Single-molecule super-resolution microscopy of membrane dynamics: application to immunology

Lead Research Organisation: University of Oxford

Abstract

Research by my group aims to unravel nanoscopic changes at the molecular level in living cells to characterise important molecular processes on the cell membrane as well as inside the cell during immunological reactions. Because many cellular responses lead to changes so subtle at the molecular level, studying them requires us to not only observe them with a superior spatial resolution but also to reach a sensitivity that is able to monitor single molecules over time and space. We are using the newest and most powerful super-resolution far-field microscopes (such as STED, RESOLFT or PALM/STORM) to image and analyse cellular structures and protein-protein and protein-lipid interactions at a level of fine detail that until now has not been possible due to the limited spatial resolution of conventional optical far-field microscopes. By combining these super-resolution microscopy techniques with single-molecule sensitive detection methods (such as fluorescence correlation spectroscopy) and fast spatio-temporal tracking tools we are able to see complex dynamic processes otherwise invisible because of the lower power of conventional far-field microscopy. These molecular interactions play an important role in the immune response to infection and cancer and so we intend to use and further develop these advanced microscopy techniques and apply them to gather new insights in immunological research.

Technical Summary

Single-molecule super-resolution microscopy of membrane dynamics: Many cellular responses lead to subtle changes on the molecular level, demanding not only for a superior spatial resolution of the analyzing method but also for the sensitivity to monitor single molecules over time and space. The combination of super-resolution optical fluorescence STED microscopy with single-molecule sensitive fluorescence-detection tools such as Fluorescence Correlation Spectroscopy (FCS) as well as the fast spatio-temporal tracking of single labeled molecules (single-particle tracking, SPT) allows for the disclosure of complex dynamic processes otherwise impeded by the limited spatial resolution of conventional far-field microscopy. For example, STED-FCS or SPT offer us the possibility to gain novel insights into important cellular processes, such as lipid-lipid, lipid-protein, and protein-protein interactions and the formation of so-called “lipid-rafts” in the cellular plasma membrane. These molecular interactions play an important role in the cellular immune response. We will therefore apply and further develop the STED-FCS and SPT microscopy techniques to highlight important molecular processes on the plasma membrane as well as inside the cell during immunological reactions. For example, these techniques will be used to shed new light on different molecular pathways triggered at the cell surface and intracellularly during antigen presentation by dendritic cells and T cell activation.

Publications

10 25 50
 
Description Access to the Advanced Imaging Facility at Janelia, Howard Hughes Medical Institute Janelia, entitled: Cytoskeleton induced microcluster dynamics lead to T-cell synapse formation
Amount $1,000 (USD)
Organisation Howard Hughes Medical Institute 
Department Janelia Research Campus
Sector Academic/University
Country United States
Start 11/2014 
End 12/2014
 
Description Access to the Central Laser Facility, STFC Rutherford Appleton Laboratory, Harwell Oxford Didcot, UK, Advanced single-molecule tracking in immune cells at the Weatherall Institute of Molecular Medicine
Amount £1 (GBP)
Organisation Rutherford Appleton Laboratory 
Sector Public
Country United Kingdom
Start 07/2014 
End 08/2016
 
Description Advancing Image Analysis at the Wolfson Imaging Centre Oxford
Amount £45,000 (GBP)
Organisation University of Oxford 
Department E P A Cephalosporin Fund
Sector Charity/Non Profit
Country United Kingdom
Start 09/2016 
End 04/2018
 
Description BBSRC research grant (BB/L00433X/1) Dynamics of Peroxisomal Protein Transport
Amount £167,308 (GBP)
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 01/2014 
End 12/2016
 
Description Dynamics of Peroxisomal Protein Transport
Amount £1,730,400 (GBP)
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 05/2013 
End 04/2016
 
Description Dynamics of Peroxisomal Protein Transport National Research Unit FOR1905-PerTrans
Amount € 207,800 (EUR)
Organisation German Research Foundation 
Sector Public
Country Germany
Start 01/2017 
End 12/2019
 
Description EMBO Long-Term Fellowship (contribution to group member Erdinc Sezgin)
Amount £26,000 (GBP)
Organisation European Molecular Biology Organisation 
Sector Learned Society
Country European Union (EU)
Start 01/2014 
End 12/2015
 
