figure 24] Schematic diagram of a multi-capillary electrophoresis device [Figure 25] Wavelength dispersion image of Raman scattered light from four capillaries using the optical system of Patent Document 1 [Figure 26] A diagram showing the setting of the detection areas of 20 wavelength bands for each capillary in FIG. [Figure 27] Schematic diagram of the optical system of Patent Document 2, in which the emitted fluorescence from the four capillaries is divided into four wavelength bands and imaged. [Figure 28] An optical system that splits the emitted fluorescence from five light-emitting points arranged on a plane into four wavelength bands and forms an image. [Figure 29] Flowchart of the conventional method [Figure 30] Flowchart of this method [Figure 31] Flowchart for determining the matrix for color conversion and spatial correction of this method [Figure 32] Flowchart for multiple analysis sessions [Figure 33] Computer configuration diagram DETAILED DESCRIPTION OF THE INVENTION

[0041] [Example 1] To investigate the characteristics of spatial crosstalk in detail, we constructed a simple optical system shown in Figure 1. The optical system is composed of a pinhole plate 1-1, a light-emitting point side aperture plate 1-2, a condenser lens 1-3, and a sensor side aperture plate 1-4. , color glass filter 1-5, two-dimensional sensor 1-6, halogen lamp irradiating halogen lamp light 1-7 Specifically, the optical system shown in Figure 1 was configured as follows: The pinhole plate 1-1 with the pinhole is irradiated from below with halogen lamp light 1-7. Therefore, a light-emitting point 1-8 with a diameter of 0.05 mm was formed. Plate 1-2 is placed 0.2 mm above light-emitting point 1-8, and a focusing lens with a focal length of f = 1.4 mm is used. The lens 1-3 was placed 1.54 mm above the light-emitting point 1-8, and the lens 1-3 had an aperture of 0.7 mm. The sensor-side aperture plate 1-4 was placed directly above the condenser lens 1-3. The colored glass filter 1-5 is placed 15 mm above the two-dimensional sensor 1-6. The pinhole plate 1-1, the light-emitting point side aperture plate 1-2, the sensor side aperture plate 1-4, and the color The glass filter 1-5 and the two-dimensional sensor 1-6 are arranged parallel to each other. 1-9 passes through a φ0.2 mm aperture, is focused by a condenser lens 1-3, passes through a φ0.7 mm aperture, The light passes through a colored glass filter 1-5 and forms a 0.5 mm diameter light image 1-10 on a two-dimensional sensor 1-6. Here, the light emitting point 1-8 is focused on the two-dimensional sensor 1-6 and is imaged at a magnification of 10. was done.

[0042] Figure 2 shows a luminescence image including image 1-10 obtained by two-dimensional sensor 1-6 in the simple optical system of Figure 1. The sensor size of the two-dimensional sensor 1-6 is 13 x 13 mm, and the signal range of each pixel is 0 to 65536. Figure 2(a) and Figure 2(b) are the same luminescence images, but the signal display scale (grayscale) of Figure 2(a) is The signal display scale in Figure 2(b) is 0 to 500. The maximum signal value of the luminescence image is approximately 50,000, and in Figure 2(a), the signals of all pixels are displayed almost without saturation, whereas in Figure 2 In (b), the luminescence image is saturated. According to Fig. 2(a), the luminescence of φ0.5 mm is as expected. The image is obtained, and the signal appears to be zero outside the φ0.5 mm emission image. However, as shown in Figure 2(b), It can be seen that a low intensity tail extends outside the φ0.5 mm emission image.

[0043] The dotted line α in Figure 3(a) to (c) indicates the horizontal direction that passes through the center of the luminescence image in Figure 2. Figures 3(a), 3(b), and 3(c) show the signal intensity distribution of the same data. The horizontal axis is the same and indicates the distance from the center of the light emission image. The plus and minus axes indicate the right and left sides of the luminescence image, respectively. The vertical axis in Figures 3(a) and (b) shows the absolute signal strength, and the vertical axis scale in Figure 3(a) is 0 to 60,000. In Figure 3(b), the vertical axis scale is 0 to 100. On the other hand, in Figure 3(c), the vertical axis is a logarithmic scale. The vertical axis scale is 0.001% to 100% as the relative signal strength with the maximum signal strength being 100%. %. β (solid line) and γ (dashed line) in Figure 3(a) to (c) are obtained under the conditions of acquiring the luminescence image in Figure 2. ,When the output intensity of the halogen lamp light 1-7 is gradually reduced, the signal intensity is As can be seen from Figure 3(a), the maximum signal intensity of the α, β, and γ emission images is On the other hand, looking at Figure 3(b), the distances from the center of the luminous image are approximately 50,000, 25,000, and 10,000, respectively. The signal intensity tends to decrease as the distance increases, but the size of the luminescence image ( It can be seen that the tail is much wider than the peak width (approximately 0.5 mm). It can be seen that as the strength of the tension decreases, the strength of the tail also decreases. This is a new discovery and brings several important implications. For example, at a position ±1 mm away from the center of the light emission image, i.e., the position shown in Figure 2(a), At positions outside the luminescence image, a signal intensity of approximately 0.1% of the maximum signal intensity was observed. If there are adjacent detection areas of the light emission image at a distance of ±1 mm, this is approximately 0.1%. This means that spatial crosstalk exists. Even though the light emitting point is just focused using the optical system, the It is surprising that there exists a spatial crosstalk of such magnitude. What is even more surprising is that α, β Since γ and γ are superimposed, as long as the optical system and the position of the light emitting point are fixed, the light The relative signal intensity distribution of the imaged point is uniform regardless of the light intensity of the light spot or the signal intensity of the imaged point. It was found to be constant.

[0044] Figure 4(a) and Figure 5(a) are the results derived from Figure 3. Figure 4(a) shows the image at -1 and -2 from the center of the image point. ,-3,-4,-5,-6 mm away,Figure 5(a) shows the positions +1,+2,+3,+4,+5,+6 mm away from the center of the image point. The vertical axis represents the absolute signal strength at a distance, and the vertical axis represents the absolute signal strength at the center of the luminescence image. The absolute signal strength is shown in Figure 3, with the horizontal axis being the center. The absolute signal intensity was calculated as the average value of the 0.1 mm width. As a result, three plots (Fig. 3) were obtained at each position. It was found that all of the α, β, and γ values ​​fit on an approximate line passing through the origin. The slope of the line indicates the spatial crosstalk ratio at the corresponding position, i.e., the center of the imaging point. As the distance from the center becomes larger, the slope of the approximated line becomes smaller, and the spatial crosstalk ratio decreases. For comparison, the straight lines for spatial crosstalk of 0.1% and 0.01% are superimposed as dotted lines. For example, the spatial crosstalk ratio at a position 1 mm away from the center of the imaging point is just over 0.1%. In contrast, the spatial crosstalk ratio at a distance of -3 mm was approximately 0.01%. The above results are based on the spatial crosstalk at any position away from the center of the image formation point. The absolute signal strength of the image is linear with respect to the absolute signal strength at the center of the image. This shows that the image ratio is constant. From the absolute signal strength at the center of the image, multiply the absolute signal strength at the center by the spatial crosstalk ratio. By subtracting the value, spatial crosstalk at any position can be eliminated or reduced. This is because the processes in Figures 4(a) and 5(a) are Equivalent to subtracting the value of the corresponding fitted line at the same position from the absolute signal strength of the lot. do.

[0045] Figure 4(b) and Figure 5(b) show the results of Figure 4(a) and Figure 5(a) after the subtraction operation. The types of plots showing each position and the horizontal axis are shown in Fig. 4(a) and 4(b), The vertical axis in Figures 4(b) and 5(b) is the value after the subtraction calculation. Absolute signal intensities are shown and scaled up compared to Figures 4(a) and 5(a). Therefore, a straight line representing spatial crosstalk of ±0.01% is superimposed on the dotted line. The absolute signal intensity after subtraction in the image center becomes almost zero, regardless of the absolute signal intensity at the image center. It was found that the spatial crosstalk at each position was less than ±0.01%. When comparing at a position 1 mm away from the center of the The absolute signal intensity after subtraction at each position was also reduced by more than one order of magnitude. The reason it does not become zero is the deviation of the approximation line corresponding to each plot in Figure 4(a) and Figure 5(a). , because there is an error. The coefficient of determination (R 2 )but The higher (closer to 1), the greater the absolute signal intensity at each position relative to the absolute signal intensity at the center of the image. The higher the linearity of the signal intensity, the smaller the error. The absolute signal intensity after subtraction is The absolute signal strength after subtraction can be negative. However, as long as the above linearity exists, the magnitude of the absolute signal strength at each position can be calculated by subtraction. The smaller the slope of the approximation line, that is, the smaller the space before the subtraction operation. The smaller the crosstalk between the sensors, the smaller the absolute value of the difference between the absolute signal strength at each position and the approximate line. Therefore, the above error also becomes smaller.

[0046] Figures 6 and 7 show the same experimental results as Figures 4 and 5. In the optical system of Figure 1, After removing and reinstalling the Hall Plate 1-1, the same results as in Figures 2 and 3 were obtained. The results shown in Figures 6 and 7 were derived using this method. The pinhole plate 1-1 was attached at approximately The results in Figures 6 and 7 are the same as those in Figures 4 and 5. The results are equivalent to those in Figure 5 and Figure 6, and the spatial crossover at any position can be calculated by subtraction processing. It was shown that the method for eliminating or reducing noise is highly reproducible.

