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.
| Original language | English |
|---|---|
| Pages (from-to) | 6454-6461 |
| Number of pages | 8 |
| Journal | Nano Letters |
| Volume | 22 |
| Issue number | 15 |
| DOIs | |
| State | Published - 10 Aug 2022 |
| Externally published | Yes |
Keywords
- DNA-PAINT
- fluorescence lifetime
- fluorescence lifetime imaging microscopy
- metal-induced energy transfer
- single-molecule localization microscopy
- super-resolution microscopy
ASJC Scopus subject areas
- Bioengineering
- General Chemistry
- General Materials Science
- Condensed Matter Physics
- Mechanical Engineering