Interferometric fluorescent super-resolution microscopy resolves 3D cellular ultrastructure

TLDR

Cellular nanometer‑scale architecture demands 3D protein localization with nanometer accuracy, yet current electron and light microscopy lack either molecular specificity or resolution. The study introduces interferometric photoactivated localization microscopy (iPALM) to achieve sub‑20‑nm 3D protein localization with high molecular specificity. iPALM merges photoactivated localization microscopy with single‑photon, simultaneous multiphase interferometry to provide sub‑20‑nm 3D localization. iPALM measured a 25‑nm microtubule diameter, resolved dorsal and ventral plasma membranes, visualized integrin organization in the ER and adhesion complexes, thereby bridging the resolution gap between electron tomography and light microscopy.

Abstract

Understanding molecular-scale architecture of cells requires determination of 3D locations of specific proteins with accuracy matching their nanometer-length scale. Existing electron and light microscopy techniques are limited either in molecular specificity or resolution. Here, we introduce interferometric photoactivated localization microscopy (iPALM), the combination of photoactivated localization microscopy with single-photon, simultaneous multiphase interferometry that provides sub-20-nm 3D protein localization with optimal molecular specificity. We demonstrate measurement of the 25-nm microtubule diameter, resolve the dorsal and ventral plasma membranes, and visualize the arrangement of integrin receptors within endoplasmic reticulum and adhesion complexes, 3D protein organization previously resolved only by electron microscopy. iPALM thus closes the gap between electron tomography and light microscopy, enabling both molecular specification and resolution of cellular nanoarchitecture.