JP2009019961A - Fluorescence detecting system - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a fluorescence detecting system that easily detects a plurality of fluorescences at the same time and easily detects the fluorescences emitted from a plurality of measuring targets at the same time. <P>SOLUTION: The fluorescence detecting system 10 includes light sources 7A, 7B and 7C for emitting exciting light, optical fiber groups 1A, 1B and 1C for exciting light optically connected to the respective light sources 7A, 7B and 7C each at one end, photodetectors 8a, 8b and 8c, a leading end part 20 wherein the other end of each of the optical fiber groups 2a, 2b and 2c for fluorescence respectively optically connected to the photodetectors 8a, 8b and 8c and one end of each of the optical fiber groups 1A, 1B and 1C for exciting light and each of the optical fiber groups 2a, 2b and 2c for fluorescence are formed into one bundle, a lens 4 and a capillary 3 for integrally holding the leading end part 20 and the lens 4. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a fluorescence detection system, and more particularly to a fluorescence detection system including an optical fiber.

  In recent years, as a fluorescence detection apparatus that irradiates an object to be measured with excitation light and detects fluorescence generated from the object to be measured, in particular, a plurality of fluorescence can be detected, and also generated from a plurality of objects to be measured. There is a demand for a fluorescence detection apparatus that can detect fluorescence.

  As a fluorescence detection device that detects a plurality of fluorescence, a fluorescence microscope that detects a plurality of fluorescence by switching a filter cassette including an excitation filter, a dichroic mirror, and a barrier filter according to the wavelength of the fluorescence to be detected is disclosed. (For example, refer to Patent Document 1).

  Furthermore, as a method for detecting a plurality of fluorescence, a method using lens aberration (for example, see Patent Document 2), a method for processing a detected spectrum (for example, Patent Document 3), or diffraction. A method using a lattice (for example, see Patent Document 4) is disclosed.

  On the other hand, as a fluorescence detection device that detects fluorescence generated from a plurality of objects to be measured, the excitation light is diffused along the array of wells in the microwell plate, and the objects to be measured in the wells are irradiated with the excitation light. Thus, a microplate reader that detects fluorescence generated from a plurality of objects to be measured has been disclosed (see, for example, Patent Document 5).

Furthermore, as a method of detecting fluorescence generated from a plurality of objects to be measured, a method of rotating a plurality of objects to be measured arranged in an arc shape and sequentially measuring them (see, for example, Patent Document 6), a plurality of objects. A method of sequentially scanning an object to be measured (for example, see Patent Document 7) is disclosed.
JP 2003-29155 A JP 2006-308403 A JP-T 8-506419 Special 3866940 JP-A-2005-55219 Special 3855485 JP 2001-255272 A

  However, the above-described fluorescence microscope can detect only one fluorescence at a time and cannot detect a plurality of fluorescence simultaneously. For this reason, in order to measure a plurality of fluorescence, it is necessary to switch the filter cassette, and the operation becomes complicated.

  Further, the above-described method for detecting a plurality of fluorescences requires expensive parts such as a highly accurate fine movement mechanism, a complicated signal processing device, or a diffraction grating.

  On the other hand, in the above-described microplate reader, when the objects to be measured are arranged at high density, the optical sensitivity of the fluorescence generated from the adjacent objects to be measured overlaps, resulting in low measurement sensitivity.

  In the above-described method for detecting fluorescence generated from a plurality of objects to be measured, the plurality of objects to be measured are sequentially measured or sequentially scanned, so that the fluorescence generated from the plurality of objects to be measured is simultaneously detected. I can't do it.

  SUMMARY OF THE INVENTION An object of the present invention is to provide a fluorescence detection system that can easily and simultaneously detect a plurality of fluorescences, and that can easily and simultaneously detect fluorescence generated from a plurality of objects to be measured.

  In order to achieve the above object, a fluorescence detection system according to claim 1 includes a light source that emits excitation light and a light receiving unit that receives fluorescence generated from a measurement object irradiated with the excitation light. In the detection system, the excitation light is incident from one end optically connected to the light source, and the excitation light is emitted from the other end arranged opposite to the measurement target. The excitation light enters the plurality of optical fibers for excitation light and one end arranged to face the object to be measured, and the fluorescence is emitted from the other end optically connected to the light receiving unit. A plurality of fluorescent optical fiber groups composed of at least one optical fiber, a distal end portion in which the other ends of the plurality of excitation light optical fiber groups and one end of the plurality of fluorescent optical fiber groups are bundled, Front and front A lens disposed between objects to be measured; and a holding portion that holds the tip portion and the lens apart and integrally, and at the tip portion, the plurality of excitation-light optical fiber groups Each of them is arranged not to be adjacent to each other.

  The fluorescence detection system according to claim 2 is the fluorescence detection system according to claim 1, wherein a plurality of optical fibers constituting each of the fluorescence optical fiber groups is centered on each of the excitation light optical fiber groups at the tip portion. And the plurality of fluorescent optical fiber groups are centered on different optical fiber groups for excitation light.

  The fluorescence detection system according to claim 3 is the fluorescence detection system according to claim 1 or 2, wherein the light constituting the plurality of fluorescence optical fiber groups and the plurality of excitation light optical fiber groups at the tip portion. Of the fibers, adjacent optical fibers are in contact with each other.

  The fluorescence detection system according to claim 4 is the fluorescence detection system according to claim 2 or 3, wherein the optical fibers constituting each of the fluorescence optical fiber groups arranged in a substantially concentric circle have a central angle of at least 90. It is characterized by being arranged in a substantially arcuate shape of at least degrees.

  The fluorescence detection system according to claim 5, wherein the first distance between the lens and the tip is a back focus distance of the lens. And a distance at which the transmittance of the excitation light through the lens is 70% or more.

  The fluorescence detection system according to claim 6 is the fluorescence detection system according to any one of claims 1 to 5, wherein the second distance between the lens and the object to be measured is the object to be measured. The diameter of the irradiation spot of the excitation light formed on the surface is smaller than the distance between the centers of the adjacent irradiation spots.

  The fluorescence detection system according to claim 7 is the fluorescence detection system according to claim 2, wherein the second distance between the lens and the object to be measured is incident on one fluorescence optical fiber group. About the light quantity of fluorescence, from the optical fiber group for excitation light corresponding to the center with respect to the light quantity of fluorescence caused by the excitation light emitted from the optical fiber group for excitation light not corresponding to the center of the optical fiber group for fluorescence The distance is such that the ratio of the amount of fluorescent light caused by the emitted excitation light is 20 dB or more.

