Light detection device and signal processing method

Abstract

This light detection device 500 comprises: an exchangeable capillary array 501 in the interior of which a plurality of types of light-emitting body emit light; an optical sensor 504 which has a plural

Description

Photodetection device and signal processing method

[0001] The present invention relates to a photodetector and a signal processing method, and more particularly to a photodetector and a signal processing method that reduce crosstalk that occurs when multiple types of light emitters emit light at multiple light-emitting points.

[0002] There is an optical measurement method in which multiple types of light-emitting substances exist at multiple spatial positions, and a light detection device that separates and detects the wavelengths of light for each spatial position is used to detect the location and concentration ratio of each light-emitting substance.One example of this method is the light detection device of a capillary electrophoresis apparatus that has multiple analytical capillaries and analyzes samples labeled with multiple types of fluorescent dyes.

[0003] In capillary electrophoresis, the sample to be analyzed is injected into a capillary filled with a separation medium, and a voltage is applied across both ends to separate the analytes based on their differences in mobility. The analytes are detected, for example, by fluorescence detection. The analytes are labeled with fluorescent dyes, and the separated analytes are detected by detecting the fluorescence emitted by irradiation with excitation light.

[0004] One example is a technique in which DNA labeled with a fluorescent dye is electrophoresed in a capillary filled with a polymer and separated by chain length. Excitation light is irradiated onto a detection site on the capillary, and the resulting fluorescence is detected. DNA molecules in the sample move through the capillary and pass through the detection site at different times depending on their chain length. As a result, the chain length distribution of DNA molecules in the sample is obtained as a fluorescence intensity waveform. When multiple types of DNA molecules exist in a sample and these need to be analyzed separately, multiple types of fluorescent dyes may be used to label the DNA. A photodetector separates and detects the fluorescent wavelengths using a grating or similar device, and identifies the type of fluorescent dye from the shape of the resulting spectrum.

[0005] Capillary electrophoresis instruments are sometimes equipped with multiple capillaries to improve measurement throughput. In such cases, for example, the separation capillaries are aligned in a row, and excitation light is irradiated onto all capillaries simultaneously. The emitted fluorescence is acquired by an image sensor. The signal intensity is calculated from the fluorescence image of each capillary, and the electrophoretic waveform of the sample is obtained.

[0006] As mentioned above, when multiple types of fluorescent dyes and multiple capillaries are used, signals other than those that should be detected may be detected, resulting in deviations from the true concentration ratio of the measured target. For example, fluorescent dye A may be mistakenly recognized as fluorescent dye B even though it is emitting light in a certain capillary, or the jth capillary may be mistakenly recognized as emitting light even though it is the ith capillary. Here, the signal output due to misidentification is called crosstalk; the former, caused by misidentification of dyes, is called spectral crosstalk, and the latter, caused by misidentification of emitting capillaries, is called spatial crosstalk.

[0007] There are various possible causes of crosstalk. For example, spectral crosstalk can occur due to the width of the emission spectrum of a fluorescent dye. Generally, fluorescent dyes have emission spectra with a wavelength width of about several tens of nanometers. When multiple dyes are used, the emission spectrum of dye A and the emission spectrum of dye B can overlap. If the emission spectra of dye A and dye B overlap, even if dye A is emitting light, the part of dye A's emission spectrum that falls within the detection wavelength band of dye B may be detected as the emission signal of dye B.

[0008] As an example, spatial crosstalk can occur due to surface reflections from adjacent capillaries. Assume that multiple capillaries are arranged in a row within the device, and a detector is positioned facing this capillary array. When a fluorescent dye emits light in the i-th capillary (hereafter referred to as capillary i), the fluorescence is emitted in all directions, and some of it is introduced into the detector. However, some of the light strikes the nearby j-th capillary (hereafter referred to as capillary j), and some of this light is reflected back toward the detector. In such a case, capillary j, which reflected the fluorescence, appears to be emitting fluorescence on the detector, and is detected as the emission signal of capillary j.

[0009] Crosstalk can have various adverse effects on capillary electrophoresis analysis. For example, if spatial crosstalk causes the fluorescence from capillary i to be recognized as the fluorescence from capillary j, a component of the sample being analyzed in capillary i may be mistakenly recognized as being contained in the sample being analyzed in capillary j. Furthermore, spectral crosstalk can cause the component b labeled with fluorescent dye B to be mistakenly recognized as being present in the sample, even though only component a labeled with fluorescent dye A actually exists in the sample.

[0010] Crosstalk can be reduced by adjusting the measurement conditions and instrument configuration. For example, spectral crosstalk can be reduced by widening the interval between the emission wavelengths of the fluorescent dyes used, and spatial crosstalk will not occur if completely independent measurement optical systems are prepared for each capillary. However, because there are upper and lower limits to the wavelengths that a detector can detect, widening the interval between the dye emission spectra will reduce the number of fluorescent dyes that can be used. Furthermore, while spatial crosstalk will not occur if the optical systems measuring each capillary are independent, this requires a light source and detector for each capillary, which is disadvantageous in terms of cost and size of the instrument.

[0011] Meanwhile, a method for reducing crosstalk through data processing is also known (Patent Document 1). In this method, crosstalk when a certain fluorescent dye emits light in a certain capillary is comprehensively acquired in advance, and an inverse matrix of a matrix listing the crosstalk components is generated. Crosstalk is reduced by applying this inverse matrix to the actually acquired signal. This method makes it possible to reduce crosstalk without changing the device configuration or measurement conditions.

[0012] Patent No. 7282880 International Publication No. 2018 / 151843 International Publication No. 2023 / 276078

[0013] SeqStudioTMGenetic Analyzer Instrument and Software USER GUIDEhttps: / / assets.thermofisher.com / TFS-Assets / LSG / manuals / MAN0018646_SeqStudioInstSW_UG.pdf

[0014] The method described in Patent Document 1 assumes that the ratios of spectral and spatial crosstalk between dyes and capillaries remain unchanged between the time the matrix used in the crosstalk reduction process is generated and the time the sample is analyzed. This condition is basically met when the same capillary array is used to generate the matrix used in the crosstalk reduction process and to analyze the sample. However, in capillary electrophoresis, capillary arrays are consumables and must be replaced after a certain number of uses.

[0015] When a capillary array is replaced, the ratios of spectral crosstalk and spatial crosstalk change. In a capillary array, the capillaries are lined up in a row, but each capillary has a placement error of several μm to several dozen μm, so the light reflection pattern on the capillary surface can change for each capillary array. In addition, the position and inclination of the center of the capillary array relative to the detector can also change before and after replacement. These factors change the ratio of spatial crosstalk.

[0016] Because the emission spectrum of the dyes does not change with capillary replacement, the change in spectral crosstalk due to capillary array replacement is expected to be smaller than spatial crosstalk. However, spectral crosstalk can also change with capillary array replacement. For example, if multiple reflections occur within the detector optical system and crosstalk occurs along a path where the signal from dye A enters the detection channel of dye B, changing the position of the capillary array can change the state of multiple reflections, resulting in a change in spectral crosstalk.

[0017] For this reason, when adopting the method of Patent Document 1, after replacing the capillary array, it is necessary to generate a matrix to be used in the crosstalk reduction process for the new array. Generating a matrix to be used in the crosstalk reduction process requires calculating the crosstalk ratios for all dyes and capillaries. The crosstalk ratio when a certain dye A in a certain capillary i emits light can be calculated 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, generating a matrix to be used in the crosstalk reduction process requires 4 × 8 = 32 electrophoresis runs. Considering that a single electrophoresis run takes several tens of minutes or more, including preparatory operations such as polymer replacement and preliminary electrophoresis to remove unnecessary ions, generating a matrix to be used in the crosstalk reduction process takes a long time. This time increases with the number of dyes and capillaries.

[0018] Patent Document 1 also describes a method for reducing the effort and time required to generate a matrix used in crosstalk reduction processing. Specifically, the method describes a method in which the timing of sample injection into each capillary is shifted to shift the timing of dye emission, so that when one capillary is emitting light, the other capillaries do not emit light. Similarly, the method describes a method in which fluorescently labeled DNAs with different chain lengths are introduced into each capillary, so that when one capillary is emitting light, the other capillaries do not emit light.

[0019] However, the former requires the device to be equipped with an operation sequence different from that of normal analysis, and the latter poses hurdles such as the need to prepare a reagent containing DNA with controlled chain length to generate the matrix used in the crosstalk reduction process.Furthermore, when the dye used is changed, the matrix used in the crosstalk reduction process must be regenerated even if the capillary array is not replaced.

[0020] Patent Literature 2 discloses a method for reducing spectral crosstalk (synonymous with the process of determining the ratio of multiple dyes with overlapping spectra from their fluorescence spectra) by optimizing a matrix for reducing spectral crosstalk from information obtained by analysis during sample analysis, without performing electrophoresis for calibration with the aim of obtaining information on the matrix used for reducing spectral crosstalk. This method optimizes the matrix used for the process of reducing spectral crosstalk by repeatedly changing the matrix based on correlation information between any two estimated dye concentrations, evaluating the correlation between the dye concentrations, and determining whether to update the matrix, starting from an initial matrix.

