Raster Image Correlation Spectroscopy (RICS): Principles, Applications, and Advances in Studying Molecular Dynamics in Live Cells

Raster image correlation spectroscopy (RICS) has become an advanced fluorescence imaging technique that has high spatiotemporal resolution to examine the molecular dynamics, transport, and interactions in live cells. In contrast to the traditional point-scanning fluorescence correlation spectroscopy (FCS), RICS provides high accuracy to determine biological interactions and molecular diffusion across many regions of a live cell, utilizing the raster scanning mode of a confocal microscope to get information about spatial and temporal correlation. RICS is applied to get global information about diffusion and dynamic in systems, such as living cells, polymer networks, suspension of nanoparticles, where conventional FCS is not useful to capture decay of correlation function based on spatial location. In this review, the fundamental principle and applications of RICS at the molecular level in a heterogeneous environment are thoroughly covered, e.g., examining the protein diffusion and dynamics in cell. Finally, limitation of this imaging methods and new developments in combination of other imaging techniques are also highlighted. © 2026 Wiley-VCH GmbH.