Multiparametric advanced fluorescence imaging strategies for in situ analysis of live cell signalling

Lead Research Organisation: King's College London
Department Name: Randall Div of Cell and Molecular Biophy

Abstract

To understand and combat the causes of human disease, we must understand the basic structure and function of the individual cells that make up the tissues and organs of the human body. For example, to allow the design of effective therapies to target cancer we first need to answer fundamental questions about how the growth, division and movement of cells are controlled. Robert Hooke was the first to use microscopes to describe cell structure in 1665, and since then microscopy has become one of the most powerful tools for cell biologists across the world. The power of light microscopes has of course continued to increase since their invention but, remarkably, the most dramatic improvement has come in the last ten years or so. In that period physicists have worked out how to measure the location of a single protein in a cell with a precision about ten times better that was previously thought possible. This is important because we can now see the internal structure and organisation of cells in much more detail. In parallel, physicists working together with biologists developed microscopical methods that, instead of just producing a map of the locations of one particular protein inside a cell, can produce a map of precisely where protein A is bound to protein B. This is a fundamental advance, because cell function is controlled by pathways and networks of such interactions between specific proteins. Potentially then, these new microscopes provide a window into the internal workings of a cell that allow us to see these protein networks. However, at the moment, the most detailed images can only be obtained from chemically preserved rather than living cells, and each image takes many minutes to record. This is a serious problem, because the interactions between proteins that control cell function take place transiently on the time scale of seconds. To understand cell function, we need movies rather than still images. In the present proposal, biologists and physicists will work together to develop the technology to allow us to record the detailed maps of protein locations and interactions in live cells in milli-seconds rather than minutes or hours. We think that these new developments will unlock the potential of these microscopes to show us how cells work at the molecular level.

Technical Summary

The power of optical imaging technology to drive major discoveries in cell biology and medicine has increased dramatically over the last decade. This proposal focuses on two areas of fluorescence microscopy where the potential for such applications is clear but has not yet been fully realised. The first relates to functional imaging modalities that can map specific protein-protein interactions in cells, in particular using Forster resonance energy transfer (FRET) and fluorescence lifetime imaging microscopy (FLIM). The second relates to super-resolution microscopy (SRM) methods that break the conventional resolution limit imposed by the wavelength of light. In recent years, many different SRM technologies have been developed that typically promise a spatial resolution of 50 nm, an order-of-magnitude improvement over conventional methods. However the commercially available FLIM and SRM instruments are limited by technical constraints of sub-optimal detector sensitivity, speed, data analysis and interpretation. We will develop new FLIM and SRM configurations and detectors to overcome these limitations, and allow protein location, orientation, environment, interactions and dynamics to be analysed in living cells and organisms. These instruments will all be built within a new Microscopy Development Centre (MDC) on the Guy's Hospital Campus of King's that is adjacent to the new Nikon Imaging Centre. These two Centres will share technical support, training, data storage and image analysis facilities/expertise. The MDC physicists will work closely with biomedical scientists in the co-I's teams to refine the new instruments and apply them to a series of exemplar biological questions in the fields of immunology, stem cell biology, cancer, cardiovascular and muscle biology. Once developed, the new FLIM and SRM technologies will be disseminated within the biomedical research community, initially within King's and subsequently to other UK Universities, institutes and companies.

Planned Impact

This proposal represents a core multidisciplinary partnership between academics with very strong links to commercial collaborators. Our goal is to design and build novel instruments to analyse direct protein-protein interactions and image three-dimensional complexes, structures and dynamic processes in living cells at the nanometer scale to reveal previously hidden details of biological structure and function. The proposed multidisciplinary partnership between several laboratories has been formed through a shared common goal to use novel imaging approaches to unravel complex biological signalling events in detail in living cells or organisms. The partnership brings together biologists, biophysicists and physicists within an environment that is ideal for training, technology development and image analysis. The team already has a track record of joint publications and research funding, as well as group meetings, journal clubs and joint PhD student supervision. The significant advances that are made in microscopy development here will eventually also be applied across multiple laboratories with different biological questions and thus provide an innovative and collaborative training environment that will broaden the knowledge base of our physics and biology postgraduate/postdoctoral staff as well as the participating faculty. We anticipate that the programme of novel optical instrument development detailed here will also have a significant impact on the research community at King's and more broadly within the UK. The alignment of King's College London with partner NHS Hospitals in King's Health Partners (KHP) also offers unparalleled opportunities for translational and clinical research. King's is also a partner in the Francis Crick Institute (FCI), and as the biophysical/medical interface is a key strategic development area of the FCI, the state-of-the-art developments made possible through our partnership will also likely extend to collaborations in the FCI in future. For the current proposal, we have established collaborations with Universities of Sussex and Edinburgh who will benefit directly from the technology arising from this project for their own research purposes. We additionally propose to host a conference/workshop in year5, at the end of the current proposed development plan, to share our findings and progress with the national and international research community. Microscopy instrument development represents one of the growing technology areas in biomedicine and our existing strong working relationship with imaging instrument manufacturers places us in an excellent position to fully realize the commercial potential of our novel developments arising from this study. Similarly, the importance of microscopy-based assays and screens to analyse cell function and test potential drugs is increasingly recognised by the pharmaceutical industry and will place us at the forefront of emerging technologies leading to the design and validation of new therapies.

