SPRINT: A SuPer-Resolution time-resolved ImagiNg and specTroscopy facility for rapid biomolecular analysis

It is essential to understand the structure and dynamics of biomolecules if we are to exploit them for medical or industrial applications. Molecules such as proteins and enzymes are dynamic, and understanding their interactions within cell pathways and biological processes can provide new insight into fundamental mechanisms and support the development of new therapeutic diagnostics and drugs. We usually require the use of a range of complex equipment to understand and characterise biomolecules fully. One common way to visualise molecular interactions is through the use of specialised microscopes; however, this approach can only resolve structures that are greater than 250 nm apart (0.00025 mm). Proteins and other molecules found in cells are much smaller than this (around 0.00001 mm), and therefore to resolve single proteins and their interactions requires access to more advanced microscopy techniques, commonly referred to as "super-resolution" microscopy.

This application proposes to establish a Scottish microscopy facility that adopts super-resolution techniques, in particular a 'quantum-ruler' (called FLIM-FRET, capable of measuring molecular interactions happening within 0.00001 mm) and a method called FCS that allows single molecules and their behaviours to be studied in microscopic regions of living cells. Access to this new facility will have a significant impact on academic researchers across Scotland. This facility will also facilitate the training of young early-stage researchers in cutting-edge technology. It will also support the training of doctoral students ensuring the next generation of scientists have the skills required to deliver significant new scientific breakthroughs to help build the UK capability and sustainability in the area of biomolecular interactions.

We request support to purchase and install an integrated SuPer-Resolution (< 50nm) time-resolved (< 10ps) ImagiNg and specTroscopy (SPRINT) system at the Strathclyde Institute of Pharmacy and Biomedical Sciences imaging facility at the University of Strathclyde. SPRINT adopts a stimulated emission depletion (STED) laser to enhance the optical resolution and also provides fluorescence lifetime imaging (FLIM) and fluorescence correlation spectroscopy (FCS) functionality. SPRINT is essential to support the research requirements of many groups at Strathclyde and Glasgow. They increasingly require access to equipment that provides enhanced resolution to study the spatiotemporal dynamics of molecular interactions in live cells, but no system with this capability is presently available.

SPRINT will enable two research themes: 1) STED-FLIM and 2) STED-FCS.
FLIM is a powerful imaging technique for in-vivo measuring how molecules interact with their microenvironments or interact with each other in combination with Förster resonance energy transfer (FRET) techniques. FLIM-FRET is a 'quantum ruler' for its ability to measure protein-protein interactions that happen within 10nm. With STED illumination, STED-FLIM or STED-FLIM-FRET offers a much better resolution to observe these behaviours that confocal FLIM is unable to resolve. Compared with traditional confocal FCS techniques, STED-FCS can distinguish spatially heterogeneous diffusion behaviour of molecules, for example, in the plasma membrane, caused by molecular interactions happening at spatial scales much smaller than 100nm.

SPRINT represents a step-change in capacity to underpin BBSRC-relevant research. Importantly the applicants have expertise in time-resolved instrumentation, optical physics and data analysis which will facilitate optimal data acquisition strategies for biological users. SPRINT will transform the way molecular interactions are quantified and explored for a wide range of biologists.