Light detection device and signal processing method

Abstract

A light detection device (500) is provided with: a replaceable capillary array (501) in which a plurality of types of light-emitting bodies emit light; a light sensor (504) having a plurality of detection channels for detecting light emitted from the plurality of types of light emitters in a plurality of wavelength bands; an optical fiber array (510) that fixes the incident position of light emitted from the plurality of types of light emitting bodies with respect to the light sensor (504); and a computer (550). A computer (550) has crosstalk information therein, and reduces crosstalk existing between detection channels of an optical sensor (504) by calculating the output from the optical sensor (504) corresponding to a plurality of light-emitting points and (each) a plurality of wavelength bands that emit light by a plurality of types of light-emitting bodies using the crosstalk information. Concentration ratios or semaphore ratios for each of the plurality of light-emitting bodies are derived for each of the plurality of capillaries, and crosstalk information is used for calculation even if the capillary array (501) is replaced with another capillary array.

Description

Technical Field

[0001] The present invention relates to a light detection device and a signal processing method, and in particular to a light detection device and a signal processing method that reduce crosstalk generated when multiple light-emitting bodies emit light at multiple light-emitting points. Background Art

[0002] There are light measurement methods that use a light detection device that separates and detects the wavelengths of light at each spatial location, using multiple luminophores at multiple spatial locations. This device can detect the location and concentration ratio of each luminophore. An example of this method is a light detection device in a capillary electrophoresis apparatus equipped with multiple analytical capillaries that analyzes samples labeled with multiple fluorescent dyes.

[0003] In capillary electrophoresis, the sample to be analyzed is injected into a capillary filled with a separation medium. A voltage is applied across both ends, and separation is achieved by utilizing differences in the mobility of the analytes. Detection of the analytes is achieved, for example, using fluorescence detection. The analytes are labeled with a fluorescent dye, and the fluorescence generated by irradiation with excitation light is detected, thereby detecting the separated analytes.

[0004] As an example, there is a method in which DNA labeled with a fluorescent dye is electrophoresed in a capillary filled with a polymer to separate it by chain length. Excitation light is irradiated to a detection site set on the capillary, and the resulting fluorescence is detected. The DNA molecules in the sample move in the capillary, passing through the detection site at different times depending on their chain length. As a result, the chain length distribution of the DNA molecules in the sample is obtained as a fluorescence intensity waveform. When there are multiple types of DNA molecules in the sample and they need to be analyzed separately, multiple fluorescent dyes are sometimes used to label the DNA. A light detection device uses a grating or the like to separate and detect the wavelengths of the fluorescence, and the type of fluorescent dye is determined based on the shape of the resulting spectrum.

[0005] To increase measurement throughput, capillary electrophoresis devices sometimes incorporate multiple capillaries. In such cases, for example, the separation capillaries are arranged in a row and all are illuminated with excitation light. The resulting fluorescence is captured by an image sensor. The signal intensity is calculated from the fluorescence image of each capillary, yielding the sample's electrophoretic waveform.

[0006] As mentioned above, when using multiple fluorescent dyes and multiple capillaries, signals different from the intended signals may be detected, causing the measured concentration of the target object to deviate from the true value. For example, fluorescent dye A may emit light in a certain capillary, but it may be mistakenly identified as fluorescent dye B. Or, although the i-th capillary emits light, it may be mistakenly identified as the j-th capillary, which is different from the i-th capillary. Signals output due to misidentification are referred to as crosstalk. The former misidentification of the dye is called spectral crosstalk, and the latter misidentification of the emitting capillary is called spatial crosstalk.

[0007] Can consider the generation cause of various crosstalk.As an example, spectral crosstalk can be produced by the width of the emission spectrum of fluorescent pigment.Usually, fluorescent pigment has the luminous spectrum of the wavelength width of about tens of nanometers.When using multiple pigments, the luminous spectrum of pigment A and the luminous spectrum of pigment B can overlap.When there is overlapping in the luminous spectrum of pigment A and pigment B, even if pigment A is luminous, the part of the detection band of the pigment B of the luminous spectrum of pigment A is detected as the situation of the luminous signal of pigment B.

[0008] As an example, spatial crosstalk may be caused by surface reflections of adjacent capillaries. In the device, multiple capillaries are arranged in a row, and the detector is arranged in a form opposite to the capillary array. If the fluorescent pigment emits light in the i-th capillary (hereinafter, appropriately referred to as capillary i), the fluorescence is radiated in all directions, and a portion is introduced into the detector. However, a portion of the light is irradiated to the nearby j-th capillary (hereinafter, appropriately referred to as capillary j), and a portion of it is reflected toward the detector. In this case, capillary j, which appears to have reflected fluorescence on the detector, emits fluorescence, which is detected as a luminescent signal of capillary j.

[0009] Crosstalk can cause various adverse effects in capillary electrophoresis-based analyses. For example, if fluorescence from capillary i is misidentified as fluorescence from capillary j due to spatial crosstalk, components of the sample originally analyzed by capillary i may be mistakenly interpreted as being contained in the sample analyzed by capillary j. Furthermore, spectral crosstalk can lead to the misinterpretation of component b labeled with fluorescent dye B, even though only component a labeled with fluorescent dye A is present in the sample.

[0010] Crosstalk can be reduced by designing the measurement conditions and device structure. For example, spectral crosstalk can be reduced by widening the spacing between the emission wavelengths of the fluorescent pigments used. If a completely independent measurement optical system is prepared according to the number of capillaries, spatial crosstalk will not occur. However, since the wavelengths that the detector can detect have upper and lower limits, widening the spacing between the pigments' emission spectra will reduce the number of fluorescent pigments that can be used. Alternatively, if the optical systems used to measure each capillary are independent, spatial crosstalk will not occur, but the number of light sources and detectors required will be proportional to the number of capillaries, which is disadvantageous in terms of device cost and size.

[0011] On the other hand, a method for reducing crosstalk through data processing is also known (Patent Document 1). In this method, the crosstalk generated by a particular fluorescent dye when it emits light through a particular capillary tube is comprehensively acquired beforehand. The inverse matrix of the matrix representing the crosstalk components is then generated. This inverse matrix is ​​then applied to the actual acquired signal to reduce crosstalk. This method can reduce crosstalk without changing the device structure or measurement conditions.

[0012] Prior art literature

[0013] Patent Literature

[0014] Patent Document 1: Japanese Patent No. 7282880

[0015] Patent Document 2: International Publication No. 2018 / 151843

[0016] Patent Document 3: International Publication No. 2023 / 276078

[0017] Non-patent literature

[0018] Non-Patent Literature 1: SeqStudio™ Genetic Analyzer Instrument and Software USERGUIDE https: / / assets.thermofisher.com / TFS-Assets / LSG / manuals / MAN0018646_SeqStudioInstSW_UG.pdf Summary of the Invention

[0019] Problems to be solved by the invention

[0020] The method described in Patent Document 1 is based on the premise that the ratio of spectral and spatial crosstalk between the pigments and capillaries remains unchanged between the time when the matrix used for the crosstalk reduction process is generated and the time when the sample is analyzed. This condition is essentially met when the same capillary array is used for both the generation of the matrix used for the crosstalk reduction process and the analysis of the sample. However, in capillary electrophoresis, the capillary array is a consumable item and needs to be replaced after a certain number of uses.

[0021] When replacing a capillary array, the ratio of spectral crosstalk to spatial crosstalk changes. In a capillary array, the capillaries are arranged in a row, but each capillary has a configuration error of several to tens of micrometers. Therefore, the light reflection pattern on the capillary surface can vary for each capillary array. Furthermore, the position and tilt of the capillary array center relative to the detector may also change between before and after replacement. These factors can alter the ratio of spatial crosstalk.

[0022] The luminescence spectrum of the pigment does not change with the replacement of the capillary tubes, so it can be expected that the change in spectral crosstalk associated with the replacement of the capillary array will be smaller than that in spatial crosstalk. However, spectral crosstalk may also change with the replacement of the capillary array. For example, if multiple reflections occur within the optical system of the detector and crosstalk occurs in a path such as the signal of pigment A entering the detection channel of pigment B, the position change of the capillary array will cause the state of the multiple reflections to change, and as a result, the spectral crosstalk may also change.

[0023] Therefore, when using the method of Patent Document 1, it is necessary to generate a matrix for crosstalk reduction processing for the new array after replacing the capillary array. To generate the matrix used in the crosstalk reduction process, it is necessary to calculate the crosstalk ratio relative to all pigments and capillaries. The crosstalk ratio when a particular capillary i, namely pigment A, emits light can be calculated by injecting pigment A into capillary i and performing electrophoresis. Therefore, for example, when using four pigments and a capillary array with eight capillaries, 4×8=32 electrophoresis cycles are performed to generate the matrix used in the crosstalk reduction process. Since one electrophoresis cycle also includes preparatory steps such as polymer replacement and preliminary electrophoresis to remove unnecessary ions, which take more than tens of minutes, generating the matrix used in the crosstalk reduction process requires a long time. The greater the number of pigments and capillaries, the longer this time is.

[0024] Patent Document 1 also describes a method for reducing the effort and time required to generate the matrix used in crosstalk reduction processing. Specifically, it describes a method for staggering the timing of sample injection into each capillary, thereby staggering the timing of pigment emission. This method prevents other capillaries from emitting light when one capillary emits light. Furthermore, a method is described in which fluorescently labeled DNA of different chain lengths is introduced into each capillary, preventing the other capillaries from emitting light when one capillary emits light.