Description EP Abraham Cephalosporin Trust Fund 2014 Nanoscopy Oxford: Super-resolution optical STED microscope facility at the WIMM
Amount £62,000 (GBP)
Organisation University of Oxford 
Department E P A Cephalosporin Fund
Sector Charity/Non Profit
Country United Kingdom
Start 06/2014 
End 04/2016
 
Description EP Abraham Cephalosporin Trust Fund 2015 BioISCAT: Biomedical application of novel fast molecular tracking. Partially funded. £
Amount £74,200 (GBP)
Organisation University of Oxford 
Department E P A Cephalosporin Fund
Sector Charity/Non Profit
Country United Kingdom
Start 07/2015 
End 07/2016
 
Description EPSRC/MRC Oxford-Nottingham Centre for Doctoral Training in Biomedical Imaging. Member of the management board. 15 students per year across all imaging modalities, developing and applying novel image analysis and imaging hardware approaches
Amount £1 (GBP)
Organisation University of Leeds 
Department Faculty of Engineering
Sector Academic/University
Country United Kingdom
Start 10/2014 
End 10/2020
 
Description Enabling enhanced optical imaging of whole organs by optimized tissue clearing apparatus at University of Oxford light microscopy imaging facilities
Amount £15,000 (GBP)
Organisation University of Oxford 
Department E P A Cephalosporin Fund
Sector Charity/Non Profit
Country United Kingdom
Start 01/2017 
End 01/2018
 
Description Establishment of force measurements in biomedical research
Amount £57,500 (GBP)
Organisation University of Oxford 
Department John Fell Fund
Sector Academic/University
Country United Kingdom
Start 06/2016 
End 05/2017
 
Description John Fell OUP Research Fund Matching funds for external applications for acquisition of a Zeiss 880 upright confocal microscope with novel Airy-scan super-resolution detector
Amount £100,000 (GBP)
Organisation University of Oxford 
Department John Fell Fund
Sector Academic/University
Country United Kingdom
Start 06/2015 
End 12/2016
 
Description MRC project grant (MR/L018942/1): The impact of IFTIM3 genetic variation on Influenza virus infection, immune responses and disease outcome
Amount £200,000 (GBP)
Funding ID MR/L018942/1 
Organisation Medical Research Council (MRC) 
Sector Academic/University
Country United Kingdom
Start 03/2014 
End 02/2017
 
Description MRC/BBSRC/EPSRC Next generation imaging grant (MR/K01577X/1).
Amount £2,000,000 (GBP)
Organisation Medical Research Council (MRC) 
Sector Academic/University
Country United Kingdom
Start 04/2013 
End 03/2018
 
Description Marie Curie Career Integration Fund (contribution to group member Jorge Bernardino de la Serna)
Amount € 100,000 (EUR)
Organisation Marie Sklodowska-Curie Actions 
Sector Academic/University
Country Global
Start 10/2013 
End 09/2017
 
Description Marie Curie Intra-European Fellowship (Erdinc Sezgin)
Amount € 221,606 (EUR)
Organisation Marie Sklodowska-Curie Actions 
Sector Academic/University
Country Global
Start 01/2015 
End 12/2016
 
Description Marie-Curie Fellowship (contribution to group member Iztok Urbancic)
Amount £164,000 (GBP)
Organisation Marie-Sklodowska University 
Sector Academic/University
Country Poland
Start 09/2016 
End 08/2019
 
Description Newton Fund Researcher Links Workshop Grant 2015
Amount £30,000 (GBP)
Organisation British Council 
Sector Charity/Non Profit
Country United Kingdom
Start 11/2015 
End 05/2016
 
Description Newton Fund Researcher Links Workshop Grant 2016
Amount £30,000 (GBP)
Organisation British Council 
Sector Charity/Non Profit
Country United Kingdom
Start 04/2017 
End 12/2017
 
Description Newton Institutional Links Fund (Turkey/UK): Investigation of Wnt-catenin signalling at the plasma membrane in health and disease
Amount £340,000 (GBP)
Organisation British Council 
Sector Charity/Non Profit
Country United Kingdom
Start 02/2018 
End 01/2020
 
Description Oxford Medical Sciences Internal Bridging Salary Fund Silvia Galiani
Amount £7,332 (GBP)
Organisation University of Oxford 
Department Medical Sciences Division
Sector Academic/University
Country United Kingdom
Start 01/2017 
End 03/2017
 
