LIGHT DETECTION DEVICE AND SIGNAL PROCESSING METHOD

Abstract

A light detection device 500 includes a replaceable capillary array 501 in which a plurality of types of light emitters emit light, an optical sensor 504 having a plurality of detection channels for respectively detecting the light emitted by the plurality of types of light emitters in a plurality of wavelength bands, a light guide array 510 for fixing an incident position of the light emitted by the plurality of types of light emitters on the optical sensor 504, and a computer 550.The computer 550 includes crosstalk information therein, performs calculation using the crosstalk information on an output of the optical sensor 504 corresponding to a plurality of light emission points at which the plurality of types of light emitters emit light and a plurality of wavelength bands (of the respective light emission points) to reduce crosstalk between the detection channels of the optical sensor 504 and derive a concentration ratio or a signal amount ratio of each of the plurality of types of light emitters for each of a plurality of capillaries, and performs the calculation using the crosstalk information even when the capillary array 501 is replaced with another one.

Description

Technical area

[0001] The present invention relates to a light detection device and a signal processing method, and more particularly to a light detection device and a signal processing method that reduce crosstalk that occurs when a plurality of types of light emitters emit light at a plurality of light emission points. State of the art

[0002] There is a light measurement method in which a variety of light emitters are present at a variety of spatial positions, and a light detection device that separates and detects a wavelength of light for each spatial position, detecting the position and concentration ratio of each light emitter. An example of this is a light detection device of a capillary electrophoresis apparatus that includes a variety of analytical capillaries for analyzing a sample labeled with a variety of fluorescent dyes.

[0003] In capillary electrophoresis analysis, a sample to be analyzed is injected into a capillary filled with a separation medium, and a voltage is applied to both ends to perform separation based on a difference in the mobility of the analysis target. Fluorescence detection, for example, is used to detect the analysis target. The analysis target is labeled with a fluorescent dye, and the fluorescence generated by irradiation with excitation light is detected to detect the separated analysis target.

[0004] For example, there is a method in which DNA labeled with a fluorescent dye is electrophoresed in a polymer-filled capillary and separated according to strand length. A detection site provided on the capillary is irradiated with excitation light, and the generated fluorescence is detected. DNA molecules in a sample move in the capillary and pass through the detection site at different times depending on the strand length. As a result, a strand length distribution of the DNA molecules in the sample is obtained as a fluorescence intensity waveform. When a variety of DNA molecules are present in the sample and the DNA molecules are to be differentiated and analyzed, a variety of fluorescent dyes can be used to label the DNA.The light detecting device separates and detects a fluorescence wavelength with a grating or the like, and determines a type of the fluorescent dye based on a shape of an obtained spectrum.

[0005] A capillary electrophoresis device can accommodate a large number of capillaries to improve measurement throughput. In such a case, for example, capillaries are arranged in a row for separation, and all capillaries are irradiated together with excitation light. The generated fluorescence is captured by an image sensor. A signal quantity is calculated based on a fluorescence image of each capillary to obtain an electrophoresis waveform of the sample.

[0006] When using a variety of fluorescent dyes and a variety of capillaries as described above, a signal different from the signal originally intended to be detected may be detected, and a measured concentration ratio of a target may deviate from the true value. For example, there may be a case where the light emission of a fluorescent dye B is erroneously detected even though a fluorescent dye A in a particular capillary is emitting light, or a case where the light emission of a j-th capillary, which is different from an i-th capillary, is erroneously detected even though the i-th capillary is emitting light.Here, a signal output due to false detection is referred to as crosstalk, a signal output due to false detection of the dye in the former case is referred to as spectral crosstalk, and a signal output due to false detection of the light-emitting capillary in the latter case is referred to as spatial crosstalk.

[0007] Various causes for the occurrence of crosstalk are conceivable. For example, spectral crosstalk may occur due to the width of the emission spectrum of a fluorescent dye. Generally, a fluorescent dye has an emission spectrum with a wavelength width of about a few tens of nanometers. When a plurality of dyes are used, an emission spectrum of dye A and an emission spectrum of dye B may overlap. If the emission spectra of dye A and dye B overlap, even if dye A emits light, a part of the emission spectrum of dye A corresponding to a detection wavelength band of dye B may be detected as the emission signal of dye B.

[0008] As an example, spatial crosstalk can be caused by surface reflection from a neighboring capillary. Assume that a plurality of capillaries are arranged in a row in the device, and a detector faces such a capillary array. When a fluorescent dye in the i-th capillary (hereinafter referred to as capillary i, if appropriate) emits light, fluorescence is emitted in all directions, and a portion of it is introduced into the detector. However, a portion of the light strikes the j-th capillary nearby (hereinafter referred to as capillary j, if appropriate), and a portion of it is further reflected toward the detector. In such a case, capillary j, which reflects the fluorescence, appears to emit fluorescence on the detector, and a light emission signal from capillary j is detected.

[0009] Crosstalk has various adverse effects on capillary electrophoresis analysis. For example, if the fluorescence of capillary i is detected as the fluorescence of capillary j due to spatial crosstalk, a component in a sample originally analyzed in capillary i may be falsely detected as being present in a sample analyzed in capillary j. Furthermore, due to spectral crosstalk, even though only component a labeled with fluorescent dye A is originally present in a sample, component b labeled with dye B may be falsely detected as being present in the sample.

[0010] Crosstalk can be reduced by designing measurement conditions and instrument configuration. For example, spectral crosstalk can be reduced by increasing the distance between the emission wavelengths of the fluorescent dyes to be used, and spatial crosstalk does not occur when completely independent measurement optical systems are prepared according to the number of capillaries. However, since a detection wavelength detectable by the detector has an upper and lower limit, the number of usable fluorescent dyes decreases when an emission spectrum interval between the dyes is increased. In addition, if the optical systems for measuring each capillary are independent, spatial crosstalk does not occur, but light sources and detectors corresponding to the number of capillaries are required, which is disadvantageous in terms of cost and device size.

[0011] Meanwhile, a method for reducing crosstalk through data processing is also known (PTL 1). In this method, the crosstalk when a specific fluorescent dye in a specific capillary emits light is comprehensively detected in advance, and an inverse matrix of a matrix in which the crosstalk components are arranged is generated. The crosstalk is reduced by applying the inverse matrix to an actually detected signal. With this method, it is possible to reduce crosstalk without changing the device configuration and measurement conditions. Citation listPatent applications PTL 1: JP 7 282 880 B PTL 2: WO 2018 / 151 843 A1 PTL 3: WO 2023 / 276 078 A1 Non-patent literature

[0012] NPL 1: SeqStudio™Genetic Analyzer Instrument and Software USER GUIDE https: / / assets.thermofisher.com / TFS-Assets / LSG / manuals / MAN0018646_SeqStudioInstSW_UG.pdf Summary of the inventionTechnical problem

[0013] The method disclosed in PTL 1 is based on the premise that the spectral and spatial crosstalk ratios between dyes and between capillaries do not change between the time the matrix used for crosstalk reduction processing is generated and the time a sample is analyzed. This condition is basically met when the same capillary array is used to generate the matrix used for crosstalk reduction processing and to analyze the sample. However, in capillary electrophoresis, the capillary array is a consumable, and the capillary array must be replaced after a certain number of uses.

[0014] When the capillary array is replaced, the ratios of spectral crosstalk and spatial crosstalk change. In the capillary array, the capillaries are arranged in a row, and since each capillary has an alignment error of approximately a few µm to a few tens of µm, the light reflection pattern on the capillary surface may change for each capillary array. The position and inclination of the center of the capillary array relative to the detector may also change before and after replacement. These change the ratio of spatial crosstalk.

[0015] Since the emission spectrum of a dye is not altered by replacing the capillary, it can be expected that the change in spectral crosstalk associated with replacing the capillary array is smaller than the change in spatial crosstalk. However, spectral crosstalk can also change by replacing the capillary array. For example, if crosstalk occurs in a path where multiple reflections occur in a detector optics system and a signal from dye A enters a detection channel of dye B, a change in the position of the capillary array will change a state of multiple reflections, and as a result, the spectral crosstalk can be changed.

[0016] For this reason, when applying the method in PTL 1, it is necessary to generate the matrix used for crosstalk reduction processing for a new array after replacing the capillary array. When generating the matrix used for crosstalk reduction processing, it is necessary to obtain crosstalk ratios for all dyes and capillaries. A crosstalk ratio when dye A emits light in the specific capillary i can be obtained by injecting only dye A into capillary i and performing electrophoresis. Therefore, for example, if four types of dyes are used and a capillary array with eight capillaries is used, electrophoresis is performed 4 × 8 = 32 times to generate the matrix used for crosstalk reduction processing.Given that a single electrophoresis run, including preparatory operations such as polymer replacement and pre-electrophoresis to remove unnecessary ions, takes approximately several tens of minutes or more, it takes a long time to generate the matrix used for crosstalk reduction processing. This time increases with the number of dyes and the number of capillaries.

[0017] PTL 1 also discloses a method for reducing the effort and time required to generate the matrix used for crosstalk reduction processing. Specifically, a method is disclosed in which the timing at which a sample is injected into each capillary is staggered to offset the light emission timing of a dye, so that when a specific capillary emits light, another capillary does not emit light. A method is also disclosed in which fluorescently labeled DNA with different strand lengths is introduced into each capillary to similarly prevent another capillary from emitting light when a specific capillary emits light.

[0018] However, in the former case, the instrument must be equipped with an operating sequence different from normal analysis, and the latter case presents hurdles such as the need to prepare a reagent containing DNA whose strand length is controlled to generate the matrix used for crosstalk reduction processing. If a dye to be used is changed, it is necessary to regenerate the matrix used for crosstalk reduction processing, even if the capillary array is not replaced.

