A multiuser confocal laser scanning microscope for live and 3-D imaging of craniofacial cellular dynamics

Birth defects, cancer, regeneration and degeneration are all processes that can be better understood by the study of normal development of embryos. Recent advances in the field have shifted the focus from individual genes to complex regulatory cascades that operate in diverse sub-cellular compartments, between cells and between tissues. This, in turn, requires the study of the distribution of gene products (proteins) and their biological effects at a very fine scale: the scale of individual cells and even within cells. Fortunately, this is facilitated by new types of microscope. The one being requested in this application is a confocal laser scanning microscope that, by illuminating only one tiny spot at a time within a specimen (but scanning the illuminating laser beam progressively across and through it), eliminates most of the glare and fuzz of conventional microscopes. Such instruments enable images from deep within tissues to be gathered and different colours that identify different proteins to be detected simultaneously. The department requesting this instrument does not have its own confocal microscope, and several members of the department currently have to go to remote sites to collect these crucial images. The provision of the requested instrument will thus result in greatly increased efficiency in using Research Council and other funds for project work.

Twelve different project areas in the Department of Craniofacial Development at King's College London require significant use of laser-scanning confocal immunofluorescent imaging. The high spatial resolution, optical sectioning and multispectral capabilities of this type of instrument are needed for three main types of imaging application. These are (1) the analysis of multiple lineage and differentiation markers at cellular resolution in fixed material; (2) the tracking of cell trajectories and motile behaviours in real time in two- and three dimensions; and (3) high resolution subcellular analysis of protein localisation for the study of cell polarity, signal transduction and cytoarchitectural changes during development. Details of each of the relevant projects illustrate the diversity and substance of the proposed uses, and show that uses of out-of-department instruments are now rapidly becoming unsustainable. An instrument configured for these multiple uses offers substantial increases in efficiency and capability, especially because it enables analysis of delicate, experimentally manipulated embryonic material on-site.