Z Stack

Generally the thickness of a single focal plane will be smaller than that of the sample, for example when imaging cells with a high magnification high numerical aperture (NA) objective.  Thus, by recording images at different focal planes the entire sample volume can be rendered and visualized.  These “Z stacks” are generated by incrementally stepping through a sample using a focal drive.  The optimal “step size” can often be calculated by the image acquisition software, however sometimes in order to increase the rate of acquisition or decrease the amount of light exposure to the sample larger step sizes are employed.  Z stacks are usually required for deconvolution of epi-fluorescence microscopy data to remove the out-of-focus signal collected within each individual image.  Furthermore, Z stacks acquired through confocal microscopy represent a very straightforward means to analyze the entirety of a sample.