[0019] PTL 2 discloses a method for optimizing a spectral crosstalk reduction matrix (synonymous with processing for obtaining a ratio of a plurality of dyes having an overlapping spectrum based on a fluorescence spectrum) in spectral crosstalk reduction based on information obtained by analysis during sample analysis without performing electrophoresis for calibration for the purpose of obtaining information on a spectral crosstalk reduction matrix.In this method, based on an initial matrix, the matrix used for spectral crosstalk reduction processing is optimized by repeating a matrix change, evaluating the dye concentration correlation, and determining whether to update the matrix so that a correlation based on correlation information between any two estimated dye concentrations decreases.

[0020] In this method, it is necessary to remove an outlier to correctly obtain a correlation between two dyes caused by spectral crosstalk. That is, it is necessary to distinguish between spectral crosstalk related to dye B when dye A is present and a case where dye B is actually present in a sample. It is generally expected that such a distinction can be made regarding spectral crosstalk. Generally, when a plurality of dyes are used, dye A and dye B mark different analysis targets (molecules) to distinguish the different analysis targets.Therefore, when a sample containing both the analysis target labeled with dye A and the analysis target labeled with dye B is electrophoresed to obtain fluorescence spectrum time series data, it can be expected that dye A and dye B will emit light at different times in many cases. Meanwhile, the spectral crosstalk related to dye B, which occurs due to dye A, occurs at the same time that dye A emits light. Since it is rare that the analysis target labeled with dye A and the analysis target labeled with dye B coincidentally emit light at the same time, when calculating correlation information between estimated dye concentrations of dye A and dye B, it is sufficient to remove a rare value as an outlier whose trend is different from that of the majority of other data.

[0021] However, even if the method in PTL 2 is extended to spatial crosstalk, the prerequisite conditions for outlier removal described above are not met. When the method in PTL 2 is extended to spatial crosstalk, it is necessary to distinguish between spatial crosstalk related to dye A in capillary j, when dye A in capillary i emits light, and a signal when dye A in capillary j actually emits light. Similar to the above case, the fluorescence of dye A caused by spatial crosstalk in capillary j occurs at the same time point as dye A in capillary i emits light. Here, it is assumed that dye A labels the same analysis target α in a sample analyzed in capillary i and a sample analyzed in capillary j.In this case, the time at which dye A in the sample actually emits light is the same between capillary i and capillary j. Therefore, there is no guarantee that the spatial crosstalk that occurs with respect to dye A in capillary j when dye A in capillary i emits light will always be distinguished from the signal actually generated by the light emission of dye A in capillary j.

[0022] This is a serious obstacle in the following cases, for example. As an example, consider analysis target α labeled with dye A and analysis target β labeled with dye B. Only analysis target α is present in sample 1. It is assumed that analysis target β is contained in sample 2, and it is to be determined whether a tiny amount of analysis target α is present in sample 2. This is, for example, a case where sample 1 is a pure substance, and a trace component (analysis target α) present in sample 2 is analyzed based on data from sample 1.

[0023] Here, sample 1 is analyzed in capillary i, and sample 2 is analyzed in capillary j. At this time, a strong signal of dye A is obtained in capillary i, and a strong signal of dye B is obtained in capillary j. Here, we aim to determine whether a weak signal of dye A is generated in capillary j, but since it is common for the analysis target α to be labeled with dye A, the fluorescence of dye A is obtained at essentially the same time between capillary i and capillary j, regardless of whether the fluorescence is due to spatial crosstalk or an actual signal of the analysis target α.Therefore, if it is assumed that a weak signal of dye A is obtained in capillary j, it cannot be distinguished based only on a data trend whether the signal is generated due to spatial crosstalk or whether a tiny amount of the analysis target α labeled with dye A is actually present.

[0024] Therefore, by combining the method in PTL 1 with the method in PTL 2, it is not possible to optimize a matrix for reducing both spectral crosstalk and spatial crosstalk during analysis and eliminate the need to re-acquire a crosstalk reduction matrix at the time of replacing the capillary array.

[0025] A capillary electrophoresis device disclosed in NPL 1 demonstrates an option for using a matrix initially set as a spectral crosstalk reduction matrix (Factory Calibration, disclosed on p. 217). However, this matrix is ​​not optimized for each individual device and can only be used for applications where residual spectral crosstalk due to incomplete information is tolerable.

[0026] Therefore, the invention has been made in view of such circumstances, and an object thereof is to provide a light detection device and a signal processing method that can perform crosstalk reduction processing using once generated crosstalk information used for the crosstalk reduction processing even when a plurality of light emitter holders (for example, a capillary array) are replaced. Solution to the problem

[0027] To solve the above-mentioned problems, a light detection device according to the invention comprises: a plurality of interchangeable light emitter holders in which a plurality of types of light emitters emit light; an optical sensor having a plurality of detection channels for respectively detecting the light emitted from the plurality of types of light emitters in a plurality of wavelength bands; a spatial filter configured to fix an incident position of the light emitted from the plurality of types of light emitters on the optical sensor;and a computer configured to process a signal output from the optical sensor, the computer including crosstalk information for reducing crosstalk between the plurality of detection channels therein, performing, using the crosstalk information, a calculation on an output of the optical sensor corresponding to the plurality of wavelength bands for each of a plurality of light emission points at which the plurality of types of light emitters emit light, to reduce crosstalk between the detection channels of the optical sensor and deriving a concentration ratio or a signal amount ratio of each of the plurality of types of light emitters for each of the plurality of light emitter holders, and performing the calculation using the crosstalk information even when each light emitter holder is replaced with a different light emitter holder.

[0028] A signal processing method according to the invention comprises: preparing a light detection device including a plurality of interchangeable light emitter holders in which a plurality of types of light emitters emit light, an optical sensor having a plurality of detection channels for respectively detecting the light emitted by the plurality of types of light emitters in a plurality of wavelength bands, a spatial filter configured to fix an incident position of the light emitted by the plurality of types of light emitters on the optical sensor, and a computer configured to process a signal output from the optical sensor; measuring the light emission of the plurality of types of light emitters by the optical sensor;Performing, using crosstalk information stored in the computer to reduce crosstalk between the plurality of detection channels, a calculation for an output of the optical sensor corresponding to the plurality of wavelength bands for each of a plurality of light emission points at which the plurality of types of light emitters emit light to reduce crosstalk between the detection channels of the optical sensor and deriving a concentration ratio or a signal amount ratio of each of the plurality of types of light emitters for each of the plurality of light emitter holders; and performing the calculation using the crosstalk information even when each light emitter holder is replaced. Advantageous effects of the invention

[0029] According to the light detection device and signal processing method of the invention, even when replacing a plurality of light emitter holders, it is possible to perform crosstalk reduction processing using once-generated crosstalk information. Therefore, it is possible to reduce the effort and time required to regenerate the crosstalk information used for crosstalk reduction processing when replacing the plurality of light emitter holders.

[0030] Problems, configurations and effects not described above will be clarified by the following description of the embodiments. Brief description of the drawings [ Fig. 1A] Fig. 1A shows a schematic diagram of a light detecting device to which a crosstalk reduction method disclosed in PTL 1 can be applied. [ Fig. 1B] Fig. Fig. 1B shows a schematic diagram of a spectrum image on a light image sensor of the light detecting device in Fig. 1A. [ Fig. 2A] Fig. Figure 2A shows a schematic diagram of a crosstalk simulation model. [ Fig. 2B] Fig. Figure 2B shows a flowchart of a simulation performed using the simulation model in Fig. 2A is executed. [ Fig. 3A] Fig. Figure 3A shows a diagram showing a result of a crosstalk simulation. [ Fig. 3B] Fig. 3B is a diagram showing a result when crosstalk reduction processing according to PTL 1 is applied to the simulation result in Fig. 3A is applied. [ Fig. 4A] Fig. Figure 4A shows a result of a crosstalk simulation assuming a replacement of the capillary array. [ Fig. 4B] Fig. Fig. 4B is a diagram showing a result when the crosstalk reduction processing according to PTL 1 is applied to the simulation result in Fig. 4A is applied. [ Fig. 5] Fig. 5 shows a schematic diagram of a light detecting device according to a first embodiment. [ Fig. 6] Fig. Figure 6 shows a schematic diagram illustrating a generation path of crosstalk occurring on the capillary side relative to an optical fiber. [ Fig. 7A] Fig. 7A is a diagram showing a simulation model of the light detecting device to which crosstalk reduction processing according to the first embodiment is applied. [ Fig. 7B] Fig. Figure 7B shows a result of a crosstalk simulation in the simulation model in Fig. 7A. [ Fig. 7C] Fig. Figure 7C shows a diagram showing a result when the crosstalk reduction processing is applied to the simulation result in Fig. 7B is applied. [ Fig. 8A] Fig. 8A is a schematic diagram showing a connection method of a calibration light source for detecting a matrix used for crosstalk reduction processing according to the first embodiment. [ Fig. 8B] Fig. 8B is a schematic diagram showing a structural example of the calibration light source in Fig. 8A shows. [ Fig. 9] Fig. 9 is a diagram showing an influence of detector saturation on the crosstalk reduction processing according to the first embodiment. [ Fig. 10] Fig. Figure 10 shows a diagram showing elements of a matrix G and a matrix F. [ Fig. 11] Fig. 11 is a schematic diagram showing a structural example of a calibration light source for detecting a matrix used for crosstalk reduction processing according to a second embodiment. [ Fig. 12] Fig. 12 is a flowchart showing an operation of a light detecting device in detecting the matrix used for the crosstalk reduction processing according to the second embodiment. [ Fig. 13] Fig. 13 shows a schematic diagram of a light detecting device according to a modification. Description of the embodiments

[0031] In the following embodiments, the description may be divided into a plurality of sections or embodiments for convenience, but unless otherwise stated, they are not unrelated to each other, and one has a relationship with all or part of modifications, details, supplementary explanations, and the like of the others. In the following embodiments, when referring to the number of elements (including the number, a numerical value, an amount, a range, or the like) or the like, the number of elements is not limited to a specific number, and may be the specific number or more, or the specific number or less, unless otherwise stated or except in a case where the number is obviously limited to a specific number in principle.

