Imaging intracellular lipid heterogeneity with fluorogen-activating coincidence encounter sensing

Here we describe techniques for the implementation of fluorogen-activated coincidence sensing (FACES), a new chemical genetic tool for quantitatively imaging lipids in organelle membranes and reporting their transbilayer orientation in living cells. FACES combines bioorthogonal chemistry with genetically encoded fluorogen-activating proteins (FAPs) for reversible proximity sensing of fluorogen-conjugated azido-phospholipids in target membranes. Here, we provide and discuss method details and experimental considerations for the approach. We focus on the investigation of phosphatidylcholine in azido-choline fed HeLa cells, but the techniques described are broadly applicable to other biomolecules labeled with azido-metabolites in live cells.