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

Abstract

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]
+ Show Author Affiliations
  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:
Research Org.:
Los Alamos National Laboratory (LANL), Los Alamos, NM (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Basic Energy Sciences (BES)
OSTI Identifier:
1438130
Report Number(s):
LA-UR-18-20822
Journal ID: ISSN 0003-2700
Grant/Contract Number:  
AC52-06NA25396
Resource 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)
Country of Publication:
United States
Language:
English
Subject:
37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY; Material Science; Fluorescence, tracking, microscopy

Citation Formats

Keller, Aaron M., DeVore, Matthew S., Stich, Dominik G., Vu, Dung M., Causgrove, Timothy, and Werner, James H. Multicolor Three-Dimensional Tracking for Single-Molecule Fluorescence Resonance Energy Transfer Measurements. United States: N. p., 2018. Web. doi:10.1021/acs.analchem.8b00244.
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Keller, Aaron M., DeVore, Matthew S., Stich, Dominik G., Vu, Dung M., Causgrove, Timothy, & Werner, James H. Multicolor Three-Dimensional Tracking for Single-Molecule Fluorescence Resonance Energy Transfer Measurements. United States. https://doi.org/10.1021/acs.analchem.8b00244
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Keller, Aaron M., DeVore, Matthew S., Stich, Dominik G., Vu, Dung M., Causgrove, Timothy, and Werner, James H. Thu . "Multicolor Three-Dimensional Tracking for Single-Molecule Fluorescence Resonance Energy Transfer Measurements". United States. https://doi.org/10.1021/acs.analchem.8b00244. https://www.osti.gov/servlets/purl/1438130.
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@article{osti_1438130,
title = {Multicolor Three-Dimensional Tracking for Single-Molecule Fluorescence Resonance Energy Transfer Measurements},
author = {Keller, Aaron M. and DeVore, Matthew S. and Stich, Dominik G. and Vu, Dung M. and Causgrove, Timothy and Werner, James H.},
abstractNote = {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.},
doi = {10.1021/acs.analchem.8b00244},
journal = {Analytical Chemistry},
number = 10,
volume = 90,
place = {United States},
year = {Thu Apr 19 00:00:00 EDT 2018},
month = {Thu Apr 19 00:00:00 EDT 2018}
}
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Journal Article:
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Publisher's Version of Record
https://doi.org/10.1021/acs.analchem.8b00244
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Cited by: 10 works
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Figures / Tables:

Figure 1 Figure 1: Layout of excitation and emission paths for the smFRET 3D tracking microscope.
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    Works referencing / citing this record:

    Super-resolution photoluminescence lifetime and intensity mapping of interacting CdSe/CdS quantum dots
    journal, January 2020

    • Dunlap, Megan K.; Ryan, Duncan P.; Goodwin, Peter M.
    • Applied Physics Letters, Vol. 116, Issue 2
    • DOI: 10.1063/1.5132563

    Real-Time 3D Single Particle Tracking: Towards Active Feedback Single Molecule Spectroscopy in Live Cells
    journal, August 2019

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