Single-Molecule Fluorescence Lifetime Imaging Using Wide-Field and Confocal-Laser Scanning Microscopy: A Comparative Analysis
2022-08-10 | journal article; research paper
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Single-Molecule Fluorescence Lifetime Imaging Using Wide-Field and Confocal-Laser Scanning Microscopy: A Comparative Analysis
Oleksiievets, N.; Mathew, C.; Thiele, J. C.; Gallea, J. I.; Nevskyi, O.; Gregor, I. & Weber, A. et al. (2022)
Nano Letters, 22(15) pp. 6454-6461. DOI: https://doi.org/10.1021/acs.nanolett.2c01586
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Details
- Authors
- Oleksiievets, Nazar; Mathew, Christeena; Thiele, Jan Christoph; Gallea, José Ignacio; Nevskyi, Oleksii; Gregor, Ingo; Weber, André; Tsukanov, Roman; Enderlein, Jörg
- Abstract
- A recent addition to the toolbox of super-resolution microscopy methods is fluorescence-lifetime single-molecule localization microscopy (FL-SMLM). The synergy of SMLM and fluorescence-lifetime imaging microscopy (FLIM) combines superior image resolution with lifetime information and can be realized using two complementary experimental approaches: confocal-laser scanning microscopy (CLSM) or wide-field microscopy. Here, we systematically and comprehensively compare these two novel FL-SMLM approaches in different spectral regions. For wide-field FL-SMLM, we use a commercial lifetime camera, and for CLSM-based FL-SMLM we employ a home-built system equipped with a rapid scan unit and a single-photon detector. We characterize the performances of the two systems in localizing single emitters in 3D by combining FL-SMLM with metal-induced energy transfer (MIET) for localization along the third dimension and in the lifetime-based multiplexed bioimaging using DNA-PAINT. Finally, we discuss advantages and disadvantages of wide-field and confocal FL-SMLM and provide practical advice on rational FL-SMLM experiment design.
- Issue Date
- 10-August-2022
- Journal
- Nano Letters
- Project
- EXC 2067: Multiscale Bioimaging
- Working Group
- RG Enderlein
- ISSN
- 1530-6984
- eISSN
- 1530-6992
- Language
- English