Optics: measuring and testing – By dispersed light spectroscopy – With sample excitation
Reexamination Certificate
2001-01-11
2004-03-30
Evans, F. L. (Department: 2677)
Optics: measuring and testing
By dispersed light spectroscopy
With sample excitation
C356S318000, C250S458100
Reexamination Certificate
active
06714297
ABSTRACT:
The invention relates to an optical arrangement for detecting light emitted by a sample.
The detection of molecules with the help of optical biosensors is done as a rule by detecting a specific binding between the molecules and what are called “receptor molecules”, which are immobilized on a surface. The detection of molecules can be performed very selectively, even from a mixture, by way of such a specific binding. Antibodies or DNA molecules are typical receptor molecules. Typical surfaces are on optical fibers or transparent microscopic slides or cover sheets.
Often, a fluorescent signal specific to the binding between the molecule sought and the receptor molecule is detected in order to detect the molecules. This can be generated by the molecules sought themselves, if they are able to fluoresce.
However, as a rule, what is called a “sandwich test” is performed, in which the molecule sought is not able to fluoresce, but a third, fluorescence labeled “probe molecule” binds selectively to the binding complex of the molecule sought and the receptor molecule, after this complex has formed.
In order to obtain quantitative information on the quantity of the molecules sought bound to the receptor molecules, only that fluorescence light must be detected that indicates a binding complex between the molecule sought and the receptor molecule. Such a complex is always located close to that surface on which the receptor molecules are immobilized. From this, it follows that the measurement task is to detect selectively fluorescent signals from molecules bound close to the surface, that is, from near-surface layers.
Recently, the detection of near-surface fluorescence with the help of the detection of what is called “evanescent radiation” in optical sensors has proven effective. The following physical principle is exploited for this:
When a ray of light strikes an interface between a medium with a higher refractive index and one with a lower refractive index, total internal reflection can occur if the angle of incidence of the ray of light with respect to the surface normal is larger than the critical angle of total internal reflection ax, which is determined by
sin &agr;=Error! (1)
(where n
2
=index of refraction of the medium with a lower one, and n
1
=index of refraction of the medium with a higher one). Rays of light that strike the interface at an angle larger than &agr; cannot leave the medium with the higher refractive index, according to considerations of geometrical optics.
And vice-versa, it is not possible according to geometrical optics for a ray of light to penetrate from the medium with a lower index of refraction into the one with a higher index, if it propagates in the medium with the higher refractive index at an angle to the interface that is larger than the critical angle of total internal reflection &agr;.
However, more precise electromagnetic considerations show that the field strength of the light wave striking the interface from the medium with the higher refractive index and being totally internally reflected does not drop abruptly to zero at the interface to the medium with the lower refractive index, but rather decreases exponentially from its value in the medium with the higher refractive index as a function of the distance from the interface. Typical decay constants for this exponential decline in the field strength lie in the range of the wavelength of the incident light. Thus despite total internal reflection, a certain portion of the light enters the medium with the lower refractive index.
Likewise, light from a location in the medium with the lower refractive index that is near the interface can also enter the medium with the higher refractive index at an angle that is greater than the critical angle of total internal reflection &agr;.
This effect is exploited in optical fiber sensors. In these sensors, receptor molecules are immobilized on the surface of the fiber. Exciting light, usually laser light, is launched into the fiber. The laser light propagates along the fiber by total internal reflection. It also passes the regions of the fiber on whose surface the molecules able to fluoresce are bound. Due to the evanescent field of the exciting light at the surface of the fiber, molecules able to fluoresce can be excited. In the bound state, the distance of the molecules from the surface of the fiber is only a few nanometers, so that the exponential drop of the exciting light's field strength has little effect. Therefore, the molecules bound to the surface are effectively excited by the exciting light. The light emitted by the molecules can in turn be launched into the fiber, in part at angles at which the fluorescence light, once inside the fiber, propagates along the fiber due to total internal reflection. The propagated portion of fluorescence light from the bound molecules can then be detected at one end of the fiber.
A disadvantage of optical fiber sensors that detect evanescent radiation is that the efficiency with which they can collect (capture and transmit) emitted fluorescence photons is very limited. In particular, the sensitivity of such an arrangement is not sufficient to detect individual fluorescence molecules.
The object of the invention is to improve the light-collecting efficiency for light launched evanescently into a medium with a higher refractive index.
According to the present invention, this object is achieved by an optical arrangement.
The optical arrangement according to the invention can detect both light that has been generated on an interface, for example due to chemoluminescence, and also light scattered on the interface, for example fluorescence light due to excitation by a light source.
The light thus generated is collected by a light-collecting device. Besides the optical waveguide according to the invention, this can also include additional lenses, mirrors, filters, and other usual optical components.
The light collected by the light-collecting device is detected by the detection device. This also can include, in addition to an ordinary detector, additional lenses or filters for directing the light onto the detector.
The first end face may be entirely planar, or only flat in a region where the sample is located. The interface in front of which the sample is located can be formed directly by the first end face itself. Or alternatively, the first end face can be optically coupled to an interface between the sample medium and the medium with a high index of refraction. In this case, the medium with the high index of refraction can be the medium of an microscopic slide that is coupled to the first end face with the help of an immersion oil with a high refractive index. In the latter case, the diameter of the microscopic slide and the dimensions of the first end face should be selected so that light emitted by the sample, and radiated into the half-space facing the medium with a high index of refraction, is not prevented from entering the first end face by purely geometrical limitations.
If light is emitted by the sample near the interface, for example by generating fluorescence light, then a portion of the light enters the optical waveguide through the first end face, and propagates at an angle (with respect to the surface normal of the first end face at the site of the sample) within the optical waveguide which is larger than the critical angle of total internal reflection corresponding to the ratio of indices of refraction of the medium with a high index of refraction and the sample medium. In fiber sensors, this “evanescent” radiation could only propagate in part to the end of the fiber, and thus onto a detector. According to the invention, the arrangement and construction of the shell surface ensures that the evanescent radiation at the shell surface is totally reflected back into the optical waveguide. This can be done on the one hand by means of total internal reflection, or on the other hand by an appropriate mirror-coating of the shell surface. The evanescent radiation can then b
Ruckstuhl Thomas
Seeger Stefan
Baldwin Stephen E.
Evans F. L.
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