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

Lead Research Organisation: University of Cambridge
Department Name: Physiology Development and Neuroscience

Abstract

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.

Technical Summary

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.

Planned Impact

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.

Publications

10 25 50
 
Description 2 photon nano lithography in soft polymers (Tuomas Knowles) 
Organisation Cancer Research UK Cambridge Institute
Country United Kingdom 
Sector Academic/University 
PI Contribution Designed a two photon confocal lithography system for the writing of 3D nanostructures in PDMS.
Collaborator Contribution Microfabrication, modelling fluid flow in nano channels.
Impact Paper submitted to Nature Photonics Multidisciplinary: Chemistry, Physics
Start Year 2016
 
Description Collaboration with Tuomas Knowles 
Organisation University of Cambridge
Country United Kingdom 
Sector Academic/University 
PI Contribution Supervision of a joint Nano CDT student project on Microfluidics for TIRG amyloid elongation assay
Collaborator Contribution Supervision of a joint Nano CDT student project on Microfluidics for TIRG amyloid elongation assay
Impact Exchange of ideas, equipment and technical skills between the groups.
Start Year 2016
 
Description Combining light-sheet microscopy and magnetic tweezing to probe tissue mechanics in developing Embryos (Craig Russell/Richard Adams) 
Organisation University of Cambridge
Department Virology
Country United Kingdom 
Sector Academic/University 
PI Contribution Live, multicolour 3D imaging of Zebrafish developing and data analysis
Collaborator Contribution Biological question, provided zebrafish, bnuilt magnetic tweezer system
Impact Collaboration ongoing/paper in prep Multidisciplinary: Physics, chemistry, developmental biology
Start Year 2014
 
Description Correlative STED and atomic force microscopy on live astrocytes reveals plasticity of cytoskeletal structure and membrane physical properties during polarized migration (Prof. Nathalie Rouach) 
Organisation College of France
Country France 
Sector Academic/University 
PI Contribution Developed stimulated emission depletion microscope and imaging protocols for connexin proteins in asrocytes. Developed atomic force microscopy for mechanical stiffness measurements to test connecin function on cytoskeletal function.
Collaborator Contribution Provision of brain slices from model systems and preparation for correlative imaging. Provision of biological reagents and expertise in neurophysiology.
Impact 2 papers in review Multidisciplinary: neuroscience, biophysics
Start Year 2016
 
Description De novo design of a biologically active amyloid (Frederic Rousseau and Joost Schymkowitz) 
Organisation University of Leuven
Department Laboratory of Angiogenesis and Vascular Metabolism
Country Belgium 
Sector Academic/University 
PI Contribution Verification of protein colocalisation betwen designer amyloid and native protein via two colour dSTORM imaging. Analysis of colocalisation data
Collaborator Contribution Synthetic amyloid design, provision of biological questions, modelling of protein misfolding and aggregation behaviour.
Impact Publication: DOI:10.1126/science.aah4949 Multidisciplinary: Physics, structural biology
Start Year 2015
 
Description Designed biologically active peptide amyloids (Frederic Rousseau and Joost Schymkowitz) 
Organisation University of Leuven
Country Belgium 
Sector Academic/University 
PI Contribution Performed and assisted with sample preparation, super resolution microscopy and data analysis of protein aggregates in cells.
Collaborator Contribution Initiated, designed and directed the research project.
Impact One publications: Gallardo et al, Science 2016 The disciplines involved are pharmacology, oncology, biochemistry, chemistry, biophysics (+ chemical engineering/optics)
Start Year 2014
 
Description Dynamics of intracellular HTT fibrils revealed by high spatiotemporal resolution microscopy (Laurence Young with Prof Alan Tunnacliffe/Dr Meng Lu) 
Organisation University of Cambridge
Department Department of Physiology, Development and Neuroscience
Country United Kingdom 
Sector Academic/University 
PI Contribution High speed multicolour super resolution imaging using custom built structured illumination microscopy
Collaborator Contribution Set up fluorescent cell line, devised experiments
Impact Collaboration ongoing/paper in prep Multidisciplinary: Physics, cell biology
Start Year 2016
 
Description High resolution imaging of HSV1 virus structure with optical techniques (Colin Crump) 
Organisation Cancer Research UK Cambridge Institute
Country United Kingdom 
Sector Academic/University 
PI Contribution Developed a structural imaging technique based on single molecule localisation microscopy to visualise protein distributions in different assembly state of virus at 1 nm transition. Developed dSTORM imaging into a tool for structural biology.
Collaborator Contribution Crump lab developed all biological models, labelling protocols and defined biological questions in the collaboration.
Impact Submitted joint application to Wellcome Trust. Publications: DOI:10.1111/tra.12340 DOI: 10.1038/ncomms6980 Multidisciplinary: pathology, biophysics
Start Year 2014
 
