There is more to scanning than meets the eye: Raster Image Correlation Spectroscopy

• Published in: Biochimica et biophysica acta. General subjects Vol. 1869; no. 8; p. 130818
• Main Authors: Gialdini, Irene, Hendrix, Jelle, Lamb, Don C.
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.07.2025
• Subjects: Animals; Confocal imaging; Cross-correlation RICS (ccRICS); Fluorescence fluctuation imaging; Fluorescent Dyes - chemistry; Humans; Image Processing, Computer-Assisted - methods; Live-cell imaging; Microscopy, Confocal - methods; Molecular diffusion; Molecular interactions; Raster Image Correlation Spectroscopy (RICS); Spectrum Analysis - methods; Molecular diffusion; Molecular interactions; Cross-correlation RICS (ccRICS); Live-cell imaging; Confocal imaging; Raster Image Correlation Spectroscopy (RICS); Fluorescence fluctuation imaging
• ISSN: 0304-4165; 1872-8006; 1872-8006
• DOI: 10.1016/j.bbagen.2025.130818

Raster Image Correlation Spectroscopy (RICS) is a confocal image analysis method that can measure the diffusion and interactions of fluorescently labeled molecules in real time in solution and in living cells. RICS is easy to implement on commercial confocal microscopes and allows detailed investigations of complex biological systems and pathways. The method is especially robust for measurements in living cells using commonly used labels such as fluorescent proteins. Moreover, since its invention in 2005, the robustness and applicability of RICS has been significantly increased to allow, e.g., straightforward kinetic analyses, advanced image segmentation, parameter mapping, and multi-species analysis. In this review, we describe the methodological principles of RICS in a manner that is accessible to a broad readership, position RICS in relation to other fluorescence fluctuation techniques, highlight recent methodological advances and present exemplary applications of the method. With this review, we hope to facilitate the implementation of this powerful method into the everyday repertoire of confocal imaging approaches. •Raster scanned confocal images contain quantitative info about molecular behavior.•Cross-correlation RICS can be used to precisely quantified molecular interactions.•Detailed maps of diffusion and concentration are achievable with RICS.•Multicolor implementation of RICS enable robust multi-species analyses.•RICS is a straightforward method to unravel the architecture of biological systems.