Optics: measuring and testing – By dispersed light spectroscopy – With sample excitation
Reexamination Certificate
2000-06-12
2003-04-29
Font, Frank G. (Department: 2877)
Optics: measuring and testing
By dispersed light spectroscopy
With sample excitation
C356S338000, C250S458100, C250S459100
Reexamination Certificate
active
06556296
ABSTRACT:
The present invention relates to a method for characterizing samples by determination of a function of at least one specific physical property of units of said sample.
The essence of a number of pharmacological, biological and chemical problems is to detect substances in a sample or to measure the interaction or reaction of these substances. In order to measure the substances in a sample more specifically, usually at least one of the reactants is radioactively or luminescently labelled. A convenient and sensitive type of labels are fluorescent labels.
Widely used methods to monitor interactions by fluorescence are the determination of changes in overall fluorescence intensity or in anisotropy of fluorescence. However, a number of side effects, such as surface binding or fluorescence from impurities, often lead to interpretation problems and artifacts. A second reason which has induced interest towards refined methods of analysis is the need to work with small amounts of a large number of samples in the field of high throughput screening and large capacity diagnostics.
New opportunities for assay development were opened when the technology for monitoring fluorescence from single fluorophore molecules became available. The first successful studies on fluorescence intensity fluctuations were performed by Magde, Elson and Webb (Biopolymers, Vol. 13, 29-61, 1974) who demonstrated the possibility to detect number fluctuations of fluorescent molecules and established a research field called fluorescence correlation spectroscopy (FCS). FCS was primarily developed as a method for determining chemical kinetic constants and diffusion coefficients. The experiment consists essentially in measuring the variation of the number of molecules of specific reactants in time in a defined open volume of solution. Microscopic fluctuations of the concentration of the reactant are detected as fluorescence intensity fluctuations from a small, open measurement volume. The measurement volume is defined by a focussed laser beam, which excites the fluorescence, and a pinhole in the image plane of the microscope collecting fluorescence. Intensity of fluorescence emission fluctuates in proportion with the changes in the number of fluorescent molecules as they diffuse into and out of the measurement volume and as they are created or eliminated by the chemical reactions. Technically, the direct outcome of an FCS experiment is the calculated autocorrelation function of the measured fluorescence intensity.
An important application of FCS is to determine concentrations of fluorescent species having different diffusion rates in a mixture. In order to separate the two terms corresponding to translational diffusion of two kinds of particles in the autocorrelation function of the fluorescence intensity, at least about a two-fold difference in diffusion time is needed, which corresponds generally to an eight-fold difference in the mass of the particles. Furthermore, if one succeeds in separating the two terms in the autocorrelation function of fluorescence intensity, it is yet not sufficient for determining the corresponding concentrations except if one knows the relative brightness of the two different types of particles.
Possible biophysical applications further demand the ability to analyze complex mixtures of different species. For that purpose, Palmer and Thompson studied higher order correlation functions of fluorescence intensity fluctuations and have outlined methods for determining the number densities and relative molecular brightness of fluorescence of different fluorescent species (Biophys. J., Vol. 52, 257-270, August 1987). Their technique may in principle proof useful in detecting and characterizing aggregates of fluorescently labelled biological molecules such as cell surface receptors, but has a major disadvantage of being rather complex, so that data processing of an experiment including the calculation of high-order correlation functions last hours.
A considerably less complicated method than calculation of high order auto-correlation functions for characterizing mixtures of fluorescent species of different specific brightness is a calculation of higher order moments of fluorescence intensity out of experimentally determined distribution of the number of photon counts. This method was presented by Qian and Elson (Biophys. J., Vol. 57, 375-380, February 1990; Proc. Natl. Acad. Sci. USA, Vol. 87, 5479-5483, July 1990). The method of moments, however, is hardly suitable for characterizing complex samples or selecting between competing models of the sample or checking whether the given model is appropriate.
Further improvements were made according to the disclosure of WO-A-98/16814. This publication describes a method for analyzing samples by measuring numbers of photon counts per defined time interval in a repetitive mode from light emitted, scattered and/or reflected by particles in said sample, and determining the distribution of the number of photon counts per said time intervals, characterized in that the distribution of specific brightness of said particles is determined from said distribution of the number of photon counts. The method can also be applied to study fluorescent samples. This special embodiment is the so called fluorescence intensity distribution analysis (FIDA). While FCS distinguishes between different species according to their diffusion time, FIDA distinguishes between them according to their specific brightness.
Kask et al. describe possibilities for the use of fluorescence correlation spectroscopy in the nanosecond time range (Eur. Biophys. J., 12: 163-166, 1985). However, there is no discussion on determining a distribution of a specific physical property from the measured photon interval distribution function in their report.
Keller et al. (Applied Spectroscopy, vol. 50, no. 7, p. 12A-32A, 1996) disclose methods for single-molecule fluorescence analysis in solution.
Madrazo et al. (Applied Optics, vol. 33, no. 21, p. 4899-4905, 1994) disclose a time-interval-statistics method that is based on the measurement of the Laplace transform of the probability function of the time intervals between two successive photoelectrons. This method has been applied to experiments of light diffusion from low-polydispersity samples from which the scattered intensity is weak.
An object of the invention is to provide a reliable and fast method for characterizing samples.
The object of the present invention is solved with the method having the features of claim 1.
It is to be understood that the following description is intended to be illustrative and not restrictive. Many embodiments will be apparent to those of skill in the art upon reviewing the following description. By way of example, the invention will be described primarily with reference to monitoring numbers of photon counts from light emitted by fluorescently labelled particles in a sample. This is because fluorescence is a very sensitive means allowing to monitor single molecules, and still rather selective allowing to distinguish between different species. However, in some embodiments it may be desirable to monitor numbers of photon counts of other origin than fluorescence.
The term “unit of a sample” refers, in general, to subparts of the sample which are capable of emitting, scattering and/or reflecting radiation. A sample might contain a number of identical units or different units which preferably can be grouped into species. The term “different species” refers also to different states, in particular different conformational states, of a unit such as a molecule. Fluorescently labelled or naturally fluorescent molecules, molecular complexes, vesicles, cells, beads and other particles in water or other liquids are examples of fluorescent units in liquid samples, while examples of fluorescent units or particles of a solid sample are impurity molecules, atoms or ions, or other fluorescence centers.
What is meant by the term “specific physical property” is generally a physical measurable property having a certain value or interval of v
Evotec BioSystems AG
Font Frank G.
Lauchman Layla
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