JP5314204B2 - Light measuring device - Google Patents

Description

The present invention relates to an optical measurement device.

Conventionally, as a technique for irradiating irradiation light (excitation light) to a well of a microplate, techniques described in Patent Documents 1 to 3 are known. In the case of Patent Document 1, the irradiation light is irradiated from the back surface of the microplate to the well in parallel to the depth direction of the well of the microplate. A well such as a culture solution, a fluorescent indicator, and an evaluation compound and a measurement object such as a cell are injected into the well of the microplate.

JP 2007-108146 A JP-A-10-197449 JP-A-10-281994

In the irradiation method of irradiation light described in Patent Document 1, since irradiation light is irradiated to the well in parallel with the depth direction of the well, not only a measurement object but also a solution is irradiated in a large amount There is. In this case, background light noise from the solution becomes relatively large. Accordingly, an object of the present invention is to provide an optical measurement device that can reduce background light noise from a microplate.

The light measurement device of the present invention is a light measurement device that measures light from a microplate provided with a plurality of wells for containing a measurement object, and a main surface on which a plurality of recesses are formed, A light guide member having a back surface opposed to the main surface, a side surface between the main surface and the back surface, a light source that enters the light guide member from the side surface of the light guide member, and a light source from the back surface of the light guide member And a detector for detecting measurement light, wherein the light guide member is disposed such that a main surface thereof faces the microplate, and a bottom surface of the recess is flat.

According to the light measurement apparatus of the present invention, the irradiation light is refracted and reflected on the side surface of the recess and is emitted from the opening of the recess, so that the well is irradiated with an inclination with respect to the depth direction of the well. For this reason, there is comparatively little irradiation light irradiated to solutions, such as a culture solution, a fluorescent indicator, and an evaluation compound. Therefore, it is possible to reduce background light noise from the microplate that is generated by irradiating the solution with irradiation light.

In the optical measurement device of the present invention, it is preferable that the recess is a cylindrical recess. In this case, since there is no corner on the side surface of the recess, random light scattering is easily induced inside the recess. Therefore, irradiation unevenness of irradiation light to each well of the microplate can be reduced.

In the optical measurement device of the present invention, the bottom surface of the recess is flat. In this case, since the bottom surface of the recess is flat, a part of the irradiation light that enters the light guide member and reaches the bottom surface of the recess is totally reflected on the bottom surface of the recess. For this reason, incidence of irradiation light parallel to the depth direction of the well is reduced.

In the optical measurement device of the present invention, it is preferable that the bottom surface of the recess is mirror-finished. In this case, since the bottom surface of the recess is mirror-finished, most of the irradiation light that enters the light guide member and reaches the bottom surface of the recess is totally reflected on the bottom surface of the recess. For this reason, it is possible to further reduce the incidence of irradiation light parallel to the depth direction of the well.

The light measurement device of the present invention further includes a light source device having a plurality of light sources, a filter, and a frame, and the light sources are arranged along the side surface of the light guide member and held by the frame. It is preferable that the light guide member is disposed between the side surface and the light source. In the light measurement device of the present invention, the light source preferably includes two types of LEDs that emit light having different wavelengths. In the light measurement device of the present invention, it is preferable that the light source enters the irradiation light from a portion corresponding to a region between the bottom surface of the concave portion and the back surface of the light guide member among the side surfaces of the light guide member. In the light measurement device of the present invention, the recess is preferably a prismatic recess. In the optical measurement device of the present invention, the recess is preferably a depression having a trapezoidal cross section.

ADVANTAGE OF THE INVENTION According to this invention, the optical measuring apparatus which can reduce the background light noise from a microplate can be provided.

