Mining Molecular Noise via Image Correlation Spectroscopy to Map Molecular Transport and Interactions in Living Cells

Abstract

Image correlation spectroscopy (ICS) methods are an extension of fluorescence correlation spectroscopy (FCS) that can measure cellular biomolecule interactions and transport properties from input fluorescence microscopy images of living cells. Various ICS methods have been developed that are based on space/time or k-space/time correlation analysis of fluctuations in fluorescence intensity within images recorded as a time series on a laser scanning, total internal reflection fluorescence (TIRF) or super resolution microscope. In this talk I will briefly introduce the essentials for understanding ICS analysis. I will then describe two applications of these methods. The first will focus on a combination of two channel structured illumination microscopy (SIM) and spatio-temporal image cross correlation spectroscopy (STICCS) analysis of fluorescent actin and membrane lipid fluorophores in Jurkat T cells plated on activating antibody substrates. The STICCS analysis revealed a coupling between retrograde actin flows and the lipids in activated immunological synapse regions of the membrane. Further STICCS measurements revealed alpha-actinin as a likely molecular linker for this coupled flow. The second application will focus on image correlation and pair vector correlation measurements on adhesion and cytoskeletal protein dynamics in podosomes in human dendritic cells. The image correlation analysis revealed a coupling between the dynamics of podosomes in local clusters which varied depending on whether the dendritic cells were on soft versus stiff substrates.