Multiplexed multiphoton fluorescence lifetime microscopy: Real time 3D imaging of protein-protein interactions by FRET

Lead Research Organisation: King's College London
Department Name: Cancer Studies

Abstract

From the earliest invention of the camera, humans have been seeking to observe processes that are too fast or too complicated for the human eye and brain to determine. The first time-laspe images of a running horse allowing us to understand its motion, the moment a bullet ripped through an apple - images, freezing a moment in time so that we can examine minute details. In cellular biology our understanding of cellular function continues to evolve as we observe complex dynamic processes played out under a microscope, captured by a camera at high speed and slowly revealing its hidden intricacy. As biologists ask every more complex questions, so we must develop more sophisticated tools to rationalise the complex data that we observe. Our current understanding of protein interaction in cells is informed, principally, through the use of microscopical tools to delineate localisation and compartmentalisation of signalling events within cellular organelles such as mitochondria. Further insight can be gained regarding protein association utilising the so called Förster resonance energy transfer (FRET) technique. FRET acts as a molecular ruler, enabling us to measure the relative separation between proteins or protein-domains on the nanometer length scale. Our work has focused on the determination of protein-protein interactions by FRET and high-resolution fluorescent lifetime imaging (FLIM). Unfortunately, these advanced techniques are relatively slow in capturing cellular events so that our desire to observe real-time the processes involved in, for example cell migration (directed motion often under the action of chemical gradient) are stymied. With this project, we seek to significantly speed up the acquisition of protein-interaction data to allows us to observe cellular signalling as it happens. This can be achieved through multiplexing of our excitation and detection channels to such an extent that we envisage a 1000-fold improvement in imaging with no loss of spatial resolution in the image. This work represents the state-of-the-art in functional imaging with the opportunity to observe complex cellular events in unpreceded detail: capturing an image of a T-cell as it surveys a cancer cell, forming dynamic 3-dimensional contacts and observing the protein signalling events that drive these processes; observing the moment a cell responds to a chemical stimulus at the level of single-proteins. The technology we will develop will drammatically improve our understanding of dynamic events within cells offering insight into drug interactions in diverse applications throughout the lifesciences.

Technical Summary

Protein interaction networks are highly regulated by spatio-temporal context within the cell. Recent methodologies for mapping these interactions using sophisticated imaging techniques have offered us considerable insight into these networks. Among the plethora of available techniques, FRET monitoring of protein-protein interactions is atractive for live cell imaging but is often stymied by limitations in dynamic range leading us to apply the most sensitive techniques such as fluorescence lifetime imaging (FLIM) to monitor both interaction distance and the fraction of molecules interacting. In this regard, we are often forced to choose trade-off resolution, for speed in an attempt to follow the temporal dynamics of evolving cellular dynamics. In order to follow fast processes in live cells we propose to develop a video rate multifocal multiphoton system for fluorescence lifetime imaging based on state-of-the-art single-photon avalanche photodiode (SPAD) arrays with on-pixel 55ps time-to-digital converter. The new SPAD array technology developed by Henderson and co-workers at the University of Edinburgh offers a huge advantage over existing fluorescence lifetime measurement tools and could present a paradigm shift in our approach to protein-interaction monitoring. We aim to exploit the advantages of this technology to, not only develop a sophisticated lifetime imaging technique, but to make it accessible to a wide variety of biological imaging problems from monitoring high-speed protein interaction dynamics to high-content screening applications using FRET readouts of cellular function or perturbation. The instrument will be characterised and exemplified in a biological context to monitor dynamic GTPase activity in cell motility using FRET biosensors. Implementation of the imaging system in a high content imaging platform will enable rapid imaging of GTPase activity in response to stimulation in a time-dependent manner.