Description Wellcome Trust Enhancement of Strategic Award (WT 091911) Advanced Microscopy for Chromosome and RNA dynamics
Amount £178,795 (GBP)
Organisation Wellcome Trust 
Department Wellcome Trust Strategic Award
Sector Charity/Non Profit
Country United Kingdom
Start 05/2014 
End 12/2015
 
Description Wellcome Trust Institutional Strategic Support Fund (WTISSF) 2013-2014 Containment level 3 super-resolution STED imaging of live virus host-cell interactions £
Amount £70,000 (GBP)
Organisation Wellcome Trust 
Department Wellcome Trust Strategic Award
Sector Charity/Non Profit
Country United Kingdom
Start 01/2014 
End 12/2015
 
Description Wellcome Trust Institutional Strategic Support Fund (WTISSF) Enhancement of technological capacity in biomedical research: Wolfson Imaging Centre Oxford
Amount £101,789 (GBP)
Organisation Wellcome Trust 
Department Wellcome Trust Strategic Award
Sector Charity/Non Profit
Country United Kingdom
Start 06/2015 
End 05/2017
 
Description Wellcome Trust Multi-User Equipment Grant (WT104924AIA) Advanced super-resolution fluorescence STED microscopy of the cellular interior
Amount £389,000 (GBP)
Organisation Wellcome Trust 
Department Wellcome Trust Strategic Award
Sector Charity/Non Profit
Country United Kingdom
Start 01/2014 
End 12/2018
 
Description Wellcome Trust Strategic Award Micron Oxford: super-resolution imaging of cellular dynamics
Amount £4,679,473 (GBP)
Organisation Wellcome Trust 
Department Wellcome Trust Strategic Award
Sector Charity/Non Profit
Country United Kingdom
Start 01/2016 
End 12/2021
 
Description Advancing fluorescence microscopy 
Organisation University of Oxford
Department Micron Oxford Advanced Bioimaging Unit
Country United Kingdom 
Sector Academic/University 
PI Contribution Fluorescence microscopy development and servicing
Collaborator Contribution Complementary fluorescence microscopy development and servicing
Impact One publication, three major grants, ongoing technological and infrastructural development, positive evaluation as a Eurobioimaging node
Start Year 2013
 
Description Antigen Presenting Cells 
Organisation University of Oxford
Country United Kingdom 
Sector Academic/University 
PI Contribution Fluorescence microscopy, biophysical techniques, data analysis
Collaborator Contribution APC biology
Impact DPhil studentship
Start Year 2014
 
Description Bcell membrane antibody interaction 
Organisation University of Manchester
Country United Kingdom 
Sector Academic/University 
PI Contribution Fluorescence microscopy
Collaborator Contribution Cells, antibody
Impact Ongoing
Start Year 2015
 
Description BioiScat - molecular membrane dynamics 
Organisation University of Oxford
Department Department of Chemistry
Country United Kingdom 
Sector Academic/University 
PI Contribution Setup and application of iSCAT setup - study of molecular membrane dynamics
Collaborator Contribution Setup of iSCAT setup, data analysis
Impact Two publications EPA grant DPhil student
Start Year 2014
 
Description Data analysis 
Organisation University of Warwick
Department Warwick Systems Biology Centre
Country United Kingdom 
Sector Academic/University 
PI Contribution Supply of experimental data for analysis
Collaborator Contribution Data analysis of experimental data
Impact Studentship
Start Year 2010
 
Description Engineering 
Organisation University of Oxford
Department Department of Engineering Science
Country United Kingdom 
Sector Academic/University 
PI Contribution Application and development of 3D super-resolution microscopy
Collaborator Contribution Development of 3D super-resolution microscopy
Impact Project planning
Start Year 2012
 
Description Epidermal tissue development 
Organisation University of Oxford
Country United Kingdom 
Sector Academic/University 
PI Contribution Fluorescence microscopy, data analysis
Collaborator Contribution skin cell biology
Impact One publication close to submission
Start Year 2013
 
Description Fluorescence Correlation Spectroscopy 
Organisation Royal Institute of Technology
Country Sweden 
Sector Academic/University 
PI Contribution Fluorescence Correlation Spectroscopy and photophysics: Experiments and Discussions
Collaborator Contribution Fluorescence Correlation Spectroscopy and photophysics: Experiments and Discussions
Impact Several publications.
 