[0021] This method requires the removal of outliers to accurately determine the correlation between the two dyes due to spectral crosstalk. In other words, we must distinguish between spectral crosstalk caused by dye A in the presence of dye B and the actual presence of dye B in the sample. This distinction can generally be expected to be possible with respect to spectral crosstalk. When multiple dyes are used, the goal is generally to distinguish between different analytes, so dyes A and B label different analytes (molecules). Therefore, when electrophoresing a sample containing both analytes labeled with dye A and analytes labeled with dye B and obtaining time-series fluorescence spectral data, we can expect dyes A and B to often emit light at different times. On the other hand, spectral crosstalk caused by dye A in relation to dye B occurs at the same time as the emission of dye A. Since it is rare for analytes labeled with dye A and analytes labeled with dye B to emit light at the same time by chance, we can simply remove these rare outliers when calculating the correlation between the estimated dye concentrations of dye A and dye B.

[0022] However, even if the method of Patent Document 2 is extended to spatial crosstalk, the prerequisite for removing outliers described above is not met. When the method of Patent Document 2 is extended to spatial crosstalk, it is necessary to distinguish between spatial crosstalk with respect to dye A in capillary j when dye A emits light in capillary i and the signal actually emitted by dye A in capillary j. As in the previous case, the fluorescence of dye A due to spatial crosstalk in capillary j occurs at the same time as the emission of dye A in capillary i. Here, assume that dye A labels the same analyte α in the sample analyzed in capillary i and the sample analyzed in capillary j. In this case, the emission of dye A actually contained in the sample in capillary i and capillary j occurs at the same time. Thus, there is no guarantee that spatial crosstalk occurring with respect to dye A in capillary j when dye A emits light in capillary i can always be distinguished from the signal actually generated by the emission of dye A in capillary j.

[0023] This becomes a major obstacle in the following cases, for example. As an example, consider analyte α labeled with dye A and analyte β labeled with dye B. Sample 1 contains only analyte α. Sample 2 contains analyte β, and we want to determine whether sample 2 contains trace amounts of analyte α. This would be the case, for example, if sample 1 is a pure substance and we were to analyze a trace component (analyte α) present in sample 2 using the data from sample 1 as the reference.

[0024] Here, sample 1 is analyzed using capillary i, and sample 2 is analyzed using capillary j. At this time, a strong signal from dye A is obtained in capillary i, and a strong signal from dye B is obtained in capillary j. We want to determine whether a weak signal from dye A is generated in capillary j. However, because dye A labels analyte α in both capillaries, dye A fluorescence is obtained from capillary i and capillary j at approximately the same time, whether due to spatial crosstalk or the actual signal from analyte α. Therefore, even if a weak signal from dye A is obtained in capillary j, it is not possible to determine from the data trend alone whether it is due to spatial crosstalk or whether a trace amount of analyte α labeled with dye A is actually present.

[0025] In this way, by combining the method of Patent Document 1 with the method of Patent Document 2, it is not possible to optimize a matrix that reduces both spectral crosstalk and spatial crosstalk during analysis and eliminate the need to re-acquire the crosstalk reduction matrix when replacing the capillary array.

[0026] Furthermore, the capillary electrophoresis device described in Non-Patent Document 1 provides an option to use a matrix that is initially set as a spectral crosstalk reduction matrix (Factory Calibration on page 217). However, this matrix is ​​not optimized for each individual device, and can only be used for applications that can tolerate residual spectral crosstalk due to incomplete information.

[0027] Therefore, the present invention has been made in consideration of the above, and aims to provide a photodetector and a signal processing method that are capable of performing crosstalk reduction processing using crosstalk information used for crosstalk reduction processing that has been generated once, even if multiple light-emitting body holders (e.g., capillary arrays) are replaced.

[0028] In order to solve the above problems, the photodetection device of the present invention comprises a plurality of replaceable light-emitting element holders in which a plurality of types of light-emitting elements emit light, an optical sensor having a plurality of detection channels that detect the light emitted from the plurality of types of light-emitting elements in a plurality of wavelength bands, a spatial filter that fixes the incident position of the light emitted from the plurality of types of light-emitting elements on the optical sensor, and a computer that processes the signal output from the optical sensor, wherein the computer has internal crosstalk information for reducing crosstalk that exists between the plurality of detection channels, and reduces crosstalk that exists between the detection channels of the optical sensor by performing calculations using the crosstalk information on the outputs from the optical sensor that correspond to the plurality of wavelength bands of each of a plurality of light-emitting points from which the plurality of types of light-emitting elements emit light, derives the concentration ratio or signal amount ratio of each of the plurality of types of light-emitting elements for each of the plurality of light-emitting element holders, and performs calculations using the crosstalk information even if the light-emitting element holder is replaced with another light-emitting element holder.

[0029] Furthermore, the signal processing method of the present invention includes preparing a light detection device including a plurality of replaceable light-emitting element holders inside which a plurality of types of light-emitting elements emit light, an optical sensor having a plurality of detection channels that detect the light emitted from the plurality of types of light-emitting elements in a plurality of wavelength bands, a spatial filter that fixes the incident position of the light emitted from the plurality of types of light-emitting elements on the optical sensor, and a computer that processes the signal output from the optical sensor; measuring the light emitted by the plurality of types of light-emitting elements using crosstalk information for reducing crosstalk existing between the plurality of detection channels stored in the computer, performing calculations on outputs from the optical sensor corresponding to the plurality of wavelength bands of each of a plurality of light-emitting points from which the plurality of types of light-emitting elements emit light, thereby reducing crosstalk existing between the detection channels of the optical sensor, and deriving the concentration ratio or signal amount ratio of each of the plurality of types of light-emitting elements for each of the plurality of light-emitting element holders; and performing calculations using the crosstalk information even when the light-emitting element holder is replaced.

[0030] According to the photodetector and signal processing method of the present invention, even if a plurality of light emitter holders are replaced, crosstalk reduction processing can be performed using crosstalk information used for crosstalk reduction processing that has been generated once. Therefore, it is possible to reduce the effort and time required to regenerate crosstalk information used for crosstalk reduction processing when a plurality of light emitter holders are replaced. Problems, configurations, and effects other than those described above will be made clear by the following description of the embodiments.

[0031] 1A is a schematic diagram of a photodetector to which the crosstalk reduction method described in Patent Document 1 can be applied. FIG. 1B is a schematic diagram of a spectral image on a light image sensor of the photodetector of FIG. 1A. FIG. 2B is a schematic diagram of a crosstalk simulation model. FIG. 2C is a diagram showing a flowchart of a simulation performed using the simulation model of FIG. 2A. FIG. 3A is a diagram showing a crosstalk simulation result. FIG. 3B is a diagram showing a result when the crosstalk reduction process of Patent Document 1 is performed on the simulation result of FIG. 3A. FIG. 4A is a diagram showing a crosstalk simulation result assuming replacement of a capillary array. FIG. 4B is a diagram showing a result when the crosstalk reduction process of Patent Document 1 is performed on the simulation result of FIG. 4A. FIG. 5A is a schematic diagram of a photodetector according to the first embodiment. FIG. 6A is a schematic diagram showing a generation path of crosstalk that occurs on the capillary side of the optical fiber. FIG. 7B is a diagram showing a simulation model of a photodetector to which the crosstalk reduction process according to the first embodiment is applied. FIG. 7A is a diagram showing a crosstalk simulation result in the simulation model of FIG. 7A. FIG. 7B is a diagram showing a result when the crosstalk reduction process is performed on the simulation result of FIG. 7B. FIG. 8A is a schematic diagram showing a method of connecting a calibration light source for obtaining a matrix used in the crosstalk reduction process according to the first embodiment. FIG. 8A is a schematic diagram showing an example structure of the calibration light source of FIG. FIG. 10 is a diagram showing the influence of detector saturation on the crosstalk reduction process according to the first embodiment. FIG. 11 is a diagram showing the components of matrix G and matrix F. FIG. 12 is a schematic diagram showing an example of the structure of a calibration light source for acquiring a matrix used in the crosstalk reduction process according to the second embodiment. FIG. 13 is a flowchart showing the operation of the photodetector when acquiring a matrix used in the crosstalk reduction process according to the second embodiment. FIG. 14 is a schematic diagram of a photodetector according to a modified example.

[0032] In the following embodiments, when necessary for convenience, the description will be divided into multiple sections or embodiments, but unless otherwise specified, they are not unrelated to each other, and one is a partial or complete modification, detail, supplementary explanation, etc. of the other. Furthermore, in the following embodiments, when the number of elements (including the number, numerical value, amount, range, etc.) is mentioned, it is not limited to that specific number, and may be more or less than the specific number, unless otherwise specified or when it is clearly limited in principle to a specific number.

[0033] Furthermore, in the following embodiments, it goes without saying that the components (including element steps, etc.) are not necessarily essential unless otherwise specified or considered to be clearly essential in principle. Similarly, in the following embodiments, when referring to the shape, positional relationship, etc. of components, etc., it is intended to include those that are substantially similar or similar to the shape, etc., unless otherwise specified or considered to be clearly not essential in principle. The same applies to the above numerical values ​​and ranges.