It is a figure showing the cross-sectional structure of the optical measurement apparatus which concerns on this embodiment. It is a figure for demonstrating the microplate shown by FIG. It is a figure for demonstrating the light irradiation apparatus shown by FIG. It is a figure for demonstrating the light irradiation apparatus shown by FIG. It is a figure showing the relationship between the well of the microplate shown by FIG. 1, and the recessed part of a light guide member. It is a figure for demonstrating the light irradiation apparatus shown by FIG. It is a figure for demonstrating the light irradiation apparatus shown by FIG. It is a figure for demonstrating the light irradiation apparatus shown by FIG. It is a figure showing the optical path of the irradiation light in the microplate and light guide member which were shown by FIG. It is a figure showing the result of having measured the measurement light (fluorescence) from the measuring object and solution which were hold | maintained at the microplate shown by FIG.

Hereinafter, preferred embodiments of the present invention will be described in detail with reference to the drawings. In the description of the drawings, if possible, the same elements are denoted by the same reference numerals, and redundant description is omitted. FIG. 1 is a diagram schematically illustrating a cross-sectional configuration of the light measurement apparatus according to the present embodiment. As shown in FIG. 1, the light measurement device 10 includes a microplate 20, microplate stockers 30 and 40, a transport belt 50, a light irradiation device 60, an excitation light cut filter 70, a detector 80, Is provided. The light irradiation device 60 includes a light guide member 61 and a light source device 62. The light measurement device 10 is a device that detects measurement light (fluorescence) emitted from a measurement target by irradiating the measurement target held on the microplate 20 with irradiation light (excitation light).

2A is a plan view of the microplate 20, and FIG. 2B is a diagram illustrating a cross-sectional configuration of the microplate 20 taken along the line II in FIG. 2A. . As shown in FIGS. 2A and 2B, the microplate 20 has a plurality (eg, 96) of wells 21 having a cylindrical shape. In the main surface 22 of the microplate 20, the openings of the plurality of wells 21 are arranged in, for example, 8 columns and 12 rows. In addition, a measuring object A such as a cell and a solution B such as a culture solution, a fluorescent indicator, and an evaluation compound are injected and held in each of the plurality of wells 21. The measurement object A is deposited on the bottom of the well 21. The well of the microplate 20 is not limited to the hollow well 21 having a cylindrical shape. For example, the well of the microplate 20 may be a prism-shaped well 21a as shown in FIG.

A description will be given with reference to FIG. 1 again. The microplate stocker 30 stores the microplate 20 before measurement. The microplate stocker 40 stores the microplate 20 after measurement. The transport belt 50 transports the microplate 20 before measurement from the microplate stocker 30 to a predetermined measurement position (position facing the light irradiation device 60). Further, the transport belt 50 transports the measured microplate 20 from the predetermined measurement position to the microplate stocker 40.

The light irradiation device 60 includes a light guide member 61 and a light source device 62. The light guide member 61 is made of, for example, quartz glass. The light source device 62 makes irradiation light incident from the side surface 61 b of the light guide member 61. The light irradiation device 60 irradiates the well 21 with irradiation light from the back surface 23 side of the microplate 20. Here, the light measurement device 10 has a mechanism for moving the light irradiation device 60 in a direction perpendicular to the back surface 23 (the depth direction of the well 21 of the microplate 20) (not shown). In addition, the light irradiation apparatus 60 may be arrange | positioned and fixed so that the main surface 61a of the light guide member 61 and the back surface 23 of the microplate 20 may have predetermined spacing (for example, about 5 mm). In this case, the macro plate 20 is transported to a predetermined measurement position without colliding with the light irradiation device 60.

The excitation light cut filter 70 blocks the transmission of the irradiation light and transmits the measurement light from the measurement object A or the like. The detector 80 is disposed on the back surface 23 side of the microplate 20, and an optical system (not shown) for imaging the measurement light that has passed through the excitation light cut filter 70 and an image for imaging the formed image It has a light detection device such as a two-dimensional CCD camera, and detects measurement light from the measurement object A or the like. The detector 80 can also be disposed on the main surface 22 side of the microplate 20. In this case, the detector 80 is, for example, a plurality of photomultiplier tubes arranged for each well 21 of the microplate 20 or a two-dimensional imaging device capable of imaging the plurality of wells 21 of the microplate 20. be able to. When a photomultiplier tube is used for the detector 80, the photomultiplier tube can be moved above the main surface 22 side of the microplate 20 to detect the measurement light from each well 21.