[0047] On the other hand, a comparison of Figures 4 to 7 revealed another important finding: Compare the approximate lines for -1 mm in Fig. 4(a), +1 mm in Fig. 5(a), -1 mm in Fig. 6(a), and +1 mm in Fig. 7(a). The slopes of these four approximate lines are all at a position 1 mm away from the center of the image point. The spatial crosstalk ratios are shown, but they are found to be different from each other. For example, use the approximate line of -1 mm in Figure 4(a) to approximate +1 mm in Figure 5(a), -1 mm in Figure 6(a), or When subtraction processing is performed for each plot of +1 mm, the above error becomes large and the spatial crosstalk This phenomenon was also observed at other positions. In some cases, the subtraction process can have the opposite effect, increasing spatial crosstalk. The causes of this phenomenon are as follows: (1) Figure 1 Even in such a simple optical system, the spatial crosstalk caused by imaging does not necessarily have point symmetry. (For example, in Figure 2, the spatial crosstalk ratio is different between the left and right directions of the imaging point.) (2) The image formation is affected by subtle changes in the optical system, such as a slight shift in the position of the light-emitting point. It is thought that the spatial crosstalk caused by the image changes significantly. The spatial crosstalk ratio between the center and an arbitrary position can be expressed as a function of the distance between them. This is because the method shown in Patent Document 3 does not function effectively. In this disclosure, the optical system and the multiple image points, i.e., the multiple detection points, are used. The spatial crosstalk ratios of the two antennas are shown in Figure 4. As shown in each of Figures 7 and 8, it differs from Patent Document 3 in that it is derived through experiments. If this occurs, the spatial crosstalk ratio must be reacquired each time.

[0048] The findings obtained from the above experiments, namely, the sky at any position away from the center of the image formation point, The absolute signal strength of the inter-channel crosstalk is linear with the absolute signal strength at the center of the imaging point. By expanding and generalizing the fact that the spatial crosstalk ratio is constant, we derive equations (9) to (17). It is served.

[0049] [Example 2] The fluorescence of multiple types of phosphors emitted from multiple emission points is detected in multiple wavelength bands. A model experiment was conducted on the simplest case of an optical system for detecting in the exit area. In other examples using the same model experimental system, for example, (Equation 9) to (Equation 12) In this case, for each of A = 2 light-emitting points, in the detection region of B = 2 wavelength bands, C = 2 types When detecting the fluorescent light emitted from a fluorescent material, if A, B, and C are different values, It goes without saying that the same effect can be obtained by the same method for the above.

[0050] Figure 8 shows a model experimental system. The model experimental system in Figure 8 consists of two capillaries, Cap(1) and Cap(2). ) and a light source ( A laser beam LB is irradiated onto the two capillaries Cap (1) and Cap (2). The light-emitting points P(1) and P(2) are provided at the positions where the capillary Cap(1) is located. The substances labeled with two fluorophores, D(1,1) and D(1,2), were electrophoresed and then separated into capillaries (Cap(2)). Substances labeled with two types of fluorophores, D(2,1) and D(2,2), are electrophoresed. D(1,1) and D(2,1) are the same type of phosphor, and D(1,2) and D(2,2) are the same type of phosphor. The same type of phosphor means that the fluorescence spectrum of the emitted fluorescence is the same. D(1,1) and D(1,2) emit fluorescence at the emission point P(1) when irradiated with the laser beam LB. The sensor S detects the light in the detection areas W(1,1) and W(1,2). The detection area W(1,1) is The detection area W(1,2) mainly detects the emitted fluorescence of the phosphor D(1,1), and the detection area W(1,2) mainly detects the emitted fluorescence of the phosphor D(1,2). The detection wavelength bands are designed so that they are compatible with each other, but there is mutual spectral crosstalk. Similarly, phosphors D(2,1) and D(2,2) emit laser light at the emission point P(2). The light emitted by the laser beam LB is detected in the detection areas W(2,1) and W(2 , 2). The detection area W(2,1) detects the emitted fluorescence of the fluorophore D(2,1), and the detection area W(2,2) detects the fluorescent light. Each detection wavelength band is designed to primarily detect the emission fluorescence of the D(2,2) complex. However, there is significant spectral crosstalk between them. The fluorescence emission of photons D(1,1) and D(1,2) is also detected in the detection regions W(2,1) and W(2,2). Similarly, the fluorescence emission of the fluorophores D(2,1) and D(2,2) at the emission point P(2) is detected in the detection area W(1,1) and The detection area W(1,1) is also detected in W(1,2), and spatial crosstalk is significantly present. ,W(1,2),W(2,1), and W(2,2) are the signal intensities (fluorescence intensities) at X(1,1), X(1,2), and X(2, 1), and X(2,2), as shown in the lower part of Figure 8, the light emitting points P(1) and P(2) For each, the signal intensities X(1,1) and X(1,2), X(2,1) and X(2,2) are obtained with electrophoresis. Time series data can be obtained.

[0051] In FIG. 8, the detection areas W(1,1), W(1,2), W(2,1), and W(2,2) are set on one sensor S. Although it is depicted as if they are separated, this is not necessarily the case. The detection areas W(1,1) and W(1,2) is provided on one sensor, and detection areas W(2,1) and W(2,2) are provided on the other sensor. Alternatively, the detection areas W(1,1), W(1,2), W(2,1), and W(2,2) may be on four different sensors. Furthermore, there may be no need to provide any other components between the light emitting points P(1) and P(2) and the sensor S. Some imaging means and spectroscopic means are required, but in this example, no imaging means or spectroscopic means are used. Since it does not matter whether the means is optical or not, the imaging means and the spectroscopic means are omitted in FIG.

[0052] Figure 9 shows a schematic diagram of the relationship between spectral crosstalk and spatial crosstalk in Figure 8. In FIG. 9(a), the fluorescent light emitted by the phosphor D(1,1) at the light-emitting point P(1) is detected in the detection area W(1 (1) is detected primarily in the detection area W(1,2), and (ii) is detected secondary in the detection area W(1,2). Photofluorescence was also detected in the detection region W(2,1) (iii) at a much lower intensity compared to (i) and (ii). Furthermore, (iv) the detection area W(2,2) is detected with an intensity even smaller than that of (iii). i) shows the spectral crosstalk, (iii) and (iv) show the spatial crosstalk and the spectral In other words, the conventional method only considered (i) and (ii), In this disclosure, all of (i) to (iv) are taken into consideration. In FIG. 9, the relationship between the strength of the detection strength and The relationship between the crosstalk and the signal is shown by the thickness of the arrow, the solid or dotted line, and the numbers (i) to (iv). Similar to Fig. 9(a), Fig. 9(b) shows the fluorescence emission of phosphor D(1,2) at emission point P(1). 9(c) shows the fluorescence emission of phosphor D(2,1) at the emission point P(2), and Fig. 9(d) shows the fluorescence emission of phosphor D(2,1) at the emission point P(2). In actual analysis, the fluorescence emission of D(2,2) is plotted in Fig. 9(a)-(c). (d) occurs simultaneously at the same time, and each has a different fluorescence intensity.

[0053] Figure 10 shows a model experiment system similar to that shown in Figure 8. However, in the model experiment system shown in Figure 10, two The lamp light LL is irradiated from the light source perpendicular to the arrangement direction of the capillaries Cap(1) and Cap(2). Therefore, the fluorescent light emitted by the irradiation of the laser beam LB is not detected, but the fluorescent light emitted by the laser beam LB is detected. The absorption of light LL due to its transmission, or the absorbance, is detected. Absorption points P(1) and P(2) are provided on ap(2) at the positions where the lamp light LL is irradiated. Two types of absorbers, D(1,1) and D(1,2), are electrophoresed in the capillary Cap(1). Two types of absorbers, D(2,1) and D(2,2), are electrophoresed in the lab cap (2). Here, D(1,1) and D(2,1) are the same type of absorber, and D(1,2) and D(2,2) are the same type of absorber. The same absorber means that the light absorption spectrum is the same. 2) is the light that is absorbed by the irradiation of the lamp light LL at the absorption point P(1) and the light that is not absorbed. The absorption is detected in the detection areas W(1,1) and W(1,2) provided on the sensor S. The detection area W(1,1) is The detection area W(1,2) mainly detects the absorption of the light absorber D(1,2), and the detection area W(1,1) mainly detects the absorption of the light absorber D(1,2). Although each detection wavelength band is designed, there is significant mutual spectral crosstalk. Similarly, the light absorbers D(2,1) and D(2,2) are illuminated by the lamp light LL at the absorption point P(2). The light that is not absorbed is reflected by the detection area W(2,1) and the sensor S. The detection area W(2,1) detects the absorption of the light absorber D(2,1), and the detection area W(2,2) detects the absorption of the light absorber D(2,1). Each detection wavelength band is designed to primarily detect the absorption of the D(2,2) molecule. There is significant mutual spectral crosstalk. The absorbance of D(1,1) and D(1,2) is also detected in the detection areas W(2,1) and W(2,2), and similarly, The absorption of the light absorbers D(2,1) and D(2,2) at the absorption point P(2) is detected in the detection areas W(1,1) and W(1,2). This is because the light passing through the absorption point P(1) is significantly different from the light passing through the absorption point P(1). A part of the transmitted light of the lamp light LL is also detected in the detection areas W(2,1) and W(2,2), and the absorption points A portion of the lamp light LL transmitted through P(2) is also detected in the detection areas W(1,1) and W(1,2). The absorbance in the detection areas W(1,1), W(1,2), W(2,1), and W(2,2) is expressed as X( 1,1), X(1,2), X(2,1), and X(2,2), as shown in the lower part of Figure 8, the absorption point P (1) and absorption point P(2), the absorbances X(1,1), X(1,2), and X(2 ,1) and X(2,2) are obtained as time series data. In the following, we will explain the case using Figure 8. However, it goes without saying that the same effect can be obtained when using FIG.