[0034] In all the drawings for explaining the embodiments, the same components are generally designated by the same reference numerals, and repeated explanations thereof will be omitted.

[0035] In the following embodiments, the photodetector of the present invention will be described using a capillary electrophoresis apparatus as a typical application example, in order to more specifically explain the configuration and effects of the present invention, and the photodetector of the present invention is not limited to a capillary electrophoresis apparatus.

[0036] (Overview of the Capillary Electrophoresis Device of the Present Embodiment) In the configuration of the capillary electrophoresis device of the present embodiment, for example, a spatial filter is installed in the light intake section of a spectroscope that separates fluorescence from a fluorescent dye to acquire a spectrum, and the light intake position into the spectroscope is fixed. For example, the spectroscope has a first lens that collimates the incident light, a grating that separates the light into wavelengths, a second lens that forms an image of the light that has passed through the grating on an optical sensor, and an optical image sensor that detects the light. For example, the spatial filter is a multimode optical fiber, and one end of the optical fiber is fixed at the light input position of the spectroscope. The other end of the optical fiber is positioned near the capillary and captures the fluorescence emitted from inside the capillary. For example, the optical image sensor is a CCD or CMOS image sensor.

[0037] Crosstalk can be caused by several factors. However, by adopting the above-described configuration, the crosstalk ratio generated on the spectrometer side by the optical fiber can be fixed without being affected by replacement of the light emitter holder (capillary array). Fluorescence generated in the capillaries is taken in by the optical fiber and guided to the spectrometer. Possible causes of crosstalk within the spectrometer include the broadening of the spatial profile of the fluorescence spectrum, multiple reflections between the surfaces of elements such as lenses, and grating anomalies. If there were no optical fiber and the capillaries were directly installed at the light introduction position of the spectrometer, the misalignment of each capillary due to replacement of the capillary array could affect the spatial profile of the fluorescence and the occurrence of multiple reflections and anomalies, potentially changing the crosstalk ratio.

[0038] By fixing the incident position on the spectrometer using the optical fiber, the spatial profile of the fluorescence and the occurrence of multiple reflections and anomalies are fixed. Changing the position of the capillary relative to the other end of the optical fiber opposite the spectrometer affects the amount of light incident on the optical fiber, but does not affect the rate of crosstalk generated inside the spectrometer. Because changing the position of the capillary does not change the rate of crosstalk, even if the capillary array is replaced, the same information as before the replacement can be used for crosstalk reduction processing.

[0039] On the other hand, crosstalk occurring on the capillary side of the optical fiber can still change when the capillary array is replaced. For example, crosstalk occurring due to the reflection of fluorescence on the capillary surface is affected by the placement error of the capillaries that make up the capillary array, and therefore changes when the capillary array is replaced. This type of crosstalk cannot be completely eliminated simply by providing the optical fiber described above. It is also possible to sufficiently reduce the crosstalk that occurs on the capillary side and fluctuates when the capillary array is replaced. This can be achieved, for example, by separating each capillary with a light-shielding wall to make them independent. Alternatively, the distance between the capillaries can be increased to the extent that crosstalk is negligible. A method is also known in which a pinhole is placed at the exit of the optical fiber to limit the incident angle of the light to be detected (Patent Document 3).

[0040] Although the optical fiber alone as described above cannot eliminate crosstalk that occurs on the capillary side and fluctuates when the capillary array is replaced, this does not reduce the effect of the present invention. Even if the crosstalk that occurs on the capillary side of the optical fiber is not completely eliminated, it is sufficient if the residual components are low enough to be acceptable for the purpose of analysis.

[0041] (Regarding the known technology of Patent Document 1) Prior to a detailed description of the embodiments, the known technology of Patent Document 1, which forms the background of the present invention, and its problems will be summarized. FIG. 1A is a configuration diagram of a photodetector 100 to which the technology described in Patent Document 1 can be applied. The photodetector 100 includes a capillary array 101, two lenses 102, a grating 103, and an optical image sensor 104. The photodetector 100 disperses and detects fluorescence emitted from the capillary array 101. The arrows in the figure indicate the irradiation direction of excitation laser light L that excites the fluorescence. The laser light L is irradiated from the lateral direction (side direction) of the capillary array 101 so as to pierce all of the capillaries in the capillary array, and excites the dye inside all of the capillaries.

[0042] In the photodetector 100, wavelengths are dispersed in the direction perpendicular to the plane of the drawing by a grating 103. As shown in FIG. 1B, spectral images 105 corresponding to each capillary are arranged on the optical image sensor 104. The optical image sensor 104 outputs light intensity measurement results for each pixel. The light intensity measurement results are summed for each wavelength section of each capillary. For example, if the measurement wavelength range is 500 nm to 700 nm and light intensity values ​​for each wavelength are summed over a 10-nm width to obtain 20 data points, the wavelength sections refer to the sections from 500 nm to 510 nm, 510 nm to 520 nm, ..., and 690 nm to 700 nm. In other words, a set of values ​​obtained by summing the outputs from pixels present in each section (channel 106) separated by dotted lines in FIG. 1B is obtained as spectral information for each capillary.

[0043] The conventional crosstalk reduction method described in Patent Document 1 will be summarized with the photodetector 100 in mind. As previously defined, crosstalk includes two types: spectral crosstalk, in which a signal from dye A is mistakenly recognized as a signal from dye B, and spatial crosstalk, in which a signal emitted from capillary i is mistakenly recognized as being emitted from capillary j. The crosstalk reduction methods described in Patent Document 1 and the present invention are based on the premise that this crosstalk is linear with respect to the signal vector (a vector that lists the signals corresponding to each capillary and each wavelength range obtained by the optical image sensor 104). This is usually satisfied because the photodetector 100 does not include any elements that exert a nonlinear effect on the light to be detected and is not used under conditions where nonlinear effects are present (generally requiring very high light intensity).

[0044] The above content is S Ai The crosstalk detected in the area where dye B is supposed to be detected in capillary j when dye A emits light in capillary i is expressed as S CT Bj←Ai When the constant c is used, the relationship between the two can be expressed as the following equation (1): CT Bj←Ai =cS Ai (1)

[0045] In the conventional crosstalk reduction method described in Patent Document 1, a process for determining the constant c is first performed. Assume that dye A is injected into capillary i and nothing is injected into the other capillaries. In this situation, the signal S when dye A emits light in capillary i is Ai and the crosstalk S detected in the area where dye B is supposed to be detected in capillary j when dye A emits light in capillary i. CT Bj←Ai From the ratio of these two, we obtain the constant c using the following equation (2): c=S CT Bj←Ai / S Ai (2)

[0046] Next, the 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. The signal acquired by capillary i is S Ai In capillary j, the signal S Bj It is desirable to obtain crosstalk S CT Bj←Ai Since the signal is included in the signal, the actual measured signal S act Bj is calculated by the following formula (3): S act Bj =S Bj +S CT Bj←Ai (3)

[0047] Here, the crosstalk component of equation (3) is expressed by equation (1). In equation (1), the constant c is obtained by equation (2). Also, S Ai is obtained as a signal acquired at capillary i. Therefore, using equations (1) and (3), the true signal S that should be obtained in the absence of crosstalk can be calculated by the following equation (4): Bj You can get S Bj =S act Bj -S CT Bj←Ai =S act Bj -cS Ai (4)

[0048] The above explanation is a simplification of the method described in Patent Document 1. In reality, crosstalk may also include components generated from capillaries other than capillary i and capillary j, and from dyes other than dye A and dye B. In the above explanation, the fluorescence generated from dye A and dye B is referred to as a signal. In reality, the optical image sensor 104 outputs the light intensity in each wavelength range for each capillary as a signal.

[0049] The method described in Patent Document 1 will be summarized based on the above. For example, assume that an L-color fluorescent dye is used for measurement, that there are N capillaries to be measured, and that the optical image sensor 104 measures M wavelength intervals. For example, the wavelength interval is M=20, and when the detection wavelength range is 500 to 700 nm, the optical image sensor 104 outputs 20 signals for each capillary: a fluorescent signal with a wavelength of 500 to 510 nm, a fluorescent signal with a wavelength of 510 to 520 nm, ... a fluorescent signal with a wavelength of 690 to 700 nm. Hereinafter, the signal (or crosstalk) generated for wavelength interval k of capillary i when the l-th dye is introduced into capillary j is defined as S ik←jl It is written as follows.

[0050] where S ik←jl The matrix having elements S is written as C. ik←jl has four types of subscripts, but the ζ and η components of matrix C ζη are arranged in two dimensions so that ζ=Mi+k,η=Mj+l. ik←jl The specific value of S can be obtained by passing the dyes used only in the capillary j one by one and obtaining the output of the optical image sensor 104. ik←jl The value of may be properly normalized by the signal when the dye used is at a reference concentration. The matrix C is a matrix in which the spectrum of each dye used is arranged at a position corresponding to each capillary in the matrix. Here, before actually measuring the sample to be measured, the generalized inverse matrix C of the matrix C is calculated. - is obtained from the following equation (6): C - =[C t C] -1 C t (6)

[0051] Next, let us consider a situation where a sample to be analyzed is analyzed. At this time, the signal of wavelength division k of capillary i is S ik Here, the signal vector s is expressed as S ik Specifically, the ζ component s of the signal vector s is ζ For S, ζ=Mi+k ik At this time, the vector d, which has the ratio of each dye as its component, can be obtained as the following formula (7): d=C - s (7)

[0052] Equation (7) is an extension of the above-mentioned explanation regarding the removal of a single crosstalk component to the case where spatial crosstalk exists between multiple capillaries and spectral crosstalk exists between multiple dyes. The above is the conventional crosstalk reduction method described in Patent Document 1. In addition, since a vector s is output from the optical image sensor at each time in an analysis using capillary electrophoresis, the signal waveform of each dye in each capillary can be obtained by calculating equation (7) at each time.

