Multi-capillary electrophoresis device

Abstract

To achieve both high sensitivity and a high dynamic range in an analysis method and analysis device that perform electrophoresis using one or more capillaries and measure fluorescence emitted by multiple types of phosphors using an image sensor or a line sensor while identifying each fluorescence.SOLUTION: In a device that performs fluorescence measurement collectively using an image sensor by splitting fluorescence emitted from a plurality of capillaries, when the number of pixels in a binning region on the image sensor in which a predetermined wavelength band component of each fluorescence is projected is set to Bm, the number of pixels for hard binning is set to Bh, the number of pixels for soft binning is set to Bs, the total noise measured in a case where Bm=Bh×Bs, Bm=Bh=Bs=1 is set to N, the readout noise is set to Nr, the dark current noise is set to Nd, and the shot noise is set to Ns, fluorescence measurement with both high sensitivity and a high dynamic range is achieved by performing control such that Bm, Bh, Bs, N, Nr, Nd and Ns satisfy a predetermined relationship.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present disclosure relates to a multi-capillary electrophoresis apparatus.

Background Art

[0002] Capillary electrophoresis apparatuses that perform electrophoresis analysis by filling a single or multiple capillaries with an electrophoresis separation medium such as an electrolyte solution, or an electrolyte solution containing a polymer gel or polymer, are widely used. The analysis targets range widely from low-molecular-weight substances to high-molecular-weight substances such as proteins and nucleic acids. Also, there are numerous 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 that occurs when the analysis target passes through the absorption point is detected, or a mode in which laser light is irradiated onto the emission point of each capillary and fluorescence or scattered light that occurs when the analysis target passes through the emission point is detected. Hereinafter, a capillary electrophoresis apparatus for DNA analysis will be described in detail as an example.

[0003] In a capillary electrophoresis apparatus for DNA analysis, by collectively irradiating a laser beam at a location where E (E is an integer of 1 or more) capillaries are arranged on the same plane, E emission points arranged in a straight line are formed. When DNA fragments labeled with G (G is an integer of 1 or more) types of phosphors pass through the emission points by electrophoresis, the phosphors are excited by the laser beam irradiation and emit fluorescence. Since these G types of phosphors have mutually different fluorescence spectra, the type of phosphor passing through the emission point can be identified by spectroscopically measuring the fluorescence. That is, in the capillary electrophoresis apparatus for DNA analysis, the fluorescence emitted from the E emission points arranged in a straight line is spectroscopically measured, and further the time change thereof is measured. To achieve this, the capillary electrophoresis apparatus for DNA analysis is equipped with a multi-color detection optical system as follows. The fluorescence emitted from the E emission points is collectively collimated by the first camera lens, the light of the laser wavelength is cut by a long-pass filter, wavelength-dispersed by a transmission diffraction grating, and imaged on an image sensor by the second camera lens. As the image sensor, a CCD, a CMOS, or other types of devices can be used. Here, the direction of wavelength dispersion is parallel to the major axis of each capillary, that is, perpendicular to the direction in which each emission point is arranged. For this reason, on the image sensor, the wavelength-dispersed images of the fluorescence from each emission point are arranged parallel to each other without mixing with each other. Therefore, the fluorescence from each emission point can be independently spectroscopically measured. Also, the image sensor is arranged such that one axis of the two-dimensional grid arrangement of the pixels of the image sensor is parallel to the wavelength dispersion direction, and the other axis is parallel to the emission point arrangement direction. As a result, the intensity distribution of the pixel array along each wavelength-dispersed image gives the fluorescence spectrum. By performing a collective imaging of the E wavelength-dispersed images by the image sensor at a constant exposure time and continuously performing this at a constant time interval, it is possible to analyze the time-series changes while spectroscopically analyzing the fluorescence from the E emission points.

[0004] The spectral dispersion image is divided into F wavelength bands (hereinafter referred to as bins), and the fluorescence intensities received by a plurality of pixels belonging to each bin are integrated. Such integration is called binning. This is called F-color detection. The wavelength width of each wavelength band can be 1 nm, 10 nm, 100 nm, etc., and can be arbitrarily set. Also, the wavelength widths may be different for each of the F wavelength bands. Generally, in order to quantify while identifying G types of phosphors, F≧G is required. For each time in the time series, color conversion is performed on the F-color detection result, and the individual fluorescence intensities of each of the G types of phosphors, that is, the concentrations of each of the G types of phosphors, can be obtained. In the present disclosure, the fluorescence intensity proportional to the concentration of each phosphor, that is, the individual fluorescence intensity of each phosphor, will simply be referred to as the concentration.

[0005] For each light-emitting point P(e) (e = 1, 2,..., E), the fluorescence emission of the phosphor D(g) (g = 1, 2,..., G) is detected in the bin W(f) (f = 1, 2,..., F). At an arbitrary time, let the concentration of the phosphor D(g) at the light-emitting point P(e) be Z(g), and let the signal intensity integrated in the bin W(f) for the light-emitting point P(e) be X(f). Here, let the F-row 1-column vector with the signal intensity X(f) as an element be X, the G-row 1-column vector with the concentration Z(g) as an element be Z, and the F-row G-column matrix with Y(f)(g) as an element be Y. Then, the following equations (1) to (4) hold. Equations (1) to (4) are relational expressions of (f) and (g), not relational expressions of (e), and hold independently for each light-emitting point P(e). In the case of single-color detection with F = 1, due to F≧G, G = 1, and X, Y, and Z are no longer vectors and matrices.

[0006]

Equation

[0007]

Equation

[0008]

Equation

[0009] [Numerical]

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

[0011] [Numerical]

[0012] Then, by performing the above process individually for all G types of phosphors D(g), all columns of the matrix Y can be determined. The matrix Y is determined only by the characteristics of the phosphor D(g) and the different bins W(f), and does not change during the electrophoresis analysis. Also, as long as the conditions such as the optical system, the phosphor D(g), and the bin W(f) are fixed, the matrix Y is kept constant for different electrophoresis analyses. Therefore, for each emission point, the concentration Z(g) of the phosphor D(g) at each time can be obtained from the signal intensity X(f) of the bin W(f) at each time by the following formula (6).

[0013] [Numerical]

[0014] Here, Y- is the generalized inverse matrix of Y in row G and column F, where Y - =(Y T ×Y) -1 ×Y T ) is obtained. 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 F-color fluorescence intensities, which are known. Equation (6) corresponds to obtaining the solution thereof. Therefore, generally, as described above, the condition F ≧ G is necessary. If F < G, the solution cannot be uniquely obtained (that is, since multiple solutions may exist), color conversion as in Equation (6) cannot be performed.

[0015] It is also possible to use a multi-color detection optical system that does not use wavelength dispersion. For example, in Patent Document 1, fluorescence emitted from E light-emitting points is individually collimated by E lenses, and after the light of the laser wavelength is cut by a long-pass filter, it is split into light beams in F wavelength bands by an F-piece dichroic mirror array and imaged on an image sensor. Here, the splitting direction 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, E×F multi-color split images are arranged two-dimensionally without mixing with each other. Therefore, fluorescence from each light-emitting point can be spectroscopically measured independently. Also, the image sensor is arranged so that one axis of the two-dimensional grid-like arrangement of the pixels of the image sensor is parallel to the splitting direction and the other axis is parallel to the light-emitting point arrangement direction. By performing a batch imaging of the E×F split images by the image sensor at a constant exposure time and continuously performing this at a constant time interval, it is possible to analyze the time-series changes while spectro-analyzing the fluorescence from the E light-emitting points. The fluorescence intensities of a plurality of pixels that receive each of the F split images for each light-emitting point are integrated. Similar to the multi-color detection optical system using wavelength dispersion, the integrated pixel region is called a bin, and this integration is called binning. Here, there may be a region that is not included in any bin among the split images. The rest is the same as the multi-color detection optical system using wavelength dispersion, and Equations (1) to (6) also hold similarly. Hereinafter, the case of using a multi-color detection optical system using wavelength dispersion will be examined, but the case of using a multi-color detection optical system that does not use wavelength dispersion as described above can be examined similarly.

[0016] As described above, the signal intensity X(f) of the bin W(f) is obtained by integrating (binning) the signal intensities of the individual pixels constituting the bin W(f). Let the number of pixels constituting the bin W(f) be B m (f). B m (f) is an integer of 1 or more. Here, when the signal intensity of pixel j constituting the bin W(f) is Q(j) (j = 1, 2,..., B m (f)), the signal intensity X(f) of the bin W(f) is expressed by the following equation (7).

[0017] [Number]

[0018] Here, the integration method (binning method) includes hard binning and soft binning. However, Equation (7) is common to both binning methods. Hard binning is a method of obtaining the signal intensity X(f) by adding up the charges accumulated in B m (f) pixels on the image sensor, converting them into voltage, and performing AD conversion. On the other hand, soft binning is performed on the circuit or computer, where B mA method of obtaining a signal intensity X(f) by summing the signal values of (f) pixels. For example, soft binning is a method of obtaining the signal intensity X(f) by converting the charge accumulated in each pixel on the image sensor into a voltage, performing AD conversion, and then integrating the digital data obtained on a computer. As will be described later, it is also possible to obtain the signal intensity X(f) by combining hard binning and soft binning. Generally, compared with soft binning, hard binning can reduce read noise and improve sensitivity, so it is known to be suitable for high-sensitivity measurement. In particular, when measuring weak light, hard binning is an extremely advantageous method. Also, compared with soft binning, hard binning can shorten the time to read the signal intensity X(f) of the bin W(f), so it is suitable for high-speed imaging. On the other hand, compared with soft binning, in hard binning, the saturation level of the light emission amount from the light-emitting point is reduced, so it is known that the dynamic range is reduced. 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, since sensitivity is more important than the dynamic range, the signal intensity X(f) is obtained by hard binning rather than soft binning. However, in recent years, in capillary electrophoresis devices for DNA analysis, it has been required to achieve both high sensitivity and a wide dynamic range. To achieve this, various prior arts have been developed as shown below.

[0019] In Patent Document 2, instead of making the exposure time in imaging by the image sensor constant as described above, long exposure times and short exposure times are alternately repeated. Under the condition that the amount of light emitted from the light-emitting point is constant, in the case of a long exposure time, since the image sensor receives more fluorescence, the sensitivity is improved. Conversely, in the case of a short exposure time, since less fluorescence is received, the sensitivity decreases, but the saturation level of the amount of light emitted from the light-emitting point increases. That is, 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, unlike the case of a single mode (for example, either the high-sensitivity mode or the low-sensitivity mode), it is possible to achieve both high sensitivity and a high dynamic range.

[0020] In Patent Document 3, an asymmetric image splitting element is added to the above-described multi-color detection optical system, and the fluorescence emitted from each light-emitting point is split into a wavelength-dispersed image with strong fluorescence intensity (hereinafter referred to as a strong split image) and a wavelength-dispersed image with weak fluorescence intensity (hereinafter referred to as a weak split image), and they are imaged and measured simultaneously. The above bins are set for both wavelength-dispersed images. Under the condition that the light emission amount from the light-emitting point is constant, in the strong split image, since the corresponding bin of the image sensor receives more fluorescence, the sensitivity is improved. Conversely, in the weak split image, since less fluorescence is received, the sensitivity decreases, but the saturation level of the light emission amount from the light-emitting point increases. That is, the strong split image functions as a high-sensitivity mode, and the weak split image functions as a low-sensitivity mode. When the light emission amount from the light-emitting point is small, the fluorescence emitted cannot be detected in the low-sensitivity mode, but the fluorescence emitted can be measured well in the high-sensitivity mode. On the other hand, when the light emission amount from the light-emitting point is large, the fluorescence emitted cannot be measured well in the high-sensitivity mode because the fluorescence emitted exceeds the saturation level, but the fluorescence emitted 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 high dynamic range, which is different from the case of a single mode (for example, either the high-sensitivity mode or the low-sensitivity mode).

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

[0022] In Patent Document 5, without fixing the bin W(f) as described above, the bin W(f) is appropriately changed. Specifically, when none of the F signal intensities X(f) (f = 1, 2, …, F) exceeds the saturation level, the bins W(f) (f = 1, 2, …, F) are set in the same manner as above (hereinafter referred to as full hard binning). However, when any one 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-th row of the matrix Y in Equation (3) are set to zero. Under the condition that the light emission amount from the light emitting point is constant, in full hard binning, since the entire bin receives more fluorescence, the sensitivity is improved. Conversely, in partial hard binning, since the entire bin receives less fluorescence, the sensitivity decreases, but the saturation level of the light emission amount from the light emitting point increases. That is, full hard binning functions as a high-sensitivity mode, and partial hard binning functions as a low-sensitivity mode. When the light emission amount 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 light emission amount 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 becomes possible to achieve both high sensitivity and a high dynamic range, which is different from 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 shown above achieve a high dynamic range while maintaining high sensitivity by combining a high-sensitivity mode and a low-sensitivity mode. Although there are differences in the combination methods of the two modes, such as alternately switching the modes, appropriately switching the modes based on the measured signals, or implementing both modes simultaneously, the basic features are common. Further, this method is effective not only for capillary electrophoresis apparatuses for DNA analysis, but also for general analysis methods and analysis apparatuses that perform electrophoresis using one or more capillaries and measure fluorescence emitted by multiple types of phosphors, scattered light scattered by multiple types of scatterers, or absorption of light absorbed by multiple types of absorbers while respectively identifying them using an image sensor or a line sensor.

[0024] On the other hand, in many commercially available digital cameras, such as the digital cameras used in smartphones, a high dynamic range can be achieved while maintaining high sensitivity by combining a high-sensitivity mode and a low-sensitivity mode. Generally, this is called HDR (High Dynamic Range) imaging. Typically, similar to Patent Document 2, imaging is performed with a long exposure time in the high-sensitivity mode and with a short exposure time in the low-sensitivity mode, and a high-dynamic-range image is synthesized by combining those images.

Prior Art Documents

Patent Documents

[0025]

Patent Document 1

Patent Document 2

Patent Document 3

Patent Document 4

Patent Document 5

Summary of the Invention

Problems to be Solved by the Invention

[0026] A method of achieving both high sensitivity and a high dynamic range by combining a high-sensitivity mode and a low-sensitivity mode, as shown in Patent Documents 2 to 5, functions when the measurement target is a single phosphor (G = 1). Alternatively, this method functions even when the measurement target is a plurality of phosphors (G ≥ 2) and there is no need to identify them. However, as will be shown below, it has been clarified by detailed studies by the present inventors that it does not function when the measurement target is a plurality of phosphors (G ≥ 2) and they are to be identified and analyzed.

