JP6448933B2 - Optical device for fluorescence detection - Google Patents

Description

  The present invention relates to an optical device for fluorescence detection that irradiates an object to be detected with excitation light and detects fluorescence generated by the object to be detected.

  In recent years, in the field of life science, optical devices for fluorescence detection have begun to be used frequently. Since the optical apparatus for fluorescence detection is simple and has high detection sensitivity, it is effective in quantitative detection of nucleic acids such as DNA labeled with a chemical substance that emits fluorescence, sometimes in combination with an amplification process.

  For example, the light detection device disclosed in Patent Document 1 is guided by a laser beam guided by a first optical waveguide, condensed by a first lens, and irradiated on a specimen from substantially obliquely above, and excited by the irradiated light. And a light receiving optical system that condenses the fluorescence from the sample by the second lens and guides the fluorescence to the measuring device through the second optical waveguide. In this photodetection device, the irradiation optical system and the light receiving optical system are separate light guide paths.

International Publication No. 2007/091530

  However, the fluorescence detection optical apparatus in which the irradiation optical system and the light receiving optical system are separated as in Patent Document 1 has the following problems.

  FIG. 1 shows an example of a conventional fluorescence detection optical device. An optical device for fluorescence detection 100 shown in FIG. 1 condenses the irradiation optical system 102 for irradiating the sample S in the container C with the excitation light EL and the fluorescence FL generated from the sample S on the fluorescence condensing point 103. And a light receiving optical system 104. The irradiation optical system 102 includes an excitation light source 106 and an excitation light lens 108 that condenses the excitation light EL on the sample S. The light receiving optical system 104 includes an objective lens 110 and condenses the fluorescent light FL emitted from the fluorescent light emitting point 101 on the sample S on a fluorescent light condensing point 103 conjugate with the fluorescent light emitting point 101. As shown in FIG. 1, the optical axis 112 of the irradiation optical system 102 is inclined with respect to the optical axis 114 of the light receiving optical system 104 (the optical axis 112 of the excitation light EL and the optical axis 114 of the light receiving optical system 104). Is the angle formed by θi).

  In the fluorescence detection optical device 100 configured as shown in FIG. 1, in order to improve detection sensitivity, it is necessary to capture more fluorescence FL into the light receiving optical system 104. For this purpose, it is necessary to increase the numerical aperture (NA) of the light receiving optical system 104, but the optical device 100 for fluorescence detection has a limit. In order to increase the NA of the light receiving optical system 104, the objective lens 110 may be enlarged. However, if the objective lens 110 is made too large, the excitation light EL irradiated at an angle θi and the objective lens 110 interfere with each other. Because.

  Further, when an attempt is made to avoid interference between the excitation light EL and the objective lens 110 while ensuring a sufficient NA of the light receiving optical system 104, the excitation light source is positioned away from the sample S or at a position where the angle θi becomes large. 106 and the excitation light lens 108 must be arranged. In this case, it is difficult to reduce the size of the fluorescence detection optical device 100.

  Further, particularly when the container C is deep as shown in FIG. 1, the excitation light EL may be blocked by the side wall of the container C if the angle θi is large. In this case, the sample S located at the bottom of the container C cannot be irradiated with sufficient excitation light EL, and as a result, there is a possibility that fluorescence cannot be detected from the sample S.

  The present invention has been made in view of such circumstances, and an object thereof is to provide a small fluorescence detection optical device with high detection sensitivity.

  In order to solve the above problems, an optical device for fluorescence detection according to an aspect of the present invention irradiates an object to be detected with excitation light, and detects fluorescence generated from the object to be detected by the irradiation. The optical device for fluorescence detection includes a first lens group that directly receives fluorescence generated from an object to be detected, and a second lens group that is disposed coaxially with the first lens group and receives fluorescence that has passed through the first lens group. And a second lens group in which a part of the off-axis is cut off to form a gap, and an excitation light optical system that emits excitation light, and the emitted excitation light is off-axis of the first lens group. And an excitation light optical system arranged to irradiate the object to be detected. In this fluorescence detection optical device, a part of the excitation light optical system is disposed in the gap portion of the second lens group.

  The first lens group may convert the fluorescence generated from the object to be detected into parallel light. The second lens group may condense fluorescent parallel light from the first lens group.