[0032] Furthermore, in the following embodiments, it is understood that components (including element stages and the like) are not necessarily essential unless otherwise specified or clearly considered essential in principle. Similarly, in the following embodiments, when referring to a shape, positional relationship, or the like of a component or the like, the shape or the like is substantially approximate or similar to the shape or the like unless otherwise specified or clearly considered inappropriate in principle. The same applies to the numerical value and range described above.

[0033] In all the drawings for describing the embodiments, the same elements are denoted by the same reference numerals in principle, and repeated descriptions thereof are omitted.

[0034] In the following embodiments, use in a capillary electrophoresis apparatus will be described as a typical application example of a light detection device of the invention. This serves to more specifically describe the configuration and effect of the invention, and the light detection device of the invention is not limited to the capillary electrophoresis apparatus. (Overview of the capillary electrophoresis device of the embodiment)

[0035] In the configuration of the capillary electrophoresis apparatus of the embodiment, a spatial filter is provided at a light collecting section of a spectrometer, for example, which detects a spectrum by dispersing fluorescence from a fluorescent dye, and the light collecting position relative to the spectrometer is fixed. For example, the spectrometer includes a first lens that collimates incident light, a grating that separates light by wavelength, a second lens that forms an image of the light passing through the grating on an optical sensor, and a light image sensor that detects light. The spatial filter is a multimode optical fiber, for example, and one end of the optical fiber is fixed at a light incident position of the spectrometer. The other end of the optical fiber is arranged near a capillary to collect fluorescence emitted from the inside of the capillary. The light image sensor is a CCD or CMOS image sensor, for example.

[0036] Although crosstalk can occur due to a variety of factors, the configuration described above allows a fixed ratio of the crosstalk occurring on the spectrometer side relative to the optical fiber, without being affected by replacing a light emitter holder (capillary array). Fluorescence generated in the capillary is collected into the optical fiber and guided to the spectrometer. Examples of causes of crosstalk within the spectrometer include the spatial profile spread of a fluorescence spectrum, multiple reflections between surfaces of elements such as lenses, and grating anomalies.When there is no optical fiber and the capillary is directly provided at a light introduction position of the spectrometer, a positional shift of each capillary due to the replacement of the capillary array may affect a spatial profile of fluorescence, an occurrence state of multiple reflections and anomalies, and the like, and a crosstalk ratio may change.

[0037] By fixing the incident position of the optical fiber to the spectrometer, the spatial profile of the fluorescence, the occurrence of multiple reflections, anomalies, and the like are fixed. Changing the position of the capillary relative to the other end of the optical fiber on the opposite side from the spectrometer affects the amount of light incident into the optical fiber and does not affect the crosstalk ratio occurring in the spectrometer. Since changing the position of the capillary does not change the crosstalk ratio, the same information as before the replacement can be used for crosstalk reduction processing even if the capillary array is replaced.

[0038] Meanwhile, the crosstalk occurring on the capillary side relative to the optical fiber may still change by replacing the capillary array. For example, since the crosstalk occurring due to fluorescent reflection on a capillary surface is affected by an arrangement error of the capillaries constituting the capillary array, the crosstalk is changed by replacing the capillary array. Such crosstalk cannot be completely eliminated only by providing the optical fiber described above. It is also possible to sufficiently reduce the crosstalk occurring on the capillary side and varying due to the replacement of the capillary array. This is achieved, for example, by making each capillary independent by a light-shielding wall. Alternatively, a distance between the capillaries can be increased to such an extent that the crosstalk can be ignored.A method is also known in which an angle of incidence of the light to be detected is limited by placing a pinhole at an output of an optical fiber (PTL 3).

[0039] Although the crosstalk occurring on the capillary side and varying due to the replacement of the capillary array cannot be eliminated solely by the optical fiber as described above, the effect of the invention is not diminished. Even if the crosstalk occurring on the capillary side of the optical fiber is not completely eliminated, a residual component may be low enough to be acceptable for analysis purposes. (Known technology in PTL 1)

[0040] Before the detailed description of the embodiments, a known technique in PTL 1, which is the background of the invention, and its problems are organized. Fig. 1A is a configuration diagram of a light detection device 100 to which the technique disclosed in PTL 1 can be applied. The light detection device 100 includes a capillary array 101, two lenses 102, a grating 103, and a light image sensor 104. The light detection device 100 disperses and detects fluorescence emitted from the capillary array 101. An arrow in the drawings indicates an irradiation direction of excitation laser light L that excites the fluorescence. The laser light L is emitted to penetrate all the capillaries of the capillary array from a lateral direction (side surface direction) of the capillary array 101 to excite dyes inside all the capillaries.

[0041] In the light detection device 100, the grating 103 disperses a wavelength in a direction perpendicular to the sheet. As shown in Fig. 1B, spectrum images 105 corresponding to the respective capillaries are arranged on the light image sensor 104. The light image sensor 104 outputs a light intensity measurement result for each pixel. Light intensity measurement results are summed for each wavelength interval of each capillary. The wavelength interval means each interval from 500 nm to 510 nm, 510 nm to 520 nm, ..., 690 nm to 700 nm, for example, when a measurement wavelength range is 500 nm to 700 nm and light intensity values ​​for each wavelength are summed at 10 nm intervals to obtain 20 data points. That is, a set of values ​​obtained by summing outputs from pixels in each indicated by dashed lines in Fig. 1B delimited section (channel 106) is obtained as spectrum information of each capillary.

[0042] A crosstalk reduction method in the related art disclosed in PTL 1 is organized considering the light detection device 100. As defined above, crosstalk includes two types: spectral crosstalk, in which a signal of dye A is mistakenly recognized as a signal of dye B, and spatial crosstalk, in which a signal emitted from capillary i is mistakenly recognized as a signal emitted from capillary j. Crosstalk reduction methods of PTL 1 and the invention are based on the premise that crosstalk is linear with respect to a signal vector (a vector in which signals corresponding to each capillary and each wavelength interval obtained from the light image sensor 104 are arranged).This is normally satisfied because the light detecting device 100 does not include an element that induces a nonlinear effect on the light to be detected and is not used under a condition where the nonlinear effect occurs (generally, a fairly high light intensity is required).

[0043] In the above contents, if a signal when the dye A in the capillary i emits light, with S Ai and the crosstalk detected in an area where the dye B in the capillary j is to be detected when the dye A in the capillary i emits light is denoted by S CT Bj←Ai a relationship between them can be expressed by the following formula (1) using a constant c. SCTBj←Ai=cSAi

[0044] In the crosstalk reduction method disclosed in PTL 1, a process to obtain the constant c is first performed. It is assumed that dye A is injected into capillary i and nothing is injected into the other capillaries. In this situation, the signal S Ai , when the dye A in the capillary i emits light, and the crosstalk S CT Bj←Ai , which is detected in the region where dye B in capillary j is to be detected when dye A in capillary i emits light, is detected. Based on a relationship between them, the constant c is obtained by the following formula (2). c=SCTBj←Ai / SAi

[0045] Next, a sample to be analyzed is measured. A sample labeled with dye A is injected into capillary i. A sample labeled with dye B is injected into capillary j. A signal detected in capillary i is S Ai . In the capillary j it is desirable to have a signal S Bj to obtain when the dye B emits light, but since the crosstalk S CT Bj←Ai superimposed on the signal, an actually measured signal S act Bj calculated by the following formula (3). SactBj=SBj+SCTBj←Ai

[0046] Here, the crosstalk component in formula (3) is expressed by formula (1). In formula (1), the constant c is obtained by formula (2). In addition, S Ai than the signal detected in capillary i. Therefore, the true signal S Bjto be obtained when there is no crosstalk can be obtained by the following formula (4) using formula (1) and formula (3). SBj=SactBj−SCTBj←Ai=SactBj−cSAi

[0047] The above description is a simplified version of the method disclosed in PTL 1. In practice, the crosstalk may include a component generated by a capillary other than capillary i and capillary j and a dye other than dye A and dye B. In the above description, the fluorescence generated by dye A and dye B is referred to as a signal. In fact, the light image sensor 104 outputs a light intensity in each wavelength band as a signal for each capillary.

[0048] Based on the above, the method disclosed in PTL 1 is arranged. For example, assume that fluorescent dyes of L colors are used for measurement, there are N capillaries for measurement, and the light image sensor 104 measures M wavelength bands. Regarding the wavelength band, for example, when M = 20 and a detection wavelength range is 500 nm to 700 nm, the light image sensor 104 outputs 20 outputs for each capillary, that is, a signal of fluorescence with a wavelength of 500 nm to 510 nm, a signal of fluorescence with a wavelength of 510 nm to 520 nm, ..., and a signal of fluorescence with a wavelength of 690 nm to 700 nm. Here, a signal (or crosstalk) generated for a wavelength interval k of the capillary i when an I-th dye is introduced into the capillary j is represented by S ik←ji designated.

