Fast, three-dimensional super-resolution imaging of live cells

Abstract

Judicious choice of probes and imaging conditions allows two-dimensional super-resolution imaging of live cells at speeds up to 2 Hz with ~25-nm resolution and three-dimensional super-resolution imaging at ~1 Hz with ~30 nm x-y and ~50 nm z dimension resolution using stochastic optical reconstruction microscopy (STORM). We report super-resolution fluorescence imaging of live cells with high spatiotemporal resolution using stochastic optical reconstruction microscopy (STORM). By labeling proteins either directly or via SNAP tags with photoswitchable dyes, we obtained two-dimensional (2D) and 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 s. We also demonstrated live-cell 3D super-resolution imaging. We obtained 3D spatial resolution of ∼30 nm in the lateral direction and ∼50 nm in the axial direction at time resolutions as fast as 1–2 s with several independent snapshots. Using photoswitchable dyes with distinct emission wavelengths, we also 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.