[0054] Figure 11 shows the results of the experiment using the model experimental system shown in Figure 8. ) and obtained by electrophoretic analysis. In both cases, the horizontal axis represents the electrophoretic time ( The vertical axis represents the fluorescence intensity (arbitrary units). Figures 11(a) and 11(b) show the fluorescence intensity at the emission point P(1). The same raw data was obtained, and (b) is an expanded vertical scale of (a). ,Figure 11(c) and Figure 11(d) are the same raw data obtained at the light-emitting point P(2), and Figure 11(d) is the same as Figure 11(c). The vertical scale is enlarged. The horizontal scale in Figure 11(a) to (d) shows the time from 0 to 500. The vertical scale of Fig. 11(a)(c) is the fluorescence intensity from 0 to 250, and the vertical scale of Fig. 11(b)(d) is the fluorescence intensity from -0.2. X(1,1) and X(2,1) are represented by solid lines, and X(1,2) and X(2,2) are represented by dotted lines. As shown in FIG. 11, at time 100, phosphor D(1,1) (in the state of FIG. 9(a)) is emitted at light-emitting point P(1). At time 200, phosphor D(2,1) (state of Fig. 9(c)) is emitted at light-emitting point P(2), and at time 300, phosphor D(2,1) (state of Fig. 9(c)) is emitted at light-emitting point P(2). At the light point P(1), phosphor D(1,2) (as shown in Figure 9(b)), and at time 400, at the light-emitting point P(2), phosphor D(2,2) The sample was prepared so that each of the two (Fig. 9(d)) emits fluorescence independently. The sample was prepared so that no other fluorescence was emitted. As far as c) is concerned, only four large peaks corresponding to the above-mentioned fluorescence emission are observed ( (This corresponds to (i) and (ii) in Figure 9.) Looking at Figures 11(b) and 11(d), four small peaks indicated by arrows are clearly visible. That is, at time 100, fluorescent material D(2,1) is emitted at the emission point P(2), and at time 200, fluorescent material D(2,1) is emitted at the emission point P(1). At time 300, fluorescent substance D(2,2) emits light at the emission point P(1). At time 400, fluorescent substance D(2,2) emits light at the emission point P(1). A photon D(1, 2) was observed (corresponding to (iii) and (iv) in Figure 9). Therefore, these four small peaks The noise can be attributed to spatial and spectral crosstalk. For example, the small peak of phosphor D(2,1) at the emission point P(2) at time 100 is A part of the fluorescence emission of the phosphor D(1,1) at the point P(1) is due to spatial crosstalk and spectral crosstalk. The results are detected as X(2,1) and X(2,2) by Stokes (corresponding to (iii) and (iv) in Figure 9(a)). Response).

[0055] If spatial crosstalk is not taken into account, i.e., the four small pins in Fig. 11(c) and Fig. 11(d) When ignoring the peaks (i.e., when only (i) and (ii) in Fig. 9 are considered), the four peaks in Fig. 11(a) and Fig. 11(c) are Elimination of the spectral crosstalk of the large peaks, i.e., performing color conversion by the conventional method In this case, (Equation 6) can be expressed as follows:

[0056]

number

number

[0057] Here, Z(1,1), Z(1,2), Z(2,1), and Z(2,2) are the phosphors D(1,1), D( The concentrations of D(1,2), D(2,1), and D(2,2) are shown in Equation 18 and Equation 19. In the example, we have a 2x2 matrix Y and its inverse matrix Y -1 was the same at the emission points P(1) and P(2). However, in general, the matrix Y and the inverse matrix Y are given by the emitting point P(1) and the emitting point P(2). -1 is different Each element of the matrix Y for the light-emitting point P(1) is the fluorescence intensity X( Intensity ratio of X(1,1) and X(1,2), and the fluorescence intensity of fluorescent substance D(1,2) at time 300 Intensity ratio of X(1,1) and X(1,2) Each element of the matrix Y for the light-emitting point P(2) is determined by the phosphor D(2,1) at time 200. The ratio of the fluorescence intensities X(2,1) and X(2,2), and the fluorescence intensities X(2,1) and X(2,2) from fluorophore D(2,2) at time 400 ) was determined by the intensity ratio.

[0058] Figure 12 shows the results of applying color conversion (Equation 18) and (Equation 19) to the raw data in Figure 11 at each time point. The color transformation data obtained by the above method is the same as that in Figure 11. As expected, the color transformation data in Figure 11(a) and The spectral crosstalk of the four large peaks in Fig. 11(a) and Fig. 11(c) is shown in Fig. 12(a) and Fig. 12(c). It can be seen that the four large peaks have been resolved. On the other hand, the four peaks in Figures 11(b) and 11(d) The spectral crosstalk of the small peaks in Fig. 12(c) and Fig. 12(d) is also Although the peaks are eliminated by the chromatogram (also indicated by arrows), these peaks still remain. It can be seen that spatial crosstalk has not been eliminated. This is the problem with the conventional method. .

[0059] Next, we consider both spectral crosstalk and spatial crosstalk in Fig. 11. (Consider all of (i) to (iv) in Figure 9) and perform both color conversion and spatial correction. (Equation 14) is expressed by the following equation.

[0060]

number

[0061] Matrix Y and inverse matrix Y -1 has been expanded from 2 rows and 2 columns in (Equation 18) and (Equation 19) to 4 rows and 4 columns. In addition, in (Equation 18) and (Equation 19), color conversion is performed independently at the light emitting point P(1) and the light emitting point P(2). In contrast, in (Equation 20), the light emitting point P(1) and the light emitting point P(2) are mixed together and color conversion + sky Each element of the matrix Y in (Equation 20) is the phosphor D(1, 1) The intensity ratio of the fluorescence intensities X(1,1), X(1,2), X(2,1), and X(2,2) by the fluorescent substance D(2 , 1) and the intensity ratio of the fluorescent intensities X(1,1), X(1,2), X(2,1), and X(2,2), and the fluorescent substance D( 1,2), the intensity ratio of the fluorescence intensities X(1,1), X(1,2), X(2,1), and X(2,2), and the intensity at time 400 Fluorescence intensity of fluorophore D(2,2) is determined by the intensity ratio of X(1,1), X(1,2), X(2,1), and X(2,2) The upper left 2nd row and 2nd column and the lower right 2nd row and 2nd column of the matrix Y in (Equation 20) are expressed as (Equation 18) and (Equation 19). ) and the values ​​of each element are almost equal. Similarly, the matrix Y -1 The upper left 2 rows and 2 columns and the lower right 2 rows and 2 columns of -1 Corresponding to The values ​​of each element are almost equal. In other words, these elements are It is responsible for color transformation that eliminates spectral crosstalk. Here, the values ​​of these elements are ( The reason why (20) does not completely match (18) and (19) is due to the spatial crosstalk that will be explained next. The difference is whether or not the matrix Y and matrix Y in (Equation 20) are taken into account. -1 2 on the top right The second row and the second row and the second column on the bottom left are different light-emitting points that do not exist in (Equation 18) and (Equation 19). Spatial correction and color transformation to eliminate spatial and spectral crosstalk between The above becomes easier to understand if we transform (Equation 20) into the following equation:

[0062]

number

number

[0063] The first term on the right side of (Equation 21) and the second term on the right side of (Equation 22) are responsible for the color conversion in the conventional method, and are respectively This corresponds to (Equation 18) and (Equation 19). On the other hand, the second term on the right-hand side of (Equation 21), The first term on the right side of (Equation 22) is responsible for spatial correction and color conversion, which are not handled in conventional methods. In this way, color conversion and spatial correction and color conversion may be performed separately. Alternatively, for example, the light emitting point P(1) may be subjected to spatial correction and The processing is changed depending on the light-emitting point, for example, color conversion is performed on the light-emitting point P(1) and only color conversion is performed on the light-emitting point P(2). It is also possible.

[0064] Figure 13 shows the results obtained by performing color conversion and spatial correction of (Equation 20) on the raw data in Figure 11 at each time. The notation is the same as in Figure 11. First, as in Figure 12, In addition, the spectral crosstalk of the four large peaks in Figs. 11(a) and 11(c) is shown in Figs. 13(a) and 13(c). It can be seen that the four large peaks in Figure 13(c) are resolved. Spatial and spectral crosstalk of the four small peaks in Fig. 11(b) and Fig. 11(d) The talk is also resolved in Figure 13(c) and Figure 13(d), and the peaks disappear (indicated by arrows). ) can be understood.

[0065] As described above, the present disclosure allows the fluorescent light emitted from multiple types of phosphors from multiple light-emitting points to be The spectrum for the same light-emitting point generated in any optical system that detects Crosstalk, spatial crosstalk between multiple light-emitting points, and spectral crosstalk It has been shown that the problem can be eliminated or reduced by computational processing.

