This proposal requests support for the acquisition of a high resolution light microscope known as Deltavision to an existing shared imaging resource, the Keck Imaging Facility, at the University of Washington. Deltavision refers to an integrated microscopy system which achieves high resolution of fluorescently labeled samples by mathematical deconvolution. The resolution obtained is the highest available by light microscopy (to 75nm point source detection), and the applications detailed by the users demonstrates a clear need for this resolution. Quantitation is excellent, because Deltavision offers a linear range of data acquisition and display 10-fold greater than confocal microscopy. In addition, low level UV light illumination is used to excite the sample, minimizing the effects of photobleaching which can mean loss of the signal altogether and/or death of cells being monitored with live cell recording techniques. These latter effects are significant drawbacks of confocal microscopy, due to the high energy lasers employed. Because a range of filters are used in combination with the UV light source, sampling is multiwavelength and unlimited by constraints such as laser lines. Finally, the microscope with its precise stage micromanipulator and the Deltavision software offers the best opportunity to reconstruct biological samples in three dimensions. NIH-funded users and other funded groups propose to use the microscope to visualize centromere structure in living cells, living mitochondria, collagen fibril structure, transcription units within cell nuclei neurotransmitter transport and secretion, the organization of the neuromuscular junction, and hair cell structure. This broad range of uses indicates the many applications of the instrument which will be available to other investigators through the Keck Center. The operation and management of the equipment, the internal advisory committee, and institutional support for Deltavision are described.
