Multi-capillary electrophoresis device

Abstract

A device that disperses rays of fluorescent light emitted from a plurality of capillaries and uses an image sensor to perform fluorescence measurements in bulk, wherein, where Bm is the number of pixels in a binning area on an image sensor on which a predetermined wavelength band component of each ray of fluorescent light is projected, Bh is the number of hard-binning pixels, Bs is the number of soft-binning pixels, Bm = Bh × Bs, N is the total noise measured when Bm = Bh = Bs = 1, Nr is the readout noise, Nd is the dark current noise, and Ns is the shot noise, a fluorescence measurement with high sensitivity and a high dynamic range is achieved by performing a control such that Bm, Bh, Bs, N, Nr, Nd and Ns satisfy a predetermined relationship.

Description

[Technical field]

[0001] The present disclosure relates to a multi-capillary electrophoresis device. [Background technology]

[0002] Capillary electrophoresis devices are widely used in which a single or multiple capillaries are filled with an electrophoretic separation medium such as an electrolyte solution or an electrolyte solution containing a polymer gel or a polymer, and electrophoretic analysis is performed. The objects of analysis are wide-ranging, from low molecular weight molecules to high molecular weight molecules such as proteins and nucleic acids. There are also many measurement modes, such as a mode in which lamp light is irradiated onto the absorption point of each capillary and the absorption of the lamp light generated when the object of analysis passes through the absorption point is detected, or a mode in which laser light is irradiated onto the emission point of each capillary and the fluorescence or scattered light generated when the object of analysis passes through the emission point is detected. In the following, a detailed explanation will be given using a capillary electrophoresis device for DNA analysis as an example.

[0003] In the capillary electrophoresis device for DNA analysis, a laser beam is irradiated at a location where E capillaries (E is an integer of 1 or more) are arranged on the same plane, forming E light-emitting points arranged on a line. When DNA fragments labeled with G types (G is an integer of 1 or more) of fluorophores pass through the light-emitting points by electrophoresis, the fluorophores are excited by the laser beam irradiation and emit fluorescence. Since these G types of fluorophores have different fluorescence spectra, the type of fluorophor passing through the light-emitting points can be identified by spectroscopic measurement of the fluorescence. That is, in the capillary electrophoresis device for DNA analysis, the fluorescence emitted from the E light-emitting points arranged on a line is spectroscopically measured, and its time change is measured. To achieve this, the capillary electrophoresis device for DNA analysis is equipped with a multi-color detection optical system as follows. The fluorescence emitted from the E light-emitting points is collimated at once by a first camera lens, the light of the laser wavelength is cut by a long-pass filter, and then the fluorescence is dispersed by a transmission type diffraction grating and imaged on an image sensor by a second camera lens. The image sensor may be a CCD, a CMOS, or any other type of device. Here, the direction of wavelength dispersion is parallel to the long axis of each capillary, i.e., perpendicular to the direction in which each light-emitting point is arranged. Therefore, on the image sensor, the wavelength-dispersed images of the fluorescence from each light-emitting point are arranged parallel to each other without being mixed with each other. Therefore, the fluorescence from each light-emitting point can be measured spectroscopically independently. In addition, the image sensor is arranged so that one axis of the two-dimensional lattice-like arrangement of the pixels of the image sensor is parallel to the wavelength dispersion direction, and the other axis is parallel to the arrangement direction of the light-emitting points. As a result, the intensity distribution of the pixel arrangement along each wavelength-dispersed image gives a fluorescence spectrum. The image sensor captures E wavelength-dispersed images at once with a constant exposure time, and by continuously performing this at constant time intervals, the fluorescence from the E light-emitting points can be spectrally analyzed while analyzing their time-series changes.

[0004] Each wavelength dispersion image is divided into F wavelength bands (hereinafter referred to as bins), and the fluorescence intensity received by a plurality of pixels belonging to each bin is integrated. This integration is called binning. This is called F color detection. The wavelength width of each wavelength band may be 1 nm, 10 nm, or 100 nm, and can be set arbitrarily. In addition, the wavelength width may be different for each of the F wavelength bands. In general, in order to distinguish and quantify G types of fluorophores, F≧G must be satisfied. For each time in the time series, color conversion is performed on the F color detection results, and the individual fluorescence intensity of each of the G types of fluorophores, that is, the concentration of each of the G types of fluorophores, can be obtained. In this disclosure, the fluorescence intensity proportional to the concentration of each fluorophore, that is, the individual fluorescence intensity of each fluorophore, is simply referred to as the concentration.

[0005] For each light-emitting point P(e) (e=1,2,...,E), the emitted fluorescence of phosphor D(g) (g=1,2,...,G) is detected in bin W(f) (f=1,2,...,F). At any time, the concentration of phosphor D(g) at light-emitting point P(e) is Z(g), and the signal intensity integrated in bin W(f) for light-emitting point P(e) is X(f). Here, X is an F-row, 1-column vector with signal intensity X(f) as an element, Z is a G-row, 1-column vector with concentration Z(g) as an element, and Y is an F-row, G-column matrix with Y(f)(g) as an element, and the following formulas (1) to (4) hold. Formulas (1) to (4) are relational expressions between (f) and (g), but are not relational expressions for (e), and hold independently for each light-emitting point P(e). In the case of monochromatic detection with F=1, since F≧G, G=1, and none of X, Y, and Z are vectors or matrices.

[0006]

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[0007]

number

[0008]

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[0009]

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[0010] Here, the element Y(f)(g) of the matrix Y with F rows and G columns represents the signal intensity ratio of the fluorescence emitted by the phosphor D(g) detected in the bin W(f) due to the spectral crosstalk. The element Y(f)(g0) (f=1,2,...,F) of one column of the matrix Y can be determined by making any one type of phosphor D(g0) emit fluorescence alone. Here, since it is generally difficult to strictly control the concentration of the phosphor D(g0), it is convenient to normalize the element Y(f)(g0) of one column. For example, it is good to set the maximum element of the F elements as 1, and to express the other elements as a ratio to the maximum value. Alternatively, it is good to determine the ratio of each element so that the sum of the F elements is 1. In other words, it is good to use the following formula (5).

[0011]

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[0012] Then, by performing the above steps individually for all G types of phosphors D(g), all columns of matrix Y can be determined. Matrix Y is determined only by the characteristics of phosphors D(g) and different bins W(f), and does not change during electrophoretic analysis. Furthermore, as long as the conditions of the optical system, phosphors D(g), bins W(f), etc. are fixed, matrix Y remains constant even for different electrophoretic analyses. Therefore, for each light-emitting point, the concentration Z(g) of phosphor D(g) at each time can be calculated from the signal intensity X(f) of bin W(f) at each time by the following formula (6).

[0013]

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[0014] Here, Y- is the general inverse matrix of Y in row G and column F, and Y - =(Y T ×Y) -1 ×Y T ) is obtained by. When the matrix Y is a square matrix with F = G, Y - is equal to the inverse matrix Y -1 . The operation of Equation (6) is called color conversion or spectral crosstalk cancellation. Equation (1) is a system of simultaneous equations showing the relationship between the concentrations of G types of phosphors, which are unknown, and the known F-color fluorescence intensities, and Equation (6) corresponds to obtaining its solution. Therefore, generally, as described above, the condition of F ≧ G is necessary. If F < G, the solution cannot be uniquely obtained (that is, since multiple solutions may exist), color conversion cannot be performed as in Equation (6).

[0015] A multicolor detection optical system that does not use wavelength dispersion can also be used. For example, in Patent Document 1, the fluorescence emitted from E light-emitting points is individually collimated by E lenses, the light of the laser wavelength is cut by a long-pass filter, and then the fluorescence is divided into light beams of F wavelength bands by F dichroic mirror arrays and imaged on an image sensor. Here, the direction of division is parallel to the long axis of each capillary, that is, perpendicular to the direction in which each light-emitting point is arranged. Therefore, on the image sensor, the E×F multicolor divided images are arranged two-dimensionally without mixing with each other. Therefore, the fluorescence from each light-emitting point can be spectroscopically measured independently. In addition, the image sensor is arranged so that one axis of the two-dimensional lattice-like arrangement of the pixels of the image sensor is parallel to the division direction, and the other axis is parallel to the light-emitting point arrangement direction. The image sensor captures the E×F divided images at once with a constant exposure time, and by continuously performing this at constant time intervals, the fluorescence from the E light-emitting points can be spectroscopically analyzed while analyzing their time-series changes. The fluorescence intensities of the multiple pixels receiving the F divided images for each light-emitting point are integrated. As with the multicolor detection optical system using wavelength dispersion, the pixel area to be integrated is called a bin, and this integration is called binning. Here, it does not matter if there is an area among the divided images that is not included in any bin. The rest is the same as with the multicolor detection optical system using wavelength dispersion, and formulas (1) to (6) also hold. In the following, we will consider the case where a multicolor detection optical system using wavelength dispersion is used, but the same can be said for the case where a multicolor detection optical system not using wavelength dispersion as described above is used.

[0016] As described above, the signal intensity X(f) of the bin W(f) is the sum (binning) of the signal intensities of the individual pixels that make up the bin W(f). The number of pixels that make up the bin W(f) is B m (f). B m (f) is an integer equal to or greater than 1. Here, the signal intensity of pixel j that composes bin W(f) is expressed as Q(j) (j=1, 2, ..., B m (f)), the signal strength X(f) of bin W(f) is expressed by the following equation (7).

[0017]

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[0018] The binning method here can be classified into hard binning and soft binning. However, formula (7) is common to both binning methods. Hard binning is a method in which the image sensor detects B m This is a method to obtain the signal intensity X(f) by adding up the charges accumulated in (f) pixels, converting them into a voltage, and then performing AD conversion. On the other hand, soft binning is a method to obtain the signal intensity X(f) by adding up the charges accumulated in (f) pixels, converting them into a voltage, and then performing AD conversion. mA method of obtaining signal intensity X(f) by adding up the signal values ​​of (f) pixels. For example, soft binning is a method of obtaining signal intensity X(f) by converting the electric charge accumulated in each pixel on an image sensor into a voltage, and integrating the digital data obtained by AD conversion on a computer. As described later, hard binning and soft binning can also be combined to obtain signal intensity X(f). In general, hard binning is known to be suitable for high-sensitivity measurement because it can reduce readout noise and improve sensitivity compared to soft binning. In particular, hard binning is an extremely advantageous method when measuring weak light. Also, hard binning is suitable for high-speed imaging because it can shorten the time to read out the signal intensity X(f) of bin W(f) compared to soft binning. On the other hand, it is known that hard binning reduces the saturation level of the amount of light emitted from the light-emitting point compared to soft binning, thereby reducing the dynamic range. Therefore, by switching from hard binning to soft binning, it is possible to expand the dynamic range, but at the same time, there is a problem that the detection sensitivity decreases. In currently commercially available capillary electrophoresis devices for DNA analysis, sensitivity is more important than dynamic range, so the signal intensity X(f) is obtained by hard binning, not soft binning. However, in recent years, there has been a demand for capillary electrophoresis devices for DNA analysis that achieve both sensitivity and dynamic range. To achieve this, various prior art technologies have been developed, as shown below.

[0019] In Patent Document 2, the exposure time in the image capturing by the image sensor is not constant as described above, but long and short exposure times are alternately repeated. Under the condition that the amount of light emitted from the light emitting point is constant, the image sensor receives more fluorescence in a long exposure time, and therefore the sensitivity is improved. Conversely, in a short exposure time, the image sensor receives less fluorescence, and therefore the sensitivity is reduced, but the saturation level of the amount of light emitted from the light emitting point is increased. In other words, the long exposure time functions as a high sensitivity mode, and the short exposure time functions as a low sensitivity mode. When the amount of light emitted from the light emitting point is small, the emitted fluorescence cannot be detected in the low sensitivity mode, but the emitted fluorescence can be measured well in the high sensitivity mode. On the other hand, when the amount of light emitted from the light emitting point is large, the emitted fluorescence cannot be measured well in the high sensitivity mode because the emitted fluorescence exceeds the saturation level, but the emitted fluorescence can be measured well in the low sensitivity mode. Therefore, by combining the high sensitivity mode and the low sensitivity mode, it is possible to achieve both high sensitivity and a high dynamic range, unlike the case of a single mode (for example, either the high sensitivity mode or the low sensitivity mode).

[0020] In Patent Document 3, an asymmetric image splitting element is added to the above multi-color detection optical system, and the emitted fluorescence from each light-emitting point is split into two wavelength dispersion images of strong fluorescence intensity (hereinafter, strong split image) and weak fluorescence intensity (hereinafter, weak split image), which are imaged and measured simultaneously. The above bins are set for both wavelength dispersion images. Under conditions where the amount of light emitted from the light-emitting point is constant, in the strong split image, the corresponding bin of the image sensor receives more fluorescence, so the sensitivity is improved. Conversely, in the weak split image, the sensitivity is reduced because less fluorescence is received, but the saturation level of the amount of light emitted from the light-emitting point is increased. In other words, the strong split image functions as a high sensitivity mode, and the weak split image functions as a low sensitivity mode. When the amount of light emitted from the light-emitting point is small, the emitted fluorescence cannot be detected in the low sensitivity mode, but the emitted fluorescence can be measured well in the high sensitivity mode. On the other hand, when the amount of light emitted from the light-emitting point is large, the emitted fluorescence cannot be measured well in the high sensitivity mode because the emitted fluorescence exceeds the saturation level, but the emitted fluorescence can be measured well in the low sensitivity mode. Therefore, by combining the high-sensitivity mode and the low-sensitivity mode, it is possible to achieve both high sensitivity and a wide dynamic range, unlike the case of a single mode (for example, either the high-sensitivity mode or the low-sensitivity mode).

[0021] In Patent Document 4, the signal intensity X(f) of the bin W(f) is not obtained only by hard binning as described above, but the signal intensity X(f) of the bin W(f) is obtained by appropriately switching between hard binning and soft binning. Considering the characteristics of the hard binning and soft binning described above, the hard binning functions as a high sensitivity mode, and the soft binning functions as a low sensitivity mode. When the amount of light emitted from the light-emitting point is small, the emission fluorescence cannot be detected in the low sensitivity mode, but the emission fluorescence can be measured well in the high sensitivity mode. On the other hand, when the amount of light emitted from the light-emitting point is large, the emission fluorescence cannot be measured well in the high sensitivity mode because the emission fluorescence exceeds the saturation level, but the emission fluorescence can be measured well in the low sensitivity mode. Therefore, by combining the high sensitivity mode and the low sensitivity mode, it is possible to achieve both high sensitivity and a wide dynamic range, unlike the case of a single mode (for example, either the high sensitivity mode or the low sensitivity mode).

[0022] In Patent Document 5, the bin W(f) is not fixed as described above, but is changed as appropriate. Specifically, when none of the F signal intensities X(f) (f=1, 2, ..., F) exceeds the saturation level, the bin W(f) (f=1, 2, ..., F) is set in the same manner as described above (hereinafter referred to as full hard binning), but when any of them exceeds the saturation level, the corresponding bin W(f) is replaced with zero and invalidated (hereinafter referred to as partial hard binning). For example, when the bin W(f0) exceeds the saturation level, all elements Y(f0)(g) (g=1, 2, ..., G) in the f0 row of the matrix Y in Equation (3) are set to zero. Under the condition that the amount of light emitted from the light-emitting point is constant, in full hard binning, the entire bin receives more fluorescence, so the sensitivity is improved. Conversely, in partial hard binning, the entire bin receives less fluorescence, so the sensitivity is reduced, but the saturation level of the amount of light emitted from the light-emitting point is increased. In other words, full hard binning functions as a high sensitivity mode, and partial hard binning functions as a low sensitivity mode. When the amount of light emitted from the light-emitting point is small, the emitted fluorescence cannot be detected in the low sensitivity mode, but can be measured well in the high sensitivity mode. On the other hand, when the amount of light emitted from the light-emitting point is large, the emitted fluorescence exceeds the saturation level, so the emitted fluorescence cannot be measured well in the high sensitivity mode, but can be measured well in the low sensitivity mode. Therefore, by combining the high sensitivity mode and the low sensitivity mode, it is possible to achieve both high sensitivity and a wide dynamic range, unlike the case of a single mode (for example, either the high sensitivity mode or the low sensitivity mode).

[0023] All of Patent Documents 2 to 5 described above combine a high sensitivity mode and a low sensitivity mode to achieve a high dynamic range while maintaining high sensitivity. Although there are differences in the way the two modes are combined, such as alternately switching the modes, switching the modes appropriately based on the measured signal, or performing both modes simultaneously, the basic features are common to all of them. Furthermore, this method is effective not only for capillary electrophoresis devices for DNA analysis, but also for general analysis methods and analysis devices that perform electrophoresis using a single or multiple capillaries and measure the fluorescence emitted by multiple types of fluorescent materials, the scattered light scattered by multiple types of scatterers, or the absorbance absorbed by multiple types of absorbers while identifying each of them using an image sensor or a line sensor.

[0024] On the other hand, many commercially available digital cameras, such as digital cameras used in smartphones, are able to achieve a high dynamic range while maintaining high sensitivity by combining a high sensitivity mode and a low sensitivity mode. This is generally called HDR (High Dynamic Range) imaging. Typically, as in Patent Document 2, an image is captured with a long exposure time in the high sensitivity mode and a short exposure time in the low sensitivity mode, and these images are combined to synthesize an image with a high dynamic range. [Prior art documents] [Patent documents]

[0025] [Patent Document 1] Patent No. 6820907 [Patent Document 2] Patent No. 4823522 [Patent Document 3] Patent No. 6286028 [Patent Document 4] Patent No. 6093274 [Patent Document 5] U.S. Patent No. 10902593 Summary of the Invention [Problem to be solved by the invention]

[0026] The method of achieving both high sensitivity and a wide dynamic range by combining a high sensitivity mode and a low sensitivity mode as shown in Patent Documents 2 to 5 works when the measurement target is a single phosphor (G=1). Or, even if the measurement target is multiple phosphors (G≧2), this method works when there is no need to distinguish between them. However, detailed studies by the present inventors have revealed that this method does not work when the measurement target is multiple phosphors (G≧2) and they need to be distinguished and analyzed, as shown below.

