A159 - Metal-Induced Energy Transfer Single-Molecule Localization Microscopy (MIET-SMLM)

Samrat Basak (samrat.basak@phys.uni-goettingen.de)
Oleksii Nevskyi (oleksii.nevskyi@phys.uni-goettingen.de)

Fluorescence lifetime imaging microscopy is an important technique that adds another dimension to intensity and color acquired by a conventional microscopy. Nowadays, single molecule localization microscopy (SMLM) techniques have become one of the most successful and widely applied methods of super-resolution fluorescence microscopy. Unfortunately, all SMLM techniques which utilize wide-field cameras completely lack the lifetime dimension. Here, we demonstrate the combination of fluorescence-lifetime confocal laser-scanning microscopy (CLSM) with popular single-molecule localization microscopy (SMLM) approaches such as dSTORM and DNA-PAINT. This combination enables the realization of single-molecule localization-based fluorescence-lifetime super-resolution imaging, which can be used for multiplexing on samples with different labels that differ only by fluorescence lifetime but not by their spectral properties. The technique is straightforward to be implemented on a commercial confocal scanning microscope setup with TCSPC capability and fast laser scanning unit. The method combines all the advantages of CLSM with those of SMLM: axial sectioning, shot-noise limited single-photon detection, pixel-free continuous position data, and fluorescence lifetime information acquired by CLSM with the exceptional spatial resolution and single-molecule identification of SMLM, moreover, method is conceptually free of chromatic aberrations. Additionally, we combine the extreme axial resolution of metal-induced energy transfer (MIET) imaging with the extraordinary lateral resolution of single-molecule localization microscopy utilizing confocal setup. This combination allows us to achieve isotropic three-dimensional super-resolution imaging of subcellular structures.