Optics: measuring and testing – Of light reflection
Patent
1999-05-14
2000-10-24
Font, Frank G.
Optics: measuring and testing
Of light reflection
446448, 446213, G01N 2155
Patent
active
061375845
DESCRIPTION:
BRIEF SUMMARY
The invention relates to a method according to the preamble of claim 1 and a method according to the preamble of claim 15 and to an arrangement according to the preamble of claim 18.
Time-correlated single-photon counting is a versatile spectroscopic technique whereby a number of parameters of particles under investigation can be determined. These properties include the fluorescence life or the various fluorescence lives of multi-exponential decays, rotation diffusion constants, kinetic transition rates, among others. In addition, the high time resolution associated with time-correlated single-photon counting, in conjunction with pulsed excitation, is an effective means of separating non-delayed scattered excitation light from delayed fluorescent light. By this means, the desired signal (fluorescence) can be separated from undesired noise (non-delayed scattered light) and the signal-to-noise ratio in many applications can be increased.
Other properties, particularly the diffusion constant of target particles, can be obtained by the method of fluorescence correlation spectroscopy (FCS). A knowledge of the diffusion constant yields a variety of information about the size of the target particles or the bonding thereof to other large molecules.
In conventional FCS measuring methods (compare WO 94/16313), however, there is no possibility of discriminating between non-delayed scattered excitation light and delayed fluorescence light, so that the signal-to-noise ratio is too low.
Both techniques, i.e. time-correlated single-photon counting and fluorescence correlation spectroscopy, are suitable for highly sensitive measurements on very dilute solutions of fluorescent particles, preferably in the sub-nanomolar range. These techniques can even detect individual molecules in sample media (compare C. Zander et al., Applied Physics B, Volume 63, pages 516-523, 1996). Hitherto, however, it has not been possible to determine the autocorrelation function of signals from individual particles.
A method and an arrangement of the kind mentioned in the preamble were described by Richard A Keller et al. in Applied Spectroscopy, Volume 50, No. 7, 1996, on pages 12A to 32A. The possibilities of data analysis by the arrangement described are limited to isolated use of the evaluation possibilities either of time-correlated single photon counting or of FCS. The advantages of both techniques are not used in combination. Furthermore the evaluating device in the arrangement known from the citation uses a complicated and expensive CAMAC frame and needs a multi-channel scaler (MCS).
The object of the invention is to extend the possible uses of fluorescence correlation spectroscopy.
According to the invention this problem is solved by a method having the features of claim 1, a method having the features of claim 15 and an arrangement having the features of claim 18.
In the first embodiment of the method according to the invention for determining predetermined properties of target particles in a sample medium, the sample medium is initially irradiated with periodically modulated light having a predetermined period. Preferably the sample medium is irradiated with a regular sequence of uniformly spaced light pulses. The distance between two light pulses (the period) is e.g. 12 ns. Next, light in the form of individual photons scattered in the sample medium is detected by a detection device. On the one hand, the time interval between the time of detection of each photon and a reference time within the associated period of the irradiating light is determined and defined as the delay time. On the other hand the time of detection of each photon is determined. This is usually done by means of two different measuring arrangements adapted to the particular measurement requirements. The delay time, which is in the nanosecond range, requires an analog measuring arrangement for measuring extremely short-duration processes. The times of detection on the other hand are usually determined by a digital measuring arrangement, adapted to measure the corres
REFERENCES:
patent: 5434667 (1995-07-01), Hutchins et al.
Brand Leif
Gunther Rolf
Seidel Claus
Evotec BioSystems Aktiengesellschaft
Font Frank G.
Max-Planck-Gesellschaft zur
Ratliff Reginald A.
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