[0027] As the simplest example, let us assume the following: E=1, and one capillary is the measurement target. F=2, and the wavelength dispersion image of the fluorescence emitted from the emission point on the capillary is measured in two bins, W(1) and W(2), and two-color detection is performed. G=2, and the measurement targets are fluorophores D(1) and D(2). Due to the difference in the fluorescence spectra of fluorophores D(1) and D(2), the emitted fluorescence of fluorophores D(1) is measured in a ratio of 3:2 in bins W(1) and W(2), and the emitted fluorescence of fluorophores D(2) is measured in a ratio of 2:3 in bins W(1) and W(2). In high sensitivity mode, the fluorescence intensity of bins W(1) and W(2) can each be measured in the range of 10 to 100, and in low sensitivity mode, the fluorescence intensity of bins W(1) and W(2) can each be measured in the range of 100 to 1000. The low sensitivity mode is realized by multiplying the exposure time of the high sensitivity mode by 1 / 10. Here, the fluorescence intensity is in arbitrary units. However, to align the measurement results in the high sensitivity mode and the low sensitivity mode, the measurement signal in the low sensitivity mode is multiplied by 10 on the computer. In this case, the dynamic range of the high sensitivity mode and the low sensitivity mode is only one digit (10-100 and 100-1000), respectively, but by combining these modes, it is expected that the dynamic range can be expanded to two digits (10-1000) while maintaining the sensitivity (lower detection limit = 10).

[0028] First, when the emission fluorescence intensity of phosphor D(1) is 50 and the emission fluorescence intensity of phosphor D(2) is 0, in the high sensitivity mode the measured fluorescence intensity of bin W(1) is 30 and the measured fluorescence intensity of bin W(2) is 20, and in the low sensitivity mode the measured fluorescence intensity of bin W(1) is 0 and the measured fluorescence intensity of bin W(2) is 0. At this time, by performing color conversion, phosphor D(1) is measured as having an emission fluorescence intensity of 50 in the high sensitivity mode, but is not measured in the low sensitivity mode because it is below the lower detection limit (emission fluorescence intensity is measured as 0). Of course, the emission fluorescence of phosphor D(2) is not measured in either mode (emission fluorescence intensity is measured as 0).

[0029] Next, when the emission fluorescence intensity of phosphor D(1) is 500 and the emission fluorescence intensity of phosphor D(2) is 0, in the high-sensitivity mode, the measured fluorescence intensities of bins W(1) and W(2) are both saturated, and in the low-sensitivity mode, the measured fluorescence intensity of bin W(1) is 300 and the measured fluorescence intensity of bin W(2) is 200. At this time, phosphor D(1) is unmeasurable (emission fluorescence intensity is unknown) in the high-sensitivity mode because bins W(1) and W(2) are saturated, but by performing color conversion, it is measured as having an emission fluorescence intensity of 500 in the low-sensitivity mode. Phosphor D(2) is unmeasurable (emission fluorescence intensity is unknown) in the high-sensitivity mode because bins W(1) and W(2) are saturated, and is not measured in the low-sensitivity mode (it is measured as having an emission fluorescence intensity of 0).

[0030] In contrast, when the emission fluorescence intensity of fluorophore D(1) is 500 and the emission fluorescence intensity of fluorophore D(2) is 50, in the high-sensitivity mode, the measured fluorescence intensity of bin W(1) and bin W(2) are both saturated, and in the low-sensitivity mode, the measured fluorescence intensity of bin W(1) is 300 and the measured fluorescence intensity of bin W(2) is 200. At this time, in the high-sensitivity mode, fluorophore D(1) is unmeasurable (emission fluorescence intensity is unknown) because bins W(1) and W(2) are saturated, but by performing color conversion, it is measured as having an emission fluorescence intensity of 500 in the low-sensitivity mode. In the high-sensitivity mode, fluorophore D(2) is unmeasurable (emission fluorescence intensity is unknown) because bins W(1) and W(2) are saturated, and it is not measured in the low-sensitivity mode (its emission fluorescence intensity is measured as 0).

[0031] From the above, when the emission fluorescence intensity of phosphor D(1) is 500, the measurement result is the same when the emission fluorescence intensity of phosphor D(2) is 0 and when it is 50, and it is not possible to distinguish between the two. In general, when the emission fluorescence intensity of phosphor D(1) is 100-1000 and the emission fluorescence intensity of phosphor D(2) is 10-100, phosphor D(2) cannot be measured. Similarly, when the emission fluorescence intensity of phosphor D(2) is 100-1000 and the emission fluorescence intensity of phosphor D(1) is 10-100, phosphor D(1) cannot be measured. In other words, when the measurement target is only either phosphor D(1) or phosphor D(2), the emission fluorescence intensity of 10-1000 can be measured, but when the measurement target is phosphor D(1) and phosphor D(2), only the emission fluorescence intensity of 100-1000 (or the emission fluorescence intensity of 10-100) can be measured. Therefore, it has become clear that the method of achieving both high sensitivity and wide dynamic range by combining a high sensitivity mode and a low sensitivity mode as shown in Patent Documents 2 to 5 works when the measurement target is a single fluorophore (G=1), but does not work when the measurement target is multiple fluorophores (G≧2) that need to be identified and analyzed.

[0032] On the other hand, in the case of HDR installed in a digital camera of a smartphone, a wide variety of light emitters, light absorbers, and scatterers are measured, so G≧2 may occur. However, these measurement objects are not usually identified and analyzed. For example, when a digital camera captures a landscape containing a yellow car, yellow light is incident on the pixel at the position of the image of the car on the image sensor, and the color of the car is recognized as yellow. However, the combination of light emitters, absorbers, and scatterers from which the yellow light originates is not resolved, and the composition ratio of each is not measured. For example, it is not identified whether yellow light is a combination of red light and green light, or pure yellow light. In other words, in the case of HDR installed in a digital camera, G≧2 may occur, but since multiple measurement objects are not identified and analyzed, the expansion of the dynamic range is not hindered.

[0033] Therefore, in this disclosure, we propose a method for achieving both high sensitivity and a wide dynamic range in an analysis method and analysis device, such as a capillary electrophoresis device for DNA analysis, that performs electrophoresis using a single or multiple capillaries and measures the fluorescence emitted by multiple types of fluorescent materials, the scattered light scattered by multiple types of scattering materials, or the absorbance absorbed by multiple types of absorbers while identifying each of them using an image sensor or a line sensor.We propose a method that achieves both high sensitivity and high dynamic range in a single mode, rather than the methods of combining a high sensitivity mode and a low sensitivity mode as in Patent Documents 2 to 5. [Means for solving the problem]

[0034] Specifically, in the multi-capillary electrophoresis device of the present disclosure, the composition of the noise measured by controlling the composition of the electrophoretic separation medium filled in the capillaries, the composition of the sample, the wavelength and intensity of the laser beam, the configuration of the multi-color detection optical system, the exposure time, the bin settings, the type and temperature of the image sensor, and the like are set to predetermined conditions, and the number of pixels B constituting the bin W(f) is determined. m (f) and the number of soft-binned pixels in bin W(f) B sBy setting each of (f) within a predetermined optimum range, it is possible to achieve both high sensitivity and a wide dynamic range.

[0035] Further features related to the present disclosure will be apparent from the description of the present specification and the accompanying drawings. Also, the aspects of the present disclosure are achieved and realized by the elements and combinations of various elements and the following detailed description and the appended claims. The description of the present specification is merely exemplary and is not intended to limit the scope or application of the present disclosure in any way. Effect of the Invention

[0036] According to the present disclosure, in an analysis method and an analysis device, including a capillary electrophoresis device for DNA analysis, in which electrophoresis is performed using a single or multiple capillaries, and fluorescence emitted by multiple types of fluorescent materials, scattered light scattered by multiple types of scattering materials, or light absorbed by multiple types of absorbers are measured while being identified by an image sensor or a line sensor, it is possible to achieve both high sensitivity and a wide dynamic range. This makes it possible to analyze samples with a wide range of concentrations without adjusting the concentration. Alternatively, it makes it possible to analyze samples containing multiple components with significantly different concentrations.

[0037] Problems, configurations and effects other than those described above will become apparent from the following description of the embodiments. [Brief description of the drawings]

[0038] [Figure 1] FIG. 1 is a diagram illustrating the configuration of a multi-capillary electrophoresis apparatus. [Figure 2A] FIG. 1 is a diagram showing an example of the configuration of a multi-color detection optical system using wavelength dispersion. [Figure 2B] 2 is a schematic diagram of an image captured by an image sensor. FIG. [Diagram 3] FIG. [Figure 4] FIG. 2 is a diagram showing an enlarged view of a wavelength dispersion image and a pixel configuration in the vicinity thereof. [Diagram 5] FIG. 13 is a diagram showing a pixel configuration around an enlarged view of a wavelength dispersion image. [Figure 6] FIG. 13 is a diagram showing a bin configuration around an enlarged view of a wavelength dispersion image. [Figure 7] FIG. 13 is a diagram showing a hard binning configuration around an enlarged view of a wavelength dispersion image. [Figure 8] FIG. 13 shows another hard binning configuration around the enlarged view of a wavelength dispersion image. [Figure 9] FIG. 13 shows another hard binning configuration around the enlarged view of a wavelength dispersion image. [Figure 10] FIG. 13 shows another hard binning configuration around the enlarged view of a wavelength dispersion image. [Figure 11A] FIG. 13 is a diagram showing the relationship between the number of hard binning pixels and the lower limit of detection, the upper limit of detection, and the dynamic range. [Figure 11B] FIG. 13 is a diagram showing the relationship between the number of hard binning pixels and the lower limit of detection, the upper limit of detection, and the dynamic range. [Figure 11C] FIG. 13 is a diagram showing the relationship between the number of hard binning pixels and the lower limit of detection, the upper limit of detection, and the dynamic range. [Figure 12A] FIG. 13 is a diagram showing the relationship between the number of soft binning pixels and the lower limit of detection, the upper limit of detection, and the dynamic range. [Figure 12B] FIG. 13 is a diagram showing the relationship between the number of soft binning pixels and the lower limit of detection, the upper limit of detection, and the dynamic range. [Figure 12C] FIG. 13 is a diagram showing the relationship between the number of soft binning pixels and the lower limit of detection, the upper limit of detection, and the dynamic range. [Figure 13A] FIG. 1 is a diagram showing the relationship between the soft binning rate and the lower limit of detection, the upper limit of detection, and the dynamic range. [Figure 13B] FIG. 1 is a diagram showing the relationship between the soft binning rate and the lower limit of detection, the upper limit of detection, and the dynamic range. [Figure 13C] FIG. 1 is a diagram showing the relationship between the soft binning rate and the lower limit of detection, the upper limit of detection, and the dynamic range. [Figure 14A] FIG. 13 is a diagram showing the relationship between the number of hard binning pixels and the lower limit of detection, the upper limit of detection, and the dynamic range under other conditions. [Figure 14B] FIG. 13 is a diagram showing the relationship between the number of hard binning pixels and the lower limit of detection, the upper limit of detection, and the dynamic range under other conditions. [Figure 14C] FIG. 13 is a diagram showing the relationship between the number of hard binning pixels and the lower limit of detection, the upper limit of detection, and the dynamic range under other conditions. [Figure 15A] FIG. 13 is a diagram showing the relationship between the number of soft binning pixels and the lower limit of detection, the upper limit of detection, and the dynamic range under other conditions. [Figure 15B] FIG. 13 is a diagram showing the relationship between the number of soft binning pixels and the lower limit of detection, the upper limit of detection, and the dynamic range under other conditions. [Figure 15C] FIG. 13 is a diagram showing the relationship between the number of soft binning pixels and the lower limit of detection, the upper limit of detection, and the dynamic range under other conditions. [Figure 16A] FIG. 13 is a diagram showing the relationship between the number of hard binning pixels and the lower limit of detection, the upper limit of detection, and the dynamic range under other conditions. [Figure 16B] FIG. 13 is a diagram showing the relationship between the number of hard binning pixels and the lower limit of detection, the upper limit of detection, and the dynamic range under other conditions. [Figure 16C] FIG. 13 is a diagram showing the relationship between the number of hard binning pixels and the lower limit of detection, the upper limit of detection, and the dynamic range under other conditions. [Figure 17A] FIG. 13 is a diagram showing the relationship between the shot noise ratio and the number of soft-binning pixels that satisfies high sensitivity conditions and high dynamic range conditions. [Figure 17B] FIG. 13 is a diagram showing the relationship between the shot noise ratio and the number of soft-binning pixels that satisfies high sensitivity conditions and high dynamic range conditions. [Figure 17C] FIG. 13 is a diagram showing the relationship between the number of binning pixels and the soft binning rate that satisfies a first high sensitivity condition and a first high dynamic range condition. [Figure 17D] FIG. 13 is a diagram showing the relationship between the number of binning pixels and the soft binning rate that satisfies a first high sensitivity condition and a first high dynamic range condition. [Figure 17E] FIG. 13 is a diagram showing the relationship between the number of binning pixels and the soft binning rate that satisfies a first high sensitivity condition and a first high dynamic range condition. [Figure 17F] FIG. 13 is a diagram showing the relationship between the number of binning pixels and the soft binning rate that satisfies a second high sensitivity condition and a second high dynamic range condition. [Figure 17G] FIG. 13 is a diagram showing the relationship between the number of binning pixels and the soft binning rate that satisfies a second high sensitivity condition and a second high dynamic range condition. [Figure 17H] FIG. 13 is a diagram showing the relationship between the number of binning pixels and the soft binning rate that satisfies a second high sensitivity condition and a second high dynamic range condition. [Figure 18A] FIG. 13 is a diagram showing the relationship between the number of soft binning pixels in consideration of spatial crosstalk and the lower limit of detection, the upper limit of detection, and the dynamic range. [Figure 18B] FIG. 13 is a diagram showing the relationship between the number of soft binning pixels in consideration of spatial crosstalk and the lower limit of detection, the upper limit of detection, and the dynamic range. [Figure 18C] FIG. 13 is a diagram showing the relationship between the number of soft binning pixels in consideration of spatial crosstalk and the lower limit of detection, the upper limit of detection, and the dynamic range. [Figure 18D] FIG. 13 is a diagram showing the relationship between the number of soft binning pixels in consideration of spatial crosstalk and the lower limit of detection, the upper limit of detection, and the dynamic range. [Figure 18E] FIG. 13 is a diagram showing the relationship between the number of soft binning pixels in consideration of spatial crosstalk and the lower limit of detection, the upper limit of detection, and the dynamic range. [Figure 18F] FIG. 13 is a diagram showing the relationship between the number of soft binning pixels in consideration of spatial crosstalk and the lower limit of detection, the upper limit of detection, and the dynamic range. [Figure 19A] FIG. 13 is a diagram showing the relationship between the number of soft binning pixels and the lower limit of detection, the upper limit of detection, and the dynamic range in consideration of spatial crosstalk under other conditions. [Figure 19B] FIG. 13 is a diagram showing the relationship between the number of soft binning pixels and the lower limit of detection, the upper limit of detection, and the dynamic range in consideration of spatial crosstalk under other conditions. [Figure 19C] FIG. 13 is a diagram showing the relationship between the number of soft binning pixels and the lower limit of detection, the upper limit of detection, and the dynamic range in consideration of spatial crosstalk under other conditions. [Figure 19D] FIG. 13 is a diagram showing the relationship between the number of soft binning pixels and the lower limit of detection, the upper limit of detection, and the dynamic range in consideration of spatial crosstalk under other conditions. [Figure 19E] FIG. 13 is a diagram showing the relationship between the number of soft binning pixels and the lower limit of detection, the upper limit of detection, and the dynamic range in consideration of spatial crosstalk under other conditions. [Figure 19F] FIG. 13 is a diagram showing the relationship between the number of soft binning pixels and the lower limit of detection, the upper limit of detection, and the dynamic range in consideration of spatial crosstalk under other conditions. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0039] Sensitivity and dynamic range analysis An embodiment of the present disclosure will be described by taking the above-mentioned capillary electrophoresis device for DNA analysis as an example. In this device, the bins W(f) (f=1, 2, ..., F) for each light-emitting point are designed to measure the fluorescence of a desired specific wavelength band in the wavelength dispersion image at a fixed exposure time (for example, the bin W(1) is designed to measure the fluorescence of a wavelength band of 500 to 510 nm, the bin W(2) is designed to measure the fluorescence of a wavelength band of 510 to 520 nm, etc.). Here, there may be an area (pixel) in each wavelength dispersion image that is not included in any bin. Alternatively, depending on the binning conditions described later, the same area (pixel) may be included in multiple bins. In other words, different multiple bins may overlap each other on the image sensor. On the other hand, the size and shape of the wavelength dispersion image of the fluorescence emitted from each light-emitting point or the wavelength dispersion image of the fluorescence of a specific wavelength band can be changed by the configuration of the multi-color detection optical system. For example, by making the focal length of the second camera lens longer (or shorter) than the focal length of the first camera lens, each wavelength dispersion image can be enlarged (or reduced) (when both focal lengths are equal, the image is formed at the same magnification). In addition, by increasing (or decreasing) the grating frequency of the transmission diffraction grating, the wavelength dispersion distance on the image sensor can be enlarged (or reduced). In other words, the size and shape of the image on the image sensor of the fluorescence of a specific wavelength band emitted from the light emitting point can be changed by the configuration of the multi-color detection optical system. Furthermore, by using an image sensor with a large (or small) pixel size, the wavelength width per pixel on the wavelength dispersion image can be increased (or decreased). As described above, by controlling the configuration of the multi-color detection optical system, the pixel area and the number of pixels B on the image sensor onto which the image of the fluorescence of a specific wavelength band of bin W(f) is projected can be changed. m (f) can be changed. Such a study has never been done before and is unique to this disclosure. In the following, for simplicity, the bin W(f) and the number of pixels B m The (f) in (f) is omitted, but the meaning is the same.