Planned Impact

Academic Impact: The proposal will demonstrate an eagerly sought after and novel imaging modality, providing a platform technology compatible with live cell imaging and high content screening methodologies for protein interaction screening. These developments will be of direct benefit to researchers at the life-science interface particularly the biophysics community. We seek to address a fundamental problem in biological imaging- acquisition of fluorescence lifetime imaging data with high spatio-temporal resolution at video rate. Development of the instrument at the Randall Division of cell and molecular biophysics (KCL) will allow the technology to be exploited directly by bio-imaging experts in a variety of different biological systems, including unravelling G-protein-coupled receptor conformational and signalling dynamics in cells, this has major implications for drug discovery. Ameer-Beg, Ng and Suhling are currently working within larger consortia (Joint KCL/UCL Comprehensive Cancer Imaging Centre, Optical Proteomics Network and a BBSRC funded consortium (Unravelling supra-molecular rules in signal receptor network-systems using single-molecule imaging, led by Prof Peter Parker) all of which would benefit from having this technology available. Commercial Impact: If the technology developed in this proposal is taken up by the biophotonics research community, or the pharmaceutical industry for drug discovery and high-throughput screening, it will directly benefit the commercial private sector, i.e. UK industry. The pharmaceutical industry, as a beneficiary, will find utility in the biological assays for screening potential therapeutics that have effects on chemokine receptor trafficking and the associated signalling dynamics to ultimately support the discovery, validation and development of novel therapeutics. With the emergence of molecule-targeted therapies in the clinical setting there is an increasing demand for cell-based assays which can accurately report on the changes in receptor traffic, conformation and downstream signalling through the cytoskeleton in response to treatment. Health / Societal benefits: The application of the technology to understanding cell signalling is ultimately relevant for research into diseases. The GTPase activity plays a role in cancer, AIDS, multiple sclerosis, rheumatoid arthritis, allergic disorders, asthma, psoriasis, inflammatory bowel disease and nephritis. The diversity and wide range of diseases in which these proteins are involved is of great interest to the pharmaceutical industry. The results of this proposal will thus aid drug discovery and development as a significant benefit to wellbeing and in due course will be relevant for the long-term aim of addressing in healthcare in society. Exploitation and Application: Commercially exploitable output from the proposed programme may also include establishing novel fluorescence assays based on time-resolved fluorescence spectroscopy and imaging and will be managed by KCL Business or the equivalent office at University of Edinburgh. With a background in industry, Dr Henderson is well versed in these issues. Further exploitation will occur through ongoing cell imaging research at the Randall Division of Cell & Molecular Biophysics, King's College London and via COSMIC, University of Edinburgh. Moreover, since the consortium is closely linked to one of the strongest biomedical research activities in the UK, there exist significant opportunities for the exploitation of the proposed technology. Scientific data arising from this project will be disseminated at research symposia, international conferences and peer-reviewed journals.
 
Description This project has enabled us to develop a high speed multiphoton fluorescence lifetime imaging platform. The system enables 64-fold increased acquisition speed over more conventional systems. The system has enabled us to image in real time the recruitment of the adapter protein GRB2 to the cellular receptor EGFR when stimulated with its ligand. Furthermore, we have been able to image the dynamics of protein-protein interaction for other members of the ErbB family at a speed unprecidented in the field. Recent work that has followed on from the early 2-photon proposal has led to the development of a system which allows video rate FLIM of dynamic biological samples. Whilst not directly funded this has contributed to the further development of the techniques currently used in my laboratory.
Since the project completed we have developed a fully automated fluorescence lifetime imaging platform utilising the full 32x32 SPAD array provided by the Megaframe camera in what we call the Swept Array Microscope (SWARM). This involved additional firmware programming to enable a number of key features including on-chip lifetime calculation using the rapid lifetime determination method and migration onto a USB3 communication protocol. USB3 firmware completely removes the data bottleneck we suffered during the early stages of this grant and has enabled us to fully exploit the camera capability. We have now demonstrated imaging at up to 10 frames per second in a sustained manner and acquisition times down to 25ms. This is fully compatible with live cell imaging. We now have one of the world's leading lifetime imaging facilities which has opened as part of the KCL Microscopy Innovation Centre as a direct result of this BBSRC funding.
Outcomes: The Edinburgh Postdoctoral researcher funded on this project (Nikola Krstajic) has gone on to a lectureship (in part due to the success of this project) and the KCL PDRA now holds a Cancer Research UK Centre Fellowship at KCL due, largely to this project and the work that directly led from this funding.
Exploitation Route We are continuing to develop the imaging platform through new grants (for example CR UK Multidisciplinary award directly applies this microscope platform). The next steps are scaling up to super-resolution via STED/RESOLFT and SMLM. In addition, we have made the system available to our academic colleagues within the university as a collaborative platform through the Microscope Innovation Centre on Guy's Campus. Furthermore, we have developed a new imaging technique based on the original principle of multifocal FLIM which is implemented as a high content imaging platform for detection of protein-protein interactions in histological sections. We are working with Photon Force Ltd to develop a new version of the microscope with cooled detection for demonstration purposes prior to consideration for a commercial prototype.
Sectors Education,Electronics,Healthcare,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology

 
Description Academic Impact: Following development of the instrument, we have used the technology to image a variety of biological samples including cells, tissues (human archived histology) and zebrafish. In particular, we have focused on the imaging of biosensors for cell motility (RhoA biosensor) which has been imaged both in isolated live cells and zebrafish xenografts. Furthermore, we have imaged the interaction of proteins in the ErbB network showing dynamics previously unobservable due to both the speed of the biological process and the stoichiometry of the interaction. Commercial Impact: We are currently working with Andor Instruments via a BBSRC Sparking Impact Award (via KCL) to develop a preliminary prototype high speed FLIM system with a view to comercialisation. We have preliminary filing of a patent that broadly stems from the research undertaken during this project. The project also spawned another collaboration with Photon Force Ltd to develop a commercial demonstrator device for the company and investigate new cooled sensors funded by the EPSRC Quantum Hub, Quantic.
First Year Of Impact 2016
Sector Education,Electronics,Healthcare,Pharmaceuticals and Medical Biotechnology
Impact Types Societal,Economic