[0049] Here a matrix with S ik←jldescribed as an element as C. S ik←jl has four indices and is arranged two-dimensionally such that a ζ, η-element Cζη of the matrix C satisfies ζ = Mi + k and η = Mj + l. A specific value of S ik←jl can be obtained as an output from the light image sensor 104 by sequentially supplying dyes to be used only to the capillary j, one type at a time. The value of S ik←jl can be normalized by a signal or the like if a dye used has a reference concentration. The matrix C is a matrix in which spectra of the dyes to be used are arranged at positions corresponding to the respective capillaries in the matrix. Here, a generalized inverse matrix C is calculated before measuring a sample to be measured. - of the matrix C from the following formula (6). C−=[CtC]−1Ct

[0050] Next, a situation is assumed in which the sample to be analyzed is analyzed. At this time, the signal of the wavelength interval k of the capillary i is calculated with S ik Here, a signal vector s is generated by arranging S ik. In particular, S ik arranged so that a ζ-component s ζ of the signal vector s ζ = Mi + k. At this time, a vector d with a ratio of each dye as an element can be obtained by the following formula (7). d=C−s

[0051] Formula (7) is obtained by extending the description regarding the removal of a single crosstalk component described above to a case where spatial crosstalk between a plurality of capillaries and spectral crosstalk between a plurality of dyes exist. The above is the crosstalk reduction method in the related art disclosed in PTL 1. In analysis by capillary electrophoresis, since the vector s is output from the light image sensor at each time point, a signal waveform of each dye in each capillary is obtained by performing a calculation of Formula (7) at each time point.

[0052] When applying the crosstalk reduction method described above, it is necessary that the matrix C does not change between a time when the matrix C is obtained and a time when the signal is actually measured. PTL 1 discloses that the above is satisfied when the same capillary array is used to detect the crosstalk matrix C and to measure the actual sample. However, in capillary electrophoresis, the capillary array is a consumable and must be replaced with a new array after a certain number of uses. PTL 1 does not describe whether the above prerequisite condition is still satisfied at this time or whether the crosstalk reduction method works. (Simulation)

[0053] When various parameters such as a diameter of each capillary and an installation position and angle of each capillary are completely the same before and after replacing the capillary array, the above condition is satisfied. However, it is practically impossible to satisfy this condition, and the above condition is invalid when the capillary array is replaced. The following is a specific description using a simulation. In the following simulation, an influence on crosstalk is evaluated when an error in a relative position of the capillaries in each capillary array changes due to the replacement of the capillary array. The error in the relative positions of the capillaries means a deviation from ideal positions of the capillaries, which should be arranged at equal intervals on the same plane.The capillary array is manufactured by arranging and fixing the capillaries on a substrate or the like, and a distance between the capillaries and a distance to each capillary from the substrate are not completely the same for each capillary array due to individual differences between substrates or a variation in a fixing process.

[0054] Fig. Figure 2A shows a simulation model. To simplify the description, no wavelength separation is performed in this simulation and only spatial crosstalk is considered. This model includes a capillary array 201, two lenses 202, and a light image sensor 203. The capillary array 201 is set to have an inner diameter of 50 µm and an outer diameter of 343 µm, and the capillaries are arranged at a pitch of 370 µm. A light-emitting region with a length of 44 µm is set within each capillary. An image of the capillary array 201 is generated by the two lenses 202 on the light image sensor 203. The number of capillaries in the capillary array 201 is eight.

[0055] Fig. Figure 2B shows a simulation flow. When crosstalk reduction processing is performed (S200: Yes), the matrix C described above is first -calculated. A capillary arrangement to achieve the matrix C -is assumed, and a relative position error is adjusted in the capillary array (S201). After a position of each capillary is adjusted, a fluorescence image on the light image sensor 203 is calculated by a ray tracing method (S202). In this step, ray tracing is performed by generating fluorescence from only one of the capillaries 1 to 8. This step is repeated eight times by changing a light-emitting capillary, and an output from the light image sensor 203 when each of the capillaries 1 to 8 emits light is calculated. Next, a point spread function is convolved with a calculation result of the ray tracing (S203). The point spread function is actually measured by a spectrometer including a lens, a grating, and a light image sensor, and represents a "blur" of an optical system with respect to a capillary array direction (X direction in Fig. 2A). When ray tracing using the model in Fig. 2A, the lens 202 is treated as an ideal thin lens, and a deviation from an ideal situation in an actual optical system is expressed by the point spread function. Next, with respect to the output from the light image sensor 203 obtained as a calculation result, signals in regions corresponding to the respective capillaries are integrated to obtain crosstalk values. The generalized inverse matrix C - is calculated for the matrix C generated by arranging the crosstalk values ​​(see S204). Since this model does not perform wavelength separation, the matrix C is a square matrix in which spatial crosstalk between the capillaries is arranged two-dimensionally.

[0056] Next, a capillary array actually used for sample analysis is assumed, and the crosstalk is calculated. If crosstalk reduction processing is not performed (S200: No), the calculation starts from this step. Adjustment of the relative position error for the capillary array (S205), calculation by ray tracing (S206), and convolution of the point spread function (S207) are the same as those described in S201, S202, and S203. After that, the signal vector s is calculated based on an obtained output from the light image sensor 104 (S208). If crosstalk reduction processing is performed (S209: Yes), crosstalk reduction processing is performed by Formula (7) (S210). If crosstalk reduction processing is not performed (S209: No), this step (S210) is skipped. After the calculation, a result is output (S211).The vector d is output when crosstalk reduction processing is performed, and the vector s is output when processing is not performed. When there are a plurality of types of dyes and a dye concentration ratio is calculated based on a spectrum, the vector d and the vector s are different types of information (a dye ratio for each capillary and a light intensity of each wavelength interval), but when light separation in a wavelength direction is not considered, the two become the same type of information (a light intensity of each capillary directly represents a dye concentration ratio), and thus an effect of the presence or absence of crosstalk processing can be evaluated by comparing the two. (Result of crosstalk reduction processing without relative position error)

[0057] Fig. 3A is a diagram showing the crosstalk obtained by the simulation method described above in a case where the crosstalk reduction processing is not performed, and Fig. Figure 3B is a diagram showing the crosstalk in a case where crosstalk reduction processing is performed. In Fig. 3A and Fig. 3B it is assumed that there is no relative position error in the capillary arrangement and the generation of the matrix C - and sample analysis are carried out in the same capillary arrangement. Fig. In Figure 3A, a horizontal axis represents a light-emitting capillary. Each bar with different hatching indicates a value of crosstalk detected in each of the regions (labeled channels 1 to 8) for detecting capillaries 1 to 8.

[0058] According to Fig. 3A shows that in the current model, crosstalk in a channel is maximized for detecting a neighboring capillary, resulting in a crosstalk of approximately more than 1%. The crosstalk is mainly caused by light reflection on a capillary surface and fluorescence leakage to the neighboring channel due to blur (represented by a point spread function) of an imaging optical system. In addition to the above, it is conceivable that an actual cause for the occurrence of spatial crosstalk may be multiple reflections between elements constituting a spectroscopic system, a lattice anomaly, scattering by dust, or the like, but this model only reflects the above two causes.

[0059] According to Fig. 3B, with the same capillary arrangement, it can be seen that the crosstalk is reduced by the crosstalk reduction processing in the related art. Crosstalk of approximately more than 1% is reduced to 0.01% or less by the crosstalk reduction processing. Generally, the dynamic range of the light image sensor is about 3 to 4 orders of magnitude, and the crosstalk of 0.01% or less means that the crosstalk is equal to or less than a lower detection limit. This time, the parameters used to generate the matrix C are - and models used to calculate the crosstalk are completely the same, and one reason why the crosstalk value in Fig. 3B does not reach zero, is due to a calculation error in the ray tracing process. (Result of crosstalk reduction processing with relative position error)

[0060] Fig. 4A and Fig. 4B are diagrams showing the crosstalk when the crosstalk reduction processing is not performed and the crosstalk when the crosstalk reduction processing is performed, similar to Fig. 3A and Fig. 3B. However, in this calculation, each capillary is given an error in an XY direction in Fig. 2A based on a Gaussian distribution with a standard deviation of 10 µm. Assuming that different capillary arrangements are used to generate the matrix C - and used to analyze the sample, the values ​​of the relative position errors of the capillaries set in the capillary arrays are different when the matrix C -generated and when the crosstalk is calculated. Considering that the outer diameter of the capillary is 343 μm, it can be said that the above relative position error is relatively small. In this model, the outer diameter of the capillary is completely uniform, but the outer diameter of the capillary generally has a tolerance of about ± 10 μm. Assuming that a change in the diameter of the capillary causes a shift in a center position depending on a fixing method, it can be seen that the above relative position error has a value that can actually occur.

[0061] According to Fig. 4A, it can be seen that even when a placement error is introduced, there is a crosstalk of about more than 1%, as in the case where the placement error is zero. Meanwhile, according to Fig. 4B shows that in the presence of an array error, a maximum of approximately 0.2% of crosstalk remains even when crosstalk reduction processing is performed in the related art. In addition, it can be seen that the crosstalk in some channels has a negative value, and the crosstalk is excessively corrected. The calculation is repeated nine times with different array errors, and an average of the maximum values ​​of the residual crosstalk has a value of 0.28%. This shows that, since the crosstalk value varies due to the array error, an effect of the crosstalk reduction processing in the related art cannot be correctly obtained when arrays with different array errors are used to generate the matrix C. - and used to analyze the sample.

[0062] This time, the influence of array error on crosstalk reduction processing is demonstrated using a model in which one cause of crosstalk occurrence is limited. In practice, as described above, there are causes of crosstalk occurrence that are not incorporated into the model. In addition, when replacing capillaries, there are factors that increase a change in crosstalk in addition to array errors, such as a change in a position of the entire capillary array, a change in an inclination of the capillary array, a variation in the inner and outer diameters of capillaries, and dust on capillaries. As the variation of crosstalk for each capillary array increases, it becomes more difficult to achieve the effect of crosstalk reduction processing in the related art.