[0053] When applying the above-mentioned crosstalk reduction method, it is necessary that matrix C does not change between the timing at which matrix C is obtained and the timing at which the signal is actually measured. Patent Document 1 shows that the above condition is met when the same capillary array is used to obtain crosstalk matrix C and to measure the actual sample. However, in capillary electrophoresis, capillary arrays are consumables and must be replaced with new arrays after a certain number of uses. Patent Document 1 does not state whether the above prerequisites are met and the crosstalk reduction method works in this case.

[0054] (Simulation) If various parameters such as the diameter of each capillary, as well as the installation position and angle of each capillary, were exactly the same before and after replacing the capillary array, the aforementioned condition would be met. However, it is impossible to actually satisfy this condition, and the aforementioned condition does not hold when the capillary array is replaced. Below, we use a simulation to specifically demonstrate this. In the following simulation, we evaluate the effect on crosstalk when the error in the relative position of the capillaries in each capillary array changes due to replacement of the capillary array. The error in the relative position of the capillaries refers to the deviation from the ideal position of the capillaries, which should be aligned at equal intervals on the same plane. Capillary arrays are fabricated by arranging and fixing capillaries on a substrate or the like. However, due to individual differences in the substrate and variations in the fixing process, the spacing between the capillaries and the distance from the substrate of each capillary are not exactly the same for each capillary array.

[0055] A simulation model is shown in Figure 2A. For simplicity, this simulation does not perform wavelength separation and only considers spatial crosstalk. This model includes a capillary array 201, two lenses 202, and an optical image sensor 203. The capillary array 201 has 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 inside each capillary. An image of the capillary array 201 is formed on the optical image sensor 203 by the two lenses 202. The number of capillaries in the capillary array 201 is eight.

[0056] The flow of the simulation is shown in Figure 2B. When performing crosstalk reduction processing (S200: Yes), first, the above-mentioned matrix C - Calculate the matrix C -A capillary array for which a relative position error is to be calculated is assumed (S201), and a relative position error is set for the capillary array. After the capillary positions are set, a fluorescent image on the optical image sensor 203 is calculated using ray tracing (S202). In this step, ray tracing is performed by generating fluorescence from only one of capillaries 1 to 8. This step is repeated eight times, changing the capillary that emits light, to calculate the output of the optical image sensor 203 when capillaries 1 to 8 emit light. Next, a point spread function is convolved with the ray tracing calculation results (S203). This point spread function is actually measured using a spectrometer composed of a lens, a grating, and an optical image sensor, and represents the "blur" of the optical system in the capillary array direction (X direction in Figure 2A). In ray tracing using the model of Figure 2A, the lens 202 is calculated as an ideal thin lens, and the deviation from the ideal situation in the actual optical system is expressed by the point spread function. Next, for the output of the optical image sensor 203 obtained as a result of the calculation, the signal of the area corresponding to each capillary is integrated to obtain the value of crosstalk. - is calculated (S204). In this model, wavelength separation is not performed, so the matrix C is a square matrix in which the spatial crosstalk between each capillary is arranged two-dimensionally.

[0057] Next, assuming the capillary array to be actually used for sample analysis, crosstalk is calculated. If crosstalk reduction processing is not performed (S200: No), calculation begins from this step. The setting of the relative position error to the capillary array (S205), calculation by ray tracing (S206), and convolution of the point spread function (S207) are the same as S201, S202, and S203 described above. Then, a signal vector s is calculated from the obtained output of the optical image sensor 104 (S208). If crosstalk reduction processing is to be performed (S209: Yes), crosstalk reduction processing is performed using equation (7) (S210). If crosstalk reduction processing is not to be performed (S209: No), this step (S210) is skipped. After calculation is completed, the result is output (S211). If crosstalk reduction processing is to be performed, vector d is output; if crosstalk reduction processing is not performed, vector s is output. When there are multiple types of dyes and the dye concentration ratio is calculated from the spectrum, vector d and vector s represent different types of information (the dye ratio for each capillary and the light intensity for each wavelength range). However, if separation in the wavelength direction of light is not considered, they represent the same type of information (the light intensity for each capillary is the dye concentration ratio), and therefore the effect of using crosstalk processing can be evaluated by comparing the two.

[0058] (Results of crosstalk reduction processing when there is no relative position error) Fig. 3A shows the crosstalk obtained by the above-mentioned simulation method when the crosstalk reduction processing is not performed, and Fig. 3B shows the crosstalk when the crosstalk reduction processing is performed. In Figs. 3A and 3B, it is assumed that there is no relative position error in the capillary array, and the matrix C - The assumption is that the generation of luminescence and the analysis of the sample will be performed. In Figure 3A, the horizontal axis represents the emitting capillary. Each bar with different hatching represents the crosstalk value detected in the region where capillaries 1 to 8 are detected (referred to as channels 1 to 8).

[0059] Figure 3A shows that in this model, crosstalk is greatest in the channel that detects adjacent capillaries, with a crosstalk of just over 1% occurring. This crosstalk is primarily caused by the leakage of fluorescence into adjacent channels due to light reflection on the capillary surface and blurring of the imaging optical system (expressed by the point spread function). Other possible causes of actual spatial crosstalk include multiple reflections between the elements that make up the spectroscopic system, grating anomalies, and scattering by dust, but this model only reflects the above two factors.

[0060] Figure 3B shows that, for the same capillary array, crosstalk is reduced by conventional crosstalk reduction processing. Crosstalk that existed at just over 1% is reduced to 0.01% or less by crosstalk reduction processing. Generally, the dynamic range of an optical image sensor is about 3 to 4 digits, and crosstalk of 0.01% or less means that the crosstalk is below the detection limit. This time, the matrix C - The model used for generating the crosstalk and for calculating the crosstalk is completely the same, and the reason why the crosstalk value in FIG. 3B is not zero is due to a calculation error in the ray tracing method.

[0061] (Results of Crosstalk Reduction Processing When Relative Position Errors Exist) Figures 4A and 4B, like Figures 3A and 3B, are diagrams showing crosstalk when crosstalk reduction processing is not performed and when crosstalk reduction processing is performed. However, in this calculation, errors in the X and Y directions of Figure 2A are given to each capillary using a Gaussian distribution with a standard deviation of 10 μm. Matrix C - Assuming that different capillary arrays are used for generating and analyzing samples, the matrix C -The value of the relative position error of the capillaries set in the capillary array is different when the model is generated and when the crosstalk is calculated. Considering that the outer diameter of the capillaries is 343 μm, the above error can be said to be a relatively small relative position error. Furthermore, in this model, the outer diameters of the capillaries are exactly the same, but capillaries generally have a tolerance of about ±10 μm. If we assume that changes in the diameter of the capillaries can cause deviations in the center position depending on the fixing method, we can see that the above relative position error is a value that can actually occur.

[0062] Figure 4A shows that even when an array error is introduced, there is a crosstalk of just over 1%, the same as when there is no array error. On the other hand, Figure 4B shows that when there is an array error, a maximum of about 0.2% of crosstalk remains even after conventional crosstalk reduction processing. Furthermore, the crosstalk in some channels is a negative value, indicating that excessive crosstalk correction has been performed. The calculation was repeated nine times with different array errors, and the average of the maximum remaining crosstalk value was found to be 0.28%. This is because the crosstalk value fluctuates depending on the array error, and the matrix C - This indicates that the effect of conventional crosstalk reduction processing cannot be properly obtained if an array with different alignment errors is used for generating the sample and analyzing the sample.

[0063] Here, we used a model in which the causes of crosstalk were limited to demonstrate the effect of alignment errors on crosstalk reduction processing. In reality, as mentioned above, there are some crosstalk causes that are not incorporated into the model. Furthermore, when replacing capillaries, there are other factors that can increase crosstalk changes in addition to alignment errors, such as changes in the position of the entire capillary array, changes in the inclination of the capillary array, variations in the inner and outer diameters of the capillaries, and dirt on the capillaries. The greater the variation in crosstalk between capillary arrays, the more difficult it becomes to achieve the effects of conventional crosstalk reduction processing.

[0064] As mentioned above, the inventors have found that the conventional crosstalk reduction method, when replacing the capillary array, - We have newly discovered that we need to regenerate the matrix C- The crosstalk information used to generate the matrix C is obtained by injecting samples labeled with each dye into each capillary one by one, performing electrophoresis, and measuring, as described in Patent Document 1, for example. Therefore, if L fluorescent dyes are used and there are N capillaries to be measured, it is necessary to perform L×N electrophoresis runs. Since one electrophoresis run takes several tens of minutes, the matrix C - It takes a long time to generate the matrix C - Methods for reducing the labor and time required for production have also been described, but in order to utilize the described methods, it is necessary to use special samples and operate special equipment that is not used in normal analysis.

