Single-Molecule Fluorescence Lifetime Imaging Using Wide-Field and Confocal-Laser Scanning Microscopy: A Comparative Analysis

• Published in: Nano letters 2022-08, Vol.22 (15), p.6454-6461
• Main Authors: Oleksiievets, Nazar, Mathew, Christeena, Thiele, Jan Christoph, Gallea, José Ignacio, Nevskyi, Oleksii, Gregor, Ingo, Weber, André, Tsukanov, Roman, Enderlein, Jörg
• Format: Article
• Language: English
• Subjects: DNA; Letter; Microscopy, Confocal - methods; Microscopy, Fluorescence - methods; Nanotechnology; Single Molecule Imaging - methods
• ISSN: 1530-6984; 1530-6992; 1530-6992
• DOI: 10.1021/acs.nanolett.2c01586

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.