  The gap portion may be formed at the peripheral edge of the second lens group.

  The pumping light optical system may include a pumping light source that emits pumping light, and a pumping light lens group that makes the pumping light from the pumping light source parallel light and incident off the axis of the first lens group.

  The excitation light source may include a light emitting element that emits excitation light.

  The excitation light source may include an emission end face of an excitation light optical fiber that guides excitation light.

  The excitation light source may include a light emitting element provided in the image side space of the second lens group. The excitation light lens group may include a relay lens system arranged to form an intermediate image of the light emitting region of the light emitting element in the gap portion.

  A plurality of excitation light optical systems may be provided. A plurality of gap portions may be formed in the second lens group, and a part of the excitation light optical system may be disposed in each of the plurality of gap portions.

  According to the present invention, it is possible to provide a small fluorescence detection optical device with high detection sensitivity.

It is a figure which shows an example of the conventional optical apparatus for fluorescence detection. It is a figure for demonstrating the optical device for fluorescence detection which concerns on this embodiment. FIGS. 3A to 3C are diagrams for explaining the shape and size of the gap portion formed in the second lens group. It is a figure for demonstrating the optical apparatus for fluorescence detection which concerns on 1st Example. It is a figure for demonstrating the optical apparatus for fluorescence detection which concerns on 2nd Example. It is a figure for demonstrating the optical apparatus for fluorescence detection which concerns on 3rd Example. It is a figure for demonstrating the optical apparatus for fluorescence detection which concerns on another embodiment of this invention. FIG. 8 is a plan view of the second lens group viewed from the optical axis direction in the fluorescence detection optical device shown in FIG. 7.

  Hereinafter, an optical device for fluorescence detection according to an embodiment of the present invention will be described. The same or equivalent components, members, and processes shown in the drawings are denoted by the same reference numerals, and repeated descriptions are omitted as appropriate. The embodiments do not limit the invention but are exemplifications, and all features and combinations thereof described in the embodiments are not necessarily essential to the invention.

  FIG. 2 is a diagram for explaining the fluorescence detection optical device 10 according to the present embodiment. The optical device for fluorescence detection 10 irradiates a sample S as an object to be detected with excitation light and detects fluorescence generated from the sample S by the irradiation. The sample S is placed in the container C.

  As shown in FIG. 2, the fluorescence detection optical apparatus 10 includes an irradiation optical system 12 for irradiating the sample S in the container C with the excitation light EL, and a light reception for condensing the fluorescence FL generated from the sample S. And an optical system 14. The fluorescence detection optical device 10 may include a holding member (not shown) for integrally holding the irradiation optical system 12 and the light receiving optical system 14. The fluorescence detecting optical device 10 in which the irradiation optical system 12 and the light receiving optical system 14 are integrated as described above is also referred to as an “optical head”.

  The light receiving optical system 14 condenses the fluorescent light FL emitted from the fluorescent light emission point 11 on the sample S on a fluorescent light condensing point 13 conjugate with the fluorescent light emission point 11. The light receiving optical system 14 directly receives the fluorescence FL generated from the fluorescence emission point 11 on the sample S and converts it into parallel light, and the parallel light of the fluorescence FL that has passed through the first lens group 20. The second lens group 22 is configured to receive light and condense on the fluorescence condensing point 13. In this specification, the “lens group” represents one or more lenses. In the specification of the present application, in the light receiving optical system 14, a space where the sample S is located is an object point side space, a space where an image of the sample S is formed by the light receiving optical system 14 is an image side space, and Is designated by the terms “object point side” or “image side”. In this case, the fluorescence emission point 11 substantially coincides with the object point side focal point of the first lens group 20, and the fluorescence condensing point 13 substantially coincides with the image side focal point of the second lens group 22.

  By configuring the light receiving optical system 14 using the first lens group 20 and the second lens group 22 in this manner, the conjugate length can be easily adjusted, and the lens barrel length of the optical head can be easily set. Such a configuration is also advantageous in reducing aberrations. The second lens group 22 is preferably arranged coaxially with the first lens group 20. In this case, the aberration due to the lens system can be reduced and the light collection efficiency to the fluorescent light collection point 13 can be improved. The first lens group 20 and the second lens group 22 need not have the same specifications.