Next, the light irradiation device 60 will be described in detail. FIG. 3A is a diagram illustrating a cross-sectional configuration of the light irradiation device 60 taken along the line II-II in FIG. FIG. 3B is a plan view of the light irradiation device 60. 3A and 3B, the light guide member 61 of the light irradiation device 60 includes a main surface 61a, and two side surfaces 61b that are substantially orthogonal to the main surface 61a and face each other. And two side surfaces 61c substantially orthogonal to the main surface 61a and facing each other. The light guide member 61 has a plurality of recesses 61e provided in a two-dimensional array on the main surface 61a. The recess 61e is a cylindrical recess having an opening in the main surface 61a of the light guide member 61, and includes a flat bottom surface 61f that is substantially parallel to the light emitting surface 61a. The bottom surface 61f is mirror-finished.

A light source device 62 is disposed on the side surface 61 b of the light guide member 61. The light source device 62 includes a frame 62a, an LED 62b as a light source, and a filter 62c. A plurality of LEDs 62b are arranged along the side surface 61b of the light guide member 61 and are held by the frame 62a. The plurality of LEDs 62b make incident light having directivity incident on the light guide member 61 from the side surface 61b of the light guide member 61 through the filter 62c. The filter 62c is a short-pass filter, a band-pass filter, or the like that transmits only light in a specific wavelength band, and transmits only irradiation light having a wavelength suitable for measurement from the light emitted from the LED 62b. In this manner, by combining the LED 62b and the filter 62c, light having a wavelength more suitable for measurement can be incident on the light guide member 61, so that measurement accuracy can be improved.

In addition, each recessed part of the light guide member 61 is not limited to the recessed part 61e having a cylindrical shape, and may be a recessed part 61g having a prismatic shape as shown in FIG. Further, each concave portion (the concave portion 61e and the concave portion 61g) of the light guide member 61 has a cross section (along the line II-II in FIG. 3B) as shown in FIGS. 4A and 4B. A trapezoidal depression may be used. In this case, the angle φ formed between the side surface of each concave portion (the concave portion 61e and the concave portion 61g) of the light guide member 61 and the main surface 61a is preferably an acute angle, but may be an obtuse angle of about 90 to 120 degrees. Good. As shown in FIG. 4C, the light guide member 61 can also be formed by arranging a plurality of prismatic optical fibers 61i provided with a plurality of recesses 61e. The light guide member 61 described above can be arranged so that the bottom surface of the well 21 and the opening of the recess 61e face each other as shown in FIG. In addition, it is preferable that the depth of each recessed part (the recessed part 61e and the recessed part 61g) of the light guide member 61 is about 2 mm.

FIG. 5 is a diagram schematically showing a variation in the arrangement pattern of the plurality of recesses 61 e for the plurality of wells 21. As shown in FIG. 5A, each recess 61e is preferably provided so as to correspond to each well 21 on a one-to-one basis. However, as shown in FIG. It may be provided so as to correspond to one well 21. In addition, as shown in FIG. 5C, each recess 61 e may correspond to each well 21 on a one-to-one basis and may be provided wider than the well 21. Further, as shown in FIG. 5D, each recess 61e corresponds to a plurality of recesses 61e corresponding to one well 21, and a part of each recess 61e is a well when viewed from above the main surface 61a. 21 (and so that the remaining portion of each recess 61 e overlaps the well 21).

