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
Yu CH
(2013)
Integrin-matrix clusters form podosome-like adhesions in the absence of traction forces.
in Cell reports
Rosten E
(2013)
ImageJ plug-in for Bayesian analysis of blinking and bleaching.
in Nature methods
Cox S
(2013)
Imaging cells at the nanoscale.
in The international journal of biochemistry & cell biology
Cullup T
(2013)
Recessive mutations in EPG5 cause Vici syndrome, a multisystem disorder with defective autophagy.
in Nature genetics
Poland SP
(2014)
Development of a doubly weighted Gerchberg-Saxton algorithm for use in multibeam imaging applications.
in Optics letters
Walde M
(2014)
Vinculin binding angle in podosomes revealed by high resolution microscopy.
in PloS one
Poland SP
(2014)
Time-resolved multifocal multiphoton microscope for high speed FRET imaging in vivo.
in Optics letters
Ribeiro Ede A
(2014)
The structure and regulation of human muscle a-actinin.
in Cell
Fox-Roberts P
(2014)
Fixed pattern noise in localization microscopy.
in Chemphyschem : a European journal of chemical physics and physical chemistry
Krstajic N
(2015)
0.5 billion events per second time correlated single photon counting using CMOS SPAD arrays.
in Optics letters
Hirvonen L
(2015)
Sub- µ s time resolution in wide-field time-correlated single photon counting microscopy obtained from the photon event phosphor decay
in New Journal of Physics
Pernigo S
(2015)
The crystal structure of the human titin:obscurin complex reveals a conserved yet specific muscle M-band zipper module.
in Journal of molecular biology
Klapholz B
(2015)
Alternative mechanisms for talin to mediate integrin function.
in Current biology : CB
Poland SP
(2015)
A high speed multifocal multiphoton fluorescence lifetime imaging microscope for live-cell FRET imaging.
in Biomedical optics express
Krstajic N
(2015)
256 × 2 SPAD line sensor for time resolved fluorescence spectroscopy.
in Optics express
Shivalingam A
(2015)
The interactions between a small molecule and G-quadruplexes are visualized by fluorescence lifetime imaging microscopy.
in Nature communications
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
Levitt JA
(2015)
Simultaneous FRAP, FLIM and FAIM for measurements of protein mobility and interaction in living cells.
in Biomedical optics express
Pipalia TG
(2016)
Cellular dynamics of regeneration reveals role of two distinct Pax7 stem cell populations in larval zebrafish muscle repair.
in Disease models & mechanisms
Poland SP
(2016)
New high-speed centre of mass method incorporating background subtraction for accurate determination of fluorescence lifetime.
in Optics express
Foxall E
(2016)
Significance of kinase activity in the dynamic invadosome.
in European journal of cell biology
Yu H
(2016)
Estimation of Fluorescence Lifetimes Via Rotational Invariance Techniques.
in IEEE transactions on bio-medical engineering
Hastings R
(2016)
Combination of Whole Genome Sequencing, Linkage, and Functional Studies Implicates a Missense Mutation in Titin as a Cause of Autosomal Dominant Cardiomyopathy With Features of Left Ventricular Noncompaction.
in Circulation. Cardiovascular genetics
Jayo A
(2016)
Fascin Regulates Nuclear Movement and Deformation in Migrating Cells.
in Developmental cell
Hirvonen LM
(2016)
Photon counting imaging and centroiding with an electron-bombarded CCD using single molecule localisation software.
in Nuclear instruments & methods in physics research. Section A, Accelerators, spectrometers, detectors and associated equipment
Rafiq NB
(2017)
Podosome assembly is controlled by the GTPase ARF1 and its nucleotide exchange factor ARNO.
in The Journal of cell biology
Fox-Roberts P
(2017)
Local dimensionality determines imaging speed in localization microscopy.
in Nature communications
Pollpeter D
(2017)
Deep sequencing of HIV-1 reverse transcripts reveals the multifaceted antiviral functions of APOBEC3G
in Nature Microbiology
Randall TS
(2017)
A small-molecule activator of kinesin-1 drives remodeling of the microtubule network.
in Proceedings of the National Academy of Sciences of the United States of America
Pfisterer K
(2017)
Control of nuclear organization by F-actin binding proteins.
in Nucleus (Austin, Tex.)
Peddie CJ
(2017)
Correlative super-resolution fluorescence and electron microscopy using conventional fluorescent proteins in vacuo.
in Journal of structural biology
Fili N
(2017)
NDP52 activates nuclear myosin VI to enhance RNA polymerase II transcription.
in Nature communications
Suhling K
(2017)
Handbook of Photonics for Biomedical Engineering
Staszowska A
(2017)
Investigation of podosome ring protein arrangement using localization microscopy images
in Methods
Pernigo S
(2017)
Binding of Myomesin to Obscurin-Like-1 at the Muscle M-Band Provides a Strategy for Isoform-Specific Mechanical Protection.
in Structure (London, England : 1993)
Marsh RJ
(2018)
Artifact-free high-density localization microscopy analysis.
in Nature methods
Staszowska AD
(2018)
The Rényi divergence enables accurate and precise cluster analysis for localization microscopy.