  The irradiation optical system 12 includes an excitation light optical system 24 that emits the excitation light EL, an off-axis portion 20a of the first lens group 20 that refracts the excitation light EL from the excitation light optical system 24 and collects the light on the sample S. Consists of The excitation light optical system 24 is disposed on the image side from the first lens group 20. The excitation light optical system 24 is arranged so that the emitted excitation light EL is condensed on the fluorescence emission point 11 of the sample S through the off-axis portion 20 a of the first lens group 20. The optical axis 19 of the excitation light EL incident on the fluorescence emission point 11 is inclined with respect to the optical axis 17 of the first lens group 20 (the optical axis 19 of the excitation light EL and the optical axis 17 of the light receiving optical system 14). The angle between the optical axis 19 and the optical axis 19 is θi).

  The excitation light optical system 24 emits the excitation light EL, and the excitation light lens group 25 that makes the excitation light EL from the excitation light source 16 parallel light and enters the off-axis portion 20a of the first lens group 20. It consists of. The excitation light lens group 25 is arranged so that its optical axis 21 is parallel to the optical axis 17 of the first lens group 20. Thereby, the excitation light EL that has passed through the off-axis portion 20a of the first lens group 20 can be condensed on the fluorescence emission point 11 (that is, the object point side focal point of the first lens group 20).

  In the optical device 10 for fluorescence detection according to the present embodiment, a part of the second lens group 22 outside the axis is cut off to form a gap portion 30. An excitation light source 16 that is a part of the excitation light optical system 24 is disposed in the gap portion 30. The excitation light lens group 25 of the excitation light optical system 24 is disposed between the first lens group 20 and the second lens group 22.

  In the fluorescence detection optical device 10 configured as described above, the excitation light EL emitted from the excitation light source 16 is converted into parallel light by the excitation light lens group 25 and then the off-axis portion of the first lens group 20. The light passes through 20a and enters the fluorescence emission point 11 of the sample S at an angle θi. The fluorescence FL generated at the fluorescence emission point 11 of the sample S is taken into the first lens group 20 to be converted into parallel light, and then collected by the second lens group 22 at the fluorescence collection point 13. By placing a light receiving surface of a light receiving element such as a PD (Photodiode) at the fluorescence condensing point 13, the fluorescence FL generated in the sample S can be detected.

  In the fluorescence detection optical device 10 according to the present embodiment, the excitation light EL is condensed on the sample S by using the off-axis portion 20 a of the first lens group 20 that defines the NA of the light receiving optical system 14. In other words, the irradiation optical system 12 is disposed in the light receiving optical system 14. According to this configuration, unlike the fluorescence detection optical device 100 described with reference to FIG. 1, even if the first lens group 20 is enlarged, the first lens group 20 and the excitation light EL do not interfere with each other. In other words, the NA of the light receiving optical system 14 is not limited by the presence of the irradiation optical system 12. Therefore, according to the fluorescence detection optical device 10 according to the present embodiment, since the NA of the light receiving optical system 14 can be increased, a large amount of fluorescence can be taken into the light receiving optical system 14, and as a result, the fluorescence Detection sensitivity can be improved.

  Further, in the fluorescence detection optical device 10 according to the present embodiment, a part of the second lens group 22 outside the axis is cut off to form a gap part 30, and a part of the excitation light optical system 24 is formed in the gap part 30. (Excitation light source 16 here) is arranged. If the second lens group in which no gap is formed is used, in order to dispose the excitation light optical system between the first lens group and the second lens group, the distance between the first lens group and the second lens group. It is difficult to reduce the size of the optical device for fluorescence detection. As in the present embodiment, the gap between the first lens group 20 and the second lens group 22 is reduced by adopting a configuration in which a part of the excitation light optical system 24 is disposed in the gap portion 30 of the second lens group 22. Therefore, it is possible to reduce the size of the fluorescence detection optical device 10. Further, since there is a margin in the arrangement of the excitation light optical system 24, the assembly of the fluorescence detection optical device 10 can be facilitated.

  In the present embodiment, the excitation light source 16 is disposed in the gap portion 30 of the second lens group 22, but another element of the excitation light optical system 24, such as the excitation light lens group 25 and the excitation light source 16, is provided in the gap portion 30. A necessary power supply line, a mechanical configuration for holding the excitation light source 16, and the like may be arranged.