Description Fluorescent lipid analog development 
Organisation University of Birmingham
Department School of Chemistry
Country United Kingdom 
Sector Academic/University 
PI Contribution Fluorescence microscopy
Collaborator Contribution Lipid synthesis and delivery
Impact One publication in preparation
Start Year 2014
 
Description Labels for super-resolution microscopy 
Organisation University of Göttingen
Country Germany 
Sector Academic/University 
PI Contribution Application and development of labels for super-resolution microscopy,
Collaborator Contribution Development of Labels for super-resolution microscopy
Impact Two Nature papers
 
Description Lipid rafts 
Organisation Max Planck Society
Department Max Planck Institute for Molecular Cell Biology and Genetics
Country Germany 
Sector Public 
PI Contribution Super-resolution experiments on proteins and lipids in membranes.
Collaborator Contribution Delivery of protein constructs and lipids. Know-how transfer on lipid rafts. Workshops.
Impact One publication. Design of experiments.
Start Year 2009
 
Description Lipid synthesis 
Organisation University of Bonn
Country Germany 
Sector Academic/University 
PI Contribution Measurements and characterization of synthesized fluorescent lipid analogs
Collaborator Contribution Synthesis of fluorescent lipid analogs
Impact Several publications including a Nature paper.
Start Year 2008
 
Description Live-cell labeling 
Organisation Swiss Federal Institute of Technology in Lausanne (EPFL)
Department Institute of Chemical Sciences and Engineering
Country Switzerland 
Sector Academic/University 
PI Contribution Microscope experiments
Collaborator Contribution Development of live-cell fluorescent labels
Impact Publication in Nature Chemistry
Start Year 2011
 
Description Lymphatic cell receptor organization 
Organisation University of Oxford
Country United Kingdom 
Sector Academic/University 
PI Contribution Fluorescence microscopy, data analysis
Collaborator Contribution Lymphatic Receptor Biology
Impact One publication, one DPhil studentship
Start Year 2013
 
Description Micron 
Organisation University of Oxford
Country United Kingdom 
Sector Academic/University 
PI Contribution Exchange of microscopy knowhow.
Collaborator Contribution Exchange of microscopy know-how.
Impact Design of imaging facility. NanOxford imaging initiative. Further developments in super-resolution microscopy.
Start Year 2012
 
Description Photoswitchable label development 
Organisation University of Oxford
Department Department of Chemistry
Country United Kingdom 
Sector Academic/University 
PI Contribution Fluorescence microscopy
Collaborator Contribution Organic chemistry, organic dye synthesis
Impact One publication two grants
Start Year 2014
 
Description Physics 
Organisation University of Oxford
Department Department of Physics
Country United Kingdom 
Sector Academic/University 
PI Contribution Applications and developments of microscopy.
Collaborator Contribution Sample preparations and analysis software for microscopy
Impact Experimental design of experiments. Partnership with physics department.
Start Year 2012
 
Description STED/RESOLFT microscopy 
Organisation Max Planck Society
Department Max Planck Institute for Biophysical Chemistry Goettingen
Country Germany 
Sector Public 
PI Contribution Application of super-resolution STED/RESOLFT microscopy
Collaborator Contribution Development of super-resolution STED/RESOLFT microscopy
Impact Design of setups/microscopes Several publications on developments in STED/RESOLFT microscopy
Start Year 2012
 
Description T-cell activation 
Organisation University of Oxford
Country United Kingdom 
Sector Academic/University 
PI Contribution Fluorescence microscopy of T-cell during activation, receptor and actin dynamics - our contribution microscopy and analysis
Collaborator Contribution T-cell biology
Impact 3 publications, one Wellcome Trust Grant
Start Year 2013
 
Description Tracking 
Organisation University of Oxford
Country United Kingdom 
Sector Academic/University 
PI Contribution Super-resolution measurements on membrane dynamics.
Collaborator Contribution Single-molecule tracking experiments of membrane dynamics.
Impact Verification of experimental results.
Start Year 2011
 
Description Viral entry 
Organisation Chalmers University of Technology
Country Sweden 
Sector Academic/University 
PI Contribution Delivery of lipids, discussion of data/experiments
Collaborator Contribution Substances for experiments
Impact Publication in Angewandte Int Ed
Start Year 2011
 
Title CalQuo - quantifying cellular calcium release 
Description Development of a software to quantify calcium release from an ensemble of cells on a single-cell level 
IP Reference  
Protection Copyrighted (e.g. software)
Year Protection Granted 2015
Licensed No
Impact Advances general data analysis
 