[0066] [Example 3] Next, based on the experimental results of [Example 2], an unknown sample was analyzed. 4. Using the model experimental system shown in Figure 8, an unknown sample is injected into capillaries Cap(1) and Cap(2). The raw data was obtained by electrophoretic analysis using the same model as in Example 2. Since an experimental system is used, (Equation 18) to (Equation 22) can be used as is. The results are the same as those in Figure 11. However, the vertical scales of Figure 11(b) and (d) are reduced to fluorescence intensity -0.5 to 2.0. At times 100, 200, 300, and 400, a large peak appears at the emission point P(1). Meanwhile, a small peak (indicated by an arrow) was observed at the emission point P(2).

[0067] Figure 15 shows the results of applying color conversion (Equation 18) and (Equation 19) to the raw data in Figure 14 at each time point. The color transformation data obtained is the same as in Figure 14. As shown in Figure 15(a), At the emission point P(1), the emission fluorescence of the phosphor D(1,1) was detected alone at times 100 and 300. At 200 and 400 minutes, the fluorescence emitted by the phosphor D(1,2) was detected alone. As shown, four small particles were detected at the emission point P(2) at times 100, 200, 300, and 400. The origin of the peaks (indicated by arrows) was unknown. Is it the fluorescence emission of the mixture of phosphors D(2,1) and D(2,2) at (2) the emission point P(2)? Is it the fluorescence of impurities other than phosphors D(2,1) and D(2,2)? (3) Is it the fluorescence of the emission point P(1)? It was unclear whether this was due to spatial crosstalk between the fluorescence emissions of the phosphors D(1,1) and D(1,2).

[0068] Figure 16 shows the results obtained by performing color conversion and spatial correction of (Equation 20) on the raw data in Figure 14 at each time point. The notation is the same as in Figure 14. As shown in Figure 16(a), Similarly to Figure 15(a), the fluorescent light emitted by phosphor D(1,1) at the emission point P(1) was measured at times 100 and 300. Light was detected independently, and the emission fluorescence of fluorophore D(1,2) was detected independently at times 200 and 400. In addition, as shown in Figure 16(d), the phosphors D(1,1) and D(1,2) at the light-emitting point P(1) Spatial crosstalk and spectral crosstalk for the detection regions W(2,1) and W(2,2) of fluorescence emission As a result of the elimination of the stalk, the minute phosphor D(2,2) is emitted at the emission point P(2) at times 100 and 300. Weak emission fluorescence was detected alone, and at times 200 and 400, weak emission fluorescence of fluorophore D(2,1) was detected alone. The peak intensity of these weak fluorescent emissions was detected at the emission point P( On the other hand, as can be seen from the results in Figure 12, As shown in Figure 8, the spatial crosstalk ratio occurring in the model experimental system is just under 1%. In the case of 15, true weak fluorescence and false weak fluorescence due to spatial crosstalk are mixed. , and it became impossible to distinguish between the two.

[0069] The above results indicate that spatial crosstalk pushes the detection limit for the emission from each emission point. As in the example above, if the spatial crosstalk ratio is 1%, Even if the detection limit for one luminous point is 0.1%, a signal below 1% is is a true signal or a spurious signal due to spatial crosstalk. Therefore, the effective detection limit increases to 1%. In the case of detecting light from multiple emission points, both the detection sensitivity and the dynamic range are one order of magnitude lower. The present disclosure solves such a problem and improves the detection sensitivity in detecting light emission from multiple light-emitting points. This avoids a decrease in image quality and dynamic range.

[0070] [Example 4] Next, based on the experimental results of [Example 2], phosphor D(1,1) and phosphor D(1 , 2) and the unknown substance labeled with fluorophore D (1, 3) were analyzed by electrophoresis. Using a model experimental system, the sample was injected only into capillary cap (1) and into capillary cap (2). The results were obtained by electrophoretic analysis of both capillaries Cap(1) and Cap(2) without sample injection. The notation is the same as in Figure 14. At time 100, a firefly was emitted at the light-emitting point P(1). Light source D(1,1), light emitting point P(1) at time 200, phosphor D(1,2), light emitting point P(1) at time 300 At time 400, phosphor D(1,1) and phosphor D(1,3) emit fluorescence independently at emission point P(1). The sample is prepared so that other fluorescence is not emitted. As shown in Figure 17(a), four samples corresponding to the above fluorescence emissions were prepared. Only a large peak was observed. The spectral crosstalk ratio of phosphor D(1,3) was It is found that the spectral crosstalk ratio is similar to that of D(1,2), but slightly different. On the other hand, the four small peaks indicated by arrows in Figure 17(d) correspond to the four peaks in Figure 17(a). The large peaks are due to spatial and spectral crosstalk in the emission and fluorescence. It can be judged that this is a result.

[0071] Figure 18 shows the results obtained by performing color conversion and spatial correction of (Equation 20) on the raw data of Figure 17 at each time. The notation is the same as in Figure 14. As shown in Figure 18(a), At the emission point P(1), the fluorescence emitted from the phosphor D(1,1) was detected alone at times 100 and 300. At time 200, the fluorescent light emitted by fluorophore D(1,2) was detected alone. However, as indicated by the arrow, At time 400, the emission fluorescence of fluorophore D(1,3) was detected alone, but the spectral crosstalk The ratio is different from that of both phosphor D(1,1) and phosphor D(1,2), so the spectral crosstalk On the other hand, as shown in Figure 18(d), the problem at times 100, 200, and 300 The spatial crosstalk and the fluorescence emission of the phosphors D(1,1) and D(1,2) at the emission point P(1) Even though the spectral crosstalk has been eliminated, as shown by the arrow, at time 400 The spatial crosstalk and the spectrum due to the fluorescence emission of the phosphor D(1,3) at the emission point P(1) It was found that the crosstalk was not eliminated and the peak remained. ], phosphors D(1,1), D(1,2) at the emission point P(1), and phosphors D(2,1), D(2, 2) for the emission fluorescence of D(1,3) at the emission point P(1). Fluorescence has different spatial and spectral crosstalk characteristics from (Eq. 20). Therefore, for the emitted fluorescence of the phosphor D(1,3) at the emission point P(1), we obtain (Equation 20). If we do this again, the above problem will be solved. The above phenomenon represents one aspect of the present disclosure.

[0072] [Example 5] Next, based on the experimental results of [Example 2], the light-emitting point P(1) is labeled with the fluorescent substance D(1,1). An experiment was conducted in which the concentration of the substance was gradually increased to induce fluorescence. Using the experimental system, the sample was injected only into capillary cap (1), and the sample was not injected into capillary cap (2). The results were obtained by electrophoretic analysis of both capillary caps (1) and (2) without injecting any liquid. The notation is the same as in Figure 14. However, the vertical scale of Figures 19(a) and 19(c) is changed to fluorescent. The light intensity is reduced to 0 to 400. At times 100, 200, 300, and 400, the light intensity is The fluorescent substance D(1,1) emits fluorescence independently, and the concentration and fluorescence intensity of the fluorescent substance D(1,1) change gradually. The sample is prepared so that the fluorescence intensity rises to 1000 kJ / s. As shown in Figure 19(a), the sample was prepared as follows: Four large peaks corresponding to the fluorescence were observed. However, the sensor S used in the model experiment system in Figure 8 was Since the intensity saturates at 200, three peaks at times 200, 300, and 400 were detected. In contrast, the spatial correlation of the emitted fluorescence of the phosphor D(1,1) at the emission point P(1) shown in Figure 19(d) is Four small peaks caused by spectral crosstalk and spectral stalk are Due to their low intensity, none of them were observed to be saturated.

[0073] Figure 20 shows the results of applying color conversion (Equation 18) and (Equation 19) to the raw data in Figure 19 at each time point. The color transformation data obtained is the same as that in Figure 19. As shown in Figure 20(a), The fluorescence emitted from phosphor D(1,1) at time 100 at emission point P(1) is However, the emission of phosphor D(1,1) at times 200, 300, and 400 The fluorescence was saturated, so the spectral crosstalk was not eliminated. This is shown in Figure 19(a) As is clear from the graph, when the fluorescence intensity saturates, the signal intensity derived from the intensity ratio of X(1,1) and X(1,2) decreases. The spectral crosstalk ratio changes and the spectral This is because the crosstalk ratio deviates from the normal crosstalk ratio. Since the fluorescence intensity of the fluorophore D(2,1) is not saturated, each of the four small peaks indicated by the arrows The spectral crosstalk was eliminated. The ratio is the intensity ratio of the emitted fluorescence of phosphor D(1,1) at the emission point P(1) at times 100, 200, 300, and 400. represents.

[0074] Figure 21 shows the results obtained by performing color conversion and spatial correction of (Equation 20) on the raw data of Figure 19 at each time. The notation method is the same as in Figure 19. In Figure 21(a), For the same reason as in (a), the same results were obtained. For the emission fluorescence of phosphor D(2,1) at time 100, spatial crosstalk and spectral However, the fluorescent substance D at times 200, 300, and 400 For the emission fluorescence of (2,1), spatial crosstalk and spectral crosstalk have not been resolved. This is because at times 200, 300, and 400, X(1,1) or X(1, As a result of the saturation of the fluorescence intensity of X(1,1) and X(1,2), the fluorescence intensity of X(1,1) and X(1,2) is shown in Figure 19(d The ratio of the fluorescence intensities of X(2,1) and X(2,2) shown in (20) is the space This is because the crosstalk ratio deviates from the actual value. In Figure 21(d), the time increases from time 200 to time 300, and from time 300 to time 40. The fluorescence intensity of the fluorophore D(2,1) in 0 increased.