[0025] However, the former requires the device to be equipped with an operational sequence different from that of conventional analysis, while the latter presents obstacles such as the necessity to prepare reagents containing DNA with controlled chain lengths in order to generate the matrix used in the crosstalk reduction process. Furthermore, even without replacing the capillary array, the matrix used in the crosstalk reduction process must be regenerated when the pigment used is changed.

[0026] Patent Document 2 discloses a method for reducing spectral crosstalk (equivalent to the process of determining the ratio of multiple pigments with spectral overlap based on fluorescence spectra). Instead of performing calibration electrophoresis to obtain information about the matrix used for spectral crosstalk reduction, the method optimizes the matrix based on information obtained during sample analysis. This method optimizes the matrix used for spectral crosstalk reduction by repeatedly modifying the matrix, evaluating the correlation between any two estimated pigment concentrations, and determining whether to update the matrix, based on an initial matrix and the correlation between any two estimated pigment concentrations, in such a way that the correlation decreases. This process then evaluates the correlation between the pigment concentrations and determines whether to update the matrix.

[0027] In this method, in order to correctly obtain the correlation between the two pigments caused by spectral crosstalk, outliers must be removed. That is, it is necessary to distinguish the spectral crosstalk relative to pigment B when pigment A is present and the situation that pigment B is actually present in the sample. About spectral crosstalk, it is usually expected that this distinction can be made. Usually when using multiple pigments, the purpose is to distinguish different analytes, so pigment A and pigment B mark different analytes (molecules). Therefore, when the sample containing the analyte marked with pigment A and the analyte marked with pigment B is subjected to electrophoresis and the time series data of the fluorescence spectrum is obtained, it is expected that pigment A and pigment B will emit light at different timings. On the other hand, the spectral crosstalk to pigment B produced by pigment A is produced at the same moment as the luminescence of pigment A. The situation that the analyte marked with pigment A and the analyte marked with pigment B accidentally emit light at the same moment is rare, so when calculating the relevant information between the estimated pigment concentrations of pigment A and pigment B, rare values ​​different from the tendency of such other multiple data can be removed as outliers.

[0028] However, even if one were to expand the method of Patent Document 2 into spatial crosstalk, it would not satisfy the prerequisites associated with the aforementioned outlier removal. If the method of Patent Document 2 were to be expanded into spatial crosstalk, it would be necessary to distinguish between the spatial crosstalk caused by capillary j when pigment A emits light in capillary i and the signal actually emitted by pigment A in capillary j. As in the previous case, the fluorescence of pigment A caused by the spatial crosstalk in capillary j and the luminescence of pigment A in capillary i occur at the same timing. Here, in the sample analyzed with capillary i and the sample analyzed with capillary j, pigment A labels the same analyte α. In this case, the timing of the luminescence of pigment A actually contained in the sample in capillary i and capillary j is the same. Thus, it is impossible to ensure that, when pigment A emits light in capillary i, the spatial crosstalk caused by capillary j and the signal actually emitted by the luminescence of pigment A in capillary j can be consistently distinguished.

[0029] This becomes a significant obstacle in the following situation. For example, consider analyte α labeled with pigment A and analyte β labeled with pigment B. Sample 1 contains only analyte α. Sample 2 also contains analyte β, and it is desired to determine whether a trace amount of analyte α is present in sample 2. This occurs when, for example, sample 1 is a pure substance, and the trace component (analyte α) present in sample 2 is analyzed using the data from sample 1 as a benchmark.

[0030] Here, sample 1 is analyzed using capillary i, and sample 2 is analyzed using capillary j. A strong signal from dye A is obtained in capillary i, while a strong signal from dye B is obtained in capillary j. The goal is to determine whether a weak signal from dye A is generated in capillary j. However, since dye A labels analyte α, fluorescence from dye A is obtained at approximately the same time in capillaries i and j, regardless of whether it is due to spatial crosstalk or the actual signal from analyte α. Therefore, the weak signal from dye A in capillary j cannot be distinguished based solely on the data trend as to whether it is due to spatial crosstalk or the presence of a trace amount of analyte α actually labeled with dye A.

[0031] In this way, by combining the method of Patent Document 1 with the method of Patent Document 2, the matrix for reducing both spectral and spatial crosstalk during analysis is optimized, eliminating the need for re-acquisition of the crosstalk reduction matrix when replacing the capillary array.

[0032] Furthermore, the capillary electrophoresis device described in Non-Patent Document 1 includes an option to use an initially set matrix as a spectral crosstalk reduction matrix (Factory Calibration described on page 217). However, this matrix is ​​not optimized for each device and can only be used in applications where residual spectral crosstalk due to incomplete information can be tolerated.

[0033] Therefore, the present invention is completed in view of such a situation, and its purpose is to provide a light detection device and signal processing method that can use the crosstalk information used for the crosstalk reduction processing generated once to perform crosstalk reduction processing even if multiple light-emitting body holders (for example, capillary arrays) are replaced.

[0034] Means for solving problems

[0035] In order to solve the above-mentioned problems, the light detection device of the present invention comprises: a plurality of replaceable light-emitting body holders, inside which a plurality of light-emitting bodies emit light; a plurality of light sensors, which have a plurality of detection channels for detecting light emission from the plurality of light-emitting bodies in a plurality of wavelength bands respectively; a spatial filter, which fixes the incident position of light emission from the plurality of light-emitting bodies relative to the light sensor; and a computer, which processes the signal output from the light sensor, the computer having crosstalk information inside for reducing the crosstalk existing between the plurality of detection channels, and reducing the crosstalk existing between the detection channels of the light sensor by performing calculations on the outputs from the light sensor corresponding to the plurality of wavelength bands of the plurality of light-emitting points respectively emitted by the plurality of light-emitting bodies, and deriving the concentration ratio or signal quantity ratio of the plurality of light-emitting bodies for each of the plurality of light-emitting body holders, and using the crosstalk information for calculations even if the light-emitting body holder is replaced with another light-emitting body holder.

[0036] In addition, the signal processing method of the present invention has the following steps: preparing a light detection device, the light detection device including: a plurality of replaceable light-emitting body holders, inside which a plurality of light-emitting bodies emit light; a light sensor having a plurality of detection channels for detecting light emission from a plurality of light-emitting bodies in a plurality of wavelength bands respectively; a spatial filter, which fixes the incident position of light emission from a plurality of light-emitting bodies relative to the light sensor; and a computer, which processes a signal output from the light sensor; measuring the light emission of a plurality of light-emitting bodies by the light sensor; using crosstalk information stored in the computer for reducing crosstalk between a plurality of detection channels, calculating the output from the light sensor corresponding to a plurality of wavelength bands of a plurality of light-emitting points of a plurality of light-emitting bodies, reducing the crosstalk between the detection channels of the light sensor, and deriving a concentration ratio or signal quantity ratio of each of the plurality of light-emitting bodies for each of the plurality of light-emitting body holders; and using the crosstalk information for calculation even if the light-emitting body holder is replaced.

[0037] Effects of the Invention

[0038] According to the light detection device and signal processing method of the present invention, even if multiple light-emitting element holders are replaced, crosstalk reduction processing can be performed using the crosstalk information generated once for the crosstalk reduction process. This reduces the effort and time required to regenerate the crosstalk information used in the crosstalk reduction process when multiple light-emitting element holders are replaced.

[0039] Other problems, structures, and effects than those described above will become clear from the following description of the embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1A This is a diagram of a photodetection device to which the crosstalk reduction method described in Patent Document 1 can be applied.

[0041] Figure 1B yes Figure 1A Schematic diagram of a spectral image on a light image sensor of a light detection device.

[0042] Figure 2A is a schematic diagram of the simulation model of crosstalk.

[0043] Figure 2B Is to use Figure 2A A diagram of a flow chart of a simulation performed by a simulation model.

[0044] Figure 3A It is a graph of the simulation results of crosstalk.

[0045] Figure 3B Yes Figure 3A FIG is a diagram showing simulation results when the crosstalk reduction process of Patent Document 1 was performed.

[0046] Figure 4A This figure shows the simulation results of crosstalk assuming replacement of the capillary array.

[0047] Figure 4B Yes Figure 4A FIG is a diagram showing simulation results when the crosstalk reduction process of Patent Document 1 was performed.

[0048] Figure 5 It is a schematic diagram of the light detection device of the first embodiment.

[0049] Figure 6 This is a schematic diagram showing the generation path of crosstalk generated on the capillary side of the optical fiber.

[0050] Figure 7A This is a diagram showing a simulation model of a photodetection device to which the crosstalk reduction process according to the first embodiment is applied.

[0051] Figure 7B yes Figure 7AFigure 2 is a diagram of the simulation results of crosstalk in the simulation model.

[0052] Figure 7C Yes Figure 7B The figure shows the simulation results when the crosstalk reduction process is performed.

[0053] Figure 8A This is a schematic diagram showing a method of connecting correction light sources for obtaining a matrix used in the crosstalk reduction process of the first embodiment.

[0054] Figure 8B Yes Figure 8A Schematic diagram of a structural example of a correction light source.

[0055] Figure 9 This is a diagram showing the influence of detector saturation on the crosstalk reduction process of the first embodiment.

[0056] Figure 10 It is a diagram showing the components of matrix G and matrix F.

[0057] Figure 11 This is a schematic diagram showing a configuration example of a correction light source for obtaining a matrix used in the crosstalk reduction process of the second embodiment.

[0058] Figure 12 This is a flowchart showing the operation of the photodetection device when a matrix used in the crosstalk reduction process of the second embodiment is acquired.