[0040] In the present disclosure, any image sensor in which pixels are arranged two-dimensionally can be used. As a representative example, a CCD image sensor or a CMOS image sensor can be used. In addition, any AD conversion can be used to convert the amount of charge accumulated according to the amount of light irradiated on each pixel of these image sensors into a digital signal, regardless of the binning conditions described below. In general, if the number of bits of AD conversion used is small, the resolution or accuracy of the digital signal may be insufficient, and the dynamic range may be small. On the other hand, if the number of bits of AD conversion is large, the resolution or accuracy of the digital signal can be increased, but high-speed analysis becomes difficult because the time required for AD conversion increases. In addition, if the number of bits of AD conversion is large, there is a problem that the power consumption of the image sensor and the control board increases and the manufacturing cost of these increases. In the present disclosure, as described below, a method is proposed to improve the sensitivity and dynamic range of the analysis by optimizing the binning conditions. This means that the resolution or accuracy of the digital signal can be increased by optimizing the binning conditions. Therefore, by using this method, it is possible to overcome the above-mentioned disadvantages due to the small number of bits of AD conversion and at the same time enjoy the above-mentioned advantages. The number of bits of AD conversion of image sensors used for various analyses is usually 16 bits or more. In contrast, according to the present disclosure, it is possible to achieve both analytical sensitivity and dynamic range while using AD conversion of 14 bits or less. In addition, it is possible to achieve both analytical sensitivity and dynamic range while using AD conversion of 12 bits or less, or even 10 bits or less, to enable high-speed analysis, and further to reduce the power consumption and manufacturing costs of the image sensor and control board.

[0041] As mentioned above, the number of pixels in bin W is B m There are two binning methods for accumulating the signal of B: hard binning and soft binning. s There are hard binning areas, and the number of pixels in each hard binning area is B hIn this case, the number of pixels for soft binning is B s Then, B m =B h ×B s In other words, the number of pixels for soft binning, B s does not necessarily represent the number of physical pixels in the bin W, but represents the number of pixel information (read count) output from the image sensor for the bin W. Also, 1≦B h ≦B m , 1≦B s ≦B m And B h and B s are all integers. Here, B s Each hard binned region has an equal number of pixels, B h It is assumed that the system is composed of B s The hard binning regions may be composed of different numbers of pixels, in which case B s The average number of pixels in the hard binned areas is B. h In this case, the number of pixels B h is not necessarily an integer. The formulas shown below are valid in this case as well. However, in the following, unless otherwise specified, B s Each hard binned region has an equal number of pixels, B h It is assumed that the system is configured as follows.

[0042] Based on the above, we perform noise analysis and sensitivity analysis. The following applies to any bin W. Noise in optical measurement using an image sensor is classified into three types: readout noise, dark current noise, and shot noise. In addition, when analyzing a sample, such as in capillary electrophoresis, there is also sample noise derived from the sample, but sample noise will be ignored here. Assume that imaging using an image sensor is performed repeatedly with a constant exposure time. Let the readout noise of one pixel be N r The dark current noise of one pixel is N d Let N be the shot noise of the background light emitted from the light source and measured in bin W. sHere, background light is the light emitted from the light emitting point and measured, minus the fluorescence to be measured. The above noise does not depend on the binning method, so it does not change even if the binning method is changed. In this case, the number of pixels in bin W is B m Let the number of pixels in the hard binning of bin W be B h Let the number of pixels in bin W be B s And B m =B h ×B s Then, the total noise N of the signal intensity X integrated in the bin W is expressed by equation (8).

[0043]

number

[0044] B m =B h =B s When B = 1, i.e., when the bin W consists of a single pixel, the total noise is the square root of the sum of the squares of the three noise types, as is well known. However, when B m =B h =B s When B is not 1, each noise is multiplied by its own coefficient, as shown in equation (8). First, the read noise is added each time data is read from the image sensor. Therefore, the number of pixels for hard binning B h Regardless of the number of N in Equation (8), the readout noise is added for each hard binning area. r 2 In the section, "Number of hard binning areas" = "Number of readouts" corresponds to the number of soft binning pixels B s The number of pixels B of hard binning is independent of the number of readouts. h Next, the dark current noise is added by the number of pixels binned, regardless of whether it is hard binning or soft binning. Therefore, N in equation (8) d 2 In the section, B h and B sFinally, shot noise is a noise inherent in the light emitted from the light source and measured, and is therefore unrelated to binning, whether hard binning or soft binning. For example, if light in a specific wavelength range is emitted from a light source, the B m =B h =B s Even when the light is incident on one pixel with B = 1, m =B h ×B s If the light is incident on 100 pixels, i.e., 100 pixels = 10 × 10 = 100, the total amount of light and the inherent noise should be the same. s 2 In the section, B h and B s None of the above is applied.

[0045] Another important point is that, as mentioned above, the number of pixels in bin W is B m The total amount of light in a specific wavelength band within bin W is constant, regardless of the amount of light in the specific wavelength band, i.e., the signal S is constant. Here, the signal S indicates the amount of light in a specific wavelength band emitted from the measurement target and measured with a constant exposure time. As described above, the total noise N and signal S of the integrated signal X of bin W and the number of pixels B m , of which B is the number of pixels for hard binning h and soft binning pixel count B s This disclosure is the first to formulate the relationship between the above.

[0046] Here, the dark current noise ratio b is expressed by equation (9).

[0047]

number

[0048] Moreover, the shot noise ratio c is expressed by equation (10).

[0049]

number

[0050] In this case, equation (8) is expressed as the following equation (11).

[0051]

number

[0052] As is clear from equations (8) and (11), B m =B h =B s When = 1, the total noise is smallest, the S / N is largest, and the sensitivity is highest. The S / N of the signal intensity X integrated in bin W is expressed by the following equation (12) based on equation (11).

[0053]

number

[0054] Here, S indicates a constant amount of light obtained by integrating within bin W, as described above. Of course, this formulation of S / N is the first time that this disclosure has been made. If the amount of light S at which S / N=3 is the lower detection limit LLOD of bin W, then the lower detection limit LLOD is expressed by the following formula (13).

[0055]

number

[0056] Next, we perform dynamic range analysis. If the saturation light amount per pixel of the image sensor is M, the saturation light amount of bin W is B s × M. In other words, the number of reads, i.e. the number of pixels for soft binning, B s The amount of saturated light increases in proportion to the number of pixels B hIt becomes independent. This is because the saturation light amount at the stage where hard binning is performed is M regardless of the number of pixels of hard binning. However, depending on the type of image sensor, there are cases where the saturation light amount read out after performing hard binning is set to be larger than the saturation light amount per pixel, and in such cases, this does not apply. For example, there are cases where the saturation charge amount of the horizontal shift register or the sampling gate is set to be about k = 1 to 10 times larger than the saturation charge amount of the vertical shift register of the CCD. Let the maximum value of the ratio of the saturation light amount read out after performing hard binning to the saturation light amount per pixel be the saturation light amount ratio k (k ≧ 1), then B h When = 1, the saturation light amount of bin W is B s ×M, and when 1 < B h < k, the saturation light amount of bin W is B h ×B s ×M, and when k ≦ B h the saturation light amount of bin W is k × B s ×M. Therefore, generalizing the above, the saturation light amount of bin W is set to α × B s ×M. α is called the saturation light amount coefficient. Here, when B h = 1, α = 1, and when 1 < B h < k, α = B h and when k ≦ B h α = k. However, when the saturation light amount ratio is k = 1, α = 1 regardless of the number of pixels B h of hard binning. These also apply to all the formulas containing α shown later. However, generally k is not an integer, so the above k may be replaced with the integer closest to k. The detection upper limit ULOD of bin W is the same as the saturation light amount of bin W and is represented by the following formula (14).

[0057]

Equation

[0058] At this time, the dynamic range DR is represented by ULOD / LLOD and is represented by formula (15).

[0059]

number

[0060] Similarly, if the number of bits of the AD conversion of the image sensor is BN, the resolution of the digital signal is B s ×BN, B s The accuracy is improved accordingly.

[0061] <Conditions for high sensitivity and high dynamic range> As mentioned above, (B m =)B h =B s The highest sensitivity is achieved when B = 1. However, depending on the configuration of the multi-color detection optical system, h =B s It is generally difficult to make B = 1. h =B s It is important not to significantly reduce the sensitivity compared to the case of B = 1. h =B s It is necessary to obtain an S / N ratio that is at least 1 / 3 of the S / N ratio obtained when φ=1, and the condition for achieving this is defined as the first high sensitivity condition. The first high sensitivity condition is expressed by equation (16) based on equation (12).

[0062]

number

[0063] Here, B h When = 1, equation (16) can be expressed as equation (17).

[0064]

number

[0065] Also, B s When = 1, equation (16) can be expressed as equation (18).

[0066]

number

[0067] Furthermore, to obtain a more practical sensitivity, B h =B s It is necessary to obtain an S / N ratio that is at least 2 / 3 of the S / N ratio obtained when φ=1, and the condition for this is set as the second high sensitivity condition. The second high sensitivity condition is expressed by equation (19) based on equation (12).

[0068]

number

[0069] Here, B h When = 1, equation (19) can be expressed as equation (20).

[0070]

number

[0071] Also, B s When = 1, equation (19) can be expressed as equation (21).

[0072]

number

[0073] On the other hand, by devising the configuration of the multi-color detection optical system and the settings of bins, hard binning, and soft binning, h =B s Compared with the case of B = 1, it is possible to expand the dynamic range DR. In order to obtain a practical dynamic range DR, B h =B sIt is necessary to obtain a dynamic range DR that is three times or more the dynamic range DR obtained when = 1, and the condition for achieving this is defined as the first high dynamic range condition. The first high dynamic range condition is expressed by equation (22) based on equation (15).

[0074]

number

[0075] Here, B h When B = 1, equation (22) is expressed as equation (23). s When = 1, equation (22) has no solution.

[0076]

number

[0077] Furthermore, to obtain a more practical dynamic range DR, B h =B s It is necessary to obtain a dynamic range DR that is 10 times or more the dynamic range DR obtained when = 1, and the condition for this is set as the second high dynamic range condition. The second high dynamic range condition is expressed by equation (24) from equation (15).

[0078]

number

[0079] Here, B h When B = 1, equation (24) is expressed as equation (25). s When = 1, equation (24) has no solution.

[0080]

number

[0081] By satisfying both the high sensitivity condition and the high dynamic range condition, it is possible to achieve both high sensitivity and a wide dynamic range. For example, by satisfying both equation (16) and equation (22), that is, by satisfying the following equation (26), the first high sensitivity condition and the first wide dynamic range condition are satisfied.

[0082]

number

[0083] Alternatively, the second high sensitivity condition and the second wide dynamic range condition are satisfied by satisfying both equation (19) and equation (24), that is, by satisfying the following equation (27).

[0084]

number

[0085] Of course, the same effect can be obtained by satisfying both the formula (16) and the formula (24), or by satisfying both the formula (19) and the formula (22). However, when the dark current noise ratio b, the shot noise ratio c, and the number of pixels B are satisfied, m Depending on the conditions, there may be no solution that satisfies both of these.

[0086] In the above equations (16) to (27), B h and B. s However, at the same time, the preferred range of "1≦B h ≦B m And B h is an integer, 1≦B s ≦B m And B s is an integer' and 'B m =B h ×B s " must be satisfied.

[0087] Next, Bh Let us consider the case where = 1. Equation (8) is transformed into the following equation (28).

[0088]

number

[0089] Here, the combined noise N x is defined as equation (29).

[0090]

number

[0091] Moreover, the shot noise mixing ratio a is expressed by equation (30).

[0092]

number

[0093] Here, a 2 is expressed as equation (31).

[0094]

number

[0095] In this case, equation (28) is transformed into equation (32).

[0096]

number

[0097] Therefore, the S / N ratio of equation (12) is expressed by equation (33).

[0098]

number

[0099] Moreover, the dynamic range DR of equation (15) is expressed by equation (34).

[0100]

number

[0101] In this case, the first high sensitivity condition of equation (17) is expressed by equation (35).

[0102]

number

[0103] Moreover, the second high sensitivity condition of the formula (20) is expressed by the formula (36).

[0104]

number

[0105] On the other hand, the first high dynamic range condition of equation (23) is expressed by equation (37).

[0106]

number

[0107] Moreover, the second high dynamic range condition of equation (25) is expressed by equation (38).

[0108]

number

[0109] <Bin integration> In the above, we have examined the conditions under which high sensitivity and a wide dynamic range can be obtained for each of F bins W(f) (f = 1, 2, ..., F) that measure the fluorescent components of different wavelength bands of the fluorescent light emitted from one light-emitting point. The signal intensity X(f) (f = 1, 2, ..., F) of each bin W(f) is obtained by integrating the signal intensities of the individual pixels that make up the bin W(f) according to equation (7). The obtained signal intensity X(f) constitutes X in equation (2), and the concentration Z(g) (g = 1, 2, ..., G) of G types of fluorophores is derived by the calculation (color conversion) of equation (6). Equation (6) shows that the concentration Z(g) is a linear combination of the signal intensities X(1), X(2), ..., X(F), that is, the concentration Z(g) is a kind of integration of the signal intensities X(1), X(2), ..., X(F). Furthermore, when multiple signal intensities among the signal intensities X(1), X(2), ..., X(F) for the emission of the phosphor D(g) show values ​​close to each other, those signal intensities are integrated equally by formula (6). Therefore, it may be more appropriate to consider conditions for obtaining high sensitivity and a wide dynamic range based on an integrated bin WW combining multiple bins W(f) (or multiple pixels) rather than conditions for obtaining high sensitivity and a wide dynamic range based on individual bins W(f) (or individual pixels). In this case, the integrated bin WW may be simply called bin W. A bin integration method for determining which bins W(f) (or which pixels) are to be incorporated into the integrated bin WW will be considered next. The same bin W(f) may be incorporated into the integrated bins WW for different phosphors.

[0110] The elements Y(1)(g), Y(2)(g), …, Y(F)(g) in the gth column of the matrix Y in equation (3) indicate the ratio of the emission of the fluorophore D(g) to the signal intensity X(1), X(2), …, X(F) of each bin W(f). These elements are normalized so that their sum is 1 according to equation (5), but here we normalize them so that their maximum value is 1. In this case, the elements are denoted as [Y(1)(g)], [Y(2)(g)], …, [Y(F)(g)].

[0111] In the first bin integration method, when [Y(f1)(g)]=1 for each phosphor D(g), only bin W(f1) is incorporated into the integration bin WW. This method follows the previous method of not introducing the integration bin WW. However, unlike the previous methods, for any bin W(f0) where [Y(f0)(g)]≠1, there is no need to consider the conditions under which high sensitivity and high dynamic range can be obtained.

[0112] In the second bin integration method, for each fluorophore D(g), [Y(f j )(g)]≧0.9(j=1,2,…,J), then bin W(f j ) (j = 1, 2, ..., J) into integration bin WW. Similarly, for any bin W(f0) where [Y(f0)(g)] < 0.9, there is no need to consider the conditions for high sensitivity and high dynamic range.

[0113] In the third bin integration method, for each fluorophore D(g), [Y(f j )(g)]≧0.8(j=1,2,…,J), then bin W(f j ) (j = 1, 2, ..., J) into integration bin WW. Similarly, for any bin W(f0) where [Y(f0)(g)] < 0.8, there is no need to consider the conditions for high sensitivity and high dynamic range.

[0114] In the fourth bin integration method, for each fluorophore D(g), [Y(f j )(g)]≧0.5(j=1,2,…,J), then bin W(f j ) (j = 1, 2, ..., J) are incorporated into the integration bin WW. Similarly, for any bin W(f0) where [Y(f0)(g)] < 0.5, there is no need to consider conditions for obtaining high sensitivity and a wide dynamic range. That is, in the first to fourth bin integration methods, bins W(f) that contribute the highest ratio of the emission of phosphor D(g) to the signal intensity X(1), X(2), ..., X(F) are incorporated into the integration bin WW in order.

[0115] In contrast, in the fifth bin integration method, all bins W(f) (f=1, 2, ..., F) for each phosphor D(g) are incorporated into an integration bin WW. Whichever of the first to fifth bin integration methods described above is adopted, it is sufficient that the formulas (8) to (38) derived to achieve both high sensitivity and high dynamic range conditions are established for the adopted integration bin WW. In other words, the integration bin WW is regarded as a virtual single bin, and the number of pixels constituting the integration bin WW is set to B. m The average number of pixels in the (multiple) hard binned regions included in the integration bin WW is B h The number of pixels in the integrated bin WW soft binning is B s And B m =B h ×B s As a result, the high sensitivity condition and the high dynamic range condition are both satisfied as in the past. The fifth bin integration method will be described below.

[0116] For each light-emitting point P(e) (e = 1, 2, ..., E), the number of pixels in the bin W(f) (f = 1, 2, ..., F) is calculated as B m (f), and the number of pixels in the hard binning of bin W(f) is B h (f), and the number of pixels in the soft binning of bin W(f) is B s (f). As before, B m (f)=B h (f)×B s (f), 1≦B h (f)≦B m (f), 1≦B s (f)≦B m (f) and B h (f) and B s (f) is an integer. Assuming that the signal strength XX is integrated in the integration bin WW obtained by integrating bins W(1), W(2), ..., W(F), the total noise N is expressed by equation (39) by modifying equation (11).

[0117]

number

[0118] Here, the dark current noise ratio b, the shot noise ratio c, and the readout noise N r are the same definitions and values ​​as before. If the total amount of light measured in the integration bin WW is the signal S, the S / N ratio in the integration bin WW is expressed by equation (40) by modifying equation (12).

[0119]

number

[0120] Furthermore, if the light amount S at which S / N=3 is the lower detection limit LLOD of the integrated bin WW, then equation (13) can be modified to be expressed as equation (41).

[0121]

number

[0122] Furthermore, the upper detection limit ULOD of the bin WW, that is, the saturation light amount of the bin WW, is expressed by equation (42) by modifying equation (14).

[0123]

number

[0124] Here, the saturation light amount coefficient α and the saturation light amount M per pixel are defined and have the same values ​​as before. At this time, the dynamic range DR is expressed by ULOD / LLOD, and is expressed by equation (43) by modifying equation (15).

[0125]

number

[0126] Here, by defining equations (44) to (47) once again as follows, equations (8) to (38) hold as they are. In fact, by using equations (44) to (47), equations (11) and (39), equations (12) and (40), equations (13) and (41), equations (14) and (42), and equations (15) and (43) become the same equations. These are merely examples, and the same applies to other equations. In other words, when a set of multiple bins W(f) (or multiple pixels) is defined as one integrated bin WW, the conditions for the integrated bin WW to satisfy the high sensitivity condition and the high dynamic range condition are expressed by equations (8) to (38) by defining equations (44) to (47).

