Isotropic three-dimensional dual-color super-resolution microscopy with metal-induced energy transfer.

Abstract

Over the past two decades, super-resolution microscopy has seen a tremendous development in speed and resolution, but for most of its methods, there exists a remarkable gap between lateral and axial resolution, which is by a factor of 2 to 3 worse. One recently developed method to close this gap is metal-induced energy transfer (MIET) imaging, which achieves an axial resolution down to nanometers. It exploits the distance-dependent quenching of fluorescence when a fluorescent molecule is brought close to a metal surface. In the present manuscript, we combine the extreme axial resolution of MIET imaging with the extraordinary lateral resolution of single-molecule localization microscopy, in particular with direct stochastic optical reconstruction microscopy (<i>d</i>STORM). This combination allows us to achieve isotropic three-dimensional super-resolution imaging of subcellular structures. Moreover, we used spectral demixing for implementing dual-color MIET-<i>d</i>STORM that allows us to image and colocalize, in three dimensions, two different cellular structures simultaneously.