Imaging Fluorescence Correlation Spectroscopy Measures Dynamics and Structure in Live Samples

Abstract

Fluorescence Correlation Spectroscopy (FCS) has been developed more than 40 years ago and has evolved into a major tool in the biophysical sciences for the quantitative measurement of biomolecular dynamics and interactions. In the past FCS was mainly used as a confocal, single spot measurement technique. Imaging FCS, i.e. the recording of FCS measurements at all pixels in an image, became feasible only recently with the advent of fast, sensitive array detectors and the development of new illumination modes in microscopy, in particular light sheet microscopy. Despite the fact that the time resolution of imaging FCS is lower than for single spot measurements, imaging FCS has several advantages. The most important being i) that it contains much more information since spatial and temporal correlations can be evaluated between any pixels or group of pixels in the image, and ii) the illumination modes (TIRFM or SPIM) expose the sample to much lower light doses and allow more measurements per sample.We demonstrate the capabilities of imaging FCS using total internal reflection (TIR) and single plane illumination microscopy (SPIM) as illumination modes. We investigate the membrane active human Islet Amyloid Polypeptide (hIAPP) on cell membranes using imaging TIR-FCS, FCS diffusion laws, and time-lapse FCS videos, to elucidate the membrane action of monomeric hIAPP. With the aid of the FCS diffusion laws, Imaging FCS can access information about sample structure even below the diffraction limit. And light sheet illumination allows single cell observations over up to one hour (total effective illumination time ∼10 minutes) without visible cell damage. Second, we use SPIM-FCCS to demonstrate the measurement of interactions of biomolecules in vitro and in live cells. These experiments show that Imaging FCS provides quantitative images of biological samples under physiological conditions.