[0127]

number

[0128]

number

[0129]

number

[0130]

number

[0131] For the first to fourth bin integration methods, only the integration range of the bin W(f) (f=1, 2, ..., F) changes, and by changing the integration range of equations (44) to (47) accordingly, equations (8) to (38) remain valid as they are.

[0132] <Consideration of exposure time> In the above, we have analyzed the sensitivity and dynamic range when fluorescence measurements using an image sensor are repeatedly performed at a constant exposure time, and clarified the conditions for achieving both high sensitivity and a high dynamic range. In this study, we further control the exposure time to make it easier to achieve both high sensitivity and a high dynamic range, thereby expanding the range of suitable conditions. In general, shortening the exposure time makes it possible to measure stronger luminescence intensity without saturation, and therefore the upper limit of detection ULOD of bin W can be increased. On the other hand, shortening the exposure time increases the number of readings, which increases noise and also increases the lower limit of detection LLOD of bin W. Alternatively, if the number of readings is not increased while shortening the exposure time, the measured signal decreases, and the lower limit of detection LLOD also increases. In other words, shortening the exposure time reduces the sensitivity and, in some cases, the dynamic range may also decrease. Therefore, it is not enough to simply shorten the exposure time, but by setting the optimal exposure time, it is possible to achieve both high sensitivity and a high dynamic range.

[0133] The constant exposure time assumed up until now is the standard exposure time T. The standard exposure time T is equally divided into shortened exposure times t with a division number μ. Alternatively, the standard exposure time T is extended to an extended exposure time t that is 1 / μ times larger. Here, assuming that the data read time from the image sensor is zero and that there is no time loss due to division, the standard exposure time T is expressed by equation (48).

[0134]

number

[0135] μ is positive but not necessarily an integer. When μ ≥ 1, since T ≥ t, t represents the shortened exposure time. When 0 < μ < 1, since T < t, t represents the extended exposure time. Hereafter, whether μ ≥ 1 or 0 < μ < 1, it holds. For simplicity, t will be called the shortened exposure time. In the case of μ ≥ 1, by integrating the signal measured at the shortened exposure time t μ times with a computer, a signal equal to the signal measured at the standard exposure time T can be obtained. Here, the read noise of one pixel per unit exposure time is n r is denoted as, and the dark current noise of one pixel is n d is denoted as, and the shot noise of the background light emitted from the light-emitting point and measured in bin W is n s is denoted as. Further, the dark current noise ratio b0 per unit exposure time is expressed by Equation (49).

[0136]

Equation

[0137] Similarly, the shot noise ratio c0 per unit exposure time is expressed by Equation (50).

[0138]

Equation

[0139] At this time, the read noise of one pixel at the shortened exposure time t is n r and the dark current noise of one pixel is t × n d = t × b0 × n r and the shot noise of all the light emission measured in bin W is t 0.5 × n s = t 0.5 × c0 × n r Therefore, the total noise n at the shortened exposure time t of bin W is expressed by Equation (51) by transforming Equations (8) and (11).

[0140]

Equation

[0141] In contrast, the read noise of one pixel at the standard exposure time T is N r =n r The dark current noise of one pixel is N d = T × n d The shot noise of the total light emission measured in bin W is N s =T 0.5 ×n s In this case, the dark current noise ratio b is expressed by equation (52).

[0142]

number

[0143] Moreover, the shot noise ratio c is expressed by equation (53).

[0144]

number

[0145] Therefore, the total noise N of the signal corresponding to the standard exposure time T obtained by integrating the signals measured for bin W at the shortened exposure time t μ times by a computer is expressed by equation (54) by modifying equations (8) and (11).

[0146]

number

[0147] Thus, the S / N ratio of bin W is expressed by equation (55) by modifying equation (12).

[0148]

number

[0149] Therefore, the lower limit of detection LLOD of bin W is expressed by equation (56) by modifying equation (13).

[0150]

number

[0151] On the other hand, the upper detection limit ULOD for bin W is expressed by equation (57) by modifying equation (14).

[0152]

number

[0153] Therefore, the dynamic range DR is expressed by equation (58) by modifying equation (15).

[0154]

number

[0155] Based on the above, we clarify the conditions for achieving both high sensitivity and high dynamic range by controlling the exposure time. h =B s The comparison targets are the S / N and DR when = 1. The first high sensitivity condition is expressed by equation (59) by modifying equation (16).

[0156]

number

[0157] Also, B h The first high sensitivity condition when =1 is expressed by equation (60) by modifying equation (17).

[0158]

number

[0159] In addition, B s The first high sensitivity condition when =1 is expressed by equation (61) by modifying equation (18).

[0160]

number

[0161] The second high sensitivity condition is expressed by equation (62) by modifying equation (19).

[0162]

number

[0163] Also, B h The second high sensitivity condition when =1 is expressed by equation (63) by modifying equation (20).

[0164]

number

[0165] In addition, B s The second high sensitivity condition when =1 is expressed by equation (64) by modifying equation (21).

[0166]

number

[0167] On the other hand, the first high dynamic range condition is expressed by equation (65) by modifying equation (22).

[0168]

number

[0169] Also, B h The first high dynamic range condition when =1 is expressed by equation (66) by modifying equation (23).

[0170]

number

[0171] The second high dynamic range condition is expressed by equation (67) by modifying equation (24).

[0172]

number

[0173] Similarly to the above, by appropriately combining the above equations (59) to (67), noise conditions and binning conditions for achieving both high sensitivity and a wide dynamic range can be derived.

[0174] <Variations of high dynamic range conditions> In the above, the first high dynamic range condition and the second high dynamic range condition have been considered, but in addition to these, a method of specifying the absolute value of the dynamic range is extremely effective in practice. In currently commercially available capillary electrophoresis devices for DNA analysis, the dynamic range DR is about 1000, and the range of application is limited. In order to obtain a practical dynamic range and expand the range of application, a dynamic range DR of 3000 or more is necessary, and the condition for this is set as the third high dynamic range condition. The third high dynamic range condition is expressed by equation (68).

[0175]

number

[0176] For example, in the above formulas (15), (34), (43), and (58), and the following formulas (81) and (85), when formula (68) holds, the third high dynamic range condition is satisfied. In order to obtain a more practical dynamic range DR and further expand the range of application, a dynamic range DR of 10,000 or more is necessary, and the condition for this is the fourth high dynamic range condition. The fourth high dynamic range condition is expressed by formula (69).

[0177]

number

[0178] For example, in equations (15), (34), (43), and (58) shown above, and equations (81) and (85) shown below, when equation (69) holds, the fourth high dynamic range condition is satisfied.

[0179] [Example 1] <Basic conditions> FIG. 1 is a configuration diagram of a multi-capillary electrophoresis apparatus, which is an example of an analytical apparatus. The multi-capillary electrophoresis apparatus is widely used as an analytical apparatus for performing DNA sequence and DNA fragment analysis. The multi-capillary electrophoresis apparatus includes a capillary 1, a cathode 4, an anode 5, a cathode side buffer 6, an anode side buffer 7, a power supply 8, a pump block 9, a valve 10, a syringe 11, a laser light source 12, a multi-color detection optical system 15, and a computer 100. The computer 100 controls the overall operation of the multi-capillary electrophoresis apparatus. The computer 100 includes a user interface, and can set binning conditions, which will be described later. The computer 100 also executes a program stored in a memory (not shown) to analyze the time series data of the fluorescence detected by the multi-color detection optical system 15, and analyzes the DNA sequence sample.

[0180] In this example, E=4 capillaries 1 were used, and DNA sequencing of a different sample was performed in each capillary 1. The outer diameter of each capillary 1 was 360 μm, and the inner diameter was 50 μm. The DNA sequencing sample consisted of DNA fragments labeled with G=4 types of fluorophores.

[0181] One analysis session was carried out by the following steps (1) to (6). (1) First, the sample injection ends 2 of the E=4 capillaries 1 were immersed in the cathode buffer solution 6, and the sample elution ends 3 were immersed in the anode buffer solution 7 via the polymer block 9. (2) Next, the valve 10 of the pump block 9 was closed, and the piston of the syringe 11 connected to the pump block 9 was pushed down to pressurize the polymer solution Ω1 inside, and the polymer solution Ω1 was filled into each capillary 1 from the sample elution end 3 toward the sample injection end 2. (3) Next, the valve 10 was opened, and different samples were injected into each capillary 1 from the sample injection end 2, and then capillary electrophoresis was started by applying a high voltage between the cathode 4 and the anode 5 by the power source 8. DNA fragments labeled with G=4 types of fluorophores were electrophoresed from the sample injection end 2 to the sample elution end 3. (4) In parallel, a position of each capillary 1 electrophoresed a certain distance from the sample injection end 2 was set as a light emitting point 14, and a laser beam 13 with an output of 5 mW and a wavelength of 505 nm, generated from a laser light source 12, was irradiated simultaneously to each light emitting point 14. Here, the coating of each capillary 1 near the light emitting point 14 was removed in advance, and each capillary 1 near the light emitting point 14 was arranged on the same plane, and the laser beam 13 was narrowed to about φ50 μm and then introduced along the array plane from the side of the above-mentioned array plane. (5) Then, the DNA fragments labeled with G=4 kinds of fluorophores were electrophoresed inside each capillary 1, and when passing through the light-emitting points 14, each labeled fluorophore was excited by irradiation with the laser beam 13 to emit fluorescence. In other words, G=4 kinds of fluorophores were made to emit fluorescence from E=4 light-emitting points 14, and the fluorescence intensity of each was made to change from moment to moment with electrophoresis. (6) Finally, the fluorescence emitted from each light-emitting point 14 was measured by the multicolor detection optical system 15, and the obtained time-series data was analyzed by the computer 100 to perform DNA sequencing of the sample injected into each capillary 1. Here, the size and shape of each light-emitting point 14 is 50 μm square, since the inner diameter of each capillary 1 is 50 μm and the diameter of the laser beam is 50 μm. The multicolor detection optical system 15 is located behind each light-emitting point 14 in FIG. 1.

[0182] Fig. 2A is a diagram showing an example of the configuration of the multi-color detection optical system 15. Fig. 2A depicts the multi-color detection optical system 15 from the side of the arrangement plane of the four capillaries 1, that is, from the direction of the laser light source 12 in Fig. 1. The multi-color detection optical system 15 includes a first camera lens 16, a long-pass filter 17, a transmission diffraction grating 18, a second camera lens 19, and an image sensor 20.

[0183] The fluorescence 22 emitted from the light-emitting point 14 was collimated by a first camera lens 16 with a focal length f1=50 mm, and the light with a laser wavelength of 505 nm was cut by a long-pass filter 17. The fluorescence was then wavelength-dispersed by a transmission diffraction grating 18 with a grating frequency N=600 lines / mm, and imaged at 1:1 magnification on an image sensor 20 by a second camera lens 19 with a focal length f2=50 mm. In this embodiment, a CCD with a pixel size of 24 μm square was used as the image sensor 20. The saturated light amount ratio of the image sensor 20 was k=1. Here, the direction of wavelength dispersion was parallel to the long axis of each capillary 1, that is, perpendicular to the direction in which each light-emitting point 14 was arranged. However, as shown in FIG. 2A, the optical axis 21 of the multicolor detection optical system 15 is bent in the direction of the first-order diffracted light of the transmission diffraction grating 18. The collimated fluorescence 22 has different wavelength components that are wavelength-dispersed as dispersed fluorescence 23, 24, and 25.

[0184] FIG. 2B is a schematic diagram of an image 26 captured by the image sensor 20. FIG. 2B shows a wavelength dispersion image 27 of light emitted from E=4 light-emitting points 14. On the image sensor 20, the wavelength dispersion images 27 of the fluorescence 22 from each light-emitting point 14 are arranged parallel to each other without being mixed with each other. Therefore, the fluorescence 22 from each light-emitting point 14 can be independently spectroscopically measured. In addition, the image sensor 20 is arranged so that one axis of the two-dimensional lattice-like arrangement of the pixels of the image sensor 20 is parallel to the wavelength dispersion direction, and the other axis is parallel to the light-emitting point arrangement direction. In FIG. 2B, the vertical direction is the wavelength dispersion direction, and the horizontal direction is the light-emitting point arrangement direction. As a result, the intensity distribution of the pixel arrangement along each wavelength dispersion image 27 gave the fluorescence spectrum of the fluorescence 22. In the following, a method of analyzing one wavelength dispersion image 27 out of multiple wavelength dispersion images 27 measured simultaneously will be described, but the same analysis method was also applied to the other wavelength dispersion images 27.

[0185] In general, the dispersion angle θ of a wavelength λ (nm) is expressed by equation (70) using a grating frequency N (1 / mm).

[0186]

number

[0187] The dispersion angle per nm of wavelength is given by equation (71).

[0188]

number

[0189] In this case, the dispersion distance (mm) per 1 nm of wavelength on the image sensor 20 is given by equation (72) from equations (70) and (71).

[0190]

number

[0191] In this embodiment, the dispersion distance on the image sensor 20 per 1 nm of light with λ=600 nm is 0.032 mm, or 32 μm, when N=600 lines / mm and f2=50 mm in equation (72). Since the pixel size of the image sensor 20 is 24 μm, a wavelength resolution of 0.75 nm / pixel is obtained.

[0192] The wavelength range of 180 nm from 520 to 700 nm in the wavelength dispersion image 27 is the measurement target, and this wavelength range is divided into 20 wavelength ranges of 9 nm width at equal intervals, and each is set to F = 20 bins W(f) (f = 1, 2, ..., 20). Since the wavelength resolution is 0.75 nm / pixel, light of the 9 nm wide wavelength range is received by 12 pixels in the wavelength dispersion direction. In addition, since the 50 μm square light emitting point 14 is imaged at the same magnification and the pixel size is 24 μm square, light of the 9 nm wide wavelength range is received by 3 pixels in the light emitting point array direction. Therefore, as the binning conditions, each bin W(f) is set to an area of ​​12 pixels in the wavelength dispersion direction and 3 pixels in the light emitting point array direction, and the number of pixels constituting each bin W(f) is set to B m = 12 × 3 = 36. In each bin W(f), the number of pixels in hard binning is B h = 36, soft binning pixels B s = 1. Since the pixel size of the image sensor used in this embodiment is 24 μm square, the size of the above bin W(f) on the image sensor is 0.288 mm × 0.072 mm.

[0193] FIG. 3 shows an enlarged view of the upper end portion of one wavelength dispersion image 27 in the image 26 in FIG. 2B. The following FIGS. 4 to 10 show the same area as FIG. 3 with the same scale. All of them show only a small portion of the image 26 acquired by the image sensor 20. FIG. 4 shows the configuration of pixels 28 in the same area. FIG. 5 shows FIG. 4 without showing the wavelength dispersion image 27. A total of 175 pixels 28 are depicted, 5 pixels in the horizontal direction and 35 pixels in the vertical direction. FIG. 6 shows FIG. 5 with the area of ​​bin W(f) added with a thick line, and shows parts of bins W(1), W(2) and W(3). According to the above binning conditions, each bin W(f) is a B pixel of 3 pixels in the light-emitting point array direction (horizontal direction) and 12 pixels in the wavelength dispersion direction (vertical direction). m In FIG. 7, a hard binning area 7-1 is added to FIG. 6 in gray and diagonal lines in accordance with the above binning conditions. Each bin W(f) is made up of the number of pixels B s = 1 B h = 36 pixels 28 constitute the hard binning area 7-1.

[0194] On the other hand, each of Figs. 8 to 10 shows a binning condition different from the above binning condition. In Fig. 8, each bin W(f) has a pixel count B s = 12 B h In FIG. 9, each bin W(f) is made up of hard binning regions 8-1 to 8-12 each made up of 3 pixels 28. s = 36 B h 8 and 9, each bin W(f) is made up of hard binning regions 9-1 to 9-36 each consisting of one pixel 28. In FIG. 8 and FIG. 9, the number of pixels B h is constant (B h = 3, in Fig. 9 B h = 1), but it does not necessarily have to be constant, as shown in the example in Figure 10. In Figure 10, each bin W(f) is the number of pixels B s = 8 B hThe hard binning regions 10-1 to 10-8 are each made up of 4, 5, 6, 5, 4, 6, 5, and 1 pixel 28. In this case, the average number of pixels constituting the multiple hard binning regions is B h In the case of Figure 10, B h =(4+5+6+5+4+6+5+1) / 8=36 / 8=4.5.

[0195] Figures 7 to 10 show B m Four types of binning conditions for the bin W(f) of B = 36 are shown, but these are merely examples, and various other binning conditions can be set. In addition, the number of pixels 28 constituting the bin W(f) is also set to B m = 36, and can be set to any value.

[0196] The above binning conditions are set by software or firmware, so the current binning conditions can be checked. However, even if the setting value is unknown, the current binning conditions can be checked. For example, assume that only the signal intensity X(f) of bin W(f) can be referenced, or only the concentration Z(g) of phosphor D(g) can be referenced. By irradiating light to each pixel and changing the intensity of the incident light, the response of the signal intensity X(f) or concentration Z(g) to the amount of incident light at each pixel can be checked. As a means of irradiating light to each pixel, there are a method of narrowing a laser beam and irradiating it on an image sensor, and a method of emitting light of a single wavelength from a very small light-emitting point and forming an image on the image sensor. This allows each pixel to be classified into each bin W(f), and the number of pixels B in each bin W(f) can be determined. m In addition, the number of pixels for hard binning B(f) for each bin W(f) can be determined from the response of the signal intensity X(f) or concentration Z(g) when the number of pixels for which light is incident is increased to two, three, etc. h , soft binning pixel count B sIt is possible to identify the binning conditions. Of course, other means may be used to check the binning conditions. From the above results, it is possible to determine whether the current binning conditions are appropriate for achieving both high sensitivity and a wide dynamic range, and to change to appropriate binning conditions as necessary.

[0197] Imaging by the image sensor 20 was repeated with an exposure time of 100 ms and a sampling interval of 150 ms. The signal intensity X(f) (f=1, 2, ..., 20) integrated in bin W(f) was calculated for each time, to obtain the 20-row, 1-column matrix X shown in equation (2) for each time. Also, the 20-row, 4-column matrix Y shown in equation (3) was calculated in advance. Then, the 4-row, 1-column matrix Z shown in equation (4) was calculated for each time by equation (6). That is, G=the concentration Z(g)( g The time-series data was analyzed to determine the change over time in the DNA sequence of the sample injected into each capillary 1.