[0027] Assume the following as the simplest example. E = 1 and a single capillary is the measurement target. F = 2 and the wavelength dispersion image of the fluorescence emitted from the light-emitting point on the capillary is measured with two bins W(1) and W(2), and two-color detection is performed. Assume G = 2 and the measurement targets are phosphor D(1) and phosphor D(2). Due to the difference in the fluorescence spectra of phosphor D(1) and phosphor D(2), the emitted fluorescence of phosphor D(1) is measured at a ratio of 3:2 in bins W(1) and W(2), and the emitted fluorescence of phosphor D(2) is measured at a ratio of 2:3 in bins W(1) and W(2). In the high-sensitivity mode, the fluorescence intensities of bin W(1) and bin W(2) can be measured in the range of 10 to 100 respectively, and in the low-sensitivity mode, the fluorescence intensities of bin W(1) and bin W(2) can be measured in the range of 100 to 1000 respectively. The low-sensitivity mode is realized by making the exposure time in the high-sensitivity mode 1 / 10 times. Here, the fluorescence intensities are all in arbitrary units. However, in order to align the measurement results in the high-sensitivity mode and the measurement results in the low-sensitivity mode, the measurement signal in the low-sensitivity mode is multiplied by 10 on a computer. At this time, the dynamic ranges of the high-sensitivity mode and the low-sensitivity mode are only 1 digit (10 to 100 and 100 to 1000) respectively, but it is expected that by combining these modes, the dynamic range can be expanded to 2 digits (10 to 1000) while maintaining the sensitivity (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. 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 to have 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 detection limit (measured to have an emission fluorescence intensity of 0). Of course, the emission fluorescence of phosphor D(2) is not measured in either mode (measured to have an emission fluorescence intensity of 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 both bin W(1) and bin W(2) are saturated. 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) cannot be measured in the high-sensitivity mode because bin W(1) and bin W(2) are saturated (the emission fluorescence intensity is unknown), but by performing color conversion, it is measured to have an emission fluorescence intensity of 500 in the low-sensitivity mode. Phosphor D(2) cannot be measured in the high-sensitivity mode because bin W(1) and bin W(2) are saturated (the emission fluorescence intensity is unknown) and is not measured in the low-sensitivity mode (measured to have an emission fluorescence intensity of 0).

[0030] In contrast, when the emission fluorescence intensity of phosphor D(1) is 500 and the emission fluorescence intensity of phosphor D(2) is 50, in the high-sensitivity mode, the measured fluorescence intensities of bins W(1) and W(2) are both saturated. 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) cannot be measured in the high-sensitivity mode because bins W(1) and W(2) are saturated (the emission fluorescence intensity is unknown), but by performing color conversion, it is measured as having an emission fluorescence intensity of 500 in the low-sensitivity mode. Phosphor D(2) cannot be measured in the high-sensitivity mode because bins W(1) and W(2) are saturated (the emission fluorescence intensity is unknown), and it is not measured in the low-sensitivity mode (measured as having an emission fluorescence intensity of 0).

[0031] From the above, when the emission fluorescence intensity of phosphor D(1) is 500, the same measurement results are obtained whether the emission fluorescence intensity of phosphor D(2) is 0 or 50, and the two cannot be distinguished. Generally, 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. That is, when the measurement target is only one of 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, the method of achieving both high sensitivity and high dynamic range by combining the high-sensitivity mode and the low-sensitivity mode as shown in Patent Documents 2 - 5 functions when the measurement target is a single phosphor (G = 1), but it has been clarified that it does not function when the measurement target is a plurality of phosphors (G ≧ 2) and they need to be identified and analyzed.

[0032] On the other hand, in the case of HDR mounted on the digital camera of a smartphone, since a wide variety of light emitters, light absorbers, and light scatterers are the measurement targets, a state where G≧2 can occur. However, it is not usually done to identify and analyze those measurement targets. For example, when taking a picture of a landscape containing a yellow car with a digital camera, yellow light is incident on the pixels at the position of the image of the car on the image sensor, and it is recognized that the color of the car is yellow. However, it is not done to decompose what combination of light emitter, absorber, and scatterer the yellow light is derived from and measure the respective composition ratios. For example, it is not identified whether the yellow light is a combination of red light and green light or pure yellow light. That is, in the case of HDR mounted on a digital camera, a state where G≧2 can occur, but since multiple measurement targets are not identified and analyzed, the expansion of the dynamic range is not hindered.

[0033] Therefore, in the present disclosure, in an analysis method and an analysis apparatus that perform electrophoresis using one or more capillaries, such as a capillary electrophoresis apparatus for DNA analysis, and measure fluorescence emitted by multiple types of phosphors, scattered light scattered by multiple types of scatterers, or absorption by multiple types of absorbers, while identifying each by an image sensor or a line sensor, a method that achieves both high sensitivity and a high dynamic range is proposed. A method of realizing in a single mode is proposed, rather than a method of combining a high-sensitivity mode and a low-sensitivity mode as in Patent Documents 2 to 5.

Means for Solving the Problems

[0034] Specifically, in the multi-capillary electrophoresis apparatus of the present disclosure, the composition of the electrophoresis separation medium filled in the capillary, 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 setting, the type and temperature of the image sensor, etc., are set to predetermined conditions for the composition of the noise measured by the control, and the number of pixels B m (f) constituting the bin W(f) and the number of pixels B sBy keeping (f) within a predetermined optimal range respectively, it becomes possible to achieve both high sensitivity and a high dynamic range.

[0035] Further features related to the present disclosure will become apparent from the description in this specification and the accompanying drawings. Also, aspects of the present disclosure are achieved and realized by elements and combinations of various elements and the aspects of the following detailed description and the appended claims. The description in this specification is merely a typical example and does not limit the claims or applications of the present disclosure in any sense.

Advantages of the Invention

[0036] According to the present disclosure, in an analysis method and an analysis apparatus that perform electrophoresis using one or a plurality of capillaries, such as a capillary electrophoresis apparatus for DNA analysis, and measure fluorescence emitted by a plurality of types of phosphors, scattered light scattered by a plurality of types of scatterers, or absorbance absorbed by a plurality of types of absorbers, while respectively identifying them with an image sensor or a line sensor, it becomes possible to achieve both high sensitivity and a high dynamic range. As a result, it becomes possible to analyze samples in a wide concentration range without adjusting the concentration. Alternatively, it becomes possible to analyze a sample containing a plurality of components with greatly different concentrations.

[0037] Problems, configurations, and effects other than those described above will be clarified by the description of the following embodiments.

Brief Description of the Drawings

[0038]

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Embodiments for Carrying Out the Invention

[0039] <Analysis of Sensitivity and Dynamic Range> Taking the above capillary electrophoresis apparatus for DNA analysis as an example, embodiments of the present disclosure will be described. In this apparatus, for each emission point, the bins W(f) (f = 1, 2, …, F) are each designed to measure fluorescence in a desired, specific wavelength band of the wavelength dispersion image for a certain exposure time (for example, bin W(1) is designed to measure fluorescence in the wavelength band of 500 to 510 nm, bin W(2) is designed to measure fluorescence in the wavelength band of 510 to 520 nm, etc.). Here, in each wavelength dispersion image, there may be a region (pixel) that is not included in any of the bins. Alternatively, depending on the binning conditions described later, the same region (pixel) may be included in a plurality of bins. That is, a plurality of different bins may overlap each other on the image sensor. On the other hand, depending on the configuration of the multi-color detection optical system, the size and shape of the wavelength dispersion image of the fluorescence emitted from each emission point, or the wavelength dispersion image of the fluorescence in a specific wavelength band, can be changed. 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, it is an equal-magnification imaging). Also, 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). That is, depending on the configuration of the multi-color detection optical system, the size and shape of the image of the fluorescence in a specific wavelength band emitted from the emission point on the image sensor can be changed. Further, by using an image sensor with a large (or small) size of one pixel, 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 region and the number of pixels B m (f) on the image sensor onto which the image of the fluorescence in the specific wavelength band of bin W(f) is projected can be changed. Such considerations have not been made heretofore and are unique to the present disclosure. Hereinafter, for simplicity, the (f) of bin W(f) and the number of pixels B m (f) will be omitted, but the meaning is the same.

[0040] In the present disclosure, any image sensor in which pixels are two-dimensionally arranged can be used. As a typical example, a CCD image sensor or a CMOS image sensor can be used. Also, any AD conversion that converts the amount of charge accumulated according to the amount of light irradiated to each pixel of these image sensors into a digital signal can be used regardless of the binning conditions described hereinafter. Generally, if the number of bits of the AD conversion used is small, the resolution or accuracy of the digital signal may be insufficient, and the dynamic range may become small. On the other hand, if the number of bits of the AD conversion is large, the resolution or accuracy of the digital signal can be increased, but since the time required for AD conversion becomes long, high-speed analysis becomes difficult. Also, when the number of bits of the AD conversion increases, there are problems that the power consumption of the image sensor and the control board increases and their manufacturing costs increase. In the present disclosure, as described hereinafter, a method for improving the sensitivity and dynamic range of analysis by optimizing the binning conditions is proposed. This means that by optimizing the binning conditions, the resolution or accuracy of the digital signal can be increased. Therefore, by using this method, it is possible to overcome the above-mentioned demerits due to the small number of bits of the AD conversion and at the same time enjoy the above-mentioned merits. As the number of bits of the AD conversion of the image sensor used for various analyses, usually, 16 bits or more are often used. On the other hand, according to the present disclosure, it is possible to achieve both the sensitivity and dynamic range of analysis while using an AD conversion of 14 bits or less. Also, while using an AD conversion of 12 bits or less, further 10 bits or less, it is possible to achieve both the sensitivity and dynamic range of analysis, enable high-speed analysis, and further reduce the power consumption and manufacturing costs of the image sensor and the control board.

[0041] As described above, the number of pixels B in bin W m There are hard binning and soft binning in the binning method for integrating the signals of. Within the region of bin W, B s There are hard binning regions, and the number of pixels in each hard binning region is B hLet it be so. At this time, the number of pixels of soft binning is B s and B m = B h × B s holds. That is, the number of pixels B s of soft binning does not necessarily represent the number of physical pixels in bin W, but represents the number of pixel information (number of readouts) output from the image sensor with respect to bin W. Also, 1 ≤ B h ≤ B m , 1 ≤ B s ≤ B m and B h and B s are both integers. Here, it is assumed that the B s hard binning regions are each composed of an equal number of pixels B h . Generally, the B s hard binning regions may be composed of different numbers of pixels. In that case, the average value of the number of pixels of the B s hard binning regions is taken as B h . Also, in this case, the number of pixels B h does not necessarily become an integer. The mathematical formulas and the like shown hereinafter also hold in this case. However, hereinafter, unless otherwise specified, it is assumed that the B s hard binning regions are each composed of an equal number of pixels B h .

[0042] Based on the above, noise analysis and sensitivity analysis are performed. Hereinafter, it holds for any one of the bins W. The noise in optical measurement using an image sensor is classified into three types: readout noise, dark current noise, and shot noise. In addition to these, when analyzing a sample such as capillary electrophoresis, there is also sample noise derived from the sample, but here the sample noise is ignored. It is assumed that imaging by the image sensor is repeated with a constant exposure time. Let the readout noise of one pixel be N r , the dark current noise of one pixel be N d , and the shot noise of the background light emitted from the light-emitting point and measured in bin W be N sLet it be so. Here, the background light is the light emitted from the light-emitting point and measured, minus the fluorescence that is the measurement target. Since the above noise does not depend on the binning method, it does not change even if the binning method is changed. At this time, when the number of pixels in bin W is B m let the number of pixels of hard binning in bin W be B h let the number of pixels of soft binning in bin W be B s and B m = B h × B s when it is so, the total noise N of the signal intensity X integrated in bin W is expressed by Equation (8).

[0043]

Equation

[0044] B m = B h = B s = 1, that is, when bin W is composed of a single pixel, the total noise is, as is well known, the square root of the sum of the squares of three types of noise. However, when B m = B h = B s ≠ 1, as shown in Equation (8), each noise is multiplied by a coefficient unique to it. First, the read noise is added every time data is read from the image sensor once. Therefore, no matter how many the number of pixels B h of hard binning is, the read noise is added for each hard binning area. For this reason, in the term of N r 2 in Equation (8), the number of pixels B s of soft binning corresponding to "the number of hard binning areas" = "the number of read times" is multiplied, but the number of pixels B h of hard binning that is independent of the number of read times is not multiplied. Next, the dark current noise is added by the number of pixels to be binned, regardless of whether it is hard binning or soft binning. For this reason, in the term of N d 2 in Equation (8), B h and B sBoth are applied. Finally, since shot noise is the noise inherent in the light emitted from the light-emitting point and measured, it is independent of binning regardless of whether it is hard binning or soft binning. For example, when light in a specific wavelength band emitted from the light-emitting point is incident on one pixel where B m =B h =B s =1, or when it is incident on 100 pixels where B m =B h ×B s =10×10 = 100, the total amount of light and the inherent noise should be the same. Therefore, neither B s 2 nor B h is multiplied by the term N s in Equation (8).

[0045] Another important point is that, as described above, regardless of the number of pixels B m in the bin W, the amount of light in a specific wavelength band summed within the bin W is constant, that is, the signal S is constant. Here, the signal S represents the amount of light in a specific wavelength band that is emitted from the measurement target and measured with a constant exposure time. As described above, formulating the relationship between the overall noise N and the signal S of the integrated signal X of the bin W, and the number of pixels B m of the bin W, the number of pixels B h of the hard binning among them, and the number of pixels B s of the soft binning has been made for the first time by the present disclosure.

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

[0047]

Equation

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

[0049]

Equation

[0050] At this time, Equation (8) is represented by the following Equation (11).

[0051]

Equation

[0052] As is clear from Equation (8) and Equation (11), when B m = B h = B s = 1, the total noise is minimized, the S / N is maximized, and the sensitivity is the highest. The S / N of the signal intensity X integrated in bin W is represented by the following Equation (12) from Equation (11).

[0053]

Equation

[0054] Here, S represents a certain amount of light obtained by integration within bin W as described above. Of course, the formulation of S / N in this way has been made for the first time by the present disclosure. Here, assuming that the light amount S at which S / N = 3 is the detection lower limit LLOD of bin W, the detection lower limit LLOD is represented by the following Equation (13).

[0055]

Equation

[0056] Next, dynamic range analysis is performed. Assuming that the saturation light amount per pixel of the image sensor is M, the saturation light amount of bin W is B s × M. That is, the saturation light amount increases in proportion to the number of readouts, that is, the number of pixels B of soft binning s but the number of pixels B of hard binning 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 by 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. When the maximum value of the ratio of the saturation light amount read out by performing hard binning to the saturation light amount per pixel is defined as the saturation light amount ratio k (k ≧ 1), B h When B = 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 mathematical 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 expressed by the following formula (14).

