Fluorescence correlation spectroscopy using array detectors

Fluorescence Correlation Spectroscopy (FCS) is a widely-used, powerful tool to quantitatively measure molecular dynamics and interactions. However, it was mostly used at a single point and thus could not easily be extended to a true imaging tool. Imaging FCS, i.e. the recording of FCS data across a whole plane within a 2D or 3D sample, became possible with the advent of fast and sensitive cameras (EMCCDs and sCMOS) and sensors (APD arrays) in combination with total internal reflection or light sheet microscopy. Imaging FCS allows now to extract concentration, diffusion coefficient and biomolecular interaction maps in a living biological system, providing new possibilities for quantitative bioimaging. Beyond this multiplexing of collecting thousands of FCS curves simultaneously, imaging FCS offers also additional advantages. As measurements can be performed in an almost continuous manner, so-called FCS videos can be created in which one can follow the molecular dynamics and interactions of biomolecules within a biological sample over time. Furthermore, FCS images contain not only temporal correlations for each pixel but also the spatiotemporal correlations between all pixels. This allows to extract features of spatial organization even beyond the diffraction limit. In this seminar I will first introduce the principles of imaging FCS and how it can be implemented in total internal reflection or light sheet microscopes. I will then cover a range of applications i) comparing Imaging FCS to fluorescence recovery after photobleaching (FRAP), ii) investigating the action of a membrane active peptide, the human islet amyloid polypeptide (hIAPP), on live cells, and iii) determine the plasma organization of a transmembrane protein, the epidermal growth factor receptor (EGFR). Imaging FCS is in its early stages but already provides a useful biophysical tool that can obtain information not easily accessible with other techniques.