Depth
of Field
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Depth
of Field
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Depth of field (DOF) represents that portion of the specimen which is in focus. Note that it becomes "thinner" as the NA increases and "thicker" as the wavelength increases. DOF is important when determining the increment, or step size, setting before starting a z-series. If structures at the limit of z resolution need to be distinguished, then increment steps smaller than the DOF should be used. The principle here is the same as for optimizing lateral resolution. In other words, to see an object, it must be represented by at least two pixels (or steps, in this case). Thus, "oversampling" is done (think of it as overlapping focal planes), where the increment steps are smaller than the DOF. If fine axial detail is not required, increment steps equal to or greater than the DOF may be used. This provides for adequate image quality while minimizing photobleaching and computer memory. To calculate (using
the formula below) the depth of field for an objective/dye combination
not shown in the table, |
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