[0059] Figure 13 Schematic diagram of a light detection device according to a modified example. DETAILED DESCRIPTION

[0060] In the following embodiments, for convenience, when necessary, they are divided into multiple parts or embodiments for description. However, unless otherwise specified, they are not mutually independent, but rather one is a modification, detail, supplementary explanation, etc. of part or all of the other. In addition, in the following embodiments, when the number of elements, etc. (including number, value, amount, range, etc.) is mentioned, except where otherwise specified or where it is clearly limited to a specific number in principle, it is not limited to the specific number and can be greater than or less than the specific number.

[0061] Furthermore, in the following embodiments, the constituent elements (including element steps, etc.) are not necessarily required unless otherwise specified or clearly considered to be required in principle. Similarly, in the following embodiments, when referring to the shapes and positional relationships of constituent elements, etc., except where otherwise specified or clearly considered not to be required in principle, references to substantially similar or similar shapes and the like are included. This also applies to the numerical values ​​and ranges described above.

[0062] In addition, in principle, the same components are denoted by the same reference numerals in all the drawings for describing the embodiments, and repeated description thereof is omitted.

[0063] In the following embodiments, the photodetection device of the present invention is described using a capillary electrophoresis device as a representative application example. This is to more specifically illustrate the structure and effects of the present invention, and the photodetection device of the present invention is not limited to capillary electrophoresis devices.

[0064] (Overview of Capillary Electrophoresis Apparatus of This Embodiment)

[0065] In the structure of the capillary electrophoresis device of this embodiment, as an example, a spatial filter is provided at the light introduction portion of a spectrometer that separates fluorescence from a fluorescent pigment to obtain a spectrum, and the light introduction position into the spectrometer is fixed. As an example, the spectrometer includes a first lens that collimates incident light, a grating that separates the light by wavelength, a second lens that forms an image of the light passing through the grating on a light sensor, and an optical image sensor that detects the light. As an example, the spatial filter is a multimode optical fiber, one end of which is fixed to the light entrance position of the spectrometer. The other end of the optical fiber is positioned near the capillary to capture the fluorescence emitted from the capillary. As an example, the optical image sensor is a CCD or CMOS image sensor.

[0066] Crosstalk may occur due to a variety of reasons, but by adopting the above-mentioned structure, the ratio of crosstalk generated by the optical fiber on the side of the spectrometer can be fixed without being affected by the replacement of the light-emitting body holder (capillary array). The fluorescence generated in the capillary is taken into the optical fiber and guided to the spectrometer. As the causes of crosstalk inside the spectrometer, the expansion of the spatial profile of the fluorescence spectrum, multiple reflections between the surfaces of elements such as lenses, and anomalies of the grating are considered. Assuming that there is no optical fiber and the capillary is directly set at the light introduction position of the spectrometer, the offset of the position of each capillary accompanying the replacement of the capillary array affects the spatial profile of the fluorescence, multiple reflections, anomalies, etc., and the ratio of crosstalk may change.

[0067] By fixing the incident position on the spectrometer using optical fibers, the spatial profile of fluorescence, multiple reflections, and anomalies are stabilized. While changes in the position of the capillary tube relative to the other end of the optical fiber opposite the spectrometer affect the amount of light incident on the fiber, they do not affect the crosstalk ratio generated within the spectrometer. Because changes in the capillary tube position do not alter the crosstalk ratio, even if the capillary array is replaced, the same information can be used for crosstalk reduction.

[0068] On the other hand, the crosstalk generated on the capillary side of the optical fiber may still change due to the replacement of the capillary array. For example, the crosstalk generated by the reflection of fluorescence on the capillary surface is affected by the configuration error of the capillaries constituting the capillary array, and therefore changes due to the replacement of the capillary array. Such crosstalk cannot be completely eliminated by simply providing the above-mentioned optical fiber. It is also possible to fully reduce the crosstalk generated on the capillary side and changed due to the replacement of the capillary array. As an example, this can be achieved by using a light-shielding wall to separate each capillary and make it independent. Alternatively, the spacing between the capillaries can be made large enough to ignore the crosstalk. It is also known to provide a pinhole at the outlet of the optical fiber to limit the incident angle of the detected light (Patent Document 3).

[0069] While using only the aforementioned optical fiber alone cannot eliminate the crosstalk generated on the capillary side and fluctuating with capillary array replacement, this does not diminish the effectiveness of the present invention. Even if crosstalk generated on the capillary side of the optical fiber is not completely eliminated, the remaining components can be reduced to a level acceptable for analytical purposes.

[0070] (Regarding the known technology of Patent Document 1)

[0071] Before describing the embodiments in detail, the known technology and its problems in Patent Document 1, which are the background of the present invention, will be summarized. Figure 1A This is a structural diagram of a light detection device 100 that can be used as an application target of the technology described in Patent Document 1. The light detection device 100 includes a capillary array 101, two lenses 102, a grating 103, and an optical image sensor 104. The light detection device 100 separates and detects fluorescence emitted from the capillary array 101. The arrows in the figure indicate the irradiation direction of the excitation laser light L that stimulates fluorescence. The laser light L irradiates all capillaries in the capillary array 101 in a direction transverse to the sides, exciting the pigment within each capillary.

[0072] In the light detection device 100, the wavelength is dispersed in the vertical direction of the paper by the grating 103. Figure 1BAs shown, spectral images 105 corresponding to each capillary are arranged on the optical image sensor 104. The optical image sensor 104 outputs the light intensity measurement results for each pixel. The light intensity measurement results are summed up by wavelength division for each capillary. For example, if the measurement wavelength range is 500nm to 700nm, when the light intensity value of each wavelength is summed up as 20 points with a width of 10nm, the light intensity values ​​of 500nm to 510nm, 510nm to 520nm, and 690nm to 700nm are obtained. In other words, the light intensity measurement results from each capillary are summed up. Figure 1B A set of values ​​obtained by adding the outputs of pixels existing in each section (channel 106) divided by the dotted line in is used as the spectrum information of each capillary.

[0073] The conventional crosstalk reduction methods described in Patent Document 1 are reviewed with the light detection device 100 in mind. As previously defined, crosstalk includes two types: spectral crosstalk, where the signal from pigment A is mistaken for the signal from pigment B, and spatial crosstalk, where the signal emitted by capillary i is mistaken for the signal emitted by capillary j. Patent Document 1 and the crosstalk reduction methods of the present invention are based on the premise that the crosstalk is linear with respect to the signal vector (a vector that arranges the signals corresponding to each capillary and each wavelength range obtained by the optical image sensor 104). This is because the light detection device 100 does not include elements that impart nonlinear effects to the light being detected and is not used under conditions that would produce nonlinear effects (generally requiring very high light intensities), so this condition is generally met.

[0074] The above content is that the signal when the pigment A emits light in the capillary i is set as S Ai , the crosstalk detected in the region where the pigment B should be detected in the capillary j when the pigment A emits light in the capillary i is set as S CT Bj←Ai When , the relationship between the two can be expressed by the following formula (1) using the constant c.

[0075] S CT Bj←Ai =cS Ai (1)

[0076] In the conventional crosstalk reduction method described in Patent Document 1, the process of finding the constant c is first performed. Assume that the following situation is set: dye A is injected into capillary i, and nothing is injected into the other capillaries. In this situation, the signal S is obtained when the dye A emits light in capillary i. Ai , and obtain the crosstalk S detected in the area where the pigment B should be detected in the capillary j when the pigment A emits light in the capillary i CT Bj←Ai Based on these two ratios, the constant c is obtained by the following formula (2).

[0077] c=SCT Bj←Ai / S Ai (2)

[0078] Next, measure the sample you want to analyze. Inject the sample labeled with dye A into capillary i. Inject the sample labeled with dye B into capillary j. The signal obtained from capillary i is S Ai It is preferred to obtain a signal S when the pigment B emits light in the capillary j. Bj , but due to the crosstalk S CT Bj←Ai is added to the signal, so the actual measured signal S act Bj It is calculated by the following formula (3).

[0079] S act Bj =S Bj +S CT Bj←Ai (3)

[0080] Here, the crosstalk component of equation (3) is expressed by equation (1). The constant c in equation (1) is obtained by equation (2). In addition, S Ai Therefore, using equations (1) and (3), the true signal S that should be obtained in the absence of crosstalk can be obtained by the following equation (4): Bj .

[0081] S Bj =S act Bj -S CT Bj←Ai =S act Bj -cS Ai (4)

[0082] The above description simplifies the method described in Patent Document 1. In reality, crosstalk may also occur due to components generated by capillaries other than capillaries i and j, and pigments other than pigments A and B. Furthermore, in the above description, the fluorescence generated by pigments A and B is labeled as a signal. In reality, optical image sensor 104 outputs the intensity of light in each wavelength range as a signal for each capillary.

[0083] The method described in Patent Document 1 is organized based on the above content. For example, a fluorescent dye of color L is used in the measurement, there are N capillaries to be measured, and M wavelength intervals are measured in the optical image sensor 104. For example, the wavelength interval is M = 20. When the detection wavelength range is 500-700nm, the optical image sensor 104 outputs 20 signals for each capillary: a signal of fluorescence with a wavelength of 500-510nm, a signal of fluorescence with a wavelength of 510-520nm, ... a signal of fluorescence with a wavelength of 690-700nm. In the following, the signal (or crosstalk) generated for wavelength interval k of capillary i when the first dye is introduced into capillary j is recorded as S ik←j1 .

