Developing and applying genetically encoded proteins as pre-resonant coherent Raman scattering tags for next-generation live-cell imaging.

This research project will develop next generation biological imaging methods by using synthetic biology to design and apply protein tags for Raman-based micro-spectroscopy. Optical microscopy is an indispensable tool that is pivotal to understanding biological processes in the cell and is currently the only practical means of obtaining high spatial and temporal resolution within living cells and tissues. Fluorescence microscopy has provided a highly sensitive and specific method of visualizing biomolecules and has revolutionised cell imaging, especially after the discovery of fluorescent proteins (FPs) that enabled genetic tagging of specific targets inside living systems. However, there are flaws, as fluorescent probes are prone to photo-bleaching and associated cytotoxicity which hamper their use, especially for imaging over long timescales in live cells which is critical to understanding biological process and underlying disease states. Moreover, the emission spectrum of these probes is quite broad, limiting simultaneous monitoring of multiple targets.

This project will seek to merge the advantages of genetically encoded fluorescent proteins with those of vibrational Raman tags to develop a new live cell imaging approach. The underlying idea is to incorporate Raman-active chemical bonds not normally present in biology at specific locations near the functional centre of a FP. These new genetically encoded imaging probes will allow you to exploit a process called electronic pre-resonant Raman scattering (PRRS), enhancing the Raman signal by some 2-4 orders of magnitude, and therefore enabling imaging of a small number of these tags. Protein engineering together with a reprogrammed genetic code method will be used to incorporate non-natural amino acids containing Raman active chemical bonds at optimal positions in selected FPs, selected via in-silico design. Using advanced Raman scattering micro-spectroscopy you will quantify and understand PRRS in your designed proteins and apply them to image cellular events. Initially, you will generate tubulin-FP constructs to demonstrate the technique on static (microtubules) and dynamic (assembly/disassembly) cellular structures. Crucially, PRRS is expected to surpass traditional fluorescence approaches in terms of photo-stability, and you will verify this property by long term imaging throughout the cell cycle. This will in turn allow us to better understand the dynamics of tublin assembly/disassembly throughout the life time of a cell, and how anticancer and anti-inflammatory drugs impact on these processes.