Development of single molecule techniques for nanoscale imaging of toxic protein species in vitro and in cells.

Lead Research Organisation: University of Cambridge
Department Name: Chemical Engineering and Biotechnology

Abstract

Proteins are one of the fundamental building blocks for life, but in order to work correctly, proteins need to be in the right shape, or, in scientific language, they need to maintain their fold. When proteins misfold, they can take on aberrant shapes and often collapse in the form of insoluble deposits, which can be toxic to neurons in the brain. Indeed, major diseases such as Alzheimer's or Parkinson's diseases (PD), are characterized by the formation of insoluble deposits of proteins which polymerize in the form of long filaments, called amyloid fibrils. Unfortunately, the molecular interactions that lead to fibril formation and subsequent aggregation are not yet elucidated; therefore, high resolution techniques that characterize the ultrastructure of fibrils are in high demand. Electron microscopy is widely used for this purpose, but its invasive nature prevents it from following fibril formation in time and EM requires elaborate sample fixation and mounting protocols. Atomic force microscopy can also image fibrils at high resolution, but it is limited to surface topology. Although fluorescence microscopy can complement these techniques, conventional optical methods are limited by diffraction and provide a resolution far too coarse to provide information in protein fibrils / aggregates. Recently, variants of single molecule fluorescence techniques have been developed that provide 10-20 nm resolution i.e. much smaller than the wavelength of the probing light. How do they work? A sample is tagged with so called photoactivatable fluorophores which can be switched on and off with pulses of light; one can thus think of each fluorophore as a switchable light bulb. By switching on a few individual light bulbs at any one time and taking an image, the positions of the active light bulbs can be determined with very high precision (10-20 nm). By repeating this process thousands of times, activating different fluorophores each time, and taking as many images the precise location of each fluorophore can be determined and information on the underlying structure can be obtained (as an analogy one may think of a Christmas tree, 'labelled' with lots of lightbulbs: If the positions of the individual lightbulbs are accurately known, information on the underlying shape/structure of the tree can be regained). It is with such so called single molecule fluorescence techniques we would like to gain, for the first time, dynamic information on the sizes and shapes of fibrillar structures formed by alphasynclein (AS), a protein that misfolds and lies at the heart of PD. We will complement this research with other optical techniques developed by us, which also give us molecular scale resolution of protein aggregates forming in cells, and also in test solutions. If successful we will end up with tools that give us much more direct information on the aggregation process in cells than has hitherto been possible. It would be exciting for example, if we were to see differences in the AS protein deposits formed in cells treated with potential anti-aggregation drugs compared to those which have not been treated. In any case we will end up with new knowledge and tools, with which to study protein function and 'misbehaviour'. The proposed work brings together the expertise of molecular and chemical biologists, physicists, and engineers who have put in place the building blocks to tackle this important problem in neurodegenerative disease with novel optical tools.

Planned Impact

This research will lead to the development of single molecule optical superresolution methods to a problem of fundamental imporatance in neurodegenerative disease research. Who will benefit from this research (see also academic beneficiaries section above)? The National Physics Laboratory (NPL) will benefit by applying and expanding on their expertise in single molecule biophysics area and will obtain expertise in PALM and STORM imaging The UK community as a whole will benefit from these developments through NPL's role as the national provider of metrological services to the UK R&D community. This guarantees an effective means of technology diffusion both to UK academia and industry. The UK health and pharmaceutical sectors will benefit: The possibility to quantify the presence of toxic protein species in their biological environment offers opportunities to validate the effectiveness of potential drugs and explore potential means of therapeutic intervention (e.g. anti-aggregating drugs). The research will help the UK to maintain its leading role in the applied photonics field: There is huge commercialisation potential (see letter of support from Fianium) and there are opportunities for novel array detector technologies, supercontinuum technologies, and image processing tools in all of which the UK plays leading roles. Society as a whole will benefit: Through improved tools for neurodegenerative disease research, novel therapeutic and diagnostic approaches can be developed which may ultimately help in reducing some of the most devastating, costly and humiliating age related diseases. For example, in the UK alone well over 10000 new cases of Parkinson's disease are reported every year.

