Computational optical sectioning microscopy using convex projection theory with applications.

Abstract

The need to visualize and quantitatively study 3-D biological specimens close to their living state motivated the development of a computational optical sectioning microscopy (COSMic) technique. A specimen is imaged in 3-D by serial focusing along the optical axis. Distortions in image formation are alleviated and the resolution perpendicular and parallel to the optical axis are improved using the COSMic algorithm which uses convex projection theory to simultaneously satisfy prior knowledge about image formation, specimen, and noise statistics. Mathematical structures of various published algorithms used in computational 3-D microscopy are compared. Simulation tests indicated that the COSMic algorithm provides better quantitative results than other algorithms in the sense of reducing mean squared error between the actual and estimated specimens. COSMic is applied to cases where the level of fluorescence in the specimen represents a physiological parameter or a morphological feature. In this study, two issues which explore the effects of loud sound on hearing in mammals are considered. The first issue addresses minimization of errors in absolute and relative measurements of diameters of cochlear microvessels of guinea pig exposed to loud noise. COSMic is applied to the data as a preprocessing step to obtain extended-focus images. Microvessels are assumed to be contiguous, piecewise cylindrical fragments. A quantitative, sequential 2-D vessel tracking procedure is used to compare a set of prestored intensity profiles of ideal vessel fragments with the intensity profile of each vessel fragment in extended focus images. The diameter of the best-fitting ideal profile is taken as the diameter of the vessel fragment. Results indicate that the approach alleviates overestimation of absolute diameters and underestimation of relative diameter changes in microvessels. The second issue is the application of COSMic to overcome the limitations of blur and to quantitatively study phalloidin labeled actin filaments in normal and noise damaged cochlear stereocilia. Several acoustic overstimulation-related length and intensity changes can be observed in the labeled stereocilia after processing the acquired 3-D images with the COSMic algorithm. Such changes may be related to polymerization and depolymerization of actin filaments in stereocilia.