  The fluorescence detection system according to claim 8 is the fluorescence detection system according to any one of claims 1 to 7, comprising three optical fiber groups for the excitation light and three optical fiber groups for the fluorescence. Each of the excitation light optical fiber groups includes one optical fiber, each of the fluorescence optical fiber groups includes three optical fibers, and each of the lenses includes a half drum lens.

  According to the fluorescence detection system of claim 1, each of the plurality of optical fibers for excitation light is arranged so as not to be adjacent to each other at the tip portion arranged to face the measurement object. A plurality of excitation lights emitted from the optical fiber group for light can be simultaneously irradiated to a plurality of positions of the object to be measured in a spatially separated state. In addition, a plurality of fluorescence is generated from an object to be measured that is simultaneously irradiated with excitation light at a plurality of positions, but since there are a lens and a plurality of fluorescent optical fiber groups at the tip, A plurality of fluorescent lights can be simultaneously incident on the fluorescent optical fiber group without being mixed in a spatially separated state.

  In the fluorescence detection system described above, in order to receive a plurality of fluorescences, it is only necessary to simultaneously emit a plurality of excitation lights from a plurality of excitation light optical fiber groups at the tip, for example, light that emits excitation light. There is no need to switch the fiber, and there is no need to scan the measurement object with the tip. Therefore, according to the fluorescence detection system, a plurality of fluorescence generated from the measurement target can be easily and simultaneously detected.

  Furthermore, since a plurality of excitation lights can be irradiated simultaneously to a plurality of positions in a spatially separated state, a plurality of excitation lights can be applied to a plurality of objects even if there are a plurality of objects to be measured. The object to be measured can be irradiated simultaneously. In addition, although fluorescence is generated from a plurality of objects to be measured that are simultaneously irradiated with excitation light, a lens and a plurality of fluorescent optical fiber groups are present at the tip, so that each fluorescent optical fiber is provided at the tip. Fluorescence generated from a plurality of objects to be measured can enter the group simultaneously in a spatially separated state without being mixed.

  Further, in the fluorescence detection system, in order to receive fluorescence generated from a plurality of objects to be measured, it is only necessary to simultaneously emit a plurality of excitation lights from a plurality of optical fibers for excitation light at the tip, for example, There is no need to switch the optical fiber that emits the excitation light, and there is no need to scan the measurement object with the tip. Therefore, according to the fluorescence detection system, a plurality of fluorescence generated from a plurality of objects to be measured can be easily and simultaneously detected.

  In addition, since the optical fiber group for excitation light and the optical fiber group for fluorescence are provided, the optical path for transmitting excitation light and the optical path for transmitting fluorescence can be completely separated. Thus, a highly sensitive fluorescence detection system can be provided.

  According to the fluorescence detection system of claim 2, at the tip, the plurality of optical fibers constituting each fluorescence optical fiber group are arranged in a substantially concentric circle centered on each excitation light optical fiber group, and Since a plurality of optical fiber groups for fluorescence are centered on different optical fiber groups for excitation light, fluorescence generated by irradiating the object to be measured with excitation light emitted from the central optical fiber group for excitation light Can be effectively incident on the optical fibers of the fluorescent optical fiber groups arranged substantially concentrically in the optical fiber group for excitation light.

  According to the fluorescence detection system of claim 3, since the adjacent optical fibers are in contact with each other among the optical fibers constituting the plurality of optical fiber groups for fluorescence and the plurality of optical fiber groups for excitation light at the tip, Fluorescence generated by irradiating the object to be measured with excitation light emitted from each excitation light optical fiber group can be incident on each fluorescence optical fiber group without leakage. In addition, a plurality of fluorescent optical fiber groups and a plurality of excitation light optical fiber groups can be efficiently arranged at the tip portion, and thus the tip portion can be made small.

  According to the fluorescence detection system of the fourth aspect, the optical fibers constituting each fluorescence optical fiber group arranged in a substantially concentric circle are arranged in a substantially arc shape having a central angle of at least 90 degrees or more. By passing through the lens, even if it reaches the distal end portion in a state where the distribution of the light amount is uneven due to the aberration of the lens, the fluorescence can be reliably incident on any one of the optical fibers.

  According to the fluorescence detection system of claim 5, since the distance between the lens and the tip is larger than the distance of the back focus of the lens, a plurality of excitation lights emitted from the tip and transmitted through the lens are converged. Alternatively, the object to be measured can be irradiated in a substantially parallel light state. Therefore, the object to be measured can be irradiated with each of the plurality of excitation lights in a state of being surely spatially separated. In addition, since the transmittance of the excitation light lens is 70% or more, a large amount of each excitation light can be applied to the object to be measured, and thus a large amount of each fluorescence can be generated.

  According to the fluorescence detection system of the sixth aspect, the second distance between the lens and the object to be measured is emitted from each other end of each optical fiber group for excitation light on the surface of the object to be measured. Since the diameter of the irradiation spot of each excitation light is smaller than the distance between the centers of adjacent irradiation spots, the excitation light emitted from one optical fiber group for excitation light is irradiated on the measurement object. The position where the excitation light emitted from the other optical fiber group for excitation light is irradiated does not overlap. As a result, the object to be measured is irradiated in a state where a plurality of excitation lights are reliably separated spatially, and thus the generated fluorescence is prevented from being caused by two or more excitation lights. Therefore, a fluorescence detection system with low noise and high detection sensitivity can be obtained.

  According to the fluorescence detection system of claim 7, the second distance between the lens and the object to be measured is that of the fluorescence optical fiber group with respect to the amount of fluorescence incident on one fluorescence optical fiber group. The ratio of the amount of fluorescence caused by the excitation light emitted from the excitation light optical fiber group corresponding to the center to the amount of fluorescence caused by the excitation light emitted from the excitation light optical fiber group not corresponding to the center is 20 dB. Since the distance is as described above, for example, it is possible to separate the same degree of fluorescence as a fluorescence analysis system provided with a dichroic mirror, and a large-scale apparatus and expensive parts are not required. In comparison with this, the fluorescence analysis system can be downsized and the manufacturing cost can be reduced.