Publications

10 25 50

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Arnon ZA (2018) Opal-like Multicolor Appearance of Self-Assembled Photonic Array. in ACS applied materials & interfaces

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Blacker TS (2017) Investigating State Restriction in Fluorescent Protein FRET Using Time-Resolved Fluorescence and Anisotropy. in The journal of physical chemistry. C, Nanomaterials and interfaces

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Chan FT (2011) HomoFRET fluorescence anisotropy imaging as a tool to study molecular self-assembly in live cells. in Chemphyschem : a European journal of chemical physics and physical chemistry

 
Description Many serious neurodegenerative diseaeses, including Alzheimer's disease and Parkinsons's disease, involve the misfolding, aggregation and progagation of naturally-occuring proteins within the brain.

This grant has supported original research. We performed the first work in the world using super-resolution microscopy to study neurotoxic protein aggregation processes. This technique provides new insight into the nature of protein aggregation, because it makes it possible to image the formation of potentially-toxic amyloid protein fibrils at high resolution AND under physiological conditions (and even in-vivo). This new capability supports a number of biophysical studies into related protein diseases.

We have also studied the computational basis of super-resolution microscopy techniques - these techniques are recent inventions, and we are among the researches establishing their theoretical basis. An important outcome of our computational work is the development of a theoretical resolution criteria for a type of super-resolution known as localisation microscopy. This theoretical work provided the basis for an evolutionary advance in localisation microscopy, in which fluorescent shell structures are directly inferred from image data, without the need for photoswitching images of single fluorescent molecules [http://dx.doi.org/10.1016/j.bpj.2015.09.023].
Exploitation Route Advances in understanding the molecular biochemical basis of Alzheimer's and Parkinson's disease are essential to support pharmaceutical research into therapies for these illnesses.



The imaging techniques developed in this research could also form the basis of medical diagnostic tools for neurodegereative disorders. The fundamental advances in medical knowledge obtained by this research are shared with the neurodegenerative diseases research community through our involvement in the multidisciplinary Cambridge Neuroscience consortium, as well as through publication.



Our advances in imaging technology are the basis for our participation in the Cambridge Advanced Imaging Centre (http://www.pdn.cam.ac.uk/caic/). This research centre makes cutting edge microscopy techniques available to researchers throughout the University of Cambridge. Furthermore, imaging software developed as part of programme is made available as open-source.
Sectors Healthcare,Pharmaceuticals and Medical Biotechnology

URL http://laser.ceb.cam.ac.uk/news/nobel-prize-awarded-to-super-resolution-microscopy
 
Description This grant supported the development of a super-resolution localisation microscope, and the development of related data analysis and super-resolution image reconstruction software, and the application of this system to study several biomedical research problems. The first essential outcome of this research was our demonstration (doi 10.1021/ja201651w), for the first time in the world, that super-resolution microscopy is able to quantitatively and qualitatively study the formation and structure of amyloid protein fibrils which play a key role in neurodegenerative diseases including Alzheimer's and Parkinson's disease. We have gone on to publish further, original super-resolution studies into the relation of amyloid fibrils to neurotoxicity, and these studies provide important fundamental insight into the biochemistry of Alzheimer's disease. Our further work with the Medical Research Council (MR/K02292X1) has provided biomedically significant evidence of how the propagation of toxic protein species may lead to progressive neurodegeneration. Our participation in the Cambridge Neuroscience consortium (http://www.neuroscience.cam.ac.uk/) has allowed us to effectively share the scientific results of these studies, as well as to make our super-resolution imaging instruments available for collaborators in several medical imaging projects. Spin-off projects based on the use of the instrument developed in this project include a super-resolution study into the structure of virus particles, which has now been supported by a Leverhulme grant in collaboration with researchers based at Addenbrokes hospital, and which has led to original structural virology technique being accepted for publication in Nature Communications (Nov 2014, Laine et al). Such projects supported the successful case for the establishment of the Cambridge Advanced Imaging centre, at which we play a key role in development of advanced and original imaging techniques which are available to biomedical researchers throughout the University of Cambridge (http://caic.bio.cam.ac.uk/) . We have also actively engaged in media outreach activities (in collaboration with Alzheimer's research UK, and with BBC radio) to publicise the progress being made in understanding the fundamental mechanisms of neurodegenerative diseases, and hence in development of diagnostic methods and therapies. In keeping with our commitment to making our work available to the public, the super-resolution imaging software that we developed for this project has been published as open source material (10.1088/2040-8978/15/9/094012), and we continue to publish refinements as they are developed. These developments are based on practical experience provided by access to the instrumentation supported by this grant, in collaboration with theoretical expertise of our group and collaborators (e.g. 10.1186/2192-2853-1-12). Further development of these techniques provides valuable research experience for students at the EPSRC Centre for doctoral training in sensor technologies (http://cdt.sensors.cam.ac.uk/). One of the CDT projects enabled by this research was a project in which a new type of super-resolution microscopy was developed. The technique of fluorescent shell localisation microscopy (FSLM) [http://dx.doi.org/10.1016/j.bpj.2015.09.023 Biophysical journal front cover 17 Nov 2015] is a significant advance that builds on the on single molecule localisation microscopy software developed in this grant. The FSLM technique allows models for the images of fluorescent shell structures (rather than just single molecule image models) to be fitted to microscopy data. This method makes it possible to study the structure of bacterial spores at ~20 nm resolution, without needing to use complex photoswitching techniques. A 50% industrially supported grant proposal has been submitted to the BBSRC (Jan 2016 submission), in collaboration with the global biologics research and development company MedImmune. The proposed project will study the structure of C. difficle spores, which are the leading cause of hospital-acquired diarrhea disease, with a view to identifying new drug targets for overcoming the antibiotic resistance of this pathogen.
Sector Healthcare,Pharmaceuticals and Medical Biotechnology
 
