High-speed imaging of FRET in live cells applied to investigate the role of PLCe in intracellular signal pathways

Lead Research Organisation: Imperial College London
Department Name: Dept of Physics

Abstract

This multidisciplinary joint proposal between Imperial College London and the Institute of Cancer Research is to develop new technology for imaging interactions between protein molecules in live cells, to be applied to study intracellular signal pathways that are important for cancer. These protein interactions will be imaged using the fluorescence-based technique of Forster Resonant Energy transfer (FRET). Fluorescence imaging entails 'labelling' proteins of interest with fluorescent molecules (called 'fluorophores') that absorb and emit light in a characteristic manner. A recent breakthrough has been the development of genetically expressed fluorescent proteins that can be used to tag specific proteins in living cells. Conventionally the proteins of interest would be 'excited' by irradiating them with photons that can absorbed by the fluorophore labels. These would then emit light (fluorescence) and relax back to their original state. By imaging the intensity of this fluorescence, one can visualise the distribution of the fluorophores / and therefore the proteins to which they are attached. To study interactions between different proteins, one can label each kind of protein with a different fluorophore emitting at a different wavelength (i.e. different colour light). By recording images of different colours / corresponding to the distributions of each kind of protein / and superimposing them, one can see where different proteins occur in the same place, i.e. co-localisation. The problem with this technique is that the spatial resolution of the optical microscopes that are used to image living cells is limited to ~ the wavelength of the light in question (about 400-700 nm) but the proteins themselves are much smaller (~ 1-10 nm). Therefore, even if two proteins appear to occur in the same place in the fluorescence image, they can be completely independent, on a molecular scale. FRET provides a way to determine when the fluorophores are within ~ 10 nm of each other / a distance at which the proteins would be interacting. It works by observing the transfer of 'excitation energy' from one fluorophore (called the 'donor' to another (called the 'acceptor') than only occurs over this very short distance. The most straightforward way to observe FRET is to see where the donor fluorescence intensity decreases or the acceptor fluorescence intensity increases. Unfortunately this kind of intensity-based imaging is often unreliable because of background noise. The most reliable way to image FRET is by fluorescence lifetime imaging (FLIM). In general fluorescence lifetime is measured by exciting fluorophores with a short pulse of light and observing how long it takes the fluorescence signal to decay away as they relax back to their ground state. Using very fast camera technology, it is possible to image fluorescence decays across a sample and obtain a value of fluorescence lifetime for each pixel in the image. Because FRET provides an additional way for excited fluorophores to lose their energy, one can determine where FRET is occurring by observing a reduction in the donor fluorescence lifetime. Unfortunately most FLIM technology is rather slow, taking several minutes to acquire a FLIM image (map of fluorescence lifetime values) and this makes it difficult to use FLIM-FRET to follow dynamics in live cells. The goal of this project is to combine novel high-speed FLIM and microscopy expertise at Imperial with the biological expertise at the ICR to develop new FLIM-FRET imaging systems. This will involve designing new molecules with appropriate donor and acceptor fluorophore labels and combining high-speed FLIM technology with novel microscope configurations. A particularly ambitious part of this proposal will be to design experiments in which we can 'multiplex FRET imaging, i.e. image two protein-protein interactions in parallel to study how different events in cells are associated in a signalling pathway.

Technical Summary

This multidisciplinary joint proposal between Imperial College London and the Institute of Cancer Research is to develop new technology for imaging FRET of protein interactions in live cells applied to the elucidation of the role of a new member of the phospholipase C family, PLCe, in intracellular signal pathways. In particular, we first hope to clarify the interaction between PLCe and members of the Ras family of small GTP-ases, known to be implicated in a wide variety of cancerous phenotypes. The new live cell FRET imaging technology will be based on wide-field, optically-sectioned, single photon-excited, fluorescence lifetime imaging (FLIM) and related wide-field multi-spectral polarisation-resolved imaging technology. Our extensive expertise in such technologies at Imperial, including video rate FLIM, achieved by raising the S/N to unprecedented levels using new electronics and data acquisition strategies, novel tunable visible ultrafast excitation sources and in-house software tools, will be combined with the biological expertise at the ICR to design experiments and develop new fluorophore constructs. These will include GFP tagged PLCe and small GTPases labelled with mRFP to image translocation, Raichu-type FRET probes to determine specificity of the PLCe GEF domain for different GTPases and a novel FRET probe to image conformational changes in PLCe upon binding to Ras. We then aim to extend the technology to imaging multiplexed FRET interactions, correlating translocation and conformational changes with a downstream reporter of PLCe activity such as changes in intracellular calcium, using a FRET probe like Chamelon. We would explore both simultaneous (interleaved) and sequential FRET imaging, developing new FRET probe constructs to address the issues of spectral cross-talk. Linking technology development to specific biological questions should enhance our technical progress while producing useful instrumentation for a wide range of applications.

