Fluorescence correlation spectroscopy (FCS) has become an important tool for measuring diffusion, concentration, and molecular interactions of cellular components. The interpretation of FCS data critically depends on the measurement set-up. Here, we present a rigorous theory of FCS based on exact wave-optical calculations. Six of the most important optical and photophysical factors that influence FCS are studied: fluorescence anisotropy, cover-slide thickness, refractive index of the sample, laser-beam geometry, optical saturation, and pinhole adjustment. Our theoretical framework represents a general attempt to link all relevant parameters of the experimental set-up with the measured correlation function.

An efficient algorithm for pattern matching has been developed based on least-squares analysis of fitting a discrete set of master patterns against measured images. This algorithm has been applied to determine threedimensional molecule orientations in defocused single-molecule images. The developed algorithm exploits the excellent agreement between electrodynamic calculations of single-molecule emission and experimentally measured images. The procedure is found to be reliable and simple and can be applied to any kind of pattern recognition where the patterns to be recognized are precisely known a priori. The procedure works well even for noisy and low-intensity signals as usually encountered in single-molecule experiments.

Fluorescence correlation spectroscopy (FCS) is an important technique for studying low concentrations of analyte molecules in solution. The core molecular characteristic that can be addressed by FCS is the translational diffusion coefficient of the analyte molecules, which can be used for i.e. studying molecular binding and reactions, or conformational changes of macromolecules. The present paper discusses several possible optical and photophysical effects that can influence the outcome of a FCS measurement and thus can bias the value of the derived diffusion coefficient.

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