Description 2019 exercise
Amount £400 (GBP)
Organisation European Biophysical Societies' Association 
Sector Charity/Non Profit
Country Germany
Start 02/2018 
End 02/2018
 
Description British Biophysical Society Travel Grant
Amount £309 (GBP)
Organisation British Biophysical Society 
Start 05/2018 
End 05/2018
 
Description Cambridge ARUK Travel Grant
Amount £488 (GBP)
Organisation Alzheimer's Research UK 
Sector Charity/Non Profit
Country United Kingdom
Start 05/2018 
End 05/2018
 
Description Cambridge ARUK travel grant
Amount £300 (GBP)
Organisation Alzheimer's Research UK 
Sector Charity/Non Profit
Country United Kingdom
Start 03/2019 
End 03/2019
 
Description Cambridge Infinitus Research Centre
Amount £5,000,000 (GBP)
Organisation Infinitus 
Sector Private
Country China
Start 09/2015 
End 08/2020
 
Description EBSA bursary
Amount £330 (GBP)
Organisation European Biophysical Societies' Association 
Sector Charity/Non Profit
Country Germany
Start 02/2018 
End 02/2018
 
Description Fellowship from the Cambridge-Wellcome Trust Scheme for Interdisciplinary Research
Amount £29,000 (GBP)
Organisation University of Cambridge 
Department Wellcome Trust Senior Internship for Interdisciplinary Research
Sector Academic/University
Country United Kingdom
Start 07/2013 
End 07/2014
 
Description Leverhulme research grant
Amount £95,000 (GBP)
Organisation The Leverhulme Trust 
Sector Academic/University
Country United Kingdom
Start 01/2013 
End 07/2015
 
Description Newnham College Travel Grant
Amount £731 (GBP)
Organisation University of Cambridge 
Department Newnham College
Sector Academic/University
Country United Kingdom
Start 06/2018 
End 09/2018
 
Description Research Fellowship Deutsche Forschungsgemeinschaft (DFG)
Amount € 140,400 (EUR)
Funding ID LA 3609/2-1 
Organisation German Research Foundation 
Sector Public
Country Germany
Start 04/2015 
End 03/2018
 
Description SUPUVIR Marie Curie Consortium
Amount € 4,017,699 (EUR)
Funding ID 722380 
Organisation European Commission 
Sector Public
Country European Union (EU)
Start 01/2017 
End 12/2021
 
Description Swiss National Science Foundation
Amount SFr. 72,000 (CHF)
Organisation Swiss National Science Foundation 
Sector Public
Country Switzerland
Start 10/2014 
End 04/2016
 
Description Wellcome Trust MIT Postdoctoral Research Fellowship
Amount £79,046 (GBP)
Funding ID 093831/B/10/Z 
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 09/2016 
End 08/2017
 
