Doubling the resolution of fluorescence-lifetime single-molecule localization microscopy with image scanning microscopy

In this study, we integrate a single-photon detector array into a confocal laser scanning microscope, enabling the combination of fluorescence-lifetime single-molecule localization microscopy with image scanning microscopy. This unique combination delivers a twofold improvement in lateral localization accuracy for single-molecule localization microscopy (SMLM) and maintains its simplicity. Moreover, the addition of lifetime information from our confocal laser scanning microscope eliminates chromatic aberration, particularly crucial for achieving few-nanometre resolution in SMLM. Our approach, named fluorescence-lifetime image scanning microscopy SMLM, is demonstrated through direct stochastic optical reconstruction microscopy and DNA point accumulation for imaging in nanoscale topography experiments on fluorescently labelled cells, showcasing both resolution enhancement and fluorescence-lifetime multiplexing capabilities.