Confocal Microscopy is now an essential, and widely used tool for biological research. Particularly valuable is its ability to image thick sections of intact tissue. A compelling need in this field is to improve the imaging depth, as well as quantitative accuracy, and minimize the possibility of missing important structures/phenomena due to attenuation of the imaging signal. Previous methods work by amplifying the signal from deeper parts of the specimen. In these methods, amplification of noise is an unavoidable artifact. We describe a novel method that not only corrects confocal stacks for attenuation without noise amplification, but also enhances the achievable imaging depth. Interestingly, it does not require explicit estimation of the extinction factor, It relies on a synergistic combination of specimen preparation and image reconstruction algorithms.
The key idea is to image the specimen from two angles (Fig. 1), separated by 180° and then to reconstruct the complete thick image using computational methods.