Radiant energy – Luminophor irradiation
Patent
1992-02-24
1993-12-28
Hannaher, Constantine
Radiant energy
Luminophor irradiation
2504591, 2504611, G01N 2164
Patent
active
052742407
DESCRIPTION:
BRIEF SUMMARY
to provide a continuous sampling of the capillary volume.
These and other objects of the invention are achieved by a laser-excited capillary array scanner including a plurality of capillaries having a parallel, side-by-side, coplanar relationship and a laser-excited confocal fluorescence detector for detecting fluorescence from a selected interior volumes of each of said capillaries sequentially and repetitively during electrophoresis or other separation method. The invention also relates to a method of analyzing a plurality of capillaries, with a single scanner, by scanning a plurality of capillary passages in side-by-side relationship, and periodically and repetitively detecting fluorescence from each capillary passage during electrophoresis or any other separation procedure.
BRIEF DESCRIPTION OF THE DRAWINGS
The foregoing and other objects of the invention will be more clearly understood from the following description when read in connection with the accompanying drawings, wherein:
FIG. 1 is a schematic diagram of a confocal-fluorescence capillary array scanner in accordance with one embodiment of the invention;
FIG. 2 is a view of a holder for supporting a region of the capillaries in side-by-side relationship;
FIG. 3 is an enlarged view of the focal zone;
FIGS. 4A and 4B illustrate how the excitation beam is focused to a volume in the interior of a cylindrical capillary;
FIG. 5 is an image obtained by scanning a four-capillary array during a DNA separation;
FIGS. 6 (A-D) are an electropherogram of the DNA separation of FIG. 5;
FIGS. 7 (A-D) are an expanded view of the indicated regions of the electropherograms of FIGS. 6 (A-D);
FIG. 8 is an image obtained by scanning a twenty-four capillary array; and
FIG. 9 is a schematic diagram of a four-color confocal-fluorescence capillary scanner.
DESCRIPTION OF PREFERRED EMBODIMENT(s)
In accordance with this invention, the throughput in capillary electrophoresis is increased by employing a large number of capillaries in parallel. The most important problem confronting capillary array electrophoresis is detection. In copending patent application Ser. No. 07/531,900 filed Jun. 1, 1990, and incorporated herein by reference, there is described a laser-excited confocal fluorescence gel scanner which provides enhanced detection of fluorescently labelled DNA in slab gels. This detection system uses an epi-illumination format where the laser is focused on the sample by a microscope objective and the emitted fluorescence is gathered by the same objective using a 180.degree. retro-optical geometry followed by confocal detection.
Sensitive detection of fluorescently-labeled analytes separated in small diameter capillaries is a difficult task. Because the capillaries have a 100 .mu.m I.D. or less, a small focal volume is needed. The detection system must reject potentially strong Rayleigh scattering, fluorescence, and reflections from the capillary walls. Using confocal excitation and detection, the depth of field of the optical system is sufficiently small that only the interior of the 100 .mu.m I.D. capillary is probed. The lateral resolution which is dictated by the scan stage and the laser beam diameter can be as small as a few microns. Background scattering and reflections from the capillary wall are rejected by the spatial and spectroscopic filters in front of the photodetector.
A confocal fluorescence detection system for use with capillary arrays is shown in FIG. 1. An argon ion laser (Model 2020, Spectra-Physics, Mountain View, Calif.), not shown, is used as the excitation source. The laser beam is expanded to 5 mm diameter, collimated, and then directed through a 32.times., N.A. 0.4 infinite conjugate objective 11 (LD Plan-Achromat 440850, Carl Zeiss, West Germany) by a long-pass dichroic beamsplitter 12 (480 DM, Omega Optical, Brattleboro, Vt.). The dichroic beam splitter 12 reflects the excitation laser beam into the objective 11 but transmits fluorescent light collected by the objective which is Stokes shifted to longer wavelengths. The objective focuses
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This invention was made with U.S. Government support under Grant Contract No. DIR-87-20382 awarded by the National Science Foundation, and Grant Nos. 88-ER-60706 and 91-ER-61125 awarded by the Department of Defense. The U.S. Government has certain rights in this invention.
Huang Xiaohua C.
Mathies Richard A.
Quesada Mark A.
Glick Edward J.
Hannaher Constantine
The Regents of the University of California
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