Photodetector

Abstract

This light detection device comprises: a channel array in which a plurality of tubes are arranged so that axes thereof are parallel to one another, the channel array forming a channel plane; an irradiation optical system that includes a light source and emits light from the light source so that the oscillation direction of an electric field component of the light is parallel to the channel plane, the optical axis of the light is included in the channel plane, and the optical axis forms a predetermined angle θ0 relative to a direction orthogonal to the axes of the tubes on the channel plane; and a light detection system that detects light emitted from each of the plurality of tubes. The predetermined angle θ0 satisfies predetermined equations 1 and 2 with respect to the angle θ1 of the light ray in a material of each tube and the angle θ2 of the light ray inside each tube that are determined on the basis of predetermined equations from the predetermined angle, the refractive index n0 of the surrounding environment, the refractive index n1 of the material of each tube, and the refractive index n2 of a substance inside each tube.

Description

[Technical Field]

[0001] The present invention relates to an optical detection device, and more particularly to a device for detecting fluorescence emitted from fluorescent materials in a channel array. [Background technology]

[0002] There are measurement techniques that use fluorescence to detect and analyze substances flowing through multiple channels. An example of such a technique is the analysis of molecules using a capillary electrophoresis device. For example, DNA labeled with fluorescent dyes is electrophoresed in a capillary and separated by size. Excitation light is irradiated onto a detection site on the capillary, and the emitted fluorescence is detected. These devices may be equipped with multiple channels to improve measurement throughput. Capillary electrophoresis devices may be equipped with several to several dozen capillaries.

[0003] In an apparatus that detects and measures fluorescence in multiple flow channels, it is necessary to irradiate the inside of each flow channel with excitation light in order to measure the fluorescence of the sample in the flow channel. One known method for irradiating multiple flow channels simultaneously with excitation light is to make the excitation light incident from the side of the flow channels arranged in a row (array).

[0004] Patent Document 1 shows a configuration in which excitation light is irradiated from the side of a capillary array. This method allows for more efficient use of the excitation light power than methods in which the excitation light is split and irradiated to each flow path. In fluorescence measurement, only a small portion of the excitation light is used to excite the fluorophore, while the majority passes through the flow path as is. In a configuration in which excitation light is incident from the side of the array, the unused excitation light can be reused to excite the sample in the next flow path, resulting in a lower requirement for light source output compared to methods in which the excitation light is split. Another advantage is that an optical system for splitting the excitation light is not required.

[0005] In a configuration in which excitation light is incident from the side of the channel array, the excitation light beam must pass through the interior of all capillaries without significant loss. In particular, when the channels are capillaries with circular cross sections, it is known that this state cannot be achieved under any conditions, but only if specific conditions related to the inner and outer diameters, refractive index, etc. of the capillaries are met (Patent Document 2). This condition arises because, when the light beam passes through the capillaries, the capillaries act as rod lenses due to their circular cross sections.

[0006] For example, if a capillary is placed in air and its interior is filled with air, the capillary acts as a rod lens with a negative focal length, i.e., a concave rod lens. In this case, the excitation light is diffused by the capillary, and the power of the excitation light is significantly lost as it propagates through the capillary array. When used in a fluorescence detection device, parameters such as the capillary diameter and the material filled inside are adjusted to avoid this situation. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] Japanese Patent Application Laid-Open No. 2014-194362 [Patent Document 2] Patent No. 3536851 specification Summary of the Invention [Problem to be solved by the invention]

[0008] In an apparatus that performs fluorescence detection in multiple flow paths in parallel, it is desirable that the power of excitation light irradiated to each flow path be uniform to ensure uniform measurement performance for each flow path. Furthermore, to increase detection sensitivity and reduce requirements for light source output, it is desirable that the power loss of the excitation light irradiated to the flow paths be as small as possible.

[0009] On the other hand, in the method of injecting excitation light from the side of the flow channel array mentioned above, surface reflection loss occurs at the interface between each flow channel. As a result, the power of the excitation light irradiated to a flow channel farther from the excitation light incident side becomes smaller. Methods for equalizing the power of the excitation light irradiated to each flow channel include irradiating the excitation light from two directions on the side of the array and correcting the signal amount detected by a variable optical density filter for the excitation light power. However, these methods make the device configuration complicated. Furthermore, while these methods achieve uniformity of the excitation light power for each flow channel, they do not reduce the loss of the excitation light.

[0010] Furthermore, in detection methods in which multiple flow channels are arranged in an array, crosstalk between detection channels also becomes a problem. Reflection of fluorescent signals on the flow channel surface and aberrations in the detection optical system cause crosstalk in the measurements of each flow channel. Crosstalk becomes noise in fluorescence detection, reducing detection sensitivity and dynamic range, so it is desirable to minimize it as much as possible.

[0011] The present invention has been made in consideration of the above-mentioned problems, and aims to reduce the surface reflection loss of excitation light in a photodetector, thereby making the power of excitation light irradiated to each flow path more uniform.

[0012] In one example, the objective is to configure a flow path such that a light beam passes through multiple capillaries with minimal loss, particularly when the flow path is a capillary with a circular cross section. In another example, the objective is to reduce crosstalk caused by surface reflection of fluorescent light in the above configuration. [Means for solving the problem]

[0013] An example of a light detection device according to the present invention is a channel array in which a plurality of tubes are arranged so that their axes are parallel to one another, forming a channel plane; An illumination optical system including a light source, the light from the light source being: the vibration direction of the electric field component of the light is parallel to the plane of the flow channel, the optical axis of the light is contained in the plane of the flow path, and the optical axis makes an angle θ with respect to a direction perpendicular to the axis of the tube in the plane of the flow path; an irradiation optical system for irradiating light in such a manner as to a light detection system that detects light emitted from each of the plurality of tubes; Equipped with The refractive index of the surrounding environment is n0, the refractive index of the material of the tube is n1, and the refractive index of the substance inside the tube is n2.

