Optically controlling dark state lifetimes of photoswitchable fluorescent proteins and Its application to biological systems

Abstract

Advances in fluorescence microscopy have greatly expanded applications in biology for visualization of targeted molecules in research and detection methods in diagnostics. The Dickson Laboratory has developed synchronously amplified fluorescence image recovery (SAFIRe) for selectively imaging modulatable fluorophores and eliminating same-color, non-modulatable species by utilizing photophysical dark states of fluorescent proteins (FPs). Following prior research on FPs, this thesis continues to characterize the photophysical dark state a photoswitchable fluorescent protein (PS-FPs), rsFastLime. Dual modulation SAFIRe (DM-SAFIRe) suppresses 5-fold autofluorescent background by optically controlling the dark states of rsFastLime and is applied to tracking HIV-1 entry into CD4 cells. The dark state lifetime of rsFastLime is optically controlled to be slower than diffusion time, so the diffusing rsFastLime cannot accumulate enough dark state population for demodulation, while immobilized rsFastLime show 10-fold signal more than diffusing ones. Laser intensity modulation generates amplitude of fluorescent response as well as phase differences between excitation and emission. Out-of-phase imaging after optical modulation (OPIOM) can be detected under moderate secondary laser intensity (~1 W/cm2) on camera a few seconds after the dark state is accelerated by SAFIRe. After accurate prediction from a simple three-state model, SAFIRe-OPIOM is able to discriminate the molecules with different dark state lifetimes changed by secondary laser illumination, which may be applied to resolve two molecules within diffraction limit. Dark states of FPs can serve as an additional dimension for advancing fluorescence microscopy by optically controlling dark state lifetimes of PS-FPs. This thesis demonstrated autofluorescence removal, selective imaging, and possible super resolution applications.