[0065] The present invention uses the matrix C associated with the replacement of the capillary array. - This eliminates the need for regeneration of the above-mentioned material, thereby solving the above-mentioned problems.

[0066] First Embodiment FIG. 5 is a configuration diagram of a photodetector 500 according to a first embodiment. The photodetector 500 includes a capillary array 501, an optical fiber array 510 (spatial filter), a spectrometer 505, and a computer 550. The capillary array 501 has a plurality of capillaries 501a, and a plurality of light emitters emit light inside each capillary 501a. The capillary array 501 is replaceable. The optical image sensor 504 constituting the spectrometer 505 has a plurality of detection channels (see FIG. 1B ) that detect light emitted from the plurality of light emitters in a plurality of wavelength bands. The optical fiber array 510 is a spatial filter that fixes the incident positions of the light emitted from the plurality of light emitters relative to the spectrometer 505. The computer 550 processes signals output from the optical image sensor 504.

[0067] The photodetector 500 differs from the photodetector 100 in that it includes an optical fiber array 510. The optical fiber array 510 includes a plurality of optical fibers 510a each corresponding to a light-emitting point of a plurality of capillaries 501a. The optical fiber array 510 captures fluorescence emitted from each capillary 501a in the capillary array 501 and guides the fluorescence to a spectrometer 505 composed of a lens 502, a grating 503, and an optical image sensor 504. The photodetector 500 is intended as a detection device for a capillary electrophoresis apparatus, and actually operates in conjunction with a temperature controller, a high-voltage application device, an automatic sample stage, a computer for signal processing, and the like to analyze samples by capillary electrophoresis. However, a description of parts other than the photodetector 500 will be omitted.

[0068] The crosstalk reduction process of the present invention can be applied even if the spectrometer 505 has a different configuration. Specifically, the wavelength separation of light may be performed by a prism instead of a grating, or may be performed by a plurality of dichroic mirrors.

[0069] The crosstalk reduction processing method in the first embodiment is similar to the above-mentioned formula (7) and the related description. However, processing conditions require that the position at which the fluorescence is introduced into the spectrometer 505 is fixed by the optical fiber array 510, and that crosstalk occurring on the capillary side of the optical fiber array 510 is sufficiently reduced by another method. Note that the position at which the fluorescence is introduced into the spectrometer 505 may be fixed by a pinhole array, a slit array, or the like, instead of the optical fiber array 510.

[0070] The photodetector 500 includes a computer 550. The computer 550 includes a processor 551, a main memory 552, an auxiliary memory 553, and an input / output interface 554 (hereinafter, interface will be abbreviated as I / F). The processor 551 is a central processing unit that controls the operation of each part 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 deploys a program stored in the auxiliary memory 553 in an executable manner in a working area of ​​the main memory 552. The main memory 552 stores programs executed by the processor 551, data processed by the processor, etc. The main memory 552 is, for example, a flash memory, a random access memory (RAM), or a read-only memory (ROM). The auxiliary storage unit 553 stores various programs such as an OS (Operating System) and various data. The auxiliary storage unit 553 is, for example, a solid state drive (SSD), a hard disk drive (HDD), or a combination thereof. The auxiliary storage unit 553 stores crosstalk information (for example, the above-mentioned matrix C - The input / output I / F 554 is communicably connected to the optical image sensor 504 and the display unit 560 .

[0071] The calculator 550 calculates the crosstalk information (e.g., the matrix C -By performing calculations on the outputs from the optical image sensor 504 corresponding to the multiple wavelength bands of the multiple light-emitting points from which the multiple light-emitting bodies emit light using the optical image sensor 504, crosstalk present between the detection channels of the optical image sensor 504 is reduced, and the concentration ratio or signal amount ratio of each of the multiple types of light-emitting bodies for each of the multiple capillaries is derived. Then, even if the capillary array 501 is replaced with another capillary array, the computer 550 performs the above calculations using the same crosstalk information.

[0072] This crosstalk information is expressed as the generalized inverse matrix C of the matrix C that contains information on the crosstalk existing between multiple detection channels. - or the general inverse matrix C - This is equivalent to the general inverse matrix C - The information equivalent to is, for example, information that is not in a matrix format and is information for reducing crosstalk that exists between a plurality of detection channels. The calculation performed by the computer 550 to derive the concentration ratio or the signal amount ratio is performed by calculating a matrix C - Applying from the left, C - This is the operation for deriving s (see equation (7) above), or an equivalent operation.

[0073] The computer 550 sequentially flows a plurality of light emitters into each of the plurality of capillaries 501a, records the output of the optical image sensor 504 in a state in which a single light emitter among the plurality of light emitters is emitting light in a single capillary 501a among the plurality of capillaries 501a, arranges the outputs of the optical image sensor 504 to generate a matrix C, and calculates a generalized inverse matrix C of the matrix C. － [C t C] -1 C t (see equation (6) above) or an equivalent operation, and then stored.

[0074] Crosstalk occurring on the capillary side of the optical fiber array 510 occurs, for example, in a path where fluorescence generated from one capillary directly enters an optical fiber detecting another capillary, or in a path where fluorescence is reflected from the surface of another capillary and enters the optical fiber detecting that capillary, as shown in Figure 6. Reducing crosstalk occurring in such paths can be achieved, for example, by ensuring a sufficiently large capillary pitch in the capillary array 501. Alternatively, a known method is to install a pinhole slightly smaller than the core diameter of the optical fiber 510a at the end of the optical fiber array 510 on the capillary array 501 side, thereby limiting the angle at which fluorescence is received by the optical fiber 510a and reducing crosstalk. Another known method is to install a light-blocking wall between the capillaries of the capillary array 501.

[0075] Simulation results for the effect of the crosstalk reduction method of the first embodiment will be described with reference to FIGS. 7A to 7C. FIG. 7A shows a model used in the simulation. Similar to the model in FIG. 2A, the model in FIG. 7A includes a capillary array 701, two lenses 702, and an optical image sensor 703. In FIG. 7A, an optical fiber array 710 is added to the model in FIG. 2A. The optical fiber array 710 is configured with eight optical fibers with a core diameter of 200 μm and an NA of 0.5, arranged at the same intervals as the capillaries in the capillary array 701. Furthermore, a light-shielding wall 720 is installed between the capillaries as a means for eliminating crosstalk occurring before the optical fiber array 710. The simulation flow is the same as that shown in FIG. 2B.

[0076] 7B and 7C are graphs showing the results of a crosstalk simulation using the model of FIG. 7A. FIG. 7B is a graph showing crosstalk when crosstalk reduction processing is not performed, and FIG. 7C is a graph showing crosstalk when crosstalk reduction processing is performed. In this simulation, as in the case of FIG. 4, the matrix C - Assuming that different capillary arrays are used for generating and analyzing samples, the matrix C -The value of the relative position error of the capillaries set in the capillary array 701 is different when generating the crosstalk and when calculating the crosstalk.

[0077] As can be seen from Figure 7B, the model in Figure 7A has crosstalk of about 0.5%. In contrast, when the crosstalk reduction process of Equation (7) is performed, the crosstalk is reduced to 0.01% or less, as shown in Figure 7C. The calculation was repeated nine times with different relative position errors, and the average of the maximum residual crosstalk values ​​was found to be 0.006%.

[0078] A comparison of the results in Figure 4B and Figure 7C shows that the crosstalk reduction process according to the first embodiment works correctly even when using capillary arrays with different capillary misalignment values. Note that this effect is not due to the installation of the light-shielding wall 720. To demonstrate this, the same simulation was performed with the addition of the light-shielding wall 720 to Figure 2A. The calculation was repeated nine times with different relative position errors, and the average of the maximum residual crosstalk value was found to be 0.11%, more than 10 times the value of the result in Figure 7C.

[0079] As described above, in the crosstalk reduction method of the first embodiment, when the capillary array 501 is replaced, the matrix C - The effect of the crosstalk reduction technique can be obtained without regenerating the matrix C. This is the effect of fixing the incident position of the fluorescence onto the spectrometer 505 by the optical fiber array 510. Even if the position of each capillary in the capillary array 501 changes, the incident position of the fluorescence onto the spectrometer 505 is fixed by the optical fiber array 510. Crosstalk occurring within the spectrometer 505 (for example, caused by multiple reflections or blurring of the spectral image) is not affected by the position of the capillary because the incident point of the fluorescence is fixed. Therefore, the value of crosstalk does not change when the capillary array is replaced, and the matrix C - There is no need to recreate the

[0080] However, as mentioned above, the method of the present invention requires that the crosstalk occurring on the capillary side of the optical fiber array 510 be sufficiently small. The method of the first embodiment reduces the invariant component of crosstalk (basically the crosstalk of the spectrometer 505), and therefore cannot remove the component that varies with replacement of the capillary array 501. For example, if the amount of crosstalk caused by surface reflection varies due to a change in the placement error of the capillary array, the resulting crosstalk will remain even after the crosstalk reduction process. To demonstrate this, the light-shielding wall 720 was removed from the model of FIG. 7A and the same simulation was performed. The calculation was repeated nine times with different relative position errors, and the average of the maximum residual crosstalk values ​​was found to be 0.12%. Thus, an optical system to which the method of the present invention is applied must be designed so that the variation in crosstalk occurring on the capillary side of the optical fiber array 510 is sufficiently smaller than the allowable value.

