Measuring the Lateral Diffusion of Plasma Membrane Receptors Using Raster Image Correlation Spectroscopy

Raster image correlation spectroscopy (RICS) enables detecting and quantifying diffusion in live cells using standard commercial laser scanning confocal microscopes. Here, we describe a protocol based on RICS for measuring the lateral diffusion of two immunoreceptors within the plasma membrane of the macrophage cell line RAW 264.7. The sample images and measurements presented in this chapter were obtained from RICS analysis of Toll-like receptor 2 (TLR2) and cluster of differentiation 14 (CD14), which are transmembrane and membrane-anchored receptors, respectively. A step-by-step guideline is provided to acquire raster-scanned images and to extract the diffusion coefficients using RICS analysis.