Novel Bio-Imaging Techniques by Harnessing Optical Highlighter Fluorescent Proteins

Abstract

Optical highlighters are a remarkable family of fluorescent proteins (FPs) that could change their excitation and emission spectra upon certain wavelength illumination. Optical highlighters have been extensively used in super-resolution imaging, protein dynamics, gene expression and cellular trafficking. Herein we are harnessing these unique proteins in three different imaging applications: genetically-encoded microviscosity sensor, ultra-deep tissue imaging, and light-driven fluorescent timers. First, we report that the photoswitching kinetics of the chromophore inside Dronpa, a FP with the reversible on-off switching capability, is actually slowed down by increasing medium viscosity outside Dronpa. This effect is attributed to protein-flexibility mediated coupling where the chromophore's cis-trans isomerization is accompanied by conformational motion of protein beta-barrel. Based on this effect, we developed a genetically encoded protein-specific micro-viscosity sensor. Secondly, we have demonstrated a spectroscopy concept to extend the fundamental imaging-depth limit of multiphoton microscopy by generating super-nonlinearity of photo-switchable probes. Due to the long-lived nature of these switchable states, the nonlinearity effect could accumulate as the population is being cycled through these states. Conceptually different from conventional multiphoton processes mediated by transient virtual states, our strategy constitutes a new class of super-nonlinear fluorescence microscopy mediated by real population transfer. Last, we demonstrate a novel class of light-driven fluorescent timer (FT), based on the photo-convertible fluorescent proteins. It's a new method to image the protein “age” in live cells with a simple snap-shot measurement. This tunable light-driven FT will be a valuable tool that does not only provide the spatial location information of an individual protein, but also its temporal dynamics.