[0057]

Equation

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

[0059]

Number

[0060] Similarly, when the number of bits of the AD conversion of the image sensor is BN, the resolution of the digital signal is improved to B s ×BN, and the accuracy is improved with B s along with it.

[0061] <High-sensitivity and high-dynamic range conditions> As described above, when (B m =) B h = B s = 1, the sensitivity is the highest. However, depending on the configuration of the multi-color detection optical system, it is generally difficult to set B h = B s = 1. Therefore, it is important to prevent a significant decrease in sensitivity compared to the case where B h = B s = 1. To obtain practical sensitivity, it is necessary to obtain an S / N of 1 / 3 or more of the S / N obtained when B h = B s = 1, and the conditions for this are defined as the first high-sensitivity condition. The first high-sensitivity condition is expressed by Equation (16) from Equation (12).

[0062]

Number

[0063] Here, when B h = 1, Equation (16) is expressed as Equation (17).

[0064]

Number

[0065] Also, when B s = 1, Equation (16) is expressed as Equation (18).

[0066]

Number

[0067] Furthermore, in order to obtain a more practical sensitivity, B h = B s When = 1, it is necessary to obtain an S / N that is 2 / 3 or more of the S / N obtained when = 1, and the condition for this is defined as the second high-sensitivity condition. The second high-sensitivity condition is expressed by Equation (19) from Equation (12).

[0068]

Number

[0069] Here, when B h = 1, Equation (19) is expressed as Equation (20).

[0070]

Number

[0071] Also, when B s = 1, Equation (19) is 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 binning, hard binning, and soft binning, it is possible to expand the dynamic range DR compared to the case where B h = B s = 1. In order to obtain a practical dynamic range DR, B h = B sWhen [B]=1, it is necessary to obtain a dynamic range DR that is three times or more the dynamic range DR obtained when [B]=1, and the condition for this is defined as the first high dynamic range condition. The first high dynamic range condition is expressed by Equation (22) from Equation (15).

[0074]

Number

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

[0076]

Number

[0077] Furthermore, in order to obtain a more practical dynamic range DR, it is necessary to obtain a dynamic range DR that is ten times or more the dynamic range DR obtained when B h =B s =1, and the condition for this is defined 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, when B h =1, Equation (24) is expressed as Equation (25). Also, when B s =1, Equation (24) has no solution.

[0080]

Number

[0081] By satisfying any one of the above high-sensitivity conditions and any one of the high-dynamic range conditions, it becomes possible to achieve both high sensitivity and high dynamic range. For example, by satisfying Equation (16) and Equation (22), that is, by satisfying the following Equation (26), the first high-sensitivity condition and the first high-dynamic range condition are satisfied.

[0082] [Number]

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

[0084] [Number]

[0085] Of course, effects can also be obtained by satisfying both Equation (16) and Equation (24), or by satisfying both Equation (19) and Equation (22). However, depending on the conditions of the dark current noise ratio b, the shot noise ratio c, and the number of pixels B m , there may be cases where there is no solution that satisfies both of these.

[0086] In the above Equations (16) to (27), the preferred ranges of B for obtaining high sensitivity or high dynamic range are shown. At the same time, "1 ≤ B h ≤ B s and B h is an integer", "1 ≤ B m ≤ B h and B s is an integer", and "B m = B s × B m = B h × B s " need to be satisfied.

[0087] Next, Bh Consider the case where it is equal to 1. Equation (8) is transformed as follows into Equation (28).

[0088]

Number

[0089] Here, the mixed noise N of the read noise and the dark current noise of one pixel x is defined as in Equation (29).

[0090]

Number

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

[0092]

Number

[0093] Here, a 2 is as in Equation (31).

[0094]

Number

[0095] At this time, Equation (28) is transformed as in Equation (32).

[0096]

Number

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

[0098]

Number

[0099] Also, the dynamic range DR of formula (15) is represented by formula (34).

[0100]

Number

[0101] At this time, the first high-sensitivity condition of formula (17) is represented by formula (35).

[0102]

Number

[0103] Also, the second high-sensitivity condition of formula (20) is represented by formula (36).

[0104]

Number

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

[0106]

Number

[0107] Also, the second high-dynamic range condition of formula (25) is represented by formula (38).

[0108]

Number

[0109] <Bin Integration> In the above, for each of the F bins W(f) (f = 1, 2, …, F) that measure the fluorescence components in different wavelength bands of the fluorescence emitted from a single light-emitting point, the conditions for obtaining high sensitivity and a high dynamic range were examined. 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 constituting the bin W(f) according to Equation (7). Further, the obtained signal intensity X(f) constitutes X in Equation (2), and the concentrations Z(g) (g = 1, 2, …, G) of G types of phosphors are derived by the operation (color conversion) of Equation (6). Equation (6) indicates 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 certain kind of integration of the signal intensities X(1), X(2), …, X(F). Also, when a plurality of the signal intensities among X(1), X(2), …, X(F) show values close to each other with respect to the emission of the phosphor D(g), those signal intensities are equally integrated by Equation (6). Therefore, rather than examining the conditions for obtaining high sensitivity and a high dynamic range based on individual bins W(f) (or individual pixels), it may be appropriate to examine the conditions for obtaining high sensitivity and a high dynamic range based on an integrated bin WW formed by combining a plurality of bins W(f) (or a plurality of pixels). In this case, the integrated bin WW may sometimes be simply referred to as bin W. Next, the bin integration method for which bin W(f) (or which pixel) to incorporate into the integrated bin WW will be examined. Note that the same bin W(f) may be incorporated into integrated bins WW for different phosphors respectively.

[0110] The elements Y(1)(g), Y(2)(g), …, Y(F)(g) in the g-th column of the matrix Y in Equation (3) respectively indicate the ratio at which the emission of the phosphor D(g) contributes to the signal intensities X(1), X(2), …, X(F) of each bin W(f). These elements are normalized so that their sum becomes 1 according to Equation (5), but here, they will be normalized so that the maximum value of these elements becomes 1. Each element at this time is denoted as [Y(1)(g)], [Y(2)(g)], …, [Y(F)(g)].

[0111] In the first bin integration method, for each phosphor D(g), when [Y(f1)(g)] = 1, only bin W(f1) is incorporated into the integrated bin WW. This is a method following the method of not introducing the integrated bin WW until now. However, different from the past, for any bin W(f0) where [Y(f0)(g)] ≠ 1, it is not necessary to consider the conditions for obtaining high sensitivity and high dynamic range.

[0112] In the second bin integration method, for each phosphor D(g), when [Y(f j )(g)] ≥ 0.9 (j = 1, 2, …, J), bins W(f j )(j = 1, 2, …, J) are incorporated into the integrated bin WW. Similarly, for any bin W(f0) where [Y(f0)(g)] < 0.9, it is not necessary to consider the conditions for obtaining high sensitivity and high dynamic range.

[0113] In the third bin integration method, for each phosphor D(g), when [Y(f j )(g)] ≥ 0.8 (j = 1, 2, …, J), bins W(f j )(j = 1, 2, …, J) are incorporated into the integrated bin WW. Similarly, for any bin W(f0) where [Y(f0)(g)] < 0.8, it is not necessary to consider the conditions for obtaining high sensitivity and high dynamic range.

[0114] In the fourth bin integration method, for each phosphor D(g), when [Y(f j )(g)] ≥ 0.5 (j = 1, 2, …, J), bins W(f j )(j = 1, 2, …, J) are incorporated into the integrated bin WW. Similarly, for any bin W(f0) where [Y(f0)(g)] < 0.5, it is not necessary to consider the conditions for obtaining high sensitivity and high dynamic range. That is, in the first to fourth bin integration methods, the bins W(f) with a high contribution ratio to the signal intensities X(1), X(2), …, X(F) of the emission of the phosphor D(g) are incorporated into the integrated bin WW in order.

[0115] In contrast, in the fifth bin integration method, for each phosphor D(g), all bins W(f) (f = 1, 2, …, F) are incorporated into the integrated bin WW. No matter which of the above first to fifth bin integration methods is adopted, for the adopted integrated bin WW, the equations (8) to (38) derived to achieve both high-sensitivity conditions and high-dynamic range conditions should hold as they are. That is, the integrated bin WW is virtually regarded as one bin, and the number of pixels constituting the integrated bin WW is denoted as B m Let the average value of the number of pixels in the (plural) hard binning regions included in the integrated bin WW be B h Let the number of pixels of the soft binning of the integrated bin WW be B s Let it be B m = B h × B s In this way, similar to before, both high-sensitivity conditions and high-dynamic range conditions are achieved. Hereinafter, the above will be described for the fifth bin integration method.

[0116] For each light-emitting point P(e) (e = 1, 2, …, E), let the number of pixels of the bin W(f) (f = 1, 2, …, F) be B m (f), let the number of pixels of the hard binning of the bin W(f) be B h (f), and let the number of pixels of the soft binning of the bin W(f) be B s (f). Similar to 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 both B h (f) and B s (f) are integers. Assuming the signal intensity XX integrated in the integrated bin WW obtained by integrating the bins W(1), W(2), …, W(F), the total noise N is expressed by Equation (39) by transforming Equation (11).

[0117]

Equation

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

[0119] [Number]

[0120] Also, if the light amount S at which S / N = 3 is defined as the detection lower limit LLOD of the integration bin WW, then it is expressed by Equation (41) by transforming Equation (13).

[0121] [Number]

[0122] Furthermore, the detection upper limit ULOD of the bin WW, that is, the saturation light amount of the bin WW, is expressed by Equation (42) by transforming 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 as ULOD / LLOD and is expressed by Equation (43) by transforming Equation (15).

[0125] [Number]

[0126] Here, by redefining Expressions (44) to (47) as follows, Expressions (8) to (38) hold as they are. In fact, when using Expressions (44) to (47), Expression (11) and Expression (39), Expression (12) and Expression (40), Expression (13) and Expression (41), Expression (14) and Expression (42), Expression (15) and Expression (43) each become the same expression. These are just examples, and the same applies to other mathematical expressions. That is, when a set of a plurality of bins W(f) (or a plurality of pixels) is regarded 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 shown by Expressions (8) to (38) by defining Expressions (44) to (47).

[0127] [Number]

[0128] [Number]

[0129] [Number]

[0130] [Number]

[0131] Regarding the first to fourth bin integration methods, only the integration range of the bin W(f) (f = 1, 2,..., F) changes, and if the integration ranges of Expressions (44) to (47) are changed accordingly, Expressions (8) to (38) hold as they are.

[0132] [Consideration of exposure time] In the above, regarding the case where fluorescence measurement by an image sensor is repeatedly performed with a constant exposure time, the sensitivity and dynamic range were analyzed, and the conditions for achieving both high sensitivity and high dynamic range were clarified. In this study, further, by controlling the exposure time, it becomes easier to achieve both high sensitivity and high dynamic range, and the range of suitable conditions is expanded. Generally, when the exposure time is shortened, it becomes possible to measure without saturating stronger emission intensities, so the upper detection limit ULOD of bin W can be increased. On the other hand, when the exposure time is shortened, the number of readouts increases, so the noise increases and the lower detection limit LLOD of bin W also increases. Or, if the number of readouts is not increased while shortening the exposure time, the measured signal decreases, so the lower detection limit LLOD also increases. That is, when the exposure time is shortened, the sensitivity decreases, and in some cases, the dynamic range may also decrease. Therefore, it is not simply a matter of shortening the exposure time, but by setting the optimal exposure time, it becomes possible to achieve both high sensitivity and high dynamic range.

[0133] Let the constant exposure time assumed so far be the standard exposure time T. Divide the standard exposure time T equally into μ shortened exposure times t. Or, expand the standard exposure time T to an extended exposure time t that is 1 / μ times. Here, assuming that the data readout time from the image sensor is zero and there is no time loss due to the division, the standard exposure time T is expressed by Equation (48).

[0134]

Equation

[0135] μ is positive but not necessarily an integer. When μ ≥ 1, since T ≥ t, t represents a shortened exposure time. When 0 < μ < 1, since T < t, t represents an extended exposure time. Hereinafter, whether μ ≥ 1 or 0 < μ < 1, it holds, but 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 readout 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 readout noise of one pixel at the shortened exposure time t is n r becomes, and the dark current noise of one pixel is t × n d = t × b0 × n r becomes, 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 becomes. 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 readout noise of one pixel at the standard exposure time T is N r =n r and the dark current noise of one pixel is N d =T×n d and the shot noise of the total light emission measured with binning W is N s =T 0.5 ×n s At this time, the dark current noise ratio b is expressed by Equation (52).

[0142]

Equation

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

[0144]

Equation

[0145] Therefore, the total noise N of the signal corresponding to the standard exposure time T obtained by integrating the signals measured with the shortened exposure time t for μ times with a computer for binning W is expressed by Equation (54) by transforming Equations (8) and (11).

[0146]

Equation

[0147] From this, the S / N of binning W is expressed by Equation (55) by transforming Equation (12).

[0148]

Equation

[0149] Therefore, the detection limit LLOD of binning W is expressed by Equation (56) by transforming Equation (13).

[0150]

Mathematics

[0151] On the one hand, the detection upper limit ULOD of the vial W is expressed by Equation (57) by transforming Equation (14).

[0152]

Mathematics

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

[0154]

Mathematics

[0155] Based on the above, by controlling the exposure time, the conditions that satisfy both the high-sensitivity condition and the high-dynamic range condition are clarified. Here, the S / N and DR when B h = B s = 1 at the standard exposure time T are used as the comparison targets. The first high-sensitivity condition is expressed by Equation (59) by transforming Equation (16).

[0156]

Mathematics

[0157] Also, the first high-sensitivity condition when B h = 1 is expressed by Equation (60) by transforming Equation (17).

[0158]

Mathematics

[0159] Furthermore, the first high-sensitivity condition when B s = 1 is expressed by Equation (61) by transforming Equation (18).

[0160]

Mathematics

[0161] The second high-sensitivity condition is expressed by Equation (62) by transforming Equation (19).

[0162]

Mathematics

[0163] Also, when B h = 1, the second high-sensitivity condition is expressed by Equation (63) by transforming Equation (20).

[0164]

Mathematics

[0165] Furthermore, when B s = 1, the second high-sensitivity condition is expressed by Equation (64) by transforming Equation (21).

[0166]

Mathematics

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

[0168]

Mathematics

[0169] Also, when B h = 1, the first high-dynamic range condition is expressed by Equation (66) by transforming Equation (23).