[0075] [Example 6] Here, we will explain a method for determining the matrix Y in (Equation 20) using a method different from that in [Example 2]. In Example 2, as shown in FIG. 11(a), phosphors D(1,1), D(2,1), D(1,2), and D(2,2) In this order, by making each emit fluorescence independently, that is, the emission point P(1) and the emission point The matrix Y was determined by alternating fluorescence emission at P(2). If the electrophoretic velocity of either Cap(1) or Cap(2) deviates from the expected value, the light emission point P(1) and the light emission point P( 2) There is a possibility that fluorescence may occur simultaneously, in which case the matrix Y cannot be determined.

[0076] Figure 22 shows a more practical way to avoid the above problems. First, As shown in (a), the sample was injected only into capillary cap (1), and the sample was collected in capillary cap (1) and cap (2). Electrophoretic analysis is performed, and the fluorescent substances D(1,1) and D(1,2) emit fluorescence independently. Fluorescence detection is performed at W(1,1), W(1,2), W(2,1), and W(2,2). Next, as shown in Figure 22(b), the capillary The sample was injected only into Capillary Cap (2), and electrophoresis analysis was performed using Capillary Cap (1) and Cap (2). The light sources D(2,1) and D(2,2) emit fluorescence independently, and the detection areas W(1,1), W(1,2), and W(2,1) are , W(2,2) and perform fluorescence detection. In this way, the matrix Y is determined in the same way as in [Example 2]. Furthermore, this method allows the matrix Y to be determined more simply and reliably than in [Example 2]. In Fig. 22, for ease of understanding, the capillary Cap(1) The phosphors D(1,1) and D(1,2), and the phosphors D(2,1) and D(2,2) in the capillary Cap(2) are respectively Although the illustration shows multiple fluorescent emissions, it is sufficient to emit fluorescence once for each. In the above example, the next electrophoretic analysis was performed after the first electrophoretic analysis was completed. However, it is not necessary to extend the interval between electrophoretic analyses to that extent. By appropriately shifting the timing of the sample injection into the capillary tube and the sample injection into the capillary tube (2), For example, after injecting a sample into the capillary cap (1), Electrophoresis is performed for a short time, then the sample is injected into the capillary cap (2) and electrophoresis is resumed. This reduces the time required to determine the matrix Y. In addition, according to this method, the sample injected into the capillary cap (1) and the sample injected into the capillary cap (2) are This allows the samples injected into the sample tank to be of the same composition or identical. This simplifies the samples to be used and reduces the costs involved.

[0077] Figure 23 shows a method for further simplifying the determination of the matrix Y. Here, the capillary Even though sample injection into (1) and sample injection into capillary cap (2) were performed at the same time, However, as in Figure 22, the phosphors D(1,1) and D(1,2) in the capillary Cap(1) and the cap The fluorescent substances D(2,1) and D(2,2) in the Array Cap(2) are designed to emit fluorescence at different times. The sample to be injected into the capillary cap (1) and the sample to be injected into the capillary cap (2) are prepared. By changing the composition of the sample injected into Cap (2), the electrophoretic velocity of each fluorescently labeled substance differs. For example, the sample injected into the capillary cap (1) contains a fluorescent substance. A 50-base-long DNA fragment labeled with D(1,1) and a 60-base-long DNA fragment labeled with fluorophore D(1,2). The sample containing the fragments and injected into the capillary cap (2) was labeled with 70 fluorophore D (2,1). The DNA fragments are labeled with fluorophore D(2,2) and 80 bases long. Just prepare it.

[0078] Alternatively, Figure 23 shows the same composition sample in capillary Cap (1) and Cap (2) at the same time. Even if the injection is performed in the same way, different electrophoresis conditions can be set for capillaries Cap(1) and Cap(2). For example, during electrophoresis, the applied voltage to Capi (2) can be temporarily reduced. and lowering the temperature of Capi (2) during electrophoresis.

[0079] In the above, we assumed that a dedicated sample for determining the matrix Y was prepared in advance. , but it is not necessarily required. In the raw data of the electrophoretic analysis of the actual sample to be analyzed, Therefore, there exists a state in which the fluorescence of each phosphor in each capillary occurs independently, and this state is specified. If the time can be determined, the matrix Y can be determined using the raw data at that time.

[0080] [Example 7] FIG. 24 is a diagram showing the configuration of a multi-capillary electrophoresis device, which is an example of an analytical device. Capillary electrophoresis equipment is used as an analytical device for DNA sequencing and DNA fragment analysis. As shown in Figure 24, the multi-capillary electrophoresis device is 24-1, cathode 24-4, anode 24-5, cathode buffer solution 24-6, anode buffer solution 24-7, pump block 24-9 , a syringe 24-11, and a laser light source 24-12. In this embodiment, four capillaries 24-1 are used. DNA sequencing of different samples was performed in each capillary 24-1. The sample consists of DNA fragments labeled with four types of fluorophores. One analytical session was carried out according to the following procedure: (1) First, the samples in the four capillaries 24-1 were The injection end 24-2 is immersed in the cathode buffer solution 24-6, and the sample elution end 24-3 is connected to the cathode via the polymer block 24-9. (2) Next, the valve 24-10 of the pump block 24-9 was closed, and the pump The piston of the syringe 24-11 connected to the block 24-9 is pushed down to fill the internal poly The polymer solution is pressurized and injected into the inside of each capillary 24-1 through the sample elution end 24-3. (3) Then, the valve 24-10 was opened, and each capillary 24 After different samples are injected from the sample injection terminal 24-2 into the cathode 24-4 and the anode 24-5, Capillary electrophoresis was initiated by applying a high voltage to the sample using power supply 24-8. The DNA fragments labeled with the fluorescent substance flow from the sample injection end 24-2 to the sample elution end 24-3. (4) In parallel, a sample was electrophoresed at a fixed distance from the sample injection end 24-2 of each capillary 24-1. The position where the electrophoresis occurred was designated as the light emission point 24-14, and the laser emitted from the laser light source 24-12 had a wavelength of 505 nm. The laser beam 24-13 was irradiated to each light-emitting point 24-14 at once. The coating of the capillaries 24-1 is removed in advance, and each capillary 24-1 near the light-emitting point 24-14 is placed on the same plane. The laser beam 24-13 is then focused onto the array plane from the side of the array plane. (5) Then, DNA fragments labeled with four kinds of fluorophores were introduced into each capillary 24- The particles electrophoresed inside 1 and were excited by irradiation of the laser beam 24-13 when passing through the light emitting point 24-14. In other words, four types of phosphors emitted fluorescence from four emission points, and the electrophoresis (6) Finally, the fluorescence intensity of each point changed from moment to moment. The emitted fluorescence is detected in multiple colors by a sensor (not shown), and the time series obtained by a computer (not shown) is By analyzing the sequence data, the DNA sequence of the sample injected into each capillary is determined. The analysis session consisting of steps (1) to (6) above can be repeated multiple times. For example, samples (1) to (4) can be analyzed in the first analytical session, and In this session, samples (5) to (8) are analyzed, and by ..., many different samples are analyzed. The rules can be analyzed.

[0081] In this example, the multicolor detection (6) above was performed using the optical system of Patent Document 1. Fluorescence emitted from A=4 light-emitting points P(1) to P(4) is collimated by one condenser lens, and one The first-order diffracted light of each fluorescence is transmitted through a transmission type diffraction grating and focused onto one two-dimensional sensor by one imaging lens. Figure 25 shows the results when the standard solution was filled into four capillaries, Cap(1) to Cap(4). The Raman scattered light waves emitted from the four emission points P(1) to P(4) due to the laser beam irradiation are The result of acquiring a two-dimensional sensor image including a long-dispersion image. The horizontal axis corresponds to the arrangement direction of the four capillaries. The direction of the vertical axis is the wavelength direction. The four vertical stripe images indicated by the arrows are The wavelength dispersion images of the Raman scattered light from the capillaries Cap(1) to Cap(4). For the light-emitting point, wavelength calibration of the wavelength dispersion image on the two-dimensional sensor image is performed, and the pixel position and Based on the results, the relationship between wavelength and intensity was calculated. For each of the light-emitting points P(1) to P(4) on the image, there are B = 20 detection areas with different wavelength bands. Each of the 20 detection regions was divided equally into 10 nm intervals for the emission from 500 to 700 nm. For example, the detection area W( 1,1), W(1,2), …, W(1,20) are 500–510 nm, 510–520 nm, …, 690–700 nm, respectively. The wavelength bands are set to detect the emission, and the signal intensities of each are calculated as X(1,1), X(1,2), ..., X(1, 20). The same applies to the light-emitting points P(2) to P(4). Here, the positions of the light-emitting points P(1) to P(4) are As long as the position and optical system are fixed, the image of each capillary in the two-dimensional sensor image can be Since the relationship between pixel position and wavelength is maintained for all pixels, the detection area set in Figure 26 can be used for multiple different It is also useful for luminescence detection, or for multiple different analytical sessions.