  As a result of earnest research to achieve the above object, the inventors of the present invention include a light source that emits excitation light and a light receiving unit that receives fluorescence generated from the measurement target irradiated with the excitation light. In a fluorescence detection system, excitation light is made up of at least one optical fiber from which excitation light is incident from one end optically connected to a light source and is emitted from the other end arranged to face the object to be measured. A plurality of optical fiber groups and at least one optical fiber from which fluorescence is incident from one end arranged opposite to the object to be measured and from which the other end optically connected to the light receiving unit is emitted A plurality of fluorescent optical fiber groups, a distal end portion in which the other ends of the plurality of excitation light optical fiber groups and one end of the plurality of fluorescent optical fiber groups are bundled, and between the distal end portion and the object to be measured Lens, tip and lens And a plurality of optical fibers for the excitation light are arranged so as not to be adjacent to each other at the tip, thereby easily and simultaneously detecting a plurality of fluorescence. Furthermore, it has been found that fluorescence generated from a plurality of objects to be measured can be easily and simultaneously detected.

  The present invention has been made based on the above findings.

  Hereinafter, embodiments of the present invention will be described with reference to the drawings.

  FIG. 1 is a diagram schematically showing a configuration of a fluorescence detection system according to an embodiment of the present invention.

  In FIG. 1, a fluorescence detection system 10 includes light sources 7A, 7B, and 7C that emit excitation light, and excitation light optical fiber groups 1A, 1B, and one end optically connected to the light sources 7A, 7B, and 7C, respectively. 1C, optical receivers 8a, 8b, 8c, fluorescent optical fiber groups 2a, 2b, 2c optically connected to the optical receivers 8a, 8b, 8c, and excitation optical fiber groups 1A, 1B, The other end of 1C and one end of each of the fluorescent optical fiber groups 2a, 2b, and 2c are bundled together, and a distal end portion 20 (see FIG. 2), which will be described later, and a capillary 3 (holding portion) that holds the lens 4 integrally. ). The light receivers 8a, 8b, and 8c include cut filters 9a, 9b, and 9c.

  The light sources 7A, 7B, and 7C and the light receivers 8a, 8b, and 8c are electrically connected to the drive / detection circuit 13. When the power supply 11 is turned on, the drive / detection circuit 13 The amount of excitation light emitted from the light sources 7A, 7B, and 7C is controlled, and the outputs of the light sources 7A, 7B, and 7C and the outputs of the light receivers 8a, 8b, and 8c are output to the display 12 (output unit).

  FIG. 2 is a diagram schematically showing a configuration of a tip portion which is a component of the fluorescence detection system in FIG.

  In FIG. 2, the other ends of the excitation light optical fiber groups 1A, 1B, and 1C that are arranged to face the measurement target object 5 and the measurement target objects 5 of the fluorescence optical fiber groups 2a, 2b, and 2c are arranged. Each end arranged opposite to each other constitutes a bundled tip portion 20 that is fitted to the base 22, and the end faces of the bundled optical fibers form a planar end face 21. To do.

  FIG. 3 is a diagram schematically showing the configuration of the end face in FIG.

  In the present embodiment, the excitation-light optical fiber groups 1A, 1B, and 1C are single-core optical fibers (hereinafter referred to as “single-core fibers”), and the fluorescence optical fiber groups 2a, 2b, and 2c are 3 respectively. A core optical fiber (hereinafter referred to as “three-core fiber”). Each of the three cores of the three-core fiber 2a is arranged in a substantially concentric circle centered on the single-core fiber 1A. Similarly, each of the three cores of the three-core fiber 2b has a substantially concentric shape centered on the single-core fiber 1B, and each of the three cores of the three-core fiber 2c has a substantially concentric shape centered on the single-core fiber 1C. Arranged. The single-core fibers 1A, 1B, and 1C are arranged so as not to be adjacent to each other, and among the three-core fibers 1A, 1B, and 1C and the three-core fibers 2a, 2b, and 2c, adjacent optical fibers are in contact with each other. Are arranged as follows.

  4 is a cross-sectional view showing the relationship between the capillary in FIG. 1 and its peripheral structural components.

  The alternate long and short dash line 23 indicates the central axis of the tip 20, and the alternate long and short dash line 24 indicates the central axis of the lens 4. The diameter of the base 22 is the same as the diameter of the lens 4, the distal end portion 20 fitted to the base 22 and the lens 4 are separated from each other, and the central axis 23 of the distal end portion 20 and the central axis 24 of the lens 4 are formed. It is integrally held by the capillary 3 so as to be parallel.

  With the above configuration, the excitation light emitted from the light sources 7A, 7B, and 7C is incident from each end of the single-core fibers 1A, 1B, and 1C and is emitted from the distal end portion 20. Here, since the single-core fibers 1A, 1B, and 1C are arranged so as not to be adjacent to each other, each of the emitted excitation lights is spatially separated into three lenses 4 without being mixed. The object to be measured 5 in the microwell 6 is simultaneously irradiated via the. Then, three fluorescences are generated from the measurement object 5 irradiated with the excitation light that is spatially separated into three, and the single-core fibers 1A, 1B, and 1C are centered at the tip portion 20, respectively. Since the three-core fibers 2a, 2b, and 2c exist, three fluorescent lights can be simultaneously incident on each of the three-core fibers 2a, 2b, and 2c at the distal end portion 20. In order to receive three fluorescent lights, it is only necessary to simultaneously emit three excitation lights from the single-core fibers 1A, 1B, and 1C at the distal end portion 20. For example, it is necessary to switch the optical fiber that emits the excitation light. In addition, there is no need to scan the measurement object 5 with the tip 20. Therefore, according to the fluorescence detection system 10 according to the present embodiment, it is possible to easily and simultaneously detect the three fluorescences generated from the measurement object 5.

