Confocal Fluorescence-Lifetime Single-Molecule Localization Microscopy

Abstract

Fluorescence lifetime imaging microscopy is an important technique that adds another dimension to intensity and color acquired by conventional microscopy. In particular, it allows for multiplexing fluorescent labels that have otherwise similar spectral properties. Currently, the only super-resolution technique that is capable of recording super-resolved images with lifetime information is stimulated emission depletion microscopy. In contrast, all single-molecule localization microscopy (SMLM) techniques that employ wide-field cameras completely lack the lifetime dimension. Here, we combine fluorescence-lifetime confocal laser-scanning microscopy with SMLM for realizing single-molecule localization-based fluorescence-lifetime super-resolution imaging. Besides yielding images with a spatial resolution much beyond the diffraction limit, it determines the fluorescence lifetime of all localized molecules. We validate our technique by applying it to direct stochastic optical reconstruction microscopy and points accumulation for imaging in nanoscale topography imaging of fixed cells, and we demonstrate its multiplexing capability on samples with two different labels that differ only by fluorescence lifetime but not by their spectral properties.

Luminescence lifetime imaging of three-dimensional biological objects

“…In FLI-based approaches, lifetime signatures can be successfully discriminated from unwanted spectrally overlapping autofluorescence signals (Rudkouskaya et al, 2020b;del Rosal and Benayas, 2018;Okkelman et al, 2017bOkkelman et al, , 2020cRich et al, 2013). FLIM as a contrast enhancement methodology has become more popular with the advent of super-resolution microscopy (Auksorius et al, 2008;Bückers et al, 2011;Hell and Wichmann, 1994;Niehörster et al, 2016;Masullo et al, 2020;Thiele et al, 2020), providing a new way to multiplex fluorescence imaging. 3D FLIM for contrast enhancement has been demonstrated in proof-ofconcept light-sheet microscopy with optically cleared and fixed samples Greger et al, 2011;Favreau et al, 2020;Funane et al, 2018;Mitchell et al, 2017;Weber et al, 2015;Li et al, 2020;Birch et al, 2016).…”

Recent innovations in fluorescence lifetime imaging microscopy for biology and medicine

“…A super-resolution microscope that can also acquire lifetime data is advantageous for detecting fluorophores with similar spectral properties and for lifetime-based Förster resonance energy transfer (FRET) measurements. Thiele et al 11 two-channel images. Super-resolved separation of two species was achieved by Baysian pattern matching on confocal FLIM data.…”

Illuminating amyloid fibrils: Fluorescence-based single-molecule approaches

“…This excitation light is passed through confocally aligned pinholes, which results in a small volume (200 nm × 200 nm × 500 nm in dimension) being illuminated, and the emission light from that volume being detected while excluding out-of-focus sources of signal [93] , [94] . The traditional approach to confocal microscopy utilises ‘optical sectioning’, whereby the laser is scanned through a sample in all three spatial dimensions (depths of up to 500 nm can typically be achieved), and a three-dimensional image of the sample (such as a cell) is obtained [95] , [96] .…”

“…An alternative use of a confocal microscope is to place the confocal volume inside an aqueous solution, detecting the transient bursts of fluorescence originating from individual labelled species as they diffuse through the excitation volume [97] . Following excitation of a fluorophore, photons emitted from the molecule can be detected (typically using single-photon detector arrays), thus providing single-molecule sensitivity [96] , [98] . In contrast to TIRF microscopy ( Fig.…”

“…Common methods that employ this approach include (i) fluorescence correlation spectroscopy (FCS), in which the apparent sizes of molecules as they pass through the confocal volume are calculated by analysis of their diffusion, and (ii) two-colour coincidence detection (TCCD), in which the simultaneous detection of two fluorescent signals within the confocal volume is used to detect biomolecular associations. Experiments that couple confocal microscopy with time-resolved single-photon detection enable the collection of data on fluorescence lifetimes [96] , and fast dynamics via Förster resonance energy transfer (FRET) based applications [96] , [100] , [101] vastly increase the information that can be obtained from single-molecule confocal imaging approaches. A limitation of confocal microscopy is the size of the confocal volume: molecules can only be monitored for the period of time they remain in the confocal volume (often data acquisition has to occur on the nano- to milli-second time scales) [102] .…”