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[0014] An example of a light detection device according to the present invention is a flow channel substrate having a plurality of flow channels arranged therein with their axes parallel to each other and constituting a flow channel plane; An illumination optical system including a light source, the light from the light source being: the vibration direction of the electric field component of the light is parallel to the plane of the flow channel, the optical axis of the light is contained in the plane of the flow path, and the optical axis forms an angle θ0 with respect to a direction perpendicular to the axis of the channel in the plane of the channel; an irradiation optical system for irradiating light in such a manner as to an optical detection system that detects light emitted from each of the plurality of flow paths; Equipped with The refractive index of the material of the flow path substrate is n0, the refractive index of the substance inside the flow path is n1, and the predetermined angle θ0 is

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[0015] The photodetector according to the present invention can reduce the reflection loss of the excitation light on the surface of the flow path and improve the uniformity of the power of the excitation light for a plurality of flow paths.

[0016] In one example, particularly when the flow path is a capillary with a circular cross section, the light beam can be configured to pass through multiple capillaries with minimal loss by adjusting the angle of the light beam relative to a direction perpendicular to the axis of the capillary.

[0017] In another example, crosstalk between channels measuring each capillary can be reduced by directing only the fluorescence generated from some positions on the capillaries to a fluorescence detector. [Brief explanation of the drawings]

[0018] [Figure 1] FIG. 1 is a schematic diagram of a light ray propagating through a channel array. [Figure 2] 1 is a graph plotting the left side of equation (7). [Figure 3] 1 is a schematic diagram of a light beam propagating through a channel array provided in a channel substrate. FIG. [Figure 4] 10 is a graph showing the number of flow paths and the angle range in which the pump light power uniformity effect can be obtained. [Figure 5] 1 is a configuration diagram of a fluorescence detection device 500 according to a first embodiment of the present invention. [Figure 6] 10 is a graph showing the dependency of the optical power irradiated to the rearmost capillary on the capillary pitch. [Figure 7] FIG. 1 is a diagram showing the cross-sectional shape of a capillary as viewed from a cross section including an excitation light beam. [Figure 8] 10 is a graph showing the dependency of the optical power irradiated to the rearmost capillary on the capillary pitch in the first embodiment. [Figure 9] 10 is a graph of the power of excitation light irradiated onto each capillary. [Figure 10] FIG. 1 is a configuration diagram of a fluorescence detection device 1000 according to a second embodiment of the present invention. [Figure 11] FIG. 1 is a schematic diagram showing how crosstalk occurs due to fluorescence reflection on the capillary surface. [Figure 12] 10 shows the results of a simulation of fluorescence reflection on the capillary surface. [Figure 13] 10 is a schematic diagram showing how crosstalk is reduced in the fluorescence detection device 1000 according to the second embodiment. FIG. [Figure 14] 10 is a diagram showing the crosstalk reduction effect achieved by the fluorescence detection device 1000 according to the second embodiment. FIG. [Figure 15] FIG. 10 is a diagram showing a configuration in which an optical slit is used in a fluorescence detection device 1000 according to a second embodiment. DETAILED DESCRIPTION OF THE INVENTION

[0019] Before illustrating specific embodiments, we will derive the range of light incident angles onto the array that can achieve improved uniformity of excitation light power for multiple flow channels. Figure 1 shows the structure of a flow channel array that is the subject of an embodiment of the present invention. In Figure 1, a flow channel array 100 has multiple flow channels 102 formed of a material with a refractive index n1 arranged in a surrounding environment 101 with a refractive index n0. A lumen 103 exists within the flow channel 102, and a substance (liquid, gel, etc.) with a refractive index n2 is injected into the lumen 103.

[0020] Light from the light source is incident on the side of the channel array 100. In Fig. 1, the light is incident on both sides of the same optical path, but it may be incident on only one side.

[0021] The light propagates through the flow channels and is irradiated onto the lumens 103 of all of the flow channels 102. In this configuration, the light propagates within a plane defined by the flow channel array (a plane including the central axes of all of the flow channels). Since it is desirable for the light from the light source to have high linearity in order to efficiently irradiate the flow channel array, laser light is mainly used.

[0022] The angle of incidence of light incident on the side surface of the array is θ0. That is, the optical axis of light from the light source forms a predetermined angle θ0 with respect to the direction perpendicular to the axis of the channel 102 in the channel plane. In this case, the propagation angles θ1 and θ2 in the regions of refractive index n1 and n2 are given by Snell's law as follows:

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[0023] To achieve the effects of reducing surface reflection loss and improving the uniformity of excitation light power described in this disclosure, it is preferable to make the polarization direction of the irradiated light parallel to the plane defined by the flow channel array. This can also be said to mean that the vibration direction of the electric field component of the light from the light source is parallel to the flow channel plane, and the optical axis of the light from the light source is contained in the flow channel plane. Even if the vibration direction of the electric field component is slightly deviated from the direction parallel to the plane defined by the flow channel array, the effect of improving the uniformity of excitation light power described in this disclosure can be achieved to some extent as long as the angle between the vibration direction and the plane defined by the flow channel array is small. The same applies to elliptically polarized light, as long as the majority of the electric field component is contained in the plane defined by the flow channel array.

[0024] In a channel array 100 such as that shown in FIG. 1, there are four interfaces for one channel 102. That is, there are four interfaces where the refractive index changes from n0 to n1, n1 to n2, n2 to n1, and n1 to n0. When the polarization of light is within the plane defined by the channel array, the amplitude transmittance t of light at these interfaces is given by the Fresnel equation as follows:

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[0025] In Patent Document 2, light from the light source is incident perpendicularly to the longitudinal axis of the array. In other words, θ0 = 0°. At this time, the amplitude transmittance t c teeth

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[0026] When the polarization at a certain optical interface is p-polarized, it is known that there exists an incident angle at which the transmittance is higher than when the incident angle is 0°. In particular, there exists an angle at which all light is transmitted, and this angle is known as the Brewster angle. Even in a structure like that shown in Figure 1, which has multiple interfaces, by setting the angle θ0 within an appropriate range, it is possible to increase the light transmittance of the flow path (reduce reflection loss) compared to when the incident angle is 0°, and make the power of light irradiated to each flow path more uniform.