Publications

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Görlitz F (2017) Open Source High Content Analysis Utilizing Automated Fluorescence Lifetime Imaging Microscopy. in Journal of visualized experiments : JoVE

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Kumar S (2011) FLIM FRET technology for drug discovery: automated multiwell-plate high-content analysis, multiplexed readouts and application in situ. in Chemphyschem : a European journal of chemical physics and physical chemistry

 
Description This multidisciplinary joint proposal between Imperial College London and the Institute of Cancer Research aimed to develop new technology for imaging interactions between protein molecules in live cells, to be applied to study intracellular signal pathways that are important for cancer. These protein interactions are to be imaged using the fluorescence-based technique of Forster Resonant Energy transfer (FRET).
Fluorescence imaging entails "labelling" proteins of interest with fluorescent molecules (called "fluorophores") that absorb and emit light in a characteristic manner. A recent breakthrough has been the development of genetically expressed fluorescent proteins that can be used to tag specific proteins in living cells. Conventionally the proteins of interest would be "excited" by irradiating them with photons that can absorbed by the fluorophore labels. These would then emit light (fluorescence) and relax back to their original state. By imaging the intensity of this fluorescence, one can visualise the distribution of the fluorophores / and therefore the proteins to which they are attached. To study interactions between different proteins, one can label each kind of protein with a different fluorophore emitting at a different wavelength (i.e. different colour light). By recording images of different colours / corresponding to the distributions of each kind of protein / and superimposing them, one can see where different proteins occur in the same place, i.e. co-localisation. The problem with this technique is that the spatial resolution of the optical microscopes that are used to image living cells is limited to ~ the wavelength of the light in question (about 400-700 nm) but the proteins themselves are much smaller (~ 1-10 nm). Therefore, even if two proteins appear to occur in the same place in the fluorescence image, they can be completely independent, on a molecular scale.
FRET provides a way to determine when the fluorophores are within ~ 10 nm of each other / a distance at which the proteins would be interacting. It works by observing the transfer of excitation energy from one fluorophore (called the donor) to another (called the acceptor) than only occurs over this very short distance. The most straightforward way to observe FRET is to see where the donor fluorescence intensity decreases or the acceptor fluorescence intensity increases. Unfortunately, this kind of intensity-based imaging is often unreliable because of background noise. The most reliable way to image FRET is by fluorescence lifetime imaging (FLIM). In general fluorescence lifetime is measured by exciting fluorophores with a short pulse of light and observing how long it takes the fluorescence signal to decay away as they relax back to their ground state. Using very fast camera technology, it is possible to image fluorescence decays across a sample and obtain a value of fluorescence lifetime for each pixel in the image. Because FRET provides an additional way for excited fluorophores to lose their energy, one can determine where FRET is occurring by observing a reduction in the donor fluorescence lifetime. Unfortunately, most FLIM technology is rather slow, taking several minutes to acquire a FLIM image (map of fluorescence lifetime values) and this makes it difficult to use FLIM-FRET to follow dynamics in live cells.
The main output of this project was the development of a novel fluorescence microscope in which we combined novel high-speed FLIM using wide-field time-gated imaging based on a gated image intensifier with a Nipkow "spinning disc" scanner to provide optical sectioning and developed an instrument capable of FLIM at up to 10 Hz. We applied this to live cells expressing fluorescent protein labels and showed that its low phototoxicity (compared to standard laser scanning fluorescence microscopes) was compatible with extended imaging over a time course. We also implemented the capability to acquire FLIM data in multiple spectral channels and demonstrated its ability to read out FRET signals from two different cell signalling events in parallel. This is important because disease mechanisms and drugs can impact multiple signalling pathways.
Exploitation Route Out technology for high speed FLIM and FRET of cells utilising a Nipkow disc scanner and time-gated FLIM has been copied by several groups and is the basis for our FLIM high content analysis platform, which can be used to systematically screen for protein-protein interactions or to read out FRET biosensors. This is of interest to pharma and to basic science.
Sectors Healthcare,Pharmaceuticals and Medical Biotechnology

 
Description In later projects we applied this technology in follow up collaborative studies of protein -protein interactions focused PLCepsilon and other Ras effectors. Our technology for high speed FLIM and FRET of cells utilising a Nipkow disc scanner and time-gated FLIM has been copied by several groups for rapid FLIM and FRET, particularly to study live cells. The manufacturers of the gated optical intensifier used, Kentech Instruments Ltd, and the supercontinuum excitation source, Fianium Ltd, have reported that they believe their sales increased significantly as a result of our work. The new approach to optically sectioning FLIM microscopy is also the basis of our automated FLIM high content analysis (HCA) platform that we have applied to assays of cell signalling using FRET, including SUMOylation of FOX1 and screening for binding partners of MST1 amongst the RASSF family. Our work on multiplexed FRET readouts exploiting FLIM has been taken forward by ourselves in subsequent projects and by others.
First Year Of Impact 2010
Sector Healthcare,Pharmaceuticals and Medical Biotechnology
Impact Types Economic

