Filtering out contrast reversals for microscopy autofocus

Autofocus functions based on measurement of image resolution appear to be p recise and robust for biological microscopy. However, the through-focus res ponse of these functions previously exhibited unwanted local maxima, or sid e peaks. Here we report theoretical and experimental studies showing that s ide peaks are mainly a result of contrast reversals inherent in optical sys tems at mid-range frequencies. These contrast reversals are not present in frequencies near optical cutoff. Contrast reversals thus Limit the lower cu toff for resolution measurement filters, whereas signal-to-noise Limits the upper cutoff. These improved bandpass design criteria led to sharp, unimod al autofocus responses for all tested microscopy specimens. (C) 1999 Optica l Society of America OCIS codes: 070.2590, 110.4850, 170.5810.