[0027] The range in which the above effect can be obtained is the light intensity transmittance T c is expressed as the range where the angle is greater than 0°.

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[0028] Figure 2 shows a graph showing the value of the left side of equation (7) when n0 = 1, n1 = 1.46, and n2 = 1.33, assuming that the area around the flow path is air, the flow path is made of silica glass, and the flow path lumen is filled with water. The region in Figure 2 where the value exceeds 1 is the range in which the effect of improving the uniformity of the pump light power can be achieved. This range has a lower limit of 0° and an upper limit determined by the values ​​of n0, n1, and n2, which is 69.1° under the above conditions. However, the slope of the left side of equation (7) with respect to θ0 is almost zero near 0°, and the angle range in which a significant loss reduction effect can actually be achieved is narrower than the above.

[0029] Expanding the left side of equation (6) with respect to θ0 and ignoring third-order or higher terms with respect to θ0 results in the following equation (8).

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[0030] The second term in equation (8) represents the increase in intensity transmittance. For example, a capillary electrophoresis device may have several dozen capillaries arranged in a row. In such cases, even a small change in the second term can significantly reduce overall loss and homogenize excitation light power. For example, if n0 = 1, n1 = 1.46, n2 = 1.33, and there are 20 flow paths, and θ0 = 0°, the power of the light irradiated to the last flow path is 23.7% of the power of the light before entering the flow path array. In contrast, if the transmittance improvement effect (the second term in equation (8)) is 0.01 (this is an approximation and is not exact, but is shown schematically by the dashed line in Figure 2), the power of the light irradiated to the last flow path is 28.6%, an improvement of approximately 5%.

[0031] In this way, the lower limit angle at which an improvement effect can be obtained is when the value of the second term in equation (8) exceeds 0.01. This can be expressed mathematically as the range of the angle θ0:

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[0032] Patent Document 1 shows a configuration in which excitation light is incident at an angle to the channel array. The purpose of this configuration is to prevent light from returning to the light source due to light reflection from the capillaries, and the angle is said to be several degrees. However, as calculated above, the range of angles required to homogenize the power of the irradiated light is clearly different from the range of angles of several degrees shown in Patent Document 1.

[0033] When light from a light source is incident at an angle of several degrees, the effect of reducing reflection loss due to that angle is negligible, and is essentially equivalent to an incident angle of 0°. Furthermore, to achieve the effect of uniforming the irradiated light power, the polarization of the excitation light must be within the plane defined by the flow path, but Patent Document 1 does not mention this condition. On the other hand, with the configuration of the present disclosure, the excitation light is inevitably not reflected back toward the light source, and return light is also prevented.

[0034] In the above example, when n0 = 1, n1 = 1.46, and n2 = 1.33, the angle at which the intensity transmittance peaks is 55.8°, as shown in Figure 2. At this point, the reflection loss due to the flow path is at its smallest, which is most advantageous for equalizing the power of the irradiated excitation light.

[0035] Under the above conditions, the reflection loss at the interface between air (n0 = 1) and silica glass (n1 = 1.46) is dominant, and reflecting this effect, the angle at which transmittance peaks is close to the Brewster angle of 55.6° at the interface between air and silica glass. When the reflection loss at a specific interface is significantly large as described above, setting the conditions so that the angle of incidence at that interface is the Brewster angle will be nearly the optimal angle for reducing loss.

[0036] In the above case, it is possible to further simplify equation (8). For example, suppose that the flow path 102 is made of resin, glass, or the like, is placed in the air, and is filled with an aqueous solution, gel, or the like. In such a case, n0=1, n1=approximately 1.4 to 1.5, and n2=approximately 1.3 to 1.5 in most cases. In such a case, the reflection loss at the interface between the air and the flow path becomes dominant. In this case, the number of interfaces to be considered is reduced to two: n0 → n1 and n1 → n0, and equation (7) becomes

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[0037] Equation (9) can also be simplified, and the angle range of θ0 that can be effectively achieved in combination with equation (11) is

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[0038] The above formula (12) can also be applied to a structure such as that shown in Figure 3. In the structure shown in Figure 3, the channel array 100 is provided on a channel substrate 301 having a refractive index of n0. The channel substrate 301 has multiple lumens 103 therein. The multiple lumens 103 are arranged so that their axes are parallel to one another. Each lumen 103 functions as a channel, and the channel substrate 301 forms a channel plane.

[0039] A substance (liquid, gel, etc.) with a refractive index n1 is poured into each lumen 103 (i.e., the inside of the flow path substrate 301). In this case, the angle range in which the effect of improving the pump light power uniformity is obtained is the same as that of equation (12). In this case, θ0 is defined as the propagation angle of light from the light source in the flow path substrate 301.

[0040] Patent Document 1 discloses a structure in which excitation light is irradiated from both sides of a channel array to improve the variation in power of the irradiated excitation light caused by reflection loss at the channel interface. On the other hand, the configuration of the present disclosure can achieve more uniform light irradiation even when light is irradiated from one side compared to when light is incident from both sides at θ0 = 0°.

[0041] The above conditions can be written specifically as the following equation (13).

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[0042] The right side of equation (13) represents the ratio of the power of light irradiated into the lumen of the flow path where the power of the irradiated light is the maximum to the power of light irradiated into the lumen of the flow path where the power of the irradiated light is the minimum, when light from the light source is incident from one side at an angle θ0. The power of the irradiated light is maximum in the flow path where light is irradiated first, and is minimum in the flow path located at the end on the light output side (opposite the incident side).

[0043] As described above, when θ0 satisfies the formula (13), more uniform light irradiation can be achieved.

