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Title: Enhanced information content for three-dimensional localization and tracking using the double-helix point spread function with variable-angle illumination epifluorescence microscopy

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.
 [1] ; ORCiD logo [2] ;  [3] ;  [1]
  1. Univ. of Colorado, Boulder, CO (United States). Dept. of Chemical and Biological Engineering
  2. Double Helix LLC, Boulder, CO (United States)
  3. Univ. of Colorado, Boulder, CO (United States). Dept. of Electrical, Computer, and Energy Engineering
Publication Date:
Grant/Contract Number:
SC0001854; IIP-1353638; 1556473; 1548924
Accepted Manuscript
Journal Name:
Applied Physics Letters
Additional Journal Information:
Journal Volume: 110; Journal Issue: 21; Journal ID: ISSN 0003-6951
American Institute of Physics (AIP)
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) (SC-22); National Science Foundation (NSF)
Country of Publication:
United States
42 ENGINEERING; diffusion; fluorescence; photons; total internal reflection; illumination; cameras; confocal microscopy; opal; fluorescence microscopy; three dimensional microscopy
OSTI Identifier:
Alternate Identifier(s):
OSTI ID: 1361911