[0084] Here, we will have S ik←j1 The matrix as a component is recorded as C. S ik←j1 There are 4 subscripts, but relative to the ζ and η components of the matrix C ζη , are arranged two-dimensionally so that ζ=Mi+k and η=Mj+1. ik←j1 The specific value of S can be obtained by making the pigments used only in the capillary j flow one by one in sequence and obtaining it as the output of the optical image sensor 104. ik←j1 The value of can also be appropriately normalized based on the signal when the pigment used is at the reference concentration. The matrix C becomes a matrix in which the spectra of each pigment used are arranged at the position corresponding to each capillary in the matrix. Here, before measuring the sample to be actually measured, the generalized inverse matrix C of the matrix C is obtained according to the following formula (6): - .

[0085] C - =[C t C] -1 C t (6)

[0086] Next, let the signal of wavelength range k of capillary i be S ik Here, the arrangement S ik To create the signal vector s. Specifically, the ζ component s relative to the signal vector s ζ Arrange S in such a way that ζ=Mi+k ik At this time, the vector d having the ratio of each pigment as a component can be obtained by the following formula (7).

[0087] d=CS (7)

[0088] Equation (7) expands the description of removing a single crosstalk component described above to encompass the case where spatial crosstalk exists between multiple capillaries and spectral crosstalk exists between multiple pigments. This is the conventional crosstalk reduction method described in Patent Document 1. Furthermore, in capillary electrophoresis-based analysis, the optical image sensor outputs a vector s at each moment. Therefore, by performing Equation (7) at each moment, the signal waveform of each pigment in each capillary can be obtained.

[0089] When applying the above-mentioned crosstalk reduction method, it is necessary that the matrix C does not change between the timing of obtaining the matrix C and the timing of the actual measurement signal. Patent Document 1 shows that this condition is satisfied when the same capillary array is used for obtaining the crosstalk matrix C and for actual sample measurement. However, in capillary electrophoresis, the capillary array is a consumable item and must be replaced with a new array after a certain number of uses. In this case, Patent Document 1 does not describe whether the above prerequisite is met and whether the crosstalk reduction method is effective.

[0090] (simulation)

[0091] If the various parameters such as the diameter of each capillary and the setting position and angle of each capillary are exactly the same before and after the capillary array is replaced, the above conditions are met. However, it is actually impossible to meet this condition and the above conditions do not hold when the capillary array is replaced. Below, a simulation is used to specifically illustrate. In the following simulation, the impact of crosstalk when the error of the relative position of the capillaries in each capillary array changes is evaluated by replacing the capillary array. The error in the relative position of the capillaries refers to the deviation from the ideal position of the capillaries, which should be arranged at equal intervals on the same plane. Capillary arrays are made by arranging and fixing capillaries on a substrate, etc., but due to individual differences in the substrate and deviations in the fixing operation, the spacing between the capillaries and the distance between each capillary and the substrate are not exactly the same in each capillary array.

[0092] Figure 2A Figure 2 shows a simulated model. For simplicity, this simulation does not perform wavelength separation; only spatial crosstalk is considered. The model consists of a capillary array 201, two lenses 202, and an optical image sensor 203. The inner diameter of the capillary array 201 is set to 50 μm, the outer diameter is set to 343 μm, and the capillaries are arranged with a pitch of 370 μm. A light-emitting region with a length of 44 μm is defined within the capillaries. The image of the capillary array 201 is formed on the optical image sensor 203 through the two lenses 202. There are eight capillaries in the capillary array 201.

[0093] Figure 2B The simulation process is described in . In the case of performing crosstalk reduction processing (S200: Yes), first calculate the above matrix C- . Imagine finding the matrix C - The capillary array is set to a relative position error for the capillary array (S201). After setting the position of the capillary, the fluorescence image on the optical image sensor 203 is calculated by the ray tracing method (S202). In this step, fluorescence is generated only from one capillary among capillaries 1 to 8 to perform ray tracing. Repeat this step 8 times by changing the emitting capillary, and calculate the output of the optical image sensor 203 when capillaries 1 to 8 emit light. Next, the point image distribution function is convolved with the calculation result of the ray tracing (S203). The point image distribution function is actually a function measured by a spectrometer composed of a lens, a grating, and an optical image sensor, which represents the position relative to the capillary arrangement direction ( Figure 2A The X direction) of the optical system is "blurred". Figure 2A In the ray tracing of the model, lens 202 is calculated as an ideal thin-walled lens, and the deviation from the ideal state in the actual optical system is expressed by the point image distribution function. Next, the output of the optical image sensor 203 obtained as a result of the calculation is integrated with the signal of the area corresponding to each capillary to obtain the crosstalk value. The generalized inverse matrix C is calculated for the matrix C generated by arranging the crosstalk values. - ( S204 ) In this model, wavelength separation is not performed, so the matrix C becomes a square matrix in which spatial crosstalk between capillaries is two-dimensionally arranged.

[0094] Next, imagine the capillary array actually used for sample analysis and calculate the crosstalk. If crosstalk reduction processing is not performed (S200: No), the calculation starts from this step. The relative position error setting to the capillary array (S205), the calculation based on ray tracing (S206), and the convolution of the point image distribution function (S207) are the same as the above-mentioned S201, S202, and S203. Then, the signal vector s is calculated based on the output of the obtained optical image sensor 104 (S208). If crosstalk reduction processing is performed (S209: Yes), crosstalk reduction processing is performed according to formula (7) (S210). If crosstalk reduction processing is not performed (S209: No), this step (S210) is skipped. After the calculation is completed, the result is output (S211). If crosstalk reduction processing is performed, vector d is output, and if no processing is performed, vector s is output. When there are multiple types of pigments and the pigment concentration ratio is calculated based on the spectrum, vector d and vector s are different types of information (the ratio of pigments in each capillary and the light intensity in each wavelength range). However, when the separation of the light in the wavelength direction is not considered, they become the same type of information (the light intensity of each capillary is directly the concentration ratio of the pigment). Therefore, the effect of the presence or absence of crosstalk processing can be evaluated by comparing the two.

[0095] (Result of crosstalk reduction processing when there is no relative position error)

[0096] Figure 3A is a diagram showing crosstalk obtained by the above-mentioned simulation method when no crosstalk reduction processing is performed, Figure 3B is a diagram showing crosstalk when crosstalk reduction processing is performed. Figure 3A as well as Figure 3B In the above example, it is assumed that the capillary array has no relative position error and the matrix C is used in the same capillary array. - Generation and analysis of samples. Figure 3A The horizontal axis indicates the capillary that emits light. The bars with different shades indicate the crosstalk values ​​detected in the region of detection capillaries 1 to 8 (referred to as channels 1 to 8).

[0097] according to Figure 3A In this model, crosstalk is greatest in the channels detecting adjacent capillaries, reaching approximately 1%. This crosstalk is primarily caused by reflections from the capillary surfaces and fluorescence leakage into adjacent channels due to blurring of the imaging optical system (represented by the point image distribution function). Actual spatial crosstalk can also be caused by multiple reflections between components of the spectroscopic system, grating anomalies, and scattering caused by debris, but this model only reflects these two factors.

[0098] according to Figure 3B It can be seen that, given the same capillary array, crosstalk is reduced by conventional crosstalk reduction processing. The crosstalk reduction process reduces the approximately 1% crosstalk to less than 0.01%. Generally speaking, the dynamic range of an optical image sensor is approximately 3 to 4 bits, and a crosstalk of less than 0.01% means that the crosstalk is below the detection limit. This time, the matrix C is generated. - The model is exactly the same as the one used to calculate crosstalk. Figure 3B The reason why the crosstalk value is not zero is the calculation error of the ray tracing method.

[0099] (Results of Crosstalk Reduction Processing in the Case of Relative Position Error)

[0100] Figure 4A as well as Figure 4B and Figure 3A as well as Figure 3B Similarly, this is a graph showing the crosstalk when no crosstalk reduction treatment is performed and when a crosstalk reduction treatment is performed. However, in this calculation, each capillary is given a Gaussian distribution with a standard deviation of 10 μm. Figure 2A The error in the XY direction. Assume that in the matrix C - The generation and analysis of samples using different capillary arrays in the matrix C -The relative positional error values ​​set for the capillaries in the capillary array differed during generation and crosstalk calculation. Considering the capillary outer diameter is 343 μm, this error is considered a relatively small relative positional error. Furthermore, while the capillaries in this model have identical outer diameters, capillary outer diameters typically have a tolerance of approximately ±10 μm. If the fixing method assumes that variations in capillary diameter will cause center position shifts, the relative positional error values ​​described above are realistically possible.

[0101] according to Figure 4A It can be seen that even when an alignment error is introduced, there is a strong crosstalk of 1%, just like when the alignment error is zero. Figure 4B It can be seen that even with the conventional crosstalk reduction process, a maximum of about 0.2% crosstalk remains when there is an alignment error. In addition, in some channels, the crosstalk is negative, indicating that the crosstalk correction is excessive. The calculation is repeated 9 times with different alignment errors, and the average of the maximum residual crosstalk value is 0.28%. This shows that since the crosstalk value varies according to the alignment error, when using the matrix C - When arrays with different alignment errors are generated and analyzed in samples, the effects of conventional crosstalk reduction processing cannot be properly obtained.

[0102] This paper uses a model that defines the factors that cause crosstalk to illustrate the impact of alignment errors on crosstalk reduction. In reality, as mentioned above, there are cases where the causes of crosstalk are not accounted for by the model. Furthermore, when replacing capillaries, in addition to alignment errors such as changes in the overall position of the capillary array, changes in the capillary array's tilt, variations in the inner and outer diameters of the capillaries, and dust on the capillaries, there are also factors that increase crosstalk variation. The greater the crosstalk variation within each capillary array, the less effective the conventional crosstalk reduction treatment will be.

