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.
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.
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.
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.
Organisations
- University of Cambridge (Lead Research Organisation)
- Engineering and Physical Sciences Research Council (Co-funder)
- Biotechnology and Biological Sciences Research Council (Co-funder)
- Lund University (Collaboration)
- AstraZeneca (Collaboration)
- Cancer Research UK Cambridge Institute (Collaboration)
- Wellcome Trust (Collaboration)
- University of Leuven (Collaboration)
- College of France (Collaboration)
- UNIVERSITY OF CAMBRIDGE (Collaboration)
Publications
Bali K
(2023)
Biosensor for Multimodal Characterization of an Essential ABC Transporter for Next-Generation Antibiotic Research.
in ACS applied materials & interfaces
Bali K
(2023)
Multiparametric Sensing of Outer Membrane Vesicle-Derived Supported Lipid Bilayers Demonstrates the Specificity of Bacteriophage Interactions.
in ACS biomaterials science & engineering
Laine RF
(2019)
Fast Fluorescence Lifetime Imaging Reveals the Aggregation Processes of a-Synuclein and Polyglutamine in Aging Caenorhabditis elegans.
in ACS chemical biology
Van Tartwijk FW
(2022)
Protein Condensation, Cellular Organization, and Spatiotemporal Regulation of Cytoplasmic Properties.
in Advanced biology
Pang C
(2019)
On-Chip Super-Resolution Imaging with Fluorescent Polymer Films
in Advanced Functional Materials
Mela I
(2021)
Revealing Nanomechanical Domains and Their Transient Behavior in Mixed-Halide Perovskite Films
in Advanced Functional Materials
Wunderlich LCS
(2021)
Superresolving the kidney-a practical comparison of fluorescence nanoscopy of the glomerular filtration barrier.
in Analytical and bioanalytical chemistry
Stephens AD
(2018)
Different Structural Conformers of Monomeric a-Synuclein Identified after Lyophilizing and Freezing.
in Analytical chemistry
Mela I
(2020)
DNA Nanostructures for Targeted Antimicrobial Delivery
in Angewandte Chemie
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 | 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 |