期刊
NANO LETTERS
卷 22, 期 15, 页码 6454-6461出版社
AMER CHEMICAL SOC
DOI: 10.1021/acs.nanolett.2c01586
关键词
super-resolution microscopy; single-molecule localization microscopy; fluorescence lifetime; DNA-PAINT; fluorescence lifetime imaging microscopy; metal-induced energy transfer
类别
资金
- [PST990]
- [49151]
This study compares the performance of wide-field and confocal FL-SMLM techniques in different spectral regions and provides practical advice.
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.
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