  Further, in the fluorescence detection optical device 10 according to the present embodiment, the irradiation optical system 12 is arranged in the light receiving optical system 14, so that the optical axis 19 of the excitation light EL and the optical axis 17 of the light receiving optical system 14 are arranged. Can be set small. As a result, the excitation light EL is not easily blocked by the side wall of the container C even when the container C is deep as shown in FIG. As a result, it becomes possible to irradiate the sample S located at the bottom of the container C with sufficient excitation light EL, so that the amount of fluorescence generated from the sample S increases and the fluorescence detection sensitivity can be improved. The light receiving optical system and the excitation light optical system are not limited to the present embodiment in which parallel light is formed in the middle of the lens system, and the optical system within the light receiving optical system 14 is appropriately designed. When the irradiation optical system 12 is configured so as to be installed, the angle θi formed by the optical axis of the excitation light EL and the optical axis of the light receiving optical system 14 can be set small. Needless to say, the same effects as those of the present invention can be obtained.

  FIGS. 3A to 3C are diagrams for explaining the shape and size of the gap portion 30 formed in the second lens group 22. 3A to 3C are plan views of the second lens group 22 viewed from the optical axis direction. FIGS. 3A to 3C also show the excitation light lens group 25.

  FIG. 3A shows a gap portion 30 formed by cutting off the off-axis portion of the second lens group 22 in a square shape. The gap portion 30 shown in FIG. 3A is formed in a size substantially equal to that of the excitation light lens group 25. FIG. 3B shows a gap portion 30 formed by cutting off the off-axis portion of the second lens group 22 with a plane parallel to the optical axis. The gap portion 30 shown in FIG. 3B is formed larger than the excitation light lens group 25. FIG. 3C shows a gap portion 30 formed by punching the off-axis portion of the second lens group 22. The gap portion 30 shown in FIG. 3C is formed smaller than the excitation light lens group 25.

  3A to 3C show an example of the shape and size of the gap portion 30, and naturally the shape and size of the gap portion 30 are limited to the shapes shown in FIGS. 3A to 3C. Not. The shape and size of the gap portion 30 vary depending on the shape and size of the element disposed in the gap portion 30. Since the fluorescence incident on the gap portion 30 is lost without being condensed at the fluorescence condensing point 13, it is preferable that the size of the gap portion 30 is minimized.

  A part or all of the lenses constituting the excitation light lens group 25 may be a ball lens. As for the excitation light lens group 25, the smaller the diameter or width, the better. However, a small-diameter lens is generally expensive. In that respect, ball lenses are relatively inexpensive and are produced in large quantities, and have the advantage that they can be easily obtained from materials having a wide variety of refractive indexes and transmission characteristics and those having a small diameter.

  The gap portion 30 is preferably formed at the peripheral edge of the second lens group 22. This is because the amount of fluorescence per unit lens area taken in by the light receiving optical system 14 becomes larger as it is closer to the normal optical axis. By forming the gap portion 30 at the peripheral edge where the light density is lower than that in the vicinity of the optical axis, more fluorescence can be condensed at the fluorescence condensing point 13 and the fluorescence detection sensitivity can be improved.

  Next, an embodiment of the fluorescence detection optical device 10 will be described.

  FIG. 4 is a diagram for explaining the optical device 40 for fluorescence detection according to the first embodiment. The fluorescence detection optical device 40 shown in FIG. 4 includes a light emitting element 42. The light emitting element 42 is disposed so that at least a part thereof is located in the gap portion 30 of the second lens group 22. In the first embodiment, the light emitting element 42 can be regarded as the excitation light source 16 of the fluorescence detecting optical device 10 shown in FIG. As the light emitting element 42, an LED (Light-Emitting Diode), an LD (Laser Diode), or the like that includes a wavelength suitable for excitation of the sample S in the light emission band can be used.

  In the first embodiment, the fluorescence condensed by the light receiving optical system 14 enters the fluorescent fiber 44 from the incident end face 44 a and is guided to the light receiving unit 46. The light receiving unit 46 is disposed away from the light receiving optical system 14. In the first embodiment, the incident end face 44a of the fluorescence fiber 44 can be regarded as the fluorescence condensing point 13 of the fluorescence detection optical device 10 shown in FIG.

