Filtering out contrast reversals for microscopy autofocus.
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.
Oliva
M A
MA
Department of Bioengineering and Institute for Biomedical Engineering, University of California, San Diego, La Jolla, California 92093-0412, USA.
Bravo-Zanoguera
M
M
Price
J H
JH