[0170]

Mathematics

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

[0172] [Number]

[0173] Similar to the above, by appropriately combining the above Equations (59) to (67), noise conditions and binning conditions for achieving both high sensitivity and high dynamic range are derived.

[0174] [Transformation of High Dynamic Range Condition] Above, the first high dynamic range condition and the second high dynamic range condition have been examined. In addition to these, a method for defining 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 application range is limited. To obtain a practical dynamic range and expand the application range, a dynamic range DR of 3000 or more is required, and the conditions therefor are defined as the third high dynamic range condition. The third high dynamic range condition is expressed by Equation (68).

[0175] [Number]

[0176] For example, in Equations (15), (34), (43), (58) shown above, and Equations (81), (85) shown below, when Equation (68) holds, the third high dynamic range condition is satisfied. Also, to obtain a more practical dynamic range DR and further expand the application range, a dynamic range DR of 10000 or more is required, and the conditions therefor are defined as the fourth high dynamic range condition. The fourth high dynamic range condition is expressed by Equation (69).

[0177] [Number]

[0178] For example, in the formulas (15), (34), (43), (58) shown above, and the formulas (81), (85) shown below, when the formula (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 analyzer. The multi-capillary electrophoresis apparatus is widely used as an analyzer for DNA sequence and DNA fragment analysis. The multi-capillary electrophoresis apparatus includes capillaries 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 the binning conditions described later. Further, the computer 100 analyzes the time-series data of the fluorescence detected by the multi-color detection optical system 15 and analyzes the DNA sequence sample by executing a program stored in a memory (not shown).

[0180] In this example, E = 4 capillaries 1 were used, and DNA sequences of different samples were carried out in each capillary 1. The outer diameter of each capillary 1 is 360 μm, and the inner diameter is 50 μm. The DNA sequence sample is composed of DNA fragments labeled with G = 4 types of phosphors.

[0181] One analysis session was executed by the following steps (1) to (6). (1) First, the sample injection ends 2 of E = 4 capillaries 1 were immersed in the cathode-side buffer 6, and the sample elution ends 3 were immersed in the anode-side buffer 7 via the polymer block 9. (2) Next, close the valve 10 of the pump block 9, and pressurize the internal polymer solution Ω1 by pushing down the piston of the syringe 11 connected to the pump block 9, and fill the internal of each capillary 1 with the polymer solution Ω1 from the sample elution end 3 toward the sample injection end 2. (3) Subsequently, open the valve 10, electrokinetically inject different samples into each capillary 1 from the sample injection end 2, and then start capillary electrophoresis by applying a high voltage between the cathode 4 and the anode 5 with the power supply 8. G = DNA fragments labeled with four types of phosphors were electrophoresed from the sample injection end 2 toward the sample elution end 3. (4) In parallel, the position electrophoresed a certain distance from the sample injection end 2 of each capillary 1 was defined as the emission point 14, and a laser beam 13 with an output of 5 mW and a wavelength of 505 nm oscillated from the laser light source 12 was collectively irradiated onto each emission point 14. Here, the coating of each capillary 1 near the emission point 14 was removed in advance, each capillary 1 near the emission point 14 was arranged on the same plane, and after narrowing the laser beam 13 to about φ50 μm, it was introduced along the arrangement plane from the side of the arrangement plane. (5) Then, the DNA fragments labeled with G = four types of phosphors were electrophoresed inside each capillary 1, and each phosphor labeled by the irradiation of the laser beam 13 was excited when passing through the emission point 14, emitting fluorescence. That is, G = four types of phosphors were caused to fluoresce from E = four emission points 14, and with electrophoresis, the respective fluorescence intensities were made to change moment by moment. (6) Finally, the fluorescence emitted from each emission point 14 was measured by the multi-color detection optical system 15, and the DNA sequence of the sample injected into each capillary 1 was determined by analyzing the obtained time-series data with the computer 100. Here, the size and shape of each emission point 14 are 50 μm square because the inner diameter of each capillary 1 is 50 μm and the diameter of the laser beam is 50 μm. The multi-color detection optical system 15 is located on the back side of each emission point 14 in FIG. 1.

[0182] FIG. 2A is a diagram showing a configuration example of the multi-color detection optical system 15. FIG. 2A depicts the multi-color detection optical system 15 from the side of the array 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 is collimated by the first camera lens 16 with a focal length f1 = 50 mm, and after the light with a laser wavelength of 505 nm is cut by the long-pass filter 17, it is wavelength-dispersed by the transmission diffraction grating 18 with a grating frequency N = 600 lines / mm, and is imaged on the image sensor 20 at a magnification of 1 by the 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 saturation light amount ratio of the image sensor 20 was k = 1. Here, the direction of wavelength dispersion was set to be parallel to the major axis of each capillary 1, that is, perpendicular to the direction in which the respective light-emitting points 14 are arranged. However, as shown in FIG. 2A, the optical axis 21 of the multi-color 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 wavelength-dispersed like the dispersed fluorescence 23, 24, 25.

[0184] FIG. 2B is a schematic diagram of an image 26 captured by the image sensor 20. FIG. 2B shows a wavelength-dispersed image 27 of light emitted from E = 4 light-emitting points 14. On the image sensor 20, the wavelength-dispersed images 27 of the fluorescence 22 from each light-emitting point 14 are arranged parallel to each other without mixing with each other. Therefore, the fluorescence 22 from each light-emitting point 14 could be independently spectroscopically measured. Further, the image sensor 20 was arranged such that one axis of the two-dimensional lattice arrangement of the pixels of the image sensor 20 was parallel to the wavelength dispersion direction and the other axis was 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-dispersed image 27 gave the fluorescence spectrum of the fluorescence 22. Hereinafter, a method for analyzing one of the plurality of simultaneously measured wavelength-dispersed images 27 will be described, but the same analysis method was applied to the other wavelength-dispersed images 27 as well.

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

[0186]

Equation

[0187] The dispersion angle per 1 nm of wavelength is given by Equation (71).

[0188]

Equation

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

[0190]

Equation

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

[0192] A wavelength range of 180 nm from 520 to 700 nm of the wavelength-dispersed image 27 was set as the measurement target. This wavelength range was equally divided into 20 wavelength bands with a width of 9 nm, and each was set to F = 20 bins W(f) (f = 1, 2,..., 20). Since the wavelength resolution is 0.75 nm / pixel, the light in the 9-nm-wide wavelength band is received by 12 pixels in the wavelength dispersion direction. Also, since the light-emitting point 14 with a size of 50 μm square is imaged at the same magnification and the pixel size is 24 μm square, the light in the 9-nm-wide wavelength band is received by 3 pixels in the light-emitting point array direction. Therefore, as the binning condition, each bin W(f) is set as a region of 12 pixels in the wavelength dispersion direction and 3 pixels in the light-emitting point array direction, and the number of pixels B m constituting each bin W(f) is set to 12 × 3 = 36 pixels. Also, in each bin W(f), the number of pixels for hard binning is B h = 36 pixels, and the number of pixels for soft binning is B s = 1 pixel. 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] Figure 3 shows an enlarged view of the upper end portion of one wavelength-dispersed image 27 among the images 26 in FIG. 2B. FIGS. 4 to 10 hereinafter show the same region as FIG. 3 on the same scale. All of them show only a small part of the image 26 acquired by the image sensor 20. FIG. 4 shows the configuration of the pixels 28 in the same region. FIG. 5 is obtained by hiding the wavelength-dispersed image 27 in FIG. 4. A total of 175 pixels 28, five pixels in the horizontal direction and 35 pixels in the vertical direction, are drawn. FIG. 6 is obtained by adding the region of the bin W(f) with a thick line to FIG. 5, and shows a part of the bin W(1), the bin W(2), and the bin W(3). According to the above binning condition, each bin W(f) has 3 pixels in the light-emitting point array direction (horizontal direction) and 12 pixels in the wavelength dispersion direction (vertical direction), and B m = 12×3 = 36 pixels 28. FIG. 7 is obtained by adding the hard binning region 7-1 with gray and diagonal lines to FIG. 6 according to the above binning condition. Each bin W(f) is composed of a hard binning region 7-1 composed of B s = 1, B h = 36 pixels 28.

[0194] On the other hand, FIGS. 8 to 10 show binning conditions different from the above binning condition. In FIG. 8, each bin W(f) is composed of hard binning regions 8-1 to 8-12 composed of B s = 12, B h = 3 pixels 28. In FIG. 9, each bin W(f) is composed of hard binning regions 9-1 to 9-36 composed of B s = 36, B h = 1 pixel 28. In FIGS. 8 and 9, the number of pixels B h of each of the plurality of hard binning regions constituting each bin W(f) is constant (B h = 3 in FIG. 8, B h = 1 in FIG. 9), but as shown in FIG. 10, it does not necessarily have to be constant. In FIG. 10, each bin W(f) has B s = 8, B hIt is composed of hard binning regions 10-1 to 10-8 each composed of 4, 5, 6, 5, 4, 6, 5, and 1 pixel 28. In such a case, the average value of the number of pixels constituting the plurality of hard binning regions is B h Let it be. In the case of Fig. 10, B h =(4 + 5 + 6 + 5 + 4 + 6 + 5 + 1) / 8 = 36 / 8 = 4.5.

[0195] Figs. 7 to 10 show four types of binning conditions for bin W(f) with B m = 36, but these are just examples, and various other binning conditions can be set. Also, the number of pixels 28 constituting bin W(f) is not limited to B m = 36, and it can be set to any value.

[0196] The setting of the above binning conditions is performed by software or firmware, so the current binning conditions can be examined. However, even if the setting value is unknown, the current binning conditions can be examined. For example, assume a case where only X(f), which is the signal intensity of bin W(f), can be referred to, or a case where only Z(g), which is the concentration of phosphor D(g), can be referred to. By irradiating light onto each pixel and changing the intensity of the incident light, the response of the signal intensity X(f) or concentration Z(g) to the incident light amount of each pixel can be examined. As means for irradiating light onto each pixel, there are a method of narrowing a laser beam and making it incident on an image sensor, a method of emitting light of a single wavelength from a very small light-emitting point and forming an image on the image sensor, etc. Thereby, each pixel can be classified into each bin W(f), and the number of pixels B m of each bin W(f) can be identified. Also, from the response of the signal intensity X(f) or concentration Z(g) when the number of pixels onto which light is incident is increased to 2 pixels, 3 pixels, etc., the number of pixels B h of hard binning of each bin W(f), the number of pixels B sIt can be identified. Of course, the binning conditions may be examined by other means. From the above results, it is possible to determine whether the current binning conditions are appropriate for achieving both high sensitivity and a high 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. By obtaining the signal intensity X(f) (f = 1, 2, …, 20) integrated by the bin W(f) at each time, a 20-row and 1-column matrix X shown in Equation (2) was obtained for each time. Also, a 20-row and 4-column matrix Y shown in Equation (3) was obtained in advance. Then, according to Equation (6), a 4-row and 1-column matrix Z shown in Equation (4) was obtained for each time. That is, the time changes of the concentrations Z(g) (g = 1, 2, 3, 4) of four types of phosphors G = 4 were obtained. By analyzing the obtained time-series data, the DNA sequence of the sample injected into each capillary 1 was performed.

[0198] Under the above measurement conditions and B shown in FIG. 7 m = 36, B h = 36, B s Noise analysis of the image sensor was performed under the binning conditions of = 1. There was no difference in the noise composition for each bin W(f). In a state where no light is incident on the image sensor, that is, N s = 0 count, the same measurement as above was performed, and the standard deviation of the time-series data of the signal intensity X(f) was obtained. The "count" used in the present application is the unit of the signal intensity digitally output by the image sensor.

[0199] Also, the exposure time was changed stepwise in the range of 0 ms to 1000 ms, and the standard deviation was similarly obtained. When the standard deviation with respect to the exposure time was plotted and an approximate curve was obtained, the vertical-axis intercept was 1.5 counts. At this time, the read noise per W(f) is the vertical-axis intercept of the above 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 the first term B on the right side of Equation (8) s × N r2 is given by the root. Therefore, under this condition, B s = 1, so the read noise of 1 pixel is N r = 1.5 counts.

[0200] Next, it was determined that the value of the above approximation curve at an exposure time of 100 ms was 1.55 counts. At this time, the left side of Equation (8) is N 2 = 1.55 2 , the first term on the right side is B s ×N r 2 = 1.5 2 , the third term on the right side is N s 2 = 0. Therefore, the second term on the right side that 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 this condition, B h = 36, B s = 1. Therefore, the dark current noise of 1 pixel is N d = 0.065 counts.

[0201] Finally, light was made incident on the image sensor, and it was determined that the standard deviation of the background light during electrophoresis analysis, that is, the total noise, was N = 1.6 counts. At this time, the left side of Equation (8) is N 2 = 1.6 2 , the first term on the right side is B s ×N r 2 = 1.5 2 , the second term on the right side is B h ×B s ×N d 2 = 0.39 2 . Therefore, the third term on the right side that gives the shot noise per bin W(f) is N s 2 = 1.6 2 - 1.5 2 - 0.39 2 = 0.40 2That is, the shot noise of the background light emitted from the light-emitting point and measured by the bin W(f) is N s = 0.40 counts. From the above, the dark current noise ratio of Equation (9) was found to be b = 0.043, and the shot noise ratio of Equation (10) was found to be c = 0.27.

[0202] Regarding the above, the performance of sensitivity and dynamic range was examined. Substituting b = 0.043 and c = 0.27 into Equation (18) and "1 ≤ B h ≤ B m ", since B h ≤ 36, this condition (B h = 36, B s = 1) satisfied the first high-sensitivity condition. Also, substituting b = 0.043 and c = 0.27 into Equation (21) and "1 ≤ B h ≤ B m ", since B h ≤ 36, this condition (B h = 36, B s = 1) also satisfied the second high-sensitivity condition. On the other hand, since this condition has B s = 1, it did not satisfy either the first high-dynamic range condition or the second high-dynamic range condition. That is, it was found that it is impossible to achieve both the high-sensitivity condition and the high-dynamic range condition under this condition. In the above, since the saturation light quantity ratio of the image sensor 20 is k = 1, α = 1 was set.

[0203] In the above, N r , b, and c indicating the noise composition were derived using the means of changing the exposure time, but the noise composition can also be obtained even when the exposure time cannot be changed. It is known that the shot noise of each bin W(f) is proportional to the square root of the amount of light incident on the bin W(f). Therefore, it is possible to control the shot noise by changing the incident light quantity. Thus, when plotting the square of the total noise N 2 of the signal intensity X(f) of the bin W(f) against the square of the shot noise ratio c 2 , according to Equation (11), the slope is N r 2 , and the vertical axis intercept is (Bs +b 2 ×B m )×N r 2 A straight line of is obtained, and N r and the dark current noise ratio b can be derived. Also, from the total noise N under the actual measurement conditions and the above straight line, the shot noise ratio c under the actual measurement conditions can be derived.

