Confocal scanning microscopy (CSM) is widely used for three-dimensional visualization of fixed specimens. However, living specimens are often damaged and/or bleached by the high intensity excitation light required by CSM. Moreover, temporal changes in a living specimen are often too rapid to be imaged by a CSM. This has severely limited the applications of CSM to the imaging of living specimens. The current technique of choice for imaging living specimens is computational optical sectioning (COSM), which has a much greater light-collecting capability and permits rapid image collection with less photobleaching, but suffers from severely degraded resolution along the optical-axis. This degradation in optical axis resolution can be partially compensated for via computationally intensive restoration methods. Partially confocal scanning microscopes (PCSMs) allow faster image recording at the expense of degraded optical-axis resolution. The long- term goal of the research proposed here is to use computational methods to recover this lost optical-axis resolution. With the combination of PCSM and computational methods, it will be possible to image living specimens at resolutions comparable to or better than those of strictly confocal microscopes and with computation times much shorter than those required by COSM of non-confocal images. This goal will be achieved as follows: First, the two available PCSMs, a rotating disk PCSM and a single-slit PCSM, will be characterized by deriving and validating a theoretical point spread function for each. Potential limitations in the design of these microscopes will be determined with a gold to improving their performance. Second, restoration methods developed for COSM will be applied to PCSMs and the gain in performance that they afford will be characterized. Third, in collaboration with colleagues at Washington University, the design of a rotating-disk PCSM will be improved using information gained in the first specific aim. Finally, in order to achieve these goals, the rotating disk PCSM must be modified for precise position control over the three axes (x, y, and z).
