A novel frequency domain FLIM microscope for the dynamic study of protein function in live cells

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

Abstract

Optical techniques are powerful tools to probe the function of proteins in living cells. Proteins can be fluorescently labelled with high specificity and this permits one to study where and when they are produced or degraded within living cells with high spatial and temporal resolution. The fluorescence signatures may also report on subtle changes in the molecular environment of the tagged proteins. For example interactions between proteins may lead to subtle intensity, colour and lifetime changes in the emission of the reporting fluorophores. There is therefore a constant demand in the life sciences for novel, better, and more flexible instrumentation to measure fluorescence from within living cells. The fluorescence lifetime in particular is a key parameter in such efforts. Molecular proximity can quench fluorescence and this in turn results in a reduction of the lifetime. Measuring lifetime from molecules in cells is however a difficult task: There is always a trade-off between the precision and the speed at which a lifetime measurement can be performed. In the current proposal we seek to improve on a technique called frequency domain lifetime imaging microscopy (FD FLIM): This technique is popular in conjunction with widefield fluorescence microscopy because it is simple and flexible in application but it has drawbacks, because in its conventional form, the speed of measurement and the precision are limited. In the present work we aim to overcome these limitations and vastly improve on the capability of FD FLIM for measurements of protein-protein interactions in living systems. The instrument to be developed here will be used in the study of the molecular processes, which regulate cell division and we will be able to do this in much more detail than before: The features we study are highly dynamic and occur over small spatial scales and the improved temporal resolution and precision will quantify events that we have not been able to observe so far. The capability we are developing here will be available for researchers from various departments and be useful in situations where either molecular scale events need to be monitored at rapid speed, such as signalling events in cells, or in situations where high measurement precision is required, for example to record subtle lifetime changes that may report on different oligomeric states of aggregating monomers.

Technical Summary

With the current proposal we seek funds to develop a novel and versatile Frequency Domain Fluorescence Lifetime Imaging Microscope (FD FLIM) which is flexible to operate at 1) high measurement speed by a method we term phi2FLIM, or, 2) at a precision hitherto difficult to achieve with available FD FLIM instrumentation in a variant of FD FLIM we term mh-FLIM (multi harmonic FLIM). The technology we propose is motivated by our wish to promote research on the role of key mitotic regulators during cell division. It represents a substantial improvement over existing technologies and, although specifically applied to mitosis research, will find widespread applications for biophysical studies of molecular function in living cells. Beneficiaries will include life scientists who may already have FD FLIM instrumentation available in their laboratories: The proposed technologies are relatively straight forward to implement on current, commercially available FD FLIM systems. The ideas behind the proposed research arose from ongoing collaborations between the groups of Dr. Clemens Kaminski at the department of Chemical Engineering and Biotechnology and Dr. Catherine Lindon at the department of Genetics at the University of Cambridge. We have conducted extensive feasibility studies to assess the feasibility of the proposed tools and methods and the current grant would permit us to: 1) set up a fully functioning microscopy unit which permits, at user's choice and essentially 'at the flick of a switch', either mh FLIM or phi2FLIM to be performed, 2) Validate and calibrate the developed techniques, 3) demonstrate the use of the instrumentation to generate insights into the ubiquitination of substrates in living cells. If successful, this will generate preliminary results to pump prime a larger scale research programme for which separate funding will be sought.

Planned Impact

This research will develop a capability to perform frequency domain fluorescence lifetime microscopy 1) at rapid speed without the problem of frequency aliasing and 2) at high accuracy, with the capability to resolve multiple lifetime components from a sample. Who will benefit from this research (see also academic beneficiaries section above)? The tools to be developed will help the UK maintain its leading role in applied photonics research. There is great commercialisation potential (see attached letter of support from Fianium). European manufacturers of diagnostic equipment are likely to incorporate the proposed technology into future platforms for example for drug screening. This idea is currently being explored with Astra Zeneca and the technology transfer office in Cambridge to develop a screening platform for anti aggregation drugs against neurodegenerative disease. The general potential to generate impact for the economic sector is maximised through the PI's role as director of CamBridgeSens (http://sensors.cam.ac.uk) which facilitates liaison with, and technology diffusion to, leading manufacturers of biomedical equipment. The technology proposed is enabled through the use of supercontinuum radiation generated in Photonic Crystal Fibres, an invention that originates from the UK. The leading manufacturer of supercontinuum products (Fianium Ltd, southampton) is also situated in the UK and collaborates with us, ensuring a rapid translation of concepts to products which are of general value to the community. The research will enable the study of dynamic molecular interactions in living systems and initially be used for fundamental research related to cancer and protein aggregation disease. Outcomes from such research will increase fundamental knowledge that in the longer term may lead to novel treatments of disease.

Publications

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Arnon Z (2018) Opal-like Multicolor Appearance of Self-Assembled Photonic Array in ACS Applied Materials & Interfaces

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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 We have developed of a novel, widefield frequency domain fluorescence lifetime imaging microscope (FD FLIM) that greatly improves on current capability by providing both speed of measurement (and thus reduction of sample exposure to light) and measurement precision (with a capability to measure complex multi-exponential decay characteristics). We have demonstrated and used these capabilities through application in live cell mitosis research. The system was developed through modification of an existing wide-field FD FLIM system and makes use of a pulsed supercontinuum light source for excitation and permits arbitrary phase profiles to be imposed on the gain waveform of the image collection system. In one mode of operation, which we term multiharmonic FLIM (mh FLIM), information can be retrieved from multiple harmonics present in the signal waveform. This permits multiple lifetimes to be resolved in every pixel. In another mode of operation, which we term phi2FLIM, the system will be capable of recording fluorescence lifetime images at several Hz repetition rates. phi2FLIM removes all components from higher harmonic frequencies from the signal and thus completely eliminates frequency aliasing artefacts in FD FLIM. We have applied mh FLIM for the study of the CFP-Plk1/YFP-ubiquitin pair in fixed cells to characterize mitosis-specific FRET between the substrate and ubiquitin. We have furthermore performed dynamic live cells studies using phi2FLIM to establish when and where ubiquitination of Plk1 occurs, and to determine the fate of ubiquitinated Plk1 complexes. This provides us with novel insights into how ubiquitination of Plk1 contributes to its activity, and to the organization of mitosis.

