Development of live cell imaging from single cells to single molecules

Lead Research Organisation: University of Liverpool
Department Name: Sch of Biological Sciences


A major challenge in biology is to understand how cells recognize external signals and give appropriate responses. Now that the sequence of the human genome is complete it is important to assign functions to each gene and to identify the corresponding proteins that control key cellular functions. We pioneered the development of microscopy-based methods for the visualization and timelapse measurement of biological processes in single living cells. We have used natural light-emitting proteins from fireflies, jelly fish and fluorescent corals. Synthesis (expression) of these proteins causes mammalian cells to become luminescent (light emitting in the dark) or fluorescent (change the colour of light). By placing the gene that codes for a luminescent protein next to a promoter that controls a gene of interest, we can use luminescence from living cells as a way of measuring when the gene of interest is normally switched on and off. Fluorescent proteins have also been used to genetically label proteins of interest, so that the movement of the protein can be visualized in a living cell. White, See and Spiller have previously used timelapse fluorescence and luminescence microscopy to investigate cell decision making. We discovered that a set of important signalling proteins, called NF-kappaB, move repeatedly into and out of the nucleus of the cell, suggesting that cells may use proteins as timers to encode complex messages (like Morse Code). Only timelapse measurements in single living cells were able to see this. We now propose to develop simultaneous imaging of luminescence from two different colour firefly (green) and click beetle (red) proteins in single cells to track the expression of two different genes at the same time. We will also develop and apply new fluorescent tools for more accurate measurement of protein movement rates and protein stability in single cells. One approach will explore new uses for fluorescent proteins that have been engineered so that they can be optically switched between light-emitting and non-light emitting forms (highlighters). This will include improved measurement of whether and when two proteins of interest bind to each other in cells. Until now, it has been a major challenge to track when such protein interactions occur. Levy, Fernig and Brust have considerable experience in synthesis of very small metal (gold and silver) nanoparticles, which have been coated to make them more stable in water and hence in the interior of cells. We will develop generic methods to precisely couple these nanoparticles to any protein (or sequence of DNA) of interest. We will build a novel photothermal microscope to allow the visualization and tracking of single nanoparticles in single cells. We will target the nanoparticles to particular DNA sequences in the nucleus of living cells to allow the localization of genes of interest within the nucleus. We will also target nanoparticles to bind to the artificially tagged RNA of genes of interest. This will allow the immediate visualization of when RNA is synthesized (transcription) from the gene of interest with no delay. We will use this technology to measure the movement of proteins that bind to DNA, such as NF-kappaB proteins. These approaches will for the first time allow us to study how single proteins regulate transcription at single genes. The NF-kappaB signalling system will be used as a model, since we have a great deal of experience and interest in its regulation and excellent computer models that predict how it may work. This technology will have widespread applications in cell biology and systems biology. The Centre for Cell Imaging is an ideal site for this project, since it is a resource used by a large number of scientists in the North West and is well placed to transfer technology to other labs through training and conferences. We also have close links with pharmaceutical and instrumentation industries who will benefit from these developments.

Technical Summary

Non-invasive maging of biological processes in living cells has become an important tool in molecular and cell biology research. There is an increasing need for new multiparameter imaging technologies that can provide more quantitative measurement of a greater range of dynamic biological processes in single cells. The development of such improved technologies can provide a major contribution to systems biology, analysis of cell signalling, transcription and cell fate. The aims of this project are firstly to develop new technology based on our current expertise in luminescence and fluorescence imaging. Secondly, we will develop new bionanotechnology tools for the imaging and tracking of single molecules in cells. Specifically we will develop: 1) Dual imaging of transcription in single living cells; 2) Improved assays for quantitative measurement of protein translocation rates, protein half-life and the kinetics of protein interactions; 3) Single molecule imaging in living cells based on photothermal microscopy of biomimetic nanoparticles and 4) The application of multiparameter imaging to study single gene transcription in single cells. This work will be carried out by a multidisciplinary team from Molecular Cell Biology, Chemistry and Physics backgrounds. The nanotechnology part of the project will involve the building of the world's second photothermal microscope for single metal nanoparticle imaging. This will be the first instrument to be specifically designed for imaging of nanoparticles in living cells. The work is based on our existing expertise in cell imaging (White, See and Spiller) and nanoparticle development (Levy, Brust and Fernig). The work will project will contribute to our ongoing systems biology analysis of the important NF-kappaB system, which will be used as a model for application of the technology. In addition, the new technology will have generic applications in systems and cell biology.


