Optics: measuring and testing – By dispersed light spectroscopy – With sample excitation
Reexamination Certificate
2000-04-20
2003-11-25
Evans, F. L. (Department: 2877)
Optics: measuring and testing
By dispersed light spectroscopy
With sample excitation
C356S333000, C250S458100
Reexamination Certificate
active
06654119
ABSTRACT:
TECHNICAL FIELD
This invention relates to wavelength scanning fluorescence spectrophotometers using dual grating monochromators, but not optical filters, to select excitation and emission wavelengths of light and to detect and quantify simultaneous fluorescence emission from multiple fluorophores in the same sample.
BACKGROUND
Definitions:
1) Fluorescence: The result of a multi-stage process of energy absorption and release by electrons of certain naturally occurring minerals, polyaromatic hydrocarbons and other heterocycles.
2) Excitation: photons of energy, e=hv
exc
, are supplied by a light source and absorbed by an outer electron of a fluorophore, which is elevated from the ground state, S
0
, to an excited electronic singlet state, S′
1
.
3) Excited State Lifetime: An excited electron remains in the singlet state for a finite period, typically from 1 to 20 nanoseconds, during which the fluorophore undergoes a variety of changes including conformational changes and alterations in the interaction with solvent. As a result of these changes, the energy of the S′
1
singlet electron partially dissipates to a relaxed singlet excited state, S
1
, from which fluorescence emission of energy occurs, returning the electron to the ground state, S
0
.
4) Emission: photons of energy, e=hv
em
, are released from an excited state electron, which returns the fluorophore to the ground state. Owing to energy loss during the excited state lifetime, the energy of these photons is lower than that of the exciting photons, and the emitted light is of longer wavelength. The difference in the energy (or wavelengths) is called the Stoke's shift and is an important feature in the selection of a dye for use as a label or in a probe. The greater the Stoke's shift, the more readily low numbers of photons can be distinguished from background excitation light.
5) Fluorophores: Fluorescent molecules are generally referred to as fluorophores. When a fluorophore is utilized to add color to some other molecule, the fluorophore is called a fluorescent dye and the combination is referred to as a fluorescent probe. Fluorescent probes are designed to: 1) localize and help visualize targets within a specific region of a biological specimen, or, 2) respond to a specific stimulus.
6) Electromagnetic Spectrum: the entire spectrum, considered as a continuum, of all kinds of electric and magnetic radiation, from gamma rays, having a wavelength of 0.001 Angstroms to long waves having a wavelength of more than 1,000,000 kilometers and including the ultraviolet, visible and infrared spectra.
7) Fluorescence Spectrum: Unless a fluorophore is unstable (photobleaches), excitation and emission is a repetitive process during the time that the sample is illuminated. For polyatomic molecules in solution, discrete electronic transitions are replaced by broad energy bands called the fluorescence excitation and fluorescence emission spectra, respectively.
8) Monochromator: A device which admits a wide spectral range of wavelengths from the electromagnetic spectrum via an entrance aperture, and, by dispersing wavelengths in space, makes available at an exit aperture only a narrow spectral band of prescribed wavelength(s). Optical filters differ from monochromators in that they provide wavelength selection through transmittance of selected wavelengths rather than through spatial dispersion. A second distinguishing feature of a monochromator is that the output wavelength(s), and in many cases, the output spectral bandwidth, may be continuously selectable. Typically, the minimal optical components of a monochromator comprise:
(a) an entrance slit that provides a narrow optical image;
(b) a collimator which ensures that the rays admitted by the slit are parallel;
(c) some component for dispersing the admitted light into spatially separate wavelengths;
(d) a focusing element to re-establish an image of the slit from selected wavelengths; and,
(e) an exit slit to isolate the desired wavelengths of light.
In a monochromator, wavelength selection is achieved through a drive system that systematically pivots the dispersing element about an axis through its center. Slits are narrow apertures in a monochromator which may have adjustable dimensions. Slits effect selection of the desired wavelength(s) and their dimensions may be adjustable.
9) Double Monochromator: Two monochromators coupled in series. The second monochromator accepts wavelengths of light selected by the first and further separates the prescribed wavelengths from undesired wavelengths.
10) Wavelength Scanning: Continuous change of the prescribed output wavelength(s) leaving the exit slit of a monochromator. In a spectrophotometer, wavelengths of the electromagnetic spectrum are scanned by the excitation monochromator to identify or prescribe the wavelength(s) at which a fluorophore is excited; wavelength scanning by the emission monochromator is used to identify and detect the wavelength(s) at which a fluorophore emits fluorescent light. In automated fluorescence spectrophotometers, wavelength scanning by the excitation and emission monochromators may be performed either separately or concurrently (synchronous scanning).
11) Area Scanning: Area scanning is distinct from wavelength scanning and is the collective measurement of local fluorescence intensities in a defined two dimensional space. The result is an image, database or table of intensities that maps fluorescence intensities at actual locations in a two dimensional sample. At its simplest, area scanning may be a photograph made with a camera in which all data are collected concurrently. Alternatively, the sample may be moved past a detector which measures the fluorescence in defined sub-areas of a sample. The collected information creates a matrix which relates fluorescence intensity with position from which an image, table or graphical representation of the fluorescence in the original sample can be created.
12) Fluorescence Detectors
Five elements of fluorescence detection have been established through laboratory use of fluorophores during the last two decades:
(a) an excitation source,
(b) a fluorophore,
(c) some type of wavelength discrimination to isolate emission photons from excitation photons,
(d) some type of photosensitive response element that converts emission photons into a recordable form, typically an electronic signal or a photographic image, and,
(e) a light tight enclosure to restrict ambient light.
Fluorescence detectors are primarily of four types, each providing distinctly different information:
(a) Cameras resolve fluorescence as spatial coordinates in two dimensions by capturing an image: [a] as a photographic image on highly sensitive film, or, [b] as a reconstructed image captured on arrays of pixels in a charge coupled device (CCD).
(b) Fluorescence microscopes also resolve fluorescence as spatial coordinates in two or three dimensions. Microscopes collect all of the information for an image for a prescribed visual field at the same time without any movement of either the sample or the viewing objective. A microscope may introduce qualitative estimation of fluorophore concentration through use of a camera to capture an image in which case the measure is a function of exposure time.
(c) Flow cytometers measure fluorescence per biological cell in a flowing liquid, allowing subpopulations within a mixture of cells to be identified, quantitated and in some cases separated. Flow cytometers cannot be used to create an image of a defined area or perform wavelength scanning. The excitation light source is invariably a laser and wavelength discrimination is accomplished through some combination of tunable dye lasers and filters. Although these instruments may employ photomultiplier tubes to detect a measurable signal, there are no flow cytometers that employ monochromators for wavelength scanning.
(d) Spectrofluorometers (spectrophotometer(s)) typically employ a PMT to detect fluorescence but can measure either: [a] the average current ev
Conrad Michael J.
Gould Gene
Chromagen, Inc.
Evans F. L.
Pillsbury & Winthrop LLP
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