[0198] The above measurement conditions and the B m =36, B h =36, B s We performed a noise analysis of the image sensor under the binning condition of N = 1. The noise composition was the same for each bin W(f). s The same measurement as above was performed under the condition of count = 0, and the standard deviation of the time series data of the signal intensity X(f) was calculated. The term "count" used in this application is the unit of signal intensity digitally output by the image sensor.

[0199] In addition, the exposure time was changed stepwise in the range of 0 ms to 1000 ms, and the standard deviation was calculated in the same manner. The standard deviation was plotted against the exposure time to obtain an approximate curve, and the vertical intercept was 1.5 counts. In this case, the read noise per W(f) is the vertical intercept of the approximate curve, that is, the standard deviation when the exposure time is 0 ms. On the other hand, the read noise per bin W(f) is calculated by the first term B on the right side of equation (8), s ×Nr 2 Therefore, under these conditions, B s = 1, the read noise of one pixel is N r =1.5 counts was determined.

[0200] Next, the value of the approximation curve above at an exposure time of 100 ms was found to be 1.55 counts. At this time, the left side of equation (8) is N 2 =1.55 2 , the first term on the right hand side is B s ×N r 2 =1.5 2 , the third term on the right hand side is N s 2 = 0, the second term on the right hand side, which gives the dark current noise per bin W(f), is B h ×B s ×N d 2 =1.55 2 -1.5 2 =0.39 2 Under these conditions, B h =36, B s = 1, the dark current noise of one pixel is N d =0.065 counts.

[0201] Finally, light was incident on the image sensor, and the standard deviation of the background light during electrophoretic analysis, i.e., the total noise, was determined to be N = 1.6 counts. In this case, the left side of equation (8) is N 2 =1.6 2 , the first term on the right hand side is B s ×N r 2 =1.5 2 , the second term on the right hand side is B h ×B s ×N d 2 =0.39 2 Therefore, the third term on the right hand side, which gives the shot noise per bin W(f), is N s 2 =1.6 2 -1.5 2 -0.39 2 =0.40 2In other words, the shot noise of the background light emitted from the light emitting point and measured in bin W(f) is N s From the above, the dark current noise ratio in equation (9) was calculated to be b = 0.043, and the shot noise ratio in equation (10) was calculated to be c = 0.27.

[0202] The sensitivity and dynamic range performance of the above was investigated. b = 0.043, c = 0.27 were set in equation (18) and "1 ≤ B h ≦B m ", B h ≦36, this condition (B h =36, B s = 1) satisfied the first high sensitivity condition. Also, b = 0.043 and c = 0.27 were used in equation (21) and "1 ≤ B h ≦B m ", B h ≦36, this condition (B h =36, B s =1) also satisfied the second high sensitivity condition. On the other hand, this condition s = 1, neither the first high dynamic range condition nor the second high dynamic range condition was satisfied. In other words, it was found that under this condition, it is not possible to achieve both the high sensitivity condition and the high dynamic range condition. In the above, since the saturation light amount ratio of the image sensor 20 is k = 1, α = 1 was set.

[0203] In the above, we have used the exposure time to measure the noise composition, N r We have derived , b and c, but the noise composition can be calculated even if the exposure time cannot be changed. It is known that the shot noise of each bin W(f) is proportional to the root of the amount of light incident on the bin W(f). Therefore, it is possible to control the shot noise by changing the amount of incident light. Therefore, the square of the shot noise ratio c 2 , the square of the total noise of the signal intensity X(f) of the bin W(f) is N 2 When plotting, the slope is N r 2 , the vertical axis intercept is (Bs +b 2 ×B m ) ×N r 2 A straight line of N r and the dark current noise ratio b can be derived. In addition, the shot noise ratio c under the actual measurement conditions can be derived from the overall noise N under the actual measurement conditions and the above straight line.

[0204] <Changes to Terms and Conditions> Therefore, by changing the binning conditions, specifically, the settings of the multi-color detection optical system and B m B=36 while keeping the number of pixels for hard binning fixed h and soft binning pixel count B s We investigated whether it would be possible to achieve both high sensitivity and high dynamic range conditions by changing the m = 36, b = 0.043, c = 0.27 into equation (16) and "1 ≤ B s ≦B m And B s is an integer, B s ≦9. Also, B m = 36, b = 0.043, c = 0.27 into equation (22) and "1 ≤ B s ≦B m And B s is an integer, B s ≧9. Therefore, B h =4, B s By setting the condition of B = 9, the formula (26) is satisfied, and the first high sensitivity condition and the first high dynamic range condition are satisfied. m = 36, b = 0.043, c = 0.27 into equation (19) and "1 ≤ B s ≦B m And B s is an integer, B s ≦2. Also, B m = 36, b = 0.043, c = 0.27 into equation (24) and "1 ≤ B s ≦B m And B sWhen x is substituted for an integer, there is no solution. Therefore, equation (27) is never satisfied, and the second high sensitivity condition and the second high dynamic range condition are never satisfied.

[0205] Next, the measurement conditions were changed. The separation medium filled in capillary 1 was changed from polymer solution Ω1 to polymer solution Ω2, and the laser beam power was increased from 5 mW to 20 mW. When a noise analysis similar to that described above was performed under these conditions, the noise composition changed. Furthermore, the noise composition differed depending on bin W(f). Bin W(20) had the smallest total noise of N=4 counts, and bin W(10) had the largest total noise of N=16 counts. In both cases, the readout noise of one pixel was N r = 1.5 counts, dark current noise of one pixel is N d = 0.065 counts did not change. From the above, the shot noise of the background light emitted from the light emitting point and measured in bin W(f) is N s = 3.7 counts, and N s = 16 counts. That is, in each bin W(f), N r and N d is constant, but N s changed in the range of 3.7 to 16 counts. As a result, the dark current noise ratio in equation (9) was b = 0.043, and the shot noise ratio in equation (10) changed in the range of c = 2.5 to 10.7.

[0206] These are expressed as (18) and "1 ≤ B h ≦B m ", then for any shot noise ratio c between 2.5 and 10.7, B h ≦36, this condition (B h =36, B s = 1) satisfied the first high sensitivity condition. h ≦B m ", then for any shot noise ratio c between 2.5 and 10.7, B h ≦36, this condition (B h =36, Bs =1) also satisfied the second high sensitivity condition. On the other hand, this condition s =1, so neither the first nor the second high dynamic range condition was satisfied. That is, it was found that this condition cannot satisfy both the high sensitivity condition and the high dynamic range condition. In the above, the saturated light amount ratio of the image sensor 20 is k=1, so α=1. Meanwhile, the saturated light amount per pixel of the image sensor was M=65000. When c=2.5, DR=5340 according to the formula (15). Therefore, it was found that this condition satisfies the third high dynamic range condition but does not satisfy the fourth high dynamic range condition according to the formulas (68) and (69). On the other hand, when c=10.7, DR=1344 according to the formula (15). Therefore, it was found that this condition does not satisfy either the third or the fourth high dynamic range condition according to the formulas (68) and (69).

[0207] Next, in the same way as above, the multi-color detection optical system and B m =B h ×B s B=36 while keeping the number of pixels for hard binning fixed h and soft binning pixel count B s We investigated whether it would be possible to achieve both high sensitivity and high dynamic range conditions by changing the number of pixels B h is an integer. First, the shot noise ratio is set to c=2.5. m Substituting =36 and b=0.043 into equations (16) and (19), we get "1 ≤ B s ≦B m And B s are integers" s ≦36 and B s ≦10. Also, B m Substituting =36 and b=0.043 into equations (22) and (24), we get "1 ≤ B s ≦B m And B s are integers" s ≥ 4 and Bs ≧19.

[0208] The above is summarized as follows: h , B s The second high sensitivity condition is satisfied when B is (36, 1), (18, 2), or (12, 3). h , B s When the θ is (9, 4), (6, 6), or (4, 9), the second high sensitivity condition and the first high dynamic range condition are satisfied. (B h , B s When (B) is (3, 12) or (2, 18), the first high sensitivity condition and the first high dynamic range condition are satisfied. h , B s When (B ) is (1, 36), the first high sensitivity condition and the second high dynamic range condition are satisfied. Therefore, there is no solution that satisfies the second high sensitivity condition and the second high dynamic range condition. On the other hand, from equations (15), (68), and (69), h , B s ) satisfies both the third and fourth high dynamic range conditions in any of the cases of (18, 2), (12, 3), (9, 4), (6, 6), (4, 9), (3, 12), (2, 18), and (1, 36).

[0209] Next, the shot noise ratio is c = 10.7. m Substituting =36 and b=0.043 into equations (16) and (19), we get "1 ≤ B s ≦B m And B s Under the condition that "is an integer", s ≦36. Also, B m Substituting =36 and b=0.043 into equations (22) and (24), we get "1 ≤ B s ≦B m And B s are integers" s ≥ 3 and B s ≧11. The above is summarized as follows. (Bh , B s When (B) is (36, 1) or (18, 2), the second high sensitivity condition is satisfied. h , B s When the θ is (12, 3), (9, 4), (6, 6), or (4, 9), the second high sensitivity condition and the first high dynamic range condition are satisfied. (B h , B s When (B) is (3, 12), (2, 18) or (1, 36), the second high sensitivity condition and the second high dynamic range condition are satisfied. In the above, the saturation light amount ratio of the image sensor 20 is k=1, so α=1. h, B s ) is (36, 1), and Fig. 8 shows the case of (B h , B s ) is (3, 12), and Fig. 9 shows the case of (B h , B s ) is (1, 36). As shown above, there may be multiple solutions that satisfy the high sensitivity and high dynamic range conditions at the same level. In such cases, the number of pixels for hard binning B h Selecting a solution with a large value is advantageous in that the data read speed from the image sensor can be improved. On the other hand, from equations (15), (68) and (69), (B h , B s It was found that when (B ) is (12, 3), (9, 4), or (6, 6), the third high dynamic range condition is satisfied, but the fourth high dynamic range condition is not satisfied. h , B s It was found that in the cases of (4, 9), (3, 12), (2, 18), and (1, 36), both the third and fourth high dynamic range conditions were satisfied.

[0210] As described above, it has become clear that it is possible to achieve both high sensitivity and high dynamic range conditions by adjusting the binning conditions. However, the binning conditions that satisfy both the high sensitivity and high dynamic range conditions change depending on the noise composition, represented by the dark current noise ratio b and the shot noise ratio c. Depending on the noise composition, the binning conditions may become wider, or there may be no binning conditions that provide a solution.

[0211] <Change in saturation light ratio> Next, the sensitivity and dynamic range performance were similarly examined when the image sensor 20 with a saturation light amount ratio k=1 was replaced with an image sensor 20 with k=3. Here, the number of pixels B h is a positive integer, and B h = 1, α = 1, B h = 2, α = 2, B h ≧3, then α=3. In the following, 1≦B s ≦B m And B s is an integer.

[0212] First, B m = 36, b = 0.043, and c = 0.27. Since α is not included in equations (16) and (19), the high sensitivity condition is the same as when k = 1. That is, B s ≦9, the first high sensitivity condition is satisfied, and B s By setting k = 1, the second high sensitivity condition is satisfied. In contrast, because α is included in equations (22) and (24), the high dynamic range condition changes from the case of k = 1. h If = 1, then α = 1, and from equation (22), B s ≧9. B h When = 2, α = 2, and from equation (22), B s ≧3. B h If ≧3, then α=3, and from equation (22), B s ≧1. Therefore, (B h , B s) with (1, 36), (2, 18), (3, 12), (4, 9), (6, 6), (9, 4), (12, 3), (18, 2), and (36, 1) all met the first high dynamic range condition.

[0213] On the other hand, B h If = 1, then α = 1, and from equation (24), B s ≧36. B h When = 2, α = 2, and from equation (24), B s ≧24. B h If ≧3, then α=3, and from equation (24), B s ≧11. Therefore, (B h , B s ) is the solution to the second high dynamic range condition with the combination of (1, 36) and (3, 12). h , B s The first high sensitivity condition and the first high dynamic range condition were satisfied by changing the binning condition from (4, 9), (6, 6), (9, 4), (12, 3), (18, 2), or (36, 1). It was also found that there was no binning condition that simultaneously satisfied the second high sensitivity condition and the second high dynamic range condition.

[0214] Next, B m = 36, b = 0.043, and c = 2.5. Since α is not included in equations (16) and (19), the high sensitivity condition is the same as when k = 1. That is, B s By setting the value of B to 36, the first high sensitivity condition is satisfied, and B s By setting k = 10, the second high sensitivity condition is satisfied. On the other hand, since α is included in equations (22) and (24), the high dynamic range condition changes from the case of k = 1. h If = 1, then α = 1, and from equation (22), B s ≧4. B h When = 2, α = 2, and from equation (22), B s ≧2. B h If ≧3, then α=3, and from equation (22), Bs ≧1. Therefore, (B h , B s ) with (1, 36), (2, 18), (3, 12), (4, 9), (6, 6), (9, 4), (12, 3), (18, 2), and (36, 1) all met the first high dynamic range condition.

[0215] On the other hand, B h If = 1, then α = 1, and from equation (24), B s ≧18. B h When = 2, α = 2, and from equation (24), B s ≧7. B h If ≧3, then α=3, and from equation (24), B s ≧4. Therefore, (B h , B s ) is the solution for the second high dynamic range condition with the combination of (1, 36), (2, 18), (3, 12), (4, 9), (6, 6), and (9, 4). h , B s The first high sensitivity condition and the first high dynamic range condition are satisfied by changing (B ) to any one of (1, 36), (2, 18), (3, 12), (4, 9), (6, 6), (9, 4), (12, 3), (18, 2), and (36, 1). h , B s ) to any one of (4, 9), (6, 6), or (9, 4), the second high sensitivity condition and the second high dynamic range condition were satisfied.

[0216] Finally, B. m = 36, b = 0.043, c = 10.7. Since α is not included in equations (16) and (19), the high sensitivity condition is the same as when k = 1. That is, B s By setting k=36, both the first and second high sensitivity conditions are satisfied. On the other hand, since α is included in equations (22) and (24), the high dynamic range condition changes from the case of k=1. hIf = 1, then α = 1, and from equation (22), B s ≧3. B h When = 2, α = 2, and from equation (22), B s ≧2. B h If ≧3, then α=3, and from equation (22), B s ≧1. Therefore, (B h , B s ) with (1, 36), (2, 18), (3, 12), (4, 9), (6, 6), (9, 4), (12, 3), (18, 2), and (36, 1) all met the first high dynamic range condition.

[0217] On the other hand, B h If = 1, then α = 1, and from equation (24), B s ≧11. B h When = 2, α = 2, and from equation (24), B s ≧6. B h If ≧3, then α=3, and from equation (24), B s ≧4. Therefore, (B h , B s ) is the solution for the second high dynamic range condition with the combinations (1, 36), (2, 18), (3, 12), (4, 9), (6, 6) and (9, 4). h , B s The first high sensitivity condition and the first high dynamic range condition are satisfied by changing (B ) to any one of (1, 36), (2, 18), (3, 12), (4, 9), (6, 6), (9, 4), (12, 3), (18, 2), and (36, 1). h , B s The second high sensitivity condition and the second wide dynamic range condition were satisfied by changing k=1 to any of (1, 36), (2, 18), (3, 12), (4, 9), (6, 6), and (9, 4). As described above, by changing the saturation light ratio from k=1 to k=3, the range of binning conditions that satisfy both the high sensitivity condition and the wide dynamic range condition is expanded, making it easier to obtain the desired effect.

[0218] In this example, we found noise and binning conditions that satisfy both high sensitivity and high dynamic range conditions. However, when sensitivity is more important than dynamic range, the original conditions (B h , B s ) back to (36, 1). Therefore, it is effective to enable the use of a mode that prioritizes high sensitivity over a high dynamic range and a mode that achieves both high sensitivity and a high dynamic range in the same multi-capillary electrophoresis device according to the purpose, that is, to enable the selection of an appropriate binning condition from among multiple binning conditions. It is preferable to enable the user to select a desired binning condition from among multiple binning conditions using a user interface. Alternatively, it is effective to enable the software to select an appropriate binning condition from among multiple binning conditions without the user having to consciously select the binning condition.

[0219] <Image sensor change> Next, under the conditions defined in the <Basic Conditions> above, the image sensor was changed from a CCD to a CMOS with a pixel size of 3.63 μm square. The AD conversion of the CCD was BN=16 bits, while the AD conversion of the CMOS was BN=12 bits. Here, the settings of the multi-color detection optical system were not changed, so the size on the image sensor of the image of the light in the wavelength range that each of the bins W(1) to W(20) measures remains 0.288 mm x 0.072 mm. Therefore, since each bin W(f) measures light in the same wavelength range, the number of pixels that make up each bin W(f) was changed to B m = 79 × 20 = 1580. In other words, the number of pixels in the bin W(f) has been increased by more than 40 times compared to the previous example. Also, since the CMOS cannot perform hard binning, the number of pixels for hard binning in each bin W(f) is set to B h = 1, the number of pixels for soft binning is B s = 1580. Also, since the saturated light ratio is k = 1, α = 1. Although the AD conversion of CMOS is only BN = 12 bits, B s= 1580, so the resolution of the digital signal is B s ×BN=1580×12 bits=23 bits, which effectively gives a resolution far exceeding 16 bits.

[0220] As a result of noise analysis under the above conditions, the readout noise of one pixel was N r = 1.06 counts, the dark current noise ratio was b = 0.21, and the shot noise ratio was c = 10. At this time, the binning condition that satisfies the first high sensitivity condition is given by the formula (17): s ≦775. The binning condition that satisfies the second high sensitivity condition is, according to equation (20), B s On the other hand, the binning condition that satisfies the first high dynamic range condition is, according to equation (23), B s ≧3. Furthermore, the binning condition that satisfies the second high dynamic range condition is, from equation (25), s ≧11. m =B s It was found that the condition of =1580 satisfies both the first high dynamic range condition and the second high dynamic range condition, but does not satisfy either the first high sensitivity condition or the second high sensitivity condition.

