Optics: measuring and testing – For light transmission or absorption – Of fluent material
Reexamination Certificate
1999-12-22
2001-02-06
Font, Frank G. (Department: 2877)
Optics: measuring and testing
For light transmission or absorption
Of fluent material
C356S441000, C356S410000, C250S574000
Reexamination Certificate
active
06184990
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Area of the Art
The present invention relates generally to the technology and device for fluorescence detection and more specifically to optical systems for laser-induced fluorescence detection in capillary electrophoresis, and methods of use thereof.
2. Description of the Prior Art
In fluorescence detection devices and technologies, it is important to improve the sensitivity of the fluorescence detectors. One of the key factors in improving the sensitivity of the fluorescence detectors is to collect maximum fluorescing radiation, i.e., providing the largest solid angle for collecting re-emitted fluorescence light from a flow cell containing moving analyte (i.e., CE and HPLC, etc.), while minimizing the background (i.e., collected excitation light intensity).
Therefore, in order to obtain optimum detection limit in a fluorescence detector, the fluorescence generated from the illuminated sample stream must be collected with high efficiency, while scattered excitation light reaching the detector must be minimized. In practice, high numerical aperture objectives or reflectors may be used to collect fluorescence. The fraction of light collected by a lens is related to the numerical aperture (N.A.) of the lens, and refractive index (n) of the surrounding medium. The following equation expresses this relationship:
Collection Efficiency=Sin
2
(
Arc
Sin(
N.A.
)/2) [1]
where collection efficiency of 1 implies that the lens collects all of the light emitted by the sample.
Usually the light collecting lens is surrounded by air, which has a refractive index of 1 (n=1). From Equation [1], it can be seen that a lens of very high numerical aperture is required to obtain a high collection efficiency. A lens with a numerical aperture of 1 will collect 50% of the light emitted by the sample. Although lenses immersed in a liquid medium (oil, water, etc.) can have a numerical aperture (N.A.) greater than 1, they collect less than 50% of the emitted light because the refractive index of the immersion fluid is usually larger than the refractive index of the lens material. In addition, according to Equation [1], a lens with a numerical aperture of 0.5 and immersed in air collects only 7% of the emitted light.
In conventional art, refractive and/or reflective optical collectors are utilized in laserinduced fluorescence (LIF) detectors with or without optical fibers for collecting the emitted fluorescing radiation. However, the capability of these collectors to collect fluorescence light is limited by their maximum collection angle. A typical high collector will have a collection cone angle of about 90 degrees. This corresponds to a 0.7 N.A., or 14% collection efficiency, (according to Equation [1]) in the optical system for a fluorescence based detector.
One example of a conventional 6-7% collection efficiency optical system is an LIF detector disclosed by U.S. Pat. No. 5,614,726 issued to Kaye et al. on Mar. 25, 1997 for “Automated Optical Alignment System and Method Using Raman Scattering of Capillary Tube Contents.” The fluorescence detector disclosed by Kaye uses a single excitation fiber which is the means for delivering the excitation light from the laser source to the capillary. As laser light is launched into a section of the capillary, due to the tubular nature of fused silica capillary, an isotropic (i.e., equally in all direction) excitation light scattering in the same plane as the incident light is produced. The fluorescence is collected by an ellipsoidal mirror and focused on to a photo-multiplier tube (PMT) detector. A collection mirror (i.e., the ellipsoidal reflector) with a through hole at the center is located in the opposite side and on the same plane as the excitation fiber which allows most of the unwanted excitation light from the fiber to pass into an empty dump hole. This dump hole is large enough to allow the passage of the excitation fiber's light cone, which is about 25 degrees (N.A.=0.22) for this case. The scattered (unwanted) excitation light is further attenuated by a beam block. With all these baffles and beam dumps, the collection efficiency capability of this system is about 6-7% (0.7 N.A. for the mirror minus 0.2 N.A. is equal to 0.5 N.A. or approximately 7% collection efficiency).
Therefore, a need exists to develop a new LIF detector in capillary electrophoresis that has a higher collection efficiency optics and enhanced signal strength.
SUMMARY OF THE INVENTION
It is an object of the present invention to improve the collection efficiency and emission signal strength of laser-induced fluorescence detectors in capillary electrophoresis.
It is also an object of the present invention to provide a novel miniature multiple wavelength excitation and emission optical system for laser-induced fluorescence detectors in capillary electrophoresis.
These and other objects and advantages are achieved in a miniature multiple wavelength excitation and emission optical system for laser-induced fluorescence detectors in capillary electrophoresis. The system of the present invention comprises a first spherical reflector and collector on one side of the capillary flow cell, and a second sapphire ball (or modified lens) collector and collimator on the opposite side of the flow cell, where excitation light from multiple sources are delivered by optical fibers arranged in the plane orthogonal to the axis of the collection optics.
Such an arrangement has been found to provide a number of advantages. As explained in greater detail below, it has been found that by providing a first spherical reflector and collector on one side of the capillary flow cell, and a second ball (or modified) lens collector and collimator on the opposite side of the flow cell, a larger solid collection angle can be obtained from each side of the flow cell, which results in increased collection efficiency and enhanced signal strength. It has also been found that by arranging the optical fibers which deliver the excitation lights in the plane orthogonal to the axis of the collection optics, the leakage of back-scattered excitation light to the collection optics is reduced, which results in a lower scattered background and a higher signal-to-noise fluorescence detection. Furthermore, the design is self aligning with respect to the flow cell, without any moving optical parts.
The present invention is well suited for use in detection of fluorescence in a capillary flow cell of a capillary zone electrophoresis instrument such as, but not limited to, the P/ACE MDQ™ Capillary Electrophoresis System, available from Beckman Coulter, Inc. It can also be applied to multiple capillary fluorescence detectors. The arrangement of the present invention can easily accommodate multiple excitation wavelengths to excite multiple fluorescence species in a sample. Furthermore, it can be readily coupled to a filter based or dispersive spectrophotometer (
FIG. 9
) instrument for performing analysis of the spectral information to quantify the amount of fluorescing species in a given sample in the flow cell.
The invention is defined in its fullest scope in the appended claims and is described below in its preferred embodiments.
REFERENCES:
patent: 5037199 (1991-08-01), Hlousek
patent: 5089714 (1992-02-01), Ludlow et al.
patent: 5484571 (1996-01-01), Pentoney, Jr. et al.
patent: 5491765 (1996-02-01), Matsumoto
patent: 5584557 (1996-12-01), Alexay
Amirkhanian Varouj
Deliwala Sunil S.
Franck Ronald W.
Tepermeister Gary
Wanders Bart J.
Beckman Coulter Inc.
Font Frank G.
Kivinski Margaret A.
May William H.
Nguyen Sang H.
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