MICA: Imaging of cellular dynamics from single molecules to tissues

Microscopy has been a key tool for giving insights into the structure and function of cells and tissues. The use of microscopy depends on achieving appropiate labelling/contrast of the molecules/structures of interest so that they can be visualised. In the past 20 years there have been enormous developments in microscopy. These have included the use of naturally glowing enzymes and proteins to visualise cellular processes. In addition, new "super-resolution" microscopes allow smaller structures to be visualised than ever before.
This project will build upon our established track record in timelapse microscopy for the generation of cell movies of biological processes of interest. In the new Systems Microscopy Centre, we have state-of-the-art facilities and air-conditioned rooms specifically designed for microscopy experiments to generate movies of cells. We use the enzyme luciferase that makes fireflies glow to make cells glow (bioluminescence) when a chemical substrate is added. We also use a large number of different fluorescent proteins that glow in different colours when they are illuminated with different coloured light (fluorescence). The project is a close collaboration with the leading microscopy company Carl Zeiss, who will loan equipment which later will be purchased by the University once it has been optimised for the scientific applications.
In this project we will combine bioluminescence and fluorescence so that different biological processes can be measured at the same time in single cells. This will allow us to watch cell signal timing at the same time as watching whether and when a gene becomes switched on to make a protein.
We will also study a new super-resolution microscopy technique called SOFI where single molecules are located by watching the flickering in their light emission. This requires mathematical analysis to identify the exact localisation.
Finally, we will use a microscopy method called "SPIM" that allows 3-dimensional biological structures to be studied. The principle is that a sheet of light is shone through the biological sample and then light emission by fluorescence is detected at 90 degrees to the plane of the light sheet. Often cells in the lab are cultured on glass which is clearly different to what normally happens in the body. Ultimately we hope to improve SPIM and to combine it with bioluminescence and the SOFI super-resolution approach.
The project involves a large number of researchers with interests in a range of normal biomedical processes and diseases. Examples include inflammation, cancer, cardiovasular disease, and studies of how to grow new tissues from stem cells and the processes of normal embryo development. This project will act as a focus for future microscopy developments in Manchester University.

The aim of the project is to enhance synergistic interactions between microscopy technology developers and users in Manchester. Following the recruitment of the PI, M. White & D. Spiller to Manchester, we established a new Systems Microscopy Centre (SMC) to focus on development and exploitation of live cell imaging. We believe that microscopy developments depend on 1) biological developments and probes; 2) new microscopy instrumentation and 3) software development and mathematical analysis.

Here, we plan a close collaboration with the leading microscopy company Carl Zeiss to develop new technologies over a 4-year period. The aim is to ensure that relevent skills and resources are fully ultilised to tackle biomedically important problems.

We propose development of 3 novel technologies.
1. Automated confocal fluorescence and bioluminescence microscopy. These imaging modalities have not previously been combined automatically in timelapse microscopy. (We previously used a manual strategy that led to a major publication but was labour intensive). The new microsope would be developed in collaboration with Zeiss and we will purchase the optimised system at the end of year 1.
2. Super-resolution fuctuation imaging. The great advantage of this technique is that it involves low illumination leading to less photo-toxicty than some other super-resultion approaches. With fast imaging and sophisticated data analysis, SOFI offers promise for timelapse super-resolution imaging in living cells.
3. We will access new Selective plane illumination microscopy (SPIM) technology from Zeiss (working with C Power, a Zeiss staff-member who recently relocated to UK, after managing the Zeiss SPIM development team for 6 years. Zeiss will loan a prototype system and we will purchase an optimised system in year 3. We will also investigate the potential for luminescence tomography and SPIM.
There is an active community of multidisciplinary users for these technologies in Manchester

The application of quantitative microscopy offers great potential for a better understanding of cell signalling and decision-making pathways. This in turn creates the opportunity for the identification of better drug targets and more efficacious modes of treatment of disease. This application is relevant to important processes in human and animal disease. For example, in inflammation and innate immunity, NF-kappaB is a critically important regulator. This project is important for applied research and is of relevance to healthcare and to the pharmaceutical industry.

In this respect several of the applicants have ongoing collaborations with AstraZeneca, GSK and other companies. Dr John Unitt from AstraZeneca wasa collaborator for several years and a co-author on several of our papers including two Science papers. All the pharmaceutical companies are looking for developments in systems biology and systems medicine that prove its utility for new drug target identification and validation. We will at all stages in this project consider whether the results are of commercial value and will seek to establish specific collaborations with pharmaceutical companies when appropriate.

The major technologies being used in this project are based on microscopy. This is an important and currently growing area. We have close relationships with instrumentation companies and in particular with Carl Zeiss and Hamamatsu Photonics with whom MW has collaborated for 16 and 20 years respectively. This involves the loan and testing of equipment and the exchange of ideas, for new developments in microscopy and detection. Both companies (outside the present project) currently sponsor support for an annual nicroscopy training course. The companies provide speakers for the course from Germany and Japan, the loan of demonstration equipment and financial support. The current project establishes an even stronger collaboration with Carl Zeiss that will lead to close and regular interactions with the company

The use of microscopy generates movies and images that are colourful and visual. They represent an excellent resource for the development of public understanding of science. MW has given lectures at Public Understanding of Science meetings (e.g. ASE lecture in 2008). In 2006 a group from the CCI led by MW and DS, presented an exhibit entitled "The Language of Cells" at the Royal Society exhibitions in London, Glasgow and at Science Day at Buckingham Palace. The whole group are very keen apply to exhibit at future Royal Society exhibitions and this project may well give us a useful theme from which to develop a new exhibit. This offers a specific opportunity and we will seek others through talking to schools and other groups. When publicity of outcomes from this project are important, we will engage with the University of Manchester Press Office to coordinate this. We have good experience of media publicity and have previously worked with funding body and University Press offices in publicising high impact publications.