Methods for imaging thick specimens: confocal microscopy, deconvolution, and structured illumination

When a thick specimen is viewed through a conventional microscope, one sees the sum of a sharp image of an in-focus region plus blurred images of all of the out-of-focus regions. High background, scattering, and aberrations are all problems when viewing thick specimens. Several methods are available to deal with these problems in living samples. These methods can be grouped into three classes: primarily optical (e.g., confocal microscopy, multiphoton microscopy), primarily computational (e.g., deconvolution techniques), and mixed (e.g., structured illumination) approaches. This article describes these techniques, which make it possible to see details within thick specimens (e.g., the interiors of cells within living tissue) by optical sectioning, without the artifacts associated with physically sectioning the specimen.