Two-photon fluorescence imaging and correlation analysis applied to protein dynamics in C. elegans embryo

Two-photon fluorescence imaging of proteins labelled with GFP or its analogues provides information on the localization of the molecules in cells and tissues, and their redistribution on timescales as short as milliseconds. Fluorescence correlation spectroscopy (FCS) analyzes fluctuations of the fluorescence signal in order to yield information about the motion of the molecules on timescales considerably shorter than those accessible with imaging, allowing the determination of diffusion coefficients, estimation of aggregate size, molecular concentrations, etc., i. e., parameters that can be difficult to determine with imaging alone. Scanning FCS (sFCS) is a modification of FCS that provides information about molecular dynamics and type of motion, which is too slow for standard FCS, and not resolvable with imaging. We have applied two-photon imaging, FCS and sFCS to study the localization and redistribution of GFP-labelled proteins involved in the asymmetric first division of C. elegans embryos. While the distribution of the investigated proteins in the cytoplasm is homogeneous on the scale limited by the optical resolution and their fast motion can be well characterized with conventional FCS, the proteins localized in the cortex exhibit patterns evolving on the ms-s temporal scale. We use sFCS and explore the applicability of spatial correlation analysis (image correlation, STICS) to the qualitative and quantitative description of the dynamics of the cortex-localized proteins.