Next generation live super-resolution microscopy: development and application at the Cambridge Advanced Imaging Centre

The Cambridge Advanced Imaging Centre (CAIC) will collaborate with internationally leading laboratories to develop two instruments that surpass currently available commercial solutions in terms of resolution, versatility and depth of recording. These instruments will enable us to observe at nanometer scale the trafficking of individual molecules in living tissue and determine the structure of protein complexes in live cells.
A investment from the Wolfson Foundation together with significant contributions from University funds have enabled us to build a modern, purpose-built facility that fosters collaboration of physical scientists and biologists. Dedicated staff imaging specialists and a computer programmer will assist scientists with the use of new instruments and data analysis. From the start, the instrument development is driven from end user need, arising from research topics in cancer, developmental biology, neuroscience and neurodegeneration. Due to the wide range of biological and optical physics expertise present at CAIC, biologists will be able to quickly test the suitability of imaging platforms for their specific experimental requirements and establish what will be needed to perform these experiments successfully.
This proposal addresses some of the most pressing needs in modern biological imaging identified by world-class biologists at Cambridge University. Biomedical scientists at Cambridge interested in the molecular and cell biology of diseases such as cancer and neurodegeneration, and biological scientists interested in basic questions of developmental biology and neurobiology, will help physical scientists devise and refine these microscopes so that they can ask the most fundamental questions about cellular structure and function and gain new insights into cellular functions in health and disease.
CAIC is a top priority initiative of the University of Cambridge. The new CAIC facility will consists of two areas: the multi-user imaging facility and a separated optics lab to allow the development of prototype equipment requiring open laser path arrangements.
Strong links exist between CAIC and the other imaging hubs at Cambridge, which allows synergising on complementary expertise. CAIC is not just a physical hub for advanced microscopy at the University of Cambridge, it is the basis of a pipeline for the advancement of imaging technology. In the optical development area, new microscopes are built and trialled. From there they will be moved to the service area, where they enable biologists to use state-of-the-art imaging methods without the need to find funds to buy or build these instruments themselves. But as this happens new space becomes available to build and test new machines in the optical development area.

A training programme is a key component in the long-term success of the centre. New developments in imaging technologies will feed through to enhancing training in the theoretical and practical aspects of biological microscopy, and in image processing techniques. Graduate students and postdoctoral fellows will work alongside the physical scientists building the new machines and the biological scientists trialing them. This new breed of interdisciplinary microscopists will, it is hoped, be inspired to build the next generation of advanced imaging instruments.

We will custom-develop 2 separate instruments for 1) far-field individual molecule localization with Total Internal Reflection Fluorescence (TIRF) and 2) gated Stimulated Emission Depletion microscopy (g-STED) & Reversibly Switchable Optical Fluorescence Transition using reversible photoswitching GFP (rsGFP-RESOLFT).

Instrument 1) will implement the Double Helix Point Spread Function detection (DH-PSF) technique to axially encode the position of single emitters from direct Stochastic Optical Reconstruction Microscopy (dSTORM) or PhotoActivation Localization Microscopy (PALM). DH-PSF can typically attain a resolution of 10-15 nm laterally and 20-25 nm axially.

Instrument 2) will readily achieve a planar resolution of 70 nm, and 300 nm axially in fixed tissue in g-STED operation. This instrument will also serve as development platform for rsGFP-RESOLFT. In collaborative research with the inventors of RESOLFT, we will implement 1) parallel STED beam excitation and readout using microlens arrays and EMCCD detectors to shorten overall acquisition time; 2) improve z- resolution through the use of two individual phase plates to generate a 3-dimensional RESOLFT 'doughnut'.

Building on our expertise in Fluorescence Light Sheet Microscopy (FLSM), we will combine FLSM with PALM/dSTORM and DH-PSF detection to enable super-resolved imaging of large cells (>5 um diameter) at estimated 20 nm planar and 40 nm axial resolution.

We will use novel microfluidic devices to effectively trap individual cells at multiple predefined position as to make them available for automated and parallelised super-resolution microscopy. This technique will allow efficient use of microscopy time and promises to shorten the overall experimental time.

We will develop software to control our above microscopy instruments as modules for the open-source platform microManager and will make the source-code publically available.

Super-resolution microscopy has the potential to extend the resolution of optical microscopy from around 200-300 nm down to 10-20 nm, which is close to the size of single protein molecules. It could, therefore, bridge the gap between conventional life cell imaging and structural studies of proteins and protein complexes using X-ray crystallography, NMR spectroscopy and electron microscopy. It is hard to overstate the potential impact of this - at best, it has the possibility of revolutionising biological studies allowing widespread detailed studies of nuclear processes at a single molecule level in intact live cells. Studies using our present super-resolution microscopes (in the Klenerman and Kaminski groups, and in the Gurdon Institute) are showing, however, that considerable innovation and optimisation will be necessary before these instruments yield the hoped for gains in resolution and deliver the impact that they promise.
There is enormous strength in biological studies in Cambridge and establishing a thriving community developing and utilising super-resolution microscopy will have a very considerable impact, not just in the science we can carry out, but in a number of other areas:
1. Benefitting the UK population through improved healthcare.
An improved understanding of neurodegenerative processes and epigenetic processes to control, for example, stem cell differentiation and diseases such as cancer will have a direct impact on healthy aging in our society. This increased understanding, in combination with personalised approaches to medicine, will directly benefit the pharmaceutical industry, and its efforts to develop new drugs.
2. Providing a scientifically well-trained workforce.
The training of a new generation of researchers who can use super-resolution imaging in both academia and industry will contribute directly to scientific and ultimately societal development. In collaboration with colleagues in Oxford and London, we will also organise regular one-day workshops to foster the development of super resolution microscopy and help biological scientists in the community work out how best to apply it.
3. General public.
Life cell imaging, and the spectacular insights which can be gained from this, have enormous potential to excite a new generation of students to take up science. We will provide images and movies, which can be used in lectures and demonstrations for A-level students in schools and colleges.