  Furthermore, even if there are a plurality of, for example, three objects to be measured, the excitation light emitted from the light sources 7A, 7B, and 7C is incident from one end of each of the single-core fibers 1A, 1B, and 1C, and the tip portion 20 is emitted. Here, since each single-core fiber 1A, 1B, 1C is arrange | positioned so that it may not mutually adjoin, each emitted light is not mixed, but in the state separated into three spatially, it is a lens. The three objects to be measured are simultaneously irradiated via 4. And three fluorescences generate | occur | produce from the three to-be-measured objects irradiated with the excitation light spatially isolate | separated into three, but the single core fibers 1A, 1B, and 1C are respectively attached to the front-end | tip part 20. Since the three-core fibers 2a, 2b, and 2c are present at the center, three fluorescent lights can be simultaneously incident on each of the three-core fibers 2a, 2b, and 2c at the distal end portion 20. In order to receive three fluorescent lights, it is only necessary to simultaneously emit three excitation lights from the single-core fibers 1A, 1B, and 1C at the distal end portion 20. For example, it is necessary to switch the optical fiber that emits the excitation light. In addition, there is no need to scan the measurement object with the tip 20. Therefore, according to the fluorescence detection system 10 according to the present embodiment, it is possible to easily and simultaneously detect the three fluorescences generated from the three objects to be measured.

  In the fluorescence detection system 10 described above, each of the three cores of the three-core fiber 2a is arranged substantially concentrically around the single-core fiber 1A, and each of the three cores of the three-core fiber 2b is centered on the single-core fiber 1B. Since each of the three cores of the three-core fiber 2c is arranged in a substantially concentric circle centered on the single-core fiber 1C, for example, the excitation light emitted from the single-core fiber 1A is the object to be measured. 5 can be effectively incident on the three-core fibers 2a arranged substantially concentrically around the single-core fiber 1A.

  Further, when the adjacent optical fibers are in contact with each other, the fluorescence generated by the excitation light emitted from the single-core fibers 1A, 1B, and 1C being irradiated onto the object to be measured 5 is obtained. The light can be incident on 2a, 2b, and 2c without leakage. In addition, each optical fiber can be efficiently arranged at the distal end portion 20, thereby reducing the distal end portion.

  Further, by arranging the three-core fibers 2a, 2b, and 2c in a substantially arc shape having a central angle of at least 90 degrees or more, each single-core fiber is caused by the aberration of the lens 4 due to the fluorescence passing through the lens 4. Even when the light reaches the tip 20 with unevenness in the distribution of the amount of light around 1A, 1B, and 1C, each fluorescent light can be reliably incident on the three-core fibers 2a, 2b, and 2c.

  In the fluorescence detection system 10, the optical fibers constituting the excitation light optical fiber group and the fluorescence optical fiber group may be single-core or multi-core. Furthermore, the fluorescence detection system 10 includes at least two excitation light optical fiber groups, and the fluorescence optical fiber groups may be arranged so that the excitation light optical fiber groups are not adjacent to each other.

  If the distance between the lens 4 and the tip portion 20 (the distance between the surface facing the tip portion 20 of the lens 4 and the end surface 21) is equal to or less than the back focus distance of the lens 4, the excitation light is diffused light. The measurement object 5 is irradiated through the lens 4 in the state. In this case, the fluorescence generated from the object 5 to be measured is blurred and reaches the tip 20 in a largely diffused state, so that the incident light quantity of the fluorescence of the three-core fibers 2a, 2b, 2c is reduced. End up. Therefore, the distance between the lens 4 and the tip portion 20 is set larger than the back focus distance of the lens 4. Thereby, excitation light can be emitted from the lens 4 as substantially parallel light or convergent light.

  Further, as the distance between the lens 4 and the tip portion 20 increases, the transmittance of the excitation light of the lens decreases and the amount of excitation light reaching the measurement object 5 decreases, and the amount of generated fluorescence light Therefore, effective fluorescence measurement cannot be performed. In addition, if the transmittance of the excitation light of the lens is low, that is, if the excitation light is scattered by the lens, the shape of the irradiation spot on the surface of the object to be measured is distorted. Overlapping causes each excitation light to mix. Here, the irradiation spot refers to an irradiation spot on the measurement target object 5 of the excitation light emitted from each of the single-core fibers 1A, 1B, and 1C and irradiated on the measurement target object 5 through the lens 4. is there.

  Therefore, in order to prevent a decrease in sensitivity due to a decrease in the amount of excitation light incident on the object to be measured 5 and a decrease in the separation ability of the excitation light, the distance between the lens 4 and the tip portion 20 is determined by the distance of the excitation light. The distance is set such that the transmittance of the lens is 70% or more.

  Further, the detection sensitivity of the fluorescence detection system 10 can be increased by adjusting the distance between the lens 4 and the object 5 to be measured (hereinafter referred to as “WD (Working Distance)”). This will be described in detail below.

  As a first factor for adjusting the WD, there is “spatial separation of irradiation spots”. As described above, the irradiation spot is an irradiation spot on the measurement target object 5 of the excitation light emitted from each of the single-core fibers 1A, 1B, and 1C and irradiated on the measurement target object 5 through the lens 4. That is.

In the fluorescence detection system 10, the position where the irradiation light amount of the excitation light is the largest in each irradiation spot on the surface of the measurement object 5 is the center of the irradiation spot, and the irradiation light amount of the excitation light is 1 / e ² of the irradiation light amount at the center. When the distance between the position and the center is the radius of the irradiation spot, by adjusting the “irradiation spot diameter” to be smaller than the “distance between the centers of adjacent irradiation spots”, For example, the emission position of the fluorescence generated when the measurement object 5 is irradiated with the excitation light emitted from 1A and the emission of the fluorescence generated when the measurement object 5 is irradiated with the excitation light emitted from 1B. The position can be prevented from overlapping. As a result, it is possible to prevent the generated fluorescence from being caused by two or more excitation lights, so that a fluorescence detection system with low noise and high detection sensitivity is obtained.

  As a second factor for adjusting the WD, there is an “S / N ratio”. The S / N ratio is, for example, “generated by the excitation light emitted from the single-core fiber 1A” with respect to “the amount of light generated by the excitation light emitted from each of the single-core fibers 1B and 1C and incident on the three-core fiber 2a”. It is a ratio to “the amount of light incident on the three-core fiber 2a”.

  That is, for example, the smaller the amount of fluorescence caused by the excitation light from the single-core fibers 1B and 1C different from the single-core fiber 1A located at the center of each three-core fiber 2a, that is, the fluorescence Can be said to be a system with lower noise and higher detection sensitivity, as the S / N ratio in each light receiver of the fluorescence detection system 10 is higher.

  In the fluorescence detection system 10, the S / N ratio is adjusted to 20 dB or more, more preferably 30 dB or more. Thereby, for example, when the S / N ratio is 20 dB or more, it is possible to separate the same degree of fluorescence as the fluorescence analysis system provided with the dichroic mirror. In this case, the fluorescence detection system 10 has high detection sensitivity, and does not require a large apparatus or expensive parts. Therefore, the fluorescence detection system can be downsized and manufactured at a lower cost than the conventional fluorescence detection system. Reduction is possible.

