The investigators are requesting instrumentation to establish a confocal microscope and 3D imaging analysis system dedicated to the observation and sophisticated 3D image analysis of living specimens. All the contributing investigators are experienced at various levels with advanced light microscopy methods as applied to their own research. The Principal Investigator is experienced in confocal microscopy, application of image analysis methods and supervising complex multiuser facilities. Eight of the 10 investigators have experience with the present Bio-Rad MRC 600 supervised by the PI. The proposed projects have grown out of experience with this system mainly on fixed specimens. The investigators' work in support of 6 NIH and 2 NSF grants has advanced to the point that they need to examine living specimens at fast frame rates and to analyze their data with sophisticated image processing software. Thus, a fast scan confocal attachment, inverted light microscope, robotic manipulator/injector and computer system are requested. The equipment will be integrated into a single set-up for optimized 3D imaging of living and photosensitive specimens, and for 3D quantitative analysis. Ten users, 5 in neurobiology and 5 in cell biology, will utilize the facility. In addition to needing faster scan rates, it is clear from our preliminary work with living specimens that we also need improved sensitivity. Our glial cultured cells survived for over 1,000 frames of laser exposure in a standard NORAN confocal microscope, but only for a maximum of 10 frames in our Bio-Rad. This increased sensitivity is also required for quantitative studies comparing different pools of enzymes and imaging single microtubules in fixed cells. The quantitative analysis will be based on our unique software that automatically segments, counts, traces and montages 3D image structures. However, due to their mathematics, the algorithms are computationally intensive requiring a new dedicated computer system.
