A sharper light from gSTED microscopy on biological structure and molecular interactions

One of the central goals of 21st century cell biologists is to provide a seamless link of structural understanding between the macroscopic level of tissue organization to the molecular and even atomic level organization of the building blocks of cells and tissues. As these building blocks are nanoscale objects, applying superresolution microscopy to cell biology intuitively makes sense. The material sciences have also advanced in to the nanometre range as well, readily producing nowadays synthetic particles of nanometre proportions. Studying these particles and their interactions with equally small cellular components has also become important in many biomedical applications, from targeted therapeutics to bone repair. As cellular reactions are often associated with specific subcellular organelles and compartments it is also important to study the transport of particles and pinpoint their destination within the cell with high accuracy even at the sub-organelle level.

For centuries a fundamental limitation of light microscopy was that is resolution was insufficient to resolve the nanoscale processes underpinning biology. Despite this, through the availability of many organic labels and the discovery of green fluorescent protein, fluorescence microscopy has been fundamental for decades to many of the in vitro-based key discoveries in the biomedical sciences. The 'resolution limit' of light microscopy was broken at the end of the last millennium using a challenging technique, stimulated emission depletion (STED) microscopy, which showed up to ~20 nm resolution.

In recent years, STED microscopy has matured from an exclusive and highly specialised method for superresolution imaging of a limited set of suitable sample types, to a widespread, general purpose mode of fluorescence microscopy. STED microscopy can now been performed in multi-colour, live cells and even at video rate. By achieving very high resolution, STED microscopy has opened up a field of application for fluorescence microscopy that had been previously been an exclusive domain of electron microscopy, the advantage being that STED allows investigating intracellular physiological processes in the nanoscale in almost any organelle of a living cell, and in real time.
High resolution imaging is critical to understand basic cell biology. We have formed an interdisciplinary partnership that seeks to exploit STED microscopy and testing in a range of relevant samples within a multidisciplinary environment. After commissioning the microscope, our experience will help other scientists and collaborators to apply this method to answer their scientific questions. The concentration of scientists at the Harwell Campus will help in our efforts to underpin fundamental discoveries in the next decade.

The super-resolution techniques offered commercially are structured illumination microscopy (SIM), fluorophore localisation microscopies (fLM) such as stochastic optical reconstruction microscopy (STORM), and STED microscopy. Since no single microscopy provides all of these capabilities, different questions must be addressed using different techniques.

STED is a good system to investigate dynamic processes. The basic idea behind STED microscopy is the engineering of the point spread function (PSF) of the microscope, which is the image through the microscope of an infinitely small light source. By illuminating the sample with a Gaussian beam, quickly followed by a beam with a 'doughnut-shape' and longer wavelength, the lateral PSF size is reduced because the fluorophores excited by the 'tails' of the Gaussian beam don't emit fluorescence as they return to the ground state via stimulated emission. As the size of the PSF determines the resolution of the microscope, using these beams together increases the resolution.

Today, STED microscopy is a prominent and versatile form of superresolution light microscopy. STED can be performed in multi-colour, live cells and at video rate. This is eminently suitable to studying protein interactions or the structural-functional dependencies that require the recording of at least two separate channels.

STED is suitable to be used in a scanning mode, like a confocal microscope. By combining it with FLIM we can extend to the nanoscale regime the functional studies currently undertaken using the combination of confocal microscope, FLIM and Forster resonance energy transfer (FRET). This is important towards bridging the resolution gap between FRET (2-8 nm) and the resolution of light microscopy (>250 nm), allowing us to follow protein interactions within organelles in the cell. By combining STED with FCS analysis, we can investigate subtle differences in the diffusion characteristics of different proteins and nanoclusters.

The immediate beneficiaries of the partnership will be the academic user community of the new imaging capability. Collaborative programmes are outlined in this proposal, and in the long term, as access to the gSTED microscope becomes available through open access peer-review, we expect many beneficiaries in the academic community. These academics will be largely from the life-sciences research community, although other disciplines (e.g. biomedical materials research) will also benefit from the availability of a cluster of super-resolution imaging facilities. Users of the microscope will be trained in the use of the gSTED technique, and this expertise will be transferred to their home institutions, expanding the UK's base of experts in new imaging technologies. Ultimately, there will be societal benefits in the form of new medical treatments and diagnostic techniques, the collaborative nature of the Research Complex and Harwell Oxford campus speeding up the process of translating research findings into medical benefits.

The research that will be enabled by the availability of gSTED is expected to benefit other commercial sectors, such as pharmaceuticals and medical diagnostics. The applicants have a track record of working with these sectors (e.g. MMF's current collaborations with Evotec and Illumina, and the successful spin-out from the Central Laser Facility, Cobalt Light Systems, SC's collaborations with Giltech, Pfizer, and Stryker, IJ's work with Wyeth and Pfizer). STFC provides a high level of support for identification and support of commercial opportunities, and this will be drawn upon to ensure maximum economic impact is derived from the work of the partnership. Finally, the research outputs will be of significant public interest because of the healthcare connections, and the high visual impact of microscopy work. There is already an extensive public engagement programme operated by STFC and RCaH, with regular organised visits from members of the public, schools, and undergraduates. The microscopy suite is a regular feature on these visits, and the new microscope will be seen and demonstrated. We actively encourage the dissemination of research outputs through additional routes to the "conventional" scientific literature, and regular press releases are issued when potentially high impact findings are published.