Optically Modulated Photoswitchable Fluorescent Proteins Yield Improved Biological Imaging Sensitivity

Abstract

Photoswitchable fluorescent proteins (PS-FPs) open grand new opportunities in biological imaging. Through optical manipulation of FP emission, we demonstrate that dual-laser modulated synchronously amplified fluorescence image recovery (DM-SAFIRe) improves signal contrast in high background through unambiguous demodulation and is linear in relative fluorophore abundance at different points in the cell. The unique bright-to-dark state interconversion rates of each PS-FP not only enables discrimination of different, yet spectrally indistinguishable FPs, but also allows signal rejection of diffusing relative to bound forms of the same PS-FP, rsFastLime. Adding to the sensitivity gains realized from rejecting non-modulatable background, the selective signal recovery of immobilized vs diffusing intracellular rsFastLime suggests that DM-SAFIRe can detect weak protein-protein interactions that are normally obscured by large fractions of unbound FPs.