Live-Cell Super-Resolution Fluorescence Imaging at High Spatiotemporal Resolutions

Abstract

Super-resolution fluorescence microscopy accomplished nanometer resolutions in optical imaging along with multi-color and three-dimensional capability in fixed specimens. For live cells, time and spatial resolutions has been limited by the intrinsic tradeoff between the two resolutions. Using bright, fast switching cyanine dyes, we achieved high spatiotemporal resolutions with stochastic reconstruction microscopy (STORM). We obtained two-dimensional (2D) and three-dimensional (3D) super-resolution images of clathrin-coated pits and their transferrin cargo labeled with photoswitchable dyes either directly or via SNAP tags in living cells. The high photon output and fast switching rate of the cyanine dye enabled us to demonstrate 2D imaging at spatial resolutions of ∼25 nm and temporal resolutions as fast as 0.5 sec, which represent a 2-3 fold improvement in spatial resolution and 50-100 fold improvement in temporal resolution compared to previously reported resolutions obtained using single-molecule-localization based super-resolution approaches. Furthermore, we demonstrate live-cell 3D volumetric super-resolution imaging for the first time, with a spatial resolution of ∼30 nm in the lateral directions and ∼50 nm in the axial direction at time resolutions down to ∼1 sec. Two-color 3D super-resolution imaging in live cells was also achieved using photoswitchable probes with distinct emission spectra. In addition, we obtained similarly high spatial and temporal resolutions imaging other important cellular structures. These imaging capabilities open a new window for resolving ultrastructures in living cells.