Z axis (astigmatism) - UU-cellbiology/DoM_Utrecht GitHub Wiki

This submenu deals with the calculation of axial (Z-axis) localization position for the astigmatic point spread function (PSF). It is usually introduced by cylindrical lens. For the historical background and applications of this method, check these references [1],[2],[3]. In short, depending on the defocus value (distance in z from "true" focal plane), astigmatic PSF changes its shape from symmetrical Gaussian to asymmetrical, i.e. being more elongated either horizontally or vertically:

example of astigmatic PSF

A typical workflow for this is the following: first you would need to Make Z calibration (click for instruction) using images of beads. This calibration will be used to Calculate Z values for the XY localizations. In addition, for multiple calibrations/setups, you can Save/Load or Show Z calibration.

NB: After making a calibration, it will be stored in "ImageJ registry" and so further it will be used for Z position calculation. Keep in mind, that after you restart ImageJ/FIJI, the transform/calibration will still be there. For example, when you run Calculate Z values, the plugin will not ask you for the calibration file. The current stored version of the calibration will be used for registration. But again, you can look at it, save it to a disk or load "to registry" another calibration file.