Description 2-photon microlithography for microscopic lab on chip devices with Tuomas Knowles and Jerome Charmet 
Organisation University of Cambridge
Department Department of Chemistry
Country United Kingdom 
Sector Academic/University 
PI Contribution We designed the 2-photon microscope used in this nanolithography work and led the optimisation of the photo-lithograpjhy conditions (dwell time, intensity). Currently we are working on software to allow the design of arbitrary structures
Collaborator Contribution They proposed the initial idea for the project and have optimised the photoresist preparation conditions and the development conditions. They have also tested the devices
Impact Project student from sensor CDT shared between groups (principally LAG)
Start Year 2015
 
Description Building of a dSTORM set up with Prof Marcus Sauer 
Organisation University of Wurzburg
Country Germany 
Sector Academic/University 
PI Contribution We contributed by building our own dSTORM set up and exchanging on the use of this technique for the study of neurodegenerative diseases
Collaborator Contribution Prof Marcus Sauer contributed by exchanging on his knowledge of the dSTORM technique and the photochemistry associated with it
Impact This collaboration resulted in our group having its own dSTORM set up Kaminski-Schierle GS, Sauer M, Kaminski CF, "Probing Amyloid Aggregation and Morphology In Situ by Multiparameter Imaging and Super-Resolution Fluorescence Microscopy," (2014) in Uversky VN, Lyubchenko YL, (eds) Bio-nanoimaging: Protein Misfolding & Aggregation, Academic Press, pp. 105-120 Kaminski Schierle GS, van de Linde S, Erdelyi M, Esbjörner EK, Klein T, Rees E, Bertoncini CW, Dobson CM, Sauer M, and Kaminski CF, "In Situ Measurements of the Formation and Morphology of Intracellular ß-Amyloid Fibrils by Super-Resolution Fluorescence Imaging", J. Am. Chem. Soc., 133 (33), pp 12902-12905, (2011).
Start Year 2008
 
Description Connexin regulation of perisynaptic astroglial processes: molecular and cellular mechanisms with Nathalie Rouach 
Organisation College of France
Country France 
Sector Academic/University 
PI Contribution The STED microscope built by the group is used to investigate connexion locations in astroglial processes. Reccomendations were made by the group regarding mounting media and choice of fluorescent labels for super-resolution. We optimise the imaging conditions for thick samples. The AFM microscope is also used to measure force curves and this will be correlated with images from our STED microscope
Collaborator Contribution They proposed the initial idea for the project and have performed all sample prep for imaging. Our work fits into a broader piece of work of which all bits are performed by the collaborator
Impact Prof Nathalie Rouach currently spending her sabbatical in our group for 8 months
Start Year 2015
 
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 Developing super-resolution microscopy for the analysis of virus replication analysis of virus replication with Dr Colin Crump 
Organisation University of Cambridge
Country United Kingdom 
Sector Academic/University 
PI Contribution The team in the LAG was then able to perform nanometre-scale imaging of the virus to study its assembly and replication. The spatial resolution of the localization specific proteins in the virus is unprecedented in the context of virology.
Collaborator Contribution The team of Dr Colin Crump provided relevant samples to be used for studying the structure of the virus both in-vitro in purified viruses as well as in cells.
Impact This collaboration allows interdisciplinary work by joining expertise in virology biochemistry and that of advanced microscopy techniques, essentially based on super-resolution techniques. This collaboration enabled the investigation of the structure of the Herpes Simplex Virus type-1 (HSV-1) via fluorescence optical super-resolution techniques. Update in 2016: Paper published in Traffic
Start Year 2013
 
Description Development of super-resolution microscopy with dSTORM with Miklos Erdelyi 
Organisation University of Szeged
Country Hungary 
Sector Academic/University 
PI Contribution The Laser Analytics Group continues to collaborate with Dr Miklos Erdelyi on the development of our dSTROM microscope and the algorithms associated with data analysis
Collaborator Contribution The Laser Analytics Group continues to collaborate with Dr Miklos Erdelyi on the development of our dSTROM microscope and the algorithms associated with data analysis
Impact This collaboration has led to the publication of the following papers: Sinkó J, Kákonyi R, Rees E, Metcalf D, Knight AE, Kaminski CF, Szabó G, Erdélyi TestSTORM: Simulator for optimizing sample and image acquisition in localization based super-resolution microscopy, Biomed. Opt. Exp. (2014), 5 (3), pp. 778-787 Erdelyi M, Rees EJ, Metcalf D, Kaminski-Schierle GS, Dudas L, Sinko J, Knight AE, Kaminski CF, "Correcting chromatic offset in multicolor super-resolution localization microscopy", Optics Express 21 (9), 10978-10988 (2013)
Start Year 2013
 
