Confocal Microscope for Centre for Cell Imaging
Lead Research Organisation:
University of Liverpool
Department Name: Sch of Biological Sciences
Abstract
Live cell imaging has emerged as a very important technology for the study of the functions of genes and cells. In the Centre for Cell Imaging we have established a facility that specialises in timelapse microscopy looking at the same cells as they change, over time periods from seconds to days. This work involves the use of fluorescent labels to follow biological processes. Our existing microscopes are ageing but are extremely heavily used by a very large number of scientists from different disciplines, Departments and Institutions. We need a new system to take advantage of improvements that have occurred since the last confocal microscope was purchased 6 years ago. Since then, electronics and detection capabilities have been improved substantially. During this period, we have developed new methods that allow us to perform many experiments in a single dish containing a 'cell array' which consists of spots of around one tenth of a millimetre which are placed on a glass surface next to each other. Each spot has about 50 cells on it which have had different genes (or drugs) introduced into them. The new microscope will allow us to look at more spots every day . The improved speed is mainly because the new microscope will spend less time automatically setting the focus as it moves from spot to spot. We will also connect this microscope to a new computer controlled laser (purchased separately) that can excite fluorescence using intense pulses of light, where each photon carries half the energy normally used to excite fluorescence. Compared to our previous system, this microscope will be far more flexible and powerful, since the intensity of light that can be shone onto the cells will be far greater and there is new equipment for more efficient detection of the signal. The microscope will be used to study a variety of processes including: the timing of protein movement in cells, protein stability and protein interactions and gene expression. Protein interactions have been difficult to study in timelapse experiments and it is becoming very important that we understand which proteins bind to each other and when and where they do this inside cells. The research objectives lie in a broad area of applied biology. We are interested in how the timing of signals may change when genes (and which genes) are switched on in cells and how this affects cell division and cell death. We have discovered that NF-kappaB, one of the most important signals in the cell moves into the cell nucleus repeatedly suggesting that the timing of movement may control cell functions. We want to study this further and to relate this to other important signalling systems in the cell such as p53, the 'guardian of the genome' a tumour suppressor protein that is changed in most cancers. The information that we get will be fed into computer models of these systems because they are so complicated that it is difficult to understand in any other way, exactly what is going on. We will also study related projects in the areas of neurobiology, endocrinology and glycobiology. The research that this equipment will support is substantially funded by BBSRC and also by other organisations. There is a strong record of collaboration with industry and excellent strategic support for the Centre for Cell Imaging from Liverpool University.
Technical Summary
The Centre for Cell Imaging has an established track record in pioneering the development and application of novel timelapse live cell imaging techniques. The facility at present consists of 3 widefield microscopes (mainly used for luminescence imaging) and 3 confocal microscopes. Our existing multiphoton microscope is housed in a darkened room for luminescence imaging, with the lasers outside the room to allow dual luminescence imaging. The current manually tunable multiphoton laser is therefore fibre-optically coupled to the microscope. This meant a compromise with incident light intensity compared to a direct laser connection. The microscope was purchased before direct descanned detectors became available. Therefore both illumination and detection efficiencies are lower than could now be achieved. Zeiss made available to us a Becker and Hickl fluorescence lifetime system. However, this ideally needs direct coupled multiphoton laser and direct detection. Since the last two confocal microscopes in the Centre were funded in 2000, we have concentrated on maximising the efficient use of time on the microscopes. The three confocal microscopes are used almost 100% for fluorescence and luminescence timelapse experiments, 7 days per week 24 h per day and time is allocated up to a month in advance. Through a DTI Beacon project we have developed live cell imaging of reverse transfected cell arrays as a method of increasing the number of parallel timelapse experiments that can be performed at the same time. We have substantially improved the computer facilities for data capture, handling and storage by establishing a local gigabit local area network and our own RAID storage. We have also written new automated timelape image analysis software. We request funds for a new LSM510 confocal microscope equipped for live cell imaging. Through an associated but independent purchase we will directly link the microscope to a computer controlled chameleon multiphoton laser.
Publications

Sillitoe K.
(2007)
Single-cell time-lapse imaging of the dynamic control of NF-kappa B signalling
in BIOCHEMICAL SOCIETY TRANSACTIONS

Cesbron Yann
(2010)
Intracellular Delivery and Fate of Peptide-Capped Gold Nanoparticles
in BIOPHYSICAL JOURNAL

Meley D
(2010)
p53-mediated delayed NF-?B activity enhances etoposide-induced cell death in medulloblastoma.
in Cell death & disease

Davies L
(2011)
PERP expression stabilizes active p53 via modulation of p53-MDM2 interaction in uveal melanoma cells.
in Cell death & disease

Featherstone K
(2011)
Pulsatile patterns of pituitary hormone gene expression change during development
in Development

Featherstone K.
(2010)
Prolactin Gene Expression Patterns Change during Pituitary Development and Are Influenced by Tissue Architecture.
in ENDOCRINE REVIEWS

Adamson AD
(2008)
Human prolactin gene promoter regulation by estrogen: convergence with tumor necrosis factor-alpha signaling.
in Endocrinology

Mullassery D
(2008)
Single live-cell imaging for systems biology.
in Essays in biochemistry

