Autofocus functions based on measurement of image resolution appear to be precise and robust for biological microscopy. However, the through-focus response of these functions previously exhibited unwanted local maxima, or side peaks. Here we report theoretical and experimental studies showing that side peaks are mainly a result of contrast reversals inherent in optical systems at mid-range frequencies. These contrast reversals are not present in frequencies near optical cutoff. Contrast reversals thus limit the lower cutoff for resolution measurement filters, whereas signal-to-noise limits the upper cutoff. These improved bandpass design criteria led to sharp, unimodal autofocus responses for all tested microscopy specimens.
© 1999 Optical Society of America
Michael A. Oliva, Miguel Bravo-Zanoguera, and Jeffrey H. Price, "Filtering Out Contrast Reversals for Microscopy Autofocus," Appl. Opt. 38, 638-646 (1999)