  Further, WD is set to a distance at which the excitation light reflected by the measurement object 5 is not coupled to the end face 21. Thereby, noise caused by excitation light can be reduced.

  A lens can be used regardless of the material and shape, such as a half drum lens, a rod lens, a plano-convex lens, a biconvex lens, an achromatic lens, and an aspheric lens, for example. Particularly, since the half drum lens and the rod lens have a thick edge, it is easy to integrate with the tip portion 20 by the capillary 3, and further downsizing of the fluorescence detection system 10 and reduction in manufacturing cost can be achieved.

  Further, since the half drum lens has small chromatic aberration, it is possible to suppress variations in the focal length of each excitation light when the measurement object 5 is irradiated with excitation light having different wavelengths. Therefore, the half drum lens is suitable for irradiating the measurement object 5 with a plurality of excitation lights having different wavelengths.

  On the other hand, since the coma aberration of the rod lens is small, the diameter of the irradiation spot of the excitation light on the measurement target object 5 is small, the irradiation spot is not blurred, and fluorescence generated from a plurality of locations on the measurement target object 5 is generated. Do not mix. Therefore, it is suitable for irradiating a plurality of positions of the object to be measured or a plurality of objects to be measured with excitation light having the same wavelength.

  Further, in the present embodiment, the tip portion 20 is bundled with the base 22, but if the diameter of the tip portion 20 when bundled is the same as the diameter of the lens 4, the base 22 is not provided. The bundle may be held directly by the capillary 3. Further, the central axis 23 of the distal end portion 20 and the central axis 24 of the lens 4 may not be parallel.

  In addition, although the cut filters 9a, 9b, and 9c are provided in the respective light receivers 8a, 8b, and 8c, the present fluorescence detection system is sufficiently sensitive, so that the cut filters need not be used.

  In the fluorescence detection system 10 described above, the object 5 to be measured may be put in a microtube as shown in FIG. 5A, or may be put in a flow path on a slide glass as shown in FIG. Alternatively, it may be placed in a cell as shown in FIG.

  In the fluorescence detection system 10 described above, each excitation light optical fiber group and each fluorescence optical fiber group are adjacent to each other, but the measurement target is irradiated with the excitation light more spatially separated. In this case, as shown in FIG. 6, an optical fiber 40 that does not receive fluorescence may be arranged at the boundary between fluorescence optical fibers having different wavelengths of fluorescence to be measured.

  Next, examples of the present invention will be specifically described.

Example 1
In the fluorescence detection system 10, the excitation light optical fiber groups 1A, 1B, 1C and the fluorescence optical fiber groups 2a, 2b, 2c have a core diameter of 200 μm, a cladding diameter of 250 μm, and a numerical aperture of 0.22. Step index fibers are used, the excitation light optical fiber groups 1A, 1B, and 1C are each single-core fibers, and the fluorescence optical fiber groups 2a, 2b, and 2c are three-core fibers, respectively, so that the arrangement shown in FIG. Designed.

  The lens 4 is a half drum lens made of BK7 having a diameter of 2.4 mm, a length of 2.4 mm, a radius of curvature of 1.5 mm, and an edge thickness of 1.5 mm in order to reduce excitation light and fluorescence reflection. An antireflection film was applied to the surface of the lens 4. The tip portion 20 was fitted with a base having a diameter of 2.4 mm, and the tip portion 20 and the lens 4 were bonded and fixed by a cylindrical capillary 3.

  LED light sources (manufactured by Nichia Corporation) are used as the light sources 7A, 7B, and 7C, and the light sources 7A, 7B, and 7C have bands having transmission wavelength ranges of 470 nm ± 10 nm, 532 nm ± 10 nm, and 632 nm ± 10 nm, respectively. A pass filter was provided.

  Further, photomultiplier tubes (manufactured by Hamamatsu Photonics) are used for the light receivers 8a, 8b, and 8c, and further, the light receivers 8a, 8b, and 8c are respectively provided with a transmission wavelength region corresponding to the wavelength of fluorescence to be detected. Are provided with bandpass filters of 532 nm ± 5 nm, 580 nm ± 5 nm, and 680 nm ± 5 nm.

Study 1
First, the optimum distance between the lens 4 and the tip 20 was examined.

  While changing the distance between the lens 4 and the tip 20, excitation light having a main wavelength of 470 nm was irradiated from the single-core fiber 1 </ b> A, and the transmitted light amount of the excitation light of the lens 4 was measured. Similarly, the excitation light having the main wavelength of 632 nm was irradiated from the single-core fiber 1B, and the transmitted light amount of the excitation light of the lens 4 was measured. The transmittance of the excitation light was calculated from each measurement result, and the relationship between the excitation light transmittance and the distance between the lens 4 and the tip portion 20 is shown in FIG.

  In FIG. 7, the vertical axis represents the excitation light transmittance (%) of the lens 4, and the horizontal axis represents the distance (mm) between the lens 4 and the tip portion 20. The solid line in the graph indicates the change in the transmittance of the excitation light having the main wavelength of 470 nm, and the dotted line in the graph indicates the change in the transmittance of the excitation light having the main wavelength of 632 nm. It has been found that the transmittance of both the excitation lights is higher as the distance between the lens 4 and the tip 20 is smaller, and the transmittance is 70% or more when the distance is 4.5 mm or less.

  Therefore, in consideration of the result and the back focus of the half drum lens used in the present embodiment is about 1 mm, the distance between the lens 4 and the tip portion 2 is designed to be 2 mm. 4 was done.

Study 2
Next, “irradiation spot” which is a first factor for adjusting WD was examined.

  In the fluorescence detection system 10, while changing the WD, the excitation light having the main wavelength of 470 nm is irradiated from the single-core fiber 1 </ b> A, and the excitation light having the main wavelength of 532 nm is irradiated from the single-core fiber 1 </ b> B. The diameter of each irradiation spot and the distance between the centers of each irradiation spot were measured. When the excitation light is irradiated from the single-core fibers 1A and 1C and the excitation light is irradiated from the single-core fibers 1B and 1C, the same measurement is performed, and the relationship between the diameter of the irradiation spot and WD, between the centers of the irradiation spots 8A to 8C show the relationship between the distance and WD.

