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This content will become publicly available on April 19, 2019

Title: Multicolor Three-Dimensional Tracking for Single-Molecule Fluorescence Resonance Energy Transfer Measurements

Single-molecule fluorescence resonance energy transfer (smFRET) remains a widely utilized and powerful tool for quantifying heterogeneous interactions and conformational dynamics of biomolecules. However, traditional smFRET experiments either are limited to short observation times (typically less than 1 ms) in the case of “burst” confocal measurements or require surface immobilization which usually has a temporal resolution limited by the camera framing rate. We developed a smFRET 3D tracking microscope that is capable of observing single particles for extended periods of time with high temporal resolution. The confocal tracking microscope utilizes closed-loop feedback to follow the particle in solution by recentering it within two overlapping tetrahedral detection elements, corresponding to donor and acceptor channels. We demonstrated the microscope’s multicolor tracking capability via random walk simulations and experimental tracking of 200 nm fluorescent beads in water with a range of apparent smFRET efficiency values, 0.45-0.69. We also demonstrated the microscope’s capability to track and quantify double-stranded DNA undergoing intramolecular smFRET in a viscous glycerol solution. In future experiments, the smFRET 3D tracking system will be used to study protein conformational dynamics while diffusing in solution and native biological environments with high temporal resolution.
Authors:
 [1] ;  [2] ;  [3] ;  [4] ;  [5] ; ORCiD logo [4]
  1. William Jewell College, Liberty, MO (United States)
  2. Evangel Univ., Springfield, MO (United States)
  3. Univ. of Colorado, Boulder, CO (United States)
  4. Los Alamos National Lab. (LANL), Los Alamos, NM (United States)
  5. Texas A & M Univ., Corpus Christi, TX (United States)
Publication Date:
Report Number(s):
LA-UR-18-20822
Journal ID: ISSN 0003-2700
Grant/Contract Number:
AC52-06NA25396
Type:
Accepted Manuscript
Journal Name:
Analytical Chemistry
Additional Journal Information:
Journal Volume: 90; Journal Issue: 10; Journal ID: ISSN 0003-2700
Publisher:
American Chemical Society (ACS)
Research Org:
Los Alamos National Lab. (LANL), Los Alamos, NM (United States)
Sponsoring Org:
USDOE Office of Science (SC), Basic Energy Sciences (BES) (SC-22)
Country of Publication:
United States
Language:
English
Subject:
37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY; Material Science; Fluorescence, tracking, microscopy
OSTI Identifier:
1438130