Single Molecule Detection


Fluorescence Correlation Spectroscopy (FCS) in its modern embodiment measures and exploits temporal fluctuations in the fluorescence emission of small numbers of molecules in an open excitation volume (of ca 1 fl) defined by confocal optics. Fluctuations arise from changes in the number of molecules observed as they diffuse in and out of the volume element, as well as by the fluorescent intensity or yield, particularly manifest via the autocorrelation function of the time series. Since small ligands have an autocorrelation function which falls off much more rapidly when they are free than when they are bound to (say) proteins or nucleic acids, analysis of the shape of the autocorrelation function allows one to discriminate bound (macromolecular) and free ligands non-invasively and simultaneously. The autocorrelation function is dominated by the diffusion coefficient, is thus known as a 'mass-dependent' method, and is the simplest version of this general principle of fluctuation analysis. However, the same apparatus may be used to generate data which may be subjected to much more powerful and more recently developed analyses such as brightness-dependent fluorescence correlation spectroscopy as represented by Fluorescence Histogram/Fluorescence Intensity Distribution Analysis (FHA/FIDA) and related methods. As part of a program in the Analytical Biotechnology of single molecules, we are developing and exploiting variants of these strategies in a variety of areas.