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Updated: Mar 21, 2026
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Single-Molecule Tracking Microscopy - A Tool for Determining the Diffusive States of Cytosolic Molecules
Published on: September 5, 2019
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Investigating the usage of point spread functions in point source and microsphere localization
Jerry Chao1, Sripad Ram2, E Sally Ward3
1Dept. of Biomedical Engineering, Texas A&M University, College Station, TX 77843, USA; Dept. of Molecular and Cellular Medicine, Texas A&M Health Science Center, College Station, TX 77843, USA.
Summary
Accurate localization of point-like objects in microscopy is crucial. This study reveals that integration step size impacts accuracy, and point spread function (PSF) fitting is suitable for microsphere localization within specific size limits.
Area of Science:
- Microscopy
- Image Analysis
- Computational Biology
Background:
- Accurate localization of point-like objects is essential in single-molecule microscopy.
- Point spread function (PSF) fitting is a common method for this task.
Purpose of the Study:
- To evaluate the impact of integration step size on PSF localization accuracy.
- To assess the suitability of PSF fitting for microsphere localization.
Main Methods:
- Two simulation studies were conducted.
- Maximum likelihood fitting of a PSF to image data was employed.
- Standard errors of the mean and variance were used as criteria.
Main Results:
- Coarse integration step sizes significantly reduce localization accuracy, especially at low magnifications.
- PSF fitting is suitable for microspheres up to 1 µm in diameter when fitting width simultaneously.
- PSF fitting is suitable for microspheres up to 100 nm in diameter when fitting width is not simultaneous.
Conclusions:
- Optimizing integration step size is critical for accurate PSF-based localization.
- PSF fitting is a viable technique for microsphere localization within defined size parameters, depending on fitting strategy.