 
Description BBSRC iCASE
Amount £100,000 (GBP)
Funding ID BB/L015773/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 10/2014 
End 09/2018
 
Description Next Generation Optical Imaging Initiative
Amount £1,647,763 (GBP)
Funding ID MR/K015664/1 
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start 03/2013 
End 03/2018
 
Description Sparking Impact
Amount £10,000 (GBP)
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 11/2014 
End 05/2015
 
Title Multifocal Multiphoton Fluorescence lifetime imaging Microscope 
Description We have developed a novel microscope that enables high speed fluorescence lifetime imaging at depth within a biological structure. This microscope is essential 64 parallel FLIM microscopes, acquiring data at >0.5 s per frame - ~64 times faster than a conventional single beam scanning FLIM instrument. 
Type Of Material Technology assay or reagent 
Year Produced 2013 
Provided To Others? Yes  
Impact We are making this technology available to other researchers which will ultimately create impact. We have discussed a route to commercialisation of this research tool with several companies and will work towards realisation of a prototype production model for demonstration purposes. Ultimately, this technology may be provided in an "OPEN" format, depending on the demand or desire to commercialise. 
 
Description FRET imaging of cellular receptors 
Organisation UCB Pharma
Department UCB Celltech
Country United Kingdom 
Sector Private 
PI Contribution Contribute expertise in FLIM/FRET and high content assay development. In addition our research groups has developed a quantitative acceptor anisotropy imaging methodology.
Collaborator Contribution Provided CASE research studentship. Provided materials (covered under non-disclosure). Provide research expertise for target receptor (Covered under non-disclosure)
Impact CASE studentship award. Collaborative research award (Fee for Service arrangement - confidential) Rolling 3 year Research project (Confidential) Multidisciplinary collaboration Biophysics, FRET, optics and pharmaceutical science.
Start Year 2012
 
Description Feasibility study for STED imaging with multifocal arrays 
Organisation University College London
Country United Kingdom 
Sector Academic/University 
PI Contribution This project has been conceived to determine the feasibility of applying a new STED technique to multifocal multiphoton beam arrays. In the course of the project we have considered potential improvements to the technique using exisitng technology and as part of that have provided a hybrid photomultiplier tube and controller to UCL Physics (Dr Angus Bain). In addition we have had helpful discussions regarding the complex photophysics of STED.
Collaborator Contribution Dr Angus Bain has provided expertise with respect to his reported novel method of using stimulated emission depletion whereby the temporal evolution of the fluorophore population on the nanosecond time-scale is manipulated to obtain super-resolved imaging data.
Impact Multidisciplinary collaboration between physics department and biophysics/cancer biology. Grant application submitted (FLIP) to BBSRC - Bain/Ameer-Beg/Cox
Start Year 2014
 
Description High Speed In vivo imaging of Zebrafish Larvae 
Organisation King's College London
Department Dental Institute
Country United Kingdom 
Sector Academic/University 
PI Contribution With Dr Robert Knight, we have developed imaging methods which are applicaple to live zebrafish lavae imaging of transfected fluorescent proteins. In addition, we have developed methods to specifically laser ablate regions of interest within the Zebrafish muscle to enable studies of macrophage recruitment. We have further provided expertise in specifying a multiphoton imagign system appropriate for deep tissue imaging of Zebrafish and small mammals.
Collaborator Contribution Our partners have provided samples to enable us to optimise our imaging platform and expertise in the form of animal husbandry. Access will be available to the newly installed multiphoton microscope facility we have helped to specify.
Impact This reseearch collaboration has led to the publication of a research paper (http://dx.doi.org/10.1364/OL.39.002431). Furthermore, a recent application has been made to BBSRC for responsive mode funding (result pending). This is a multidisciplinary collaboration consisting Biophysics, Optics, laser physics and biology.
Start Year 2013
 
Description Sparking Impact 
Organisation Andor Technology
Country United Kingdom 
Sector Private 
PI Contribution We have successfully partnered with Andor Technology as part of a postdoc led initiative through BBSRC Sparking Impact funding at KCL. Researchers at KCL (PI and Dr Simon Poland (PDRA for grant)) are developing a prototype platform for multifocal multiphoton fluorescence lifetime imaging which will be used for demonstration to stake-holders. The imaging platform will be a simplified version of the highly flexible imaging sytstem that was developed as part of the BBSRC grant.
Collaborator Contribution Andor have offered no financial support but have offered industrial mentoring and significant advice and interest in development of the project to a working prototype.
Impact This is a multidisciplinary collaboration. The KCL experience is primarily in development of biological assays for protein-protein interaction analysis and instrument development. Andor Technology are principly an electrical enginneering SME.
Start Year 2014