A spinning disk confocal microscope for live cell imaging in plant animal and fungal cells.

Robert Hooke in 1665 was the first to introduce the term 'cell' when he was viewing the 'boxes' he saw in slices of cork using one of the earliest optical compound microscopes (two lenses: an objective lens and an eye piece) that he developed. He probably didn't quite realise the significance of this discovery, as it was only when it became apparent that the great majority of organisms are composed of cells that Cell Theory was born. Cell Theory, first proposed by M.J. Schleiden and Theodore Schwann in 1839, states that cells are of universal occurrence and are the basic units of an organism. This theory in still undisputed, although in those days rivals tried. Over 300 years of microscope improvements have led to fascinating discoveries of how cells function. Electron microscopes use beams of electrons rather than light and can magnify by hundreds of thousands of times. However the material being examined although fixed in time is dead. Light microscopes have evolved with the development of laser technology, imaging solutions, computer hardware/software and the use of fluorescent biological/chemical probes. One of the greatest developments in the last few decades has been the confocal laser scanning microscope, which allows the user to generate 3D images of fixed material and short live cell movies in one plane. With the development of confocal microscopy have come parallel developments/improvements. One such development is the spinning disk technology coupled with laser illumination, and high resolution imaging allowing the user to visualise fluorescent probes in cells in 3D with time (4D) i.e in living cells. To understand the role and function of particular constituents of a cell these features are a desirable advantage to a scientist.

In the projects outlined here, we describe how the SDCM properties are fit for our purpose; why it is important for us to have low phototoxicity and photobleaching so that we can keep our samples alive and visualise them for longer periods. Importantly, the speed of acquisition with the SDCM will allow visualisation of rapid events, as this is not dependent on the time taken to mechanically scan the sample, as it is with a CLSM. This feature also allows us to generate data sets for 3D monitoring over time (4D) so that organelle/protein localisations can be visualised. Moreover, with Andor Technology as our partner, we will be a test site for their cameras and as a result we will have access to all their latest developments. In our projects we will visualise cytoskeletal and nucleoskeletal protein dynamics using existing and novel sets of associated proteins (Hussey, Goldberg, Hutchison, Maatta, Quinlan) and intra-cellular membrane dynamics (Goldberg and Hutchison). We will generate data sets for modelling by members of our Mathematics Department who are particularly interested in microtubules as a self-assembly system (Hussey). Signalling mechanisms will also be visualised, particularly the second messengers Ca2+ and reactive oxygen species (ROS) (Knight) and proteins that are effectors of these pathways. Proteins involved in auxin and ethylene signalling pathways will also be assessed (Lindsey) and we will test the feasibility of using new fluorophores developed and being developed in our Chemistry Department for studying enzyme dynamics in vivo (Roberts). Stem cell growth and differentiation in culture and its interaction with other cell types using GFP-positive lineages will also be monitored (Przyborski, Hutchison). The School of Biological Sciences at Durham has an Imaging Centre with dedicated staff. This proposal outlines how an SDCM will be used and maintained and how this will significantly enhance our research capabilities and our research infrastructure.