[0082] In this example, dR110, dR6G, dTAMRA, and dROX were used as C=4 types of phosphors. Each of these is targeted to DNA fragments with terminal bases of T, C, A, and G prepared by the Sanger reaction. The maximum fluorescence emission wavelengths of dR110, dR6G, dTAMRA, and dROX were 541 nm and 568 nm, respectively. The emission fluorescence is 540-550 nm, 560-570 nm, and 595 nm, respectively. The strongest intensity is detected in the wavelength ranges of 590-600 nm, 610-620 nm. For example, the emission fluorescence of dR110, dR6G, dTAMRA, and dROX at the emission point P(1) is ,It is mainly detected in the detection areas W(1,5), W(1,7), W(1,10), and W(1,12). However, Each emitted fluorescence is detected in other detection areas of the emission point P(1) due to spectral crosstalk. The spatial and spectral crosstalks also lead to the emission points P(2)-P(3). The dR110, dR6G, and dR11G at the emission point P(1) are also detected with weak intensity in the detection area. TAMRA and dROX are respectively designated as D(1,1), D(1,2), D(1,3), and D(1,4). The concentrations of the luminous points P(2) to P(4) were set to Z(1,1), Z(1,2), Z(1,3), and Z(1,4). It seems that

[0083] In this case, (Equation 10) to (Equation 12) in (Equation 9) become the following equations.

number

number

number

[0084] Here, X is a matrix with 80 rows and 1 column, Y is a matrix with 80 rows and 16 columns, and Z is a matrix with 16 rows and 1 column. The spatial correction and color conversion obtained by substituting into (Equation 14) are performed, and the spatial crosstalk and the spectrum The crosstalk between the two was eliminated, and the time series data of the concentration of the four types of phosphors in each capillary was obtained. As a result, the DNA of different samples injected into each capillary was obtained. The sequence was successfully performed. The conventional method corresponding to this example requires separate capillaries. The conventional method cannot eliminate spatial crosstalk. .

[0085] (23) to (25), and (14) derived from them, are the positions of the light-emitting points, the optical As long as the system and the fluorophores used are fixed, multiple different emission detections or multiple It is also valid for different analysis sessions.

[0086] The method for determining the matrix Y has already been described. However, all 80 × 16 elements are set. For example, it is not necessary to set an element whose absolute value is sufficiently small compared to other elements to zero. It is also possible to simplify the calculations involved by replacing If the range is limited, it is acceptable to define (Equation 9) to (Equation 12) within the limited range. In this case, if necessary, the limited range can be gradually slid to perform a wider analysis. For example, if the range of spatial crosstalk can be limited to the adjacent capillaries, The spatial crosstalk and spectral crosstalk between two or more separated capillaries are In this case, we can ignore the matrix Y and skip the associated calculations. In order to achieve this, the process of making each light-emitting point emit light independently is performed in a way that prevents the influence of spatial crosstalk on each other. It is also possible to simplify the process to a process in which multiple light-emitting points are simultaneously made to emit light.

[0087] [Example 8] In this example, similar to Example 7, a multi-capillary electrophoresis apparatus shown in FIG. NA sequence was performed. However, the fluorescence emission of four types of phosphors emitted from four emission points was At each time, the light was detected in multiple colors by the optical system of Patent Document 2. Figure 27 shows the structure of this optical system. The optical system in Figure 27 shows the composition of the four light-emitting points and the four-part image of the four light-emitting points obtained by this optical system. Array 27-1, long-pass filter 27-3, dichroic mirrors 27-4, 27-5, 27-6, and As shown in Figures 27(a) and 27(b), first, A The fluorescent light emitted from the four light-emitting points P(1) to P(4) is focused onto the condenser lens array 27-1. The four condenser lenses 27-2 collimate the light individually to form four beams 27-9, and one long-pass The laser beam was cut off by passing it through filter 27-3. A set of dichroic mirrors consisting of dichroic mirrors 27-4, 27-5, 27-6, and 27-7 By collectively inputting each light beam 27-9 to the mirror array, each light beam 27-9 is divided into B=4 divided light beams 2 7-10, 27-11, 27-12, and 27-13, respectively, and Here, the spectral characteristics of each dichroic mirror are adjusted to separate the light. The light beams 27-10, 27-11, 27-12, and 27-13 are 520-550 nm, 550-580 nm, and 580-6 The wavelengths of the light were set to 10 nm and 610-640 nm. Finally, the generated A × B = The 4 × 4 = 16 split beams are incident on the two-dimensional sensor 27-8, and as shown in Figure 27(c), the 16 split beams are The images W(1,1) to W(4,4) were obtained. Here, the arrangement direction of the four capillaries Cap(1) to Cap(4), i.e., The direction of the arrangement of the light-emitting points P(1) to P(4) and the direction of division by the dichroic mirror array, In other words, the wavelength directions are perpendicular to each other, so as shown in Figure 27(c), the two-dimensional sensor image 27-14 During this process, the divided images W(1,1) to W(4,4) were detected in an aligned state without overlapping each other. For example, the fluorescence emitted from the emission point P(1) is divided into W(1,1), W(1,2), W(1,3), and W(1,4). and detected as 520-550 nm, 550-580 nm, 580-610 nm, and 610-640 nm, respectively. The same is true for the light-emitting points P(2) to P(4). The divided images W(1,1) to W(4,4) in the sensor image 27-14 were set as the detection areas. The signal intensities detected in the emission areas are designated as X(1,1) to X(4,4), respectively. As long as the position of 4) and the optical system are fixed, each cap in the two-dimensional sensor image 27-14 The relationship between pixel position and wavelength for the array is maintained, so the detection area set in Figure 27(c) is valid for multiple different emission detections or multiple different analytical sessions .

[0088] In this example, as in Example 7, dR110, dR6G, and dTAMRA were used as C=4 types of phosphors. , and dROX are used, and the terminal base species prepared by Sanger reaction are T, C, and A. The maximum fluorescence emission wavelengths of dR110, dR6G, dTAMRA, and dROX were measured. are 541 nm, 568 nm, 595 nm, and 618 nm, respectively, so the emission fluorescence is , 520-550 nm, 550-580 nm, 580-610 nm, and 610-640 nm wavelength bands For example, dR110 and dR110 at the light-emitting point P(1) are detected with the strongest intensity in the detection area of ​​the divided image. The emission fluorescence of R6G, dTAMRA, and dROX is detected in the detection regions W(1,1), W(1,2), W(1,3), and W(1,4), respectively. However, each emission fluorescence is mainly detected by W(1,4) due to spectral crosstalk. Therefore, the light is also detected in other detection areas of the light emitting point P(1), and spatial crosstalk and spectral Due to vector crosstalk, it is also detected in the detection area of ​​light-emitting points P(2) to P(3). The dR110, dR6G, dTAMRA, and dROX at the emission point P(1) were replaced by the phosphors D(1,1) and D(1,2), respectively. ), D(1,3), and D(1,4), and their concentrations are 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).

[0089] In this case, (Equation 10) to (Equation 12) in (Equation 9) become the following equations.

number

number

number

[0090] Here, X is a matrix with 16 rows and 1 column, Y is a matrix with 16 rows and 16 columns, and Z is a matrix with 16 rows and 1 column. By substituting into (Equation 14), both spatial and spectral crosstalk can be calculated. This allowed us to obtain time-series data on the concentrations of the four types of phosphors in each capillary. As a result, DNA sequencing of different samples injected into each capillary was performed. In the conventional method corresponding to this embodiment, the color conversion of (Equation 8) was performed for each capillary. Conventional methods cannot eliminate spatial crosstalk.

[0091] (26) to (28) and (14) derived from them are the positions of each light emitting point, the optical As long as the system and the fluorophores used are fixed, multiple different emission detections or multiple It is also valid for different analysis sessions.

[0092] [Example 9] Figure 28 shows a multi-channel reactor with multiple reactors arranged on a plane, with different flow rates in each channel. The complementary strand is elongated in a single base unit using the DNA fragment as a template, and the resulting complementary strand is incorporated into the elongation reaction. By detecting the fluorescence emitted by the four C=4 fluorophores labeled on the four bases, The diagram of the multi-channel extension reaction device for DNA sequencing of each DNA fragment, and the A schematic diagram of a two-dimensional sensor image obtained with a two-dimensional sensor is shown. 28-1, a laser light source 28-2, a dichroic mirror 28-3, a lens 28-4, and a dichroic mirror 28 -8, 28-9 and 28-10, lens 28-11, first two-dimensional sensor 28-12, lens 28-13, second two-dimensional The sensor 28-14, the lens 28-15, the third two-dimensional sensor 28-16, the lens 28-17, and the fourth two-dimensional sensor The maximum fluorescence emission wavelengths of the fluorophores labeled with the bases T, C, A, and G are The wavelengths were set to 535 nm, 565 nm, 595 nm, and 625 nm, respectively. First, the laser source 28-1 oscillated. The laser beam 28-2 is transmitted through a dichroic mirror 28-3 and then focused by a lens 28-4. The laser beam was then irradiated onto the multi-channel sample 28-5. The fluorescent light 28-6 from each phosphor excited by the beam irradiation was collimated collectively by the lens 28-4. After that, the resulting light beam 28-7 is reflected by a dichroic mirror 28-3. 28-3 has the spectral characteristics of transmitting the laser beam light and reflecting the emitted fluorescent light. Three types of dichroic mirrors 28-8, 28-9, and 28-10 are used to direct the light beam 28-7 into four different beams. The first split beam is then passed through lens 28-11. The second split light beam is focused onto the first two-dimensional sensor 28-12 by the lens 28-13. The third split beam is focused on the first sensor 28-14, and the third split beam is focused on the third two-dimensional sensor 28- The fourth split beam is imaged on the fourth two-dimensional sensor 28-18 by the lens 28-17. made him do so.

