Two-photon image correlation spectroscopy: dynamic measurements of molecular aggregation and transport on living cells

Summary form only given. 85% of biochemical reactions within cells occur at membrane surfaces. Indirect experimental evidence suggests that many of these biochemical processes are regulated through cellular control of the transport properties and oligomerization state of proteins within the membranes. Consequently, our being able to visualize and measure protein motions and aggregation state on living cells would tremendously enhance our understanding of cell function. The spatial and temporal scales that govern the biochemical reactions make measuring these membrane phenomena on living cells experimentally challenging. We use the image correlation spectroscopy (ICS) technique to obtain such information from measurements on living cells. To achieve the necessary spatial resolution we use two-photon fluorescence microscopy. This also has the additional advantage that the imaging process does not bleach non-imaged membranes contained within the cell volume. The image field can be acquired at rates up to video rate, which makes it possible to measure dynamic events occurring over a range of time scales.