[0044] Although equation (13) assumes that light is incident only from one side of the channel array, light may be incident from both sides of the channel array in opposing directions, as shown in Figure 1. This allows for more uniform light irradiation. Furthermore, the light incident from both sides may be split from a single light source, or light from multiple light sources may be used.

[0045] In Figure 4, the angle range that satisfies the above formula (13) for each number of flow channels when n0 = 1, n1 = 1.46, and n2 = 1.33 is shown by the diagonal lines. In Figure 4, formula (13) was evaluated for each number of flow channels and incident angle, and the region that satisfies formula (13) is shown by the diagonal lines. When θ0 is within this angle range, light irradiation from one side can achieve irradiation with more uniform optical power than when light is incident from both sides of the array at an incident angle of 0°.

[0046] In applications where uniformity of the excitation light power in each flow path is particularly important, the configuration of the present disclosure may be used and the excitation light may be irradiated from both sides of the flow path to achieve higher uniformity of the excitation light power. In this case, the angle range only needs to satisfy Equation (7).

[0047] Although the configuration of the present disclosure is described as an example of a fluorescence detection device that detects fluorescence, the configuration of the present disclosure can also be used in a light detection device that detects light other than fluorescence, as long as the detection principle is that signal light is generated by irradiating excitation light. For example, the object to be detected may be phosphorescence, Raman scattered light, or scattered light caused by an object flowing in a flow path.

[0048] <Embodiment 1> 5 is a configuration diagram of a fluorescence detection device 500 according to the first embodiment of the present invention. Here, an example of the configuration is described in which fluorescence detection of a sample in a capillary array is performed.

[0049] The fluorescence detection device 500 is an example of a light detection device, and includes a capillary array 501, a fluorescence excitation optical system 502, and a fluorescence detection optical system 503. The fluorescence detection device 500 is, as an example, a detection device in an electrophoresis system. In this case, it is preferable to include, in addition to the configuration of Fig. 5, a power supply for performing electrophoresis, a device for injecting gel into the capillaries, and the like, but this drawing shows only the detection portion according to this embodiment.

[0050] The capillary 504 is an example of a tube that constitutes a flow path, and may be what is called a thin-diameter tube. The cross-sectional shape of the tube is, for example, circular, but is not limited to this. Generally, the capillary is coated with polyimide or the like, but the coating is removed at the detection site.

[0051] The capillary array 501 is a channel array that forms a channel plane. The capillary array 501 has a structure in which a plurality of capillaries 504 are arranged with their axes parallel to each other.

[0052] The fluorescence excitation optical system 502 functions as an irradiation optical system and irradiates light (laser light in this embodiment) onto the capillary array 501. The fluorescence excitation optical system 502 includes a laser light source 505, a polarization control element 506, and a lens 507. The laser light source 505 is an example of a light source, and a light source other than a laser may also be used.

[0053] A laser beam 508 emitted from a laser light source 505 is controlled by a polarization control element 506 so that the electric field oscillation direction thereof is within the plane defined by the capillary array 501. The laser beam 508 is then focused by a lens 507 and incident on the side surface of the capillary array 501.

[0054] The laser beam 508 propagates through the capillary array 501 and is irradiated onto the measurement target inside the capillary. In addition to the mechanism shown in Fig. 5, the fluorescence excitation optical system 502 may be provided with a mechanism for adjusting the optical axis position using a reflecting mirror, a mechanism for attenuating the laser output and adjusting its power, and the like.

[0055] The fluorescence detection optical system 503 is a detection optical system. Fluorescence emitted from the measurement target is detected by the fluorescence detection optical system 503. The fluorescence detection optical system 503 includes lenses 509 and 510, a wavelength filter 511, a grating 512, and a camera 513.

[0056] The fluorescence emitted from the measurement object is collimated by lens 509. A wavelength filter 511 removes excitation light and transmits the fluorescence. The propagation angle of the fluorescence is changed according to the wavelength by a grating 512. The fluorescence is then focused onto a camera 513 by a lens 510. In this way, the fluorescence detection optical system 503 guides the light from each capillary 504 to the camera 513. The camera 513 is an example of an optical detection system, and detects the light from each capillary 504. By performing spectroscopy, the spectrum of the fluorescence emitted from the measurement object is observed on the camera 513.

[0057] 5 shows an example in which the fluorescence detection optical system 503 is a spectroscopic optical system, with the detection of multiple types of fluorescent dyes by electrophoresis in mind, but the fluorescence detection optical system 503 does not necessarily have to be a spectroscopic optical system. If spectroscopic detection is not necessary, such as when only one type of fluorescent dye is detected, the generated fluorescence can be detected directly with a detector. Furthermore, even when spectroscopic detection is performed, the spectroscopic optical element does not necessarily have to be a grating, and spectroscopic detection can be performed by other spectroscopic optical elements such as a prism.

[0058] The light irradiation onto the capillary array 501 can be performed in the manner shown in Fig. 1. In this case, the angle range of θ0 that can obtain the effect of uniforming the excitation light power on the capillaries can be found by the method described above.

[0059] In the calculation of the angle range described above, each interface was treated as a plane, but the capillary 504 has a circular cross section, and the interface onto which the excitation light is incident is not a plane. However, if the laser beam 508 is focused on the capillary array 501 to a size smaller than the inner and outer diameters of the capillary 504, the above discussion can be applied approximately.

[0060] On the other hand, when the flow channel has a circular cross section, additional restrictions are imposed on its inner diameter, outer diameter, and pitch. This is because the flow channel acts as a rod lens, and Patent Document 2 shows the conditions when the laser light is incident perpendicularly (θ0 = 0°) on the capillary array. However, when the laser light is incident at an angle to the capillary array (θ0 > 0°), the restrictions change from the perpendicular case.

