Image acquisition in 80 ps steps to monitor fluorescence lifetime, diffusion and flow of single molecules.

A novel ultrafast 64x32 SPAD-based CMOS image sensor for single molecule spectroscopy is presented. The detector triggers a laser and allows the shift of the detection gate with 80 ps steps quick enough image propagating light in water with a spatial resolution of 1.8 cm. We show measurements of fluorescence intensity and lifetime of single diffusing molecules and the enhanced Green Fluorescent Protein (eGFP) expressed in mammalian cells. The precision of the setup is suitable to measure Foerster resonance energy transfer in less than 1 ms. Fluorescence amplitudes and lifetimes are determined simultaneously with subsequent correlation of the fluorescence signal for each pixel delivering the information of fluorescence lifetime imaging microscopy and fluorescence correlation spectroscopy. The system is suitable to spatially resolve diffusion constants and concentrations of single molecules in total internal reflection excitation of the sample. Applications are particle size measurements, imaging of dynamic molecular processes, spectroscopy of single molecule chemistry and diffusion and flow of fluorophores.