Patent Grant
7405824
1. Field of the Invention
The present invention relates to an optical coupling system comprising a light measuring device and a sample. More specifically, the present invention relates to an optical coupling system wherein a coupling lens is arranged ahead of an optical fiber and a lens portion is integrally formed at the bottom of a sample container, and excitation light, transmitted by the optical fiber, is efficiently coupled with the sample by the coupling lens and the lens portion, and an optical coupling system wherein light generated by a sample is efficiently coupled with an optical fiber by a lens portion and a coupling lens. This technology can be applied for a light measuring device, for example, that irradiates a sample with excitation light, and measures the intensity of fluorescence generated by the excited sample.
2. Related Art
As a technique that physically or chemically examines or analyzes a material, a light measuring device is available that excites a sample through the radiation of light, and measures light generated by the excited sample to obtain various data concerning the sample. Example light measuring devices are an apparatus that measures the intensity of fluorescence having a specified wavelength that is emitted by an excited sample, or that measures a transient change in fluorescence and detects a specified material or monitors a change in density, and an apparatus that measures the spectrum of fluorescence emitted by a sample to perform a quantitative or qualitative analysis. Since both apparatuses are capable of conducting highly sensitive measurements and are appropriate for the performance of microanalysis, they are widely employed. When a liquid sample is used, during the measurement process the sample is retained in a transparent sample container that permits irradiation with an excitation light and fluorescence.
For these light measuring devices, excitation light is projected onto a sample that is positioned at a location to which light from a light source is emitted, and fluorescence generated by the excited sample is appropriately collected and enters an optical detection system. Recently, another structure has been proposed wherein, using an optical fiber, excitation light is transmitted to a sample through a coupling lens, and by using another optical fiber, fluorescence output by the sample is guided through the coupling lens to an optical detection system (see, for example, JP-A-11326210). When the optical fibers are employed in this manner, the degree of freedom is increased for the positional relationships of the sample, the light source and the optical detection system. Further, since the fluorescence to be detected is transmitted through the optical fiber, disturbance light seldom enters the optical system, so that the sensitivity of an analysis of the fluorescence, which in many cases is weak, can be increased.
However, according to the conventional example, fluorescence cannot be efficiently extracted from a sample. As one reason, fluorescence output by the sample container is dispersed. As another reason, since there is a long distance between the sample and the coupling lens, it is difficult for fluorescence to be collected at the coupling lens, and since the irradiation area for excitation light is large and the radiation density is low, the surface emission of fluorescence occurs. Further, in the conventional example, a sample cannot be irradiated efficiently using excitation light. The reason for this is that since an excitation light optical fiber, that has a comparatively large diameter is employed in order to increase the intensity of the excitation light, the excitation light can not be controlled by a coupling lens and directed to a predetermined location for a sample, and thus, the radiation density of the excitation light is lowered.
Additionally, in the conventional example, since the amount of excitation light and the amount of fluorescence are increased as much as possible, an optical fiber having a large diameter, or multiple optical fibers, must be employed, and accordingly, the diameter of the coupling lens must be increased, and to connect the excitation light with the fluorescence, a plurality of lenses are required. Thus, the size of the optical system is increased.
The objective of the present invention is to efficiently irradiate a sample with excitation light, and to efficiently couple, with an optical fiber, light discharged by the sample.
To achieve this objective, according to the present invention, there is provided an optical system coupling a light measuring device and a sample, wherein excitation light emitted by a light source is guided through an excitation light optical fiber to irradiate the sample, and excited light emitted by the sample is guided to an optical detection system through a receiving light optical fiber, the optical system comprising:
a light measuring device including
a sample container including a cylindrical body for retaining the sample to which the excitation light is irradiated, and
wherein the excitation light is changed to parallel light or converged light by the coupling lens and collected at the sample by the lens portion. The present invention can be applied not only for measuring fluorescence, but also for measuring any other type of light, such as phosphorescence, emitted by a sample that is irradiated with excitation light.
