Abstract
Membrane proteins often need to be inserted into or attached to the cell membrane to perform their functions. Understanding their transmembrane topology and conformational dynamics during insertion is crucial for elucidating their roles. However, it remains challenging to monitor nanoscale changes in the insertion depth of individual proteins in membranes. Here, we introduce two single-molecule imaging methods, SIFA and LipoFRET, designed for in vitro observation of the nanoscale architecture of membrane proteins within membranes. These methods have demonstrated their efficacy in studying biomolecules interacting with bio-membranes with sub-nanometer precision.