[0103] The inventors have newly discovered that, as described above, the conventional crosstalk reduction processing method needs to regenerate the matrix C when the capillary array is replaced. - In the matrix C - For example, as described in Patent Document 1, the crosstalk information used in the generation of the matrix C is to inject one sample labeled with each dye into each capillary, perform electrophoresis, and then measure. Therefore, when using L fluorescent dyes and there are N capillaries for measurement, L×N electrophoresis cycles are required. One electrophoresis cycle takes about tens of minutes, so the matrix C is - The generation of takes a long time. Patent document 1 also describes a method for reducing the generation matrix C - However, the use of the described methods requires the use of special samples or the operation of special equipment that is not performed in ordinary analysis.

[0104] The present invention does not require the replacement of the matrix C with the capillary array. - The above issues can be solved by regeneration.

[0105] <First embodiment>

[0106] Figure 5 This is a structural diagram of a light detection device 500 according to the first embodiment. The light detection device 500 includes a capillary array 501, an optical fiber array 510 (spatial filter), a spectrometer 505, and a computer 550. The capillary array 501 includes a plurality of capillaries 501a, and within each capillary 501a, a plurality of light emitters emit light. The capillary array 501 is replaceable. The light image sensor 504 constituting the spectrometer 505 includes a plurality of detection channels (see FIG. 5 ) for detecting light emitted from the plurality of light emitters in a plurality of wavelength bands. Figure 1B The optical fiber array 510 is a spatial filter that fixes the incident position of light emitted from a plurality of light emitters with respect to the spectrometer 505. The computer 550 processes the signal output from the optical image sensor 504.

[0107] The light detection device 500 differs from the light detection device 100 in that it includes an optical fiber array 510. The optical fiber array 510 comprises a plurality of optical fibers 510a arranged corresponding to the light-emitting points of the plurality of capillaries 501a. The optical fiber array 510 receives the fluorescence emitted from each capillary 501a in the capillary array 501 and guides the fluorescence toward a spectrometer 505 composed of a lens 502, a grating 503, and an optical image sensor 504. Furthermore, the light detection device 500 is conceived as a detection device for a capillary electrophoresis apparatus. In practice, it operates integrally with a temperature control device, a high voltage application device, an automatic sample stage, a computer for signal processing, and other components to perform analysis based on capillary electrophoresis of the sample. However, descriptions of components other than the light detection device 500 are omitted.

[0108] Furthermore, the crosstalk reduction process of the present invention can also be applied to different forms of the optical splitter 505. Specifically, the wavelength separation of light can be performed by a prism instead of a grating, or by a plurality of dichroic mirrors.

[0109] The crosstalk reduction processing method in the first embodiment is the same as the above-mentioned formula (7) and its related description. However, as a condition for the processing, it is required that the position of the fluorescence introduction to the spectrometer 505 be fixed by the optical fiber array 510, and that the crosstalk generated on the capillary side of the optical fiber array 510 be sufficiently reduced by other methods. In addition to the optical fiber array 510, the position of the fluorescence introduction to the spectrometer 505 can also be fixed by a pinhole array, a slit array, etc.

[0110] The light detection device 500 includes a computer 550. The computer 550 includes a processor 551, a main memory unit 552, an auxiliary memory unit 553, and an input / output interface 554 (hereinafter referred to as the interface by abbreviation I / F). The processor 551 is a central processing unit that controls the operation of each component of the computer 550. The processor 551 is, for example, a CPU (Central Processing Unit), a GPU (Graphics Processing Unit), a DSP (Digital Signal Processor), an ASIC (Application Specific Integrated Circuit), etc. The processor 551 expands the program stored in the auxiliary memory unit 553 in an executable manner in the working area of ​​the main memory unit 552. The main memory unit 552 stores programs executed by the processor 551, data processed by the processor, etc. The main memory unit 552 is a flash memory, a RAM (Random Access Memory), a ROM (Read Only Memory), etc. The auxiliary storage unit 553 stores various programs such as the OS (Operating System) and various data. The auxiliary storage unit 553 is, for example, a solid state drive (SSD), a hard disk drive (HDD), or a combination thereof. The auxiliary storage unit 553 has crosstalk information (for example, the matrix C mentioned above) for reducing crosstalk between multiple detection channels. - The input / output I / F 554 is connected to the optical image sensor 504 and the display unit 560 so as to be communicable therewith.

[0111] The computer 550 uses the crosstalk information (eg, the matrix C - ) is calculated based on the output from the optical image sensor 504 corresponding to the multiple wavelength bands of the multiple light-emitting points emitted by the multiple light-emitting bodies, thereby reducing the crosstalk between the detection channels of the optical image sensor 504 and deriving the concentration ratio or signal intensity ratio of each of the multiple light-emitting bodies for each of the multiple capillaries. Furthermore, even if the capillary array 501 is replaced with another capillary array, the computer 550 performs the above calculations using the same crosstalk information.

[0112] The crosstalk information is the generalized inverse matrix C - Or the generalized inverse matrix C - Equivalent information, the matrix C contains the information of crosstalk between multiple detection channels. -Equivalent information is, for example, information in a non-matrix format, which is information used to reduce crosstalk between multiple detection channels. Furthermore, the operation of deriving the concentration ratio or the signal quantity ratio performed by the computer 550 is to make the matrix C - C is derived from the vector s acting on the output from the optical image sensor 504 - s operation (refer to the above formula (7)), or an equivalent operation.

[0113] The computer 550 sequentially flows the plurality of luminous bodies through each of the plurality of capillaries 501a, records the output of the optical image sensor 504 when a single luminous body among the plurality of luminous bodies emits light through a single capillary 501a among the plurality of capillaries 501a, arranges the output of the optical image sensor 504 to generate a matrix C, and calculates the matrix C by [C t C] -1 C t (Refer to the above formula (6)) or its equivalent operation to calculate and save the generalized inverse matrix C of the matrix C - .

[0114] Crosstalk generated on the capillary side of the optical fiber array 510 is, for example, Figure 6 As shown, crosstalk occurs along paths where fluorescence generated from one capillary directly enters the optical fiber detecting another capillary, or along paths where fluorescence reflects off the surface of another capillary and enters the optical fiber detecting that capillary. Reducing crosstalk along these paths can be achieved, for example, by sufficiently increasing the spacing between the capillaries in capillary array 501. Alternatively, a known method is to provide a pinhole slightly smaller than the core diameter of optical fiber 510a at the end of optical fiber array 510 on the capillary array 501 side, thereby limiting the angle at which fluorescence enters optical fiber 510a and reducing crosstalk. Furthermore, a method is also known in which light-blocking walls are provided between the capillaries in capillary array 501.

[0115] Reference Figures 7A to 7C , describing simulation results on the effect of the crosstalk reduction method of the first embodiment. Figure 7A is a diagram showing a model used for simulation. Figure 7A The model and Figure 2A The model of has a capillary array 701, two lenses 702 and an optical image sensor 703. Figure 7A In, yes Figure 2A The model adds an optical fiber array 710. The optical fiber array 710 is set with a core diameter of 200μm and NA0.5 optical fibers, and 8 fibers are arranged at the same intervals as the capillaries of the capillary array 701. In addition, as a means of eliminating crosstalk generated in front of the optical fiber array 710, a light shielding wall 720 is set between the capillaries. The simulation process is the same as Figure 2B The process shown is the same.

[0116] Figure 7B and Figure 7C It means using Figure 7A A graph of the results of the simulation of the crosstalk of the model. Figure 7B is a graph showing crosstalk when no crosstalk reduction processing is performed. Figure 7C 4. In this simulation, it is assumed that in the matrix C - The generation and analysis of samples using different capillary arrays in the matrix C - When generating and when calculating crosstalk, different values ​​of relative position errors of the capillaries set in the capillary array 701 are used.

[0117] from Figure 7B It can be seen that in Figure 7A In the model of , there is about 0.5% crosstalk. In contrast, if the crosstalk reduction process of formula (7) is implemented, then Figure 7C As shown, the crosstalk is reduced to less than 0.01%. The calculation is repeated 9 times with different relative position errors, and the average of the maximum residual crosstalk values ​​is 0.006%.

[0118] according to Figure 4B The results and Figure 7C The comparison of the results shows that even when the capillary arrays with different capillary position offset values ​​are used by the method of the first embodiment, the crosstalk reduction process works correctly. In addition, this effect is not caused by the provision of the light shielding wall 720. To illustrate this, Figure 2A The same simulation was performed with the addition of a light shielding wall 720. The calculation was repeated 9 times with different relative position errors, and the average of the maximum value of the residual crosstalk was 0.11%, which is Figure 7C The result is 10 times more than the value.

[0119] As described above, in the crosstalk reduction method of the first embodiment, when the capillary array 501 is replaced, even if the matrix C - The effect of the crosstalk reduction method can be achieved by regenerating the optical fiber array 510. This is an effect produced by fixing the incident position of the fluorescence on the spectrometer 505 by the optical fiber array 510. Even if the position of each capillary of the capillary array 501 changes, the incident position of the fluorescence relative to the spectrometer 505 is fixed by the optical fiber array 510. The crosstalk generated in the spectrometer 505 (for example, due to multiple reflections and blurring of the spectral image) is not affected by the position of the capillary because the incident point of the fluorescence is fixed. Therefore, the crosstalk value will not change due to the replacement of the capillary array, and there is no need to recreate the matrix C. - .