According to an example structure for the optical system provided for the light measuring device, an optical fiber bundle is formed of a single, centered excitation light optical fiber and a plurality of receiving light optical fibers surrounding this optical fiber, a capillary is attached to the distal end of the optical fiber bundle, and a coupling lens is inserted into, positioned within the capillary, and used both for excitation light and for receiving light. As another example structure, one excitation light optical fiber and another receiving light optical fiber may be adjacently fixed through a ferrule, a capillary may be attached to the distal ends of the optical fibers, and a coupling lens, used both for excitation light and for receiving light, may be inserted into and fixed within the capillary. It is preferable that the outer diameter of the optical fiber bundle be smaller than the diameter of the coupling lens, and that the diameter of the excitation light optical fiber be equal to or smaller than the diameter of the receiving light optical fiber. The coupling lens and the lens portion at the bottom of the sample container are arranged at a distance to prevent the formation of a light collecting point between them.
Normally, the sample container has a cylindrical body, but it may have a square barrel shape. And typically, the lens portion at the bottom of the cylindrical member is semi-spherical.
According to the invention, in the optical coupling system of the light measuring device and the sample, the lens portion, for collecting excitation light and fluorescence, is integrally formed with the bottom of the sample container in which the sample is retained, so that parallel light or converged light, obtained by the coupling lens, is collected at the sample by the coupling lens. Therefore, excitation light can be radiated, and fluorescence can be efficiently extracted. That is, as the effects provided by this invention, since the irradiation of excitation light can be concentrated more at the sample by using the lens portion, the radiation density of excitation light is increased, so that the amount of light output by the sample is increased. Further, since the state of fluorescence can be similar to the state of a point source in consonance with the condensed state of excitation light that is radiated, light can be easily detected. Furthermore, since the lens portion can be located nearer the sample, light to be dispersed can be reduced, and light can be easily extracted. Therefore, the sensitivity of the device can be increased. Additionally, since the lens portion is integrally formed with the bottom of the sample container, this contributes to the downsizing of the optical system.
An example optical coupling system of the invention for a fluorescence measuring device, and a sample, is shown in
In this example, six optical fibers 18, used for receiving light, are disposed around the periphery of one optical fiber 12, as the excitation light optical fiber, in the center, and these optical fibers are formed into a single optical fiber bundle (bundled fiber) 26 (see
The sample container 22, wherein the lens portion 24 is integrally formed with the cylindrical body, can be obtained by employing the following manufacturing method. For example, a cylindrical body having a thick bottom is prepared and is inserted into a pipe-shaped holding tool. In this state, the cylindrical body is ground into a semispherical shape and finished by a spherical grindstone. Or, as another method, a semispherical lens portion is separately fabricated and adhered to the bottom of the cylindrical body.
In this example, the optical coupling system of the light measuring device and the sample is constituted by the optical fibers 12 and 18, the coupling lens 22 and the lens portion 24 of the sample container 22. Excitation light, emitted by the light source 10, is guided through the excitation light optical fiber, and is collected by the coupling lens 14 and the semispherical lens portion 24, so that the sample 16 in the sample container 22 is irradiated with the excitation light. Fluorescence output by the sample 16 is then passed through the lens portion 24 and the coupling lens 14, and is guided to the optical detection system 20 by the light fiber 18 used for receiving light.
Since the sample container 22 is so designed that the semispherical lens portion 24 is integrally formed with the cylindrical bottom, the sample 16 can be efficiently irradiated with excitation light, and fluorescence can be efficiently extracted. Compared with when only the coupling lens 14 is employed, the excitation light used to irradiate the sample 16 is more concentrated, and the radiation density of the excitation light is increased, so that a greater amount of florescence is output by the sample 16. Further, since the lens portion 24 can be arranged so it is nearer the sample 16, the dispersal of fluorescence is reduced, and since the state of fluorescence can be similar to the state of a point source in consonance with the collected state of excitation light to be emitted, fluorescence can be more easily detected. Furthermore, since the lens portion 24 is integrally formed with the sample container 22, the size of the system can be reduced.