[0081] In the photodetection device 500 and the photodetection method of the first embodiment, the matrix C - Since is basically unchanged, for example, after the equipment is manufactured or installed at the place of use, this matrix C - The matrix generation can be performed in the same manner as the method described in Patent Document 1. For example, a sample labeled with a certain dye is injected into a certain capillary, electrophoresis is performed, and the signal of the optical image sensor 504 is recorded. This is repeated for all dyes in all capillaries. Then, the measurement results are arranged to form matrix C. Specifically, the outputs of each wavelength range of each capillary when the signal of the target dye in the target capillary is maximized are arranged. After that, the generalized inverse matrix C of matrix C is calculated. - Generate.

[0082] During sample analysis, the output of the optical image sensor 504 at each time is processed using equation (7) to obtain information on the ratio of each dye at each time. By arranging the ratios of each dye in chronological order, the electrophoretic waveform of the analyte labeled with each dye is obtained.

[0083] matrix C - is basically unchanged, but for some reason, the matrix C- For example, the positions of the components of the spectrometer 505 may be shifted due to the relocation of the device or long-term use. In such a case, the crosstalk ratio inside the spectrometer may also change, so the matrix C - You may also regenerate the

[0084] In order to reduce the time and effort required for generating the matrix C, the matrix C may be constructed from partial information. That is, the matrix C may be constructed from a plurality of matrices C containing only crosstalk information between some of the illuminant carriers or dyes. n For example, the simulation results of Figures 7A to 7C show that spatial crosstalk occurs strongly in the channel measuring the adjacent capillary. In such a case, matrix C can be generated by ignoring the crosstalk from capillaries that are two or more distances away.

[0085] Take the configuration of Figure 7A as an example. n is a matrix generated by arranging signals acquired from channels measuring each capillary when dye is injected into capillary n. In generating matrix C, dye is simultaneously injected into each set of capillaries: capillary 1, capillary 4, and capillary 7; capillary 2, capillary 5, and capillary 8; and capillary 3 and capillary 6. Then, matrix C n is generated using the signals of the channels corresponding to capillaries n+1, n, and n-1, and the signals of the channels corresponding to the other capillaries are set to zero. That is, when dye is injected into capillaries 1, 4, and 7 simultaneously, C1 is generated using the values ​​of channels 1 and 2, C4 is generated using the values ​​of channels 3, 4, and 5, and C7 is generated using the values ​​of channels 6, 7, and 8. All matrices C n After generating (C1 to C8 in the above case), matrix C n The matrix C is obtained by arranging these in order. By generating matrix C in this way, the number of electrophoresis runs required to obtain information can be reduced.

[0086] Note that the above ignores spatial crosstalk between capillary pairs with small spatial crosstalk values, but the same method can be used when the emission spectra of a dye pair are significantly different and have almost no overlap. In other words, for dye pairs with no overlapping fluorescence spectra, matrix C can be generated using data measured simultaneously for the dye pair.

[0087] It should be noted that the matrix C does not necessarily have to be generated by injecting a dye into the illuminant holder, i.e., the capillary. - ) may be acquired by connecting a calibration light source 801 to the spatial filter. The calibration light source 801 sequentially introduces calibration light to each emission capture position of the spatial filter. This calibration light has the same spectrum as the emission spectra of multiple light emitters. Figure 8A shows a diagram of generating matrix C using the calibration light source 801. The calibration light source 801 sequentially introduces light having the same spectrum as the emission spectrum of each dye into the optical fibers of the optical fiber array 510. Matrix C may be generated from the output of the spectrometer 505 in the same manner as described above.

[0088] An example structure of the calibration light source 801 is shown in Figure 8B. The calibration light source 801 has an excitation light source 802 inside. The excitation light source 802 irradiates a flow path 803 with fluorescence excitation light. Each dye is injected into the flow path 803 in order 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 one fiber of an output fiber array 809 by a mirror 807 and a lens 808. The angle of a portion of the mirror 807 can be changed, allowing the fiber that introduces the fluorescence to be changed.

[0089] The crosstalk reduction method of the first embodiment is based on the premise that there is a linear relationship between crosstalk and the signal that is the source of that crosstalk. Therefore, if this relationship is disrupted due to detector saturation, the method of the first embodiment will no longer function. Figure 9 shows a schematic graph illustrating the effects of detector saturation. In the graph of Figure 9, the horizontal axis represents the concentration of the dye to be measured, and the vertical axis represents the amount of crosstalk. In a spectroscopic system with crosstalk, the dye concentration and crosstalk are proportional to each other. In an ideal situation where there is no crosstalk at all, crosstalk is always zero, regardless of the dye concentration.

[0090] According to the crosstalk reduction method of the first embodiment, ideally, crosstalk is completely eliminated. Therefore, in a spectroscopic system where crosstalk exists but the crosstalk reduction process of the first embodiment is performed, crosstalk is ideally zero regardless of the dye concentration up to a certain level (even under realistic conditions where some crosstalk remains even after the reduction process, the reduced crosstalk value is smaller than the original value). However, if the detector in the capillary measuring the target dye becomes saturated, the output signal remains constant even if the dye concentration increases further. On the other hand, since crosstalk itself is proportional to the fluorescence intensity, it continues to increase in proportion to the concentration. As a result, the actual amount of crosstalk exceeds the amount of crosstalk estimated from the detector output. In this case, the crosstalk reduction method of the first embodiment cannot completely eliminate crosstalk, and the crosstalk value will no longer be zero. Therefore, the crosstalk reduction method of the first embodiment must be used within a range that does not saturate the detector. If a certain channel of the detector becomes saturated, it is useful to warn the user that the original performance may not be achieved even for other channels that are not saturated.

[0091] Specifically, when the output from the optical image sensor 504 becomes saturated, the computer 550 may display a warning on the display unit 560 .

[0092] <Signal Processing Method> Here, a signal processing method using the photodetector device 500 of the first embodiment will be described.

[0093] The signal processing method of this embodiment includes the steps of: preparing a photodetector 500; measuring light emitted from a plurality of light emitters by an optical image sensor 504; and calculating crosstalk information (matrix C - ) to perform calculations on outputs from an optical image sensor 504 corresponding to a plurality of wavelength bands for each of a plurality of light-emitting points from which a plurality of light-emitting bodies emit light, thereby reducing crosstalk present between detection channels of the optical image sensor 504 and deriving concentration ratios or signal amount ratios of each of a plurality of types of light-emitting bodies for each of a plurality of light-emitting body holders (capillaries 501 a); and performing the calculations using the same crosstalk information even when the capillary array 501 is replaced.

[0094] Crosstalk information (matrix C - ) is the generalized inverse matrix C of the matrix C containing crosstalk information. - The density ratio or signal amount ratio is calculated by dividing the vector s of the output from the optical image sensor 504 by the matrix C - Applying from the left, C - This is the operation for deriving s (see equation (7) above), or an equivalent operation.

[0095] <Summary of First Embodiment> The photodetector 500 according to the first embodiment includes a capillary array 501, an optical fiber array 510, a lens 502, a grating 503, and an optical image sensor 504. The photodetector 500 also includes a computer 550 that processes the output of the optical image sensor 504. The computer 550 calculates a generalized inverse matrix C of a matrix C generated from the output of the optical image sensor 504 when a sample labeled with the dye to be used is injected into each of all capillaries in the capillary array 101 in the photodetector 500 and measured. - When analyzing a measurement object, the computer 550 calculates the generalized inverse matrix C - The ratio of each dye is obtained by applying the general inverse matrix C - The same value is used even when the capillary array 501 is replaced.

[0096] Second Embodiment In the first embodiment, the outputs from the optical image sensor 104 are collectively calculated as a general inverse matrix C - The concentration ratio of each dye was obtained by processing the data as follows. In the second embodiment, spectral crosstalk caused by the dyes, spectral crosstalk caused by the spectrometer, and spatial crosstalk are processed separately. In the crosstalk reduction method according to the second embodiment, the crosstalk generated inside the spectrometer is reduced using a matrix obtained using a calibration light source, and the spectral crosstalk caused by overlapping fluorescence spectra of the dyes is reduced using a matrix obtained by measuring the fluorescence spectra from the dyes introduced into the capillary. Therefore, the crosstalk reduction process takes a form different from that of equation (7). This processing format makes it possible to easily change or add dye types.

