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Abstract

Membranes are dynamic lipophilic structures that constitute a selective barrier essential for the smooth functioning of cellular machinery. Their complex and highly heterogeneous composition, as well as the intricate diffusion patterns that they display, have been intensely studied in the past decades. Several innovative microscopy techniques have recently been developed to gain insight into the basis of membrane dynamics, interactions, and molecular organization. In this article, we introduce four pioneer fluorescence-based approaches that have substantially broadened our overall understanding of membranes: fluorescence correlation spectroscopy (FCS), single particle tracking (SPT), fluorescence recovery after photobleaching (FRAP), and 6-lauroyl-2-(dimethylamino)-naphtalene (LAURDAN) microscopy. We discuss each technique in detail, explaining their principles, methodology, and applications in the research of the dynamic processes controlled by membranes.