The microscope developed in this project is now supporting an PhD project supported by industry. Our industrial partners (MedImmune) are supporting the development of an image texture analysis technique that incorporates fluorescence lifetime data as well as spectrally-resolved image data, in order to determine whether solid pharmaceutical samples are genuine products or counterfeit.
Exploitation Route The platforms developed can be used for drug screening applications and biomedical diagnostics. Development of hardware:

1) A prototype instrument incorporating mhFLIM and phi2FLIM with a supercontinuum excitation source and fast gated intensifier has been built. An optimised version of the instrument will be made available in the Cambridge Advanced Imaging Center (CAIC), a multiuser imaging facility to serve the life science research community. Software for mhFLIM and phi2FLIM methods has been developed and is available free of charge from our web page.

2) A new tool for quantitative analysis of ubiquitination, in vivo-biotinylatable ubiquitin, was developed that outperforms current assays for endogenous ubiquitin in human cells. The method permits the timing and location of ubiquitination of specific mitotic substrates.



We have collaborated with Fianium UK and could show that their supercontinuum technology is ideally matched for high fidelity frequency domain FLIM applications, that are both fast and possess a high photon economy. This opens new commercial opportunities for supercontinuum light sources. We have collaborated with LaVision to obtain a custom made intensifier unit that is optimised for mhFLIM and phi2FLIM applications. We have also been collaborating with Lambert Instruments (Netherlands) on the implementation of mhFLIM on their commercial frequency domain FLIM platforms .



The developed widefield techniques are faster and more precise than current techniques and, although this was unanticipated at the time of writing this grant, they are proving useful also in the study of protein aggregation phenomena in live cells and small organism models of Alzheimer's disease



We have participated in the Cambridge Science Festival to educate the broader public on the use of advanced microscopic imaging in medical research. The PI has also given a public lecture organised by the Kelvin Club in Cambridge on the history and use of microscopy.
Sectors Healthcare,Pharmaceuticals and Medical Biotechnology

URL http://laser.ceb.cam.ac.uk
 
Description This grant was used to develop a novel frequency domain FLIM instrument based on a theoretical concept developed earlier by us. The technique, mhFLIM and phi2FLIM, were implemented experimentally and are now available for the Cambridge biology community. The instrument permits rapid screening of protein protein interactions in cellular models of disease at unprecedented speed and resolution. The instrument is also being used to develop a new image texture analysis technique, in collaboration with the global biologics research and development company MedImmune, to determine whether pharmaceutical samples are genuine products or counterfeits.
Sector Education,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 EBSA bursary
Amount £330 (GBP)
Organisation European Biophysical Societies' Association 
Sector Charity/Non Profit
Country Germany
Start 02/2018 
End 02/2018
 
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 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 BRCA2 dynamics experiment in cancer research with Prof. Ashok Venkitaraman 
Organisation Hutchison/MRC Research Centre
Country United Kingdom 
Sector Multiple 
PI Contribution The Laser Analytics Group performed fluorescence lifetime imaging for research on BRCA2 dynamics
Collaborator Contribution The group of Prof. Ashok Venkitaraman is expert in the study of BRCA2
Impact This collaboration has led to the following publication: Jeyasekharan AD, Liu Y, Hattori H, Pisupati V, Jonsdottir AB, Rajendra E, Lee M, Sundaramoorthy E, Schlachter S, Kaminski CF, Rosenfeld Y, Sato K, Savill J, Ayoub N, Venkitaraman AR, "A cancer-associated BRCA2 mutation reveals masked nuclear export signals controlling localization", Nature Structural & Molecular Biology (2013), 20,1191-1198(2013)
Start Year 2011
 
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 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 Quantitative Affinity Determination by Fluorescence Anisotropy Measurements of Individual Nanoliter Droplets (Florian Hollfelder) 
Organisation University of Cambridge
Department Department of Chemistry
Country United Kingdom 
Sector Academic/University 
PI Contribution integrated fluorescence anisotropy imaging (FAIM) with microfluidic platforms to look at protein-protein interactions in nanolitre samples. Developed quantification algorithms to determine binding constants from FAIM data.
Collaborator Contribution Hollfelder provided microfluidic technology platform and contributed expertise in high content screening.
Impact Publication: DOI: 10.1021/acs.analchem.6b02528 Multidisciplinary: biochemistry, biophysics
Start Year 2013
 
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 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 TriPer, an optical probe tuned to the endoplasmic reticulum, tracks H2O2 consumption by glutathione (Prof. David Ron / Dr. E. Avezov) 
Organisation University of Cambridge
Department Mechanics, Materials and Design
Country United Kingdom 
Sector Academic/University 
PI Contribution Developed FRET protocol to measure hydrogen peroxide and redox state in live cells via fluorescence lifetime imaging microscopy.
Collaborator Contribution Developed TriPer fusion protein technology that responds to H2O2 concentration changes in ER
Impact Paper accepted in BMC Biology Multidisciplinary: Cell biology, Physics
Start Year 2014
 
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 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 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 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