[0204] <Change of various conditions> Therefore, by changing the binning conditions, specifically, while fixing the settings of the multi-color detection optical system and B m = 36, while varying the number of pixels B of hard binning h and the number of pixels B of soft binning s we examined whether it is possible to achieve both high-sensitivity conditions and high-dynamic range conditions. B m = 36, b = 0.043, c = 0.27 are substituted into Equation (16) and "1 ≤ B s ≤ B m and B s is an integer", then B s ≤ 9. Also, when B m = 36, b = 0.043, c = 0.27 are substituted into Equation (22) and "1 ≤ B s ≤ B m and B s is an integer", then B s ≥ 9. Therefore, by setting the conditions of B h = 4, B s = 9, Equation (26) is satisfied, and the first high-sensitivity condition and the first high-dynamic range condition are satisfied. On the other hand, when B m = 36, b = 0.043, c = 0.27 are substituted into Equation (19) and "1 ≤ B s ≤ B m and B s is an integer", then B s ≤ 2. Also, when B m = 36, b = 0.043, c = 0.27 are substituted into Equation (24) and "1 ≤ B s ≤ B m and B sWhen substituting into "is an integer", there is no solution. Therefore, Equation (27) is not satisfied, and the second high-sensitivity condition and the second high-dynamic range condition are not satisfied.

[0205] Next, the measurement conditions were changed. The separation medium filled in the capillary 1 was changed from the polymer solution Ω1 to the polymer solution Ω2, and the output of the laser beam was increased from 5 mW to 20 mW. When the same noise analysis as above was performed under these conditions, the noise composition changed. Also, the noise composition differed depending on the bin W(f). The total noise of bin W(20) became the minimum N = 4 counts, and the total noise of bin W(10) became the maximum N = 16 counts. In any case, the readout noise of 1 pixel was N r = 1.5 counts, and the dark current noise of 1 pixel was N d = 0.065 counts and did not change. From the above, the shot noise of the background light emitted from the light-emitting point and measured by the bin W(f) is N s = 3.7 counts in the bin W(20), and it was found that it increased to N s = 16 counts in the bin W(10). That is, in each bin W(f), N r and N d are constant, but N s changed in the range of 3.7 to 16 counts. As a result, the dark current noise ratio of Equation (9) is b = 0.043, and the shot noise ratio of Equation (10) changed in the range of c = 2.5 to 10.7.

[0206] Substituting these into Equation (18) and "1 ≤ B h ≤ B m ", for any shot noise ratio c of c = 2.5 to 10.7, B h ≤ 36, so this condition (B h = 36, B s = 1) satisfied the first high-sensitivity condition. Also, substituting these into Equation (21) and "1 ≤ B h ≤ B m ", for any shot noise ratio c of c = 2.5 to 10.7, B h ≤ 36, so this condition (B h = 36, Bs = 1) also satisfied the second high-sensitivity condition. On the other hand, since this condition has B s = 1, it did not satisfy either the first high-dynamic range condition or the second high-dynamic range condition. That is, it was found that under this condition, it is impossible to achieve both the high-sensitivity condition and the high-dynamic range condition. As described above, since the saturation light quantity ratio of the image sensor 20 is k = 1, α = 1 was set. On the other hand, the saturation light quantity per pixel of the image sensor was M = 65000. When c = 2.5, from Equation (15), DR = 5340. Therefore, it was found from Equations (68) and (69) that this condition satisfies the third high-dynamic range condition but does not satisfy the fourth high-dynamic range condition. In contrast, when c = 10.7, from Equation (15), DR = 1344. Therefore, it was found from Equations (68) and (69) that this condition does not satisfy either the third high-dynamic range condition or the fourth high-dynamic range condition.

[0207] Subsequently, in the same manner as above, by changing the settings of the multi-color detection optical system and B m = B h × B s = 36 while fixing it, and varying the number of pixels B h of hard binning and the number of pixels B s of soft binning, it was examined whether it is possible to achieve both the high-sensitivity condition and the high-dynamic range condition. Here, the number of pixels B h of hard binning is an integer. First, set the shot noise ratio to c = 2.5. Substituting B m = 36 and b = 0.043 into Equations (16) and (19), under the condition of "1 ≤ B s ≤ B m and B s is an integer", B s ≤ 36 and B s ≤ 10 are obtained respectively. Also, substituting B m = 36 and b = 0.043 into Equations (22) and (24), under the condition of "1 ≤ B s ≤ B m and B s is an integer", B s ≥ 4 and Bs becomes ≧19.

[0208] The above is summarized as follows. (B h , B s ) When it is (36, 1), (18, 2) or (12, 3), the second high-sensitivity condition is satisfied. (B h , B s ) When it 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 it is (3, 12) or (2, 18), the first high-sensitivity condition and the first high-dynamic range condition are satisfied. And, (B h , B s ) When it is (1, 36), the first high-sensitivity condition and the second high-dynamic range condition are satisfied. Therefore, there was no solution that satisfied the second high-sensitivity condition and the second high-dynamic range condition. On the other hand, from Equation (15), Equation (68) and Equation (69), (B h , B s ) In any of the cases of (18, 2), (12, 3), (9, 4), (6, 6), (4, 9), (3, 12), (2, 18) and (1, 36), it was found that both the third high-dynamic range condition and the fourth high-dynamic range condition were satisfied.

[0209] Next, let the shot noise ratio be c = 10.7. B m = 36 and b = 0.043 are substituted into Equation (16) and Equation (19). Then, under the condition of "1 ≦ B s ≦ B m and B s is an integer", B s ≦ 36 in all cases. Also, when B m = 36 and b = 0.043 are substituted into Equation (22) and Equation (24), under the condition of "1 ≦ B s ≦ B m and B s is an integer", B s ≧ 3 and B s ≧ 11 respectively. The above is summarized as follows. (Bh , B s ) When it is (36, 1) or (18, 2), the second high-sensitivity condition is satisfied. (B h , B s ) When it 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 it is (3, 12), (2, 18) or (1, 36), the second high-sensitivity condition and the second high-dynamic range condition are satisfied. Above, since the saturation light quantity ratio of the image sensor 20 is k = 1, α = 1 was set. Incidentally, FIG. 7 shows the case where (B h, B s ) is (36, 1), FIG. 8 shows the case where (B h , B s ) is (3, 12), and FIG. 9 shows the case where (B h , B s ) is (1, 36). As described above, there may be a plurality of solutions in which the high-sensitivity condition and the high-dynamic range condition are satisfied at the same level. In such a case, when selecting a solution with a large number of hard-binning pixels B h from among the plurality of solutions, it is advantageous in terms of improving the data readout speed from the image sensor. On the other hand, from equations (15), (68) and (69), (B h , B s ) In any of the cases of (12, 3), (9, 4) and (6, 6), it was found that the third high-dynamic range condition is satisfied, but the fourth high-dynamic range condition is not satisfied. Also, (B h , B s ) In any of the cases of (4, 9), (3, 12), (2, 18) and (1, 36), it was found that both the third high-dynamic range condition and the fourth high-dynamic range condition are satisfied.

[0210] As described above, it has been clarified that by devising the binning conditions, it is possible to achieve both high-sensitivity conditions and high-dynamic range conditions. However, depending on the noise composition represented by the dark current noise ratio b and the shot noise ratio c, the binning conditions that satisfy the high-sensitivity conditions and the high-dynamic range conditions change. 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 quantity ratio> Subsequently, when the image sensor 20 with a saturation light quantity ratio of k = 1 was replaced with the image sensor 20 with k = 3, the sensitivity and dynamic range performance were similarly examined. Here, the number of pixels B for hard binning h is a positive integer, and when B h = 1, α = 1, when B h = 2, α = 2, and when B h ≧ 3, α = 3. In the following, 1 ≦ B s ≦ B m and B s is an integer.

[0212] First, the case where B m = 36, b = 0.043, and c = 0.27 was examined. Since α is not included in equations (16) and (19), the high-sensitivity conditions do not change from the case where k = 1. That is, the first high-sensitivity condition was satisfied by setting B s ≦ 9, and the second high-sensitivity condition was satisfied by setting B s ≦ 2. On the other hand, since α is included in equations (22) and (24), the high-dynamic range conditions change from the case where k = 1. When B h = 1, α = 1, and from equation (22), B s ≧ 9. When B h = 2, α = 2, and from equation (22), B s ≧ 3. When B h ≧ 3, α = 3, and from equation (22), B s ≧ 1. Therefore, (B h , B s) were all combinations of (1, 36), (2, 18), (3, 12), (4, 9), (6, 6), (9, 4), (12, 3), (18, 2), and (36, 1) and became solutions to the first high dynamic range condition.

[0213] On the other hand, for B h = 1, α = 1, and from Equation (24), B s ≥ 36. For B h = 2, α = 2, and from Equation (24), B s ≥ 24. For B h ≥ 3, α = 3, and from Equation (24), B s ≥ 11. Therefore, the combinations of (B h , B s ) being (1, 36) and (3, 12) became solutions to the second high dynamic range condition. From the above, by setting (B h , B s ) to any of (4, 9), (6, 6), (9, 4), (12, 3), (18, 2), and (36, 1), the first high sensitivity condition and the first high dynamic range condition were satisfied. Also, it was found that there was no binning condition that simultaneously satisfied the second high sensitivity condition and the second high dynamic range condition.

[0214] Next, the case where B m = 36, b = 0.043, and c = 2.5 was examined. Since α is not included in Equations (16) and (19), the high sensitivity condition does not change from the case of k = 1. That is, by setting B s ≤ 36, the first high sensitivity condition was satisfied, and by setting B s ≤ 10, the second high sensitivity condition was satisfied. In contrast, since α is included in Equations (22) and (24), the high dynamic range condition changes from the case of k = 1. For B h = 1, α = 1, and from Equation (22), B s ≥ 4. For B h = 2, α = 2, and from Equation (22), B s ≥ 2. For B h ≥ 3, α = 3, and from Equation (22), Bs is equal to or greater than 1. Therefore, (B h , B s ) for all combinations of (1, 36), (2, 18), (3, 12), (4, 9), (6, 6), (9, 4), (12, 3), (18, 2) and (36, 1) became solutions to the first high dynamic range condition.

[0215] On the other hand, when B h = 1, α = 1, and from Equation (24), B s is equal to or greater than 18. When B h = 2, α = 2, and from Equation (24), B s is equal to or greater than 7. When B h is equal to or greater than 3, α = 3, and from Equation (24), B s is equal to or greater than 4. Therefore, (B h , B s ) for combinations of (1, 36), (2, 18), (3, 12), (4, 9), (6, 6), (9, 4) became solutions to the second high dynamic range condition. From the above, by setting (B h , B s ) to any of (1, 36), (2, 18), (3, 12), (4, 9), (6, 6), (9, 4), (12, 3), (18, 2) and (36, 1), the first high sensitivity condition and the first high dynamic range condition were satisfied. Also, by setting (B h , B s ) to any of (4, 9), (6, 6), (9, 4), the second high sensitivity condition and the second high dynamic range condition were satisfied.

[0216] Finally, the case where B m = 36, b = 0.043, and c = 10.7 was examined. Since α is not included in Equation (16) and Equation (19), the high sensitivity condition does not change from the case where k = 1. That is, by setting B s to be equal to or less than 36, both the first high sensitivity condition and the second high sensitivity condition were satisfied. In contrast, since α is included in Equation (22) and Equation (24), the high dynamic range condition changes from the case where k = 1. B hWhen α = 1 when = 1, from Equation (22), B s ≧ 3. B h When α = 2 when = 2, from Equation (22), B s ≧ 2. B h When α ≧ 3, α = 3, from Equation (22), B s ≧ 1. Therefore, (B h , B s ) being all combinations of (1, 36), (2, 18), (3, 12), (4, 9), (6, 6), (9, 4), (12, 3), (18, 2) and (36, 1) became the solutions of the first high dynamic range condition.

[0217] On the other hand, when = 1, α = 1, from Equation (24), B h ≧ 11. B s When = 2, α = 2, from Equation (24), B h ≧ 6. B s When ≧ 3, α = 3, from Equation (24), B h ≧ 4. Therefore, (B s , B h , B s ) being combinations of (1, 36), (2, 18), (3, 12), (4, 9), (6, 6) and (9, 4) became the solutions of the second high dynamic range condition. From the above, by setting (B h , B s ) to any of (1, 36), (2, 18), (3, 12), (4, 9), (6, 6), (9, 4), (12, 3), (18, 2) and (36, 1), the first high sensitivity condition and the first high dynamic range condition were satisfied. Also, by setting (B h , B s ) to any of (1, 36), (2, 18), (3, 12), (4, 9), (6, 6) and (9, 4), the second high sensitivity condition and the second high dynamic range condition were satisfied. As described above, by changing the saturation light amount ratio from k = 1 to k = 3, the range of the binning condition that satisfies both the high sensitivity condition and the high dynamic range condition is expanded, and it was found that the effect is more easily obtained.

[0218] In this embodiment, noise conditions and binning conditions that satisfy both high-sensitivity conditions and high-dynamic-range conditions were found. However, when sensitivity is more important than dynamic range, it may be better to return the original conditions (B h , B s ) to (36, 1). Therefore, within the same multi-capillary electrophoresis device, it is effective to be able to selectively use a mode that emphasizes high sensitivity over high dynamic range and a mode that satisfies both high sensitivity and high dynamic range according to the application, that is, to be able to select an appropriate binning condition from among multiple binning conditions. It is preferable that the user can select a desired binning condition from among multiple binning conditions using a user interface. Alternatively, it is also effective to enable software to select an appropriate binning condition from among multiple binning conditions even if the user does not consciously select a binning condition.

[0219] <Change of Image Sensor> Next, under the conditions defined by the above <basic conditions>, 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, since the settings of the multi-color detection optical system were not changed, the size of the images of the light in the wavelength bands to be measured by each of the bins W(1) to W(20) on the image sensor remained 0.288 mm × 0.072 mm. Therefore, since each bin W(f) measures light in the same wavelength band, the number of pixels B m = 79 × 20 = 1580 was set. That is, compared with before, the number of pixels in the bin W(f) was increased by more than 40 times. Also, since the CMOS cannot perform hard binning, in each bin W(f), the number of pixels for hard binning was B h = 1, and the number of pixels for soft binning was B s = 1580. Also, since the saturated light quantity ratio was k = 1, α = 1 was set. Although the AD conversion of the CMOS is only BN = 12 bits, B sSince it is 1580, the resolution of the digital signal is B s ×BN = 1580×12 bits = 23 bits, and a resolution far exceeding 16 bits can be substantially obtained.

