Diffusion, Transport, and Cell Membrane Organization Investigated by Imaging Fluorescence Cross-Correlation Spectroscopy

摘要: Cell membrane organization is dynamic and assumed to have different characteristic length scales. These scales, which are influenced by lipid protein composition as well the cytoskeleton, can range from below optical resolution limit (as with rafts or microdomains) far above capping phenomena formation of “platforms”). The measurement these features poses a significant problem because dynamics on millisecond timescale thus beyond time conventional imaging approaches. Fluorescence correlation spectroscopy (FCS), widely used spectroscopic technique measure dynamics, has required but lacks capabilities. A promising solution recently introduced method known total internal reflection (ITIR)-FCS, probe diffusion in membranes good temporal spatial resolution. In this work, we extend ITIR-FCS perform ITIR fluorescence cross-correlation (ITIR-FCCS) between pixel areas arbitrary shape derive generalized expression that applicable active transport diffusion. ITIR-FCCS applied model systems exhibiting diffusion, transport, combination two. To demonstrate its applicability live cells, observe marker, sphingolipid-binding domain (SBD) derived amyloid peptide Aβ, neuroblastoma cells. We investigate SBD-bound microdomains under conditions cholesterol removal cytoskeleton disruption.