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Simultaneous Multicolor Imaging of Biological Structures with Fluorescence Photoactivation Localization Microscopy

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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.

|February 29, 2008

Summary

Accurate autofocus in biological microscopy was hindered by side peaks. New bandpass criteria, addressing contrast reversals, create sharp, reliable autofocus responses for all specimens.

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Area of Science:

Optical microscopy
Image processing

Background:

Autofocus functions in biological microscopy rely on image resolution measurements.
These functions often exhibit undesirable local maxima (side peaks) in their through-focus response.

Purpose of the Study:

To investigate the cause of side peaks in autofocus functions.
To develop improved autofocus algorithms for biological microscopy.

Main Methods:

Theoretical and experimental studies were conducted.
Analysis focused on contrast reversals in optical systems at mid-range frequencies.
Bandpass design criteria were optimized based on optical cutoff and signal-to-noise ratio.

Main Results:

Side peaks are primarily caused by contrast reversals at mid-range frequencies.
Contrast reversals are absent near the optical cutoff frequency.
Optimized bandpass filters established lower and upper cutoffs based on contrast reversals and signal-to-noise ratio, respectively.

Conclusions:

The study identified contrast reversals as the source of side peaks in autofocus.
Improved bandpass design criteria effectively eliminate side peaks.
The refined method yields sharp, unimodal autofocus responses across diverse microscopy specimens.