The point spread function (PSF) of the scanning electron microscope (SEM) can be determined using a recently developed nanoparticle calibration method. Many parameters are involved in PSF determination and introduce a previously unstudied amount of uncertainty into the PSF size and shape. Signal type, support material thickness, reference particle size, PSF smoothing (K), and background correction were investigated regarding their effect on the PSF. Experimental data were complemented by CASINO simulations. Differences in detector position between the observed particles and the method's simulated reference particles caused shifting between secondary electron PSFs and backscattered electron PSFs. Support material thickness did not have a practical effect on the PSF at the tested voltages. Uncertainty in reference particle size varied the PSF full width at half maximum (FWHM) within ±0.7 nm at 2σ, with virtually no uncertainty in some cases. K and background correction within a reasonable range of values resulted in PSF FWHM differences within ±0.9 nm, except at 2 kV for K with an upper bound of ±1.9 nm due to increased noise. Tailoring K and background correction case-by-case would result in smaller differences. The interconnection of these parameters may help in future efforts to calculate their best selection.