Radiant energy – Luminophor irradiation – Methods
Reexamination Certificate
2003-06-04
2004-09-21
Evans, F. L. (Department: 2877)
Radiant energy
Luminophor irradiation
Methods
Reexamination Certificate
active
06794659
ABSTRACT:
BACKGROUND OF THE INVENTION
The invention relates to improvements in a multiple channel spectrometer capable of quickly analyzing large volumes of samples by the fluorescence emitted by the samples. While many different systems for causing fluorescence emission and processing of the fluorescence may be used, one currently preferred method is known as fluorescence correlation spectroscopy (also known as fluorescence fluctuation spectroscopy). Examples of this technique are as described in Schrof et al. U.S. Pat. No. 5,815,262. Other examples of fluorescence cross-correlation spectroscopy are shown in the articles by Andre Koltermann et al. from the
Proceedings of the National Academy of Science
, Volume 95, pages 1421-1426, February 1998 entitled “Rapid Assay Processing by Integration of Dual Color Fluorescence Cross-Correlation Spectroscopy: High Throughput Screening for Enzyme Activity”; and the article by Petra Schwille et al. from the
Biophysical Journal
, Volume 72, pages 1878-1886, April 1997, entitled “Dual Color Fluorescence Cross-Correlation spectroscopy for Multi Component Diffusional Analysis Solution”.
In the Schrof et al. U.S. Pat. No. 5,815,262, fluorescence correlation spectroscopy (FCS) is described in which an excitation laser beam passes through a series of wells (sample chambers) arranged in linear array. The beam is refocused prior to entering each sample chamber, so that fluorescence takes place in a very small area, which area is monitored and sensed to obtain fluorescence data simultaneously from a plurality of samples.
With such a technique, difficulties have been found in the refocusing of light after the laser beam has passed through the first well or sample chamber. Also, possible change of the laser light beam may take place as it passes through the various, separate samples, which may effect the data obtained from particularly the “downstream” samples in the linear array. Also, two photon excitation, as is used in the above cited patent, generally requires a very expensive laser, while one photon excitation permits the use of cheaper lasers.
Additionally most of the prior art systems utilize a conventional hardware correlator to receive the raw data from multiwell plates in fluorescence correlation spectroscopy. Data from the hardware correlator is passed to the computer processing unit for determination of the parameters of interest, which may include diffusion coefficients and the concentrations of the components. However, several limitations hinder the use of a hardware correlator in this manner. Specifically, when a particulate or an aggregate is present in the solution being analyzed, and it passes through the tiny region illuminated by the focused laser beam, the entire calculated autocorrelation function resulting from the data has to be rejected, as it is altered by the temporary presence of the particulate or aggregate. As the result, the measurement on that specific well from where the data comes has to be rejected, and, more likely, measurement of the entire multiwell plate has to be acquired again.
Also, the raw data may contain useful information and features that are completely and definitively lost once the data has been processed through the hardware correlator. By system of this invention, the user can examine the raw data after they have been acquired. Specifically, it may turn out that higher order correlation functions may be of more interest in describing the molecular interactions occurring in the sample solutions being analyzed through the spectrometer. Thus, if one can keep the raw data, this permits the user to further analyze it with more complicated analysis models when that is desired.
The analysis can be easily implemented in an automated fashion by properly designed software for a spectrometer, particularly for high throughput screening instruments.
Also, the first order autocorrelation function of fluorescence correlation spectroscopy is typically determined by using the time-mode, which is the traditional way of calculating the function. See particularly Thompson, Fluorescence Correlation Spectroscopy, in “
Topics in Fluorescence Spectroscopy
”, Volume 1 (J. R. Lakowicz, Editor), Plenum Press, New York 1991, pages 337-410. Time mode operations tend to limit the precision in determining concentrations, and increase the data acquisition time.
It is desired to perform drug and other screening at very large rates of analysis. For example, current techniques can allow screening up to 50,000 to 100,000 compounds a day. However, it should be desirable in the field of high throughput screening to significantly increase the capacity of spectrometers to process large numbers of compounds. Many of the current high throughput screening apparatus are manufactured by L. J. L. Biosystems of Sunnyvale, Calif.; Aurora Biosciences of San Diego, Calif.; Molecular Devices of Sunnyvale, Calif.; and Packard Instruments of Meriden, Conn.
Fluorescence emission is usually preferred for such high throughput uses due to the overall sensitivity when compared to other techniques such as absorption measurements. Another advantage of using fluorescence is the detection method of availability of a range of fluorophores that can be used as extrinsic probes. Typically, five parameters can be measured when using a fluorescence technique, namely the intensity of the excitation spectra, the intensity of the emission spectra (each at selected wavelengths), the polarization of the excitation spectrum, the quantum yield of fluorescence, and the decay time of the excited level.
Fluorescence correlation spectroscopy was originally proposed by Magde et al., Thermodynamic Fluctuations in a Reacting System: Measurements by Fluorescence Correlation Spectroscopy,
Physical Review Letters
, Volume 29 (1972), pages 705-708. In this technique, the temporal fluctuations of the detected fluorescence signal (that is time-dependent, spontaneous intensity fluctuations of the fluorescence signal in the typically tiny observation volume) are detected and analyzed to obtain information about the processes occurring on a molecular scale. These intensity fluctuations and the volume under observation may arise from Brownian motion, flow, and chemical reactions. During the past years, fluorescence correlation spectroscopy has been utilized to measure transitional diffusion coefficients, rotational diffusion coefficients, kinetic rate constants, molecular aggregation, and molecular weights. An article by Thompson et al., presents a review of the technique (N. L. Thompson et al., Fluorescence Correlation Spectroscopy, in “
Topics in Fluorescence Spectroscopy
”, Volume 1 (J. R. Lakowicz, Editor Pleanum Press, New York 1991, pages 337-410).
DESCRIPTION OF THE INVENTION
By this invention, an apparatus and method are provided for carrying out high throughput screening of active compounds, typically using fluorescence correlation spectroscopy, although other techniques may be utilized making use of this invention. A fluorescence probe is excited through a one photon or multi photon excitation process. The light source may be lamp such as an xenon arc or deuterium lamp, or a laser such as continuous wave lasers: i.e., argon-ion, krypton-ion, helium-neon, helium-cadmium, or other lasers. Pulsed lasers may also be used such as nitrogen lasers or mode-locked lasers, diode lasers, or lasers placed in an array. In each of the possible radiation sources, the light source should be capable of delivering radiation at a particular wavelength or wavelengths that excite the fluorescence probe through one photon or multi photon excitation processes. Typically, such excitation wavelengths may range from 200 nm. to 5,000 nm.
By this invention, a fluorescence spectrometer is provided which comprises a laser; and at least one beam splitter (which may be a prism-type beam splitter, a fiber optic system, or similar device for accomplishing the beam splitting function) positioned to receive a light beam from the laser and to divide the beam into a plurality of separate, first light portions. Thus, mult
Barbieri Beniamino
Gratton Enrico
Ellis Garrettson
Evans F. L.
I.S.S. (USA) Inc.
Seyfarth Shaw LLP
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