Ratnayaka A
(2007)
A dual Golgi- and mitochondria-localised Ala25Ser precursor cystatin C: an additional tool for characterising intracellular mis-localisation leading to increased AMD susceptibility.
in Experimental eye research

Featherstone K
(2011)
Pulsatile patterns of pituitary hormone gene expression change during development.
in Journal of cell science

Harper CV
(2008)
Dynamic resolution of acrosomal exocytosis in human sperm.
in Journal of cell science

Harper CV
(2010)
Dynamic organisation of prolactin gene expression in living pituitary tissue.
in Journal of cell science

Davies L
(2009)
P53 apoptosis mediator PERP: localization, function and caspase activation in uveal melanoma.
in Journal of cellular and molecular medicine

Semprini S
(2009)
Real-time visualization of human prolactin alternate promoter usage in vivo using a double-transgenic rat model.
in Molecular endocrinology (Baltimore, Md.)

Spiller DG
(2010)
Measurement of single-cell dynamics.
in Nature

Mullasserv Dhanva
(2007)
NF kappa B inhibitors promote cell death in both S and N type neuroblastoma cell lines
in PEDIATRIC BLOOD & CANCER

Harper CV
(2011)
Dynamic analysis of stochastic transcription cycles.
in PLoS biology

Thomas LW
(2012)
Serine 162, an essential residue for the mitochondrial localization, stability and anti-apoptotic function of Mcl-1.
in PloS one

Ashall L
(2009)
Pulsatile stimulation determines timing and specificity of NF-kappaB-dependent transcription.
in Science (New York, N.Y.)

Pennington SR
(2007)
Cell shape-dependent Control of Ca2+ influx and cell cycle progression in Swiss 3T3 fibroblasts.
in The Journal of biological chemistry

Moulding DA
(2007)
Unregulated actin polymerization by WASp causes defects of mitosis and cytokinesis in X-linked neutropenia.
in The Journal of experimental medicine
Description | Appointed to Pool of Experts for BBSRC Panels, 2012-2013. |
Geographic Reach | National |
Policy Influence Type | Participation in advisory committee |
Description | BBSRC Bioscience for Industry Committee (single meeting for presentation) |
Geographic Reach | National |
Policy Influence Type | Participation in advisory committee |
Description | BBSRC Panel: Enabling Theme 2, the Exploiting New Ways of Working Strategy Panel (ENWW) |
Geographic Reach | National |
Policy Influence Type | Participation in advisory committee |
Description | BBSRC Strategic Tools and Resources Development Fund (sTRDF) Panel |
Geographic Reach | National |
Policy Influence Type | Participation in advisory committee |
Description | External Reviewer for FRISYS-ZBSA (Zentrum fur biosystemanalyse), Freiberg, Germany, 26th May 2011. |
Geographic Reach | Asia |
Policy Influence Type | Membership of a guidance committee |
Description | German Virtual Liver Scientific Advisory Board (42m euros programme ) |
Geographic Reach | Asia |
Policy Influence Type | Participation in advisory committee |
URL | http://www.virtual-liver.de/wordpress/en/media/handling-biological-complexity-using-systems-approach... |
Description | Invited to attend Research Sponsors' Consultation Meeting (BBSRC &MRC) on Imaging for the biological and biomedical sciences, 14 May 2012. |
Geographic Reach | National |
Policy Influence Type | Membership of a guidance committee |
Description | MRC Microbiome |
Geographic Reach | National |
Policy Influence Type | Participation in advisory committee |
Description | MRC New Microscopy Initiative |
Geographic Reach | National |
Policy Influence Type | Participation in advisory committee |
Description | MRC Systems Immunology |
Geographic Reach | National |
Policy Influence Type | Participation in advisory committee |
Description | Member of BBSRC Research Equipment Initiative (REI) Review Panel |
Geographic Reach | National |
Policy Influence Type | Participation in advisory committee |
Description | Member of BBSRC Site visit to Imperial Centre for Systems Biology |
Geographic Reach | National |
Policy Influence Type | Participation in advisory committee |
Description | Member of BBSRC Site visit to Oxford Centre for Systems Biology |
Geographic Reach | National |
Policy Influence Type | Participation in advisory committee |
Description | Member of BBSRC Tools and Resources Development Fund Panel, Feb 2012. |
Geographic Reach | National |
Policy Influence Type | Participation in advisory committee |
Description | Member of BBSRC/EPSRC TDRI Panel |
Geographic Reach | National |
Policy Influence Type | Participation in advisory committee |
Description | Member of MRC Doctoral Training Panel |
Geographic Reach | National |
Policy Influence Type | Participation in advisory committee |
Description | Member of MRC Population and System Medicine Board, 1 April 2012 - 1 April 2014 |
Geographic Reach | National |
Policy Influence Type | Participation in advisory committee |
Description | Membership of BBSRC Interdisciplinary Systems Biology Strategy Committee |
Geographic Reach | Asia |
Policy Influence Type | Participation in advisory committee |
Description | Wellcome Trust/Wolfson Capital Awards in Biomedical Sciences Committee |
Geographic Reach | National |
Policy Influence Type | Participation in advisory committee |
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 | 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. |
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. |
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 |