Multiparameter Spectroscopy For Single-Molecule Fluorescence And Confocal Imaging

Abstract

Multiparameter fluorescence detection (MFD) is a technique that simultaneously records all information observable in a fluorescence experiment, i.e. polarization, color, photon arrival time and spatial origin. This is particularly useful in single molecule studies, where sequential measurements of different properties could not easily account for temporal variations in heterogeneous or unstable samples, but also in low light level investigations where the number of available photons is very limited. In MFD a single raw data set delivers fluorescence decay histograms, anisotropy decay histograms, intensity ratios and time traces or fluorescence correlation curves for individual molecules or sub-ensembles with spatial resolution close to the diffraction limit. The technique requires single photon sensitive detectors, high speed data acquisition and sophisticated software to analyze the data. In a confocal environment imaging can be achieved either by beam scanning or by stage scanning, depending on the requirement of the experiment.A general overview, a study of rotational Brownian motion of biomolecules by high-resolution fluorescence correlation spectroscopy (FCS) and some applications in confocal imaging will be presented.