10 25 50
Description Improvement of photothermal microscopy technology

Development of switchable FRET technology and its application to p105 (NF-kB subunitO processing

Improved dual luminescence imaging and combination with fluorescence imaging
Exploitation Route Continued collaboration with Zeiss to further develop the new technology. The technology developed in this project remains core to ongoing work and further outputs are anticipated.
Sectors Digital/Communication/Information Technologies (including Software),Education,Healthcare,Pharmaceuticals and Medical Biotechnology

Description Development of a set of microscopy tools and technologies that have underpinned developments in the Centre for Cell Imaging an Liverpool and the subsequent Systems Microscopy Centre in Liverpool. This work has resulted in improved photothermal and nanotechnology approaches in Liverpool. In Manchester the work with Zeiss has culminated in the development of dual luminescence / fluorescence imaging. This project strengthened that collaboration. New switchable fluorescent protein technology for FRET measurements
First Year Of Impact 2007
Sector Healthcare,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology
Impact Types Economic

Description An upright confocal microscope for multidisciplinary research
Amount £282,781 (GBP)
Funding ID BB/R014361/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 05/2018 
End 04/2019
Description Development of novel luciferases for real-time monitoring of protein secretion.
Amount £117,309 (GBP)
Funding ID BB/K013882/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 05/2013 
End 06/2014
Description Dynamics and function of the NF-?B signalling system
Amount £5,072,010 (GBP)
Funding ID BB/F005938/2 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 04/2008 
End 03/2013
Description Imaging of cellular dynamics from single molecules to tissues.
Amount £1,273,491 (GBP)
Funding ID MR/K015885/1 
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start 04/2013 
End 04/2017
Description Strategic Lola
Amount £4,160,524 (GBP)
Funding ID BB/K003097/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 05/2013 
End 04/2018
Description Temporal regulation of endocrine gene expression - timing in living cells and tissues
Amount £82,244 (GBP)
Funding ID 087960 
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 08/2009 
End 07/2010
Description Carl Zeiss 
Organisation Carl Zeiss AG
Country Germany 
Sector Private 
PI Contribution We have advised Zeiss on trends in bioimaging since 1996. We have provided new data and tested prototype equipment. We have spoken at Zeiss organised meetings. We have given them an opportunity to display Zeiss equipment at our training courses. We have organised symposia that have been supported by Zeiss. We have held expert discussion meetings to review microscopy trends that have involved senior Zeiss staff
Collaborator Contribution Zeiss have made a cash contribution to training courses (received) of £16,250. Zeiss estimate of total value of in-kind staff time for collaboration, training courses and other meetings (including visits of teams from Germany) ?25,000. In addition, Zeiss have also committed over £30,000 in cash and ~£80000 in in kind staff for future training meetings and collaborative visits. Zeiss helped to design the new Systems Microscopy Centre in Manchester and made a 45% discount (value ?350k) for the purchase of equipment in 2011. Zeiss are a formal MICA partner on both Liverpool and Manchester awards from the MRC/BBSRC New Microscopy Initiative. In the award to Manchester they have made a contribution of £614,314 in staff time, development costs and equipment contribution. This involves FCS (developed during this project), luminescence fluorescence imaging, light sheet microscopy and SOFI super-resolution imaging. More recently Zeiss have made a further contribution to our new clinical single cell centre. This includes over £400k in equipment discounts and £25k in cash contribution to training and symposia. Over the years the Zeiss contributions have included them helping us with public understanding of science exhibitions where they loaned equipment, provided support for professional poster preparation and used their delivery services to transport our exhibit materials and equipment to the exhibition venues. This included an exhibition in Buckingham Palace in 2006. Zeiss have sponsored 2-3 meetings per year in Manchester. In 2016 this included a session on light sheet imaging and a session on new confocal imaging technologies. In 2017 they have sponsored an image analysis daya and will sponsor a single cell biology workshop.
Impact Annual training courses MICA collaborative MRC grant MICA collaboration on new single cell centre The relationship with Zeiss has been two way. We have been given the opportunity to be early adopters f new technology and to feedback idease for improvement. We receive very favourable deals on microscope purchases and maintenance contracts. Specific areas of successful collaboration lie in improvements to higher throughput live cell imaging using the confocal microscopes; optimisation of truly dark microscopes for quantitative luminescence imaging and the development of FCS. Multiple workshops organised (2-3 per yeat)
Description Hamamatsu Photonics 
Organisation PMT Hamamatsu Photonics K.K.
Country Japan 
Sector Private 
PI Contribution We have advised Hamamatsu on trends in bio-imaging for 20 years. We have provided new data and tested prototype equipment. We have given them an opportunity to display their equipment at our training courses.
Collaborator Contribution They have loaned us equipment and provided privileged discounts for almost 20 years (in kind value well over £100k). They have advised us on new emerging technology. They have assisted by providing staff for our training courses (recent in kind value calculated as £50k. They have provided £10k in cash towards training courses since 2008. A further £4k in cash and £10k has been committed to future training courses.
Impact Annual training courses