Schematic of the light distribution in a multiple pinhole confocal microscope. (Top left) A vertical slice (i.e., x–z plane) of the distribution of illumination near the focal plane for a single spot of illumination with an objective lens of NA = 1.2. (Bottom left) The corresponding fluorescence emission with the acceptance angle of the objective superimposed. (Right) The overlap of neighboring illumination cones and acceptance regions, with a spacing between adjacent spots/pinholes of d. This limits the region of confocal behavior—that is, the ability to exclude light from out-of-focus planes—to a narrow band on either side of the focal plane. Out-of-focus light that originates from within the confocal region is excluded by the pinholes, but fluorescence coming from more distant planes is accepted, thus limiting performance in very thick samples.