Description Experimental and theoretical studies of alpha synyclein aggregation with Alessio Zaccone 
Organisation University of Cambridge
Department Department of Chemical Engineering and Biotechnology
Country United Kingdom 
Sector Academic/University 
PI Contribution Led project on experimental investigation of amyloid growth with FLIM (Fluorescence lifetime imaging microscopy) and provided data to validate computational code developed by Zaccone.
Collaborator Contribution Use of stochastic models to calcualate fibril growth kinetics and thermophsical data to help us interpret experimental data.
Impact Manuscript submitted for review.
Start Year 2016
 
Description FLIM and Anisotropy imaging of recepter protein dimerisation with Prof Eric Barnard 
Organisation University of Cambridge
Country United Kingdom 
Sector Academic/University 
PI Contribution The Laser Analytics Group performed Fluorescence lifetime imaging and anisotropy imaging for this collaboration
Collaborator Contribution The group of Prof Eric Barnard is specialised in the study of the P2Y1 receptor
Impact This collaboration led to the following publication: Erdelyi M, Simon J, Barnard EA, and Kaminski CF, "Analyzing receptor assemblies in the cell membrane using fluorescence anisotropy imaging with TIRF microscopy", PLOS One, 9.6, 2014, e100526
Start Year 2011
 
Description Imaging of amyloid proteins present in cerebrospinal fluid with Prof Mathias Jucker 
Organisation German Centre for Neurodegenerative Diseases
Country Germany 
Sector Public 
PI Contribution We contributed by imaging the samples provided by the collaborators on our dSTORM optical nanoscopy microscope.
Collaborator Contribution The group of Prof Mathias Jucker contributed samples of cerebrospinal fluid from mice and from humans
Impact An original paper has been published and further collaboration will take place. This is a collaboration between a lab which has access to rodent and human cerebrospinal fluid (our collaborators) and our lab which develops optical nanoscopy equipment.
Start Year 2013
 
Description Imaging viscosity and gel melting by single molecule tracking with Peter st George Hyslop 
Organisation University of Cambridge
Department Cambridge Institute for Medical Research (CIMR)
Country United Kingdom 
Sector Academic/University 
PI Contribution The group have performed single fluorescent molecule tracking experiments on fluorophores entrapped in gel phases, in order to study gel melting and confined diffusion processes that are relevant to intracellular trafficking of RNA.
Collaborator Contribution Preparation and fluorescent staining of biologically relevant biogel phases.
Impact The present experiments have been made possible by the development of the localisation microscope associated with this grant. We have been able to use this instrument, together with data analysis software developed with the assistance of this grant, to supply a high spatial-resolution technique for observing gel melting. So far, this has provided academic impact in the form of original observations of the melting of gel phases with key roles in cell biology. Update in 2016: Paper published in Neuron
Start Year 2014
 
Description Interactions between aSynuclein and synaptic vesicles 
Organisation Swiss Federal Institute of Technology in Lausanne (EPFL)
Country Switzerland 
Sector Public 
PI Contribution Prepared recombinant protein and purified synaptic vesicles for interaction studies
Collaborator Contribution Microfluidics-integrated nanophotonics-enhanced IR spectroscopy platform
Impact Paper in preparation. Molecular/structural biology and bionanophotonics
Start Year 2018
 
Description Investigating the role of water in protein structure and aggregation 
Organisation Swiss Federal Institute of Technology in Lausanne (EPFL)
Country Switzerland 
Sector Public 
PI Contribution Prepared recombinant protein alpha synuclein for aggregation studied in the presence of different salts. Morphology studies
Collaborator Contribution Used second harmonic scattering to determine mobility of water around fibrils
Impact Paper in preparation. Molecular/structural biology and physics/biophotonics
Start Year 2018
 
Description Investigating the role of water in protein structure and aggregation 
Organisation University of Cambridge
Country United Kingdom 
Sector Academic/University 
PI Contribution Prepared recombinant protein alpha synuclein for aggregation studied in the presence of different salts. Morphology studies
Collaborator Contribution Runing and analysis of small angle neuron scattering data
Impact Paper in preparation, application for SANS beam time. Molecular biology and structural biology
Start Year 2018
 
