Optically modulated fluorescent proteins

Abstract

Optical modulation has shown the selective and sensitive signal improvement in high background systems in cell imaging; however, cell applications are still limited due to biocompatibility and delivery issues. Fluorescent proteins have a variety of optically accessible states that make them ideal candidates for investigation of modulatability. Combining the optical modulation technique with the biocompatibility of fluorescent proteins is a major advance. This work focuses on evaluation fluorescent proteins and their optical states for modulation, as well demonstrations of cellular imaging. Herein, we evaluate a green fluorescent protein with interesting photophysical properties favorable for optical modulation. Positive for optical modulation, further investigation of the state dictating modulation reveals the presence of a slow component on the order of milliseconds. To better understand the mechanism responsible modulation, blue fluorescent proteins are created to modify the chromophore environment. Extraction of photophysics confirm the alteration timescales of the modulated state. Motivated by the ability to improve imaging and decode hidden dynamics, demodulation of these proteins demonstrates the selective recovery of signal in the presence of high cellular background. The continued investigation of several other fluorescent proteins identifies modulatable proteins across the visible wavelength region. Additionally, solvent environmental factors show varying timescales which, when combined with mutagenesis, suggest a cis/trans isomerization coupled with a proton transfer. This information of the properties dictating optical modulation allows for the engineering of improved modulatable proteins to study cellular dynamics.