[0221] Therefore, as in the above <Changes in conditions>, the measurement conditions were changed without changing the settings of the multi-color detection optical system. As a result, a noise analysis was performed and the readout noise of one pixel was N r = 1.06 counts, the dark current noise ratio was unchanged at b = 0.21, but the shot noise ratio increased from c = 24 to 52, where c = 24 was obtained in bin W(20) and c = 52 was obtained in bin W(10).

[0222] First, when the shot noise ratio is c=24, the binning condition that satisfies the first high sensitivity condition is given by equation (17): s ≦4422. The binning condition that satisfies the second high sensitivity condition is, according to equation (20), B sOn the other hand, the binning condition that satisfies the first high dynamic range condition is, according to equation (23), B s ≧3. Furthermore, the binning condition that satisfies the second high dynamic range condition is, from equation (25), s ≧11. m =B s It was found that the condition of =1580 satisfies both the first high dynamic range condition and the second high dynamic range condition, and also satisfies the first high sensitivity condition, but does not satisfy the second high sensitivity condition.

[0223] Next, when the shot noise ratio is c=52, the binning condition that satisfies the first high sensitivity condition is given by equation (17): s ≦21571. The binning condition that satisfies the second high sensitivity condition is, according to equation (20), B s On the other hand, the binning condition that satisfies the first high dynamic range condition is, according to equation (23), B s ≧3. Furthermore, the binning condition that satisfies the second high dynamic range condition is, from equation (25), s ≧10. m =B s It was found that the condition of =1580 satisfied all of the first high sensitivity condition, the second high sensitivity condition, the first high dynamic range condition, and the second high dynamic range condition.

[0224] As described above, it was found that by setting appropriate noise composition conditions, it is possible to achieve both high sensitivity and a wide dynamic range while using the same binning conditions. In other words, it was found that, depending on the noise conditions, it is possible to achieve both high sensitivity and a wide dynamic range, but conversely, it is also possible that these two cannot be achieved simultaneously.

[0225] <Changes in the configuration of the multi-color detection optical system> We investigated whether it was possible to achieve both high sensitivity and a wide dynamic range by changing the configuration of the multi-color detection optical system described in <Basic Conditions>. The image sensor was a CMOS with a pixel size of 3.63 μm square. In addition, since the saturated light amount ratio was k=1, α=1 was set. First, the grating frequency of the transmission type diffraction grating used in the multi-color detection optical system was changed from N=600 lines / mm to N=200 lines / mm. In this case, according to equation (72), the dispersion distance per 1 nm on the image sensor is 10 μm, so the size of the image of the light of the 9 nm wide wavelength band that is the measurement target of bins W(1) to W(20) is 0.10 mm × 0.072 mm. In other words, each bin W(f) is divided into 25 pixels in the wavelength dispersion direction and 100 pixels in the light emission point arrangement direction. Direction 20 pixels, B m = 25 × 20 = 500. Also, the number of pixels for hard binning was B h = 1, and the number of pixels for soft binning is B s If the measurement conditions are not changed from the basic conditions, the readout noise of one pixel is N r = 1.06 counts, dark current noise ratio b = 0.21, and shot noise ratio c = 10. In other words, the first high sensitivity condition is B s ≦775, and the second high sensitivity condition is B s ≦121, and the first high dynamic range condition is B s ≧3, and the second high dynamic range condition is B s ≧11. Therefore, B h =1, B s The binning condition of =500 satisfies the first high sensitivity condition, the first high dynamic range condition, and the second high dynamic range condition. As described above, by changing the configuration of the multi-color detection optical system under the same measurement conditions, it has become possible to achieve both the high sensitivity condition and the high dynamic range condition.

[0226] Next, we considered how to more suitably achieve both high sensitivity and a high dynamic range by making further changes to the configuration of the multi-color detection optical system described above. Specifically, in addition to changing the grating frequency of the transmission diffraction grating used in the multi-color detection optical system from N=600 lines / mm to N=200 lines / mm, we also changed the focal length of the second camera lens from f2=50 mm to f2=25 mm. At this time, the wavelength-dispersed image of the light emitted from the light-emitting point is reduced by 1 / 2. Therefore, from equation (72), the dispersion distance per 1 nm on the image sensor is 5 μm, and the image size of a 50 μm square light-emitting point without wavelength dispersion is 25 μm square. As a result, the size of the image of the light of the 9 nm-wide wavelength band to be measured by the bins W(1) to W(20) is 0.045 mm × 0.025 mm. That is, each bin W(f) is divided into 12 pixels in the wavelength dispersion direction and 12 pixels in the light-emitting point array direction. Direction 7 pixels, B m = 12 × 7 = 84. Also, the number of pixels for hard binning is B h = 1, and the number of pixels for soft binning is B s In this case, if the measurement conditions are not changed from the basic conditions, the read noise of one pixel is N r = 1.06 counts, dark current noise ratio b = 0.21, and shot noise ratio c = 10, the first high sensitivity condition, the second high sensitivity condition, the first high dynamic range condition, and the second high dynamic range condition are all satisfied. From the above, by changing the configuration of the multicolor detection optical system under the same measurement conditions, it has become possible to achieve both the high sensitivity condition and the high dynamic range condition, and it has become possible to expand the range in which the high sensitivity condition and the high dynamic range condition can be achieved simultaneously.

[0227] [Example 2] In this embodiment, the conditions for achieving both high sensitivity and high dynamic range are systematically clarified by generalizing the contents of the study in the first embodiment. In the first embodiment, mainly (except for the change in the configuration of the multi-color detection optical system in the first embodiment), the number of pixels B in the bin W is m While fixing the number of pixels B in hard binning hand soft binning pixel count B s In this embodiment, the number of pixels B h and soft binning pixel count B s Not only the number of pixels B m We also consider changing B m The change in is achieved by controlling the configuration of the multi-color detection optical system to change the pixel area on the image sensor onto which the image of the fluorescence in the specific wavelength band targeted by bin W is projected.

[0228] In addition, in Example 1, not only was the noise composition changed by changing the measurement conditions, , Total The magnitude of the combined noise was also changed. , Total In order to avoid the influence of changes in the magnitude of the combined noise, , Total The noise composition is changed while keeping the magnitude of the combined noise constant. In the study of this embodiment, formulas (11), (13), (14) and (15) are used. However, since the saturation light amount ratio of the image sensor is k=1, α=1 is used. In formula (11), B h =B s = 1, the total noise is constant at N = 1 count. Furthermore, the saturation light amount per pixel of the image sensor is set to M = 10,000 counts. In the following, the counts, which are the units of noise and light amount, will be omitted.

[0229] Figures 11A to 11C show s Under the condition of =1, the number of pixels for hard binning is B hThe results of determining the detection limit LLOD, detection limit ULOD, and dynamic range DR with respect to the change in are shown by dotted lines. Here, the detection limit LLOD is shown by a triangular plot, the detection limit ULOD is shown by a square plot, and DR is shown by a circle plot. In addition, in FIG. 11A, the noise composition is set to b=0, c=0, in FIG. 11B, the noise composition is set to b=0.1, c=0, and in FIG. 11C, the noise composition is set to b=1, c=0. On the right side of each graph, the binning conditions that satisfy the first high sensitivity condition, the second high sensitivity condition, the first high dynamic range condition (referred to as the "first high DR condition"), and the second high dynamic range condition (referred to as the "second high DR condition") are shown in a table. Here, the first high sensitivity condition was obtained from equation (18), and the second high sensitivity condition was obtained from equation (21). In addition, when there is no solution, it is represented as "-".

[0230] First, in the case of b=0 and c=0 in FIG. 11A, the lower limit of detection LLOD, the upper limit of detection ULOD, and the dynamic range DR, which is the ratio between them, are all determined by the number of pixels B h It is constant regardless of B h The lower detection limit LLOD=3, the upper detection limit ULOD=10000, and the dynamic range DR=3333 were the same as in the case of =1. b=0, c=0 means that the total noise is composed only of read noise, so the number of pixels B for hard binning is h Even if the number of pixels in the hard binning is increased, the total noise does not change and the detection limit LLOD does not change. h In FIG. 11A, the binning condition that satisfies both conditions is shown as B in accordance with the scale of the graph. h <= 10 5 Also, the number of pixels for hard binning is B h Even if M increases, the saturation light amount after hard binning remains at M = 10,000, so the upper detection limit ULOD does not change. For this reason, there was no solution for the binning conditions that satisfied the high dynamic range condition.

[0231] Next, in the case of b=0.1 and c=0 in FIG. 11B, the number of pixels for hard binning is B h As B increases, the dark current noise increases, so the total noise increases and the detection limit LLOD also increases. h When B = 1, the detection lower limit LLOD, detection upper limit ULOD, and dynamic range DR are the same as those in FIG. 11A. As a result, as shown in the table, the first high sensitivity condition is expressed by equation (18): h The second high sensitivity condition is satisfied by setting B h This was satisfied by making it ≦127.

[0232] In addition, in the case of b=1 and c=0 in FIG. 11C, the number of pixels for hard binning is B h The increase rate of the detection limit LLOD against the dark current noise ratio b increases further and follows a straight line with a slope of approximately 1 / 2. This can be explained as follows. As is clear from equation (13), when c = 0 and the dark current noise ratio b becomes sufficiently large, the number of pixels B h The lower limit of detection LLOD increases in proportion to the root of the number of pixels in hard binning B h The relationship between the lower limit of detection (LLOD) and the concentration of the sample is linear with a slope of 1 / 2. As a result, as shown in the table, the first high sensitivity condition is expressed as B h The second high sensitivity condition is satisfied by setting B h ≦4. On the other hand, from equation (14), the dark current noise ratio b, the shot noise ratio c, and the number of pixels for hard binning B h Regardless of this, the saturation light amount remains M=10,000, so the detection upper limit ULOD in Figures 11B and 11C did not change from Figure 11A.

[0233] From the above, as the dark current noise ratio b increases, the number of pixels for hard binning B h As shown in the log-log graph of FIG. 11C, when the dark current noise ratio b becomes sufficiently large, the number of pixels in hard binning B hand the dynamic range DR had a linear relationship with a slope of -1 / 2. For this reason, there was no solution for the binning conditions that satisfied the high dynamic range condition. From the above, it became clear that under the conditions in Figure 11, it was not possible to achieve both high sensitivity and high dynamic range conditions.

[0234] Figures 12A to 12C show h Under the condition of =1, the number of pixels for soft binning is B s The results of determining the detection limit LLOD, detection limit ULOD, and dynamic range DR with respect to the change in are shown by solid lines. Here, the detection limit LLOD is shown by a triangular plot, the detection limit ULOD is shown by a square plot, and the dynamic range DR is shown by a circle plot. As in Figures 11A to 11C, the noise composition was set to b = 0, c = 0 in Figure 12A, to b = 0.1, c = 0 in Figure 12B, and to b = 1, c = 0 in Figure 12C, and the results were the same for Figures 12A, 12B, and 12C. s When = 1, B in Figs. 11A to 11C h Similarly to the case of c = 1, the lower limit of detection LLOD = 3, the upper limit of detection ULOD = 10000, and the dynamic range DR = 3333. On the other hand, according to equation (13), when c = 0, the number of pixels B s Therefore, as shown in the log-log graphs of FIGS. 12A to 12C, the number of pixels in soft binning B s The relationship between the lower limit of detection (LLOD) and the concentration of the sample is linear with a slope of 1 / 2. In this case, as shown in the table, the first high sensitivity condition is expressed as B s ≦9, and the second high sensitivity condition is satisfied by B s On the other hand, from equation (14), the number of pixels for soft binning B s Therefore, as shown in the log-log graphs of FIGS. 12A to 12C, the number of pixels in soft binning B s and the upper detection limit ULOD had a linear relationship with a slope of 1.

[0235] From the above, regardless of the dark current noise ratio b and the shot noise ratio c, the number of pixels for soft binning B s As shown in the log-log graphs of FIGS. 12A to 12C, the dynamic range DR increased in proportion to the root of the number of pixels B s The dynamic range DR has a linear relationship with a slope of 1 / 2. In this case, from equations (23) and (25), as shown in the table, the first high dynamic range condition is 9≦B s The second high dynamic range condition is satisfied by 100≦B s From the above, as shown in equation (26), B s = 9, it was found that the first high sensitivity condition and the first wide dynamic range condition could be achieved simultaneously. However, as shown in equation (27), it was found that the second high sensitivity condition and the second wide dynamic range condition could not be achieved simultaneously under the conditions of Figures 12A to 12C.

[0236] In Figures 11A to 11C, B s Number of pixels for hard binning under the condition of B = 1 h (=B m ) was changed, and in Figures 12A to 12C, B h Number of pixels for soft binning under the condition of B = 1 s (=B m ) was changed, whereas in Figures 13A to 13C, the pixel area of ​​the bin was fixed, specifically B m Under the condition of =100, B m =B h ×B s Hard binning while maintaining pixel count B h and soft binning pixel count B s However, the number of pixels for hard binning, B h and soft binning pixel count B sare all positive integers. As in Figs. 11A to 11C and Figs. 12A to 12C, the noise composition is set to b=0, c=0 in Fig. 13A, to b=0.1, c=0 in Fig. 13B, and to b=1, c=0 in Fig. 13C. The lower detection limit LLOD is shown by a triangular plot, the upper detection limit ULOD is shown by a square plot, and the dynamic range DR is shown by a circle plot, all of which are indicated by dashed dotted lines. The lower detection limit LLOD was calculated from equation (13), the upper detection limit ULOD was calculated from equation (14), and the dynamic range DR was calculated from the ratio of these. The horizontal axis indicates the soft binning rate B s / B m Here, (B h , B s ) is (100, 1), B s / B m The binning conditions were set as follows: 1% for (50, 2), 2% for (20, 5), 5% for (10, 10), 10% for (5, 20), 50% for (2, 50), and 100% for (1, 100). s / B m =1% is B in Figure 11 h = 100, the same results as B s / B m =100% is B in Figure 12 s = 100, the same results as B s / B m =2% to 50% indicates the results between these. The binning conditions that satisfy the high sensitivity and high dynamic range conditions are the same as those in FIG. 12, but B s / B m The range is shown as B s / B m By setting the sensitivity at B = 9%, it is possible to achieve both the first high sensitivity condition and the first high dynamic range condition. However, it has been found that under the conditions of FIG. 13, it is not possible to achieve both the second high sensitivity condition and the second high dynamic range condition. m =100=10 2 The study was carried out under the conditions of B m =10 0 ~105 Any number of pixels B m Needless to say, a similar study can be conducted on the above.

[0237] In Figs. 11 to 13, the dark current noise ratio b was changed under the condition of c = 0, but in Figs. 14 to 16 shown below, the shot noise ratio c was changed under the condition of b = 0. Figs. 14A to 14C show the same results as Fig. 11, with respect to B s Under the condition of =1, the number of pixels for hard binning is B h The dotted lines show the results of determining the lower limit of detection LLOD, the upper limit of detection ULOD, and the dynamic range DR with respect to the change in the number of pixels B of hard binning. In Fig. 14A, the noise composition was set to b = 0 and c = 0, in Fig. 14B, the noise composition was set to b = 0 and c = 2.5, and in Fig. 14C, the noise composition was set to b = 0 and c = 10, and the results were the same in Figs. 14A, 14B, and 14C. Also, the lower limit of detection LLOD, the upper limit of detection ULOD, and the dynamic range DR all change with the number of pixels B of hard binning. h It is constant regardless of B h The detection limit LLOD was 3, the detection limit ULOD was 10000, and the dynamic range DR was 3333, which were the same as in the case of b = 1. This result is the same as the result in FIG. 11A. This is because, as shown in formula (13), under the condition of b = 0, the detection limit LLOD is the number of pixels B h In addition, as shown in equation (14), regardless of the dark current noise ratio b and the shot noise ratio c, the detection limit ULOD is h Therefore, the number of pixels for any hard binning B h The first high-sensitivity condition and the second high-sensitivity condition are satisfied for the binning condition B, as in FIG. 11A. h <= 10 5 In addition, there was no solution for the binning conditions that satisfied the high dynamic range condition. From the above, it was found that the conditions in Figures 14A to 14C cannot simultaneously satisfy both the high sensitivity condition and the high dynamic range condition.

[0238] Figures 15A to 15C are h Under the condition of =1, the number of pixels for soft binning is B s The solid lines show the results of determining the lower limit of detection LLOD, the upper limit of detection ULOD, and the dynamic range DR with respect to the change in noise composition. As in Figs. 14A to 14C, the noise composition was set to b=0 and c=0 in Fig. 15A, to b=0 and c=2.5 in Fig. 15B, and to b=0 and c=10 in Fig. 15C. The conditions and results in Fig. 15A are the same as those in Fig. 12A. In other words, the first high sensitivity condition is given by equation (17): s ≦9, and the second high sensitivity condition is satisfied by B s ≦2. Also, the first high dynamic range condition is satisfied by 9≦B s The second high dynamic range condition is satisfied by 100≦B s Therefore, B s By setting θ=9, it was possible to achieve both the first high sensitivity condition and the first high dynamic range condition, but it was found that it was not possible to achieve both the second high sensitivity condition and the second high dynamic range condition.

[0239] Compared with the results in FIG. 15A, the detection limit LLOD in FIG. 15B is lower, and the detection limit LLOD is further lower in FIG. 15C. This is because, as shown in Equation (13), as the shot noise ratio c increases, the contribution rates of readout noise and dark current noise decrease, and the number of pixels B of soft binning to the total noise decreases. s 15A, 15B, and 15C, the detection upper limit ULOD does not change with the shot noise ratio c, and therefore the dynamic range DR improves by the amount that the detection lower limit LLOD decreases.

[0240] In FIG. 15B, the first high sensitivity condition is B s ≦59, and the second high sensitivity condition is satisfied by B s≦10. Also, the first high dynamic range condition is satisfied by 4≦B s The second high dynamic range condition is satisfied by 19≦B s Therefore, 4≦B s By setting the binning condition range to 4≦B≦59, it was possible to achieve both the first high sensitivity condition and the first high dynamic range condition. This range of binning conditions is wider than that in FIG. 15A, and it was found to be advantageous for achieving both the high sensitivity condition and the high dynamic range condition. However, it was found that it was not possible to achieve both the second high sensitivity condition and the second high dynamic range condition. However, when 4≦B s By setting B≦10, it is possible to simultaneously achieve the second high sensitivity condition and the first high dynamic range condition. s By setting the value at ≦59, it is possible to simultaneously achieve the first high sensitivity condition and the second high dynamic range condition.