Description Investigating the role of water in protein structure and aggregation 
Organisation University of Vermont
Country United States 
Sector Academic/University 
PI Contribution Prepared recombinant protein alpha synuclein for aggregation studied in the presence of different salts. Morphology studies
Collaborator Contribution Molecular dynamic simulations of peptide fragments in different salts
Impact Paper in preparation. Molecular/structural biology and physics/chemistry
Start Year 2018
 
Description Liquid droplets and phase transitions in disease (Peter St G. Hyslop) 
Organisation University of Cambridge
Department Department of Biochemistry
Country United Kingdom 
Sector Academic/University 
PI Contribution Performed single particle tracking in wild type and mutant hydrogels formed of FUS protein. Performed high resolution experiments in axonal growth cones of FUS distribution and phase states. Used optical technique to look at FUS phase states in live cells.
Collaborator Contribution Hyslop set up protein expression system, defined experiments in biological models, supplied biological materials and helped in analysis of data.
Impact Successful follow on grant with WT/MRC Multidisciplinary: neuroscience, biophysics
Start Year 2012
 
Description Organisation of axonal ER in Drosophila axons, Cahir O'Kane and Belgin Yalcin 
Organisation University of Cambridge
Department Department of Genetics
Country United Kingdom 
Sector Academic/University 
PI Contribution The STED microscope built by the group is used to investigate the axonal ER in cleared drosophila tissues. In our group we proposed labelling conditions and strategies for the samples to achieve super-resolution and optimised the imaging conditions for thick samples
Collaborator Contribution They proposed the initial idea for the project and have performed all sample prep for imaging. Our work fits into a broader piece of work of which all bits are performed by the collaborator
Impact No output yet
Start Year 2015
 
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 Single molecule translation imaging with Christine Holt and Bill Harris 
Organisation University of Cambridge
Department Department of Physiology, Development and Neuroscience
Country United Kingdom 
Sector Academic/University 
PI Contribution Carried out microscopy imaging and provided tools for data analysis. Continuous feedback for improvement of the imaging and preparation conditions.
Collaborator Contribution Prepared samples of Xenopus eyes for the study of the development of neural tract in the frog. Provided the biological question.
Impact None. Ongoing collaborations
Start Year 2014
 
Description Structure of monomeric aSynuclein 
Organisation University of Antwerp
Country Belgium 
Sector Academic/University 
PI Contribution Prepared recombinant protein alpha synuclein for aggregation studied in the presence of different salts. Morphology studies
Collaborator Contribution native mass spec and ion mobility mass spec
Impact Two papers in preparation. Molecular biology and structural biology
Start Year 2018
 
Description Structure of monomeric aSynuclein 
Organisation University of Exeter
Country United Kingdom 
Sector Academic/University 
PI Contribution Prepared recombinant protein and purified synaptic vesicles for interaction studies
Collaborator Contribution Hydrogen-Deuterium Exchange Mass Spectroscopy
Impact Paper in preparation. Molecular biology and structural biology
Start Year 2016
 
Description Structure of monomeric aSynuclein 
Organisation University of Leeds
Country United Kingdom 
Sector Academic/University 
PI Contribution Prepared recombinant protein alpha synuclein for aggregation studied in the presence of different salts. Morphology studies
Collaborator Contribution native mass spec and ion mobility mass spec
Impact Two papers in preparation. Molecular biology and structural biology
Start Year 2018
 
Description Study of monomeric Tau propagation from cell to cell with Prof Eckard Mandelkow 
Organisation German Centre for Neurodegenerative Diseases
Country Germany 
Sector Public 
PI Contribution We have applied multi parametric and super resolution imaging techniques to the study of Tau propagation from cell to cell
Collaborator Contribution Prof Eckard Mandelkow provided us with purified recombinant Tau protein, both unlabelled and fluorescently labelled.
Impact Michel CH, Kumar S, Pinotsi D, Tunnacliffe A, St George-Hyslop P, Mandelkow E, Mandelkow E-M, Kaminski CF, Kaminski Schierle GS, "Extracellular Monomeric Tau is Sufficient to Initiate the Spread of Tau Pathology", J. Biol. Chem. (2014), 289: 956-967. This collaboration is multi-disciplinary and involved biochemistry and physics
Start Year 2010
 
