Optics: measuring and testing – By dispersed light spectroscopy – With sample excitation
Reexamination Certificate
2001-01-11
2003-12-09
Evans, F. L. (Department: 2877)
Optics: measuring and testing
By dispersed light spectroscopy
With sample excitation
C356S317000, C356S344000, C250S458100
Reexamination Certificate
active
06661510
ABSTRACT:
The present invention relates to an improvement in the kind of devices that are being used for detection of fluorescent species.
Fluorescence detection or fluorometry is a well established and often used method within analytical chemistry. The main features of fluorescence detection are high selectivity and very high sensitivity, and the method is often applied to detection of trace constituents in samples of various kinds. Fluorescence detectors consist, in general, of three main subsystems, i/ an excitation light source and associated optics, ii/ a sample cell, and iii/ collection optics and light detector. The light source generates the light that excites the fluorescent species. The most often used light sources are high intensity lamps, like, e.g., xenon lamps or lasers. The excitation optics transports the light from the light source to the illumination zone, where the light excites the sample. Focusing optics is most often used, but also fiber optics and other kinds of waveguides, for example, may be used. When a laser light source is used, the focusing optics may, in some cases, be omitted. The sample can contain one or several fluorescent species. The sample is, in general, present in a medium, e.g., a liquid solution, which in turn is contained in some kind of sample cell. The sample cell may, e.g., be a compartment into which the sample is first loaded, then detected while being. stationary, and finally withdrawn. The cell may also be part of some kind of conduit, through which the sample is transported to and from the illumination zone. The collection optics collects the emitted fluorescent light in an efficient way, and transports it to the light detector. Also for the collection optics, focusing elements are commonly used, but also, e.g., fiber optics may be used. The collection, as well as the excitation, optics may also comprise some kind of device, e.g., a monochromator or one or several filters, for selection or dispersion of wavelengths. The excitation is most often performed at one wavelength or a few well-defined wavelengths, or, alternatively, the excitation wavelength may be scanned. The detection may be performed at one or several discrete wavelengths or wavelength intervals, or scanned or dispersed over a wavelength interval, or the total amount of emitted light may be detected. Wavelength selective detection increases the versatility and selectivity of fluorometry, and is a prerequisite in applications like, e.g., four colour DNA sequencing. There are many different kinds of light detectors, e.g., photodiodes, diode arrays, CTD:s (charge transfer devices, including CCD:s (charge coupled devices) and CID:s (charge injection devices)), and photomultiplier tubes.
One of the most common and most important uses of fluorometry is as a detection method in connection with analytical methods wherein the sample is contained and transported in some kind of conduit. Such analytical methods include, but are not limited to, CE (capillary electrophoresis), LC (liquid chromatography), and FIA (flow injection analysis). In this context, the present invention will mainly be discussed in connection with CE, but applications to other analytical methods are obvious to the skilled person. CE is a well-established separation method with the possibility to analyse very small amounts of sample, and yielding a very high separation efficiency.
Fluorescence detection, and especially LIF (laser induced fluorescence), is a well-established detection technique for CE. Lasers have two main advantages: i/ the high intensity of the light, and ii/ the ability to focus the laser beam to a small spot within the capillary. It is important that the size of the light beam at the point of excitation does not contribute to band broadening: the width of CE peaks may require beam diameters of 100 &mgr;m or less. In the most common, and well-established, optical set-up, the orthogonal set-up, the capillary is illuminated with a laser, and the emitted light is collected at 90° to the direction of the laser beam. The main concerns, in order to maximise the sensitivity, are to maximise the light collection efficiency, and to minimise the amount of stray light reaching the light detector. High collection efficiency is, in general, obtained by using high numerical aperture collection optics. The term stray light is used here to denote all kinds of unwanted, detected light. Stray light may, to some extent, be rejected through the use of spectral and/or spatial filters. Electrophoresis capillaries are often protected by a polymer coating, e.g., polyimide, which has to be removed before fluorescence detection can be performed. Scattering of primary laser light may occur if there are polymer or other particles left on the capillary wall, if the wall is scratched, or if there are heterogeneities within the wall or the medium inside the capillary. Further, light scattering occurs at every optical interface according to Fresnel's laws of reflection. In particular, the cylindrical columns ordinarily used in CE pose a problem, since they scatter light also at 90° to the direction of the laser beam. Also, most materials scatter light by elastic (Rayleigh) Raman molecular scattering. Scattered primary light may often, but not in all cases, be efficiently rejected by spectral filtering or wavelength dispersion. Wavelength shifted secondary light may present a more severe problem. Inelastic (Stokes shifted) Raman scattering or fluorescence emission from polymer or dirt particles on the column wall, from the column wall itself, from the medium in which the sample is contained, or from impurities in the medium or in the sample itself may not be easily rejected by spectral filtering or wavelength dispersion. Spatial filtering may be obtained by, e.g., shallow focal depth collection optics and apertures. The light collection is spatially concentrated to the region of the medium, while light emanating from other regions is rejected.
For high efficiency separation methods, utilizing small diameter columns and small samples, and yielding very narrow analyte bands at the detector, like e.g., micro-LC and, in particular, CE, it is imperative that the detection is performed on column and that the detection volume is as small as possible. Use of an external detection cell with diameter larger than the column will lead to band broadening, and coupling to such a cell does, in general, cause dead volumes leading to further band broadening. The maximum allowable detection volume for, e.g., a highly efficient CE separation on a 100 &mgr;m column may be on the order of or less than 2 nl.
One proposed device for maximising light collection efficiency and minimising stray light is the confocal fluorescence microscope [Ju, J. et al., Anal. Biochem. 1995, 231, 131-40]. A laser beam is reflected by a low-pass dichroic beam splitter, and focused by a microscope objective to a very small spot, on the order of 10 &mgr;m, inside the capillary. The emitted fluorescent light is collected by the same objective, but transmitted through the beam splitter to the detection optics. By focusing the collection optics tightly inside the capillary, stray light contributions from the capillary wall are diminished. By placing an aperture at the focal point of the collected fluorescent light, stray light may be further rejected by spatial filtering. High light collection efficiency is achieved by using a high numerical aperture microscope objective. Drawbacks of this device include the need for very strict mechanical tolerances, very careful optical alignment, and the sensitivity to, e.g., vibrations. These drawbacks are a consequence of the shallow focal depth utilized. Further, for cylindrical capillaries, the problem of focusing light and light collection in the interior of a body lacking circular symmetry is encountered.
Another proposed device for optimisation of detection sensitivity is the sheath flow cell [Swerdlow, H. et al., Anal. Chem. 1991, 63, 2835-41; Chen, D. Y. et al., J. Chromatogr. 1991, 559, 237-46]. The anal
Hanning Anders
Roeraade Johan
Evans F. L.
Geisel Kara
Goldberg Joshua B.
Hanning Instruments AB
Juneau Todd L.
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