A Single-Photon Avalanche Camera for Fluorescence Lifetime Imaging Microscopy and Correlation Spectroscopy

Confocal laser scanning microscopy (CLSM) is commonly used to observe molecules of biological relevance in their native environment, the live cell, and study their spatial distribution and interactions nondestructively. CLSM can be easily extended to measure the lifetime of the excited state, the concentration and the diffusion properties of fluorescently labeled molecules, using fluorescence lifetime imaging microscopy (FLIM) and fluorescence correlation spectroscopy (FCS), respectively, in order to provide information about the local environment and the kinetics of molecular interaction in live cells. However, these parameters cannot be measured simultaneously using conventional CLSM due to damaging effects that are associated with strong illumination, including phototoxicity, photobleaching, and saturation of the fluorescence signal. To overcome these limitations, we have developed a new camera consisting of 1024 single-photon avalanche diodes that is optimized for multifocal microscopy, FLIM and FCS. We show proof-of-principle measurements of fluorescence intensity distribution and lifetime of the enhanced green fluorescent protein expressed in live cells and measurement of quantum dot diffusion in solution by FCS using the same detector.