  8A to 8C, the vertical axis represents the diameter of the irradiation spot and the distance (mm) between the centers of the irradiation spots, and the horizontal axis represents WD (mm).

  FIG. 8A shows the result of irradiating excitation light of 470 nm and 532 nm from the single-core fibers 1A and 1B, respectively, and the solid line in the graph indicates the irradiation of the measurement object 5 when the excitation light having a wavelength of 470 nm is irradiated. The change in the diameter of the spot, the dotted line in the graph indicates the change in the diameter of the irradiation spot on the object to be measured 5 when the excitation light having the wavelength of 532 nm is irradiated, and the alternate long and short dash line in the graph indicates the distance between the centers of the irradiation spots It shows the change in distance.

  FIG. 8B shows the result of irradiating the single-core fibers 1B and 1C with excitation light of 532 nm and 632 nm, respectively, and the solid line in the graph indicates the irradiation of the measurement object 5 when the excitation light with the wavelength of 532 nm is irradiated. The change in the diameter of the spot, the dotted line in the graph indicates the change in the diameter of the irradiation spot on the object to be measured 5 when the excitation light having a wavelength of 632 nm is irradiated, and the alternate long and short dash line in the graph indicates the distance between the centers of the irradiation spots It shows the change in distance.

  FIG. 8C shows the result of irradiating excitation light of 632 nm and 470 nm from the single-core fibers 1C and 1A, respectively, and the solid line in the graph indicates the irradiation of the object 5 to be measured when the excitation light having a wavelength of 632 nm is irradiated. The change in the diameter of the spot, the dotted line in the graph indicates the change in the diameter of the irradiation spot on the object to be measured 5 when the excitation light having a wavelength of 470 nm is irradiated, and the alternate long and short dash line in the graph indicates the distance between the centers of the irradiation spots. It shows the change in distance.

  8A to 8C show that when the WD is 5 mm or more, the distance between the centers of the spots is larger than the diameter of each irradiation spot.

Study 3
Next, the “S / N ratio” which is the second factor for adjusting the WD was examined.

  Fluorescein-4-isothiocyanate (hereinafter referred to as “FITC”), Cy3 (manufactured by GE Healthcare) and Cy5 (manufactured by GE Healthcare) are mixed as the object 5 to be measured, and each concentration is 10 μM. An aqueous solution prepared to 10 μM and 10 μM was used, and the aqueous solution was added to a depth of 4 mm in a microwell having a diameter of 3.5 mm and a depth of 8 mm.

  FITC absorbs excitation light having a dominant wavelength of 470 nm and emits fluorescence having a dominant wavelength of 532 nm, Cy3 absorbs excitation light having a dominant wavelength of 532 nm and emits fluorescence having a dominant wavelength of 580 nm, and Cy5 emits fluorescence having a dominant wavelength of 632 nm. It is a fluorescent material having the characteristic of absorbing the excitation light and emitting fluorescence with a dominant wavelength of 680 nm.

  In the fluorescence detection system 10, the WD is adjusted to 5.7 mm, the excitation light having the main wavelength of 470 nm is transmitted from the single core fiber 1A, the excitation light having the main wavelength of 532 nm is output from the single core fiber 1B, and the main wavelength is 632 nm from the single core fiber 1C. Excitation light was respectively emitted and irradiated on the object 5 to be measured, the amount of received light of each fluorescence generated from the object 5 to be measured was measured, and the SN ratio was calculated and shown in Table 1 below. Similarly, the WD was adjusted to 6.5 mm, each excitation light was emitted from the single-core fibers 1A, 1B, and 1C, the amount of received light of each fluorescence was measured, and the SN ratio was calculated and shown in Table 2 below.

  In Tables 1 and 2, O (au) represents the amount of light received by each of the light receivers 8a, 8b, and 8c when the object to be measured 5 is simultaneously irradiated with excitation light having wavelengths of 470 nm, 532 nm, and 632 nm. Output is shown. On (au) is the amount of light received by each of the light receivers 8a, 8b, and 8c when each of the light receivers does not emit the excitation light having the wavelength to be received, and the other two wavelengths of the excitation light are emitted. Shows the output. For example, in the case of the “light receiving device 8a”, the output of the light receiving amount of the light receiving device 8a when the excitation light with the wavelength of 470 nm is not emitted and the excitation light with the wavelengths of 532 nm and 632 nm is emitted is shown. Further, Os (au) is obtained by subtracting the On (au) value from the O (au) value, that is, only the excitation light having a wavelength that should be received by each light receiver. , And the output of the received light amount of each of the light receivers 8a, 8b, and 8c when the excitation light of the other two wavelengths is not emitted.

  In Table 1, an S / N ratio of about 20 dB was obtained for any fluorescence. Furthermore, in Table 2, an S / N ratio of about 30 dB was obtained for any fluorescence.

Study 4
Next, the “S / N ratio” which is the second factor for adjusting the WD was examined in more detail.

  In the fluorescence detection system 10, unlike Study 3, each excitation light is emitted while changing the WD, the amount of received light of each fluorescence generated from the measurement object 5 is measured, and the SN ratio is calculated. This is shown in FIGS. 9A to 9C. Other configurations were the same as those in Study 3.

  9A to 9C, the vertical axis represents the output (au) of the amount of received light of each fluorescence, and the horizontal axis represents WD (mm).

  FIG. 9A shows the relationship between the amount of light received by the light receiver 8a and WD, and the solid lines in the graph indicate the 532 nm received by the light receiver 8a when each excitation light is emitted from the single-core fibers 1A, 1B, and 1C. The change in the output of the fluorescence with the wavelength, and the dotted line in the graph shows the change in the output of the fluorescence with the wavelength of 532 nm received by the light receiver 8a when each excitation light is emitted from the single-core fibers 1B and 1C. A thick solid line indicates a change in the SN ratio of the wavelength of 532 nm of the fluorescence received by the light receiver 8a.

  FIG. 9B shows the relationship between the fluorescence received by the light receiver 8b and WD, and the solid lines in the graph indicate 580 nm received by the light receiver 8b when each excitation light is emitted from the single-core fibers 1A, 1B, 1C. The dotted line in the graph shows the change in the output of the fluorescent light having the wavelength of 580 nm and the change in the output of the fluorescent light having the wavelength of 580 nm received by the light receiver 8b when the excitation light is emitted from the single-core fibers 1A and 1C. The thick solid line indicates the change in the S / N ratio of the fluorescence having a wavelength of 580 nm received by the light receiver 8b.