As the light source of the light source device 62, two types of LEDs that emit light having different wavelengths can be used. In this case, as shown in FIG. 6A, a plurality of LEDs 62b are arranged along the side surface 61b of the light guide member 61, and light having a wavelength different from that of the LED 62b is formed along the other side surface 61c of the light guide member 61. A plurality of LEDs 62d that emit light are arranged. Thereby, the light source device 62 can enter the light guide member 61 with irradiation light having two different wavelengths. A filter 62c is disposed between the LED 62b and the light guide member 61, and a filter such as a short pass filter or a band pass filter that transmits light having a wavelength different from that of the filter 62c is provided between the LED 62d and the light guide member 61. 62e is arranged. Therefore, the filter 62e can transmit only irradiation light having a wavelength suitable for measurement from the light emitted from the LED 62d. Further, as shown in FIG. 6B, the LEDs 62 b and the LEDs 62 d can be alternately arranged along the side surface 61 b and the side surface 61 c of the light guide member 61. At this time, the filters 62 c and 62 e are alternately arranged between the LEDs 62 b and 62 d and the light guide member 61.

The light source of the light source device 62 is not limited to the LED. As the light source of the light source device 62, for example, a white light source such as a xenon lamp 62f can be used as shown in FIG. In this case, the xenon lamp 62f enters the light guide member 61 from the side surface 61b of the light guide member 61 through the wavelength switching device 62g and the optical fiber 62h. Thereby, the light of the wavelength which is not realizable with LED can be used as irradiation light. At this time, the xenon lamp 62f itself as the light source may not be disposed on the side surface 61b of the light guide member 61 as shown in FIG. Further, as shown in FIG. 7B, a predetermined light source 62k that emits light having no directivity is used as a light source of the light source device 62, and a collimator lens 62n is interposed between the light source 62k and the filter 62m. May be arranged. The collimator lens 62n can be provided integrally with the light guide member 61 on the side surface 61b of the light guide member 61, as shown in FIG. The filter 62m transmits only irradiation light having a wavelength suitable for measurement from the light emitted from the light source 62k.

FIG. 8 is a diagram illustrating a range of irradiation light incident on the light guide member 61. As shown in FIG. 8A, it is preferable that the irradiation light is incident from the portion corresponding to the region between the back surface 61f of the recess 61e and the bottom surface 61h of the light guide member 61 in the side surface 61b. As shown in FIG. 8B, the irradiation light may be incident from the entire side surface 61b.

As described above, in the light irradiation device 60, directional irradiation light is incident from the side surface 61b of the light guide member 61 on the light guide member 61 in which the plurality of recesses 61e are formed on the main surface 61a. The A part of the irradiation light incident on the light guide member 61 travels on the optical path indicated by the broken line L1 in FIG. Reflected. For this reason, it can suppress that the irradiation light which progresses in the depth direction of the well 21 enters into the recessed part 61e. Further, part of the irradiation light incident on the light guide member 61 travels along the optical path indicated by the alternate long and short dash line L2 in FIG. 9A and enters the recess 61e from the side surface of the recess 61e. The irradiation light is refracted in a direction parallel to the light emitting surface 61a due to a difference in refractive index between the light guide member 61 and the air in the recess 61e, and is irradiated to each well 21. Thereby, the irradiation light incident on the well 21 is inclined with respect to the depth direction of the well 21. Therefore, as shown in FIG. 9B, the irradiation light applied to the solution B is relatively small. Therefore, it is possible to reduce background light noise that occurs when the solution B is irradiated with the irradiation light. On the other hand, FIG. 9C is a diagram showing the optical path of the irradiation light when the irradiation light is irradiated to the well 21 in the depth direction of the well 21 by the conventional method. As shown in FIG. 9C, according to the conventional method, a large amount of irradiation light is irradiated not only on the measurement object A but also on the solution B, so that the background light noise becomes relatively large.