[0063] As described above, the inventors have newly found that the crosstalk reduction processing method in the related art involves regenerating the matrix C - required when the capillary array is replaced. Crosstalk information required to generate the matrix C - are measured by injecting a sample labeled with each dye, one type at a time, into each capillary and performing electrophoresis, as disclosed, for example, in PTL 1. Therefore, if fluorescent dyes of L colors are used and there are N capillaries to be measured, it is necessary to perform electrophoresis L × N times. Since a single electrophoresis run takes about several tens of minutes, a long time is required to obtain the matrix C - PTL 1 also discloses a method for reducing the effort and time for generating the matrix C -. However, to use the disclosed method, the use of a special sample or operation of a special device is required, which is not performed in normal analysis.

[0064] The invention eliminates the need to calculate the matrix C - in connection with the replacement of the capillary arrangement, thus solving the problems mentioned above. <Erste Ausführungsform>

[0065] Fig. 5 is a configuration diagram of a light detection device 500 according to a first embodiment. The light detection device 500 includes a capillary array 501, a light guide array 510 (spatial filter), a spectrometer 505, and a computer 550. The capillary array 501 includes a plurality of capillaries 501a, and a plurality of light emitters emit light inside each capillary 501a. The capillary array 501 is replaceable. A light image sensor 504 constituting the spectrometer 505 includes a plurality of detection channels (see Fig. 1B) for respectively detecting light emitted by the plurality of light emitters in a plurality of wavelength bands. The light guide assembly 510 is a spatial filter that fixes an incident position of light emitted by the plurality of light emitters on the spectrometer 505. The computer 550 processes a signal output from the light image sensor 504.

[0066] The light detection device 500 differs from the light detection device 100 in that it includes the optical fiber array 510. The optical fiber array 510 is a light guide array 510 comprising a plurality of optical fibers 510a, each provided corresponding to one of the light emission points of the plurality of capillaries 501a. The optical fiber array 510 collects fluorescence emitted from each capillary 501a in the capillary array 501 and guides the fluorescence to the spectrometer 505, which includes a lens 502, a grating 503, and the light image sensor 504.The light detection device 500 is assumed to be a detection device of a capillary electrophoresis apparatus, and actually works integrally with a temperature control device, a high-voltage application device, an automatic sample stage, a computer for signal processing, and the like, and performs analysis by capillary electrophoresis of a sample. However, descriptions of parts other than the light detection device 500 are omitted.

[0067] The crosstalk reduction processing of the invention can also be applied when the spectrometer 505 has a different shape. Specifically, a light wavelength separation process can be performed by a prism instead of a grating, or by a plurality of dichroic mirrors.

[0068] A crosstalk reduction processing method according to the first embodiment is the same as the above formula (7) and the related description. However, as processing conditions, it is required that a position of fluorescence introduction into the spectrometer 505 is fixed by the light guide assembly 510, and the crosstalk occurring on a capillary side relative to the light guide assembly 510 is sufficiently reduced by another method. Fixing the position of fluorescence introduction into the spectrometer 505 can be implemented by a pinhole assembly, a slit assembly, or the like instead of the light guide assembly 510.

[0069] The light detection device 500 includes the computer 550. The computer 550 includes a processor 551, a main storage unit 552, an auxiliary storage unit 553, and an input and output interface 554 (hereinafter, an interface is abbreviated as I / F). The processor 551 is a central processing unit that controls an operation of each unit of the computer 550. The processor 551 is, for example, a central processing unit (CPU), a graphics processing unit (GPU), a digital signal processor (DSP), or an application-specific integrated circuit (ASIC). The processor 551 loads a program stored in the auxiliary storage unit 553 into a work area of ​​the main storage unit 552 in an executable manner. The main storage unit 552 stores a program to be executed by the processor 551, data to be processed by the processor, and the like.The main storage unit 552 is a flash memory, a random access memory (RAM), a read-only memory (ROM), or the like. The auxiliary storage unit 553 stores various programs such as an operating system (OS) and various types of 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 contains crosstalk information (for example, the matrix C described above). - ) to reduce crosstalk between a plurality of sensing channels. The input and output I / F 554 is communicatively connected to the light image sensor 504 and a display unit 560.

[0070] The computer 550 reduces the crosstalk between the detection channels of the light image sensor 504 and derives a concentration ratio or a signal amount ratio of each of a plurality of types of light emitters for each of the plurality of capillaries by using the crosstalk information (for example, the matrix C described above). - ) performs a calculation on an output of the light image sensor 504 corresponding to a plurality of wavelength bands of each of the plurality of light emission points at which the plurality of light emitters emit light. Even if the capillary array 501 is replaced with another capillary array, the computer 550 performs the calculation described above using the same crosstalk information.

[0071] The crosstalk information is the generalized inverse matrix C - or information corresponding to the generalized inverse matrix C -correspond to the matrix C, which contains information about the crosstalk between the plurality of detection channels. The information corresponding to the generalized inverse matrix C - Examples of information that are not in a matrix format and are information for reducing crosstalk between the plurality of acquisition channels. The calculation performed by computer 550 to derive the concentration ratio or the signal quantity ratio is a calculation to derive C - s by applying the matrix C - from the left to the vector s of the output of the light image sensor 504 (see the above formula (7)) or an equivalent calculation.

[0072] The computer 550 sequentially supplies the plurality of light emitters to each of the plurality of capillaries 501a, records the output of the light image sensor 504 in a state where a single light emitter among the plurality of light emitters in a single capillary 501a among the plurality of capillaries 501a emits light, arranges the outputs of the light image sensor 504 to generate the matrix C, and calculates and stores the generalized inverse matrix C - the matrix C by [C t C] -1 C t (see formula (6) above) or an equivalent calculation.

[0073] The crosstalk occurring on the capillary side relative to the optical fiber assembly 510 occurs, for example, in a path in which fluorescence generated by a particular capillary directly enters an optical fiber for detecting another capillary, or in a path in which fluorescence is reflected at a surface of another capillary and enters the optical fiber for detecting the capillary, as in Fig. 6. The reduction of crosstalk occurring in such a path is implemented, for example, by setting a sufficiently large capillary spacing in the capillary array 501. Alternatively, a method is known in which a pinhole slightly smaller than a core diameter of the optical fiber 510a is provided at one end of the optical fiber array 510 on the capillary array 501 side to limit an angle at which fluorescence is collected into the optical fiber 510a and reduce crosstalk. A method is also known in which a light-shielding wall is provided between the capillaries in the capillary array 501.

[0074] A simulation result of the effects of the crosstalk reduction method in the first embodiment will be described with reference to Fig. 7A to 7C. Fig. Figure 7A is a diagram showing a model used for the simulation. The model in Fig. 7A comprises a capillary array 701, two lenses 702 and a light image sensor 703, similar to the model in Fig. 2A. In Fig. 7A, a light guide arrangement 710 is modeled in Fig. 2A. Optical fibers with a core diameter of 200 µm and an NA of 0.5 are set as the optical fiber array 710, and eight optical fibers are arranged at the same pitches as the capillaries in the capillary array 701. In addition, a light-shielding wall 720 is set between the capillaries as a unit for removing crosstalk that occurs in front of the optical fiber array 710. A simulation flow is the same as that in Fig. 2B shown.

[0075] Fig. 7B and Fig. 7C are graphs showing a result of the crosstalk simulation using the model in Fig. Show 7A. Fig. 7B is a graph showing the crosstalk when the crosstalk reduction processing is not performed, and Fig. Figure 7C is a graph showing the crosstalk when crosstalk reduction processing is performed. In this simulation, as in the case of Fig. 4, assuming that different capillary arrangements are used to generate the matrix C - and used to analyze the sample, values ​​of the relative position errors of the capillaries set in the capillary arrays 701 are different when the matrix C - generated and when the crosstalk is calculated.

[0076] As from Fig. As can be seen in Figure 7B, the model in Fig. 7A shows a crosstalk of about 0.5%. On the other hand, when performing the crosstalk reduction processing of formula (7), it can be seen that the crosstalk is reduced to 0.01% or less, as shown in Fig. 7C. The calculation is repeated nine times with different relative position errors, and an average of the maximum values ​​of the residual crosstalk has a value of 0.006%.

[0077] From a comparison between the result in Fig. 4B and the result in Fig. 7C, it can be seen that the crosstalk reduction processing works correctly even when using the capillary arrays with different capillary position deviation values ​​using the method in the first embodiment. Such an effect is not due to the installation of the light-shielding wall 720. To demonstrate this, the light-shielding wall 720 is Fig. 2A and performed the same simulation. The calculation is repeated nine times with different relative position errors, and an average of the maximum values ​​of the residual crosstalk has a value of 0.11%, which is 10 times or more the result in Fig. 7C is.

[0078] As described above, according to the crosstalk reduction method in the first embodiment, the effect of the crosstalk reduction method can be achieved without changing the matrix C - to be regenerated when the capillary array 501 is replaced. This is an effect achieved by fixing the incident position of fluorescence to the spectrometer 505 by the light guide array 510. Even if a position of each capillary in the capillary array 501 changes, the incident position of the fluorescence to the spectrometer 505 is fixed by the light guide array 510. The crosstalk occurring in the spectrometer 505 (e.g., due to multiple reflections or blurring of the spectrum image) is not affected by the position of the capillary because the fluorescence incident point is fixed. Therefore, the crosstalk value is not changed by replacing the capillary array, and regenerating the matrix C -is not required.

