Enhanced three-dimensional reconstruction from confocal scanning microscope images. II. Depth discrimination versus...

The enhanced depth discrimination of a confocal scanning optical microscope is produced by a pinhole aperture placed in front of the detector to reject out-of-focus light. Strictly confocal behavior is impractical because an infinitesimally small aperture would collect very little light and would result in images with a poor signal-to-noise ratio (SNR), while a finite-sized partially confocal aperture provides a better SNR but reduced depth discrimination. Reconstruction algorithms, such as the expectationmaximization algorithm for maximum likelihood, can be applied to partially confocal images in order to achieve better resolution, but because they are sensitive to noise in the data, there is a practical trade-off involved. With a small aperture, fewer iterations of the reconstruction algorithm are necessary to achieve the desired resolution, but the low a priori SNR will result in a noisy reconstruction, at least when no regularization is used. With a larger aperture the a priori SNR is larger but the resolution is lower, and more iterations of the algorithm are necessary to reach the desired resolution; at some point the a posteriori SNR is lower than the a priori value. We present a theoretical analysis of the SNR-toresolution trade-off partially confocal imaging, and we present two studies that use the expectationmaximization algorithm as a postprocessor; these studies show that a for a given task there is an optimum aperture size, departures from which result in a lower a posteriori SNR.