[0241] On the other hand, in FIG. 15C, the first high sensitivity condition is B s The second high sensitivity condition is satisfied by setting B s ≦127. Also, the first high dynamic range condition is satisfied by 3≦B s The second high dynamic range condition is satisfied by 11≦B s Therefore, 3≦B s By setting the B value at 11≦B≦809, the first high sensitivity condition and the first high dynamic range condition can be simultaneously satisfied. s By setting B≦127, it is possible to simultaneously achieve the second high sensitivity condition and the second high dynamic range condition. s By setting B≦127, it is possible to simultaneously achieve the second high sensitivity condition and the first high dynamic range condition. s≦809, it was possible to achieve both the first high sensitivity condition and the second high dynamic range condition. In other words, it was found that by setting the shot noise ratio c to a larger value, Fig. 15B is better than Fig. 15A, and Fig. 15C is better than Fig. 15B, that is, by setting the shot noise ratio c to a larger value, it was possible to provide a suitable condition for achieving both high sensitivity and a wide dynamic range.

[0242] In Figures 14A to 14C, B s Number of pixels for hard binning under the condition of B = 1 h (=B m ) was changed, and in Figures 15A to 15C, B h B = 1 s (=B m ) was changed, whereas in Figs. 16A to 16C, the pixel area of ​​the bin was fixed, specifically, B m Under the condition of =100, B m =B h ×B s Hard binning while maintaining pixel count B h and soft binning pixel count B s However, the number of pixels for hard binning, B h and soft binning pixel count B s were all positive integers. As in Figs. 14 and 15, the noise composition was set to b=0, c=0 in Fig. 16A, to b=0, c=2.5 in Fig. 16B, and to b=0, c=10 in Fig. 16C. The lower detection limit LLOD is shown by a triangular plot, the upper detection limit ULOD by a square plot, and the dynamic range DR by a circle plot, all of which are indicated by dashed dotted lines. The lower detection limit LLOD was calculated from equation (13), the upper detection limit ULOD was calculated from equation (14), and the dynamic range DR was calculated from the ratio of these. The horizontal axis indicates the soft binning rate B s / B m As in Figures 13A to 13C, (B h , B s ) is (100, 1), B s / B mThe binning conditions were set as follows: 1% for (50, 2), 2% for (20, 5), 5% for (10, 10), 10% for (5, 20), 50% for (2, 50), and 100% for (1, 100). s / B m =1% is B in Figures 14A to 14C h = 100, the same results as B s / B m = 100% is B in Figures 15A to 15C s = 100, the same results as B s / B m =2% to 50% indicates the results between these. The high sensitivity conditions and high dynamic range conditions satisfied in Figures 16A, 16B, and 16C were the same as those in Figures 15A, 15B, and 15C, respectively. However, in Figure 15C, the first high sensitivity condition was B s ≦809, and the second high sensitivity condition is B s ≦127, but in Figure 16, B s ≦100, the binning conditions that satisfy the first high sensitivity condition and the second high sensitivity condition are B s / B m ≦100%. In addition, in Figs. 16A to 16C, B m =100=10 2 The study was carried out under the conditions of B m =10 0 ~10 5 Any B m Needless to say, a similar study can be conducted on the above.

[0243] From the above investigation, it was found that the shot noise ratio c is an important factor in achieving both high sensitivity and high dynamic range conditions. In Figs. 14 to 16, only the cases of c = 0, 2.5, and 10 were investigated, but in Figs. 17A and 17B, the effect of the shot noise ratio c on the high sensitivity and high dynamic range conditions was investigated in more detail. Fig. 17A shows the relationship between the number of pixels B of soft binning that satisfies the first high sensitivity condition and the shot noise ratio c. sThe number of pixels B for soft binning that satisfies the first condition for high dynamic range and the second condition s FIG. 17B shows the condition for the number of pixels B of soft binning that satisfies the second high sensitivity condition with respect to the shot noise ratio c. s The number of pixels B for soft binning that satisfies the first condition and the second high dynamic range condition s In both graphs, the area below the curve of the circular plot is the high sensitivity condition, and the area above the curve of the triangular plot is the high dynamic range condition. Therefore, in the area between these curves, both the high sensitivity condition and the high dynamic range condition are satisfied. From FIG. 17A, in order to satisfy both the first high sensitivity condition and the first high dynamic range condition, it is sufficient that c≧0, and at least B s = 9. This is the same as the results in Figures 12 and 15A. However, if the solution is B s The only condition where =9 is the number of pixels for soft binning, B s Generally, the wider the range of solutions, the easier it is to implement and the easier it is to obtain benefits.

[0244] Therefore, from FIG. 17A, the number of pixels B of the soft binning that is the solution s Condition A is that the range is 30 pixels or more, and the number of pixels B for soft binning is the solution. s Condition B was extracted, where the range of is 100 pixels or more. From FIG. 17A, condition A is c≧1.75, and at least 4≦B s It was found that c ≧ 3.43 is sufficient. Condition B is c ≧ 3.43, and 3 ≦ B is sufficient. s On the other hand, from FIG. 17B, in order to simultaneously satisfy the second high sensitivity condition and the second high dynamic range condition, c≧3.15 is sufficient, and at least B s = 15. Conversely, it became clear that no solution exists when c<3.15. Similarly, conditions A and B were obtained for FIG. 17B. From FIG. 17B, condition A is c≧5.61, and at least 12≦B sIt was found that c ≧ 42 is sufficient. Condition B is c ≧ 9.31, and at least 11 ≦ B s I found that it is fine if it is ≦111.

[0245] 17C-17E and 17F-17H are further generalizations of the discussion in FIGS. 17A and 17B, respectively. h When the condition of 1 is met, the shot noise ratio c and the number of pixels for soft binning B that satisfy the high sensitivity and high dynamic range conditions are s In contrast, in Figs. 17C to 17E and Figs. 17F to 17H, m , Number of pixels for hard binning B h , soft binning pixel count B s B that satisfies the high sensitivity and high dynamic range conditions without fixing either m and B s / B m (=1 / B h ) relationship was clarified. The soft binning rate B s / B m is the ratio of the number of pixels in soft binning to the number of pixels in bin W, and B m =B h ×B s Therefore, B s / B m =1 / B h It can also be expressed as:

[0246] 17C to 17E show binning conditions that satisfy the first high sensitivity condition and the first high dynamic range condition. In FIG. 17C, the noise composition is set to b=0 and c=0, in FIG. 17D, the noise composition is set to b=0 and c=2.5, and in FIG. 17E, the noise composition is set to b=0 and c=10. The area below the dotted line satisfies the first high sensitivity condition, and the area above the solid line satisfies the first high dynamic range condition, so that the area between the two straight lines satisfies both the first high sensitivity condition and the first high dynamic range condition. However, B s / B mIn reality, the ratio of the shot noise ratio c to the total noise ratio b does not exceed 100%. Therefore, the bold line portion in FIG. 17C and the gray shaded portions in FIG. 17D and FIG. 17E provide binning conditions that satisfy both the first high sensitivity condition and the first high dynamic range condition. As in the result in FIG. 17A, the region or range of the allowable binning conditions expands with an increase in the shot noise ratio c. For example, s / B m =100%, that is, B h =1, B s =B m So, when c=0, B s = 9, and when c = 2.5, 4 ≦ B s ≦59, and when c=10, 3≦B s By setting the value of B to 809, the first high sensitivity condition and the first high dynamic range condition are satisfied. These can also be seen from Figs. 15A to 15C, Figs. 16A to 16C, and Fig. 17A. However, other rich information can also be seen from Figs. 17C to 17E. For example, B s / B m =50%, that is, B h =2, B s =1 / 2*B m So, when c=0, B s = 18, and when c = 2.5, 7 ≤ B s ≦118, and when c=10, 7≦B s ≦1618, the first high sensitivity condition and the first high dynamic range condition are satisfied. s / B m = 33.33%, or B h =3, B s =1 / 3*B m So, when c=0, B s = 27, c = 2.5, 11 ≤ B s ≦177, when c=10, 10≦B s By setting α=k=3, the first high sensitivity condition and the first high dynamic range condition are satisfied. Furthermore, if α=k=3, then when c=0, 3≦B s ≦27, when c=2.5, 3≦B s ≦177, when c=10, 3≦B sBy setting it to ≦2427, the first high sensitivity condition and the first high dynamic range condition are satisfied, and the range of allowable binning conditions can be expanded.

[0247] On the other hand, Figs. 17F to 17H show binning conditions that satisfy the second high sensitivity condition and the second high dynamic range condition. In Fig. 17F, the noise composition is set to b=0 and c=0, in Fig. 17G, the noise composition is set to b=0 and c=2.5, and in Fig. 17H, the noise composition is set to b=0 and c=10. The area below the dotted line satisfies the second high sensitivity condition, and the area above the solid line satisfies the second high dynamic range condition, so that the area between the two straight lines satisfies both the second high sensitivity condition and the second high dynamic range condition. In Figs. 17F and 17G, there is clearly no solution. In contrast, the gray shaded area in Fig. 17H gives binning conditions that satisfy both the second high sensitivity condition and the second high dynamic range condition. As in the results of Figs. 16A to 16C and Fig. 17B, the area and range of the allowable binning conditions expands with an increase in the shot noise ratio c. B s / B m =100%, that is, B h =1, B s =B m So, when c=10, 11≦B s By setting B to ≦127, the second high sensitivity condition and the second high dynamic range condition are satisfied. These can also be seen from Figs. 15A to 15C and Fig. 17B. However, other rich information can also be seen from Figs. 17F to 17H. For example, B s / B m =50%, that is, B h =2, B s =1 / 2*B m So, when c=10, 21≦B s By setting B to ≦254, the second high sensitivity condition and the second high dynamic range condition are satisfied. s / B m = 33.33%, or B h =3, B s =1 / 3*B mSo, when c=10, 32≦B s By setting α=k=3, the second high sensitivity condition and the second high dynamic range condition are satisfied. Furthermore, if α=k=3, then when c=10, 11≦B s By setting it to ≦381, the second high sensitivity condition and the second high dynamic range condition are satisfied, and the range of allowable binning conditions can be expanded.

[0248] As described above, the obtained sensitivity and dynamic range change depending on the configuration of the multi-color detection optical system, the noise composition, and the binning conditions. Therefore, it is effective to switch the binning conditions according to the sensitivity or dynamic range desired to be obtained under given conditions. For example, the optimal binning conditions differ when priority is given to sensitivity and when priority is given to dynamic range. It is convenient for the user if the multi-capillary electrophoresis apparatus allows the user to select the desired binning condition from among multiple binning conditions.

[0249] [Example 3] The various methods proposed in the present disclosure above have made it possible to achieve both high sensitivity and a wide dynamic range. However, it has become clear that there are cases where the expected sensitivity and dynamic range are not necessarily obtained. As a result of detailed investigations, the present inventors have discovered that the cause is spatial crosstalk inherent in the multi-color detection optical system. The newly discovered problem will be described in detail below.

[0250] As explained in [Background Art], the multi-color detection optical system is composed of multiple optical components. Even a single camera lens is a combination of multiple lenses. In order to suppress light reflection on the surfaces of these optical components, anti-reflection coatings are sometimes applied to the surfaces, but even so, it is not possible to reduce light reflection to zero. When multiple reflections of fluorescence occur between multiple optical components inside the multi-color detection optical system, false images such as ghosts or flares are generated on the image sensor, and may be measured overlapping with the true image of the light-emitting point that emits fluorescence. Here, the true image also includes the wavelength dispersion image of the fluorescence emitted from the light-emitting point. The size of the false image of the light-emitting point is generally larger than the size of the true image of the light-emitting point, and affects a wide range on the image sensor. For example, the false image of light-emitting point A can overlap not only the true image of light-emitting point A but also the true image of light-emitting point B, so spatial crosstalk occurs from light-emitting point A to light-emitting point B. However, the intensity of the false image is much smaller than the intensity of the true image, so the presence of the false image does not necessarily cause a problem. When the intensity of the true image is small, the intensity of the false image is even smaller, below the detection limit, and does not pose a problem. When the intensity of the true image is large, the intensity of the false image exceeds the detection limit and may pose a problem. Therefore, as the dynamic range of the fluorescence measurement by the image sensor becomes larger, the possibility of this problem becoming apparent increases. Since the main purpose of this disclosure is to achieve both high sensitivity and a high dynamic range, the greater the effect, the more likely it is that this problem will be encountered. In other words, the method of achieving both high sensitivity and a high dynamic range according to this disclosure may not function due to spatial crosstalk. This is a new problem that has become clear in this disclosure.

[0251] Up until now, formulas (1) to (6) have been established for each light-emitting point individually, but when spatial crosstalk is taken into consideration, these must be expanded and expressed as follows. At each light-emitting point P(e) (e=1,2,…,E), fluorescent material D(e,g) (e=1,2,…,E, and g=1,2,…,G) emits fluorescent light, and the fluorescent light received at all bins W(e,f) (e=1,2,…,E, and f=1,2,…,F) is measured. At any given time, let Z(e,g) be the concentration of fluorescent material D(e,g) at light-emitting point P(e), and X(e',f) be the signal intensity of bin W(e',f) for light-emitting point P(e'). Here, let X be a vector with (E×F) rows and 1 column whose element is the signal intensity X(e', f), let Z be a vector with (E×G) rows and 1 column whose element is the concentration Z(e, g), and let Y be a matrix with (E×F) rows and (E×G) columns whose element is Y(e', f)(e, g). Then, the following equations (73) to (78) hold corresponding to equations (1) to (6).

[0252]

number

[0253]

number

[0254]

number

[0255]

number

[0256]

number

[0257]

number

[0258] Here, the elements Y(e', f)(e, g) of the matrix Y with (E x F) rows and (E x G) columns represent the signal intensity ratio of the emitted fluorescence of the phosphor D(e, g) at the emission point P(e) detected in the bin W(e, f) for the emission point P(e) due to the spectral crosstalk for the same emission point when (i) e' = e, and represent the signal intensity ratio of the emitted fluorescence of the phosphor D(e, g) at the emission point P(e) detected in the bin W(e', f) for the emission point P(e') due to the spatial crosstalk for different emission points when (ii) e' ≠ e. Each column Y(e, f)(e0, g0) (e = 1, 2, ..., E, and f = 1, 2, ..., F) of the matrix Y can be determined by making any one type of phosphor D(e0, g0) emit fluorescence independently at any one emission point P(e0). In equation (77), the (E×F) elements of each column Y(e, f)(e0, g0) of matrix Y are normalized so that the sum of the elements is 1. As described above, among the (E×F) elements of each column Y(e, f)(e0, g0) of matrix Y, the F elements of e=e0 indicate the spectral crosstalk ratio, and the ((E-1)×F) elements of e≠e0 indicate the spatial crosstalk ratio. The former spectral crosstalk ratio is the same as the F elements of the elements Y(f)(g0) of one column of matrix Y in equation (3). However, the normalization conditions are different between equation (5) and equation (77). In general, the spatial crosstalk ratio is << the spectral crosstalk ratio, but there are cases where the spatial crosstalk ratio cannot be regarded as zero. Conversely, when all spatial crosstalk ratios can be regarded as zero, equations (73) to (78) are the same as equations (1) to (6).

[0259] The maximum spatial crosstalk ratio XR2 = Y(e m ', f m ')(e m , g m ) The light emitting point P(e m ) from phosphor D(e m , g m) is emitted at the emission point P(e m ') of bin W(e m ', f m That is, the measurement is made in bin W(e m ', f m Suppose the maximum spatial crosstalk is obtained in bin W(e m ', f m ') pixels B m ' and the number of pixels for hard binning is B h ' and the number of pixels for soft binning is B s The read noise of one pixel is N r The dark current noise of one pixel is N d With light emitted from all light-emitting points, bin W(e m ', f m The shot noise of the background light measured by s ', the dark current noise ratio is b, the shot noise ratio is c', and the saturation light coefficient is α. In this case, the bin W(e m ', f m The lower detection limit LLOD, upper detection limit ULOD, and dynamic range DR of (1′) are expressed by equations (79), (80), and (81) by modifying equations (13), (14), and (15), respectively.

[0260]

number

[0261]

number

[0262]

number

[0263] On the other hand, the element Y(e m , f m )(e m , g mThe maximum spectral crosstalk ratio XR1 = Y(e m , f m )(e m , g m ) The light emitting point P(e m ) from phosphor D(e m , g m ) emission at the emission point P(e m ) bin W(e m , f m ), that is, the bin (e m , f m ) for which the maximum spectral crosstalk is obtained. m , f m ) pixels B m The number of pixels for hard binning is B h The number of pixels for soft binning is B s The read noise of one pixel is N r The dark current noise of one pixel is N d All of the light is emitted from the light emitting point and bin W(e m , f m ) is the background shot noise measured by N s The dark current noise ratio is b, the shot noise ratio is c, and the saturation light coefficient is α. In this case, the bin W(e m , f m The lower detection limit LLOD, upper detection limit ULOD, and dynamic range DR of are given by equations (13), (14), and (15), respectively.

[0264] From the above, the light emitting point P(e m ) from phosphor D(e m , g m If the ratio of the maximum signal strength measured by spatial crosstalk to the maximum signal strength measured by spectral crosstalk based on the emission of the light from the light emission point P(e m ) for phosphor D(e m , gm When only the light emitting point P(e m ) phosphor D(e m , g m ) concentration Z(e m , g m ) signal strength at the emission point P(e m Phosphor D(e m ', g m Concentration Z(e m ', g m In other words, the crosstalk ratio XR can be derived both before and after color conversion, and is an index that represents the effect of spatial crosstalk.

[0265] Here, the light emitting point P(e m ) from phosphor D(e m , g m ) emission, bin W(e m , f m Assume that the amount of light of the detection upper limit ULOD is measured at the light emission point P(e m ') of bin W(e m ', f m The maximum spatial crosstalk light amount measured in (x') is calculated by multiplying the saturation light amount ULOD shown in equation (14) by the crosstalk ratio XR. In order to achieve high sensitivity and high dynamic range without being affected by spatial crosstalk, it is sufficient that the maximum spatial crosstalk light amount is smaller than the lower limit of detection LLOD in equation (79), and this condition is expressed by equation (82).

