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
Krstajic N
(2015)
0.5 billion events per second time correlated single photon counting using CMOS SPAD arrays.
in Optics letters
Krstajic N
(2015)
256 × 2 SPAD line sensor for time resolved fluorescence spectroscopy.
in Optics express
Poland SP
(2015)
A high speed multifocal multiphoton fluorescence lifetime imaging microscope for live-cell FRET imaging.
in Biomedical optics express
Rafiq NBM
(2019)
A mechano-signalling network linking microtubules, myosin IIA filaments and integrin-based adhesions.
in Nature materials
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
Klapholz B
(2015)
Alternative mechanisms for talin to mediate integrin function.
in Current biology : CB
Marsh R
(2018)
Artifact-free high-density localization microscopy analysis
in Nature Methods
Marsh RJ
(2018)
Artifact-free high-density localization microscopy analysis.
in Nature 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)
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
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 | 05/2016 |
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 | 08/2016 |
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 |
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 | Nanoclinical |
Description | Nanoclinical develops cancer treatments through the use of microscopy technology. |
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 |