[0061] First, consider the case where a ray of light is incident perpendicularly on a capillary array. The cross section of the capillary is circular, and a ray of light irradiated near the center of the capillary passes through four interfaces. Using the paraxial approximation, the ABCD matrix for this capillary is calculated. The ray of light incident on the capillary passes through the capillary in the following steps: (i) passes through the outer interface of the capillary, (ii) propagates inside the capillary, (iii) passes through the inner interface of the capillary, (iv) propagates through the lumen of the capillary, (v) passes through the inner interface of the capillary, (vi) propagates inside the capillary, and (vii) passes through the outer interface of the capillary. The ABCD matrix for each is

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[0062] The ABCD matrix of the entire capillary is the product of the above matrices.

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[0063] Below we describe the conditions that the derived focal length and capillary pitch must satisfy. In the following, we assume that the capillaries are arranged at equal intervals with a pitch of distance d. The focal length in the cross-sectional direction of the capillary is f. In calculations, the arrangement of equally spaced capillaries can be seen as a repeating unit of "space of distance d / 2", "capillary", and "space of distance d / 2". In the ABCD matrix, the effect of propagation at a distance d / 2 and the effect of a lens with focal length f are respectively

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[0064] In order for all the light incident from the end of the capillary array to pass through all the capillaries in the array, the light that passes through a capillary must enter the next capillary without diverging. Since the ABCD matrix describes the effect of the height and angle from the optical axis of the light ray, this condition is expressed as follows:

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[0065] Calculating the eigenvalues ​​from the matrix of equation (18)

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[0066] The results of comparing the above conditions with a ray tracing simulation are shown below. In the simulation, 10 capillaries with an outer diameter of 300 μm and an inner diameter of 100 μm were arranged. The refractive index of the material that makes up the capillaries was 1.46, the refractive index of the substance filled inside the capillaries was 1.41, and the refractive index of the surrounding environment was 1. Laser light was incident perpendicularly from the side of the array. The diameter of the irradiated beam was 20 μm. The light was polarized in the direction of the long axis of the flow path. In the simulation, the power irradiated to the inner diameter part of each capillary was calculated by ray tracing.

[0067] Figure 6 plots the power of light irradiated onto the inner diameter of the 10th (last) capillary against the capillary pitch. Due to surface reflection losses at the capillary, the power of light reaching the 10th capillary is 54% even at the smallest pitch of 300 μm (the same value as the outer diameter). It can be seen that the power irradiated onto the 10th capillary begins to decrease from a pitch of approximately 1200 μm. Meanwhile, the right-hand side of equation (20), calculated under the above conditions, is 1240 μm. The above 1240 μm is shown in the graph with a dashed line. From this result, it can be seen that the range of capillary pitches allowable under the conditions of equation (20) can be defined.

[0068] Next, we will describe the case where light is incident on the capillary array at an angle θ0. As with the calculation of transmittance mentioned above, let n0 be the refractive index of the surrounding environment, n1 be the refractive index of the capillary material, and n2 be the refractive index of the substance filling the capillary lumen. Let θ0, θ1, and θ2 be the angles of propagation through materials with each refractive index.

[0069] The capillary is assumed to have a circular cross section. In other words, in a cross section taken along a plane perpendicular to the axis of the capillary, the inner and outer circumferences of the capillary are circular. When the capillary is viewed in a cross section including a beam of light irradiated obliquely onto the capillary (dotted line in Figure 7(a)), the capillary cross section is an ellipse as shown in Figure 7(b). The outer diameter (radius R out ), the cross section of the ellipse has a simple axis radius R out , the major axis radius is R out / cosθ0. The light rays enter this ellipse from the major axis.

[0070] Taking the above into consideration, we can rewrite the previous ABCD matrix into one for an ellipse:

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[0071] To summarize the above, the outer radius of the capillary R out and inner radius R in The matrix of equation (15) can be calculated using the matrix expressed by equation (21). Then, the focal length f in the direction perpendicular to the axis of the capillary can be calculated using the 2nd row, 1st column component of the matrix of equation (15) and equation (16). When this focal length f satisfies equation (22) with respect to the pitch d of the capillaries in the channel array, the power irradiated to the capillaries can be increased.

[0072] The results of comparing the above conditions with ray tracing simulation are shown below. The model is the same as that used to calculate the graph in Figure 6 above, with only the incident angle of the light beam being different. The angle of the incident excitation light was set to θ0 = 55.6°. This is the Brewster angle when the interface is flat between an ambient environment with a refractive index of 1 and a capillary material with a refractive index of 1.46.

[0073] In Figure 8, the power of light irradiated onto the inner diameter of the 10th (last) capillary is plotted against the capillary pitch. The effect of this embodiment is to reduce the loss of light power due to capillary surface reflection. Therefore, when the capillary pitch is 300 μm, even the 10th capillary is irradiated with 99.6% of the light power before incidence into the array. When θ0 = 0°, the light power irradiated onto the 10th capillary is 54%, which shows that the effect of this embodiment reduces reflection loss.

[0074] The power irradiated to the 10th capillary begins to decrease from a pitch of approximately 600 μm. Meanwhile, the right-hand side of equation (22) calculated under the above conditions is 615 μm. The above 615 μm is shown by a dashed line in the graph. This result shows that the allowable capillary pitch range can be determined by equation (22).

[0075] Figure 9 shows a graph showing the power of excitation light irradiated onto each of the 10 capillaries when the capillary pitch is 300 μm, using the same simulation model as in Figures 6 and 8. The power of the excitation light is shown with the value before it enters the array as 100%. In a configuration in which excitation light is irradiated from both sides of the array, it is assumed that the excitation light is split into two beams with the same power.

[0076] When the incident light is incident from one side of the array at an incident angle of 0°, the irradiated power decreases depending on the capillary number. However, when the incident angle is set to 55.6°, the reflection loss is kept small and the power of the irradiated excitation light becomes more uniform.