Description Articles Written for the Leica Science Lab 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Added information to Leica website about nanoscopy. have received queries about further information

no notable impact
Year(s) Of Engagement Activity 2012,2013,2014,2015,2016
URL http://www.leica-microsystems.com/science-lab/
 
Description Conference Organizations 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact Conference/workshop Organizer: 4th International Symposium on Physics, Chemistry & Biology with Single Molecules, German Volkswagen foundation, Kloster Banz, Germany (2004); The Physics of Imaging, German Physical Society, Bad Honnef, Germany (2005); Trends in Biological Optical Microscopy, Boehringer Ingelheim foundation, Titisee, Germany (2007); SPIE Photonics West BIOS, Single Molecule Spectroscopy and Superresolution Imaging, USA (2009-); SPIE Nanoimaging and Nanospectroscopy, San Diego (2013-); EOS Frontiers in Optical Imaging, Murten, Switzerland (2013); Leica Super-Resolution User Club Meeting, Oxford, UK (2014); Coherent Bioimaging Workshop, Oxford, UK (2014); Conference on Lasers and Electro-Optics Europe (CLEO/Europe), Munich, Germany (2015); International Conference On Nanoscopy EUROPE (ICON2016), Basel, 2016; EBSA European Biophysical Congress, Madrid, 2019
Year(s) Of Engagement Activity 2012,2013,2014,2015,2016
 
Description Editorial Board Member 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Media (as a channel to the public)
Results and Impact Editorial Board Member in journals: NanoBioimaging, Frontiers in Physiology, Journal of Physics D, Scientific Reports
Year(s) Of Engagement Activity 2012,2013,2014,2015,2016
 
Description High School, undergraduate and graduate placement students in lab 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Schools
Results and Impact Visits of high school, undergraduate and graduate students (7 so far) in my lab for up to 6 months, teaching them in our research
Year(s) Of Engagement Activity 2013,2014,2015,2016
 
Description High-School visits 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact Visits of high school students to our institute/laboratory - insights into research activities
Year(s) Of Engagement Activity 2017
 
Description Lectures 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Type Of Presentation Keynote/Invited Speaker
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact Insights into general and super-resolution fluorescence microscopy and membrane bioactivity

Increased enquiry in using our imaging facilities, teach students/researchers
Year(s) Of Engagement Activity 2012,2013,2014,2015,2016
 
Description Microscopy Teaching 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact Teaching on microscopy on international/national fares, workshops, University courses, doctoral and undergraduate training centres
Year(s) Of Engagement Activity 2012,2013,2014,2015,2016
 
Description Microscopy Teaching in High Schools 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact Teach basics of microscopy to high school kids including hands-on
Year(s) Of Engagement Activity 2015,2016
 
Description Microscopy Workshop Izmir, Turkey 10.2017 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Undergraduate students
Results and Impact Teaching of basics of optical microscopy to public/scientists in Turkey - funded by the British Council
Year(s) Of Engagement Activity 2017
 
Description Microscopy Workshop Izmir, Turkey 4.2016 and 12.2016 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact Organization of microscopy course in Izmir, Turkey including hands-on sessions
Year(s) Of Engagement Activity 2016
 
Description Microscopy Workshop Lubljana, 9.2017 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Undergraduate students
Results and Impact Teaching of basics of optical microscopy to public/students
Year(s) Of Engagement Activity 2017
 
Description Microscopy in school 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact Teaching of microscopy in regional schools
Year(s) Of Engagement Activity 2015,2016
 
Description Oxford Bioimaging Festival 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Undergraduate students
Results and Impact Co-organizer Oxford Bioimaging festival

Large increase in enquiries to our group - many people wanting to know if they could obtain copies of the images we had for display in public areas.
Year(s) Of Engagement Activity 2012,2013,2014,2015,2016
URL http://www.imaging.ox.ac.uk/activities/annual-festival/imagefest12
 
Description Oxford curiosity carnival 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact Presenting general activities of the institute/University to the public at an open event
Year(s) Of Engagement Activity 2017
 
Description Webinar on STED Far-field Nanoscopy 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Media (as a channel to the public)
Results and Impact At the request of LEICA, I put together a webinar (online presentation) to discuss my current work. This was advertised to their employees, and made publicly available

Closer relationship with LEICA. They have since held a Symposium here at the WIMM to discuss Nanoscopy
Year(s) Of Engagement Activity 2013,2014,2015
URL http://view6.workcast.net/?pak=8993948753416174