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

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.

Fluorescence correlation spectroscopy and fluorescence lifetime imaging microscopy for deciphering the morphological evolution of supramolecular self-assembly

“…60 Generally, the lifetime of the emissive aggregates or the self-assembled structures can vary due to spontaneous or stimuli-responsive morphological transformation. 85–90 Hence, FLIM images, along with fluorescence lifetime histograms and in situ lifetime profiles, provide a clear idea about the evolution dynamics of self-assembled structures. 88,90…”

“…60 Generally, the lifetime of the emissive aggregates or the self-assembled structures can vary due to spontaneous or stimuli-responsive morphological transformation. [85][86][87][88][89][90] Hence, FLIM images, along with fluorescence lifetime histograms and in situ lifetime profiles, provide a clear idea about the evolution dynamics of self-assembled structures. 88,90 Cienfuegos and co-workers probed the early-stage selfassembly process leading to the evolution of supramolecular nanofibers of fluorenylmethyloxycarbonyl-diphenylalanine (Fmoc-FF), a short peptide, in the presence of metal salts of Ca 2+ (Fmoc-FF Ca 2+ ) and Cs + (Fmoc-FF Cs + ).…”

Shrinking gate fluorescence correlation spectroscopy yields equilibrium constants and separates photophysics from structural dynamics

“…The fact that sg-FCS could directly reveal new insights for two projects of the laboratory, including a fluctuating tether on graphene and a new lipid-surface membrane-potential sensor, indicates the broad applicability of the method and suggests implementation of the method whenever TCSPC-data are available in combination with the autocorrelation analysis. Recent developments of commercial lifetime cameras suggest this approach can also be applied to widefield illumination where several molecules can be measured in parallel ( 52 ). In addition, smFRET experiments are now being performed in living cells (e.g., ref.…”

22 nm Resolution Achieved by Femtosecond Laser Two-Photon Polymerization of a Hyaluronic Acid Vinyl Ester Hydrogel

“…The cells were stained by Hoechst 33342 (λ ex = 405 nm, 2.5 μL of 17.8 μM in 2.5 mL DMEM with L929), MitoTracker Green (λ ex = 488 nm, 2.5 μL of 1 μM in 2.5 mL DMEM with L929), and PI (λ ex = 561 nm, 13 μL of 1.5 mM in 2.5 mL of DMEM with L929) for 15 min. Confocal fluorescence microscopy (AIR MP, Nikon) was employed to observe fluorescence images using a 60× objective lens. − …”

“…Hyclone DMEM/HIGH GLUCOSE culture medium (GE Healthcare Life Sciences) containing 10% fetal bovine serum (FBS) and 1% penicillin−streptomycin was used. L929 cells (fibroblast cells) 45 were cultured on the glass-bottom cell culture dish (35 mm × 12 mm Style; NEST, Cat. No.…”