[0220] As a result of performing noise analysis under the above conditions, the read noise of one pixel is N r = 1.06 counts, the dark current noise ratio is b = 0.21, and the shot noise ratio is c = 10. At this time, the binning condition that satisfies the first high-sensitivity condition is, from Equation (17), B s ≦775. Also, the binning condition that satisfies the second high-sensitivity condition is, from Equation (20), B s ≦121. On the other hand, the binning condition that satisfies the first high-dynamic range condition is, from Equation (23), B s ≧3. Furthermore, the binning condition that satisfies the second high-dynamic range condition is, from Equation (25), B s ≧11. From the above, the above B m = B s = 1580 condition satisfies both the first high-dynamic range condition and the second high-dynamic range condition, but it was found that it does not satisfy either the first high-sensitivity condition or the second high-sensitivity condition.

[0221] Therefore, similar to the above <changes in various conditions>, the measurement conditions were changed without changing the settings of the multi-color detection optical system. As a result, when noise analysis was performed, the read noise of one pixel was N r = 1.06 counts, the dark current noise ratio was b = 0.21 and did not change, but the shot noise ratio increased to c = 24 - 52. Here, c = 24 was obtained with bin W(20), and c = 52 was obtained with bin W(10).

[0222] First, when the shot noise ratio is c = 24, the binning condition that satisfies the first high-sensitivity condition is, from Equation (17), B s ≦4422. Also, the binning condition that satisfies the second high-sensitivity condition is, from Equation (20), B swas ≤ 652. On the other hand, the binning condition that satisfies the first high dynamic range condition is, from Equation (23), B s was ≥ 3. Furthermore, the binning condition that satisfies the second high dynamic range condition is, from Equation (25), B s was ≥ 11. From the above, it was found that the condition of the above B m = B s = 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, from Equation (17), B s was ≤ 21571. Also, the binning condition that satisfies the second high sensitivity condition is, from Equation (20), B s was ≤ 3312. On the other hand, the binning condition that satisfies the first high dynamic range condition is, from Equation (23), B s was ≥ 3. Furthermore, the binning condition that satisfies the second high dynamic range condition is, from Equation (25), B s was ≥ 10. From the above, it was found that the condition of the above B m = B s = 1580 satisfies 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 the noise composition to appropriate conditions, it is possible to achieve both high sensitivity and a high dynamic range while using the same binning condition. That is, depending on the noise conditions, it is possible to achieve both high sensitivity and a high dynamic range, or conversely, it is also possible that it becomes impossible to achieve both high sensitivity and a high dynamic range.

[0225] <Change in the configuration of the multi-color detection optical system> By changing the configuration of the multi-color detection optical system described in <Basic Conditions>, we considered achieving both high sensitivity and a high dynamic range. The image sensor was a CMOS with a pixel size of 3.63 μm square. Also, since the saturated light quantity ratio was k = 1, α = 1 was set. First, the grating frequency of the transmission diffraction grating used in the multi-color detection optical system was changed from N = 600 lines / mm to N = 200 lines / mm. At this time, from Equation (72), since the dispersion distance per 1 nm on the image sensor was 10 μm, the size of the image of the light in the 9 nm-wide wavelength band to be measured by bins W(1) to W(20) became 0.10 mm × 0.072 mm. That is, each bin W(f) was set to 25 pixels in the wavelength dispersion direction, 20 pixels in the emission point array direction, and B m = 25×20 = 500. Also, the number of pixels for hard binning was set to B h = 1, and the number of pixels for soft binning was set to B s = 500. When the measurement conditions were not changed from <Basic Conditions>, the readout noise of 1 pixel was N r = 1.06 counts, the dark current noise ratio was b = 0.21, and the shot noise ratio was c = 10. That is, the first high-sensitivity condition was B s ≦775, the second high-sensitivity condition was B s ≦121, the first high-dynamic range condition was B s ≧3, and the second high-dynamic range condition was B s ≧11. Therefore, the binning conditions of B h = 1 and B s = 500 satisfied the first high-sensitivity condition, the first high-dynamic range condition, and the second high-dynamic range condition. From the above, under the same measurement conditions, by changing the configuration of the multi-color detection optical system, it became possible to achieve both the high-sensitivity condition and the high-dynamic range condition.

[0226] Next, by further modifying the configuration of the above-described multi-color detection optical system, it was considered to more suitably achieve both high sensitivity and a high dynamic range. 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, the focal length of the second camera lens was changed from f2 = 50 mm to f2 = 25 mm. At this time, the wavelength dispersion image of the light emitted from the light-emitting point becomes a reduced image that is 1 / 2 times as large. Therefore, from Equation (72), the dispersion distance per 1 nm on the image sensor is 5 μm, and the imaging size of the light-emitting point with an angle of 50 μm in the case of no wavelength dispersion becomes an angle of 25 μm. From this, the size of the image of the light in the 9-nm wavelength band that bins W(1) to W(20) measure is 0.045 mm × 0.025 mm. That is, each bin W(f) has 12 pixels in the wavelength dispersion direction, 7 pixels in the light-emitting point array direction, and 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 = 84. At this time, when the measurement conditions are not changed from the <basic conditions>, assuming that the read noise of 1 pixel is N r = 1.06 counts, the dark current noise ratio is b = 0.21, and the shot noise ratio is c = 10, 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 are satisfied. From the above, under the same measurement conditions, by changing the configuration of the multi-color detection optical system, it becomes possible to achieve both the high-sensitivity condition and the high-dynamic range condition, and it becomes possible to expand the range in which both the high-sensitivity condition and the high-dynamic range condition can be achieved.

[0227] [Example 2] In this example, by generalizing the considerations of Example 1, the conditions for achieving both the high-sensitivity condition and the high-dynamic range condition are systematically clarified. In Example 1, mainly (excluding the <change in the configuration of the multi-color detection optical system> of Example 1), while fixing the number of pixels B m of bin W, the number of pixels B h for hard binning and the number of pixels B for soft binnings changing it was considered. In contrast, in this embodiment, the number of pixels B of hard binning h and the number of pixels B of soft binning s not only, but also considering changing the number of pixels B m Here, the change of B m is performed by controlling the configuration of the multi-color detection optical system to change the pixel region on the image sensor where the image of the fluorescence in the specific wavelength band targeted by bin W is projected.

[0228] Also, in Example 1, by changing the measurement conditions, not only the composition of the noise was changed, but also the magnitude of the total noise was changed. In contrast, in this embodiment, in order to avoid the influence of the change in the magnitude of the total noise, while keeping the magnitude of the total noise constant by changing the measurement conditions, the composition of the noise was changed. In the study of this embodiment, Equations (11), (13), (14) and (15) were used. However, since the saturation light amount ratio of the image sensor is k = 1, α = 1 was set. Also, when B h = B s = 1, it was assumed that the total noise was constant at N = 1 count. Furthermore, the saturation light amount per pixel of the image sensor was set to M = 10000 counts. Hereinafter, the unit of count for noise and light amount is omitted.

[0229] FIGs. 11A to 11C show the number of pixels B of hard binning under the condition of B s = 1, the number of pixels B of hard binning hThe results of obtaining the lower detection limit LLOD, the upper detection limit ULOD, and the dynamic range DR with respect to the change are shown by dotted lines. Here, the lower detection limit LLOD is shown by triangular plots, the upper detection limit ULOD is shown by square plots, and DR is shown by round plots. Also, in Fig. 11A, the noise composition with b = 0 and c = 0 was set, in Fig. 11B, the noise composition with b = 0.1 and c = 0 was set, and in Fig. 11C, the noise composition with b = 1 and c = 0 was set. 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 (denoted as "the first high DR condition"), and the second high-dynamic range condition (denoted 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 denoted as "-".

[0230] First, in the case of b = 0 and c = 0 in Fig. 11A, the lower detection limit LLOD, the upper detection limit ULOD, and the dynamic range DR, which is the ratio of these, are all constant regardless of the number of pixels B of hard binning h , that is, the same lower detection limit LLOD = 3, upper detection limit ULOD = 10000, and dynamic range DR = 3333 as when B h = 1. Since b = 0 and c = 0 means that the total noise is composed only of readout noise, the total noise did not change and the lower detection limit LLOD did not change even when the number of pixels B of hard binning h increased. Therefore, the first high-sensitivity condition and the second high-sensitivity condition were satisfied for any number of pixels B of hard binning h . In Fig. 11A, in accordance with the scale of the graph, the binning conditions that satisfy both conditions are denoted as B h ≦10 5 . Also, since the saturated light amount M = 10000 remained the same even when the number of pixels B of hard binning h increased, the upper detection limit ULOD did not change either. Therefore, there was no solution for the binning conditions that satisfy the high-dynamic range condition.

[0231] Next, when b = 0.1 and c = 0 in FIG. 11B, as the number of pixels B of hard binning increases, the dark current noise increases, so the total noise increases and the detection lower limit LLOD also increases. However, when B h = 1, the detection lower limit LLOD, the detection upper limit ULOD, and the dynamic range DR are the same as the values in FIG. 11A, respectively. As a result, as shown in the table, the first high-sensitivity condition is satisfied by setting B h ≦809 according to Equation (18), and the second high-sensitivity condition is satisfied by setting B h ≦127 according to Equation (21). h

[0232] Also, when b = 1 and c = 0 in FIG. 11C, the increase rate of the detection lower limit LLOD with respect to the number of pixels B of hard binning further increased and increased generally according to a straight line with a slope of 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 detection lower limit LLOD increases in proportion to the square root of the number of pixels B of hard binning. At this time, since FIG. 11 is a double logarithmic graph, the number of pixels B of hard binning h and the detection lower limit LLOD have a linear relationship with a slope of 1 / 2. As a result, as shown in the table, the first high-sensitivity condition is satisfied by setting B h ≦17 according to Equation (18), and the second high-sensitivity condition is satisfied by setting B h ≦4 according to Equation (21). On the other hand, from Equation (14), since the saturation light amount remains M = 10000 regardless of the dark current noise ratio b, the shot noise ratio c, and the number of pixels B of hard binning h , the detection upper limit ULOD in FIGS. 11B and 11C did not change from FIG. 11A. h h

[0233] From the above, as the dark current noise ratio b increases, the dynamic range DR decreases with respect to the number of pixels B of hard binning. As shown in FIG. 11C of the double logarithmic graph, when the dark current noise ratio b becomes sufficiently large, the number of pixels B of hard binning h h ​​​​and 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 sAll were positive integers. Similar to FIGS. 11A to 11C and FIGS. 12A to 12C, in FIG. 13A, the noise composition was set to b = 0 and c = 0, in FIG. 13B, the noise composition was set to b = 0.1 and c = 0, and in FIG. 13C, the noise composition was set to b = 1 and c = 0. The lower limit of detection LLOD is shown by a triangular plot, the upper limit of detection ULOD is shown by a square plot, and the dynamic range DR is shown by a circular plot, each indicated by a dashed line. The lower limit of detection LLOD was obtained from Equation (13), the upper limit of detection ULOD was obtained from Equation (14), and the dynamic range DR was obtained from the ratio of these. The horizontal axis is the soft binning rate B s / B m as shown. Here, when (B h , B s ) is (100, 1), B s / B m is 1%, when (50, 2) it is 2%, when (20, 5) it is 5%, when (10, 10) it is 10%, when (5, 20) it is 20%, when (2, 50) it is 50%, and when (1, 100) it is 100%. These binning conditions were plotted. B s / B m = 1% shows the same result as B h = 100 in FIG. 11, B s / B m = 100% shows the same result as B s = 100 in FIG. 12, and B s / B m = 2% to 50% shows the results in between. The binning conditions that satisfy the high-sensitivity condition and the high-dynamic-range condition are equivalent to the results in FIG. 12, but are shown in the range of B s / B m . By setting B s / B m = 9%, the first high-sensitivity condition and the first high-dynamic-range condition can be achieved simultaneously. However, under the conditions of FIG. 13, it was found that the second high-sensitivity condition and the second high-dynamic-range condition cannot be achieved simultaneously. In FIG. 13, as an example, the study was conducted under the condition of B m = 100 = 10 2 , but B m = 10 0 ~105 For any number of pixels B m it goes without saying that a similar examination can be carried out.

[0237] In FIGS. 11 to 13, under the condition of c = 0, an examination was carried out when the dark current noise ratio b was changed. However, in FIGS. 14 to 16 shown hereinafter, under the condition of b = 0, an examination was carried out when the shot noise ratio c was changed. FIGS. 14A to 14C are, similar to FIG. 11, under the condition of B s = 1, the results of obtaining the lower detection limit LLOD, the upper detection limit ULOD, and the dynamic range DR with respect to the change in the number of pixels B of hard binning are shown by dotted lines. 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. As a result, FIGS. 14A, 14B, and 14C all showed the same result. Also, the lower detection limit LLOD, the upper detection limit ULOD, and the dynamic range DR were all constant regardless of the number of pixels B of hard binning, that is, the same lower detection limit LLOD = 3, upper detection limit ULOD = 10000, and dynamic range DR = 3333 as in the case of B h = 1. This result is the same as the result of FIG. 11A. This is because, as shown in Equation (13), under the condition of b = 0, the lower detection limit LLOD does not depend on the number of pixels B of hard binning. Also, as shown in Equation (14), regardless of the conditions of the dark current noise ratio b and the shot noise ratio c, the upper detection limit ULOD does not depend on B h . Therefore, for any number of pixels B of hard binning h = 1, the first high-sensitivity condition and the second high-sensitivity condition are satisfied. Similar to FIG. 11A, the binning condition that satisfies both conditions is expressed as B h ≤ 10 h . Also, there was no solution for the binning condition that satisfies the high dynamic range condition. From the above, it was found that under the conditions of FIGS. 14A to 14C, it is impossible to achieve both the high-sensitivity condition and the high dynamic range condition. h For any number of pixels B of hard binning h ≦ 10 5 is expressed. Also, there was no solution for the binning condition that satisfies the high dynamic range condition. From the above, it was found that under the conditions of FIGS. 14A to 14C, it is impossible to achieve 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 sIt was satisfied by setting it to ≦ 10. Also, the first high dynamic range condition is 4 ≦ B from Equation (23) s It was satisfied by setting it to, and the second high dynamic range condition is 19 ≦ B from Equation (25) s It was satisfied by setting it to. Therefore, 4 ≦ B s By setting it to ≦ 59, the first high sensitivity condition and the first high dynamic range condition could be made compatible. The range of this binning condition was expanded compared to the case of FIG. 15A, and it was found to be advantageous for making the high sensitivity condition and the high dynamic range condition compatible. However, it was found that the second high sensitivity condition and the second high dynamic range condition could not be made compatible. However, by setting 4 ≦ B s ≦ 10, the second high sensitivity condition and the first high dynamic range condition could be made compatible. Alternatively, by setting 19 ≦ B s ≦ 59, the first high sensitivity condition and the second high dynamic range condition could be made compatible.