  FIG. 9C shows the relationship between the fluorescence received by the light receiver 8c and WD, and the solid lines in the graph indicate 680 nm received by the light receiver 8c when each excitation light is emitted from the single-core fibers 1A, 1B, 1C. The dotted line in the graph shows the change in the output of the fluorescent light having the wavelength of 680 nm and the change in the output of the fluorescent light having the wavelength of 680 nm received by the light receiving device 8c when each excitation light is emitted from the single-core fibers 1A and 1B. The thick solid line indicates the change in the SN ratio of the fluorescence having a wavelength of 680 nm received by the light receiver 8a. From FIGS. 7A to 7C, it was found that an S / N ratio of 20 dB or more can be obtained with any fluorescence when the WD is 6 mm or more.

  FIG. 10 shows a graph in which each solid line and each thick solid line in FIGS. 9A to 9C are extracted and superimposed.

  The change in the output of each fluorescence drawn almost the same curve, and the change in each S / N ratio also drawn the same curve. This is because the chromatic aberration of the half drum lens is small, so that it is possible to suppress variations in the focal length of each excitation light when the object to be measured is irradiated with excitation light having different wavelengths. From FIG. 10, it was found that the half drum lens is suitable for irradiating the object to be measured with a plurality of excitation lights having different wavelengths.

Example 2
Next, in the fluorescence detection system 10, unlike Example 1, the FITC, Cy3 and Cy5 are not mixed as the object to be measured 5, so that the concentrations of FITC are 10 μM, Cy3 is 10 μM, and Cy5 is 10 μM. As shown in FIG. 11, the FITC aqueous solution, the Cy3 aqueous solution, and the Cy5 aqueous solution were dropped onto the slide glass so as to be at the positions of 31a, 31b, and 31c, respectively. The positions 31a, 31b, and 31c are respectively arranged at the positions that are the apexes of the triangle, and are adjusted so that the excitation light of 470 nm is irradiated to 31a, the excitation light of 532 nm is irradiated to 31b, and the excitation light of 632 nm is irradiated to 31c. did. In addition, the WD was adjusted to 6 mm, and the other configurations were the same as those of the fluorescence detection system 10 of Example 1.

  Then, in the same manner as in Study 3 of Example 1, excitation light with a main wavelength of 470 nm is emitted from the single-core fiber 1A, excitation light with a main wavelength of 532 nm is emitted from the single-core fiber 1B, and excitation light with a main wavelength of 632 nm is emitted from the single-core fiber 1C. Each emitted light was irradiated to the objects to be measured 31a, 31b, 31c, the amount of received light of each fluorescence generated from each object to be measured was measured, and the SN ratio was calculated and shown in Table 3 below. Similarly, the WD was adjusted to 7 mm, each excitation light was emitted from the single-core fibers 1A, 1B, and 1C, the amount of received fluorescence was measured, and the SN ratio was calculated and shown in Table 4 below.

  In Table 3, a signal-to-noise ratio of about 20 dB was obtained for any fluorescence. Furthermore, in Table 4, an S / N ratio of about 30 dB was obtained for any fluorescence. From Table 3 and Table 4, it was found that an S / N ratio of 20 dB or more can be obtained with any fluorescence when the WD is 6 mm or more.

Example 3
In the fluorescence detection system 10, unlike the second embodiment, a GRIN lens (manufactured by Nippon Sheet Glass Co., Ltd.) having a diameter of 1.8 mm and a length of 4.63 mm is used as the lens 4 to reduce reflection of excitation light and fluorescence. Further, an antireflection film was applied to the surface of the lens 4. The tip portion 20 was fitted with a base having a diameter of 1.8 mm, and the tip portion 20 and the lens 4 were bonded and fixed by a cylindrical capillary 3. The distance between the lens 4 and the end face 20 was designed to be 2 mm.

  As the light sources 7A, 7B, and 7C, LED light sources (manufactured by Nichia Corporation) were used, and band pass filters having a transmission wavelength range of 470 nm ± 10 nm were further provided. Further, photomultiplier tubes (manufactured by Hamamatsu Photonics) are used for the light receivers 8a, 8b, and 8c, and further, the light receivers 8a, 8b, and 8c are respectively provided with a transmission wavelength region corresponding to the wavelength of fluorescence to be detected. Provided a band-pass filter of 532 nm ± 5 nm.

  As the object 5 to be measured, an aqueous solution prepared so that FITC had a concentration of 10 μM was used, and as shown in FIG. 11, the FITC aqueous solution was dropped on the slide glass so as to be at positions 31a, 31b, and 31c. Further, the WD was adjusted to 1.73 mm, and the other configurations were the same as those of the fluorescence detection system 10 of Example 2.

  As in the second embodiment, excitation light is emitted from each of the single-core fibers 1A, 1B, and 1C and irradiated to 31a, 31b, and 31c, and each fluorescence generated from the measurement target objects 31a, 31b, and 31c. The amount of received light was measured, and the SN ratio was calculated and shown in Table 5 below. Similarly, the WD is adjusted to 1.35 mm, each excitation light is emitted from each of the single-core fibers 1A, 1B, and 1C, the amount of received light of each fluorescence is measured, and the SN ratio is calculated and shown in Table 6 below. It was.

  Tables 5 and 6 show the amount of light received by the light receiver 8a. The meanings of O (au), On (au), and Os (au) are the same as those in Tables 3 and 4.

  In Table 5, an S / N ratio of about 20 dB was obtained. Similar results were obtained with the light receivers 8b and 8c. Furthermore, in Table 6, fluorescence having a wavelength to be received with an S / N ratio of 30 dB or more could be received.

Example 4
Next, in the fluorescence detection system 10, unlike Example 3, the light sources 7A, 7B, and 7C were provided with bandpass filters having transmission wavelength ranges of 470 nm ± 10 nm, 532 nm ± 10 nm, and 632 nm ± 10 nm, respectively.