FIG. 10 is a diagram illustrating a result of measuring fluorescence as measurement light from the measurement object A or the like. The horizontal axis represents the number of cells (measuring object A) injected into each well 21, and the vertical axis represents the fluorescence detection ratio. Cells injected into each well 21 are stained with a fluorescent dye. The measurement result represented by the broken line L3 shows that after injecting cells and a fluorescent solution (solution B: FITC) into each well 21 of the microplate 20, irradiation light parallel to the depth direction of the well 21 is applied to the well 21. It is the result obtained by the conventional method which measures the fluorescence emitted from a cell by irradiating. The measurement result represented by the alternate long and short dash line L4 is emitted from the cells by injecting the cells 21 and the fluorescent solution into each well 21 of the microplate 20 and then irradiating each well 21 with irradiation light using the light irradiation device 60. It is the result of measuring the obtained fluorescence. In this measurement, the depth of each recess 61e of the light guide member 60 is 4 mm. The measurement result represented by the two-dot chain line L5 is obtained by injecting cells and a fluorescent solution into each well 21 of the microplate 20 and then irradiating each well 21 with irradiation light using the light irradiation device 60. It is the result of measuring the emitted fluorescence. In this measurement, the depth of each recess 61e of the light guide member 61 of the light irradiation device 60 is 1 mm. The measurement result represented by the solid line L6 is a result of measuring fluorescence emitted from the cells by irradiating each well 21 with irradiation light after injecting only the cells into each well 21 of the microplate 20. Accordingly, in FIG. 10, the background light noise from the fluorescent solution is reduced as the result is closer to the result represented by the solid line L6. According to the measurement result shown in FIG. 10, the measurement result when the irradiation light is irradiated using the light irradiation device 6 represented by the one-dot chain line L4 and the two-dot chain line L5 is the conventional one represented by the broken line L3. It can be seen that it is closer to the measurement result represented by the solid line L6 than the measurement result by the irradiation method of the irradiation light. Therefore, according to the light irradiation apparatus 60, it turns out that background light noise is reduced.

DESCRIPTION OF SYMBOLS 10 ... Light measuring device, 20 ... Microplate, 21, 21a ... Well, 22, 61a ... Main surface, 23, 61h ... Back surface, 30, 40 ... Microplate stocker, 50 ... Transport belt, 60 ... Light irradiation device, 61 ... light guide member, 61b, 61c ... side face, 61e, 61g ... concave part, 61f ... bottom face, 62 ... light source device, 62a ... frame, 62b, 62d ... LED, 62c, 62e, 62m ... filter, 62f ... xenon lamp, 62g ... wavelength switching device, 61i, 62h ... optical fiber, 62k ... light source, 62n ... collimator lens, 70 ... excitation light cut filter, 80 ... detector.

Claims (8)

A light measurement device for measuring light from a microplate provided with a plurality of wells for containing a measurement object,
A light guide member having a main surface on which a plurality of recesses are formed, a back surface facing the main surface, and a side surface between the main surface and the back surface;
A light source that makes irradiation light incident on the light guide member from the side surface of the light guide member;
A detector for detecting measurement light from the back surface of the light guide member,
The light guide member is disposed such that the main surface faces the microplate,
The bottom surface of the recess is flat;
An optical measuring device characterized by that. A light source device including a plurality of the light sources, a filter, and a frame;
The light source is arranged along the side surface of the light guide member and held by the frame,
The filter is disposed between the side surface of the light guide member and the light source.
The optical measurement apparatus according to claim 1. The light source includes two types of LEDs that emit light of different wavelengths.
The optical measurement apparatus according to claim 2, wherein The light source enters the irradiation light from a portion corresponding to a region between the bottom surface of the concave portion and the back surface of the light guide member among the side surfaces of the light guide member.
The optical measurement device according to claim 1, wherein The bottom surface of the recess is mirror-finished,
The optical measurement device according to claim 1, wherein The recess is a cylindrical recess.
The optical measurement device according to claim 1, wherein The recess is a prismatic recess.
The optical measurement device according to claim 1, wherein The recess is a depression having a trapezoidal cross section.
The optical measurement device according to claim 1, wherein

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