Super-Resolution Microscopy of live cells in 3D

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Observing how and when molecules move within subcellular structures allows us to precisely pinpoint how cells make decisions; this fundamental knowledge is critical for human health, food security and biotechnology research. While Electron Microscopy can clearly resolve two structures that are separated by less than one nanometer (nm), Light Microscopy had been limited to resolving structures that are at least 220 nm apart, due to the intrinsic properties of light. Despite this dramatic limitation, biologists frequently prefer Light Microscopy as it allows the tracking and co-staining of multiple biomolecules (using differently coloured fluorescent probes) to compare and unravel their roles and fates in living cells. The 220 nm resolution barrier in Light Microscopy was recently broken by a set of revolutionary techniques which are collectively known as Super-Resolution Microscopy methods; the methods won the Nobel prize for Chemistry in 2014. Super-Resolution microscopy (SRM) however has been primarily used to generate static snapshots or tiny bursts of movies for a few minutes because of two key hurdles: (i) SRM exposes live specimens to light for longer than a few seconds inducing damage (photo-toxicity) and (ii) SRM requires the collection of a huge number of snapshots which slows 3D data acquisition, disallowing reliable tracking of fast moving structures in 3D. Hence, SRM could not be effectively used for live-cell studies of rapid biological processes that are highly sensitive to light. For example, studies of DNA damage repair pathways, cell division mechanisms and steps of photosynthesis require methods that allow fast image acquisition without inducing phototoxicity. Recently, newer Super-Resolution microscopes capable of long-term live-imaging of light sensitive processes (ie., tools rendering increased sensitivity, data acquisition speed that reduce phototoxicity) have become commercially available. We aim to take advantage of this recent development and establish a multi-user SRM facility for light sensitive live-cell studies in several model systems. The multi-user SRM facility will push forward ongoing conventional light microscopy studies into the super-resolution regime so that biological processes involving tiny sub-cellular structures, 100-130 nm in size, can be studied in greater detail. For example, studies of compartments inside cells that are all less than 150 nm in size will radically benefit from using this new facility. We expect this multi-user facility to not only enable high-impact research in 17 different research areas led by BBSRC funded investigators, it will also help share methodologies to push forward the imaging of a wide range of subcellular structures in a range of model organisms, from bacteria to human cells. The researchers of the consortium will together help identify or modify software tools to advance the analysis of Super-Resolution images and movies. Determining when changes in the levels and localisation of biomolecules occur within cells is crucial to reveal how biomolecules organize themselves, communicate with each other and control the function of living cells. For this purpose, researchers will combine super-resolution imaging with time-lapse microscopy - a method where images of sub-cellular structures (using fluorescently tagged biomolecules) are recorded through a period of time to reveal the sequence of dynamic changes within cells. In summary, the requested super-resolution microscope will allow several BBSRC funded groups to observe photosensitive processes for long period of hours to measure quantitative changes in biomolecules within 100-130 nm resolution accuracy. Thus, the multi-user SRM facility will provide researchers with the tools needed to expand our knowledge of subcellular structures and multiprotein organisation in unprecedented spatial and temporal detail.