[0120] However, as described above, in the method of the present invention, the crosstalk generated on the capillary side needs to be sufficiently small compared to the optical fiber array 510. The method of the first embodiment is a method for reducing the constant component of the crosstalk (basically the crosstalk of the optical splitter 505), and therefore cannot remove the component that changes due to the replacement of the capillary array 501. For example, in the case where the amount of crosstalk caused by surface reflection changes due to a change in the configuration error of the capillary array, the crosstalk of the changed amount will remain even after the crosstalk reduction process. To illustrate this, Figure 7A The same simulation was performed by removing the light shielding wall 720 from the model. The calculation was repeated nine times with different relative position errors, and the average of the maximum residual crosstalk values ​​was found to be 0.12%. Thus, in the optical system to which the method of the present invention is applied, the variation in crosstalk generated on the capillary side of the optical fiber array 510 must be designed to be sufficiently below an allowable value.

[0121] In the light detection device 500 and the light detection method of the first embodiment, the matrix C - Basically, it does not change, so for example, after the device is manufactured or installed at the place of use, the matrix C - The matrix generation operation can be performed using the same method as described in Patent Document 1. For example, a sample labeled with a certain pigment is injected into a certain capillary, electrophoresis is performed, and the signal from the optical image sensor 504 is recorded. This process is repeated in all capillaries with all pigments. Then, the measurement results are arranged to form a matrix C. Specifically, the output of each wavelength range of each capillary is arranged when the signal of the target pigment in the target capillary reaches the maximum. Then, the generalized inverse matrix C of the matrix C is generated. - .

[0122] During sample analysis, the output of the optical image sensor 504 at each time is processed according to equation (7) to obtain information on the ratio of each pigment at each time. By arranging the ratios of each pigment in time series, the electrophoresis waveform of the analyte labeled with each pigment is obtained.

[0123] Matrix C - Basically unchanged, but for some reason, the matrix C can be obtained again - For example, due to the relocation of the device or long-term use, the positions of the components of the optical splitter 505 may shift. In such a case, the ratio of crosstalk inside the optical splitter may also change, so the matrix C can also be used. - of regeneration.

[0124] In addition, in order to reduce the trouble of generating the matrix C, the matrix C may be formed by partial information. That is, the matrix C may be a plurality of matrices C containing only a part of the crosstalk information between the luminescent body holder or the pigment. nFor example, according to Figures 7A to 7C The simulation results show that in this case, spatial crosstalk is generated more strongly in the channel measuring adjacent capillaries. In this case, the matrix C can be generated by ignoring the crosstalk from separating two or more capillaries.

[0125] by Figure 7A As an example, let the matrix C n Let's assume that a matrix is ​​generated by arranging the signals obtained from measuring the channels of each capillary when injecting dye into capillary n. When generating matrix C, dye is simultaneously passed through the capillaries of each combination of capillary 1, capillary 4, and capillary 7, capillary 2, capillary 5, and capillary 8, and capillary 3 and capillary 6. Furthermore, matrix C n The signals of the channels corresponding to capillaries n+1, n, and n-1 are used to generate the matrix, and the signals of the channels corresponding to the other capillaries are zero. That is, when the dye is injected into capillaries 1, 4, and 7 at the same time, the values ​​of channels 1 and 2 are used to generate C1, the values ​​of channels 3, 4, and 5 are used to generate C4, and the values ​​of channels 6, 7, and 8 are used to generate C7. After all the matrices C are generated, n (Case C1 to C8 above) After that, the matrix C will be arranged in sequence n The obtained matrix is ​​defined as matrix C. By generating matrix C using this method, the number of electrophoresis operations for obtaining information can be reduced.

[0126] While the above method ignores spatial crosstalk between capillary pairs with low spatial crosstalk values, the same method can be applied even when the emission spectra of a particular pigment pair differ significantly and have little overlap. Specifically, for pigment pairs with non-overlapping fluorescence spectra, the matrix C can be generated using data obtained from simultaneous measurements of these pigment pairs.

[0127] Furthermore, the matrix C may not be generated by injecting the dye into the light-emitting body holder, ie, the capillary. - ) can also be obtained by connecting the correction light source 801 to the spatial filter. The correction light source 801 sequentially introduces correction light to each light-intake position of the spatial filter. This correction light has the same spectrum as the emission spectrum of the multiple light emitters. Figure 8A This diagram shows how matrix C is generated using calibration light source 801. Calibration light source 801 sequentially directs light having the same spectrum as the emission spectrum of each pigment into the optical fibers of optical fiber array 510. Matrix C can be generated based on the output of spectrometer 505 in the same manner as described above.

[0128] Figure 8BThis figure shows an example of the structure of calibration light source 801. Calibration light source 801 includes an excitation light source 802. Excitation light source 802 irradiates flow path 803 with fluorescence excitation light. Dye injection mechanism 804 sequentially injects each dye into flow path 803. Fluorescence generated in flow path 803 is collimated by lens 805, and the excitation light is removed by wavelength filter 806. The collimated fluorescence is guided through reflector 807 and lens 808 to a single optical fiber in output fiber array 809. By changing the angle of a portion of reflector 807, the optical fiber to which the fluorescence is directed can be changed.

[0129] The crosstalk reduction method of the first embodiment is based on the premise that there is a linear relationship between crosstalk and the signal that is the basis of the crosstalk. Therefore, if this relationship is destroyed due to detector saturation, the method of the first embodiment will not work. Figure 9 A schematic diagram illustrating the effect of detector saturation is shown. Figure 9 The horizontal axis of the graph represents the concentration of the pigment being measured, and the vertical axis represents the amount of crosstalk. In a spectroscopic system with crosstalk, the pigment concentration is proportional to the crosstalk. In an ideal situation with no crosstalk at all, crosstalk is always zero, regardless of pigment concentration.

[0130] According to the crosstalk reduction method of the first embodiment, the ideal crosstalk is completely zero. Therefore, although crosstalk exists, in the spectroscopic system performing the crosstalk reduction process of the first embodiment, under ideal conditions, up to a certain pigment concentration, the crosstalk becomes zero regardless of the concentration (even under realistic conditions where some crosstalk remains after the reduction process, the crosstalk after reduction becomes a value smaller than the original value). However, when the detector in the capillary that measures the pigment to be measured is saturated, the output signal remains constant even if the pigment concentration rises further. On the other hand, the crosstalk itself is proportional to the fluorescence intensity and therefore continues to increase in proportion to the concentration. As a result, the actual crosstalk amount exceeds the crosstalk amount estimated from the detector output. In this case, the crosstalk reduction method of the first embodiment cannot completely eliminate the crosstalk, and the crosstalk value is no longer zero. Therefore, the crosstalk reduction method of the first embodiment needs to be used within the range where the detector is not saturated. When a channel of the detector is saturated, it is useful to warn the user that other channels where the detector is not saturated may not achieve their original performance.

[0131] Specifically, when the output from the optical image sensor 504 is saturated, the computer 550 may display a warning on the display unit 560 .

[0132] <Signal processing method>

[0133] Here, a signal processing method using the photodetection device 500 according to the first embodiment will be described.

[0134] The signal processing method of this embodiment includes: preparing a light detection device 500; measuring the light emission of multiple light-emitting bodies by using a light image sensor 504; using crosstalk information (matrix C stored in a computer 550) for reducing crosstalk between multiple detection channels - ), calculating the outputs from the optical image sensor 504 corresponding to the multiple wavelength bands of the multiple luminous points emitted by the multiple luminous bodies, reducing the crosstalk between the detection channels of the optical image sensor 504, and deriving the concentration ratio or signal quantity ratio of each of the multiple luminous bodies according to the multiple luminous body holders (capillary tubes 501a); and

[0135] Even if the capillary array 501 is replaced, the above calculation is performed using the same crosstalk information.

[0136] Crosstalk information (matrix C - ) is the generalized inverse matrix C of the matrix C including the crosstalk information - Or information equivalent thereto. In addition, the operation for deriving the concentration ratio or signal ratio is to make the matrix C - C is derived from the vector s acting on the output from the optical image sensor 504 - s operation (refer to the above formula (7)), or an equivalent operation.

[0137] <First embodiment: summary>

[0138] The light detection device 500 of the first embodiment includes a capillary array 501, an optical fiber array 510, a lens 502, a grating 503, and an optical image sensor 504. Furthermore, the device includes a computer 550 for processing the output of the optical image sensor 504. The computer 550 internally stores the generalized inverse matrix C of the matrix C. - , the generalized inverse matrix C of the matrix C - It is generated based on the output of the optical image sensor 504 when the sample labeled with the dye used is sequentially injected into each capillary in the capillary array 101 in the light detection device 500 and measured. When analyzing the measurement object, the computer 550 calculates the generalized inverse matrix C - The ratio of each pigment is obtained by applying the output of the optical image sensor 504 at each time. - The same value is used even when the capillary array 501 is replaced.

[0139] <Second embodiment>

[0140] In the first embodiment, the outputs from the optical image sensor 104 are combined with the generalized inverse matrix C -Processing is performed to obtain the concentration ratio of each pigment. In the second embodiment, the spectral crosstalk caused by the pigment, the spectral crosstalk caused by the spectrometer, and the spatial crosstalk are separated and processed. In the crosstalk reduction method of the second embodiment, the crosstalk generated inside the spectrometer is reduced using a matrix obtained using a correction light source, and the spectral crosstalk caused by the overlap of the fluorescence spectra of the pigments is reduced using a matrix obtained by measuring the fluorescence spectra from the pigments introduced into the capillary. Therefore, the crosstalk reduction process becomes a different form from that of formula (7). This processing form makes it possible to easily change or add pigment types.

