Super-Resolution Microscopy of live cells in 3D

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.

Super-Resolution Microscopy (SRM) has revolutionised our ability to localise and quantify biomolecules and their interactions in cells. Yet, this revolution had not been fully transferred to live-cell studies of processes that are either exquisitely sensitive to phototoxicity or too rapid for 3-dimensional (3D) tracking through time.

Recently, Super-Resolution Microscopes capable of low light illumination and high-speed image acquisition, without inducing photo-toxicity, have become commercially available, allowing long-term 3D live imaging of cells. Static subcellular structures previously discerned using Electron Microscopy (EM) can now be observed 'live' using Super-Resolution Microscopy, allowing the tracking of dynamic subcellular events at unprecedented spatial resolution.

The proposed equipment will transform BBSRC-funded research by enabling dynamic imaging of structures less than 150nm in size. To significantly advance research outcomes in several BBSRC-funded programs in the University and beyond, three main objectives have been identified:
(1) Combatting photo-toxicity to reveal 3D-spatial regulation during cell division in yeasts, annelids, and human cells;
(2) Enabling fast SRM to study dynamic membrane and subcellular organisation in bacteria, yeasts, fish, algae or plant cells; and
(3) Bridging the gap between EM and Light microscopy to study microbial infection and cellular homeostasis in bacteria and human cells.

Part-funding a Live-cell Super-Resolution Microscope is a timely and cost-effective solution. Several ongoing research projects using conventional high-resolution microscopy can be easily extended to Super-Resolution microscopy. No new growth facilities needed for live-cell studies needs to be set up as they are available for a wide range of models within a shared building.

Thus, a local live-cell SRM facility dedicated for photosensitive studies will enable world-class discoveries in a wide spectrum of research areas.

The equipment will allow state-of-the-art Live-cell Super-Resolution Microscopy (SRM) in funded projects relevant to a wide range of BBSRC priorities: Healthy Ageing, Anti-Microbial resistance, Agriculture and Food Security and 3Rs in research using animals. The research outlined spans a wide spectrum of biological processes - Cell Division, Membrane Dynamics and Photosynthesis, transcriptional regulation and Microbial Infection; and these are studied in a variety of model systems from bacteria to human cells. This diverse Biosciences community will work together and exchange new SR methodologies, which will drive innovation (academic impact), produce highly trained bioimaging experts (economic impact) and strengthen industrial partnerships (commercial impact).

Academic impact: Innovative Cell Biology research
The foremost impact will be through accelerating world-leading studies of dynamic cellular processes at an unprecedented spatial resolution. Instead of using conventional microscopy (~220x550 nm resolution), the 3D SR-SIM regime (~100x250 nm resolution) will be promoted. This near-doubling of spatial resolution in live studies is expected to promote high-impact discoveries in Microbiology, Cell biology, and Plant biology. Similarly, studies of membrane compartments and biofilms, previously amenable to EM methods alone, will now benefit from multi-colour colocalization nanoscopy (~20 nm resolution) and also live SIM-TIRF studies at the membrane (~100 nm resolution). Finally, to enable a truly UK-wide impact, a proportion of the microscope's use will be open to external partners who apply for microscope time, based on research relevance to BBSRC priorities.

Economic impact: Interdisciplinary Imaging experts
SRM is an emerging field with applications in biotechnology and biomedical industry in addition to academic research. The second major impact of the project will be ensured by the interdisciplinary research and training environment (created through the consortium), in addition to state-of-the-art microscopy technique. As SRM blurs the boundaries between Cell and Structural biology, work in Objective-3, will create a specially talented pool of interdisciplinary biologists. A well-trained researcher pool in new imaging methodologies and image analysis tools (for example, BBSRC-LIDo-DTP students) will benefit the growth of the UK Biosciences economy.

Commercial impact: Strengthening industrial partnerships
Some of the industry research that will benefit from new nanoscopy studies are listed:
A) Drug Screening: Static structures studied using EM can be tracked live, opening new drug screening assay. For example, the action of disruptive molecules aimed at disassembling microcompartments for use as antimicrobials in urinary tract infection could be screened (Pickersgill); the precise effect of potent anti-aggregation compounds can be discerned (Caudron); small molecule inhibitors identified against mycobacterial Mce virulence proteins and host MAPK kinase modulators affecting host-pathogen interactions can be studied ex vivo. G-protein coupled receptors are also a major interest in drug screening (McCormick).
B) Bio-Imaging: High-speed images in the Millisecond regime using a liquid lens can be benchmarked using 3D-SR data of submicron particles to develop oral imaging-based noninvasive diagnostic tools (Draviam).
C) Agriculture Sector: How gene isoforms confer stress tolerance can be probed using SRM for improving crop varieties (Hanke); Genes suggested as targets for increased photosynthetic productivity can influence research in Agrobiotech companies (Ruban).
D) Genome-wide tool generation: Chromosome marker tool (Thorpe), Mitochondrial quality assessors (Campanella) can be determined using SRM.
Thus, the timely application of live-cell SRM in ongoing research will generate academic, economic and industrial impact supporting the UK's position as a global leader in biosciences.