  As the fluorescent fiber 44, a single mode fiber or a multimode fiber can be used. The light receiving unit 46 includes a photoelectric conversion element that photoelectrically converts fluorescence and an electric circuit that performs predetermined processing such as amplification on the electric signal. As a photoelectric conversion element, PD (Photodiode), APD (Avalanche Photodiode), PMT (Photomultiplier Tube), etc. can be used.

  FIG. 5 is a diagram for explaining the fluorescence detection optical device 50 according to the second embodiment. The fluorescence detection optical device 50 shown in FIG. 5 includes a light source unit 52 disposed away from the excitation light optical system 24 and an excitation light fiber 54 that guides the excitation light emitted from the light source unit 52. .

  The light source unit 52 includes a light source such as an LED, an LD, or a xenon lamp, and an electric circuit for supplying power to the light source. As the excitation light fiber 54, a single mode fiber or a multimode fiber can be used.

  In the second embodiment, the emission end face 54a of the excitation light fiber 54 is disposed in the gap portion 30 of the second lens group 22, and the excitation light emitted from the emission end face 54a of the excitation light fiber 54 is The light enters the excitation light lens group 25. In the second embodiment, the emission end face 54a of the excitation light fiber 54 can be regarded as the excitation light source 16 of the fluorescence detection optical device 10 shown in FIG.

According to the optical device 50 for fluorescence detection according to the second embodiment, a portion having only an optical system such as a lens and an optical fiber (that is, an “optical head”), an electric circuit, a photoelectric conversion type light emitting / receiving element, Can be separated. According to the configuration of the second embodiment, the following effects can be obtained.
(A) The exit end face 54a of the excitation light fiber 54 is used as an excitation light source, and the incident end face 44a of the fluorescence fiber 44 is used as a fluorescence condensing point. Since the diameter of the optical fiber is usually 1 mm or less, the optical head can be made smaller than when the photoelectric conversion type light emitting / receiving element itself is mounted on the optical head.
(B) Since the electric circuit and the light emitting / receiving element that are likely to generate electrical noise are separated from the optical head, the influence of noise can be suppressed. Since the electrical system that is easily affected by noise due to disturbance is separated from the optical head, the measurement environment is not limited.
(C) Monochromaticity is required in the field of fluorescence detection. In this case, by installing a predetermined filter in a separate light source / light receiving unit, the demand level can be easily adjusted, and the degree of freedom in design specifications is increased.
(D) Since the gap portion 30 may be provided in the second lens group 22 to such an extent that the tip of an optical fiber having a diameter of 1 mm or less can be normally disposed, the ablation region of the second lens group 22 can be set smaller. , The amount of fluorescence detected can be increased.

  FIG. 6 is a diagram for explaining the fluorescence detection optical device 60 according to the third embodiment. The excitation light optical system 24 of the fluorescence detection optical device 60 shown in FIG. 6 includes a light emitting element 42 provided on the image side of the second lens group 22 and a first lens that converts the excitation light from the light emitting element 42 into parallel light. A relay lens system 62 that is incident on the off-axis portion 20 a of the group 20. The relay lens system 62 includes a first lens 62a and a second lens 62b that are arranged so as to sandwich the second lens group 22 therebetween. The relay lens system 62 forms an intermediate image 64 of the light emitting region of the light emitting element 42 in the gap portion 30 of the second lens group 22.

In the fluorescence detection optical device 40 (see FIG. 4) according to the first example described above, at least a part of the light emitting element 42 is disposed in the gap portion 30 of the second lens group 22. However, for example, when a bullet-type LED is used as the light emitting element 42, since the outer diameter is about 3 mm, the gap portion 30 tends to be large, and the amount of fluorescence that can be collected may be reduced. Therefore, the present inventor noticed that the light emitting area of the LED is as small as, for example, 0.3 × 0.3 mm ^2, and used the relay lens system 62 to place the gap portion 30 in the middle of the light emitting area of the light emitting element 42. I thought of making a statue. According to the fluorescence detection optical device 60 according to the third embodiment, the gap portion 30 of the second lens group 22 can be reduced, and the detected fluorescence amount can be increased.

