Single-Molecule Fluorescence Lifetime Imaging Using Wide-Field and Confocal-Laser Scanning Microscopy: A Comparative Analysis.
Nazar OleksiievetsChristeena MathewJan Christoph ThieleJosé Ignacio GalleaOleksii NevskyiIngo GregorAndré WeberRoman TsukanovJörg EnderleinPublished in: Nano letters (2022)
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.
Keyphrases
- single molecule
- living cells
- high resolution
- atomic force microscopy
- optical coherence tomography
- energy transfer
- high speed
- quantum dots
- healthcare
- raman spectroscopy
- magnetic resonance imaging
- mass spectrometry
- oxidative stress
- diabetic rats
- convolutional neural network
- magnetic resonance
- health information
- contrast enhanced
- endothelial cells
- sensitive detection