Optics: measuring and testing – By dispersed light spectroscopy – With sample excitation
Reexamination Certificate
2001-02-09
2004-02-10
Nguyen, Thong (Department: 2872)
Optics: measuring and testing
By dispersed light spectroscopy
With sample excitation
C250S458100, C250S459100, C356S311000
Reexamination Certificate
active
06690463
ABSTRACT:
The present invention relates to a method for characterizing samples having fluorescent particles and applications of said method.
The utilization of fluorescence has evolved rapidly during the past decades because it offers high sensitivity in various scientific applications. New developments in instrumentation, data analysis, probes and employment have resulted in enhanced popularity for a technique that relies on a phenomenon discovered nearly 150 years ago (Stokes, Phil. Trans. R. Soc. Lond. 142, 463-562, 1852). In a number of applications in physical chemistry, biology and medicine, fluorescence is used as a sensitive means of detecting chemical binding reactions in dilute solutions. Drug screening and pharmaceutical assay development are examples of fields of applications of this kind.
In addition to classical methods based on detecting changes in macroscopic fluorescence characteristics such as overall intensity or anisotropy, a number of different fluctuation methods have been developed during the last decades distinguishing species on ground of properties characteristic to single molecules. One of the most elaborated fluorescence techniques with single molecule sensitivity is fluorescence correlation spectroscopy (FCS) that can resolve different species first of all on the basis of different translational diffusion coefficient (Magde et al., Phys. Rev. Lett. 29, 104-708, 1972; Elson et al., Biopolymers 13, 1-27, 1974; Rigler et al., Eur. Biophys. J 22, 169-175, 1993). Recently, this fluorescence fluctuation method found its counterpart in fluorescence intensity distribution analysis (FIDA) that discriminates different fluorescent species according to their specific brightness (Kask et al., Proc. Natl. Acad. Sci. USA 96, 13756-13761, 1999). The term “specific brightness” generally denotes the mean count rate of the detector from light emitted by a particle of given species situated in a certain point in the sample, conventionally in the point where the value of the brightness profile function is unity.
Aside methods like FCS and FIDA which distinguish species on the ground of a single specific physical property, two-dimensional methods have been developed, utilizing two detectors monitoring different polarization components or emission bands of fluorescence. In particular, fluorescence cross-correlation analysis and two-dimensional fluorescence intensity distribution analysis (2D-FIDA) are methods recognizing species on the ground of two types of specific brightness (Kask et al., Biophys. J. 55, 213-220, 1989; Schwille et al., Biophys J. 72, 1878-1886, 1997; Kask et al. Biophys. J. 78, 2000).
While FCS, FIDA and the mentioned two-dimensional methods are statistical methods of fluctuation spectroscopy, there is also another broad field of research having the goal to identify individual molecules. Many applications make use of the fluorescence lifetime as an intrinsic molecular property that is sensitive to any changes of the molecule's direct environment. However, different from the above mentioned fluctuation methods, fluorescence lifetime analysis (FLA) is basically a macroscopic technique that allows the discrimination of different fluorescence decay times without the need for molecular number fluctuations in the monitored sample volume. Therefore, fluorescence lifetime measurements are usually performed in cuvettes at high sample concentrations. However, the disadvantage of this implementation is that the experimentally collected excitation to detection delay time histogram has contributions from different species which are difficult to be resolved. In addition, FLA has only a low robustness—slightly wrong assumptions yield very wrong results.
On the contrary, lifetime experiments have also been applied to extremely low mean particle numbers. This approach, being opposed to conventional FLA, was introduced as burst integrated fluorescence lifetime analysis (BIFL) (Keller et al., Applied Spectroscopy 50, 12A-32A, 1996). BIFL searches for fluorescence bursts from single molecules above a certain threshold intensity. Its disadvantage is that it can only be applied at very low concentrations of significantly less than one particle per measurement volume and therefore relatively long data collection time is needed.
In fluorescence lifetime experiments, if performed in the time domain with time correlated single photon counting (TCSFC), the excitation to detection delay time, t, of single photons is recorded and collected in a histogram. To extract the fluorescence lifetime a theoretical distribution P(t) is fitted against these experimental data. Usually P(t) is described by a single- or multi-exponential decay function that is convoluted with the respective instrument response function (IRF). Whereas this kind of analysis allows to characterize constituents of the sample according to their individual lifetimes, &tgr;, it does not allow the determination of their concentrations, c, and specific brightness, q, but only the products, qc.
Therefore, it is an object of the present invention to provide a method of high accuracy and robustness which allows the characterization of individual particles based on their fluorescence properties.
According to the present invention, a method for characterizing samples having fluorescent particles is presented which comprises the following steps. At first particles in a measurement volume are excited by a series of excitation pulses and the emitted fluorescence is monitored by detecting sequences of photon counts. For this purpose, a confocal epi-illuminated microscope might preferably be used in connection with a high repetition rate (e.g. 100 MHz) laser pulse excitation. Numbers of photon counts in counting time intervals of given width are determined as well as the detection delay times of the photon counts relative to the corresponding excitation pulses. A function of said detection delay times is built—as described in detail below—and as a next step a probablity function of at least two arguments, {circumflex over (P)}(n, t, . . . ) is determined, wherein at least one argument is the number of photon counts and another argument is said function of detection delay times. Thereafter, a distribution of particles as a function of at least two arguments is determined on basis of said probability function {circumflex over (P)}(n, t, . . . ), wherein one argument is a specific brightness (or a measure thereof) of the particles and another argument is a fluorescence lifetime (or a measure thereof) of the particles. The method according to the present invention, called Fluorescence Intensity and Lifetime Distribution Analysis (FILDA) has the advantage that it is possible to determine absolute concentrations, a quantity that is not directly accessible with conventional FLA. The combined information, when used in such a correlated manner according to the present invention, results in significantly increased accuracy as compared to FIDA and fluorescence lifetime analysis alone. In contrast to BIFL, that searches for fluorescence bursts from single molecules above a certain threshold intensity, FILDA analyses preferably the relative fluctuations of the whole data stream and thus accounts for the possibility of simultaneous photon emission from different molecules. Therefore, the present invention can also be used at significantly higher concentrations than BIFL.
In the following, the underlying theory as well as preferred embodiments are elaborated and applied to simulated as well as experimental data. The outstanding power in resolving different species is shown by quantifying the binding of calmodulin to a peptid ligand, promising a broad applicability in the life sciences.
As outlined above, the present invention relies on a method which is at least two-dimensional: different fluorescent particle species in the sample are distinguished from each other by specific brightness as well as lifetime values. In some cases it might however be advantageous to take into consideration further particle properties, such as their diffusion coeffici
Evotec BioSystems AG
Lavarias Arnel C.
Nguyen Thong
LandOfFree
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