in Bioinformatics (Oxford, England)
Poland SP
(2018)
Multifocal multiphoton volumetric imaging approach for high-speed time-resolved Förster resonance energy transfer imaging in vivo.
in Optics letters
Hirvonen LM
(2018)
STORM without enzymatic oxygen scavenging for correlative atomic force and fluorescence superresolution microscopy.
in Methods and applications in fluorescence
Rafiq NBM
(2019)
Forces and constraints controlling podosome assembly and disassembly.
in Philosophical transactions of the Royal Society of London. Series B, Biological sciences
Rafiq NBM
(2019)
A mechano-signalling network linking microtubules, myosin IIA filaments and integrin-based adhesions.
in Nature materials
Morton PE
(2019)
TNFR1 membrane reorganization promotes distinct modes of TNFa signaling.
in Science signaling
Rafiq N
(2019)
Publisher Correction: A mechano-signalling network linking microtubules, myosin IIA filaments and integrin-based adhesions
in Nature Materials
Rafiq NBM
(2019)
A mechano-signalling network linking microtubules, myosin IIA filaments and integrin-based adhesions.
in Nature materials
Pedrosa AR
(2019)
Tumor Angiogenesis Is Differentially Regulated by Phosphorylation of Endothelial Cell Focal Adhesion Kinase Tyrosines-397 and -861.
in Cancer research
Foxall E
(2019)
PAK4 Kinase Activity Plays a Crucial Role in the Podosome Ring of Myeloid Cells.
in Cell reports
Tsang CM
(2020)
Integration of biochemical and topographic cues for the formation and spatial distribution of invadosomes in nasopharyngeal epithelial cells.
in Acta biomaterialia
Pfisterer K
(2020)
FMNL2 regulates dynamics of fascin in filopodia.
in The Journal of cell biology
Bennett P
(2020)
The Axial Alignment of Titin on the Muscle Thick Filament Supports Its Role as a Molecular Ruler.
in Journal of molecular biology
Levitt JA
(2020)
Quantitative real-time imaging of intracellular FRET biosensor dynamics using rapid multi-beam confocal FLIM.
in Scientific reports
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 | 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 |
Title | LUMINESCENCE IMAGING APPARATUS AND METHODS |
Description | Luminescence imaging apparatus, methods and computer program products are disclosed. A time-resolved luminescence imaging apparatus (100A) comprises: an optical assembly (2) operable to generate an array of beams; a scanner (4A) operable to scan the array of beams with respect to a sample (8), along a single scanning axis; and a detector assembly (10) having an array of detector elements, adjacent detector elements being spaced apart by an inter-element gap, each detector element being operable to detect emissions generated by the sample (8) in response to the array of beams. In this way, different locations on the sample (8) may be simultaneously scanned and imaged by the detector assembly (10) in order to image multiple parts of the sample (8) simultaneously. Also, by scanning along a single scanning axis, the complexity of the scanner (4A) is significantly reduced and the speed of scanning is increased compared to scanners which have to scan in two dimensions, such as a traditional raster scan mechanism. |
IP Reference | US2020132976 |
Protection | Patent application published |
Year Protection Granted | 2020 |
Licensed | No |
Impact | Nano Clinical Ltd has an option to licence subject to contract which will be optioned in the next 12 months. |
Title | LUMINESCENCE IMAGING APPARATUS AND METHODS |
Description | Luminescence imaging apparatus, methods and computer program products are disclosed. A time-resolved luminescence imaging apparatus (100A) comprises: an optical assembly (2) operable to generate an array of beams; a scanner (4A) operable to scan the array of beams with respect to a sample (8), along a single scanning axis; and a detector assembly (10) having an array of detector elements, adjacent detector elements being spaced apart by an inter-element gap, each detector element being operable to detect emissions generated by the sample (8) in response to the array of beams. In this way, different locations on the sample (8) may be simultaneously scanned and imaged by the detector assembly (10) in order to image multiple parts of the sample (8) simultaneously. Also, by scanning along a single scanning axis, the complexity of the scanner (4A) is significantly reduced and the speed of scanning is increased compared to scanners which have to scan in two dimensions, such as a traditional raster scan mechanism. |
IP Reference | WO2019008342 |
Protection | Patent application published |
Year Protection Granted | 2019 |
Licensed | No |
Impact | Nano Clinical Ltd has an option to Licence which will be optioned in the next 12 months. |
Company Name | NANO CLINICAL LTD |
Description | Nano Clinical Ltd is an innovation driven company that develops paradigm-shifting instruments for high content imaging of functional molecular interactions. Our innovative approach to histological screening of protein-protein interactions in cancer biopsies aims to capitalize on a short-term, global, pharma driven biomarker service business to establish an artificial intelligence augmented diagnostic testing capability to improve treatment outcomes from cancer targeted therapies. The company will partner with clients and leading academic institutions to solve intractable biology questions and generate critical application data, which, if successful will drive the demand for instruments & kits. |
Year Established | 2020 |
Impact | The company is just formed and does not yet have any impacts |
Website | https://www.nanoclinical.com/ |
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 | 2018,2019,2021 |
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 |