[0077] When excitation light is incident from both sides of the array at an incident angle of 0°, the variation in excitation light power is reduced compared to when it is irradiated from one side at an incident angle of 0°. In this case, the coefficient of variation (standard deviation divided by the average value) of the excitation light power for all 10 beams is 1.5%. On the other hand, when it is incident from one side at an incident angle of 55.6°, the coefficient of variation of the excitation light power is 0.1%, which means that the excitation light power is more uniform than when it is irradiated from both sides.

[0078] Furthermore, even when excitation light is irradiated from both sides, approximately 20% of the excitation light power is lost due to reflection losses. On the other hand, with the configuration of this embodiment, almost all of the excitation light power can be used to excite fluorescence. Furthermore, when excitation light is irradiated from both sides, a mechanism for splitting the light beam and a mechanism for adjusting the two beams so that they are incident on the array coaxially may be required, making the irradiation optical system more complex. With the configuration of this embodiment, the effect of uniforming the excitation light power can be achieved simply by angling the excitation light.

[0079] As described above, in embodiment 1, it was shown that by setting the angle θ0 to a certain angle, the reflection loss of the excitation light can be reduced and the power of the excitation light irradiated to each capillary can be made uniform. In addition to the above effects, the method of this embodiment makes it possible to irradiate the excitation light even in cases where the conditions necessary for light irradiation from the side of the capillary array cannot be met by adjusting the angle θ0.

[0080] As an example, let's say the capillary has an outer diameter of 300 μm and an inner diameter of 50 μm. The refractive index of the material that makes up the capillary is 1.46, the refractive index of the substance filled into the capillary is 1.33, and the refractive index of the surrounding environment is 1. These conditions roughly correspond to a silica glass capillary placed in air and filled with water.

[0081] If the excitation light is incident at θ0 = 0°, the focal length calculated by equation (16) is -866 μm. Because the focal length is a negative value, equation (20) cannot be satisfied regardless of the capillary pitch. In this case, the excitation light propagating through the capillary array diverges due to the concave lens effect of the capillaries, and it is not possible to irradiate all of the capillaries.

[0082] Under the above conditions, if the angle θ0 = 55.6°, the focal length becomes a positive value of 652 μm, and the condition for satisfying equation (22) exists. At the same time, the effect of reducing the surface reflection loss mentioned above can also be obtained. Even under the conventional condition where the excitation light cannot be irradiated onto all capillaries when θ0 = 0°, by adjusting the value of the angle θ0 in this way, the condition for the excitation light to propagate inside the capillaries can be satisfied.

[0083] In the configuration of this embodiment, the excitation light propagates obliquely through the capillary array. As a result, the position where the excitation light is irradiated (the position where fluorescence detection is performed) varies for each capillary. For example, if the fluorescence detection device of this embodiment is applied to an electrophoresis device, the above-mentioned influence will cause the effective length of electrophoresis to change. In such cases, it is preferable to sequentially shift the arrangement of each capillary. For example, if the fluorescence detection point is shifted 0.1 mm in the axial direction of the capillary, the capillaries in the array can be shifted in steps of 0.1 mm.

[0084] Furthermore, by propagating the excitation light obliquely through the capillary array, the length of the region where fluorescence occurs in the capillary lumen becomes longer than when the excitation light is irradiated perpendicular to the capillary axis. This effect is observed when the radius of the capillary lumen is R in When the propagation angle of light in the capillary cavity is θ2, 2R in This is about tan θ2. This results in a deterioration in the detection resolution when detecting fluorescence on the capillary.

[0085] For example, when excitation light is incident at an angle of 55.6° on a capillary with an inner diameter of 50 μm, a surrounding environment refractive index of 1, and an inner lumen refractive index of 1.41, the length of the emission region increases by approximately 36 μm. The magnitude of this effect on the overall measurement depends on the type of measurement being performed and the configuration of the flow channel array. If it is necessary to minimize this effect, the incident angle can be set small within the angle range in which the effects of this embodiment can be obtained.

[0086] In embodiment 1, a configuration is described that takes into consideration the use of the optical detection device as a detection device in a capillary electrophoresis device, but the application of the configuration of the present disclosure is not limited to the above, and it can also be applied to other measurement devices that optically measure channel arrays.

[0087] <Embodiment 1: Summary> In the fluorescence detection device 500 according to the first embodiment, when a laser beam 508 is incident on the side of the capillary array 501, the laser beam 508 is angled relative to the direction perpendicular to the axis of the capillaries in the plane defined by the capillary array. This angle satisfies the condition of formula (7) or formula (13). The polarization direction of the laser beam 508 is parallel to the plane defined by the capillaries. Furthermore, the capillary pitch satisfies the condition of formula (22). This configuration reduces surface reflection loss due to the capillaries and makes the power of the laser light irradiated on each capillary more uniform than when the laser light is incident perpendicular to the capillaries. Furthermore, adjusting the laser light incident angle allows for adjustment of limitations on the outer diameter, inner diameter, and arrangement conditions of the capillaries, enabling irradiation conditions that cannot be achieved with a configuration in which light is incident perpendicular to the capillaries.

[0088] <Embodiment 2> 10 is a configuration diagram of a fluorescence detection apparatus 1000 according to a second embodiment of the present invention. The configuration of the fluorescence detection apparatus 1000, including the capillary array 501 and the fluorescence excitation optical system 502, can be the same as that of the first embodiment shown in FIG. 5. The fluorescence detection optical system 1001 includes an optical fiber array 1002 for guiding fluorescence, and a fluorescence detection apparatus 1003. The optical fiber array 1002 is composed of a plurality of optical fibers.

[0089] A wavelength filter may be provided upstream of the fluorescence detection device 1003 to remove excitation light and introduce only fluorescence into the detector. Furthermore, while the fluorescence detection device 1000 is configured such that fluorescence guided by optical fibers is introduced directly into the detection device, the detection optical system is not limited to this configuration. For example, the rear end of the optical fiber array 1002 may be connected to the fluorescence detection optical system 503 shown in FIG. 5, where the fluorescence may be detected after being dispersed.