[0141] First, the crosstalk generated inside the spectrometer 505 is sorted out. N optical fibers for introducing light are provided in the spectrometer 505, and the optical image sensor 504 measures M wavelength intervals for each optical fiber. For example, when M = 20 and the detection wavelength range is 500 to 700 nm, the optical image sensor 504 outputs 20 outputs per optical fiber: a fluorescence signal with a wavelength of 500 to 510 nm, a fluorescence signal with a wavelength of 510 to 520 nm, and ... a fluorescence signal with a wavelength of 690 to 700 nm. Here, the output of wavelength interval m for optical fiber n of the optical image sensor 504 is referred to as S mn . S mn The one-dimensional arrangement of is called the output signal vector s of the optical image sensor 104. In addition, S mn Has two subscripts, starting with S mn The Mn+m component s becomes s Mn+m Arrange in one dimension.

[0142] On the other hand, consider the light incident on the optical fiber n of the optical splitter 505. The power of the light in the wavelength range corresponding to the wavelength interval m of the light is expressed as P mn By arranging the power P mn To generate the incident power vector p. mn As before, the Mn+m component P is used to form p Mn+m Arrange in one dimension.

[0143] The optical splitter 505 outputs a linear signal with respect to the power of the incident light. Therefore, the relationship between p and s is expressed as the following equation (8).

[0144] s=G p (8)

[0145] Matrix G is a matrix representing the crosstalk generated in the spectrometer 505. Matrix G is obtained by sequentially introducing monochromatic light corresponding to the wavelength detection range of the light sensor (e.g., the optical image sensor 504) to each light input position of the spatial filter (e.g., the optical fiber array 510). In an ideal spectrometer without crosstalk, matrix G becomes a unit matrix (in addition, s and p are normalized so that the output signal becomes 1 when the light power is 1). When crosstalk exists, Figure 10 The triangular portion of the matrix G represents the crosstalk value. In addition, when the sensitivity of the spectrometer is different depending on the position of each light input, the diagonal components of the matrix G may also take values ​​other than 1. For example, when the spectrometer 505 adopts Figure 5 In such a configuration, light loss in the spectrometer may increase the further away from the light-intake position of the lens 502 from the central axis. In this case, the value of the diagonal component corresponding to the light-intake position is smaller than 1.

[0146] Next, we will analyze the relationship between the dye in the capillary and the power of light incident on each optical fiber. L types of dyes are used in the analysis. Let the ratio of the first dye present in the nth capillary among N capillaries be D. ln . D ln The one-dimensional array vector is defined as the pigment ratio vector d. 1n Has two subscripts, but D ln The Ln+1 component d becomes d Ln+1 Arrange in one dimension.

[0147] Here, the signal generated by the nth capillary is measured by the nth optical fiber. The power P of the light measured by the nth optical fiber is mn The luminescence spectra of L fluorescent pigments in the nth capillary are divided by the ratio D ln Therefore, the relationship between the incident power vector p and the pigment ratio vector d is expressed by the following formula (9).

[0148] p=Fd (9)

[0149] Here, the matrix F is a matrix in which the fluorescence spectra of the respective dyes are arranged.

[0150] The following equation (10) is obtained from equations (8) and (9).

[0151] s=GFd(1O)

[0152] Therefore, if the inverse matrix of matrix G is set to G -1 , let the generalized inverse matrix of matrix F be F - , then we get the following formula (11). The generalized inverse matrix F - By [F tF] -1 F t or an equivalent operation thereof.

[0153] d=F - G -1 s (11)

[0154] This means that if we find G - and F - , then the pigment ratio d is calculated based on the output s of the optical image sensor. -1 The calculation of F represents the process of reducing the crosstalk occurring inside the optical splitter. - The calculation of represents a process of calculating the pigment ratio (reducing spectral crosstalk) by taking into account the overlap of the fluorescence spectra of the pigments.

[0155] Figure 10 The forms of matrix G and matrix F are shown. Matrix G is a square matrix of size M×N, with 1s arranged in the diagonal components. The areas indicated by triangles above and below the diagonal components represent crosstalk. In an ideal spectrometer without crosstalk, all elements in this area are 0. Matrix F is a matrix with M×N rows and L×N columns. Matrix F is a matrix with M rows and L columns, with N elements arranged in the diagonal part and 0 in the other areas. Matrix F1 has L spectra of pigments arranged in columns 1 to L ( Figure 10 The rectangles in the figure represent the spectra).

[0156] In the crosstalk reduction method of the second embodiment, the calculation is divided into two stages, and the acquisition of the matrix G and the matrix F is also performed separately. The acquisition of the matrix G uses the correction light source 1101 (see Figure 11 ) is performed. Calibration light source 1101 is connected to optical fiber array 510 of spectrometer 505, similarly to calibration light source 801. Calibration light source 1101 sequentially introduces light of wavelengths corresponding to M wavelength intervals into each of the N optical fibers. That is, calibration light source 1101 first introduces light of wavelengths corresponding to wavelength interval 1 into optical fiber 1. Next, it introduces light of wavelengths corresponding to wavelength interval 2, light of wavelengths corresponding to wavelength interval 3, and so on, light of wavelengths corresponding to wavelength interval M. The same operation is then performed on optical fiber 2. The same operation is then performed on optical fiber 3 and optical fiber N.

[0157] The matrix G is generated as follows. When the correction light source 1101 introduces light of wavelength range m to the optical fiber n, the value of the output to the optical fiber k of the optical fiber array and wavelength range l is represented by A. kl←mn At this time, let ζ = Mk + l, η = Mn + m, and let the η and ζ components of the matrix G be G ζη =A kl←mn / A mn←mn .

[0158] The structure of the correction light source 1101 for performing the above-mentioned operation is as follows. Figure 11 As shown. The correction light source 1101 has a white light source 1102 inside. The white light source 1102 is, for example, a white LED or a halogen lamp. The light from the white light source 1102 is separated according to each wavelength by a lens 1103 and a grating 1104. A slit 1105 is placed at the imaging point of the separated light. The slit 1105 only cuts out the component of a specific wavelength in the light of the white light source 1102. The grating 1104 can change the angle, and by adjusting the angle, the wavelength of the light passing through the slit 1105 is controlled. The light that has passed through the slit 1105 is introduced into a specific optical fiber in the output optical fiber array 1108 by a lens 1106 and a reflector 1107. The reflector 1107 can change an angle, and by adjusting the angle, it is controlled to which optical fiber in the output optical fiber array 1108 the light is introduced.

[0159] The matrix F can be measured by simultaneously injecting a dye-labeled sample into all capillaries. In the first embodiment, the matrix acquisition method requires injecting dye into only one of N capillaries to measure the signal. In contrast, the second embodiment allows simultaneous measurement in all capillaries, reducing the measurement time to 1 / N.

[0160] Matrix F is a matrix obtained by repeating matrix F1, which arranges the spectra of each pigment, by the number of capillaries. Therefore, it is also possible to measure the pigment spectrum using a specific capillary to measure matrix F1, and use the matrix obtained by repeating the number of capillaries as matrix F. In conventional crosstalk reduction methods and the crosstalk reduction method of the first embodiment, information on spectral crosstalk caused by the pigments, information on crosstalk caused by the light detection device, and information on the detection efficiency of each wavelength are mixed in a single processing matrix. Therefore, each pigment must be passed through each capillary to obtain this information. However, in the crosstalk reduction method of the second embodiment, information on the effects of the spectrometer is aggregated in matrix G, eliminating the need to measure the same sample for each capillary when measuring matrix F.

[0161] As described above, the crosstalk reduction method of the second embodiment is easier to obtain than the conventional crosstalk reduction method and the crosstalk reduction method of the first embodiment described in patent document 1, and is more convenient to obtain a matrix F containing information about a pigment spectrum. This is particularly useful when adding a pigment to be analyzed. When a new pigment that does not have information is desired to be used in a computer for crosstalk reduction processing, in the conventional crosstalk reduction method or the crosstalk reduction method of the first embodiment described in patent document 1, it is necessary to sequentially import the new pigment into all capillaries to obtain the fluorescence spectrum information of the new pigment. On the other hand, in the crosstalk reduction method of the second embodiment, the new pigment is simultaneously imported into all capillaries or into a specific capillary to measure the fluorescence spectrum.

[0162] Alternatively, matrix F can be provided in a form pre-stored in a computer. Information on crosstalk, which depends on each individual spectrometer, is contained in matrix G. Therefore, it is not necessary to measure matrix F for each individual photodetection device. Instead, the spectral information of each pigment measured in a specific device can be stored in the computers of other devices and matrix F can be generated based on this information. In other words, the spectral information of the pigment measured by a particular photodetection device can be made public online, and users can download the spectral information from the internet and store it in their computers, and then generate matrix F based on the stored spectral information.

[0163] Figure 12 This is an example of the operation of the device when the matrix F is obtained. First, the user sets the pigment or pigment group to obtain the spectrum (S1201). Next, the user selects the method for obtaining the fluorescence spectrum (S1202). When the method of measuring the pigment spectra in all capillaries is selected, the device first prepares for electrophoresis (S1203). After the preparation is completed, the user sets the sample labeled with the pigment to be measured (S1204). After the sample setting is completed, the device injects the sample into the capillary and performs electrophoresis (S1205). The spectrum of the moment when the sample swims to the fluorescence measurement point is obtained (S1206). This cycle is repeated for all the pigments whose spectra are obtained (S1207). If the sample is prepared in a way that all the pigment spectra can be obtained at the same time, the cycle can be one time. For example, this is a sample in which the measured pigments A, B, C... are respectively labeled with DNA with chain lengths of 100 bases, 150 bases, 200 bases... and mixed. During electrophoresis, the pigments A, B, C... emit light at different times in sequence. Such calibration samples are commercially available and relatively easy to obtain. Due to electrophoresis, the DNA reaches the fluorescence measurement point in order of chain length, so pigments A, B, and C emit light in a time-sequential manner. After all fluorescence spectra are measured, a fluorescence spectrum matrix F is generated (S1208).