[0097] First, let us consider the crosstalk that occurs inside the spectrometer 505. The spectrometer 505 is provided with N optical fibers that introduce light, and the optical image sensor 504 measures M wavelength intervals for each optical fiber. For example, the wavelength interval is M=20, which means that when the detection wavelength range is 500 to 700 nm, the optical image sensor 504 outputs 20 signals for each fiber: a fluorescent light signal with a wavelength of 500 to 510 nm, a fluorescent light signal with a wavelength of 510 to 520 nm, ... a fluorescent light signal with a wavelength of 690 to 700 nm. Here, the output of the optical image sensor 504 for wavelength interval m for fiber n is defined as S mn Let's say S mn The one-dimensional array of these is defined as the output signal vector s of the optical image sensor 104. mn has two subscripts, but S mn is the Mn+m component s of s Mn+m The elements are arranged one-dimensionally so that

[0098] On the other hand, consider the light incident on fiber n of the spectrometer 505. The power of the light in the wavelength range corresponding to the wavelength interval m of this light is P mn This power P mn Arrange these to create the incident power vector p. P mn As before, p is the Mn+m component p Mn+m The elements are arranged one-dimensionally so that

[0099] The spectrometer 505 outputs a linear signal relative to the power of the incident light. Therefore, the relationship between p and s can be expressed by the following equation (8): s=Gp (8)

[0100] Matrix G represents the crosstalk occurring in the spectrometer 505. Matrix G is obtained by sequentially introducing monochromatic light corresponding to the wavelength detection section of the optical sensor (e.g., the optical image sensor 504) to each emission capture position of the spatial filter (e.g., the optical fiber array 510). In an ideal spectrometer without crosstalk, matrix G is an identity matrix (note that s and p are normalized so that the output signal is 1 when the optical power is 1). If crosstalk is present, the triangular portion of matrix G in Figure 10 represents the area indicating the crosstalk value. Furthermore, if the sensitivity of the spectrometer differs for each emission capture position, the diagonal elements of matrix G may take values ​​other than 1. For example, if the spectrometer 505 has the configuration shown in Figure 5, the light loss within the spectrometer may be greater at emission capture positions farther from the central axis of the lens 502. In this case, the value of the diagonal element corresponding to that capture position is less than 1.

[0101] Next, we will organize the relationship between the dye in the capillary and the power of the light incident on each fiber. Now, let's assume that L types of dye are used for analysis. The ratio of the lth dye present in the nth capillary out of N capillaries is D. ln D ln The one-dimensional arrangement of these is called the dye ratio vector d. ln has two subscripts, but D ln is the Ln+l component of d Ln+l The elements are arranged one-dimensionally so that

[0102] Here, the signal generated in the nth capillary is measured by the nth optical fiber. The optical power P measured in the nth optical fiber is mn is the ratio D of the emission spectra of L kinds of fluorescent dyes in the nth capillary. lnTherefore, the relationship between the incident power vector p and the dye ratio vector d is given by the following equation (9): p=Fd (9) Here, the matrix F is a matrix that lists the fluorescence spectra of each dye.

[0103] From equations (8) and (9), the following equation (10) can be obtained: s = GFd (10) Therefore, the inverse matrix of matrix G is G -1 , the generalized inverse of matrix F is F - Then, the following equation (11) is obtained: - is [F t F] -1 F t or equivalently, d=F - G -1 s (11) This is G -1 , F - If is known, the dye ratio d can be obtained from the output s of the optical image sensor. -1 The calculation of F means the reduction of crosstalk that occurs inside the spectrometer. - The calculation of (a) means a process of calculating the dye ratio taking into consideration the overlap of the fluorescence spectra of the dyes (reducing spectral crosstalk).

[0104] Figure 10 shows the shapes of matrices G and F. Matrix G is a square matrix of size M × N, with 1s along the diagonal elements. The triangular areas above and below the diagonal elements represent crosstalk. In an ideal spectrometer with no crosstalk, all elements in this area would be 0. Matrix F is a matrix with M × N rows and L × N columns. Matrix F has N matrices F1, with M rows and L columns, arranged along the diagonal, and the rest of the matrix is ​​0. Matrix F1 has L dye spectra arranged in columns 1 through L (the rectangles in Figure 10 represent the spectra).

[0105] In the crosstalk reduction method of the second embodiment, calculations are divided into two stages, and matrices G and F are obtained separately. Matrix G is obtained using a calibration light source 1101 (see FIG. 11 ). The calibration light source 1101 is connected to the optical fiber array 510 of the spectrometer 505 in the same manner as the calibration light source 801. The calibration light source 1101 sequentially introduces light of wavelengths corresponding to M wavelength intervals into each of the N fibers. That is, the calibration light source 1101 first introduces light of a wavelength corresponding to wavelength interval 1 into fiber 1. Next, it introduces light of a wavelength corresponding to wavelength interval 2, light of a wavelength corresponding to wavelength interval 3, ... light of a wavelength corresponding to wavelength interval M. Next, a similar operation is performed for fiber 2. Next, a similar operation is performed for fiber 3, fiber 4, ... fiber N.

[0106] The matrix G is generated as follows: When the calibration light source 1101 introduces light of wavelength division m into fiber n, the output value for fiber k and wavelength division l of the optical fiber array is expressed as A kl←mn In this case, let ζ=Mk+l and η=Mn+m, and let the η and ζ components of the matrix G be G ζη =A kl←mn / A mn←mn Let's say.

[0107] FIG. 11 shows an example of the structure of a calibration light source 1101 that operates as described above. The calibration light source 1101 includes a white light source 1102. The white light source 1102 is, for example, a white LED or a halogen lamp. Light from the white light source 1102 is separated into wavelengths by a lens 1103 and a grating 1104. A slit 1105 is placed at the focal point of the separated light. The slit 1105 extracts only specific wavelength components from the light from the white light source 1102. The angle of the grating 1104 can be changed, and adjusting this angle controls the wavelength of the light that passes through the slit 1105. The light that passes through the slit 1105 is guided by a lens 1106 and a mirror 1107 to a specific fiber in an output fiber array 1108. The angle of one of the mirrors 1107 can be changed, and adjusting this angle controls which fiber in the output fiber array 1108 the light is guided into.

[0108] The matrix F can be measured by injecting a dye-labeled sample into all capillaries simultaneously. In the matrix acquisition method of the first embodiment, dye must be injected into only one capillary and signals must be measured for N capillaries, whereas in the matrix acquisition method of the second embodiment, simultaneous measurement is possible for all capillaries, reducing the time required for measurement to 1 / N.

[0109] Since matrix F is a matrix in which matrix F1, in which the spectra of each dye are arranged, is repeatedly arranged for the number of capillaries, matrix F1 may be measured by measuring the dye spectra in a specific capillary, and this may be repeated for the number of capillaries to form matrix F. In conventional crosstalk reduction methods and the crosstalk of the first embodiment, a single processing matrix contains a mixture of information on spectral crosstalk caused by the dyes, information on crosstalk caused by the photodetector, and information on the detection efficiency for each wavelength. Therefore, it was necessary to flow each dye into each capillary to obtain this information. However, in the crosstalk reduction method of the second embodiment, information on the effects caused by the spectrometer is collected in matrix G, so there is no need to measure the same sample for each capillary when measuring matrix F.

[0110] As described above, the crosstalk reduction method of the second embodiment makes it easier to obtain the processing matrix, particularly the matrix F containing dye spectral information, compared to the conventional crosstalk reduction method described in Patent Document 1 and the crosstalk reduction method of the first embodiment. This is particularly useful when adding a dye to be used in an analysis. When using a new dye for which information does not exist in the computer that performs crosstalk reduction processing, the conventional crosstalk reduction method described in Patent Document 1 or the crosstalk reduction method of the first embodiment requires introducing the new dye into all capillaries in sequence and acquiring fluorescence spectral information for the new dye. In contrast, the crosstalk reduction method of the second embodiment requires introducing the new dye into all capillaries simultaneously or into one specific capillary and measuring the fluorescence spectrum.

[0111] Alternatively, matrix F may be provided in a form that is stored in advance in a computer. Information on crosstalk that depends on each individual spectrometer is included in matrix G. Therefore, matrix F does not need to be measured for each individual photodetector device; the spectral information of each dye measured by a specific individual device may be stored in a separate computer, and matrix F may be generated from that information. In other words, spectral information of dyes measured by an individual photodetector device may be made public on the web, and users may download the spectral information of the dyes from the web and save it in their computers, and matrix F may be generated from the saved spectral information.

[0112] Figure 12 shows an example of the operation of the instrument when acquiring matrix F. First, the user selects the dye or set of dyes for which spectra will be acquired (S1201). Next, the user selects the method for acquiring fluorescence spectra (S1202). If the method for measuring dye spectra in all capillaries is selected, the instrument first prepares for electrophoresis (S1203). After preparation is complete, the user loads a sample labeled with the dye to be measured (S1204). After the sample is loaded, the instrument injects the sample into the capillaries and performs electrophoresis (S1205). A spectrum is acquired when the sample has migrated to the fluorescence measurement point (S1206). This cycle is repeated for all dyes for which spectra will be acquired (S1207). If the sample is prepared so that all dye spectra can be acquired simultaneously, this cycle only needs to be performed once. For example, this is a sample in which dyes A, B, C, etc. to be measured are labeled and mixed with DNA of chain lengths of 100 bases, 150 bases, 200 bases, etc. When electrophoresis is performed, dyes A, B, C, etc. emit light in sequence at different times. Such calibration samples are commercially available and relatively easy to obtain. Because the DNA reaches the fluorescence measurement point in the order of its chain length through electrophoresis, dyes A, B, and C emit light in chronological order. After all the fluorescence spectra have been measured, the fluorescence spectra are arranged to generate matrix F (S1208).

