Image correlation spectroscopy approaches to probe diffusion in cell.

Among the methods for studying molecular dynamics through fluorescence, fluorescence correlation spectroscopy (FCS) is one of the most effective in vivo experimental techniques. It returns information about the diffusion of fluorescent molecule pools in a single well-defined small volume by means of auto/cross-correlating fluorescence intensity fluctuation. Since probing a single point may not be representative of a complex landscape, we can use the spatio-temporal image correlation spectroscopy (stICS) method. Using a sequence of images taken on the same functional biological area, such a method computes the auto/cross-correlation over the entire region. Since the diffusive parameters are extracted from the autocorrelation map as an average over a region of interest, stICS can be susceptible to spatial inhomogeneities of the fluorescent moiety in the same area. However, vesicles, membranes, confinements are sometimes poorly detectable from the sole fluorescence intensity. If different diffusion pools are present, some of them may be missed from the analysis of the global autocorrelation curve on account of the signal-to-noise ratio and/or their dynamic characteristics. This work explores different independent approaches to reveal the spatial feature of the molecular dynamics inside the cell. These approaches required a sequence of images to perform FCS analysis pixel by pixel. Each single-pixel autocorrelation evaluation avoids mixing different contributions due to average dynamics pools over a large field of view. Among the developed algorithm, the most promising is related to an analysis based on Maximum Entropy, which, for each point, gives a spectrum of Brownian Diffusion coefficients. This information can help link morphological and dynamics feature, also through correlative analysis with other spatial techniques, enabling new imaging schemes. These approaches are applied to recover membrane characteristics in cell's membrane such as PDL1, a significant player in an immune-checkpoint also involved in cancer progression.