[0164] When the fluorescence spectrum measurement method using a single capillary is selected, the device operates in the same manner as when the fluorescence spectrum is measured using all capillaries (S1209-S1214). However, the sample is injected into only a single capillary. When generating the matrix F, the fluorescence spectra obtained from the single capillary are repeatedly arranged.

[0165] If the method of generating matrix F based on internal information is selected, the fluorescence spectrum (luminescence spectrum information) of the set pigment is read from the memory (S1215). The fluorescence spectrum can be initially stored in the computer, or the spectrum information can be provided and downloaded via the Internet. Matrix F is generated based on the read fluorescence spectrum (S1216). The generated matrix F is stored in the computer (S1217).

[0166] <Modification>

[0167] The present invention is not limited to the above-described embodiments and includes various variations. For example, the above-described embodiments are described in detail to facilitate understanding of the present invention and are not necessarily limited to having all the structures described. In addition, a portion of the structure of a certain embodiment can be replaced with the structure of another embodiment, and the structure of another embodiment can be added to the structure of a certain embodiment. In addition, with respect to a portion of the structure of each embodiment, other structures can be added, deleted, or replaced.

[0168] For example, in the first embodiment, the optical fiber array 510 is provided as an example of a spatial filter, but the spatial filter may also be a filter having a passage hole for passing light. The shape of the passage hole of the filter may be circular or rectangular. Figure 13 This is a structural diagram of a modified light detection device 1300. Unlike light detection device 500, light detection device 1300 includes a pinhole array 1310, which includes a plurality of pinholes (circular holes) arranged in an array corresponding to the light emission points of a plurality of light-emitting body holders (capillaries). Pinhole array 1310 may also be a slit array with slits (rectangular holes) arranged in an array.

[0169] In the above embodiment, a light detecting device having multiple light-emitting body holders for emitting multiple light-emitting bodies is used. However, if there are multiple light-emitting bodies, there may be one light-emitting body holder, and if there are multiple light-emitting body holders, there may be only one light-emitting body.

[0170] Furthermore, in the calculations of the above-described embodiment, various calculations using matrices are performed. However, as long as equivalent results can be obtained, equivalent calculations using information not in a matrix format may be used.

[0171] Explanation of symbols

[0172] 100 light detection device

[0173] 101 Capillary Array

[0174] 102 lens

[0175] 103 grating

[0176] 104 light image sensor

[0177] 105 spectral images

[0178] 106 channels

[0179] 201 capillary array

[0180] 202 lens

[0181] 203 optical image sensor

[0182] 500 light detection device

[0183] 501 capillary array

[0184] 501a capillary

[0185] 502 lens

[0186] 503 grating

[0187] 504 light image sensor

[0188] 505 optical splitter

[0189] 510 fiber array

[0190] 510a optical fiber

[0191] 550 Computer

[0192] 551 processor

[0193] 552 Main Storage Unit

[0194] 553 Auxiliary Storage Unit

[0195] 554 input and output interface

[0196] 560 display unit

[0197] 701 Capillary Array

[0198] 702 lens

[0199] 703 optical image sensor

[0200] 710 fiber array

[0201] 720 light blocking wall

[0202] 801 calibration light source

[0203] 802 excitation light source

[0204] 803 flow path

[0205] 804 pigment injection mechanism

[0206] 805 lens

[0207] 806 wavelength filter

[0208] 807 reflector

[0209] 808 lens

[0210] 809 output fiber array

[0211] 1101 calibration light source

[0212] 1102 white light source

[0213] 1103 lens

[0214] 1104 grating

[0215] 1105 slit

[0216] 1106 lens

[0217] 1107 reflector

[0218] 1108 output fiber array

[0219] 1300 optical detection device

[0220] 1310 pinhole array.

Claims

1. A light detection device, characterized in that The light detection device comprises: Multiple replaceable light-emitting body holders, inside which multiple light-emitting bodies emit light; a light sensor having a plurality of detection channels for detecting light emitted from the plurality of light-emitting bodies in a plurality of wavelength bands respectively; a spatial filter that fixes the incident position of the light emitted from the plurality of light emitters relative to the light sensor; as well as a computer that processes the signal output from the light sensor, The computer internally has crosstalk information for reducing crosstalk between the plurality of detection channels. The computer reduces crosstalk between detection channels of the light sensor by performing calculations on the outputs from the light sensor corresponding to the plurality of wavelength bands of the plurality of luminous points emitted by the plurality of light-emitting bodies using the crosstalk information, and derives a concentration ratio or a signal quantity ratio of each of the plurality of light-emitting bodies for each of the plurality of light-emitting body holders. Even if the light-emitting body holder is replaced with another light-emitting body holder, the calculation is performed using the crosstalk information.

2. The light detection device according to claim 1, wherein The crosstalk information is the generalized inverse matrix C of the matrix C including the information of the crosstalk between the plurality of detection channels. - or the equivalent information about the generalized inverse matrix, The operation for deriving the concentration ratio or the signal quantity ratio is to make the matrix C - C is derived from the vector s acting on the output from the light sensor - s, or an equivalent operation.

3. The light detection device according to claim 2, wherein: The matrix C is a plurality of matrices C including only a portion of the crosstalk information between the luminescent body holder or the pigment. n combination of ingredients.

4. The light detection device according to claim 1, wherein The spatial filter is an optical fiber array including a plurality of optical fibers provided corresponding to the light-emitting points of the plurality of light-emitting body holders.

5. The light detection device according to claim 1, wherein The spatial filter includes a plurality of pinholes or slits provided corresponding to the light-emitting points of the plurality of light-emitting body holders.

6. The light detection device according to claim 2, wherein: The plurality of light-emitting bodies are sequentially passed through each of the plurality of light-emitting body holders, the output of the light sensor when a single light-emitting body among the plurality of light-emitting bodies is emitting light is recorded, the output of the light sensor is arranged to generate a matrix C, and [C t C] -1 C t Or its equivalent operation to calculate the generalized inverse matrix C of the matrix C - and save it.

7. The light detection device according to claim 1, wherein When the matrix representing the crosstalk generated by the light detection device is G and the matrix representing the spectral crosstalk generated by the overlap of the emission spectra of the pigment is F, the crosstalk information is obtained by using the inverse matrix G of the matrix G. -1 and through [F t F] -1 F t Or the generalized inverse matrix F of the matrix F obtained by an equivalent operation - Operation F - G -1 s or an equivalent operation thereof to obtain the concentration ratio or signal quantity ratio of the pigment.

8. The light detection device according to claim 7, wherein The matrix G is obtained by sequentially introducing monochromatic light corresponding to the wavelength detection range of the light sensor into each light emission input position of the spatial filter.

9. The light detection device according to claim 7, wherein The matrix F is obtained by allowing the plurality of light-emitting bodies to flow sequentially through all or part of the plurality of light-emitting body holders.

10. The light detection device according to claim 7, wherein The matrix F is generated based on the emission spectrum information of the plurality of light-emitting bodies stored in the computer.

11. The light detection device according to claim 2 or 7, characterized in that: Connecting a correction light source to the spatial filter to obtain the crosstalk information, The correction light source sequentially introduces correction light to each light-emission intake position of the spatial filter.

12. The light detection device according to claim 11, wherein The correction light source introduces light having the same spectrum as the emission spectrum of the plurality of light-emitting bodies.

13. The light detection device according to claim 11, wherein The correction light source introduces monochromatic light corresponding to a wavelength detection range of the optical sensor.

14. The light detection device according to claim 1, wherein In the event that the output from the light sensor is saturated, a warning is displayed.

15. A signal processing method, characterized in that: The steps are as follows: A light detection device is prepared, the light detection device comprising: a plurality of replaceable light-emitting body holders, within which the plurality of light-emitting bodies emit light; a light sensor having a plurality of detection channels for detecting light emitted from the plurality of light-emitting bodies in a plurality of wavelength bands; and a spatial filter for fixing the incident position of the light emitted from the plurality of light-emitting bodies relative to the light sensor. and a computer that processes a signal output from the light sensor; measuring the luminescence of the plurality of luminophores by the light sensor; Using crosstalk information for reducing crosstalk between the plurality of detection channels stored in the computer, calculating outputs from the light sensor corresponding to the plurality of wavelength bands of the plurality of luminescent points emitted by the plurality of luminescent bodies, reducing crosstalk between the detection channels of the light sensor, and deriving a concentration ratio or signal quantity ratio of each of the plurality of luminescent bodies for each of the plurality of luminescent body holders; and Even if the light-emitting body holder is replaced, the calculation is performed using the crosstalk information.

16. The signal processing method according to claim 15, characterized in that: The crosstalk information is the generalized inverse matrix C of the matrix C including the crosstalk information. - Or information equivalent thereto, the operation for deriving the concentration ratio or the signal quantity ratio is to make the matrix C - C is derived from the vector s acting on the output from the light sensor - s or an equivalent operation.

17. The signal processing method according to claim 16, characterized in that: When the matrix representing the crosstalk generated in the light detection device is G and the matrix representing the spectral crosstalk generated by the overlap of the emission spectra of the pigment is F, the crosstalk information used for the calculation is obtained by using the inverse matrix G of the matrix G. -1 and through [F t F] -1 F t Or the generalized inverse matrix F of the matrix F obtained by an equivalent operation - Operation F - G -1 s or an equivalent operation thereof to obtain the concentration ratio or signal quantity ratio of the pigment.

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