[0090] 10, the angle of incidence of the excitation light onto the capillary array is set within the angle range shown in embodiment 1. In this case, in addition to the effects shown in embodiment 1, crosstalk between the detection channels that detect the capillaries can be reduced.

[0091] The reason why crosstalk can be reduced is explained below. Crosstalk here refers to a situation in which fluorescence is mixed into a channel other than the channel for detecting the capillary emitting the fluorescence.

[0092] There are several causes of this crosstalk. For example, in the configuration shown in Figure 10, the fluorescence generated in the lumen of capillary 504 is emitted in all directions, so some of the light may enter the optical fiber installed for the adjacent capillary. Also, in the fluorescence detection optical system 503 shown in Figure 5, the profile of the spectral image formed on camera 513 is blunted due to aberrations of lenses 509 and 510, and the profile may overlap in the part where the adjacent capillary is detected.

[0093] Among the various causes of crosstalk, the configuration of this embodiment can reduce crosstalk caused by surface reflection of the capillaries. As described above, the fluorescence generated in a capillary is irradiated in all directions, and therefore the fluorescence also irradiates adjacent capillaries. Some of the fluorescence is reflected by the capillary surface, and depending on the angle and position of reflection, it may enter the optical fiber intended to detect the adjacent capillary.

[0094] At this time, the fluorescence that travels perpendicularly from the light-emitting site toward the adjacent capillary contributes significantly to crosstalk. Figure 11 shows a schematic diagram of this situation. Fluorescence emitted from light-emitting site 504c in capillary 504a on the left side of Figure 11 is emitted with equal intensity in all directions. However, only the light that travels toward the detector, which is assumed to be located on the front side of the paper, is actually detected.

[0095] Light traveling perpendicularly from the left capillary 504a to the right capillary 504b is reflected by the left half of the right capillary 504b. However, because the capillary 504b is cylindrical, the light reflected by region 504d is more likely to enter the detector located on the front side of the paper. Region 504d can be said to be a region that has a large effect on crosstalk. Fluorescence irradiated outside region 504d does not propagate toward the detector even if it is reflected by the surface of capillary 504b.

[0096] This situation is shown in a simulation in Figure 12. In Figure 12, a capillary array with a diameter of 150 μm, an inner diameter of 50 μm, and a pitch of 150 μm is set up. The refractive index of the surrounding environment is set to 1, the refractive index of the capillary material is set to 1.46, and the refractive index of the capillary lumen is set to 1.41. In the simulation, an imaging system with an NA of 0.36 and a detection camera are set up on the front side of the page, and the simulation is performed to acquire a fluorescent image of the fluorescence generated in the capillary lumen.

[0097] The image portion corresponding to two adjacent capillaries on the camera is cut out and displayed in Fig. 12. The positional relationship of the capillaries is the same as in the schematic diagram of Fig. 11, with Fig. 12(a) corresponding to capillary 504a in Fig. 11 and Fig. 12(b) corresponding to capillary 504b in Fig. 11.

[0098] The capillary 504a on the left side is emitting light. The black parts in the image in Figure 12(a) are the emitting parts. The capillary 504b on the right side is not emitting light. Note that the image in Figure 12 was constructed so that the higher the fluorescence intensity, the darker the black becomes. The darkest parts in Figure 12(a) and Figure 12(b) are about the same intensity, but this is because a color scale is set for each image. In reality, the fluorescence intensity in the image in Figure 12(b) is lower than that in Figure 12(a).

[0099] Capillary 504b, which corresponds to Figure 12(b), does not emit light, but a black area appears on the left side of the capillary. This area corresponds to area 504d in Figure 11. In this way, the fluorescence reflected from the capillary surface enters the channel that detects the adjacent capillary, and is observed as crosstalk.

[0100] The reason why crosstalk caused by the above-mentioned factors can be reduced by injecting excitation light at an angle is explained below. Figure 13 shows the schematic diagram of Figure 11, with the detection points in the optical fiber added by dashed lines. When irradiating with light at an incident angle of 0°, as in Figure 13(a), the light-emitting points are aligned horizontally on each capillary at the same angle of 0°. In this case, the light-emitting points of adjacent capillaries are close to the points that cause crosstalk due to the surface reflection mentioned above. This makes it easier for fluorescence reflected from the surface to enter the optical fiber.

[0101] On the other hand, when excitation light is irradiated at an angle to the capillary array as shown in Figure 13(b), the emission sites are also angled on the capillaries, i.e., spaced apart in the axial direction of the capillaries. Meanwhile, the site where crosstalk occurs due to surface reflection is in the same position as when the angle is 0° in Figure 13(a), so the positions of the emission site and the site where crosstalk occurs can be spaced apart in the axial direction. As a result, it becomes difficult for fluorescence reflected from the capillary surface to enter the fiber used to measure adjacent capillaries, reducing crosstalk.

[0102] Figure 14 quantitatively shows the above effect using a ray tracing simulation. In this simulation, eight capillaries with a diameter of 150 μm are arranged at a pitch of 175 μm, and a fiber with a core diameter of 100 μm and NA of 0.4 is placed 50 μm away from the light-emitting part of each capillary.

[0103] In Figure 14, it is assumed that capillary 4 is emitting light, and the power of the excitation light introduced into the optical fiber detecting capillary 4 is set to 100%. The fluorescence detected in capillaries other than capillary 4 is crosstalk caused by capillary 4. When excitation light is incident perpendicularly to the capillary array, the crosstalk occurring in capillaries adjacent to the emitting capillary is about 2.2%. On the other hand, when excitation light is irradiated at an angle of 55.6°, the crosstalk occurring in adjacent capillaries is reduced to about 0.6%.

