Protocol for characterizing biomolecular condensates through live-cell imaging and analysis

Biomolecular condensates are important targets of investigation due to their physiological functions as well as their roles in neurodegenerative diseases, cancer, and drug delivery. Here, we present a protocol for characterizing biomolecular condensates across three confocal microscopy-based modalities, namely, subcellular localization, fluorescent recovery after photobleaching (FRAP), and time lapse analysis of mobility and fission/fusion events. We detail optimized image acquisition parameters and describe steps for analysis workflows utilizing ImageJ macros and plugins, including 2D and 3D analysis approaches.