The tilt axis angle is determined by running one iteration of tracking with a range of tilt axis angles, and choosing the angle giving the smallest residual. The fiducial marker radii for tilt series taken at different magnifications were (A) 10 pixels (54500x), (B) 7 pixels (38500x), and (C) 5 pixels (26000x). The width of the inverse peak expressed as the sigma coefficient of a Gaussian curve fitted to the profile (broken lines in A–C), is a function of the marker radius (D), determining how accurately the tilt axis angle must be specified for successful tracking.