Real-time confocal 4D imaging of fast and long-term processes in living tissues and cells

The spatiotemporal analysis of processes at the cellular and molecular level in living material has experienced an enormous progress, especially with the introduction of fluorescent protein tags (e.g. GFP). 4D image analysis with GFP fusion proteins (either alone or combined with spectral variants of GFP) is an intensely pursued research avenue, since it can provide novel insight in the behavior of proteins and other molecules and in the organization of biochemical processes. 4D image-analysis in living tissues and cells demands the need of sophisticated optical sectioning and imaging devices. In the present proposal, which is a joined initiative by 12 departments within the Erasmus university Medical Center Rotterdam (EMCR), we request NWO support for the acquisition of a Perkin-Elmer Ultraview LCI confocal system (a spinning disk confocal microscope), for fast and long-term 4D imaging of living tissues and cells. The advantage of the spinning disk microscope over the standard confocal laser-scanning microscope (CLSM) is the high scanning speed and the low amount of photobleaching and phototoxicity. This makes it possible to scan fast processes in living tissues and cells, which can survive for longer periods of time. These properties make the spinning disk microscope an indispensable research system complementing the standard CLSM for biological and medical research. We envisage using the system in three major experimental set ups: 1) analysis of (micro)vascular processes in vivo by intravital microscopy, i.e. vascular targeting, gene therapy of endothelial cells, tissue and organ development, 2) the analysis of transport processes in neurons, including measurement of fast cytoskeletal processes and vesicular transport, and 3) imaging of nuclear dynamics, as well as a number of related studies as outlined below. At the EUR CLSMs are present, with which time-lapse imaging studies are performed. Acquisition of this equipment has provided the EUR with a competitive advantage over other groups and resulted already in important publications in top journals. For analysis of processes in living tissues an advanced dorsal skinfold window model has been developed both in mice and rats (according to Dr R.K. Jain, Harvard Medical School), which enables examination of the kinetics of vascular effects, pharmacodistribution, and targeting in tumor or inflamed tissue. Next to that, developmental processes in healthy tissue are studied. In this model high numerical aperture objectives are used. The introduction of an eNOS-GFP transgenic mouse, expressing GFP in endothelial cells, allows imaging of processes in, or interaction with, the endothelial lining and single endothelial cells. The EUR has acquired a leading role with respect to the use of animal models for examination of gene therapy, tumor responses, drug delivery, and vascular processes. One of the other main research themes at the EUR focuses on neuronal function in health and disease, in particular the role of cytoskeleton and organelle transport. We have generated a number of mouse model systems for neurodegenerative and neurodevelopmental disorders. In addition, several groups have generated fluorescently tagged proteins with which intracellular dynamics can be investigated. Although CLSM is highly suitable for the study of microtubule dynamics in living non-neuronal cells (see example movies, www.eur.nl/fgg/endov/NWO), we have found that similar measurements in neurons require an even more sensitive system, in combination with faster imaging. We have devised a logistic set up which enables described methodologies to be applied to one Ultraview system. Thus, all participating departments can use the equipment. Installation of the Ultraview LCI confocal system will, to our opinion, have a major impact on biomedical research at the EMCR and will emphasize the leading role of this research center at the national level in 4D image analysis. The requested equipment in this setting is unique for the Netherlands.