Deciphering Nanoscale Protein Dynamics: Massively Parallel Functional MINFLUX

Each major progression in biological understanding is driven by advances in technology to enable novel and disruptive discovery. Super-resolution techniques such as single-molecule localisation microscopy (SMLM) have revolutionised spatial biology by enabling nanoscale imaging of biomolecules. The latest challenge for super-resolved imaging optical modalities is to achieve functional read-outs of biological activity: A new paradigm beyond the observation of spatial and temporal patterning. MINFLUX (MINimum Fluorescence photon fLUXes) microscopy has achieved near-angstrom resolution, however, significant challenges remain in making it broadly impactful for research. Limitations include access to instrumentation, low throughput, restricted fields of view, and the lack of integrated multiplexing or functional capabilities. Fluorescence lifetime imaging microscopy (FLIM), when used for measuring Förster Resonance Energy Transfer (FRET), is a powerful technique for studying protein interaction and states of activity, offering critical insights into molecular pathways that govern cellular behaviour. Acting on comparable lengthscales to MINFLUX, FRET is a multiplier in experimental precision and sophistication, potentially adding significantly to the super-resolution toolbox. No such implementation has so far been reported indicating a clear route to development for this team. This project aims to address this technology gap, increasing the speed and throughput of MINFLUX super-resolution microscopy by developing a simpler, more affordable, and user-friendly paradigm, with integrated single-photon counting FLIM capabilities. Our innovative approach replaces the single laser beam in MINFLUX imaging with an optical lattice, enabling simultaneous monitoring of a multiplicity of molecules simultaneously. This expands the field-of-view and enhances throughput, addressing two key limitations of the state-of-the-art. Leveraging our expertise in microscope design for massively-parallelised FLIM (Ameer-Beg) and super-resolution techniques (Jose), we will design an optical system reducing complexity and cost. We will employ state-of-the-art single photon avalanche diode (SPAD) arrays for detection, with embedded-FLIM imaging and on-chip processing to enable real-time analysis. Our primary biological exemplar, will be demonstration of the nanoscale integration of biochemical and mechanical signalling between the cell and the extracellular matrix within focal adhesions on the cell surface. We will focus on elucidating the dynamic architecture and functional significance of clustering of key proteins (FAK, Vinculin, and Paxillin) which are essential for cell migration. The impact of our research will resonate across multiple sectors. With only one MINFLUX system operational in the UK, to date, we believe that the developed technology will provide a valuable resource for researchers in diverse fields, helping to address complex biological questions with greater precision and speed. The addition of fluorescence lifetime contrast is unique in the world, and will provide a platform for spatio-temporal exploration of cell signalling at the nanoscale. We will make this microscope widely accessible to our collaborators within the Comprehensive Cancer Centre and, through our long term initiative, the Microscope Innovation Center (BioimagingUK node). This project has the potential to be transformative for research at the single- and few-molecule level. Our project aligns perfectly with the contemporary trend of precision medicine and targeted therapies - in particular those aimed at receptor trafficking and inhibition (EGFR: Cancer), at dysregulation of metabolism (In mitochondria: Neurodegeneration) or in cell migration (ARP2/3, Vinculin force transduction: Focal adhesions): Areas in which this research group has active collaborations and research projects. Our project will develop the tools to enable us to directly investigate the roles of focal adhesion proteins in cellular processes, such as cell migration, invasion, and signalling.