Wide-Field Fluorescence Lifetime Imaging of Single Molecules

Abstract

Fluorescence lifetime imaging (FLIM) has become an important microscopy technique in bioimaging. The two most important of its applications are lifetime-multiplexing for imaging many different structures in parallel, and lifetime-based measurements of Forster resonance energy transfer. There are two principal FLIM techniques, one based on confocal-laser scanning microscopy (CLSM) and time-correlated single-photon counting (TCSPC) and the other based on wide-field microscopy and phase fluorometry. Although the first approach (CLSM-TCSPC) assures high sensitivity and allows one to detect single molecules, it is slow and has a small photon yield. The second allows, in principal, high frame rates (by 2−3 orders of magnitude faster than CLSM), but it suffers from low sensitivity, which precludes its application for single-molecule imaging. Here, we demonstrate that a novel wide-field TCSPC camera (LINCam25, Photonscore GmbH) can be successfully used for single-molecule FLIM, although its quantum yield of detection in the red spectral region is only ∼5%. This is due to the virtually absent background and readout noise of the camera, assuring high signal-to-noise ratio even at low detection efficiency. We performed single-molecule FLIM of different red fluorophores, and we use the lifetime information for successfully distinguishing between different molecular species. Finally, we demonstrate single-molecule metal-induced energy transfer (MIET) imaging which is a first step for three-dimensional single-molecule localization microscopy (SMLM) with nanometer resolution.

Recent innovations in fluorescence lifetime imaging microscopy for biology and medicine

“…In a subsequent work, this group implemented a TCSPC-camera-based wide-field single-molecule localization approach for super-resolution imaging using a similar pattern matching method. 12…”

Lifetime-based analysis of binary fluorophores mixtures in the low photon count limit

“…Studies to date involving smFRET and FLIM either exploited the measurements of FLT in multiple color channels in order to enhance the robustness or accuracy of the measurement or used this information to gain knowledge on the photophysical behavior of the dyes ( Kaye et al., 2017 ) ( Höfig et al., 2014 )( Sheng et al., 2008 ). Other studies used laser scanning-based FLT measurements or large-area FLIM for live cell FLIM ( Oleksiievets et al., 2020 ; Raspe et al., 2016 ) ( Blacker et al., 2014 ; Grecco et al., 2010 ; Lagarto et al., 2020 ; Ma et al., 2021 ; Scipioni et al., 2021 ).…”

“…Studies to date involving smFRET and FLIM either exploited the measurements of FLT in multiple color channels in order to enhance the robustness or accuracy of the measurement or used this information to gain knowledge on the photophysical behavior of the dyes (Kaye et al, 2017) (Ho ¨fig et al, 2014 (Sheng et al, 2008). Other studies used laser scanning-based FLT measurements or large-area FLIM for live cell FLIM (Oleksiievets et al, 2020;Raspe et al, 2016) (Blacker et al, 2014;Grecco et al, 2010;Lagarto et al, 2020;Ma et al, 2021;Scipioni et al, 2021). FLIM, optical nanopore sensing, as well as other applications of FLT may nevertheless benefit from the ability to resolve multiple fluorescence species in each of the excitation/emission channels, as a way to further increase the methods' multiplexibility, while preserving single-molecule resolution.…”

Isotropic Three-Dimensional Dual-Color Super-Resolution Microscopy with Metal-Induced Energy Transfer

“…[26] The technique uses the distance-dependent coupling of the evanescent field of a fluorescent emitter to surface plasmons in a thin metallic layer deposited on the surface of the glass cover slide. The resulting energy transfer is extremely distance dependent and leads to a distance-dependent fluorescence lifetime and intensity of the emitter, which can be used to determine molecule-distance values with nanometer accuracy (single-molecule MIET or smMIET), [27][28][29] despite the unavoidable fluorescence intensity losses due to partial light absorption by the metal film. This is due to the fact that, although the fluorescence brightness of a dye is increasingly reduced the closer the dye comes to the metal surface, its photo-stability increases proportionally, so that the average number of detectable photons from one molecule until photobleaching is nearly independent on dye-metal distance.…”