Precise Nanometer Localization Analysis for Individual Fluorescent Probes
Open accessAbstract
Calculation of the centroid of the images of individual fluorescent particles and molecules allows localization and tracking in light microscopes to a precision about an order of magnitude greater than the microscope resolution. The factors that limit the precision of these techniques are examined and a simple equation derived that describes the precision of localization over a wide range of conditions. In addition, a localization algorithm motivated from least-squares fitting theory is constructed and tested both on image stacks of 30-nm fluorescent beads and on computer-generated images (Monte Carlo simulations). Results from the algorithm show good agreement with the derived precision equation for both the simulations and actual images. The availability of a simple equation to describe localization precision helps investigators both in assessing the quality of an experimental apparatus and in directing attention to the factors that limit further improvement. The precision of localization scales as the inverse square root of the number of photons in the spot for the shot noise limited case and as the inverse of the number of photons for the background noise limited case. The optimal image magnification depends on the expected number of photons and background noise, but, for most cases of interest, the pixel size should be about equal to the standard deviation of the point spread function.
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Only molecules localized with an x-y precision <10 nm, fitting R2 > 0.6, and comprising >200 photons were used for further analysis.
“Only molecules localized with an x-y precision <10 nm37, fitting R2 > 0.6, and comprising >200 photons were used for further analysis.”
From A trans-synaptic nanocolumn aligns neurotransmitter release to receptors (2016) · cites this paper as [37]
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- Authors
- Thompson, R. E., Larson, D. R., & Webb, W. W.
- Affiliations
- Cornell University (US)
- Corresponding author
- Webb, Watt W.
- ORCID
- Larson, Daniel R.
- Venue
- Biophysical Journal
- Publisher
- Elsevier BV
- Published
- 2002-05
- Volume
- 82
- Issue
- 5
- Pages
- 2775-2783
- ISSN
- 0006-3495 (ISSN-L)
- PMC
- PMC1302065
- OpenAlex
- W2062552264
- Cited by
- 2,320
- References
- 1
- Citations by year
- 2026: 42 · 2025: 59 · 2024: 57 · 2023: 68 · 2022: 84 · 2021: 104 · 2020: 106 · 2019: 115 · 2018: 143 · 2017: 157 · 2016: 123 · 2015: 153 · 2014: 195 · 2013: 164 · 2012: 158
- FWCI
- 12.58
- Type
- journal article
- Language
- en
- Open access
- bronze
- doi.org
- Licence
- Locations
- Biophysical Journal (publishedVersion)
- PubMed Central (publishedVersion)
- PubMed (publishedVersion)
- Funders
- National Institute of General Medical Sciences (GM 08267, GM 08210, T32GM008210)
- National Center for Research Resources (P41RR004224, P41RR04224)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Biophysics › Advanced Fluorescence Microscopy Techniques
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Biophysics › Cell Image Analysis Techniques
- Physical Sciences › Physics and Astronomy › Atomic and Molecular Physics, and Optics › Force Microscopy Techniques and Applications
- Keywords
- single-molecule localization, centroid calculation, least squares fitting, shot noise, background noise, photon statistics, point spread function, image magnification, fluorescent microspheres, Monte Carlo simulation, single-particle tracking
- MeSH terms
- Fluorescent Dyes, Sensitivity and Specificity, Normal Distribution, Least-Squares Analysis, Photons, Microscopy, Confocal
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