Publications

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Cox S (2013) Imaging cells at the nanoscale. in The international journal of biochemistry & cell biology

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Fox-Roberts P (2014) Fixed Pattern Noise in Localization Microscopy in ChemPhysChem

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Foxall E (2016) Significance of kinase activity in the dynamic invadosome. in European journal of cell biology

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Hirvonen L (2015) Photon counting imaging with an electron-bombarded CCD: Towards wide-field time-correlated single photon counting (TCSPC) in Nuclear Instruments and Methods in Physics Research Section A: Accelerators, Spectrometers, Detectors and Associated Equipment

 
Description European Research Council Synergy Grant
Amount € 12,000,000 (EUR)
Funding ID 856118 
Organisation European Research Council (ERC) 
Sector Public
Country Belgium
Start 01/2020 
End 02/2026
 
Description MRC Programme Grant 2016-21
Amount £1,800,000 (GBP)
Funding ID MR/N021231/1 
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start 06/2016 
End 05/2021
 
Description Research Grant
Amount £1,377,345 (GBP)
Funding ID MR/K015664/1 
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start 02/2013 
End 01/2018
 
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
 
Description Wellcome Trust Collaborative Award in Sciences
Amount £1,164,059 (GBP)
Funding ID 201543/Z/16/Z 
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 09/2016 
End 09/2020
 
Title Assessment of fluorophore dynamics using DNA origami 
Description We have developed a method to use localisation microscopy data of DNA origami to assess how the behaviour of pairs of fluorophores is related to their spatial separation. It uses simultaneous imaging in two colour channels with DNA origami that has been labelled with two different dyes. 
Type Of Material Technology assay or reagent 
Provided To Others? No  
Impact The work has revealed that the behaviour of fluorophores depends on their spatial separation. This is important for molecule counting and nanoscale imaging applications. 
 
Title Confocal optical lattice microscope for fluorescence lifetime imaging 
Description We have developed a high speed fluorescence lifetime imaging microscope based on the principle of multiple focal points arranged in an optical lattice. With this system it is possible to simultaniously image 1024 diffraction limited points within a biological sample. This is analogous to our multifocal multiphoton imaging platform but with a 1024-fold speed improvement over standard single beam scanning confocal microscopy. 
Type Of Material Technology assay or reagent 
Year Produced 2014 
Provided To Others? Yes  
Impact Impact will be created through collaboration with users. We are currently developing this system via a Sparking Impact award to provide a robust prototype which will be field tested by collaborators in the UK. 
 
Title Dual density localisation microscopy imaging 
Description We have developed a method to simultaneously image a sample tagged with a photoswitchable fluorophore such as mEOS-2 in two different colour channels. This means that a high-density widefield image and a low-density single-molecule image can be acquired at the same time. This allows either tracking of the average motion of the protein from the high density image, and reconstruction of a super-resolution image from the low density image, or tracking of single molecule motion, depending on what type of data is required. 
Type Of Material Technology assay or reagent 
Year Produced 2015 
Provided To Others? Yes  
Impact We are using this method to track the dynamics of vinculin in focal adhesions in collaboration with the group of Maddy Parsons at KCL. The work is currently unpublished. 
 
Description Collaboration for SPAD detectors 
Organisation University of Edinburgh
Country United Kingdom 
Sector Academic/University 
PI Contribution Working closely with the detector development team to optimise the use and implementation of SPAD array for lifetime analysis
Collaborator Contribution Design, engineer and building the SPAD arrays
Impact NA
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 SPAD and lifetime alogrithms 
Organisation University of Sussex
Country United Kingdom 
Sector Academic/University 
PI Contribution Implementation of SPAD detectors and algorithms for high-speed analysis of lifetime
Collaborator Contribution Development of SPAD detectors and algorithms for high-speed analysis of lifetime
Impact n/a
Start Year 2011
 
Description Rosalind Franklin Prize visit 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact 6th form visit to hospital and labs to encourage science/medical University application, associated with school Rosalind Franklin Prize.
Year(s) Of Engagement Activity 2018
 
Description School visit 2014 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact lab visit and discussion

na
Year(s) Of Engagement Activity 2014