The optimal optical arrangement and the excitation light transmission state are shown in
When, as shown in
an outer diameter φF of the optical fiber bundle 26<diameter φL of the coupling lens 14, and
the diameter of the optical fiber 12 for excitation light≦the diameter of an optical fiber 18 for receiving fluorescence, and that excitation light be transmitted inside and fluorescence be transmitted outside. Thus, eclipsing of excitation light by the coupling lens 14 can be avoided, and the sample 16 can be efficiently excited. When the diameter of the excitation light optical fiber 12 is reduced, the image of excitation light formed by the coupling lens 14 becomes smaller, so that the radiation density of the excitation light is increased and the sample 16 is efficiently excited. Further, since the shape of fluorescence becomes more similar to that emitted by a point source, the extraction of fluorescence is easy. In addition, the coupling lens 14 that employs the capillary 30 and the optical fiber bundle 26 can be integrated (assembled).
As shown in
An appropriate optical arrangement, the transmission states of excitation light and fluorescence and a beam profile were obtained by using the optical coupling system shown in
A bundle of seven step index silica fibers having core diameters of 200 μm and NA=0.2 was employed as an optical fiber bundle. As shown in
A planoconvex lens of φ=2.4 mm and f=2.9 mm was employed as the coupling lens 14. The shape of the coupling lens 14 is not especially limited to a planoconvex lens; however, it is preferable that the diameter of a lens be smaller than that of the optical fiber, and that the thickness of the round edge (thickness of the flat plate portion of a lens) also be greater, because the assembling process, for integrating a coupling lens and an optical fiber bundle, is simplified. In order to prevent excitation light from being reflected by the surface of the lens and becoming noise, and to prevent fluorescence from being reflected by the surface of the lens and causing transmission loss, reflection attenuation coatings, for an excitation wavelength and for a fluorescence wavelength, were applied to both surfaces of the lens.
As shown in
Fluoresceinisothiocyanate FITC was employed as a sample, and about 10 μL of it was enclosed in a glass sample container having a diameter of about 1.5 mm by appropriately adjusting the density. A semispherical lens portion having a diameter of about 1.5 mm, which is the same as that of the sample container, and r=about 0.7 mm was integrally formed at the bottom of the sample container. It is preferable that reflection attenuation coatings for an excitation wavelength and for a fluorescence wavelength also be applied to the lens portion of the sample container.
A wavelength selection filter was employed that permits only a wavelength 470±10 nm to pass, and light was extracted from light emitted by a light emitting diode (LED) that has a light emission peak near a wavelength 470 nm. This extracted light was employed as excitation light. This light was connected to the excitation light optical fiber included in the optical fiber bundle. Excitation light, having with a beam diameter substantially equal to the size of the lens portion, is output at the end face of the excitation light optical fiber, passes through the coupling lens, enters the lens portion at the bottom of the sample container, and is converged at a location deep inside the sample, measured from the bottom of the sample container. Fluorescence is generated from the entire range where excitation light is transmitted, primarily at the focal position of excitation light. Fluorescence, which is connected to the optical fibers by the lens portion of the sample container and the coupling lens, is separated by a wavelength filter that permits only a desired wavelength, one that is to be detected (532 nm in this case), to pass. Then, the resultant light is transmitted to an optical detector module (a photomultiplier), and is converted into an electric signal.
On the other hand, when the coupling lens and the lens portion of the sample container contact each other (z=0 mm), and when the coupling lens and the lens portion of the sample container are separated, located at a distance, and a light collecting point is formed (e.g., z=8 mm), it is found that the peak intensity of excitation light is extremely low and excitation light is almost not collected, and that the intensity of fluorescence is also low, or fluorescence is broadly spread. According to these results, the distance z between the convex face of the coupling lens and the convex face of the lens portion in the light axial direction should be set at from 1 mm to 4 mm (more preferably, 1.7±0.7 mm, while 1.7 mm is optimal).
Comparison Results
A case (the present invention) wherein the lens portion was provided for the sample container and a case (a conventional example) wherein condensing means was not provided for the sample container were compared. The present invention case was the optimal state, wherein a sample container to which a lens portion was attached was located at a distance of about 1.7 mm, in the light axial direction, from the convex face of a coupling lens.