[0104] This crosstalk reduction effect is achieved by increasing the distance in the axial direction between the fluorescence-emitting region and the region that generates crosstalk due to surface reflection, so that only the fluorescence is selectively detected. In particular, in this embodiment, in the fluorescence detection optical system 1001, the optical fiber guides only the light emitted from the region where light is emitted in the capillary 504 to the detector, thereby optically shielding the regions around that region (regions where light is not emitted), thereby reducing crosstalk.

[0105] Optical elements other than optical fibers may be used as a means for selectively extracting only the fluorescence. For example, as shown in Fig. 15, the fluorescence detection optical system 1001 may include an optical slit member 514 arranged in relation to each capillary 504 (only the shape of the slit portion is shown by a dashed line in Fig. 15). The slit of the optical slit member 514 is of a size that allows only light from the fluorescence detection site to pass through.

[0106] In the fluorescence detection optical system 1001, the slits of the optical slit member 514 may be arranged to correspond only to the portions where light emission occurs in each capillary 504. In this way, the optical slit member 514 optically shields the portions (portions where light emission does not occur) around the portions where light emission occurs in each capillary 504, so that it is possible to reduce crosstalk while efficiently transmitting the fluorescence to be detected.

[0107] <Embodiment 2: Summary> In the fluorescence detection device 1000 according to the second embodiment, when a laser beam 508 is incident on the side of the capillary array 501, the laser beam 508 is angled with respect to a direction perpendicular to the axis of the capillary. The fluorescence generated in each capillary is guided to the fluorescence detection device 1003 by the optical fiber array 1002. With the above configuration, it is possible to reduce crosstalk that occurs when fluorescence is reflected on the surface of a capillary and enters an optical fiber installed for an adjacent capillary.

[0108] <About modified examples> The present invention is not limited to the above-described embodiments and includes various modifications. For example, the above-described embodiments have been described in detail to clearly explain the present invention, and the present invention is not necessarily limited to those including all of the described configurations. Furthermore, it is possible to replace part of the configuration of one embodiment with the configuration of another embodiment, or to add the configuration of another embodiment to the configuration of one embodiment. Furthermore, it is possible to add, delete, or replace part of the configuration of each embodiment with other configurations. [Explanation of symbols]

[0109] 100 channel array 101 Surrounding Area 102 Channel 103 Lumen 301 Flow path substrate 500 Fluorescence Detector 501 Capillary Array 502 Fluorescence Excitation Optical System 503 Fluorescence detection optical system 504(504a, 504b) Capillary 505 Laser Light Source 506 Polarization control element 507 Lens 508 Laser Beam 509 Lens 510 lens 511 Wavelength Filter 512 Grating 513 Camera 514 Optical slit member 1000 Fluorescence Detector 1001 Fluorescence detection optical system 1002 Optical fiber array 1003 Fluorescence detection device

Claims

1. a channel array in which a plurality of tubes are arranged so that their axes are parallel to one another, forming a channel plane; An illumination optical system including a light source, the light from the light source being: the vibration direction of the electric field component of the light is parallel to the plane of the flow channel, the optical axis of the light is contained in the plane of the flow path, and the optical axis is at an angle θ in the plane of the flow channel with respect to a direction perpendicular to the axis of the tube; ０ to form an irradiation optical system for irradiating light in such a manner as to a light detection system that detects light emitted from each of the plurality of tubes; Equipped with The refractive index of the surrounding environment is n ０ and the refractive index of the material of the tube is n １ and the refractive index of the material inside the tube is n ２ As a result, from the refractive index and the predetermined angle, [Equation 1] The angle θ of the light ray in the material of the tube is determined as １ and the angle θ of the ray inside the tube ２ With respect to the predetermined angle θ ０ are the following equations 1 and 2: [Equation 2] A photodetector device characterized by satisfying the above.

2. When the number of the tubes is N, the predetermined angle θ ０ Further, the following formula: [Equation 3] 2. The photodetector according to claim 1, wherein the following is satisfied:

3. 2. The light detection device according to claim 1, wherein the light from the light source is incident on both sides of the channel array so as to face each other.

4. the tube is a capillary having circular inner and outer peripheries in a cross section taken along a plane perpendicular to the axis of the tube; The outer radius R of the tube ｏｕｔ and the inner radius R ｉｎ Regarding [Equation 4] Using the matrix expressed as [Equation 5] The focal length in the direction perpendicular to the axis of the tube is calculated using the second row and first column elements of the matrix calculated by the formula [Equation 6] for the pitch d of the tubes in the channel array [Equation 7] 2. The photodetector according to claim 1, wherein the following is satisfied:

5. the light detection device includes a detection optical system that guides light emitted from the plurality of tubes to the light detection system; In the detection optical system, a portion where no light emission occurs in the vicinity of a portion where light emission occurs in the plurality of tubes is optically shielded.

5. The photodetector device according to claim 4, wherein:

6. the detection optics comprises an optical fiber; The optical fiber guides only the light emitted from the area where the light is emitted to the detector, thereby optically shielding the area where the light is not emitted.

6. The photodetector device according to claim 5, wherein the photodetector device comprises:

7. the detection optical system includes an optical slit member disposed relative to the plurality of tubes; The optical slit member optically shields the area where no light is emitted.

6. The photodetector device according to claim 5, wherein the photodetector device comprises:

8. a flow channel substrate having a plurality of flow channels arranged therein with their axes parallel to each other and constituting a flow channel plane; An illumination optical system including a light source, the light from the light source being: the vibration direction of the electric field component of the light is parallel to the plane of the flow channel, the optical axis of the light is contained in the plane of the flow path, and the optical axis is at a predetermined angle θ with respect to a direction perpendicular to the axis of the flow channel in the plane of the flow channel; ０ to form an irradiation optical system for irradiating light in such a manner as to an optical detection system that detects light emitted from each of the plurality of flow paths; Equipped with The refractive index of the material of the flow path substrate is n ０ and the refractive index of the material inside the flow path is n １ As the predetermined angle θ ０ but, [Equation 8] A light detection device characterized by satisfying the above.

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