Laser scanning microscopy is playing a major role in visualization of biological structures and processes. However, as images are degraded due to blurring, noise, and color shifts, quantitative interpretation of confocal images can be difficult. In this chapter, we detail a procedure that involves acquisition of high-resolution confocal image stacks in tissue sections and the subsequent deconvolution process. Data generated using these methods can be used for reliable quantification of cell biological and tissue interactions, e.g., colocalization analyses or 3D reconstructions.