Imaging cellular structure and function beyond the diffraction limit

Super-resolved microscopy (SRM) represents a revolution in optical imaging with profound implications for the life sciences. It allows researchers to visualise biological structure and function on almost the molecular scale in live cells and organisms. Typically biological processes are visualised by attaching fluorescent molecules to proteins of interest forming images from the light emitted by these fluorescent "labels". The optical resolution, which defines the smallest structures that can be visualised, has long been regarded as fundamentally limited to >200 nm by the diffraction of light waves. Recent breakthroughs in fluorescence microscopy, however, have exploited the ability to systematically switch the emission of fluorescent molecules on and off in order to break the "diffraction limit" and achieve resolutions down to 10's of nm - almost the scale of the typical biological proteins that are involved in signalling processes that determine the fate of cells and organisms and so are essential to disease processes.

This project builds on the partnership between the MRC Clinical Sciences Centre (CSC) and Imperial College London to develop a range of SRM instruments to advance our understanding of epigenetics, regulation of genes and cells, immunology and metabolism by resolving fundamental biological mechanisms inside the nucleus, inside live cells and at the synapses of interacting cells. It also aims to establish super-resolved microscopy as a widely accessible tool for the research community at the CSC and Imperial.

The first SRM technique would be structured illumination microscopy (SIM) that entails illuminating samples with patterns of light and observing the interference patterns between these patterns and the structure of the sample. This effectively doubles the resolution of a normal microscope. The second approach is an advance on laser scanning confocal microscopy, in which as sample is imaged by scanning a focused laser beam across a sample and collecting the fluorescence pixel by pixel through a pinhole that blocks out of focus or scattered light. The size of this focussed laser beam spot defines the resolution of this microscope. By using a second, collinear, specially shaped laser beam that is scanned synchronously and can switch off the fluorescence from molecules around the outside of the first beam focus, the fluorescence excitation is restricted to a smaller spot than the diffraction limit and thus resolutions below 50 nm can be realised. Because this technique can block scattered or out of focus light, it is particularly suitable for imaging in live organisms. The third SRM modality relies on having only a small fraction of the fluorescent labels being switched on at any time such that they can be treated like arrays of single molecules. Their individual positions can be determined by locating the centres of the "blobs" that represent each molecule in an image - which is possible to a precision of a few nm. By sequentially switching on different subsets of the fluorescent labels and determining their individual positions, one can build up a super-resolved "image" that is simply the map of the locations of all the fluorescent molecules.

In this proposal, we will develop and apply these new imaging techniques to visualise biological structures and processes that are simply too small or inaccessible for conventional microscopes. Starting at the surface of cells, researchers will be able to observe how immune cells communicate and nerve cells interact with each other, and how tumour cells relate to normal cells. Looking inside cells, we will visualise signals that communicate the energy status of cells and how the genome is organised. It will be possible to resolve how chromosomes behave in developing germ cells, which is relevant to the most frequent causes of infertility and birth defects. The research will deliver new insights into both normal biological processes and disease states.

This partnership aims to combine strengths in basic science underlying human disease and imaging technology to develop capabilities in super-resolution microscopy (SRM) in order to study fundamental biological mechanisms inside cell nuclei, inside live cells and at the synapses of interacting cells.
Initially we would establish a commercial SIM microscope to provide rapid SRM of cells with existing labels. This would enable users to rapidly gain expertise in SRM, e.g. through studies of chromatin organisation and reprogramming of lymphocytes by ES cells. We would also implement rapid FLIM FRET, e.g. for directly correlation of SRM of changes in subcellular structure with cell signalling.
In parallel the Photonics team would extend their 3-D STED microscopy to live cells with GFP and YFP and also implement RESOLFT using, e.g. rsGFP. This will utilise novel fibre-laser based excitation/depletion/switching lasers developed in-house, as well as SLM technology for beam shaping and adaptive optical correction of aberrations. A key application would be to image synapses between live cells - noting that these can lie in arbitrary planes not readily accessible to other SRM techniques. Progressing to intravital imaging, we would also develop a STED/RESOLFT microscope for to study the formation and function of neurological synapses in live in mice.
We would also upgrade our in-house developed TIRF-based PALM/STORM systems to live cell imaging using emerging algorithms to separate signals from closely packed fluorophores and implement 3-D PALM/STORM using, e.g. axial localisation and oblique light sheet illumination. We would then replicate this system in the CSC for studies of nuclear reorganisation during meiosis in C. Elegans and for other subnuclear imaging applications.
The in-house development of SRM technology makes it cost effective and inherently upgradeable, enabling it to be sustained at the cutting edge for multiuser access beyond the project's end.

This significant project would have impact derived from the new technology to be developed and from the novel biology to be addressed. It would have a major impact on the CSC and Imperial College through the establishment of SRM and its development to functionality beyond the state of the art. As the partnership develops, the benefits of this project will extend beyond the initial group of investigators, with "stable" SRM technologies becoming embedded as multiuser facilities within the well-established FILM in South Kensington and within the microscopy facility at the CSC on the Hammersmith site. We would also explore the possibility of becoming a node of the ESFRI Euro-BioImaging (EuBI) initiative, which would provide another avenue for broader dissemination of our technology. Thus this project would "raise consciousness" about SRM and the new opportunities it presents and highlight expertise in its application throughout Imperial and beyond.
The technology and software tools that we propose to develop and integrate could be applied by others for SRM and related microscopy techniques. They are also likely to find diverse applications in a range of optical instrumentation. With respect to technology development, the Photonics Group has a strong track record of interacting with industry particularly for fluorescence imaging techniques and for translating novel fibre based scientific laser source concepts into technologically attractive and commercially viable prototypes. A particular example of this are the high power all-fibre-based visible light sources providing pulsed radiation with variable repetition rate and picosecond pulse durations that can find application in a wide range of instrumentation besides SRM, including, e.g. wide-field and laser scanning confocal microscopes, FLIM systems, time-resolved fluorometers, flow cytometers and displays. The SLM techniques for aberration correction could also find application in diverse optical instruments including for imaging and metrology and the software tools to be developed could be applied in, or adapted to, other contexts. We would make software tools publically available, probably via the OME platform, once they are robust. The programme is also likely to deliver a range of novel labels and probes for SRM in different systems. As we publish and disseminate our work, these probes will be made available to the scientific community and will therefore become a resource for future research.
The biological components of the proposal cover a range of areas that are important for health and disease, including immune and neurological synapse formation and function, cancer biology, lymphocyte reprogramming and chromatin reorganisation. While our research is primarily aimed at making fundamental insights into the biology of these systems, both in normal cells/ tissues and following genetic or pharmacological manipulation, the use of SRM would provide a level of detail for structures such as neurological and immune synapses that has never before been possible and this may have longer term implications for the understanding of disease mechanisms and the development of new therapeutics. Combining functional insights from FRET and FLIM with structural insights from SRM for the first time could bridge the resolution gap between biochemistry and imaging, potentially providing a step change in our understanding of structure/ function relationships in these important systems and helping us better understand both normal and disease states. Thus the immediate impact would be to the academic research community but in the longer term the project could stimulate the development of new therapies.
The knowledge and skills gained by the staff working on this project would directly benefit them in terms of increasing their potential for research impact and future employment and could benefit the biophotonics instrumentation and biomedical research communities who could employ them.