 
Description BBSRC IPA BB/M006786/1
Amount £508,815 (GBP)
Funding ID BB/M006786/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 05/2015 
End 04/2017
 
Description BBSRC Responsive mode BB/H00713X/1
Amount £473,776 (GBP)
Funding ID BB/H00713X/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 04/2010 
End 03/2013
 
Description Kentech Instruments Ltd 
Organisation Kentech Instruments Ltd
Country United Kingdom 
Sector Private 
PI Contribution We have advised Kentech on improvements to be made to their gated optical image intensifiers and related components and have tested prototypes. We have demonstrated that their gated intensifiers can be integrated into a range of optical instruments, particularly for fluorescence lifetime imaging (FLIM) for biomedical applications, and this has led to increased sales for them. We collaborated with them on an EPSRC Healthcare Partnership project that built on earlier collaborations with Kentech and also on two MRC projects to develop FLIM instrumentation.
Collaborator Contribution Kentech Instruments Ltd advised us (with no charge) on the design of FLIM systems using gated optical image intensifiers and supplied us with a custom device for handheld FLIM that we designed in collaboration. They have sold us customised gated intensifier systems and refined the specifications of their products at our request.
Impact Joint publications Feedback to Kentech's product development
 
Title High speed optically sectioning FLIM microscope 
Description The main output of this project was the development of a novel fluorescence microscope in which we combined novel high-speed FLIM using wide-field time-gated imaging based on a gated image intensifier with a Nipkow "spinning disc" scanner to provide optical sectioning and developed an instrument capable of FLIM at up to 10 Hz. We applied this to live cells expressing fluorescent protein labels and showed that its low phototoxicity (compared to standard laser scanning fluorescence microscopes) was compatible with extended imaging over a time course. We also implemented the capability to acquire FLIM data in multiple spectral channels and demonstrated its ability to read out FRET signals from two different cell signalling events in parallel. This is important because disease mechanisms and drugs can impact multiple signalling pathways. 
Type Of Technology New/Improved Technique/Technology 
Year Produced 2008 
Impact Our technology for high speed FLIM and FRET of cells utilising a Nipkow disc scanner and time-gated FLIM has been copied by several groups for rapid FLIM and FRET, particularly to study live cells. It is also the basis of our automated FLIM high content analysis (HCA) platform that has been applied to assays of cell signalling using FRET, including SUMOylation of FOX1 and screening for binding partners of MST1 amongst the RASSF family. We have also applied this FLIM HCA instrument to assay changes in cellular metabolism using FLIM of NADH. Our work on multiplexed FRET readouts exploiting FLIM has been taken forward by ourselves in subsequent projects and by others. Many other groups have adopted wide-field optically sectioned FLIM for a range of studies inspired by our work and the manufacturers of the gated optical intensifier used, Kentech Instruments Ltd, and the supercontinuum excitation source, Fianium Ltd, have reported that they believe their sales increased significantly as a result of our work. 
 
Title openFLIM HCA: Automated multiwell plate FLIM microscope for optically sectioned assays in 3-D cell culture including time-lapse FLIM/FRET studies in live cells 
Description This automated multiwell plate FLIM microscope is controlled by open source (MIcroManager) software and the component lists are shared on our website to enable other research groups to replicate our instrumentation. FLIM data analysis, including global fitting, is provided by our open source software tool: FLIMfit, written in MATLAB. Exciation is provided by a super-continuum source or a frequency-doubled mode-locked Ti:Sapphire laser. FLIM detection is realised though time-gated imaging using a gated image intensifier. This instrument typically acquires a field of view every 10 seconds, including the time for sample movement and autofocus. 
Type Of Technology New/Improved Technique/Technology 
Year Produced 2016 
Impact This instrument has been applied to a number of applications including protein interactions of the kinetochore, metabolic changes in differentiating embryonic stem cells, bromodomain-histone binding and a genetically expressed FRET biosensor for EPAC,a Rap-1 guanine exchange factor that binds specifically to cAMP. 
URL http://www.imperial.ac.uk/photonics/research/biophotonics/instruments--software/openflim-hca/
 
Title openHCA-FLIM µManager Plugin 
Description This is the (MicroManager Plugin) control software for our openFLIM HCA instrument that provides automated multiwell plate FLIM, with the option to implement optical sectioning. 
Type Of Technology Software 
Year Produced 2016 
Impact This open source software is freely available and should enable other research groups to build their own FLIM HCA instrumentation. 
URL http://www.imperial.ac.uk/photonics/research/biophotonics/instruments--software/openflim-hca/
 
Description GE HC Cellular Technologies symposium 2010 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Industry/Business
Results and Impact I was an invited speaker at GE Healthcare Cellular Technologies symposium at the GE Global Research Centre, Albany 2010

This gave me the opportunity to present our technology to key industry figures in the largest company working in the field of HCA. It strengthened dour collaboration with GEHC
Year(s) Of Engagement Activity 2010
 
Description GSK Stevenage 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Industry/Business
Results and Impact Industry visit and seminar.

