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Updated: Jul 7, 2026

Simultaneous Multicolor Imaging of Biological Structures with Fluorescence Photoactivation Localization Microscopy
Published on: December 9, 2013
Filtering out contrast reversals for microscopy autofocus
M A Oliva1, M Bravo-Zanoguera, J H Price
1Department of Bioengineering and Institute for Biomedical Engineering, University of California, San Diego, La Jolla, California 92093-0412, USA.
Abstract:
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.
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