  FIG. 7 is a view for explaining an optical device for fluorescence detection 70 according to another embodiment of the present invention. FIG. 8 is a plan view of the second lens group 22 viewed from the optical axis direction in the fluorescence detection optical device 70 shown in FIG.

  The fluorescence detection optical device 70 according to the present embodiment includes four excitation light optical systems 24. Further, four gap portions 30 are formed at 90 ° intervals on the peripheral edge of the second lens group 22. An excitation light source 16 that is a part of the excitation light optical system 24 is disposed in each of these four gap portions 30. Although FIG. 8 shows the gap-shaped gap portion 30, the shape of the gap portion 30 is not particularly limited.

  In the fluorescence detection optical device 70 according to the present embodiment, the four excitation light optical systems 24 can be optical systems having the same optical characteristics such as wavelengths. In this case, the amount of excitation light can be increased, and a detectable level of fluorescence can be generated even for a sample with a small absolute amount of emitted fluorescence.

  Alternatively, the four excitation light optical systems 24 may be optical systems having different optical characteristics such as wavelengths. By detecting the fluorescence emitted from the sample by irradiation with excitation light having different wavelengths, it becomes possible to analyze the sample from various aspects.

  In the embodiment shown in FIG. 7, the four excitation light optical systems 24 are arranged in the four gap portions 30, respectively, but the numbers of the excitation light optical systems 24 and the gap portions 30 are not particularly limited.

  The present invention has been described based on the embodiments. This embodiment is an exemplification, and it will be understood by those skilled in the art that various modifications can be made to combinations of the respective constituent elements and processing processes, and such modifications are also within the scope of the present invention. is there.

  10, 40, 50, 60, 70 Fluorescence detection optical device, 11 Fluorescence emission point, 12 Irradiation optical system, 13 Fluorescence condensing point, 14 Light receiving optical system, 16 Excitation light source, 20 First lens group, 22 Second lens Group, 24 excitation light optical system, 25 excitation light lens group, 30 gap portion, 42 light emitting element, 44 fluorescent fiber, 46 light receiving unit, 52 light source unit, 54 excitation light fiber, 62 relay lens system.

Claims (8)

An optical device for fluorescence detection that irradiates a detected object with excitation light and detects fluorescence generated from the detected object by the irradiation,
A first lens group for directly receiving fluorescence generated from the object to be detected;
A second lens group that is arranged coaxially with the first lens group and receives fluorescence that has passed through the first lens group, wherein a part off the axis is cut off to form a gap portion. When,
An excitation light optical system for emitting excitation light, the excitation light optical system arranged so that the emitted excitation light is irradiated to the object to be detected through an off-axis of the first lens group;
With
A part of the excitation light optical system is disposed in the gap portion of the second lens group ,
An optical apparatus for fluorescence detection , wherein the excitation light from the excitation light optical system is condensed on the object to be detected outside the axis of the first lens group . The first lens group converts the fluorescence generated from the object to be detected into parallel light,
The second lens group collects the fluorescent parallel light from the first lens group,
The optical device for fluorescence detection according to claim 1.   The optical device for fluorescence detection according to claim 1 or 2, wherein the gap portion is formed in a peripheral portion of the second lens group. The excitation light optical system is
An excitation light source that emits excitation light;
A pumping light lens group that makes the pumping light from the pumping light source parallel light and is incident off-axis of the first lens group;
The optical device for fluorescence detection according to any one of claims 1 to 3, further comprising:   The optical device for fluorescence detection according to claim 4, wherein the excitation light source includes a light emitting element that emits excitation light.   The optical device for fluorescence detection according to claim 4, wherein the excitation light source includes an emission end face of an optical fiber for excitation light that guides excitation light. The excitation light source includes a light emitting element provided in an image side space of the second lens group,
The fluorescence detection optical device according to claim 4, wherein the excitation light lens group includes a relay lens system arranged to form an intermediate image of a light emitting region of the light emitting element in the gap portion. A plurality of the excitation light optical system,
A plurality of the gap portions are formed in the second lens group,
The fluorescence detection optical apparatus according to claim 1, wherein a part of the excitation light optical system is disposed in each of the plurality of gap portions.

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