A super-resolution multi-scale in vitro and in vivo imaging platform at Harwell: building models of development and disease from molecules to mammals

Capturing the 'why' of a disease and the 'how' of a treatment requires building a model of the disease and its evolution during treatment. This model ideally needs to capture everything, from the interplay of a single molecule with a molecular network, to inter-cellular communication networks that underpin the development of a phenotype. To reach this goal we need to bridge the different scales on which biology operates - the molecular, the cellular and the whole organism. One way to do this is to image cells and organisms at different scales: (i) the nanoscale to show how cells are organised at the molecular level and how molecular therapies affect molecular interactions; (ii) the microscale to show the functions of cellular organelles and their reorganisation under therapeutic challenge; (iii) the mesoscale to investigate cellular behaviour during development and/or the progress of disease; (iv) and the whole organisms level, to monitor changes and the well-being of the entire organism in response to therapy (e.g. tumour remission). Only when these jigsaw pieces are put together should one be able to understand how mutated genes and proteins affect development, predict how the "next generation" therapies can deliver breakthrough advances and elucidate how therapeutic agents can be delivered to focal areas of disease to maximize clinical benefit while limiting side effects.
Cells were discovered by Robert Hooke (1635-1703) under the optical microscope. Lord Rayleigh (1842-1919) empirically determined that the resolution of a diffraction-limited microscope can be no better than ~ 1/2 of the wavelength of light (i.e. >200 nm), which defines the microscale. For centuries this was a fundamental limitation of light microscopy, as this resolution is 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 microscopy, which showed ~20 nm resolution, followed by the less damaging structured illumination microscopy with 90 nm resolution. During the last decade, 'simpler' modes of super-resolution microscopy achieved similar resolutions by using the fundamental principle that molecules are much smaller than the wavelength of light and therefore can be considered 'single point' emitters. This is very important because their position in space can therefore be determined with nanometre accuracy via deconvolution of the 'blob'-like spot image created by the microscope optics, which incidentally is the origin of the poor resolution associated with light microscopy. High resolution images at the nanoscale are formed by putting together individual molecular images, a time-consuming process which nevertheless has already delivered spectacular results.
High resolution imaging at the mesoscale is critical to understand basic mammalian biology. OCT was developed to address the need for fast imaging tools to characterise the inter-play between cells in a whole organisms in order to model human pathophysiology, and assess benefits resulting from therapeutic treatments in pre-clinical research.
We have formed an interdisciplinary partnership that seeks to exploit a new generation of world-leading super-resolution microscopy in combination with state-of-the-art in vivo imaging methods (like OCT). Our principle is to break the barriers between fields to ease the exploitation of these new technologies by the wider biomedical community and to place the UK at the imaging forefront. The interdisciplinary environment and concentration of scientists at the Harwell Campus will help in our efforts to underpin fundamental discoveries in the next decade.

We have identified three developments to our current facilities to make the proposed multi-scale in vitro-in vivo platform world-leading:

Firstly, to purchase a top-of-the-range 3D super-resolution microscope that combines structured illumination microscopy (SIM) with fluorescence localisation microscopies such as stochastic optical reconstruction microscopy (STORM) for live cell imaging to satisfy the strong demand for this technology at the earliest opportunity. Secondly, to establish a 4D ultrafast OCT system to provide much needed access to high-throughput in vivo imaging of embryonic and adult organ systems. Not only there is an urgent need in our communities to have access to these systems but their availability is also essential to make the proposed multi-scale platform internationally competitive.

Our choice of commercial super-resolution system has been directed by the ideal of imaging live cells in real-time. However, all the microscopes available have a number of technical limitations that require making one or more of the following compromises: between spatial and temporal resolution, between speed, resolution and imaged sample volume and between photodamage and resolution. The third development we propose is to build the first truly 3D super-resolution microscope that will obtain images with a resolution of <20 nm both axially and laterally and that will collect data from a 3D volume of cell dimensions in a single shot rather than imaging planes within the sample individually. This microscope will be able to image multiple targets within the entire cell volume at this resolution in all three spatial axes and subsecond temporal resolution. At this time scale a wealth of new science will become accessible as we will be able to follow with nanoscale resolution in real-time a multitude of cellular process and molecular interactions as they occur. This will make the proposed imaging platform internationally leading.

The immediate beneficiaries of the partnership will be the academic user community of the new imaging facilities. Some collaborative programmes are outlined in this proposal, and in the long term, as access to the new facilities will be available through open access peer-review, we expect many beneficiaries in the academic community. These academics will be largely from the biomedical research community, although other disciplines (e.g. biomedical materials research) will also benefit from the availability of 3D super-resolution imaging facilities. Users of the facilities will be trained in the use of the new techniques, and this expertise will be transferred to their home institutions, expanding the UK's base of experts in new imaging technologies.
The partnership aims to break the boundaries between the imaging and biomedical communities, enabling the translation of histopathological and phenotypical models into the molecular and cellular regimes. An immediate impact should be increased collaboration between these two communities, resulting in sharing of expertise and enhancement of research outputs in both areas through the application of new techniques. Ultimately, there will be societal benefits in the form of new medical treatments and diagnostic techniques, the collaborative partnership speeding up the process of translating research findings into medical benefits.
There should also be significant impact for the scientific instrument industry. The 3D super-resolution method we propose to develop will be of interest to many researchers in the UK and worldwide, and there has already been significant interest from a leading microscope manufacturer. We are working closely with all the major microscope companies, and we expect that the developments we propose will be taken up by one of the manufacturers with the aim of producing a commercial instrument based on our technology. The research that will be enabled by the partnership 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. current collaborations with Evotec and Illumina, and the successful spin-out from the Central laser facility, Cobalt Light Systems). 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 OCTOPUS facility is a regular feature on these visits, and the proposed new developments 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.