Once the focus function is calculated, the power-weighted average is used to calculate best focus. The power-weighted average has been shown to calculate best focus with low sensitivity to side peaks. Independent of the specimen generating the side peaks, it was observed that more careful inclusion of the highest frequencies near optical cutoff tended to decrease or eliminate the side peaks. 3 Exploration of the relationship between side peaks and the inclusion of lower frequencies was performed by analyzing the through-focus spectra. Images acquired well away from focus have visible frequency components only in the low end of the spectrum.
Higher frequencies are detectable as best focus is approached.
At best focus, frequencies near the optical cutoff are maximum. The lines extending through the spectrum of six of the images are experimental artifacts, which are being investigated. The high frequency components that extend through the entire focal range are probably caused by optical surface imperfections. Side peaks occur in the focus function curve when frequencies that undergo contrast reversals are included in the calculations. The lowest frequency band in Fig.