[0079] However, as described above, the method in the invention requires that the crosstalk occurring on the capillary side relative to the light guide array 510 be sufficiently small. Since the method in the first embodiment reduces an invariant component in the crosstalk (basically, the crosstalk of the spectrometer 505), a component that varies due to the replacement of the capillary array 501 cannot be removed. For example, if an amount of crosstalk caused by surface reflection changes due to a change in an arrangement error of the capillary array, crosstalk corresponding to the change remains even after the crosstalk reduction processing. To demonstrate this, the light-shielding wall 720 is removed from the model in Fig. 7A is removed and the same simulation is performed. The calculation is repeated nine times with different relative position errors, and an average of the maximum values ​​of the residual crosstalk has a value of 0.12%. Therefore, an optical system to which the method in the invention is applied must be designed such that a variation in the crosstalk occurring on the capillary side relative to the light guide assembly 510 is sufficiently smaller than an allowable value.

[0080] In the light detection device 500 and the light detection method in the first embodiment, the matrix C - fundamentally not changed, so that, for example, the matrix C -after completion of device production or after installation at the site of use. Matrix generation can be performed by a method similar to the method disclosed in PTL 1. For example, a sample labeled with a specific dye is injected into a specific capillary, and electrophoresis is performed to record a signal from the light image sensor 504. This is repeated for all dyes in all capillaries. Then, the measurement results are arranged to form the matrix C. Specifically, outputs of wavelength intervals of the capillaries are arranged when a signal of a target dye is maximized in a target capillary. After that, the generalized inverse matrix C - the matrix C is generated.

[0081] At the time of sample analysis, the processing of formula (7) is applied to the output of the light image sensor 504 at each time point to obtain information about a ratio of each dye at each time point. An electrophoretic waveform of an analysis target labeled with each dye is obtained by arranging ratios of dyes in a time series.

[0082] The matrix C - is basically not changed, but the matrix C - may be re-acquired for any reason. For example, a position of a component of the spectrometer 505 may shift due to a device relocation or long-term use. In such a case, since a crosstalk ratio within the spectrometer may also change, the matrix C - be newly created.

[0083] The matrix C may contain partial information to reduce the effort involved in generating the matrix C. That is, the matrix C may be a combination of elements of a plurality of matrices Cn, each of which contains only crosstalk information between a portion of the light emitter holders or dyes. From the simulation result in Fig. For example, in Figures 7A to 7C, it can be seen that, in this case, spatial crosstalk is strongly present in a channel measuring a neighboring capillary. In such a case, it is also possible to generate matrix C while ignoring crosstalk from capillaries two or more capillary positions away.

[0084] A configuration in Fig. 7A is described as an example. It is assumed that each matrix Cn is a matrix generated by arranging signals acquired from a channel for measuring each capillary when a dye is injected into a capillary n. When the matrix C is generated, the dye is simultaneously supplied to capillaries in each set of a set of a capillary 1, a capillary 4, and a capillary 7, a set of a capillary 2, a capillary 5, and a capillary 8, and a set of a capillary 3 and a capillary 6. Then, the matrix Cn is generated using signals from channels corresponding to a capillary n+1, the capillary n, and a capillary n-1, and signals from channels corresponding to the other capillaries are zero.That is, when the dye is simultaneously injected into capillary 1, capillary 4, and capillary 7, C1 is generated using values ​​from channel 1 and channel 2, C4 is generated using values ​​from channel 3, channel 4, and channel 5, and C7 is generated using values ​​from channel 6, channel 7, and channel 8. After all matrices Cn (in the above case, C1 to C8) are generated, the matrices Cn are arranged sequentially to obtain matrix C. By generating matrix C using this method, the number of electrophoreses required to obtain information can be reduced.

[0085] Although spatial crosstalk between a capillary pair with a small spatial crosstalk value is ignored in the above description, the same method can also be applied in a case where the emission spectra of a particular dye pair are significantly different and there is almost no overlap. That is, in the case of a dye pair whose fluorescence spectra do not overlap, the matrix C can be generated using data obtained by simultaneously measuring the dye pair.

[0086] The matrix C does not necessarily have to be generated by injecting the dye into the light emitter holder, i.e., the capillary. The crosstalk information (matrix C -) can be detected by connecting a calibration light source 801 to the spatial filter. The calibration light source 801 sequentially supplies calibration light to each emitted light collection position of the spatial filter. The calibration light is light having the same spectrum as an emission spectrum of the plurality of light emitters. Fig. 8A shows a case where matrix C is generated using calibration light source 801. Calibration light source 801 sequentially introduces light having the same spectrum as an emission spectrum of each dye into the optical fiber in optical fiber array 510. Matrix C can be generated from the output of spectrometer 505 in the same manner as the method described above.

[0087] Fig. 8B shows a structural example of the calibration light source 801. The calibration light source 801 includes an excitation light source 802 therein. The excitation light source 802 irradiates a flow path 803 with fluorescence excitation light. Dyes are sequentially injected into the flow path 803 by a dye injection mechanism 804. The fluorescence generated in the flow path 803 is collimated by a lens 805, and the excitation light is removed by a wavelength filter 806. The collimated fluorescence is introduced into a fiber in an output fiber array 809 through mirrors 807 and a lens 808. The angles of a portion of the mirrors 807 are changed to allow a fiber to be changed into which the fluorescence is introduced.

[0088] The crosstalk reduction method in the first embodiment is based on the premise that a linear relationship exists between the crosstalk and a signal that is a source of the crosstalk. Therefore, if this relationship breaks down due to detector saturation, the method in the first embodiment will not work. Fig. Figure 9 is a schematic graph showing the influence of detector saturation. In the graph in Fig. 9, a horizontal axis represents the concentration of a dye to be measured, and a vertical axis represents the amount of crosstalk. In a spectroscopic system with crosstalk, the dye concentration and crosstalk are proportional. In an ideal situation where no crosstalk is present, the crosstalk is always zero, regardless of the dye concentration.

[0089] According to the crosstalk reduction method in the first embodiment, ideally, the crosstalk is completely zero. Therefore, in the spectroscopic system in which crosstalk exists and the crosstalk reduction processing of the first embodiment is performed, the crosstalk is zero up to a certain dye concentration regardless of the concentration in an ideal state (even under realistic conditions where a slight crosstalk remains even after reduction processing, the crosstalk after reduction has a value smaller than an original value). However, when a detector in a capillary that measures the dye to be measured is saturated, an output signal is constant even if the dye concentration continues to increase. Meanwhile, since the crosstalk is proportional to a fluorescence intensity, it continues to increase proportionally to the concentration.As a result, an actual amount of crosstalk exceeds an amount of crosstalk estimated based on a detector output. In this case, the crosstalk cannot be completely removed by the crosstalk reduction method in the first embodiment, and the crosstalk value is not zero. Therefore, the crosstalk reduction method in the first embodiment must be used in a range where the detector is not saturated. When a certain channel of the detector is saturated, it is useful to warn a user that there is a possibility that other channels where the detector is not saturated may not exhibit the original performance.

[0090] In particular, when the output of the light image sensor 504 is saturated, the computer 550 may display a warning on the display unit 560. <signalverarbeitungsverfahren>

[0091] Here, a signal processing method using the light detecting device 500 of the first embodiment will be described.

[0092] The signal processing method in the present embodiment includes preparing the light detecting device 500, measuring light emitted from a plurality of light emitters by the light image sensor 504, performing, using the crosstalk information (matrix C) stored in the computer 550 - ) to reduce crosstalk between a plurality of detection channels, performing a calculation on the output of the light image sensor 504 corresponding to a plurality of wavelength bands of a plurality of light emission points at which the plurality of light emitters emit light to reduce crosstalk between the detection channels of the light image sensor 504 and deriving a concentration ratio or a signal amount ratio of each of the plurality of types of light emitters for each of the plurality of light emitter holders (capillaries 501a), and performing the calculation with the same crosstalk information even when the capillary array 501 is replaced.

[0093] The crosstalk information (matrix C - ) are the generalized inverse matrix C - the matrix C, which contains information about the crosstalk or equivalent information. The calculation for deriving the concentration ratio or the signal quantity ratio is a calculation for deriving C - s by applying the matrix C - from the left to the vector s of the output of the light image sensor 504 (see the above formula (7)) or an equivalent calculation. <Erste Ausführungsform: Zusammenfassung>

[0094] The light detection device 500 according to the first embodiment includes the capillary array 501, the light guide array 510, the lens 502, the grating 503, and the light image sensor 504. Additionally, the computer 550 is provided, which processes the output of the light image sensor 504. The computer 550 stores therein the generalized inverse matrix C - the matrix C generated based on the output of the light image sensor 504 when the sample labeled with the dye to be used is sequentially injected into each of the capillaries in the capillary array 101 and measured in the light detection device 500. When a measurement target is analyzed, the computer 550 obtains each dye ratio by calculating the generalized inverse matrix C - to the output of the light sensor 504 at any time. The same value is expressed as a generalized inverse matrix C - used even if the capillary assembly 501 is replaced. <Zweite Ausführungsform>

[0095] In the first embodiment, the concentration ratio of each dye is determined by collectively processing the output of the light image sensor 104 with the generalized inverse matrix C - obtained. In a second embodiment, spectral crosstalk caused by a dye, spectral crosstalk caused by a spectrometer, and spatial crosstalk are processed separately. In a crosstalk reduction method according to the second embodiment, processing for reducing crosstalk occurring inside a spectrometer is performed using a matrix acquired using a calibration light source, and spectral crosstalk caused by the overlap of dye fluorescence spectra is reduced using a matrix acquired by measuring a fluorescence spectrum based on a dye introduced into a capillary. Therefore, the crosstalk reduction processing has a different form from formula (7). With this processing format, it is possible to easily change or add a dye type.