[0093] Sample 28-19 is a schematic diagram of Sample 28-5 observed from the front. A = 5 channels In reality, many more channels were formed on the sample. The fluorescence emitted from each channel was detected simultaneously by the optical system described above. The light emitting point P(3) of interest and the light emitting point P(3) that directly affects the spatial crosstalk are Since only the light-emitting points P(1), P(2), P(4), and P(5) around the light point P(3) are analyzed, In Sample 28-19, only the light-emitting points P(1) to P(5) are drawn for simplicity. When observing the light emission point, the light emission point and the surrounding light emission points should be analyzed in the same way. Even when focusing on different emission points, after fluorescence detection, the same fluorescence detection data is analyzed individually. The first two-dimensional sensor image 28-20 is acquired by the first two-dimensional sensor 28-12. The divided image of sample 28-19 obtained by the experiment is shown in Fig. 1. The detection area W is located at each of the image points P(1) to P(5). The first two-dimensional sensor was set to (1,1) to W(5,1), and the signal intensities were set to X(1,1) to X(5,1). In the second 2D sensor image 28-20, fluorescent components in the wavelength range of 520 to 550 nm were detected. 1 is a divided image of the sample 28-19 acquired by the second two-dimensional sensor 28-14, and the light-emitting point P(1 The detection areas W(1,2) to W(5,2) were set at the respective image points P(1,2) to P(5). In image 28-21, fluorescent components in the wavelength range of 550 to 580 nm were detected. The third two-dimensional sensor image 28-22 , a divided image of the sample 28-19 acquired by the third two-dimensional sensor 28-16, and the light-emitting point P(1)~ The detection areas W(1,3) to W(5,3) were set at the respective image points of P(5). The fourth two-dimensional sensor image 28-23 was obtained from the fourth sensor image 28-22. The image of the sample 28-19 is obtained by the two-dimensional sensor 28-18. The light-emitting points P(1) to P(5) are The detection areas W(1,4) to W(5,4) were set at the respective image points. In this study, the fluorescent components in the wavelength range of 610 to 640 nm were detected. For example, four types of fluorescent materials at the light-emitting point P(3) were detected using two-dimensional sensors. The emitted fluorescence is mainly detected in the detection areas W(3,1), W(3,2), W(3,3), and W(3,4) in wavelength order. However, each emitted fluorescence is detected at the emission point P(3) due to spectral crosstalk. Other detection areas are also detected, and spatial and spectral crosstalk The light is also detected in the detection areas of the light-emitting points P(1), P(2), P(4), and P(5). The four phosphors in P(3) are D(3,1), D(3,2), D(3,3), and D(3,4), respectively, in order of wavelength. ), and their concentrations are Z(3,1), Z(3,2), Z(3,3), and Z(3,4). The emission points P(1), P The same applies to (2), P(4), and P(5).

[0094] In this case, (Equation 10) to (Equation 12) in (Equation 9) become the following equations.

number

number

number

[0095] Here, X is a matrix with 20 rows and 1 column, Y is a matrix with 20 rows and 20 columns, and Z is a matrix with 20 rows and 1 column. By substituting into (Equation 14), both spatial and spectral crosstalk can be calculated. This allows us to obtain time series data of the concentrations of four types of fluorescent substances in each channel. However, in this example, since we are only focusing on the light emitting point P(3), we use Z(3,1) in the matrix Z. , Z(3,2), Z(3,3), and Z(3,4). As a result, the channel of the light-emitting point P(3) We were able to perform DNA sequencing in the channel. A similar approach can be used for DNA sequencing in a channel.

[0096] [Example 10] In this example, the conventional method and the method disclosed herein are summarized in a flowchart. As shown in Figure 29, the analysis session of the conventional method is The analysis session consisted of the analytical equipment, computer, display device, and database used to analyze sample 29-1. The analysis is performed by an analysis system having a light source 29-1. First, a type A labeled with a type C (C is an integer of 1 or more) of fluorescent material is used. (A is an integer of 2 or more) sample 29-1 is input into the analyzer. By using step 29-2, each sample is analyzed in parallel, and for each analysis and at each time Then, the emitted fluorescence is detected in B types of wavelength bands (B is an integer of 1 or more), and the fluorescence intensity X(a, b) of A × B pieces is calculated. The time series raw data is acquired and sent to the computer. Here, a=1, 2, ..., and A, b = 1, 2, ..., and B. Then, in the computer, step 29-3 For analysis (a0), the matrix Y with C rows and B columns stored in the database is - Color change using (a0) The spectral crosstalk is eliminated by the conversion (a0), and the fluorescence intensity X(a0, b) is calculated at each time. From this, we calculate the concentration Z(a0, c) of type C phosphor. Here, a0 = 1, 2, ... or A, c = 1, 2, ... , and C. Finally, in the display device, the time series color transformation of Z(a0, c) is performed by step 29-4. The data is output. Steps 29-3 and 29-4 are performed for all a0. As shown, the conventional method involves A analyses and analysis being carried out independently. In (Equation 1) to (Equation 4), the fluorescence intensity X(a, b) is X(b), and the concentration Z(a, c) is Z(c ) is written as follows.

[0097] Figure 30 is a flowchart showing one analysis session of this method. The analytical session of this example shown in FIG. 0 involves the analysis of sample 29-1 using an analytical instrument, a computer, and a display. The analysis is performed by an analytical system having a device and a database. First, there are C types of fluorescent light (C is an integer of 1 or more). A sample 30-1 of type A (A is an integer of 2 or more) labeled with a photobody is input into the analyzer. In the analyzer, in step 30-2, each sample is analyzed in parallel, and At each time, fluorescent light is detected in B wavelength bands (B is an integer of 1 or more), and A x B pieces of time-series raw data of fluorescence intensity X(a, b) are acquired and sent to a computer. Here, a = 1, 2, ..., and A, and b = 1, 2, ..., and B. However, as shown in Figure 29, However, as shown by the dotted arrows in step 30-2, there is spatial crosstalk between the emission fluorescence of different assays. Subsequently, in step 30-3, all the analyses (a) are performed by computer. Regarding the matrix Y - Color conversion using sky By using time correction, spectral crosstalk and spatial crosstalk are eliminated. From the fluorescence intensity X(a, b), the concentration of the fluorophore C for the sample or analysis A is calculated. Find Z(a,c) all at once, where a=1,2,…, and A, and c=1,2,…, and C. Finally, in step 30-4, the time series of Z(a,c) for each analysis (a) is displayed on the display device. As shown in Figure 30, this method outputs column color conversion + spatial correction data. It is characterized by the fact that color conversion and spatial correction are performed simultaneously for all analyses. Color transformation + spatial correction is performed in a matrix Y common to all analyses. - Another feature is the use of

[0098] Figure 31 shows the matrix Y stored in the database, which is performed before Figure 30. - Flowchart showing how to calculate First, a type A (A is an integer of 2 or more) labeled with a type C (C is an integer of 1 or more) of fluorophores is prepared. The sample 31-1 for determining the matrix Y (number of samples) is input to the analysis device. This sample is The fluorescent material (c) is prepared so that the fluorescent emission of the fluorescent material (c) occurs independently. That is, a=1, 2, ..., and A, c=1, 2, …, and C, and for all combinations of these A × C, The fluorescent light is emitted by two or more combinations of fluorescent light, but the fluorescent light is not emitted simultaneously. Here, the above is realized by the sample 31-1 for determining the matrix Y, but the setting of the analysis device Next, in the analyzer, in step 31-2, A × C individual fluorescent emissions are detected in B types of wavelength bands (B is an integer of 1 or more), and A × B fluorescent emissions are detected. The time series raw data of intensity X(a, b) is acquired and sent to a computer. In the computer, from the (A × B) × (A × C) data obtained in step 31-3, Then, we derive a matrix with (A × C) rows and (A × B) columns, which is the general inverse matrix of Y. Column Y - Then, the matrix Y -The data is stored in a database and used in subsequent analyses such as Figure 30. matrix Y - Utilize the following.

[0099] Figure 32 shows the matrix Y obtained in Figure 31. - Using this, we investigated the sample 30-1 of type A shown in Figure 30. 10 is a flowchart for repeating the analysis session of steps 30-2 to 30-4 multiple times. Here, the samples of type A analyzed in each analysis session are different from each other. As shown in Figure 32, the database is populated with data for several different analysis sessions. The same matrix Y stored - A distinctive feature of this law is that it makes use of the following:

[0100] Figure 33 shows an example of the computer configuration. The computer is connected to an analytical device. It not only analyzes the data but also controls the analytical equipment. In Figure 33, they are depicted inside the computer. The conditions for data analysis and control of the analytical equipment are set through the keyboard. The time-series raw data of the fluorescence intensity X(a, b) output from the analyzer is stored in memory in sequence. ,The matrix Y with (A × C) rows and (A × B) columns is stored in the database inside the HDD. - is in memory The CPU calculates the fluorescence intensity X(a, b) and Y(a, b) stored in the memory. - Calculate the product of these and find the phosphor concentration Z The time series color conversion + space correction data (a, c) is derived and stored in memory sequentially, and at the same time, The analysis results are displayed on a monitor. It can be compared with information on the network.

