Confocal scanning microscopes (CSM) are now widely used for three- dimensional (3D) visualization of fixed specimens. However, living specimens are often damaged and/or bleached by the high intensity laser excitation light required by the CSM. A common solution is to open the confocal aperture slightly and thus allow more light to reach the detector. A larger confocal aperture, however, accepts also more out-of- focus light and degrades the depth resolution of the CSM. The lost resolution can be recovered using computational deconvolution algorithms. Even the strictly confocal microscope benefits from the increased resolution that deconvolution algorithms provide. Most of the modem CSM's come with a host computer that digitizes 3D stacks of confocal images. Despite the availability of a computer, deconvolution of confocal images has not been widely used. One of the key reasons is that most deconvolution algorithms require accurate knowledge of the point-spread- function (PSF) that describes the microscope. In modern CSM's, the measurement or computation of the PSF is difficult at best. In these cases, it is necessary to estimate the PSF and the specimen fluorescence distribution simultaneously from the image, an approach called blind deconvolution (BD). Current methods for the BD are slow because they estimate the PSF point by point. The long term goal of the research proposed is to provide faster and more robust computational deconvolution algorithms that do not require measuring or computing the microscope's PSF. To achieve this goal, a mathematical-physical model for the PSF is used that depends on a small number of unknown parameters, then estimation methods will be derived to obtain the values of these parameters together with the specimen fluorescent distribution. Although the long term goal is BD of partially confocal images, nonconfocal microscope users will also benefit from the results of this research.
