Multiparameter fluorescence image spectroscopy to study molecular interactions.

Multiparameter Fluorescence Image Spectroscopy (MFIS) is used to monitor simultaneously a variety of fluorescence parameters in confocal fluorescence microscopy. As the photons are registered one by one, MFIS allows for fully parallel recording of Fluorescence Correlation/Cross Correlation Spectroscopy (FCS/FCCS), fluorescence lifetime and pixel/image information over time periods of hours with picosecond accuracy. The analysis of the pixel fluorescence information in higher-dimensional histograms maximizes the selectivity of fluorescence microscopic methods. Moreover it facilitates a statistically-relevant data analysis of the pixel information which makes an efficient detection of heterogeneities possible. The reliability of MFIS has been demonstrated for molecular interaction studies in different complex environments: (I) detecting the heterogeneity of diffusion properties of the dye Rhodamine 110 in a sepharose bead, (II) Förster Resonance Energy Transfer (FRET) studies in mammalian HEK293 cells, and (III) FRET study of the homodimerisation of the transcription factor BIM1 in plant cells. The multidimensional analysis of correlated changes of several parameters measured by FRET, FCS, fluorescence lifetime and anisotropy increases the robustness of the analysis significantly. The economic use of photon information allows one to keep the expression levels of fluorescent protein-fusion proteins as low as possible (down to the single-molecule level).