[0101] [Variations] The present disclosure is not limited to the above-described embodiments, but includes various modifications. For example, the above-described embodiments have been described in detail to clearly explain the present disclosure. It is not necessary to have all the configurations described in the above. In addition, the configuration of one embodiment can be replaced with the configuration of another embodiment. In addition, a part of the configuration of each embodiment can be added to the configuration of other embodiments. Parts of the configuration may be added, deleted or replaced. [Explanation of symbols]

[0102] 1-1 Pinhole plate 1-2 Light-emitting point side opening plate 1-3 Condenser lens 1-4 Sensor side opening plate 1-5 Colored Glass Filters 1-6 2D sensor 1-7 Halogen lamp light 1-8 Light-emitting point 1-9 light 1-10 Luminous image Cap(1) Capillary 1 Cap(2) Capillary 2 P(1) Light-emitting point on Cap(1) P(2) Emission point on Cap(2) Fluorescent or absorber 1 on D(1,1) P(1) D(1,2) Fluorescent or absorber 2 on P(1) Fluorescent or absorber 1 on D(2,1) P(2) D(2,2) Fluorescent or absorber 2 on P(2) D(1,3) Fluorescent or absorber 3 on P(1) W(1,1) Detection area that mainly detects D(1,1) W(1,2) Detection area that mainly detects D(1,2) W(2,1) Detection area that mainly detects D(2,1) W(2,2) Detection area that mainly detects D(2,2) X(1,1) W(1,1) signal strength Signal strength of X(1,2) W(1,2) Signal strength of X(2,1) W(2,1) Signal strength of X(2,2) W(2,2) Concentration of Z(1,1) D(1,1) Concentration of Z(1,2) D(1,2) Cardinality of Z(2,1) D(2,1) Concentration of Z(2,2) D(2,2) S sensor LB laser beam LL Lamp Light 24-1 Capillary 24-2 Sample injection port 24-3 Sample elution end 24-4 Cathode 24-5 Anode 24-6 Cathode buffer solution 24-7 Anode side buffer solution 24-8 Power supply 24-9 polymer block 24-10 valve 24-11 Syringe 24-12 Laser light source 24-13 Laser Beam 24-14 Luminous point Cap(a) Capillary a (a=1, 2, 3, and 4) P(a) Emission point on capillary a (a = 1, 2, 3, and 4) W(a,b) is the detection area of ​​the emission from P(a) in wavelength band b (a=1, 2, …, and 4) (b=1, 2, …, and 20) 27-1 Condenser lens array 27-2 Condenser lens 27-3 Long-pass filter 27-4, 27-5, 27-6, and 27-7 Dichroic Mirrors 27-8 2D Sensor 27-9 Luminous flux 27-10, 27-11, 27-12, and 27-13 Split beam 27-14 2D sensor image P(a) Emission point a (a = 1, 2, 3, 4, and 5) W(a,b) is the detection area of ​​the wavelength band b of the emission from P(a) (a=1, 2, 3, 4, and 5) (b=1, 2, 3 , and 4) 28-1 Laser light source 28-2 Laser beam 28-3, 28-8, 28-9, and 28-10 Dichroic Mirrors 28-4, 28-11, 28-13, 28-15, and 28-17 lenses 28-5, and 28-19 Samples 28-6 Fluorescence 28-7 Luminous flux 28-12, 28-14, 28-16, and 28-18 2D Sensors 28-20, 28-21, 28-22, and 28-23 2D sensor images 29-1 Sample 29-2 Analytical equipment processes 29-3 Computerized processes 29-4 Display Device Processes 30-1 Sample 30-2 Analytical equipment processes 30-3 Computerized processes 30-4 Display Device Processes 31-1 Sample 31-2 Analytical equipment processes 31-3 Computerized processes

Claims

1. Using multiple capillaries, multiple samples containing components labeled with multiple types of fluorophores are A multi-capillary electrophoresis device that performs parallel electrophoretic analysis of pools is The plurality of types of particles are irradiated onto the plurality of capillaries and move through the plurality of capillaries. one or more excitation lights configured to excite the phosphors of the class to produce fluorescence; The positions of the capillaries are fixed, and the fluorescent light is emitted from the capillaries. A plurality of light emitting points, an optical system for detecting raw signals of the fluorescence emitted from the plurality of light-emitting points in a plurality of wavelength bands; and, The raw signals are subjected to batch processing to remove spatial crosstalk and spectral crosstalk between the raw signals. and reducing the talk, and performing a process corresponding to the concentrations of the plurality of types of phosphors at the plurality of light emitting points. A computer that outputs a logical signal; A power source for applying a voltage to both ends of the plurality of capillaries. Multicapillary electrophoresis apparatus.

2. Using A capillaries, prepare A samples S(a) (a = 1, 2, ...), where A is an integer greater than or equal to 2. , A) are electrophoretically analyzed in parallel, and the A samples are electrophoretically analyzed by electrophoresis using C types of fluorescent substances (C is an integer of 1 or more). (a,c) A multi-capillary electrophoresis device containing components labeled with (c=1, 2, ..., C), The C-type fluorescent light is irradiated onto the A-type capillary and travels through the A-type capillary. One or more excitation lights that excite the bodies D(a, c) to emit fluorescence; The positions of the A capillaries are fixed, and the fluorescent light is emitted. A light emitting points P(a) The light is emitted from the A light emitting points, and B (B is an integer equal to or greater than 1) wavelength bands W(a,b) (b=1, 2, ... an optical system for acquiring A×B raw signals X(a, b) of the fluorescence detected by the first and second light sources; The A×B raw signals X(a,b) are collectively processed to obtain the spatial correlation between the A×B raw signals X(a,b). The A number of emission spectra of the C type phosphors D(a, c) are reduced by reducing spectral crosstalk and spectral A computer that outputs A × C processed signals Z(a,c) corresponding to the densities at the light spots; A multi-capillary tube comprising: a power source for applying a voltage to both ends of the A capillaries; Capillary electrophoresis apparatus.

3. The multi-capillary electrophoresis apparatus according to claim 2, The optical system comprises: an optical element for dispersing the wavelength of the fluorescence emitted from the A light-emitting points P(a); A number of wavelength dispersion images of the fluorescence emitted from the A number of light-emitting points P(a) are formed separately from each other. and one or more image sensors or a plurality of line sensors, On the one or more image sensors or the multiple line sensors, Along each of the wavelength dispersion images, the A light emitting points are arranged in the B wavelength bands W(a, b). Corresponding pixel blocks of A × B are defined, The integrated signals in the A×B pixel blocks are made to correspond to the A×B raw signals X(a,b). A multi-capillary electrophoresis apparatus comprising:

4. The multi-capillary electrophoresis apparatus according to claim 2, The optical system comprises: The fluorescence emitted from the A light-emitting points P(a) is collimated in a lump to form A light beams. A first lens; a transmission type diffraction grating for collectively dispersing the A number of light beams to form A number of wavelength-dispersed light beams; ， a second lens for collectively collecting the A wavelength-dispersed light beams to form A wavelength-dispersed images; ， one or more image sensors onto which the A wavelength dispersion images are projected and detected; or a plurality of line sensors, On the one or more image sensors or the multiple line sensors, Along each of the wavelength dispersion images, the B wavelength bands W(a,b) for the A light emitting points are A × B pixel blocks corresponding to The integrated signals in the A×B pixel blocks are made to correspond to the A×B raw signals X(a,b). A multi-capillary electrophoresis apparatus comprising:

5. The multi-capillary electrophoresis apparatus according to any one of claims 2 to 4, Let b = b0 (b0 is a constant) and c = c0 (c0 is a constant), and let the A raw signals X(a, b0) be The spatial crosstalk between the A processed signals Z(a, c0) is relatively larger than the spatial crosstalk. A multi-capillary electrophoresis device characterized by its small size.

6. The multi-capillary electrophoresis apparatus according to any one of claims 2 to 5, The spectral crosstalk between the B raw signals X(a0, b) is calculated by a=a0 (a0 is a constant). It is also noted that the spectral crosstalk between the C processed signals Z(a0,c) is relatively small. A multi-capillary electrophoresis apparatus.

7. The multi-capillary electrophoresis apparatus according to any one of claims 2 to 4, The matrix X having (A × B) rows and 1 column, with the A × B raw signals X(a, b) as elements, is [0010] Let Z be a matrix with (A × C) rows and one column, with the A × C processed signals Z(a, c) as elements: [0025] Let X = Y × Z, and let (A × B) row ( Let Y be a matrix with A×C columns, [0030] Then, the general inverse matrix of matrix Y is (A×C) rows and (A×B) columns Y - is calculated in advance, and Z=Y - X The operation of the above is regarded as the batch processing, or a process equivalent to the above operation is regarded as the batch processing. A multi-capillary electrophoresis apparatus.

8. The multi-capillary electrophoresis apparatus according to any one of claims 2 to 4 and 7, In accordance with the electrophoretic analysis of the A samples S(a), the A×B raw signals X(a, b) are Change, The computer performs the batch processing at each time to generate the A × C processed signals Z(a,c) A multi-capillary electrophoresis apparatus which outputs a time series of

9. The multi-capillary electrophoresis apparatus according to any one of claims 2 to 4 and 7, When the analysis is performed multiple times at different times, the computer a multi-capillary electrophoresis apparatus for performing the batch processing in common for all the samples;

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