[0266]

number

[0267] Hereafter, the light emitting point P(e m ) binning conditions and the light emission point P(e m By aligning the binning and noise conditions of m '=B m , B h '=B h , Bs '=B s , N s '=N s , c'=c. In this case, equation (82) can be expressed as equation (83).

[0268]

number

[0269] Moreover, equations (79), (73), and (81) can be replaced with equations (13), (14), and (15), respectively. The right-hand side of equation (83) is the reciprocal of the dynamic range DR shown in equation (15). By achieving both equation (83) and the high sensitivity condition and high dynamic range condition proposed so far, for example, equation (26) or equation (27), it is possible to avoid the influence of spatial crosstalk and achieve both high sensitivity and high dynamic range.

[0270] Conversely, when equation (83) is not satisfied, equation (13) is replaced by the following equation (84).

[0271]

number

[0272] That is, regardless of the binning conditions and noise conditions, the lower limit of detection LLOD is determined by the crosstalk ratio XR. At this time, the upper limit of detection ULOD remains as expressed by equation (14), so the dynamic range DR is expressed by equation (85).

[0273]

number

[0274] Therefore, the dynamic range DR is also determined by the crosstalk ratio XR, regardless of the binning and noise conditions.

[0275] From the above, it is clear that keeping the crosstalk ratio XR low is important for achieving both high sensitivity and a wide dynamic range. FIGS. 18A to 18F show the relationship between the number of pixels B in soft binning and the crosstalk ratio XR when the crosstalk ratio XR is changed based on FIG. 15C. s FIG. 18A shows the results of investigating the changes in the detection limit LLOD, detection limit ULOD, and dynamic range DR with respect to XR=0. FIG. 18B shows the results for XR=10 -6 In Figure 18C, XR=10 -5 In Figure 18D, XR=10 -4 In Figure 18E, XR=10 -3 In Figure 18F, XR=10 -2 In each figure, the detection upper limit ULOD is a common value, while the detection lower limit LLOD and the dynamic range DR have the same values ​​as in FIG. 18A when equation (83) is satisfied, but show values ​​according to equations (84) and (85) when equation (83) is not satisfied. As can be seen from FIG. 18, as the crosstalk ratio XR increases, the upper limit of the dynamic range DR decreases according to equation (85), and therefore the number of pixels B for soft binning s According to the results of FIGS. 18A to 18F, in order to satisfy the first high sensitivity condition and the first high dynamic range condition, XR≦10 -4 I realized that it was necessary to do so.

[0276] In Figures 18A to 18F, B h =B s In contrast, in Figs. 19A to 19F, the total noise in the case of B h =B sThe conditions were changed so that the total noise in the case of N = 1 was constant at N = 10, and the other conditions were the same as those in FIG. 18. In FIG. 19A, the total noise has increased by an order of magnitude compared to FIG. 18A, so the detection lower limit LLOD has increased by an order of magnitude overall, and the dynamic range DR has decreased by an order of magnitude. In FIGS. 19B to 19F, as in FIGS. 18B to 18F, the number of pixels B in soft binning is changed to a constant value of N = 10. s Although the effect of increasing the dynamic range DR by increasing XR is limited, it was found that the effect was obtained over a wider range of the dynamic range DR. For example, according to the results of FIGS. 19A to 19F, in order to satisfy the first high sensitivity condition and the first high dynamic range condition, XR≦10 -3 It was found that it was necessary to set the crosstalk ratio XR to the range shown in FIG. 18A to 18F, which was an order of magnitude wider than that shown in FIG. 19A to 19F. Although the effect of increasing the total noise by an order of magnitude was examined in FIG. 19A to 19F, the same effect as shown in FIG. 19A to 19F can be obtained by reducing the saturation light amount M of one pixel by an order of magnitude instead. The above can be understood from the fact that the numerator on the right side of equation (83) contains noise and the denominator contains the saturation light amount.

[0277] As described above, in order to achieve both high sensitivity and high dynamic range, in addition to the high sensitivity and high dynamic range conditions shown in formulas (8) to (67), it is important to suppress the crosstalk rate to a low level. There are several means for reducing the crosstalk rate. The basic means is to apply an anti-reflection coating with a low reflectance to the surfaces of the constituent lenses of the camera lens, but this alone may not be sufficient. It is also important to apply an anti-reflection coating to the surfaces of each optical component of the multi-color detection optical system. For example, it is effective to apply an anti-reflection coating to the entrance and exit surfaces of a transmission diffraction grating, especially the surface on the side where no ruling is applied. In addition, since the surface of an image sensor, especially the entrance and exit surfaces of the glass window of the image sensor, is often not anti-reflection coated, it is very effective to apply an anti-reflection coating there.

[0278] Alternatively, it is also effective to reduce crosstalk by data processing. In the color conversion shown in equation (6), spatial crosstalk is not taken into account, so if spatial crosstalk is significant, its influence is reflected directly in the result. In contrast, equation (78) takes spatial crosstalk into account, so it is possible to reduce the influence of spatial crosstalk using equation (78). In other words, equation (78) is a method that cancels spatial crosstalk in a single step, in addition to canceling spectral crosstalk, which is the conventional color conversion. Therefore, the spatial crosstalk rate derived from XR = XR2 / XR1 does not change even when equation (78) is implemented. However, the light-emitting point P(e m ) for phosphor D(e m , g m When only the light emitting point P(e m ) phosphor D(e m , g m ) concentration Z(e m , g m ) signal strength at the emission point P(e m Phosphor D(e m ', g m Concentration Z(e m ', g m If the maximum ratio of the signal strengths of the two signals (x, y, z) is taken as the crosstalk ratio XR, then the crosstalk ratio XR is reduced by equation (78). In this way, by reducing spatial crosstalk by data processing according to equation (78), it is possible to achieve both high sensitivity and a wide dynamic range, which is the objective of the present disclosure.

[0279] [Variations] The present disclosure is not limited to the above-described embodiments, and includes various modified examples. For example, the above-described embodiments have been described in detail to clearly explain the present disclosure, and it is not necessary to include all of the configurations described. In addition, a part of an embodiment can be replaced with a configuration of another embodiment. In addition, a configuration of another embodiment can be added to a configuration of an embodiment. In addition, a part of the configuration of each embodiment can be added to, deleted from, or replaced with a part of the configuration of another embodiment. [Explanation of symbols]

[0280] 1…Capillary 2…Sample injection port 3…Sample elution end 4...Cathode 5...Anode 6...Cathode side buffer solution 7…Anode side buffer solution 8…Power supply 9…Pump block 10…Valve 11. Syringe 12...Laser light source 13...Laser beam 14…Light emitting point 15...Multi-color detection optical system 16…1st camera lens 17...Long pass filter 18...Transmission type diffraction grating 19…Second camera lens 20...Image sensor 21...Optical axis 22...Fluorescence 23, 24, 25… Dispersed fluorescence 26...Image 27...Wavelength dispersion image 28…pixels W(1)…Bin 1 W(2)…Bin 2 W(3)…Bottle 3 7-1…B h =36 hard binned regions 8-1~8-12…B h =3 hard binning region 9-1~9-36…B h =1 hard binning region 10-1…B h =4 hard binning region 10-2…B h =5 hard binning region 10-3…B h =6 hard binning region 10-4…B h =5 hard binning region 10-5…Bh =4 hard binning region 10-6…B h =6 hard binning region 10-7…B h =5 hard binning region 10-8…B h =1 hard binning region B h …Number of pixels for hard binning B s … Number of pixels for soft binning b…Dark current noise ratio c...Shot noise ratio LLOD: Lower limit of detection ULOD: Upper limit of detection DR: Dynamic range B s / B m …Soft binning ratio N: Total noise of one pixel XR…Crosstalk ratio

Claims

1. E capillaries into which E (≧2) samples each containing components labeled with G (≧2) fluorophores are injected and subjected to simultaneous electrophoresis; a laser light source that irradiates a laser beam onto measurement units arranged on the same plane of the E capillaries; an optical system that receives fluorescence emitted by the G phosphors passing through the measurement unit as a result of being excited by the laser beam; The optical system includes: a spectroscopic element for dispersing the fluorescence emitted from each of the E capillaries into F (≧2, F≧G) predetermined wavelength bands; The image sensor includes a plurality of pixels arranged two-dimensionally, and receives E×F dispersed fluorescent light beams in E×F different bin regions on the image sensor; The image sensor includes: Measure E×F signal intensities of the E×F spectrally separated fluorescence in the E×F bin regions; The method is configured to acquire time series data of the E×F signal intensities by continuously and repeatedly measuring the E×F signal intensities at a predetermined exposure time and a predetermined time interval; In any one of the E×F bin areas, The number of pixels in the bin area is B m (≧1) pieces, The bin area is B s is divided into (≧1) hard binned regions, The average number of pixels in the hard binning area is B h (≧1) pieces, The number of pixels in the hard binning of the bin area is B h ; The number of pixels in the soft binning of the bin area is B s As individuals, B m = B h ×B s year, A ratio of a maximum value of a saturated light amount obtained by performing hard binning to a saturated light amount per pixel of the image sensor is defined as a saturated light amount ratio k (≧1); The saturation light quantity coefficient α is B h = 1, α = 1, 1 < B h <k, then α=B h , k≦B h Then, α=k, B m = B h = B s When = 1, The total noise of the time series data is classified into three components: a read noise of the image sensor, a dark current noise of the image sensor, and a shot noise of background light; The total noise is N, and the read noise is N r and the dark current noise is N d and the shot noise is N s year, Dark current noise ratio is b = N d / N r , the shot noise ratio is c = N s / N r When B m , B h , B s , k, α, b, and c satisfy a predetermined relationship.

2. In claim 1, [0010] 13. A multi-capillary electrophoresis apparatus, comprising:

3. In claim 2, B h = 1, [0025] 13. A multi-capillary electrophoresis apparatus, comprising:

4. In claim 1, [0030] 13. A multi-capillary electrophoresis apparatus, comprising:

5. In claim 4, B h = 1, [0045] 13. A multi-capillary electrophoresis apparatus, comprising:

6. In claim 1, c≧2.5、 B h ＝1、 4≦B s ≦59 13. A multi-capillary electrophoresis apparatus, comprising:

7. In claim 1, c≧10、 B h ＝1、 3≦B s ≦809 13. A multi-capillary electrophoresis apparatus, comprising:

8. In claim 7, c≧10、 B h ＝1、 11≦B s ≦127 13. A multi-capillary electrophoresis apparatus, comprising:

9. E capillaries into which E (≧2) samples each containing components labeled with G (≧2) fluorophores are injected and subjected to simultaneous electrophoresis; a laser light source that irradiates a laser beam onto measurement units arranged on the same plane of the E capillaries; an optical system that receives fluorescence emitted by the G phosphors passing through the measurement unit as a result of being excited by the laser beam; The optical system includes: a spectroscopic element for dispersing the fluorescence emitted from each of the E capillaries into F (≧2, F≧G) predetermined wavelength bands; The image sensor includes a plurality of pixels arranged two-dimensionally, and receives E×F dispersed fluorescent light beams in E×F different bin regions on the image sensor; The image sensor includes: Measure E×F signal intensities of the E×F spectrally separated fluorescence in the E×F bin regions; When any one of the G phosphors emits fluorescence from the measurement unit of any one of the E capillaries, deriving F normalized signal intensities by normalizing the F signal intensities in the F bin regions corresponding to the one capillary such that the maximum value is 1; a combined bin area is set by combining the bin areas having the normalized signal strength of 0.5 or more among the F normalized signal strengths; It is assumed that time series data of integrated signal intensity in the integrated bin area is obtained by continuous repeated measurements at a predetermined exposure time and a predetermined time interval; The number of pixels in the integrated bin area is B m (≧1) pieces, The integrated bin area is B s is divided into (≧1) hard binned regions, The average number of pixels in the hard binning area is B h (≧1) pieces, The number of pixels in the soft binning of the integrated bin area is B. s As individuals, The number of pixels in the hard binning of the integrated bin area is B h ; B m = B h ×B s year, A ratio of a maximum value of a saturated light amount obtained by performing hard binning to a saturated light amount per pixel of the image sensor is defined as a saturated light amount ratio k (≧1); The saturation light quantity coefficient α is B h When B = 1, α = 1, and 1 < B h <k, then α=B h Let k≦B h Then, α=k, B m = B h = B s When = 1, The total noise of the time series data is classified into three components: a read noise of the image sensor, a dark current noise of the image sensor, and a shot noise of background light; The total noise is N, and the read noise is N r and the dark current noise is N d and the shot noise is N s year, Dark current noise ratio is b = N d / N r Let the shot noise ratio be c = N s / N r When B m , B h , B s , k, α, b, and c satisfy a predetermined relationship.

10. In claim 9, [0050] 13. A multi-capillary electrophoresis apparatus, comprising:

11. In claim 10, B h = 1, [006] 13. A multi-capillary electrophoresis apparatus, comprising:

12. In claim 9, [0070] 13. A multi-capillary electrophoresis apparatus, comprising:

13. In claim 12, B h = 1, [0080] 13. A multi-capillary electrophoresis apparatus, comprising:

14. E capillaries into which E (≧2) samples each containing components labeled with G (≧2) fluorophores are injected and subjected to simultaneous electrophoresis; a laser light source that irradiates a laser beam onto measurement units arranged on the same plane of the E capillaries; an optical system that receives fluorescence emitted by the G phosphors passing through the measurement unit as a result of being excited by the laser beam; The optical system includes: a spectroscopic element for dispersing the fluorescence emitted from each of the E capillaries into F (≧2, F≧G) predetermined wavelength bands; The image sensor includes a plurality of pixels arranged two-dimensionally, and receives E×F dispersed fluorescent light beams in E×F different bin regions on the image sensor; The image sensor includes: Measure E×F signal intensities of the E×F spectrally separated fluorescence in the E×F bin regions; acquiring a first time series data of the E×F signal intensities by continuously and repeatedly measuring the E×F signal intensities at a predetermined exposure time and a predetermined time interval; A second time series data of E×G concentrations of the G phosphors in the measurement unit of the E capillaries is derived by calculation using the first time series data, When any one of the G phosphors emits fluorescence from the measurement unit of any one of the E capillaries, A ratio of a maximum value of a concentration of the one phosphor in the measurement portion of the E capillaries excluding the one capillary to a concentration of the one phosphor in the measurement portion of the one capillary is defined as XR, In any one of the E×F bin areas, The number of pixels in the bin area is B m (≧1) pieces, The bin area is B s (≧1) hard binned regions, The average number of pixels in the hard binning area is B h (≧1) pieces, The number of pixels in the hard binning of the bin area is B h ; The number of pixels in the soft binning of the bin area is B s As individuals, B m = B h ×B s year, A ratio of a maximum value of a saturated light amount obtained by performing hard binning to a saturated light amount per pixel of the image sensor is defined as a saturated light amount ratio k (≧1); The saturation light quantity coefficient α is B h = 1, α = 1, 1 < B h <k, then α=B h , k≦B h Then, α=k, B m = B h = B s When = 1, The total noise of the first time-series data is classified into three components: a readout noise of the image sensor, a dark current noise of the image sensor, and a shot noise of background light; The total noise is N, and the read noise is N r and the dark current noise is N d and the shot noise is N s year, Dark current noise ratio is b = N d / N r Let the shot noise ratio be c = N s / N r When XR, B m , B h , B s , k, α, b, and c satisfy a predetermined relationship.

15. In claim 14, XR≦10 -3 and [0090] 13. A multi-capillary electrophoresis apparatus, comprising:

16. In claim 15, B h = 1, [0089] 13. A multi-capillary electrophoresis apparatus, comprising:

17. In claim 14, XR≦10 -3 and ##EQU00011## 13. A multi-capillary electrophoresis apparatus, comprising:

18. In claim 17, B h = 1, ##EQU00012## 13. A multi-capillary electrophoresis apparatus, comprising:

19. E capillaries into which E (≧2) samples each containing components labeled with G (≧2) fluorophores are injected and subjected to simultaneous electrophoresis; a laser light source that irradiates a laser beam onto measurement units arranged on the same plane of the E capillaries; an optical system that receives fluorescence emitted by the G phosphors passing through the measurement unit as a result of being excited by the laser beam; A computer that controls the device, The optical system includes: a spectroscopic element for dispersing the fluorescence emitted from each of the E capillaries into F (≧2, F≧G) predetermined wavelength bands; The image sensor includes a plurality of pixels arranged two-dimensionally, and receives E×F dispersed fluorescent light beams in E×F different bin regions on the image sensor; The image sensor includes: Measure E×F signal intensities of the E×F spectrally separated fluorescence in the E×F bin regions; The method is configured to acquire time series data of the E×F signal intensities by continuously and repeatedly measuring the E×F signal intensities at a predetermined exposure time and a predetermined time interval; In any one of the E×F bin areas, The number of pixels in the bin area is B m (≧1) pieces, The bin area is B s is divided into (≧1) hard binned regions, The average number of pixels in the hard binning area is B h (≧1) pieces, The number of pixels in the hard binning of the bin area is B h ; The number of pixels in the soft binning of the bin area is B s As individuals, B m = B h ×B s year, A ratio of a maximum value of a saturated light amount obtained by performing hard binning to a saturated light amount per pixel of the image sensor is defined as a saturated light amount ratio k (≧1); The saturation light quantity coefficient α is B h = 1, α = 1, 1 < B h <k, then α=B h , k≦B h Then, α=k, B m = B h = B s When = 1, The total noise of the time series data is classified into three components: a read noise of the image sensor, a dark current noise of the image sensor, and a shot noise of background light; The total noise is N, and the read noise is N r and the dark current noise is N d and the shot noise is N s year, Dark current noise ratio is b = N d / N r , the shot noise ratio is c = N s / N r When B m , B h , B s , k, α, b, and c satisfy a plurality of predetermined relationships; The multi-capillary electrophoresis apparatus according to claim 1, wherein the computer provides an environment that enables a desired predetermined relationship to be selected from the plurality of types of predetermined relationships.

20. In claim 19, The multi-capillary electrophoresis apparatus according to claim 1, wherein the computer provides a user interface for enabling a user to select the desired predetermined relationship.

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