Fluorescence Lifetime Single Molecule Localisation Microscopy

Abstract

In conventional fluorescence microscopy, species are distinguished by colour. While these images provide an abundance of information about the sample, they are, in many aspects limited by artefacts and resolution, and some properties of the sample remain elusive. In fluorescence-lifetime imaging microscopy (FLIM), the characteristic lifetime of a fluorophore is measured in addition to intensity. This lifetime can depend on the local environment and may be altered by energy transfer to an acceptor. Therefore, FLIM allows for lifetime-based Förster resonance energy transfer (FRET), environment sensing inside cells, and lifetime-based multiplexing. Due to the lack of single-molecule sensitive lifetime cameras, all single-molecule localisation microscopy (SMLM) techniques could not access the lifetime information so far. In this thesis, I present the implementation of SMLM based super-resolved FLIM with a confocal microscope. By rapid laser-scanning, pulsed excitation, and single photon detection, the lifetime is determined for all localised molecules. The technique provides optical sectioning and is compatible with two of the most important SMLM methods, direct stochastic optical reconstruction microscopy (dSTORM) and points accumulation for imaging in nanoscale topography (PAINT) imaging. Based on the lifetime information, two different fluorophores are distinguished in dSTORM measurements. This enables super-resolution microscopy with chromatic-aberration free multiplexing. The recent development of single-photon sensitive lifetime cameras enables single-molecule wide-field FLIM which allows for higher frame rates over a larger field of view compared to confocal FLIM. In combination with metal-induced energy transfer (MIET), single molecules can be localised axially with nanometre precision. Both methods can be readily employed for super-resolved environment sensing or 3D SMLM. The feasibility of super-resolved isotropic 3D imaging is shown in a proof of concept experiment.