Confocal Laser-Scanning Fluorescence-Lifetime Single-Molecule Localisation Microscopy

Abstract Fluorescence lifetime imaging microscopy (FLIM) is an important technique that adds another dimension to the intensity and colour information of 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 (STED) microscopy. In contrast, all Single-Molecule Localisation Microscopy (SMLM) techniques that employ wide-field cameras completely lack the lifetime dimension. Here, we combine Fluorescence-Lifetime Confocal Laser-Scanning Microscopy (FL-CLSM) with SMLM for realising single-molecule localisation-based fluorescence-lifetime super-resolution imaging (FL-SMLM). Besides yielding images with a spatial resolution much beyond the diffraction limit, it determines the fluorescence lifetime of all localised molecules. We validate our technique by applying it to direct STochastic Optical Reconstruction Microscopy (dSTORM) and Points Accumulation for Imaging in Nanoscale Topography (PAINT) 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.