Single-Molecule Tracking of RNA Polymerase In and Out of Condensates in Live Bacterial Cells.

Biomolecular condensates, first discovered in eukaryotic cells, were recently found in bacteria. The small size of these organisms presents unique challenges for identifying and characterizing condensates. Here, we describe a single-molecule approach for studying biomolecular condensates in live bacterial cells. Specifically, we outline a protocol to quantify the mobility of RNA polymerase in E. coli using HILO (highly inclined and laminated optical sheet) illumination with the photoconvertible fluorophore mMaple3. Our analysis classifies the trajectories of individual molecules by their local density, enabling a comparison of molecular mobilities between different subcellular compartments.