We demonstrate single-molecule-level features using near-field optical microscopy on bacteriorhodopsin (bR), a membrane protein that functions as a light-driven proton pump. The photophysical properties of bR are utilized in this imaging technique, using a combination of photoexcitation sources, to accurately identify the active regions and quantify the optical parameters. The studies of bR monolayers are carried out on inert quartz substrates as well as active conducting polymer (polyaniline) substrates. The substrate also plays an important role in the photocycle quantum efficiencies. We speculate on mechanisms governing the higher near-field absorption strength of bR molecules. © 2010 Optical Society of America.