Image correlation methods are an extension of fluorescence fluctuation spectroscopy that can measure protein-protein interactions and macromolecular transport properties from input fluorescence microscopy images of living cells. These approaches are based on space and time correlation analysis of fluctuations in fluorescence intensity within images recorded as a time series on a laser scanning or total internal reflection fluorescence (TIRF) microscope. We recently introduced spatio-temporal image correlation spectroscopy (STICS) which measures vectors of protein flux in cells based on the calculation of a spatial correlation function as a function of time from an image time series. Here we will describe the application of STICS and its two color extension, spatio-temporal image cross-correlation spectroscopy (STICCS), for measuring transport maps of adhesion related macromolecules such as integrin, alpha-actinin, paxillin, talin, and vinculin within, or associated with the basal membrane in living U2OS osteosarcoma and CHO cells as well as during dynamic turnover of podosomes in dendritic immune cells. We will also highlight recent advances we have made with a new form of reciprocal (k-) space ICS, called kICS, that allows us to measure unbiased transport coefficients of fluorescently labeled membrane proteins even if there is complex photophysics (such as nanoparticle emission blinking) of the probe. We will describe kICS measurements of the transport properties of quantum dot labeled GPI anchored proteins in the cell membrane. Finally we will show how an extension of kICS was used to measure the transport properties of Cystic fibrosis transmembrane conductance regulator (CFTR) and detect transiently confined and more freely diffusing populations of this ion channel in the cell membrane.


Wednesday, July 3, 2013, 10:30. Seminar Room

Hosted by Prof. María García-Parajo