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

Journal Article · · Analytical Chemistry
 [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)

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.

Research Organization:
Los Alamos National Laboratory (LANL), Los Alamos, NM (United States)
Sponsoring Organization:
USDOE Office of Science (SC), Basic Energy Sciences (BES)
Grant/Contract Number:
AC52-06NA25396
OSTI ID:
1438130
Report Number(s):
LA-UR-18-20822
Journal Information:
Analytical Chemistry, Vol. 90, Issue 10; ISSN 0003-2700
Publisher:
American Chemical Society (ACS)Copyright Statement
Country of Publication:
United States
Language:
English
Citation Metrics:
Cited by: 10 works
Citation information provided by
Web of Science

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Cited By (2)

Super-resolution photoluminescence lifetime and intensity mapping of interacting CdSe/CdS quantum dots journal January 2020
Real-Time 3D Single Particle Tracking: Towards Active Feedback Single Molecule Spectroscopy in Live Cells journal August 2019

Figures / Tables (5)