Description Super-resolution imaging of aggregating peptides with Frederic Rousseau and Joost Schymkowitz 
Organisation University of Leuven
Country Belgium 
Sector Academic/University 
PI Contribution The Laser Analytics group will perform super-resolution imaging for this project
Collaborator Contribution The group of Frederic Rousseau and Joost Schymkowitz has provided us with synthetic peptides
Impact This collaboration has only just started and has not had any output yet
Start Year 2013
 
Description Super-resolution imaging of amyloid proteins with Prof Chris Dobson 
Organisation University of Cambridge
Department Department of Chemistry
Country United Kingdom 
Sector Academic/University 
PI Contribution In this collaboration with the group of Prof Chris Dobson, we imaged amyloid proteins, in particular alpha synuclein and amyloid beta, with our multi-parametric and super-resolution microscopes
Collaborator Contribution The group of Prof Chris Dobson provided us with purified recombinant alpha-synuclein and amyloid beta protein, either unlabelled or directly labelled
Impact Esbjörner E K, Chan F, Rees EJ, Erdelyi M, Luheshi LM, Bertoncini CW, Kaminski CF, Dobson CM, Kaminski-Schierle GS, "Direct Observations of the Formation of Amyloid ß Self-Assembly in Live Cells Provide Insights into Differences in the Kinetics of Aß(1-40) and Aß(1-42) Aggregation," Chemistry and Biology (2014) Pinotsi D, Büll AK, Galvagnion C, Dobson CM, Kaminski-Schierle GS, Kaminski CF, "Direct Observation of Heterogeneous Amyloid Fibril Growth Kinetics via Two-Color Super-Resolution Microscopy," Nano Letters (2013), 14 (1), 339-345 Pinotsi D, Buell AK, Dobson CM, Kaminski Schierle GS and Kaminski CF, "A label-free, quantitative assay of amyloid fibril growth based on intrinsic fluorescence", ChemBioChem (2013), 14 (7), 846-850. Chan FTS, Kaminski Schierle GS, Kumita JR, Bertoncini CW, Dobson CM and Kaminski CF, "Protein amyloids develop an intrinsic fluorescence signature during aggregation", Analyst (2013) 138 (7), 2156-216.
Start Year 2010
 
Description Super-resolution imaging of macromolecules micelles assemblies with Ian Manners 
Organisation University of Bristol
Department School of Chemistry
Country United Kingdom 
Sector Academic/University 
PI Contribution Carried out super-resolution microscopy (dSTORM) imaging and feedbacks on how to prepare and design fluorescent probes for preparation of their micellar samples.
Collaborator Contribution Designed, prepared and synthetised the micellar structure used for the study of micelle elongation.
Impact None yet. But manuscript under preparation.
Start Year 2014
 
Description Super-resolution imaging of nanotubes involved in the transfer of Tau from neuron to neuron (Luc Buee) 
Organisation National Institute of Health and Medical Research (INSERM)
Department Lille (INSERM)
Country France 
Sector Public 
PI Contribution We would carry out super-resolution imaging on samples
Collaborator Contribution Will provide cells, plasmids for expression of Tau and expertise in nanotubes
Impact Invited talk given by Dr Gabriele Kaminski Schierle in Lille for a seminar on New technologies and Neurosciences
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 aSynuclein-lipid interaction 
Organisation University of Cambridge
Country United Kingdom 
Sector Academic/University 
PI Contribution Prepared recombinant alpha synuclein and brain extract lipid samples for aSyn/membrane interactions studies. Performed AFM analysis.
Collaborator Contribution Access to FastScan AFM in Pharmacology
Impact Paper in preparation. Molecular biology and structural biology
Start Year 2018
 
Description dSTORM imaging of DNA origami with Ulrich Keuser 
Organisation University of Cambridge
Department Cavendish Laboratory
Country United Kingdom 
Sector Academic/University 
PI Contribution Carried out super-resolution microscopy imaging using DNA origami nano-structure. Carried out analysis and simulations based on observation proving the feasibility of the imaging.
Collaborator Contribution Designed, prepared and synthetised the DNA origami structure.
Impact None. Ongoing collaborations. Proof of principle of imaging of their DNA origami structures under our microscope. Foster further collaboration leading to offering student projects.
Start Year 2014
 