[0096] First, the crosstalk occurring in the spectrometer 505 is sorted. The spectrometer 505 is provided with N optical fibers for light introduction, and the light image sensor 504 measures M wavelength bands for each optical fiber. The wavelength band means that, for example, when M = 20 and a detection wavelength range is 500 nm to 700 nm, the light image sensor 504 outputs 20 outputs for each fiber, i.e., a fluorescence signal with a wavelength of 500 nm to 510 nm, a fluorescence signal with a wavelength of 510 nm to 520 nm, ..., and a fluorescence signal with a wavelength of 690 nm to 700 nm. Here, an output in a wavelength band m for a fiber n of the light image sensor 504 is represented by S mn A one-dimensional arrangement of S mn is defined as the output signal vector s of the light image sensor 104. S mn has two indices and is arranged one-dimensionally so that S mn a Mn + m component S Mn+m of s becomes.

[0097] Meanwhile, light incident on the fiber n of the spectrometer 505 is considered. The power of the light in a wavelength range corresponding to the wavelength band m of the light is denoted by P mn A vector p of the incident power is calculated by arranging the power P mn created. As before, P mn arranged one-dimensionally to form a Mn + m component p Mn+m of p.

[0098] The spectrometer 505 outputs a signal that is linear with the power of the incident light. Therefore, a relationship between p and s can be expressed by the following formula (8). s=Gp

[0099] A matrix G is a matrix representing the crosstalk occurring in the spectrometer 505. The matrix G is detected by sequentially introducing monochromatic light corresponding to a wavelength detection interval of the optical sensor (for example, the light image sensor 504) for each emitted light collection position of the spatial filter (for example, the light guide array 510). In an ideal spectrometer without crosstalk, the matrix G is an identity matrix (s and p are normalized such that an output signal is 1 when the light power is 1). When crosstalk is present, a triangular region located in the matrix G in Fig. 10, an area indicating a value of crosstalk. If the sensitivity of the spectrometer is different for each emitted light collection position, a diagonal element in the matrix G may have a value other than 1. For example, if the spectrometer 505 has a configuration as shown in Fig. As shown in Figure 5, the optical loss in the spectrometer may increase at an emitted light collection position farther from a central axis of the lens 502. In this case, the value of the diagonal element corresponding to the collection position is less than 1.

[0100] Next, a relationship between a dye in a capillary and the power of the light incident on each fiber is established. Now, assume that L types of dyes are used for analysis. A ratio of an lth dye in an nth capillary among N capillaries is expressed as D ln A dye ratio vector d is defined by a one-dimensional arrangement of D ln received. D ln has two indices and D ln is arranged one-dimensionally to form a Ln + I element d Ln+l to be of d.

[0101] Here, it is assumed that a signal generated by the nth capillary is measured by an nth optical fiber. The light power P measured in the nth optical fiber mn is a sum of the emission spectra of L types of fluorescent dyes in the n-th capillary, weighted by a ratio D ln . Therefore, a relationship between the incident power vector p and the dye ratio vector d is expressed by the following formula (9). p=Fd

[0102] Here, a matrix F is a matrix in which fluorescence spectra of dyes are arranged.

[0103] The following formula (10) is obtained from formula (8) and formula (9). s=GFd

[0104] Therefore, assuming that an inverse matrix of the matrix GG -1 and is a generalized inverse matrix of the matrix FF - is, the following formula (11) is obtained. The generalized inverse matrix F - is replaced by [F t F] -1 F t or receive an equivalent calculation. d=F−G−1s

[0105] This means that if G -1 and F - the dye ratio d is obtained from the output s of the light image sensor. The calculation of G -1 means processing to reduce the crosstalk occurring in the spectrometer, and the calculation of F - means processing to calculate the dye ratio (reducing spectral crosstalk) taking into account the overlap of the dye fluorescence spectra.

[0106] Fig. Figure 10 shows shapes of matrix G and matrix F. Matrix G is a square matrix with a size of M × N, and 1 is arranged along the diagonal elements. Regions indicated by triangles above and below the diagonal elements represent the crosstalk. In an ideal spectrometer without crosstalk, all elements in this part are 0. Matrix F is a matrix with M × N rows and L × N columns. Matrix F is a matrix in which N matrices F1, each with M rows and L columns, are arranged in a diagonal part, and the other regions are 0. In matrix F1, spectra of L dyes are arranged from a first column to an Lth column (rectangles in Fig. 10 represent the spectra).

[0107] In the crosstalk reduction method in the second embodiment, the calculation is divided into two stages, and the matrix G and the matrix F are acquired separately. The matrix G is obtained using a calibration light source 1101 (see Fig. 11). The calibration light source 1101 is connected to the light guide assembly 510 of the spectrometer 505 in the same manner as the calibration light source 801. The calibration light source 1101 sequentially introduces light with wavelengths corresponding to M wavelength bands into each of N fibers. That is, the calibration light source 1101 first introduces light with a wavelength corresponding to wavelength band 1 into fiber 1. Next, light with a wavelength corresponding to wavelength band 2, light with a wavelength corresponding to wavelength band 3, ..., and light with a wavelength corresponding to wavelength band M are introduced. Next, the same operation is performed on fiber 2. Next, the same operation is performed on fiber 3, fiber 4, ..., and fiber N.

[0108] The matrix G is generated as follows. When the calibration light source 1101 introduces light of wavelength interval m into the fiber n, an output value for a fiber k in the optical fiber array and for a wavelength interval I is denoted by A kl←mn At this point, assuming that ζ = Mk + l and η = Mn + m, an η, ζ-element in the matrix GG ζη = A kl←mn / A mn←mn .

[0109] Fig. 11 shows a structural example of the calibration light source 1101 that performs the above-described operation. The calibration light source 1101 contains a white light source 1102 therein. The white light source 1102 is, for example, a white LED or a halogen lamp. Light from the white light source 1102 is separated for each wavelength by a lens 1103 and a grating 1104. A slit 1105 is placed at an imaging point of the separated light. The slit 1105 extracts only a component of a specific wavelength in the light from the white light source 1102. The grating 1104 can change an angle thereof, and a wavelength of the light passing through the slit 1105 is controlled by adjusting the angle. The light passing through the slit 1105 is introduced into a specific fiber in an output fiber array 1108 through a lens 1106 and mirror 1107.An angle of one of the mirrors 1107 can be changed, and the optical fiber in the output fiber array 1108 into which light is to be introduced is controlled by adjusting the angle.

[0110] The matrix F can be measured by simultaneously injecting a dye-labeled sample into all capillaries. In the matrix detection method in the first embodiment, it is necessary to perform signal measurement for N capillaries by injecting a dye into only one capillary, whereas in the matrix detection method in the second embodiment, the measurement can be performed simultaneously in all capillaries, thus reducing the time required for measurement to 1 / N.

[0111] Since the matrix F is a matrix obtained by repeatedly arranging the matrix F1 in which spectra of the respective dyes are arranged for the number of capillaries, the matrix F1 can be measured by measuring a dye spectrum with a specific capillary and repeatedly arranging it for the number of capillaries to obtain the matrix F. In the crosstalk reduction method in the related art and the crosstalk in the first embodiment, information about spectral crosstalk caused by a dye, information about crosstalk caused by the light detection device, and information about the detection efficiency of each wavelength are mixed in a single processing matrix. Therefore, the information must be acquired by supplying each dye for each capillary.However, in the crosstalk reduction method in the second embodiment, since information about an effect caused by the spectrometer is consolidated in the matrix G, it is not necessary to measure the same sample for each capillary at the time of measuring the matrix F.

[0112] As described above, in the crosstalk reduction method in the second embodiment, it is easy to acquire the processing matrix, particularly the matrix F containing the information about the dye spectrum, compared to the crosstalk reduction method in the related art disclosed in PTL 1 and the crosstalk reduction method in the first embodiment. This is particularly useful when adding a dye used for analysis. When it is desired to use a new dye for which no information is present in a computer that performs the crosstalk reduction processing, in the crosstalk reduction method in the related art disclosed in PTL 1 or the crosstalk reduction method in the first embodiment, it is necessary to sequentially introduce the new dye into all the capillaries to acquire fluorescence spectrum information about the new dye.On the other hand, in the crosstalk reduction method in the second embodiment, the fluorescence spectrum can be measured by introducing the new dye simultaneously into all capillaries or into a specific capillary.

[0113] Alternatively, the matrix F can be provided in a form in which it is stored in advance in a computer. Information about crosstalk that depends on each individual spectrometer is contained in the matrix G. Therefore, it is not necessary to measure the matrix F for each individual light-detecting device. Spectrum information about each dye measured in a particular individual device can be stored in a computer of another individual device, and the matrix F can be generated based on the information. That is, spectrum information about the dye measured by a particular individual light-detecting device can be published on the Internet. The user can download the spectrum information about the dye from the Internet and store the spectrum information in a computer, and the matrix F can be generated based on the stored spectrum information.

[0114] Fig. Figure 12 shows an example of the operation of the instrument when the matrix F is acquired. First, the user sets a dye or a set of dyes for which a spectrum is to be acquired (S1201). Next, the user selects a method for acquiring a fluorescence spectrum (S1202). If a method for measuring a dye spectrum using all capillaries is selected, the instrument first prepares for electrophoresis (S1203). After the preparation is complete, the user sets a sample labeled with a dye to be measured (S1204). After the sample setting is complete, the instrument injects the sample into a capillary and performs electrophoresis (S1205). A spectrum is acquired when the sample migrates electrophoretically to a fluorescence measurement point (S1206). This cycle is repeated for all dyes for which the spectrum is to be acquired (S1207).If the sample is prepared so that all dye spectra can be acquired together, this cycle can be performed once. For example, this is a sample in which the dyes to be measured, A, B, C, etc., are labeled and mixed with DNA with a strand length of 100 bases, 150 bases, 200 bases, etc. When electrophoresis is performed, the dyes A, B, C, etc., emit light sequentially at different times. Such a calibration sample is commercially available and relatively easy to obtain. Because the DNA reaches the fluorescence measurement point in the order of strand length due to electrophoresis, the dyes A, B, and C emit light sequentially in a time series. After all fluorescence spectra have been measured, the fluorescence spectra are arranged to generate the matrix F (S1208).

