Enhanced information content for three-dimensional localization and tracking using the double-helix point spread function with variable-angle illumination epifluorescence microscopy
Abstract
The signal-to-noise ratio (SNR) and three-dimensional localization precision of a double helix point spread function (DH-PSF) can be significantly improved by applying variable-angle illumination epifluorescence microscopy (VAI, also commonly known as “pseudo-TIRF” or “quasi-TIRF”). In this paper, we performed a quantitative analysis of the dependence of SNR and localization precision on the number of measured photons and the incident angle for static particles under both low (at a planar index-matched interface) and high (within a porous silica matrix) fluorescent background conditions. We found that under noisier imaging conditions, the SNR and localization precision obtained using VAI are up to fivefold and threefold greater, respectively, than those obtained using epi-illumination. Finally and moreover, we demonstrate that the combination of DH-PSF and VAI can significantly improve the accuracy of the measured diffusion coefficient for mobile particles, even at a relatively large distance (50 μm) from the boundary of the optical cell.
- Authors:
-
- Univ. of Colorado, Boulder, CO (United States). Dept. of Chemical and Biological Engineering
- Double Helix LLC, Boulder, CO (United States)
- Univ. of Colorado, Boulder, CO (United States). Dept. of Electrical, Computer, and Energy Engineering
- Publication Date:
- Research Org.:
- Univ. of Colorado, Boulder, CO (United States); Double Helix LLC, Boulder, CO (United States)
- Sponsoring Org.:
- USDOE Office of Science (SC), Basic Energy Sciences (BES); National Science Foundation (NSF)
- OSTI Identifier:
- 1466220
- Alternate Identifier(s):
- OSTI ID: 1361911
- Grant/Contract Number:
- SC0001854; IIP-1353638; 1556473; 1548924
- Resource Type:
- Accepted Manuscript
- Journal Name:
- Applied Physics Letters
- Additional Journal Information:
- Journal Volume: 110; Journal Issue: 21; Journal ID: ISSN 0003-6951
- Publisher:
- American Institute of Physics (AIP)
- Country of Publication:
- United States
- Language:
- English
- Subject:
- 42 ENGINEERING; diffusion; fluorescence; photons; total internal reflection; illumination; cameras; confocal microscopy; opal; fluorescence microscopy; three dimensional microscopy
Citation Formats
Wang, Dapeng, Agrawal, Anurag, Piestun, Rafael, and Schwartz, Daniel K. Enhanced information content for three-dimensional localization and tracking using the double-helix point spread function with variable-angle illumination epifluorescence microscopy. United States: N. p., 2017.
Web. doi:10.1063/1.4984133.
Wang, Dapeng, Agrawal, Anurag, Piestun, Rafael, & Schwartz, Daniel K. Enhanced information content for three-dimensional localization and tracking using the double-helix point spread function with variable-angle illumination epifluorescence microscopy. United States. https://doi.org/10.1063/1.4984133
Wang, Dapeng, Agrawal, Anurag, Piestun, Rafael, and Schwartz, Daniel K. Wed .
"Enhanced information content for three-dimensional localization and tracking using the double-helix point spread function with variable-angle illumination epifluorescence microscopy". United States. https://doi.org/10.1063/1.4984133. https://www.osti.gov/servlets/purl/1466220.
@article{osti_1466220,
title = {Enhanced information content for three-dimensional localization and tracking using the double-helix point spread function with variable-angle illumination epifluorescence microscopy},
author = {Wang, Dapeng and Agrawal, Anurag and Piestun, Rafael and Schwartz, Daniel K.},
abstractNote = {The signal-to-noise ratio (SNR) and three-dimensional localization precision of a double helix point spread function (DH-PSF) can be significantly improved by applying variable-angle illumination epifluorescence microscopy (VAI, also commonly known as “pseudo-TIRF” or “quasi-TIRF”). In this paper, we performed a quantitative analysis of the dependence of SNR and localization precision on the number of measured photons and the incident angle for static particles under both low (at a planar index-matched interface) and high (within a porous silica matrix) fluorescent background conditions. We found that under noisier imaging conditions, the SNR and localization precision obtained using VAI are up to fivefold and threefold greater, respectively, than those obtained using epi-illumination. Finally and moreover, we demonstrate that the combination of DH-PSF and VAI can significantly improve the accuracy of the measured diffusion coefficient for mobile particles, even at a relatively large distance (50 μm) from the boundary of the optical cell.},
doi = {10.1063/1.4984133},
journal = {Applied Physics Letters},
number = 21,
volume = 110,
place = {United States},
year = {Wed May 24 00:00:00 EDT 2017},
month = {Wed May 24 00:00:00 EDT 2017}
}
Web of Science
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