Description super-resolution imaging of amyloid proteins with Prof Sara Linse 
Organisation Lund University
Country Sweden 
Sector Academic/University 
PI Contribution The Laser Analytics Group is in the process of imaging amyloid protein fibril formation with super-resolution microscopy
Collaborator Contribution The group of Prof Sara Linse provided purified amyloid protein and gave a two-week tutorial to three members of the Laser Analytics group on purification of proteins
Impact This partnership is still young and we hope it will develop into publication on the amyloid fibril formation kinetics. This collaboration is multi-disciplinary, the Laser Analytics group being specialised in super-resolution imaging (physics, engineering) and the group or Prof Sara Linse in protein purification and biophysical characterisation of amyloid proteins
Start Year 2014
 
Description ARUK Spring appeal 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact We participated in the preparation of an appeal letter and follow up letter to potential funders of ARUK regarding the use of emergency funding by ARUK. This was also followed by a blog post

We expect some persons who received this appeal send fund to Alzheimer's Research UK for research on neurodegenerative diseases.
Year(s) Of Engagement Activity 2014
URL http://www.dementiablog.org/every-project-counts/
 
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 Blog post on ARUK website on dSTORM (2014) 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact Engage with patients, carers and families about research funded by ARUK

Readers of the Alzheimer's Research UK might donate towards research.
Year(s) Of Engagement Activity 2014
URL http://www.dementiablog.org/10000-times-smaller-pinhead/
 
Description CamBRAIN : Teaching children about neurons 
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 Teaching children about neurons
Year(s) Of Engagement Activity 2018
 
Description Interalia magazine Science meets Art (Florian Ströhl) 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact Artistic scientific images published in Art Online Journal
Year(s) Of Engagement Activity 2016
 
Description Interview by ARUK at the Copenhagen AAIC conference 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact This activity resulted into engaging with readers of the Alzheimer's Research UK blog about research presented by Dr Claire Michel on a poster at the Alzheimer's Association International Conference 2014

With this interview funders of Alzheimer's Research UK received information regarding how their donations are used by researchers and what results researchers obtain.
Year(s) Of Engagement Activity 2014
 
Description Interview on BBC Radio Cambridge 2014 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact This activity was a 3 minutes interview on super-resolution interview used in research on neurodegenerative diseases

Engage with the wide public about advances in research on Alzheimer's disease
Year(s) Of Engagement Activity 2014
 
Description Nano^art (Laurie Young) 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact Nanoscientist/artist collaboration during the Cambridge festival of ideas, with a public presentation, exhibition and discussion
Year(s) Of Engagement Activity 2015
URL http://www.nanodtc.cam.ac.uk/News%20and%20Events/nano-art
 
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 PreLighter 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact Writing highlight sections on interesting preprints for the PreLight website and twitter feed
Year(s) Of Engagement Activity 2018,2019
 
Description Research Photography Exhibition 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact Finalist in STEM photography competition at Hughes Hall College, Cambridge
Year(s) Of Engagement Activity 2019
 
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 STEM in Song 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Schools
Results and Impact Encouraging school-aged girls to engage with the STEM subjects through music. Awards event with science demos, song launch at the LMB, music video at https://www.youtube.com/watch?v=7c4KMOWoZW4 . Reach: 15300 (according to Facebook insights)
Year(s) Of Engagement Activity 2018
URL https://www.youtube.com/watch?v=7c4KMOWoZW4
 
Description Seminar on Science and Brewing 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact Explaining science to the public
Year(s) Of Engagement Activity 2018
 
Description So you want to be a scientist?' experiment and speaking presentation 
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 Talking to school children about life as a scientist and GCSE options to take
Year(s) Of Engagement Activity 2018
 
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 and lab tour to funders of ARUK, Feb 2016 
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 Supporters
Results and Impact Engage with funders on the research carried out thanks to their donations
Year(s) Of Engagement Activity 2016
 
Description Talk and lab tour to funders of ARUK, May 2015 
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 Supporters
Results and Impact Engage with funders on the research carried out thanks to their donations
Year(s) Of Engagement Activity 2015
 
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
 
Description Talk, tour of the lab and Q&A for 15 employees of ARUK 
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 Public/other audiences
Results and Impact This activity gave a clearer understanding to employees of ARUK and to readers of the ARUK blog of the work carried out by researchers studying dementia

One employee of ARUK wrote a blog post on his visit, which increased the number of people reached through this activity.
Year(s) Of Engagement Activity 2014
URL http://www.dementiablog.org/tau-proteins/
 
Description Tech Me Out team volunteer 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact Pint of Science Festival Cambridge
Year(s) Of Engagement Activity 2018