[0115] If a method for measuring the fluorescence spectrum with a single capillary is selected, the instrument's operation is the same as the method for measuring the fluorescence spectrum with all capillaries (S1209 to S1214). However, the sample is injected into only a single capillary. When the matrix F is generated, the fluorescence spectra acquired from the individual capillaries are repeatedly arrayed.

[0116] When a method for generating the matrix F based on internal information is selected, a fluorescence spectrum (emission spectrum information) of the set dye is read from a memory (S1215). The fluorescence spectrum may be stored in the computer from the beginning, or spectrum information may be provided and downloaded via the Internet or the like. The matrix F is generated based on the read fluorescence spectrum (S1216). The generated matrix F is stored in the computer (S1217). < Modifications>

[0117] The invention is not limited to the embodiments described above and includes various modifications. For example, the embodiments described above have been described in detail in order to facilitate understanding of the invention, and the invention is not necessarily limited to those including all of the configurations described above. A part of a configuration according to a certain embodiment may be replaced with a configuration according to another embodiment, and a configuration according to another embodiment may be added to a configuration according to a certain embodiment. In addition, another configuration may be added to a part of a configuration of each embodiment, and the part of the configuration of each embodiment may be deleted or replaced with another configuration.

[0118] For example, in the first embodiment, an example was described in which the light guide assembly 510 is provided as a spatial filter. Alternatively, the spatial filter may be a filter with a through-hole through which light passes. The shape of the through-hole of the filter may be circular or rectangular. Fig. 13 is a configuration diagram of a light detection device 1300 according to a modification. Unlike the light detection device 500, the light detection device 1300 includes a pinhole array 1310 in which a plurality of pinholes (circular through-holes) are provided in an array to correspond to light emission points of a plurality of light emitter holders (capillaries). The pinhole array 1310 may be a slit array in which slits (rectangular through-holes) are provided in an array.

[0119] In the above embodiment, the light detecting device including the plurality of light emitter holders in which the plurality of types of light emitters emit light is used, and alternatively, one light emitter holder may be used when there are a plurality of types of light emitters, and one type of light emitter may be used when there are a plurality of light emitter holders.

[0120] In the calculation in the above embodiments, various calculations are performed using matrices, and alternatively, an equivalent calculation may be performed using information not in a matrix format as long as an equivalent result is obtained. List of reference symbols 100 light detection device 101 Capillary arrangement 102 lens 103 grids 104 Light sensor 105 Spectrum image 106 Channel 201 Capillary arrangement 202 lens 203 Light sensor 500 light detection device 501 Capillary arrangement 501a Capillary 502 lens 503 grids 504 light sensor 505 spectrometers 510 fiber optic arrangement 510a optical fiber 550 computers 551 processor 552 main storage unit 553 Auxiliary storage unit 554 Input and output interface 560 display unit 701 Capillary arrangement 702 lens 703 light sensor 710 fiber optic arrangement 720 Light-shielding wall 801 Calibration light source 802 Excitation light source 803 Flowway 804 Dye injection mechanism 805 lens 806 wavelength filters 807 mirrors 808 lens 809 Output fiber arrangement 1101 Calibration light source 1102 White light source 1103 lens 1104 grids 1105 Slot 1106 lens 1107 mirrors 1108 Output fiber arrangement 1300 light detection device 1310 pinhole arrangement QUOTES CONTAINED IN THE DESCRIPTION

[0000] This list of documents submitted by the applicant was generated automatically and is included solely for the convenience of the reader. This list is not part of the German patent or utility model application. The DPMA assumes no liability for any errors or omissions. Cited patent literature

[0000] JP 7 282 880 B

[0011] WO 2018 / 151 843 A1

[0011] WO 2023 / 276 078 A1

[0011] Cited non-patent literature

[0000] https: / / assets.thermofisher.com / TFS-Assets / LSG / manuals / MAN0018646_SeqStudioInstSW_UG.pdf

[0012] < / signalverarbeitungsverfahren>

Claims

[1] Light detection device comprising: a plurality of interchangeable light emitter holders in which a plurality of types of light emitters emit light; an optical sensor having a plurality of detection channels for respectively detecting the light emitted from the plurality of types of light emitters in a plurality of wavelength bands; a spatial filter configured to fix an incident position of the light emitted from the plurality of types of light emitters on the optical sensor; and a computer configured to process a signal output from the optical sensor, wherein the computer Crosstalk information for reducing crosstalk between the plurality of detection channels therein, using the crosstalk information, performs a calculation on an output of the optical sensor corresponding to the plurality of wavelength bands for each of a plurality of light emission points at which the plurality of types of light emitters emit light, to reduce crosstalk between the detection channels of the optical sensor and derive a concentration ratio or a signal amount ratio of each of the plurality of types of light emitters for each of the plurality of light emitter holders, and performs the calculation using the crosstalk information even when each light emitter holder is replaced with a different light emitter holder. [2] A light detecting device according to claim 1, wherein the crosstalk information is a generalized inverse matrix C -a matrix C containing information about the crosstalk between the plurality of detection channels or information corresponding to the generalized inverse matrix, and the calculation for deriving the concentration ratio or the signal quantity ratio a calculation applying the matrix C - to a vector s of the output of the optical sensor from the left to C - s, or an equivalent calculation. [3] A light detecting device according to claim 2, wherein the matrix C is a combination of elements of a plurality of matrices Cn each containing only crosstalk information between a part of the light emitter holders or dyes. [4] The light detecting device according to claim 1, wherein the spatial filter is a light guide assembly comprising a plurality of optical fibers each provided corresponding to one of the light emitting points of the plurality of light emitting holders. [5] The light detecting device according to claim 1, wherein the spatial filter comprises a plurality of pinholes or slits each provided corresponding to one of the light emitting points of the plurality of light emitting holders. [6] A light detecting device according to claim 2, wherein the plurality of types of light emitters are sequentially supplied to each of the plurality of light emitter holders, the output of the optical sensor is recorded in a state where a single light emitter among the plurality of types of light emitters in a single holder among the plurality of light emitter holders emits light, outputs of the optical sensor are arranged to generate the matrix C, and the generalized inverse matrix C - the matrix C by (C t C] -1 C t or an equivalent calculation is calculated and stored. [7] The light detecting device according to claim 1, wherein, with respect to the crosstalk information, when a matrix representing crosstalk occurring by the light detecting device is G and a matrix representing spectral crosstalk occurring by the overlap of emission spectra of dyes is F, a concentration ratio or a signal amount ratio of a dye is calculated by a calculation F - G -1 s or an equivalent calculation using an inverse matrix G -1 the matrix G and a generalized inverse matrix F - the matrix F defined by [F t F] -1 F t or an equivalent calculation is obtained. [8] A light detecting device according to claim 7, wherein the matrix G is detected by sequentially introducing monochromatic light corresponding to a wavelength detection interval of the optical sensor for each emitted light collecting position of the spatial filter. [9] A light detecting device according to claim 7, wherein the matrix F is detected by sequentially supplying the plurality of types of light emitters to all or a part of the plurality of light emitter holders. [10] The light detecting device according to claim 7, wherein the matrix F is generated based on emission spectrum information of the plurality of types of light emitters stored in the computer. [11] Light detecting device according to claim 2 or 7, wherein the crosstalk information is captured by connecting a calibration light source to the spatial filter, and the calibration light source successively introduces calibration light to each emitted light collection position of the spatial filter. [12] The light detecting device according to claim 11, wherein the calibration light source introduces light having the same spectrum as an emission spectrum of the plurality of types of light emitters. [13] The light detecting device according to claim 11, wherein the calibration light source introduces monochromatic light corresponding to a wavelength detection interval of the optical sensor. [14] The light detecting device according to claim 1, wherein a warning is displayed when the output of the optical sensor is saturated. [15] Signal processing method comprising: Preparing a light detection device comprising a plurality of interchangeable light emitter holders in which a plurality of types of light emitters emit light, an optical sensor having a plurality of detection channels for respectively detecting the light emitted by the plurality of types of light emitters in a plurality of wavelength bands, a spatial filter configured to fix an incident position of the light emitted by the plurality of types of light emitters on the optical sensor, and a computer configured to process a signal output from the optical sensor; Measuring the light emitted by the plurality of types of light emitters by the optical sensor; Calculating, using crosstalk information stored in the computer to reduce crosstalk between the plurality of detection channels, an output of the optical sensor corresponding to the plurality of wavelength bands for each of a plurality of light emission points at which the plurality of types of light emitters emit light to reduce crosstalk between the detection channels of the optical sensor and deriving a concentration ratio or a signal amount ratio of each of the plurality of types of light emitters for each of the plurality of light emitter holders; and Perform the calculation using the crosstalk information even if each light emitter holder is replaced. [16] A signal processing method according to claim 15, wherein the crosstalk information is a generalized inverse matrix C -a matrix C containing information about the crosstalk or equivalent information, and the calculation to derive the concentration ratio or the signal quantity ratio is a calculation applying the matrix C - to a vector s of the output of the optical sensor from the left to C - s, or an equivalent calculation. [17] A signal processing method according to claim 16, wherein, with respect to the crosstalk information used for the calculation, when a matrix representing the crosstalk occurring in the light detecting device is G and a matrix representing the spectral crosstalk occurring due to the overlap of emission spectra of dyes is F, a concentration ratio or a signal amount ratio of a dye is calculated by a calculation F - G -1 s or an equivalent calculation using an inverse matrix G -1the matrix G and a generalized inverse matrix F - the matrix F defined by [F t F] -1 F t or an equivalent calculation is obtained.

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