Optics: measuring and testing – By dispersed light spectroscopy – With sample excitation
Reexamination Certificate
1999-06-21
2001-05-22
Evans, F. L. (Department: 2877)
Optics: measuring and testing
By dispersed light spectroscopy
With sample excitation
C250S458100, C422S082080, C436S172000
Reexamination Certificate
active
06236456
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates generally to detection systems, and more particularly, to a method and apparatus for detecting fluorescence, luminescence, or absorption in a sample.
BACKGROUND OF THE INVENTION
In biology as well as other related scientific fields, samples are routinely characterized by examining the properties of fluorescence, luminescence, and absorption. Typically in a fluorescence study, selected tissues, chromosomes, or other structures are treated with a fluorescent probe or dye. The sample is then irradiated with light of a wavelength that causes the fluorescent material to emit light at a longer wavelength, thus allowing the treated structures to be identified and to some extent quantified. The wavelength shift between the peak excitation wavelength and the peak fluorescence wavelength is defined as the Stokes shift and is the result of the energy losses in the dye molecule.
In a luminescence study, the sample material in question is not irradiated in order to initiate light emission by the material. However, one or more reagents may have to be added to the material in order to initiate the luminescence phenomena. An instrument designed to monitor luminescence must be capable of detecting minute light emissions, preferably at a predetermined wavelength, and distinguishing these emissions from the background or ambient light.
In a typical light absorption study, a dye-containing sample is irradiated by a light source of a specific wavelength. The amount of light transmitted through the sample is measured relative to the amount of light transmitted through a reference sample without dye. In order to determine the concentration of dye in a sample, both the light absorption coefficient (at the wavelength used) and the path length through the sample must be known. Other relative measurements may also be of interest, for example determining the wavelength dependence of the absorption.
In general, an instrument designed to determine the fluorescence of a sample requires at least one light source emitting at one or more excitation wavelengths and a detector for monitoring the fluorescence emissions. This same instrument can often be used for both luminescence and absorption measurements with only minor changes.
U.S. Pat. No. 4,626,684 discloses a fluorescence measurement system for use with a multi-assay plate. The disclosed system uses concave holographic gratings to control both the excitation and emission detection wavelengths. Optical fibers are used to couple the optical scanning head to both the source and detector subassemblies. The paths of both the excitation light and the fluorescent emissions are orthogonal to the surface of the material under study.
U.S. Pat. No. 4,501,970 discloses a fluorometer for use with multi-assay plates. The disclosed system directs the excitation beam of light through the open top of the sample holding vessel and receives the fluorescent emission through this same opening. The system uses a series of mirrors and masks to decouple the excitation light from the emitted fluorescence, thereby reducing the noise signal level in the detector and increasing the sensitivity of fluorescence detection.
From the foregoing, it is apparent that a high sensitivity, wavelength scanning fluorometer is desired.
SUMMARY OF THE INVENTION
The present invention provides a method and apparatus for determining the fluorescence, luminescence, or light absorption of a sample. The sample may either be contained within a cuvette or within one or more sample wells of a multi-assay plate. The system is designed to accommodate a variety of different multi-assay plates in which the plate dimensions as well as the number of sample wells varies.
In one aspect of the invention, an excitation means is provided for either fluorescence or absorption measurements. The excitation means includes a broadband light source, a monochromator, and a series of optical filters. This combination of optical components allows the excitation wavelength to be tuned to a predetermined value within a relatively wide wavelength band. Depending upon the dispersion of the components, bandpass values of approximately 10 nanometers are commonly achievable. A similar optical configuration is used to detect the emissions from the sample (i.e., fluorescence or luminescence) or the amount of light absorbed by the sample. The detection means includes a photomultipler tube detector, a diffraction grating, and a series of optical filters.
In another aspect of the invention, multiple optical fibers are coupled to the excitation source, thus allowing the system to be quickly converted from one optical configuration to another. For example, the source can be used to illuminate either the top or the bottom of a sample well within a multi-assay plate or to illuminate a single cuvette cell. Similarly, multiple optical fibers are coupled to the detector. The multiple detector fibers allow the system to be easily converted from detecting fluorescence or luminescence to detecting the amount of excitation light passing through the sample (i.e., for absorption measurements). The multiple detection fibers also allow the optical configuration to be converted to match the excitation configuration, e.g., cuvette cell versus multi-assay plate.
In another aspect of the invention, the excitation light and the detected sample emissions pass to and from an optical head assembly via a pair of optical fibers. The optical head assembly is coupled to a pair of guide rails and controlled by a step motor, thus allowing the head assembly to be driven along one axis of a multi-assay plate. The multi-assay plate is mounted to a carriage assembly that is also coupled to a pair of guide rails and controlled by a step motor. The carriage assembly drives the multi-assay plate along a second axis orthogonal to the first axis.
In another aspect of the invention, the system is designed to accommodate a wide range of sample intensities automatically, such as would be expected from a group of random samples within a multi-assay plate. In order to accommodate varying intensities, a photomultiplier tube detector is used and the voltage is automatically varied in order to change its gain. The automatic voltage adjustment is performed in three steps, each providing a nominal dynamic range of three decades. Alternatively, the voltage adjustment can be performed in more than three steps employing finer gradations of dynamic range.
In another aspect of the invention for use with a multi-assay plate configuration, the system is designed to minimize the effects of temperature drop from one sample to another that are due to evaporative cooling. Specifically, the plate holding carriage moves the multi-assay plate to a sample holding area between readings. Within the sample holding area the multi-assay plate is confined by an upper or lid surface that is close to the upper surface of the multi-assay plate. The sides of the multi-assay plate may also be confined. When the multi-assay plate is within this area the relative humidity above the plate rises to more than 90 percent, thus reducing evaporative cooling. This aspect of the invention is preferably coupled to a temperature regulation and air circulation system.
In another aspect of the invention, an optical scanning head assembly is used that includes mirrored optics for coupling an excitation source to the sample and the emitted light to a detector. An ellipsoidal focussing mirror is used to magnify and focus the source light projected from an optical fiber onto the sample. A portion of the source light is reflected by a beamsplitter onto a reference detector used to monitor the output of the source. The light from the ellipsoidal mirror passes through an aperture in a second ellipsoidal mirror prior to impinging upon the sample. The light emitted by the sample within the sample well (e.g., fluorescence) is reflected by the second ellipsoidal mirror and imaged onto the entrance aperture of an optical fiber coupled to the detector subassembly. The optical axes of
Giebeler Robert
Hafeman Dean
Kaye Roger
Ogle David G.
Beck David G.
Evans F. L.
McCutchen Doyle Brown & Enersen LLP
Molecular Devices Corporation
LandOfFree
Optical system for a scanning fluorometer does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Optical system for a scanning fluorometer, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Optical system for a scanning fluorometer will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2450921