The amount 76 mV of fluorescence detected for this invention is an actual value observed by a photomultiplier with an excitation light of 9.2 μW and an FITC density of 1 pmol/10 μL. The amount 8.6 mV of fluorescence detected in the comparative example is a value calculated based on the amount 76 mV for the invention (the amount of fluorescence detected in the case wherein a lens portion was not used/the amount of detected fluorescence in the case wherein a lens portion was used).
As shown in Table 1, when the present invention case (the lens portion was mounted on the sample container) is compared with the comparative example case (the lens portion was not provided for the sample container), it is found that, in the present invention case, about nine times the fluorescence can be detected.
Also, when a lens portion was not provided for the sample container, in order to efficiently collect fluorescence, the distance between the coupling lens and the sample container was reduced and the distance between the optical fiber and the coupling lens was extended, so that adjustment of the positions of the optical fiber and the coupling lens was difficult (since excitation light employed almost the entire face of the coupling lens, eclipsing of light tended to occur as a result of only a small position difference). On the other hand, according to this invention, since the distance between the optical fiber and the coupling lens is reduced, the positions of the optical fiber and the coupling lens could be easily adjusted.
In this embodiment, the optical fiber bundle has been employed. However, a plurality of optical fibers are not always necessary to receive fluorescence, and only a single optical fiber may be employed. Such an embodiment is shown in
Here, a step index silica fiber having a core diameter of 200 μm, a clad diameter of 250 μm and NA=0.2 is employed for an excitation light optical fiber, and a grated index fiber having a core diameter of 600 μm, a clad diameter of 750 μm and NA=0.2 is employed for a receiving light optical fiber. A cylindrical ferrule is employed and the diameter of the center through hole is 1.0 mm. Therefore, the center of the excitation light optical fiber is located at a distance of 0.375 mm from the center of the ferrule, while the center of the receiving light optical fiber is located at a distance of 0.5 mm from the center of the excitation light optical fiber, with the center of the ferrule in between. That is, the center of the receiving light optical fiber is located at a distance of 0.125 mm from the center of the ferrule.
A planoconvex lens with φ=2.4 mm and f=2.9 mm, which are the same as those in the previous embodiment, is employed as a coupling lens. On the surface of the coupling lens, reflection attenuation coatings are applied for an excitation wavelength and a fluorescence wavelength. The coupling lens and the optical fibers are positioned and secured by being inserted through a capillary. At this time, the flat face of the coupling lens is directed toward the optical fiber, while the convex face is directed toward the sample, and the distance between the coupling lens and the end face of the optical fiber is 2.1 mm in the light axial direction.
The same measurement was conducted for the same reagent used for the previous embodiment. It was found that when the distance z, in the light axial direction, was about 0.5 mm and the reagent was irradiated by excitation light of 11.3 φW, a fluorescence intensity of 8 pW was obtained by an optical power meter, and an extremely small amount of fluorescent signals could be efficiently measured. At this time, since the center of the excitation light optical fiber and the center of the receiving light optical fiber are shifted away from the center of the coupling lens, in accordance with the shifting distance, the arrangement of the components is shifted perpendicular to the z axis.
According to this embodiment, an explanation has been given only for a case wherein there is an excitation light wavelength of 470±10 nm and a detected fluorescence wavelength of 532±20 nm. The present invention is not limited to these wavelengths, and can be applied for any wavelength when a light source, a wavelength selection filter for excitation light and a wavelength selection filter for fluorescence are changed in accordance with the excitation spectrum and the fluorescence spectrum of a sample. For example, PE-Cy5 (R-Phycoerythrin/Cy5 tandem pigment) can be measured by setting a wavelength of 470±10 nm for excitation light and a wavelength of 670±20 nm for light to be detected. Further, Texas Red can be measured by setting a wavelength of 532±10 nm for excitation light and a wavelength of 640±20 nm for light to be detected.
| Number | Date | Country | Kind |
|---|---|---|---|
| P2005-326032 | Nov 2005 | JP | national |
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| Number | Date | Country |
|---|---|---|
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| Number | Date | Country | |
|---|---|---|---|
| 20070127020 A1 | Jun 2007 | US |