Description Imaging secondary nucleation of alpha synuclein (Prof Emma Sparr/Prof Sara Linse) 
Organisation Lund University
Country Sweden 
Sector Academic/University 
PI Contribution Assisted in design of experiments, performed super resolution microscopy of alpha synuclein amyloid fibrils.
Collaborator Contribution Initiated and designed research project.
Impact Outputs: Gaspar et al, Amyloid 2017 Disciplines: biochemistry, structural biology (+ chemical engineering/optics)
Start Year 2014
 
Description SIM imaging of brain slices to investigate myelination with Robin Franklin 
Organisation Wellcome Trust
Department Wellcome - MRC Cambridge Stem Cell Institute
Country United Kingdom 
Sector Academic/University 
PI Contribution Setting up the microscope, operation of the microscope, processing, reconstruction and analysis of the raw data, creation of a semi-automated image analysis protocol in Icy.
Collaborator Contribution Slicing of mouse brains, secondary anti-body staining, mounting of brain slices onto coverslip, statistical analysis of the data, biological interpretation.
Impact No publications yet, but promising results presented at various small talks - eg. in college, group meetings, to the IPES CDT
Start Year 2016
 
Description STED microscopy during early Drosophila development with Isabel Palacios 
Organisation University of Cambridge
Department Department of Zoology
Country United Kingdom 
Sector Academic/University 
PI Contribution STED microscopy on Drosophila oocytes performed by us, as well as data analyis.
Collaborator Contribution Makes all biological constructs for projects and defines biologica questions.
Impact experiments in progress
Start Year 2015
 
Description Secondary nucleation of monomers on fibril surface dominates a-synuclein aggregation and provides autocatalytic amyloid amplification (Prof Emma Sparr/Prof Sara Linse) 
Organisation Lund University
Department Department of Immunotechnology
Country Sweden 
Sector Hospitals 
PI Contribution Kinetic imaging of secondary nucleation reactions at the single molecule level with two colour direct stochastic Superresolution microscopy (dSTORM)
Collaborator Contribution Kinetic modelling of amyloid elongation reactions. Performed measurements in bulk samples.
Impact Paper in press at Quarterly Reviews of Biophysics Multidisciplinary: Chemistry, Physics
Start Year 2015
 
Description Single molecule translation imaging in axonal growth cones (Christine Holt) 
Organisation University of Cambridge
Department Department of Pathology
Country United Kingdom 
Sector Academic/University 
PI Contribution We have developed a new technique for single molecule translation imaging in neuronal growth cones.
Collaborator Contribution The biological system was developed in the Holt laboratory. Sample preparation and labelling strategies were developed by Holt et al.
Impact Publications: DOI:10.1016/j.neuron.2015.10.030 Multidisciplinary: Developmental biology, neuroscience, biophysics
Start Year 2015
 
Description Super-resolution imaging of virus vaccines and aggregation proteins with MedImmune 
Organisation AstraZeneca
Department AZ Medimmune
Country United Kingdom 
Sector Private 
PI Contribution Generated initial proof-of-concept data for imaging projects. Testing viability of these projects as PhD projects.
Collaborator Contribution Provided samples and problematic
Impact Beacon day presentation. PhD projects funding from industrial partners. Scientific paper manuscript in preparation. Highly multi-disciplinary projects: - high resolution optical imaging - high resolution contact imaging - protein folding - biochemistry - virology - neuroscience
Start Year 2015
 
Description Believing is seeing: a Cambridge Shorts film (Marcus Fantham) 
Form Of Engagement Activity A broadcast e.g. TV/radio/film/podcast (other than news/press)
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact A short film/ 'cinematic poem' exploring what it means to observe. Undertaken in collaboration with Eleanor Chan from the History of Art department, with £3000 funding from the university publicity office sponsored by the Wellcome Trust. 1 of 4 films selected for funding from abstract.
Video which premiered at the Arts Picturehouse; subsequently published on Youtube
Year(s) Of Engagement Activity 2016
URL https://www.youtube.com/watch?v=SNe65oJsOos
 
Description Passion for Knowledge 2016 festival, San Sebastian (Laurence Young) 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact Images and video used in artwork called "Breaking Boundaries" by D. Scarborough, commissioned for the opening ceremony of the Passion for Knowledge festival organized by the Donostia International Physics Center
Year(s) Of Engagement Activity 2016
URL http://p4k.dipc.org/en/performance-breaking-boundaries
 
Description Pint of Science Festival Cambridge 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact Public lecture at Pint of Science Festival.
Year(s) Of Engagement Activity 2016
 
Description Research exchange meeting with DAAD students 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Undergraduate students
Results and Impact Organised a visit (lab tours and research lectures) for German Biophysics students funded under the "Studienstiftung" programme
Year(s) Of Engagement Activity 2016
 
Description Superresolution public video 
Form Of Engagement Activity A broadcast e.g. TV/radio/film/podcast (other than news/press)
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact Video interview and research video to explain superresolution microscopy in medical research to a lay audience. Currently >14000 views.
Year(s) Of Engagement Activity 2015
URL https://www.youtube.com/watch?v=W-0GWbOFT3w
 
Description Talk on A short history of Microscopy, Cambridge 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact Public lecture on A short history of Microscopy
Year(s) Of Engagement Activity 2017