[0113] When the method of measuring fluorescence spectra using a single capillary is selected, the operation of the device is the same as the method of measuring fluorescence spectra using all capillaries (S1209 to S1214). However, the sample is injected into only one capillary. When generating matrix F, the fluorescence spectra acquired using a single capillary are repeatedly arranged.

[0114] If the method of generating matrix F from internal information is selected, the fluorescence spectrum (emission spectrum information) of the set dye is read from memory (S1215). The fluorescence spectrum may be stored in the computer from the beginning, or the spectral information may be provided and downloaded via the Internet, etc. Matrix F is generated from the read fluorescence spectrum (S1216). The generated matrix F is saved in the computer (S1217).

[0115] <Modifications> The present invention is not limited to the above-described embodiments and includes various modifications. For example, the above-described embodiments have been described in detail to clearly explain the present invention, and are not necessarily limited to those including all of the described configurations. Furthermore, it is possible to replace part of the configuration of one embodiment with the configuration of another embodiment, or to add the configuration of another embodiment to the configuration of one embodiment. Furthermore, it is possible to add, delete, or replace part of the configuration of each embodiment with other configurations.

[0116] For example, in the first embodiment, an example in which an optical fiber array 510 is provided as a spatial filter has been described, but the spatial filter may also be a filter having passage holes that allow light to pass through. The shape of the passage holes of this filter may be circular or rectangular. FIG. 13 is a configuration diagram of a light detection device 1300 according to a modified example. Unlike the light detection device 500, the light detection device 1300 has a pinhole array 1310 in which a plurality of pinholes (circular passage holes) are arranged in an array, each pinhole corresponding to a light-emitting point of a plurality of light-emitting body holders (capillaries). This pinhole array 1310 may also be a slit array in which slits (rectangular passage holes) are arranged in an array.

[0117] In the above embodiment, a light detection device was used that had multiple light-emitting body holders that emit light from multiple types of light-emitting bodies, but if there are multiple types of light-emitting bodies, there may be only one light-emitting body holder, or if there are multiple light-emitting body holders, there may be only one type of light-emitting body.

[0118] Furthermore, in the calculations of the above embodiment, various calculations are performed using matrices, but equivalent calculations may be performed using information that is not in matrix format as long as equivalent results are obtained.

[0119] 100 Optical detection device 101 Capillary array 102 Lens 103 Grating 104 Optical image sensor 105 Spectral image 106 Channel 201 Capillary array 202 Lens 203 Optical image sensor 500 Optical detection device 501 Capillary array 501a Capillary 502 Lens 503 Grating 504 Optical image sensor 505 Spectrometer 510 Optical fiber array 510a Optical fiber 550 Computer 551 Processor 552 Main memory unit 553 Auxiliary memory unit 554 Input / output interface 560 Display unit 701 Capillary array 702 Lens 703 Optical image sensor 710 Optical fiber array 720 Light-shielding wall 801 Calibration light source 802 Excitation light source 803 Flow path 804 Dye injection mechanism 805 Lens 806 Wavelength filter 807 Mirror 808 Lens 809 Output fiber array 1101 Calibration light source 1102 White light source 1103 Lens 1104 Grating 1105 Slit 1106 Lens 1107 Mirror 1108 Output fiber array 1300 Photodetector 1310 Pinhole array

Claims

1. A photodetection device comprising: a plurality of replaceable light-emitting body holders inside which a plurality of types of light-emitting bodies emit light; an optical sensor having a plurality of detection channels for detecting light emission from the plurality of types of light-emitting bodies in a plurality of wavelength bands; a spatial filter for fixing the incident position of the light emission from the plurality of types of light-emitting bodies on the optical sensor; and a computer for processing a signal output from the optical sensor, wherein the computer has internal crosstalk information for reducing crosstalk existing between the plurality of detection channels, reduces crosstalk existing between the detection channels of the optical sensor by performing calculations using the crosstalk information on outputs from the optical sensor corresponding to the plurality of wavelength bands of each of a plurality of light-emitting points from which the plurality of types of light-emitting bodies emit light, derives concentration ratios or signal amount ratios of the plurality of types of light-emitting bodies for each of the plurality of light-emitting body holders, and performs the calculations using the crosstalk information even if the light-emitting body holder is replaced with another light-emitting body holder.

2. The crosstalk information is a generalized inverse matrix C of a matrix C including information on crosstalk existing between the multiple detection channels. - or information equivalent to the generalized inverse matrix, and the calculation for deriving the concentration ratio or the signal amount ratio is carried out by: - Apply from the left and C - 2. The light detection device according to claim 1, wherein the calculation is an operation for deriving s or an equivalent operation.

3. The matrix C is a plurality of matrices C containing only crosstalk information between some of the illuminant carriers or dyes. n 3. The optical detection device according to claim 2, characterized in that the optical detection device is a combination of the following components:

4. The optical detection device according to claim 1, characterized in that the spatial filter is an optical fiber array having a plurality of optical fibers provided corresponding to the respective light emitting points of the plurality of light emitting body holders.

5. The optical detection device according to claim 1, characterized in that said spatial filter has a plurality of pinholes or slits provided corresponding to the respective light emitting points of said plurality of light emitting body holders.

6. The plurality of types of light-emitting elements are sequentially passed through each of the plurality of light-emitting element holders, and an output of the optical sensor is recorded in a state in which a single light-emitting element among the plurality of types of light-emitting elements is emitting light in a single holder among the plurality of light-emitting element holders. The outputs of the optical sensor are arranged to generate a matrix C, and a general inverse matrix C of the matrix C is calculated. － [C t C] -1 C t 3. The light detection device according to claim 2, wherein the light detection signal is calculated by a calculation equivalent thereto and stored.

7. The crosstalk information is expressed by the inverse matrix G of the matrix G, where G is a matrix representing the crosstalk generated by the photodetector, and F is a matrix representing the spectral crosstalk generated by the overlap of the emission spectra of the dyes. -1 And, [F t F] -1 F t Or the generalized inverse matrix F of the matrix F obtained by an equivalent operation - Calculation F using - G -1 2. The light detection device according to claim 1, wherein the dye concentration ratio or signal amount ratio is obtained by a calculation equivalent thereto.

8. The optical detection device according to claim 7, characterized in that the matrix G is obtained by introducing monochromatic light corresponding to a wavelength detection division of the optical sensor in sequence to each emission capture position of the spatial filter.

9. The light detection device according to claim 7, characterized in that the matrix F is obtained by sequentially passing the plurality of types of light emitters through all or a portion of the plurality of light emitter holders.

10. The photodetection device according to claim 7, characterized in that the matrix F is generated from emission spectrum information of the plurality of types of light-emitting bodies stored inside the computer.

11. The optical detection device according to claim 2 or 7, characterized in that the crosstalk information is acquired by connecting a calibration light source to the spatial filter, and the calibration light source introduces calibration light sequentially to each emission capture position of the spatial filter.

12. The optical detection device according to claim 11, wherein the calibration light source introduces light having the same spectrum as the emission spectrum of the plurality of types of light emitters.

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

14. The optical detection device according to claim 1, further comprising a warning display when the output from the optical sensor becomes saturated.

15. A signal processing method comprising: preparing an optical detection device comprising: a plurality of replaceable light-emitting body holders inside which a plurality of types of light-emitting bodies emit light; an optical sensor having a plurality of detection channels for detecting light emission from the plurality of types of light-emitting bodies in a plurality of wavelength bands; a spatial filter for fixing the incident position of the light emission from the plurality of types of light-emitting bodies with respect to the optical sensor; and a computer for processing a signal output from the optical sensor; measuring the light emission from the plurality of types of light-emitting bodies using the optical sensor; calculating outputs from the optical sensor corresponding to the plurality of wavelength bands for each of a plurality of light-emitting points from which the plurality of types of light-emitting bodies emit light, using crosstalk information for reducing crosstalk existing between the plurality of detection channels stored in the computer, thereby reducing crosstalk existing between the detection channels of the optical sensor, and deriving a concentration ratio or signal amount ratio of each of the plurality of types of light-emitting bodies for each of the plurality of light-emitting body holders; and performing the calculation using the crosstalk information even if the light-emitting body holder is replaced.

16. The crosstalk information is expressed as a generalized inverse matrix C of a matrix C containing crosstalk information. - or information equivalent thereto, and the calculation for deriving the concentration ratio or the signal amount ratio is carried out by: - Apply from the left and C - 16. The method of claim 15, further comprising: deriving s, or an equivalent operation.

17. The crosstalk information used in the calculation is the inverse matrix G of the matrix G, where G is a matrix representing the crosstalk generated in the photodetector, and F is a matrix representing the spectral crosstalk generated by the overlap of the emission spectra of the dyes. -1 And, [F t F] -1 F t Or the generalized inverse matrix F of the matrix F obtained by an equivalent operation - Calculation F using - G -1 17. The signal processing method according to claim 16, further comprising the step of: obtaining a dye concentration ratio or a signal amount ratio by a calculation equivalent thereto.

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