Fluorescence correlation spectroscopy (FCS)
FCS is a method to detect molecular parameters like e.g. diffusion time, diffusion coefficent. Threrefore FCS can be used to analyse interaction of molecules (Elson & Magde, 1974; Magde et al., 1974; overview: Schwille & Haustein, 2001). In Fluorescence Correlation Spectroscopy the fluctuation of the fluorescence intensity is recorded in a very small volume, i.e. in femtoliter range via a confocal optic system.
Fluorescence Intensity Distribution Analysis
The photon statistic method FIDA (Fluorescence Intensity Distribution Analysis) was developed in 1999 by Kask (Kask, P., Palo, K., Ullman, D., and Gall, K. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 13756-13761) and in parallel by Chen et al. (1999) Biophys. J. 77, 553-567, where it was named PCH (Photon Count Histogram).
The method FIDA was quickly developed into a family of photon statistic methods (2D-FIDA, Kask, P., Palo, K., Fay, N., Brand, L., Mets, Ü., Ullman, D., Jungmann, J., Pschorr, J., and Gall, K. (2000) Biophys. J. 78, 1703-1713, FIMDA, , Palo K., Mets, Ü., Jäger, S., Kask, P., and Gall, K. (2000) Biophys. J. 79, FILDA, Palo, K., Brand, L., Eggeling, C., Jäger, S., Kask, P., and Gall, K. (2002) Biophys. J. 83, 605-618). In all three methods additional information (2nd detector in 2D-FIDA, molecular diffusion in FIMDA and fluorescence lifetime in FILDA) is used to improve statistical accuracy and/or gain additional information. All these methods deliver detailed information about a fluorescent tracer diffusing through the open 3D volume of the size of one bacteria (roughly one femto-liter). As long as enough fluorescent molecules are available (nano-molar range) this methods are well suited for diagnostic or drug discovery.
