Fast three-dimensional super-resolution imaging of live cells

We report super-resolution fluorescence imaging of live cells with high spatiotemporal resolutions using stochastic optical reconstruction microscopy (STORM). By labeling proteins either directly or via SNAP tags with photoswitchable dyes, we obtained two-dimensional (2D) and three-dimensional (3D) super-resolution images of living cells, using clathrin-coated pits and the transferrin cargo as model systems. Bright, fast switching probes enabled us to achieve 2D imaging at spatial resolutions of ~25 nm and temporal resolutions as fast as 0.5 sec. We also demonstrated live-cell 3D volumetric super-resolution imaging. A 3D spatial resolution of ~30 nm in the lateral directions and ~50 nm in the axial direction was obtained at time resolutions down to 1 – 2 sec with several independent snapshots. Using photoswitchable dyes with distinct emission wavelengths, we further demonstrated two-color 3D super-resolution imaging in live cells. These imaging capabilities open a new window for characterizing cellular structures in living cells at the ultrastructural level.