In microscopy, the focus can be implied from image parameters, such as the contrast or intensity, that vary with the position of the objective’s plane of observation in the sample and the level of detail at a given plane. However, an algorithm that tries to detect (and maximize) such values will only return an axial position that corresponds to a plane of interest if these coincide (which is typically the case with (thin) samples with a singular discrete plane of detail).

This becomes increasingly difficult with higher numerical aperture (NA) lenses, thicker samples, and less pronounced levels of detail (modulated as change in contrast or intensity in the resulting image). Hence, SWAF is not to be understood as a focus finder, but can be used as a method for reliably searching over a given axial range and locating such a plane in a sample. Thus, although not all samples and imaging conditions will be appropriate, SWAF is an approach that allows a useful detection of a focus plane as a start for further imaging activities.