High-resolution fluorescence imaging, combined with labeling techniques available today have yet been able to provide the spatiotemporal resolution needed to document cellular processes at physiological conditions. Substituting the use of fluorescent protein tags (such as GFP) typically used in live cell applications with fluorescent dyes will improve the currently advanced imaging techniques considerably.
The superb photophysical characteristics of Fl-dyes and their small size sizes make them superior probes for quantitative high-resolution fluorescence imaging, particularly in the study of macromolecular assemblies. The implementation of a smaller fluorescent tag (GFP = 4.2 nm; Fl-dye = ~0.5 nm) will minimize potential artifacts associated with the size of the tag size, which is particularly critical in the case of for macromolecular complexes that often rely on intimate and coordinated interactions between their subunits to carry out their functionfunctions. At the same time, labeling proteins with Fl-dyes, will improve the spatiotemporal resolution of current technologies through the use of a more potent fluorophore.
We use genetic code expansion and bioorthogonal chemistry to directly and site-specifically tag proteins with fluorescent dyes in live mammalian cells. In this approach, a new (modified) amino acid carrying a unique chemical handle capable of reacting with modified fluorescent dyes (Fl-dyes) through bioorthogonal conjugation is genetically encoded into a protein during ribosomal protein synthesis. (Refer to cartoon). Over the past few years we have calibrated this approach using and demonstrated its applicability for live cell imaging and structured illumination microscopy (SIM) using tubulin as a benchmark. This project is done in collaboration with Eyal Arbely, BGU.
We are currently working to improve the robustness of the approach to adapt it for improving temporal resolution in STORM microscopy. At the same time we began to exploit this labeling approach to study the dynamics of ESCRT proteins in cells with improved spatiotemporal resolution.