[0241] On the other hand, in FIG. 15C, the first high sensitivity condition is satisfied by setting B s ≦ 809 from Equation (17), and the second high sensitivity condition is satisfied by setting B s ≦ 127 from Equation (20). Also, the first high dynamic range condition is satisfied by setting 3 ≦ B from Equation (23) s and the second high dynamic range condition is satisfied by setting 11 ≦ B from Equation (25) s It was satisfied by setting it to. Therefore, by setting 3 ≦ B s ≦ 809, the first high sensitivity condition and the first high dynamic range condition could be made compatible. Furthermore, by setting 11 ≦ B s ≦ 127, the second high sensitivity condition and the second high dynamic range condition could be made compatible. Also, by setting 3 ≦ B s ≦ 127, the second high sensitivity condition and the first high dynamic range condition could be made compatible. Alternatively, by setting 11 ≦ B 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 mis set to 1% for (50, 2), 2% for (20, 5), 5% for (10, 10), 10% for (5, 20), 20% for (2, 50), 50% for (1, 100), and 100% for (1, 100), and plotted for these binning conditions. B s / B m = 1% shows the same result as B in FIGS. 14A to 14C h = 100, and B s / B m = 100% shows the same result as B in FIGS. 15A to 15C s = 100, and B s / B m = 2% to 50% show the results in between. The high-sensitivity conditions and high-dynamic range conditions satisfied in FIGS. 16A, 16B, and 16C were the same as those in FIGS. 15A, 15B, and 15C, respectively. However, in FIG. 15C, the first high-sensitivity condition was B s ≦ 809, and the second high-sensitivity condition was B s ≦ 127, but in FIG. 16, since B s ≦ 100, in FIG. 16C, the binning conditions that satisfy the first high-sensitivity condition and the second high-sensitivity condition are B s / B m ≦ 100% respectively. In FIGS. 16A to 16C, as an example, B m = 100 = 10 2 was examined under the condition of, but B m = 10 0 ~ 10 5 For any B m it goes without saying that the same examination is possible.

[0243] From the above examination, it was found that the shot noise ratio c is an important factor for achieving both high-sensitivity conditions and high-dynamic range conditions. In FIGS. 14 to 16, only the cases of c = 0, 2.5, and 10 were examined, but in FIGS. 17A and 17B, the influence of the shot noise ratio c on the high-sensitivity conditions and high-dynamic range conditions was examined in more detail. FIG. 17A shows the number of soft-binning pixels B that satisfy the first high-sensitivity condition with respect to the shot noise ratio c sThe number of pixels B of soft binning that satisfies the condition and the first high dynamic range condition s is shown. Further, FIG. 17B shows the number of pixels B of soft binning that satisfies the second high sensitivity condition with respect to the shot noise ratio c s and the number of pixels B of soft binning that satisfies the second high dynamic range condition s is shown. In any of the graphs, the region below the curve of the round plot is the high sensitivity condition, and the region above the curve of the triangular plot is the high dynamic range condition. Therefore, the high sensitivity condition and the high dynamic range condition are compatible in the region sandwiched by these curves. From FIG. 17A, in order to make the first high sensitivity condition and the first high dynamic range condition compatible, it suffices that c ≧ 0, and at least B s = 9 suffices. This is the same as the results of FIGS. 12 and 15A. However, the condition that the solution is only B s = 9 has a narrow allowable range for the number of pixels B of soft binning s and is difficult to implement. Generally, the wider the range of the solution, the easier it is to implement and the easier it is to obtain the effect.

[0244] Therefore, from FIG. 17A, the condition A where the range of the number of pixels B of soft binning that is the solution is 30 pixels or more, and the range of the number of pixels B of soft binning that is the solution s Condition B where the range is 100 pixels or more was extracted. From FIG. 17A, it was found that condition A is c ≧ 1.75, and at least 4 ≦ B s ≦ 34 suffices. Also, for condition B, it was found that c ≧ 3.43, and at least 3 ≦ B s ≦ 103 suffices. On the other hand, from FIG. 17B, in order to make the second high sensitivity condition and the second high dynamic range condition compatible, it suffices that c ≧ 3.15, and at least B s = 15 suffices. Conversely, it became clear that there is no solution when c < 3.15. Similarly for FIG. 17B, condition A and condition B were obtained. From FIG. 17B, condition A is c ≧ 5.61, and at least 12 ≦ B s ≦... (the original text seems incomplete here) sIt was found that it only needs to be ≦42. Also, condition B is c≧9.31, and at least 11≦B s It was found that it only needs to be ≦111.

[0245] Figures 17C to 17E and Figures 17F to 17H are each a further generalization of the considerations in Figures 17A and 17B. In Figures 17A and 17B, under the condition of B h =1, the relationship between the shot noise ratio c that satisfies the high-sensitivity condition and the high-dynamic range condition and the number of pixels B of soft binning s was clarified. In contrast, in Figures 17C to 17E and Figures 17F to 17H, without fixing any of B m , the number of pixels B of hard binning h , and the number of pixels B of soft binning s , the relationship between B m that satisfies the high-sensitivity condition and the high-dynamic range condition and B s / B m (=1 / B h ) was clarified. The soft binning rate B s / B m used also in Figures 13 and 16 is the ratio of the number of pixels of soft binning to the number of pixels of bin W, and since B m =B h ×B s , it can also be expressed as B s / B m =1 / B h .

[0246] Figures 17C to 17E show binning conditions that satisfy the first high-sensitivity condition and the first high-dynamic range condition. In Figure 17C, the noise composition was set to b = 0, c = 0, in Figure 17D, the noise composition was set to b = 0, c = 2.5, and in Figure 17E, the noise composition was set to b = 0, c = 10. Since the region below the dotted line satisfies the first high-sensitivity condition and the region above the solid line satisfies the first high-dynamic range condition, the region sandwiched between the two straight lines satisfies both the first high-sensitivity condition and the first high-dynamic range condition. However, B s / B mIt does not actually exceed 100%. Therefore, the thick line portion in Fig. 17C and the shaded portions in Figs. 17D and 17E give the binning conditions that satisfy both the first high-sensitivity condition and the first high-dynamic range condition. Similar to the results in Fig. 17A, as the shot noise ratio c increases, the region and range of the allowable binning conditions expand. For example, B s / B m = 100%, that is, B h = 1, B s = B m when c = 0, B s = 9, when c = 2.5, 4 ≤ B s ≤ 59, and when c = 10, 3 ≤ B s ≤ 809, the first high-sensitivity condition and the first high-dynamic range condition are satisfied. These can also be read from Figs. 15A - 15C, 16A - 16C, and 17A. However, other rich information can also be read from Figs. 17C - 17E. For example, B s / B m = 50%, that is, B h = 2, B s = 1 / 2 * B m when c = 0, B s = 18, 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. Alternatively, B s / B m = 33.33%, that is, B h = 3, B s = 1 / 3 * B m when c = 0, B s = 27, when c = 2.5, 11 ≤ B s ≤ 177, and when c = 10, 10 ≤ B s ≤ 2427, the first high-sensitivity condition and the first high-dynamic range condition are satisfied. Further, when α = k = 3 at this time, when c = 0, 3 ≤ B s ≤ 27, when c = 2.5, 3 ≤ B s ≤ 177, and 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, c = 0. In FIG. 17G, the noise composition is set to b = 0, c = 2.5. In FIG. 17H, the noise composition is set to b = 0, c = 10. Since the lower side of the dotted line satisfies the second high-sensitivity condition and the upper side of the solid line satisfies the second high-dynamic range condition, the region sandwiched 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 shaded portion in FIG. 17H gives binning conditions that satisfy both the second high-sensitivity condition and the second high-dynamic range condition. Similar to the results of FIGS. 16A to 16C and FIG. 17B, as the shot noise ratio c increases, the region and range of allowable binning conditions expand. B s / B m = 100%, that is, B h = 1, B s = B m In the case of, when c = 10, by setting 11 ≤ B s ≤ 127, the second high-sensitivity condition and the second high-dynamic range condition are satisfied. These can also be read from FIGS. 15A to 15C and FIG. 17B. However, other rich information can also be read from FIGS. 17F to 17H. For example, B s / B m = 50%, that is, B h = 2, B s = 1 / 2 * B m In the case of, when c = 10, by setting 21 ≤ B s ≤ 254, the second high-sensitivity condition and the second high-dynamic range condition are satisfied. Alternatively, B s / B m = 33.33%, that is, B h = 3, B s = 1 / 3 * B mWhen c = 10, 32 ≤ B s ≤ 381, thereby satisfying the second high-sensitivity condition and the second high-dynamic range condition. Further, when α = k = 3 at this time, when c = 10, 11 ≤ B s ≤ 381, thereby satisfying the second high-sensitivity condition and the second high-dynamic range condition, and the range of allowable binning conditions can be expanded.

[0248] As shown above, the sensitivity and dynamic range obtained vary 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 desired sensitivity or dynamic range under the given conditions. For example, the optimal binning conditions are different when sensitivity is prioritized and when dynamic range is prioritized. In a multi-capillary electrophoresis device, it is convenient for the user to be able to select a desired binning condition from among a plurality of binning conditions.

[0249] [Example 3] By the various methods of the present disclosure proposed above, it has become possible to achieve both high sensitivity and high dynamic range. However, it has become clear that the expected sensitivity and dynamic range may not always be obtained. Therefore, as a result of the inventors' detailed studies, it was found that the cause is spatial crosstalk inherent in the multi-color detection optical system. Hereinafter, the newly clarified problem will be described in detail.

[0250] As described in the [Background Art], the multi-color detection optical system is composed of a plurality of optical components. Even a single camera lens is a combined lens of a plurality of lenses. In order to suppress light reflection on the surfaces of these optical components, an antireflection coating may be applied to the surfaces, but still, it is impossible to make the light reflection zero. Inside the multi-color detection optical system, when multiple reflections of fluorescence occur between a plurality of optical components, artifacts such as ghosts or flares may occur on the image sensor and may overlap with the original true image of the light-emitting point that emits fluorescence and be measured. Here, the true image includes also the wavelength dispersion image of the fluorescence emitted from the light-emitting point. The size of the artifact 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 artifact of the light-emitting point A can overlap not only with the true image of the light-emitting point A but also with the true image of the light-emitting point B, so that spatial crosstalk occurs from the light-emitting point A to the light-emitting point B. However, since the intensity of the artifact is much smaller compared to the intensity of the true image, the presence of the artifact does not necessarily become a problem. When the intensity of the true image is small, the intensity of the artifact is even smaller and becomes below the detection limit, so it does not become a problem. When the intensity of the true image is large, the intensity of the artifact exceeds the detection limit and may become a problem. Therefore, as the dynamic range of fluorescence measurement by the image sensor increases, the possibility of this problem becoming apparent increases. Since the main object of the present disclosure is to achieve both high sensitivity and high dynamic range, the greater the effect, the more likely it is to face this problem. That is, the method for realizing both high sensitivity and high dynamic range according to the present disclosure may become inoperative due to spatial crosstalk. This is a new problem revealed in the present disclosure.

[0251] Previously, equations (1) to (6) held individually for each light-emitting point. However, when considering spatial crosstalk, it is necessary to expand them and express them as follows. At each light-emitting point P(e) (e = 1, 2, …, E), the phosphor D(e, g) (e = 1, 2, …, E, and g = 1, 2, …, G) emits fluorescence, and the fluorescence received by all bins W(e, f) (e = 1, 2, …, E, and f = 1, 2, …, F) is measured. At any given time, let the concentration of the phosphor D(e, g) at the light-emitting point P(e) be Z(e, g), and the signal intensity of the bin W(e', f) for the light-emitting point P(e') be X(e', f). Here, let the (E×F) row 1-column vector with the signal intensity X(e', f) as an element be X, the (E×G) row 1-column vector with the concentration Z(e, g) as an element be Z, and the (E×F) row (E×G) column matrix with Y(e', f)(e, g) as an element be Y. Corresponding to equations (1) to (6), the following equations (73) to (78) hold.

[0252]

Number

[0253]

Number

[0254]

Number

[0255]

Number

[0256]

Number

[0257]

Number

[0258] Here, the element Y(e', f)(e, g) of the (E×F)-row and (E×G)-column matrix Y represents, when (i) e' = e, the signal intensity ratio at which the fluorescence emission of the phosphor D(e, g) at the light-emitting point P(e) is detected in the bin W(e, f) for the same light-emitting point due to spectral crosstalk, and when (ii) e' ≠ e, the signal intensity ratio at which the fluorescence emission of the phosphor D(e, g) at the light-emitting point P(e) is detected in the bin W(e', f) for a different light-emitting point due to spatial crosstalk. By causing any one type of phosphor D(e0, g0) to emit fluorescence alone at any one light-emitting point P(e0), each column Y(e, f)(e0, g0) (where e = 1, 2, …, E and f = 1, 2, …, F) of the matrix Y can be determined. Equation (77) normalizes such that the sum of the (E×F) elements of each column Y(e, f)(e0, g0) of the matrix Y becomes 1. As described above, among the (E×F) elements of each column Y(e, f)(e0, g0) of the matrix Y, the F elements where e = e0 indicate the spectral crosstalk ratio, and the ((E - 1)×F) elements where 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) in one column of the matrix Y in Equation (3). However, the normalization conditions are different between Equation (5) and Equation (77). Generally, the spatial crosstalk ratio ≪ the spectral crosstalk ratio, but there may be cases where the spatial crosstalk ratio cannot be regarded as zero. Conversely, when all the spatial crosstalk ratios can be regarded as zero, Equations (73) to (78) become the same as Equations (1) to (6).

[0259] Let the maximum value among the spatial crosstalk ratios indicated by the elements Y(e', f)(e, g) of the matrix Y where e' ≠ e be the maximum spatial crosstalk ratio XR2 = Y(e m ', f m ')(e m , g m ). The fluorescence from the light-emitting point P(e m ) of the phosphor D(e m , g m) emits light with the maximum spatial crosstalk ratio XR2 at the emission point P(e m ') in the bin W(e m ', f m '). That is, it is assumed that the maximum spatial crosstalk is obtained in the bin W(e m ', f m '). Let the number of pixels in the bin W(e m ', f m ') be B m ', the number of pixels in hard binning be B h ', and the number of pixels in soft binning be B s '. Let the read noise of one pixel be N r , the dark current noise of one pixel be N d , and the shot noise of the background light measured in the bin W(e m ', f m ) with all emission points emitting light be N s '. Let the dark current noise ratio be b, the shot noise ratio be c', and the saturation light amount coefficient be α. At this time, the detection lower limit LLOD, detection upper limit ULOD, and dynamic range DR of the bin W(e m ', f m ) are expressed by equations (79), (80), and (81) respectively by transforming equations (13), (14), and (15).