Paul French gave an invited seminar on FLIM and its biomedical applications including results from this project.
Year(s) Of Engagement Activity 2012
 
Description International School of Physics "Enrico Fermi": "Microscopy Applied To Biophotonics" 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact Invited lectures at the International School of Physics "Enrico Fermi": "Microscopy Applied To Biophotonics", Varenna, Lake Como, Italy, 2011



Introduction to the sources of cellular and tissue autofluorescence; Microspectrophotometry and imaging for studying celluar autofluorescence in vitro; & Autofluorescence instrumentation.
Year(s) Of Engagement Activity 2011
 
Description MDFI for HCA workshop 2014 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Industry/Business
Results and Impact This workshop on multidimensional imaging for high content analysis was intended to showcase our MDFI technology, including FLIM and FRET, implemented in automated multiwell plate readers, endoscopes, microscopes and in vivo tomographic imaging of zebrafish. Our target audience was industry, including pharma and technology developers, as well as research students and academics from Imperial and other universities.
Discussions with industry led to new collaborations with SME to develop advanced imaging technology and continued interest from pharma who gave feedback on our technology and potential applications. This event included an internet presentation from Rainer Pepperkok at EMBL.
Year(s) Of Engagement Activity 2014
 
Description MDFI user-orientated workshop 2017 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Postgraduate students
Results and Impact This one day user-orientated workshop entitled "Multidimensional fluorescence imaging technology: super-resolution, HCA and preclinical imaging" showcased the biophotonics instrumentation and applications of the technologies developed in the Photonics Group at Imperial College London. It attracted 80 participants including 11 from industry and 40 for other research organisation. The talks and posters were well received and this event led to further collaborations, including with industry.
Year(s) Of Engagement Activity 2017
 
Description MDFIM user-orientated workshop 2013 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Type Of Presentation Workshop Facilitator
Geographic Reach National
Primary Audience Industry/Business
Results and Impact Multidimensional fluorescence imaging and metrology user-orientated workshop held at Physics Department, Imperial College London on 27/09/13.
~50 external visitors attended talks, posters and networking sessions. The aims were to identify new users of our research, to reinforce relationships with existing users, to identify new research partners, sponsors and opportunities. The event was also a showcase for our research progress and students and staff and a source of feedback to inform our ongoing technology development.

Three companies expressed interest in commercialising aspects of our work
Several external research partners learned about our capabilities and suggest new research collaborations
We received excellent feedback about quality of work presented and of presentations
Year(s) Of Engagement Activity 2013
URL http://www3.imperial.ac.uk/photonics/events/photworkshop
 
Description Multidimensional fluorescence imaging - for clinical diagnosis, cell biology and drug discovery 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact 1st Europhotonics Spring School, Barcelona, Spain.
Year(s) Of Engagement Activity 2012
 
Description Multidimensional fluorescence imaging and metrology - for cell biology, high content analysis and clinical diagnosis 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact Invited lectures at Biophotonics 2011 summer school, Ven, Sweden.
Year(s) Of Engagement Activity 2011
 
Description PicoQuant workshop 2013 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact Invited oral presentation at the 3rd European Short Course on Time-Resolved Microscopy and Correlation Spectroscopy, Berlin, Germany. mainly aimed at post-graduate students

Pau French was asked to come back again to lecture on this training course
Year(s) Of Engagement Activity 2012
 
Description Plenary lecture ECBO 2013 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact Plenary lecture, presented at the European Conference on Biomedical Optics, Munich 2013, entitled: "Fluorescence lifetime imaging and metrology for cell biology, high content analysis and clinical diagnosis"

Audience was > 500 including many attendees form industry since this conference was co located with Europe's largest laser trade fair.

Subsequently I received many enquiries about our work (including an request to give a webinar) and invitations to speak at further meetings.
Year(s) Of Engagement Activity 2013
URL http://www.osa.org/en-us/meetings/osa_meeting_archives/2013/european_conferences_on_biomedical_optic...
 
Description Wellcome Trust 2009 Focus on Light Microscopy 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Supporters
Results and Impact The Wellcome Trust organised a meeting to develop strategy for the development and application of emerging new light microscopy techniques. As well as research talks, this meeting included discussions about future strategic priorities for the field. A report of the meeting was compiled and circulated to attendees.
Year(s) Of Engagement Activity 2009