Although most images captured with the microscope are two-dimensional with lateral coordinates in the plane perpendicular to the optical train, the fact remains that most biological materials (including cells, tissues, and whole organisms) are highly three-dimensional. The emergence of laser scanning confocal microscopy and related techniques, including spinning disk, structured illumination, aperture correlation, total internal reflection, multiphoton, deconvolution, and superresolution, has enabled investigators to obtain thin slices from thick specimens by removing the contribution of out-of-focus light in each image plane, rather than actual physical sectioning of the specimen. Referred to as optical sectioning, the resulting image planes provide a high level of contrast and permit three-dimensional reconstructions by computationally combining image data from a collection or stack of images.