[0260]

Equation

[0261]

Equation

[0262]

Equation

[0263] On the other hand, the element Y(e m , f m )(e m , g m) The maximum value among the spectral crosstalk ratios shown by is defined as the maximum spectral crosstalk ratio XR1 = Y(e m , f m )(e m , g m ). Let the fluorescence from the phosphor D(e m ) from the light-emitting point P(e m ,g m ) be measured at the bin W(e m ) of the light-emitting point P(e m , f m ) with the maximum spectral crosstalk ratio XR1. That is, it is assumed that the maximum spectral crosstalk is obtained in the bin (e m , f m ). Let the number of pixels in the bin W(e m , f m ) be B m , the number of pixels of hard binning be B h , and the number of pixels of soft binning be B s . Let the readout noise of one pixel be N r , the dark current noise of one pixel be N d , and the shot noise of the background light emitted from all light-emitting points and measured at the bin W(e m , f m ) be N s . Let the dark current noise ratio be b, the shot noise ratio be c, and the saturation light quantity coefficient be α. At this time, the lower detection limit LLOD, the upper detection limit ULOD, and the dynamic range DR of the bin W(e m , f m ) are represented by equations (13), (14), and (15), respectively.

[0264] From the above, based on the fluorescence from the phosphor D(e m ) from the light-emitting point P(e m , g m ), if the ratio of the maximum signal intensity measured by spatial crosstalk to the maximum signal intensity measured by spectral crosstalk is called the crosstalk ratio XR, then XR = XR2 / XR1. The crosstalk ratio XR is at the light-emitting point P(e m ) for the phosphor D(e m , gm ) when only emits light, the phosphor D(e m ) at the emission point P(e m , g m ) concentration Z(e m , g m ) signal intensity, and the phosphor D(e m ') at the emission point P(e m ', g m ') concentration Z(e m ', g m ') signal intensity ratio also coincides with the maximum value. That is, the crosstalk rate XR can be derived from either before or after color conversion and is an index representing the influence of spatial crosstalk.

[0265] Here, assume a state where the light amount at the detection upper limit ULOD is measured in the bin W(e m ) due to the emission of the phosphor D(e m , g m ) from the emission point P(e m , f m ). At this time, the maximum light amount of spatial crosstalk measured in the bin W(e m ') of the emission point P(e m ', f m ') is the saturation light amount ULOD shown in Equation (14) multiplied by the crosstalk rate XR. In order to achieve high sensitivity and high dynamic range without being affected by spatial crosstalk, it is sufficient that the above maximum light amount of spatial crosstalk is smaller than the detection lower limit LLOD of Equation (79), and the condition is represented by Equation (82).

[0266]

Equation

[0267] Hereafter, by aligning the binning conditions of the emission point P(e m ) and the binning conditions and noise conditions of the emission point P(e m '), B m ' = B m , B h ' = B h , Bs ' = B s 、N s ' = N s 、let c' = c. At this time, Equation (82) is represented by Equation (83).

[0268]

Equation

[0269] Also, Equation (79), Equation (73), and Equation (81) are respectively replaced by Equation (13), Equation (14), and Equation (15). The right side of Equation (83) is the reciprocal of the dynamic range DR shown in Equation (15). By making the above Equation (83) compatible with the previously proposed high-sensitivity condition and high-dynamic range condition, for example, Equation (26) or Equation (27), it becomes 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]

Equation

[0272] That is, regardless of the binning condition and the noise condition, the lower detection limit LLOD is determined by the crosstalk rate XR. At this time, since the upper detection limit ULOD remains as Equation (14), the dynamic range DR is represented by Equation (85).

[0273]

Equation

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

[0275] From the above, it has become clear that suppressing the crosstalk rate XR to a low level is important for achieving both high sensitivity and a high dynamic range. Figures 18A to 18F show the changes in the lower detection limit LLOD, upper detection limit ULOD, and dynamic range DR with respect to the number of soft binning pixels B when the crosstalk rate XR is changed with reference to Figure 15C. s Figure 18A shows the case of XR = 0, which is the same result as Figure 15C. In contrast, Figure 18B shows the case where XR = 10 -6 , Figure 18C shows the case where XR = 10 -5 , Figure 18D shows the case where XR = 10 -4 , Figure 18E shows the case where XR = 10 -3 , and Figure 18F shows the case where XR = 10 -2 . In each figure, the upper detection limit ULOD is a common value, while for the lower detection limit LLOD and the dynamic range DR, when Equation (83) is satisfied, they are the same values as in Figure 18A, but when Equation (83) is not satisfied, the values according to Equations (84) and (85) are shown. From Figure 18, as the crosstalk rate XR increases, the upper limit value of the dynamic range DR decreases according to Equation (85), so it was found that the effect of increasing the dynamic range DR by increasing the number of soft binning pixels B s is limited. According to the results of Figures 18A to 18F, in order to satisfy the first high-sensitivity condition and the first high-dynamic-range condition, it was found that XR ≤ 10 -4 is required.

[0276] In Figures 18A to 18F, it was assumed that the total noise is constant at N = 1 when B h = B s = 1. In contrast, in Figures 19A to 19F, B h = B sThe overall noise when N = 1 was changed to the condition where N = 10 is constant, and other conditions were the same as in Fig. 18. In Fig. 19A, since the overall noise has increased by one digit compared to Fig. 18A, the detection lower limit LLOD has increased by one digit as a whole, and the dynamic range DR has decreased by one digit. In Figs. 19B to 19F, similar to Figs. 18B to 18F, although the effect of increasing the dynamic range DR by increasing the number of pixels B of soft binning is limited, it was found that the effect can be obtained in 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, it was found that XR ≤ 10 s is necessary. This range of the crosstalk ratio XR was one digit wider compared to Figs. 18A to 18F. In Figs. 19A to 19F, the influence of increasing the overall noise by one digit was investigated. Instead, a similar effect to Figs. 19A to 19F can also be obtained by reducing the saturated light amount M of one pixel by one digit. The above can also be understood from the fact that the noise of the numerator on the right side of Equation (83) and the saturated light amount are included in the denominator. -3 As described above, in order to achieve both high sensitivity and high dynamic range, in addition to the high-sensitivity conditions and high-dynamic-range conditions shown in Equations (8) to (67), it is important to keep the crosstalk ratio low. There are several means to reduce the crosstalk ratio. Applying an antireflection coating with a low reflectivity to the surface of the constituent lenses of the camera lens is a basic means, but it may not be sufficient alone. It is also important to apply an antireflection coating to the surface of each optical component of the multicolor detection optical system. For example, it is effective to apply an antireflection coating to the input and output surfaces of the transmission diffraction grating, especially the surface on the side where no rulings are made. In addition, since the surface of the image sensor, especially the input and output surfaces of the glass window of the image sensor, is often not coated with an antireflection coating, applying an antireflection coating here is very effective.

[0277] As mentioned above, in order to achieve both high sensitivity and high dynamic range, in addition to the high-sensitivity conditions and high-dynamic-range conditions shown in Equations (8) to (67), it is important to keep the crosstalk ratio low. There are several means to reduce the crosstalk ratio. Applying an antireflection coating with a low reflectivity to the surface of the constituent lenses of the camera lens is a basic means, but it may not be sufficient alone. It is also important to apply an antireflection coating to the surface of each optical component of the multicolor detection optical system. For example, it is effective to apply an antireflection coating to the input and output surfaces of the transmission diffraction grating, especially the surface on the side where no rulings are made. In addition, since the surface of the image sensor, especially the input and output surfaces of the glass window of the image sensor, is often not coated with an antireflection coating, applying an antireflection coating here is very effective.

[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 of 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.

Description of Symbols

[0280] 1…Capillary 2…Sample injection end 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…Emission point 15…Multicolor detection optical system 16…First camera lens 17…Long pass filter 18…Transmission diffraction grating 19…Second camera lens 20…Image sensor 21…Optical axis 22…Fluorescence 23, 24, 25…Dispersed fluorescence 26…Image 27…Wavelength-dispersed image 28…Pixel W(1)…Bin 1 W(2)…Bin 2 W(3)…Bin 3 7-1…B h = 36 hard binning region 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 Hard binning region with = 4 10 - 6…B h Hard binning region with = 6 10 - 7…B h Hard binning region with = 5 10 - 8…B h Hard binning region with = 1 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…Cross - talk rate

Claims

1. E (≥2) capillaries into which E (≥2) samples containing components labeled with G (≥2) phosphors are injected and electrophoresed simultaneously, a laser light source that irradiates a measurement unit arranged in the same plane as the E capillaries with a laser beam, an optical system that receives fluorescence emitted when the G phosphors passing through the measurement unit are excited by the laser beam, and the optical system includes a spectroscopic element that spectroscopically separates the fluorescence emitted from the E capillaries into F (≥2, F≥G) predetermined wavelength bands respectively, an image sensor in which a plurality of pixels are two-dimensionally arranged and the E×F spectroscopically separated fluorescence is received in E×F different bin regions on the image sensor, and the image sensor includes measures E×F signal intensities of the E×F spectroscopically separated fluorescence in the E×F bin regions, is configured to acquire time-series data of the E×F signal intensities by continuous repetitive measurement at a predetermined exposure time and a predetermined time interval, in any one of the E×F bin regions, Let the number of pixels in the bin area be B m (≧1), and The bottle region is B s It is divided into (≧1) hard binning regions, Let the average number of pixels in the hardening region be B h = 1, and Let the number of pixels of hard binning in the bin region be B h pieces, Let the number of pixels of the soft binning in the bin area be B s pieces, B m = B h × B s = B s is set as B m = B h = B s For the case where it is set to 1 the total noise of the time-series data is classified into three components: the read noise of the image sensor, the dark current noise of the image sensor, and the shot noise of background light, Let the total noise be N, and the read noise be N r Let the dark current noise be N d Let the shot noise be N s Let the mixed noise be N x Let N x 2 = N r 2 + N d 2 be defined as When the shot noise mixing ratio is a = N s / N x then B s A multi-capillary electrophoresis device, characterized in that B and a satisfy a predetermined relationship.

2. In claim 1, 【Number 1】 A multi-capillary electrophoresis apparatus, characterized in that is satisfied.

3. In claim 1 or 2, 【Number 2】 A multi-capillary electrophoresis apparatus, characterized in that is satisfied.

4. In claim 1, [Number 3] A multi-capillary electrophoresis apparatus, characterized in that is satisfied.

5. In claim 1 or 4, 【Number 4】 A multi-capillary electrophoresis apparatus, characterized in that is satisfied.

6. E (≥2) capillaries into which E (≥2) samples containing components labeled with G (≥2) phosphors are injected and electrophoresed simultaneously, a laser light source that irradiates a measurement unit arranged in the same plane as the E capillaries with a laser beam, an optical system that receives fluorescence emitted when the G phosphors passing through the measurement unit are excited by the laser beam, and the optical system includes a spectroscopic element that spectroscopically separates the fluorescence emitted from the E capillaries into F (≥2, F≥G) predetermined wavelength bands respectively, An image sensor in which a plurality of pixels are two-dimensionally arranged and E×F spectrally separated fluorescences are received in E×F different bin regions on the image sensor. The image sensor is configured to: measure E×F signal intensities of the E×F spectrally separated fluorescences in the E×F bin regions; acquire time-series data of the E×F signal intensities by continuous repeated measurement for a predetermined exposure time and a predetermined time interval; in any one of the E×F bin regions; Let the number of pixels in the bin region be B m (≥1), and The bottle region is B s It is divided into (≧1) hard binning regions, Let the average number of pixels in the hard binning region be B h = 1, and Let the number of pixels of hard binning in the bin region be B h pieces, and Let the number of pixels of the soft binning in the bin region be B s pieces, B m = B h × B s = B s is set as B m = B h = B s when it is set to = 1 the overall noise of the time-series data is classified into three components: the read noise of the image sensor, the dark current noise of the image sensor, and the shot noise of background light; Let the total noise be N, and the read noise be N r and the dark current noise be N d and the shot noise be N s then The dark current noise ratio is b = N d / N r and the shot noise ratio is c = N s / N r When this is the case, B m and B h and B s A multi-capillary electrophoresis apparatus, characterized in that a, b, and c satisfy a predetermined relationship. **Claim 7** In claim 6, c≧1.75、 4≦B s ≦34 A multi-capillary electrophoresis apparatus, characterized in that [condition] is satisfied. **Claim 8** In claim 6, c≧3.43、 3≦B s ≦103 A multi-capillary electrophoresis apparatus, characterized in that [condition] is satisfied. **Claim 9** In claim 6, c≧5.61、 12≦B s ≦42 A multi-capillary electrophoresis apparatus, characterized in that [condition] is satisfied. **Claim 10** In claim 6, c≧9.31、 11≦B s ≦111 A multi-capillary electrophoresis apparatus, characterized in that [condition] is satisfied. **Claim 11** In claim 6, 【Number 5】 A multi-capillary electrophoresis apparatus, characterized in that [condition] is satisfied. **Claim 12** In claim 6, 【Number 6】 A multi-capillary electrophoresis apparatus, characterized in that [condition] is satisfied. **Claim 13** In claim 6, 【Number 7】 A multi-capillary electrophoresis apparatus, characterized in that [condition] is satisfied. **Claim 14** In claim 6, 【Number 8】 A multi-capillary electrophoresis apparatus, characterized in that [condition] is satisfied. **Claim 15** In claim 6, c≧2.5、 4≦B s A multi-capillary electrophoresis apparatus, characterized in that [condition] is satisfied. **Claim 16** In claim 6, c≧2.5、 B s ≦59 A multi-capillary electrophoresis apparatus, characterized in that [condition] is satisfied. **Claim 17** In claim 6, c≧10、 3≦B s A multi-capillary electrophoresis apparatus, characterized in that [condition] is satisfied. **Claim 18** In claim 6, c≧10、 B s ≦809 A multi-capillary electrophoresis apparatus, characterized in that [condition] is satisfied. **Claim 19** In claim 17, c≧10、 11≦B s A multi-capillary electrophoresis apparatus, characterized in that [condition] is satisfied. **Claim 20** In claim 18, c≧10、 B s ≦127 A multi-capillary electrophoresis apparatus, characterized in that [condition] is satisfied.

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