Cellular Module

University Of Washington, Seattle WA

Investigators

Linked publications, trials & patents

Abstract

The Morphometry/lmaging module was our most widely and heavily used module in the last funding period. Under its new name. Cellular Biology Services and Shared Instrumentation, it will continue to provide key resources that will be heavily utilized. In 2009, we purchased an Olympus FV1000 confocal microscope in order to meet the growing needs and new directions of NEI-funded investigators. The availability of fluorescent proteins with distinct spectral properties (e.g. cyan, green, yellow, red) has recently increased substantially, enabling investigators to colorize different cell types in the same retina, an approach that facilitates studies of cell-cell interactions. Importantly, fluorescent proteins are more photostable and label live cells. Several NEI-funded investigators have projects that involve labeling and visualization of cells (Wong, Reh, Brockerhoff, Clark, Chao, Neitz). The Olympus FV1000 confocal microscope was acquired because it has the appropriate combination of lasers to specifically excite cyan, green, yellow or red fluorescent protein. Currently, this confocal microscope is central to NEI-funded UW projects investigating (1) retinal function (Deeb, Detwiler, Neitz, Van Gelder), (2) retinal biology in development, disease and regeneration (Brockerhoff, Chao, Clark, Hurley, Reh, Wolf, Wong) and (3) lens organization and development (Clark). Moreover, support from this module enabled investigators to utilize multiphoton microscopy that has been, and continues to be, critical for their in vivo imaging projects in zebrafish (Brockerhoff, Hurley, Clark) and mice (Reh, Wong). Due to the limited resolution of light microscopy, there is increasing need to correlate ultrastructure to observations from light microscopy. This is facilitated by conversion of fluorescent protein labeled cells and tagged proteins into an electron dense material using protocols based on immunolabeling for GFP. Such protocols are, however, not readily transferred to visual structures, including the retina. Mr. Parker, an experienced electron microscopist, is working closely with Dr. Wong to develop protocols that will allow excellent visualization of labeled fluorescent protein and good preservation of the tissue. The benefits of the Cellular Module establishing this approach are far-reaching. For example, the Brockerhoff lab is interested in connectivity of specific types of photoreceptors labeled in live zebrafish, and the Wong lab has several mouse transgenic lines in which neurotransmitter receptors are tagged with YFP, but their localization to synapses need to be ascertained. Moreover, this 'GFP-EM' technique will open new avenues for investigators who need to establish the detailed structure and connectivity of cells identified by light microscopy in normal and diseased conditions.