  The light receivers 8a, 8b, and 8c correspond to the wavelengths of fluorescence to be detected, and the light receivers 8a, 8b, and 8c have transmission wavelength ranges of 532 nm ± 5 nm, 580 nm ± 5 nm, and 680 nm ± 5 nm, respectively. A bandpass filter was provided. Other configurations were the same as those of the fluorescence detection system of Example 3. Then, while changing the WD, each excitation light was emitted to measure the amount of received light of each fluorescence generated from the measured objects 31a, 31b, 31c, and the SN ratio was calculated and shown in FIG.

  In FIG. 12, the vertical axis represents the output (au) of the amount of received light of each fluorescence, and the horizontal axis represents WD (mm). Solid lines in the figure indicate changes in the output of fluorescence having a wavelength of 532 nm received by the light receiver 8a when each excitation light is emitted from the single-core fibers 1A, 1B, and 1C. A change in the output of fluorescence having a wavelength of 580 nm received by the light receiver 8b when each excitation light is emitted from 1B and 1C. A dotted line in the graph indicates when each excitation light is emitted from the single-core fibers 1A, 1B and 1C. A change in the output of fluorescence having a wavelength of 680 nm received by the light receiver 8c is shown. The solid line plotted with a black triangle in the graph indicates the change in the S / N ratio of the light receiver 8a, and the solid line plotted with the cross in the graph indicates the change in the S / N ratio of the light receiver 8b with a white circle in the graph. Indicates a change in the SN ratio of the light receiver 8c.

  From FIG. 12, it was found that at any wavelength, an S / N ratio of 20 dB or more can be obtained when WD is 1.2 mm or more and 1.4 mm or less.

It is a figure showing roughly the composition of the fluorescence detection system concerning an embodiment of the invention. It is a figure which shows schematically the structure of the front-end | tip part in FIG. It is a figure which shows schematically the structure of the end surface in FIG. It is sectional drawing which shows the relationship between the capillary in FIG. 1, and its peripheral structure component. It is a figure which shows the modification of the to-be-measured object in this invention. It is a figure which shows the modification of arrangement | positioning of the optical fiber in this invention. It is a graph which shows the relationship between excitation light transmittance and the distance between a lens and a front-end | tip part. Relationship between the distance between the lens and the object to be measured and the diameter of the irradiation spot of the excitation light, and relationship between the distance between the lens and the object to be measured and the distance between the center of the irradiation spot of the excitation light FIG. 8A shows a case where excitation light of 470 nm and 532 nm is irradiated, FIG. 8B shows a case where excitation light of 532 nm and 632 nm is irradiated, and FIG. This is a case where excitation light of 632 nm and 470 nm is irradiated. FIG. 9A is a graph showing fluorescence output and fluorescence S / N ratio when WD is changed in the case of using a half drum lens, and FIG. 9A shows the relationship between the amount of received light of fluorescence having a wavelength of 532 nm and WD. FIG. 9B shows a relationship between the received light amount of fluorescence having a wavelength of 580 nm and WD, and FIG. 9C shows the relationship between the received light amount of fluorescence having a wavelength of 680 nm and WD. This is the case. 10 is a graph in which each solid line and each thick solid line in FIGS. 9A to 9C are extracted and superimposed. FIG. 6 is a diagram schematically showing the arrangement of objects to be measured in Example 2. It is a graph which shows the output of fluorescence when the WD is changed, and the SN ratio of fluorescence when a rod lens is used.

Explanation of symbols

1A, 1B, 1C Excitation optical fiber group 2a, 2b, 2c Fluorescence optical fiber group 3 Capillary 4 Lens 5 Object to be measured 6 Microwell 7A, 7B, 7C Light source 8a, 8b, 8c Light receiver 9a, 9b, 9c Cut filter 20 Tip

Claims (8)

In a fluorescence detection system comprising: a light source that emits excitation light; and a light receiving unit that receives fluorescence generated from the measurement target irradiated with the excitation light.
For excitation light comprising at least one optical fiber from which the excitation light is incident from one end optically connected to the light source and the excitation light is emitted from the other end arranged opposite to the object to be measured A plurality of optical fiber groups;
An optical fiber for fluorescence comprising at least one optical fiber from which the fluorescence is incident from one end arranged to face the object to be measured and from which the other end optically connected to the light receiving unit is emitted A plurality of groups;
A tip portion in which the other end of the plurality of optical fibers for excitation light and one end of the plurality of optical fibers for fluorescence are bundled;
A lens disposed between the tip and the object to be measured;
A holding portion that separates and holds the tip portion and the lens integrally;
Each of the plurality of optical fibers for excitation light is arranged so as not to be adjacent to each other at the tip portion.   At the tip, the plurality of optical fibers constituting each of the fluorescence optical fiber groups are arranged in a substantially concentric circle centered on each of the excitation light optical fiber groups, and the plurality of fluorescence optical fiber groups are 2. The fluorescence detection system according to claim 1, wherein each of the different excitation light optical fiber groups is centered.   The adjacent optical fibers are in contact with each other among the optical fibers constituting the plurality of fluorescent optical fiber groups and the plurality of excitation light optical fiber groups at the tip. 2. The fluorescence detection system according to 2.   The optical fiber constituting each of the fluorescent optical fiber groups arranged in the substantially concentric circles is arranged in a substantially arc shape having a central angle of at least 90 degrees or more. Fluorescence detection system.   The first distance between the lens and the tip is larger than the back focus distance of the lens, and is a distance at which the transmittance of the excitation light is 70% or more. The fluorescence detection system according to any one of claims 1 to 4.   The second distance between the lens and the object to be measured is such that the diameter of the irradiation spot of the excitation light on the surface of the object to be measured is smaller than the distance between the centers of the adjacent irradiation spots. The fluorescence detection system according to claim 1, wherein the fluorescence detection system is a distance.   The second distance between the lens and the object to be measured is for the excitation light that does not correspond to the center of the fluorescent optical fiber group with respect to the amount of the fluorescent light incident on one fluorescent optical fiber group. At a distance where the ratio of the amount of fluorescence caused by the excitation light emitted from the excitation light optical fiber group corresponding to the center to the amount of fluorescence caused by the excitation light emitted from the optical fiber group is 20 dB or more. The fluorescence detection system according to claim 2, wherein:   Three excitation light optical fiber groups and three fluorescence optical fiber groups are provided. Each of the excitation light optical fiber groups includes one optical fiber, and each of the fluorescence optical fiber groups includes three light beams. The fluorescence detection system according to any one of claims 1 to 7, wherein the fluorescence detection system is made of a fiber, and the lens is a half drum lens.

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