Video-Rate Far-Field Optical Nanoscopy Dissects Synaptic Vesicle Movement

TLDR

The study introduces video‑rate far‑field optical imaging achieving 62‑nm resolution in living cells. Using STED microscopy, fluorescently labeled synaptic vesicles in cultured neurons were imaged at 28 fps over a 2.5 × 1.8‑µm field, with the focal spot reduced to 260 nm to map vesicle mobility within synaptic boutons. Vesicle movement was markedly faster outside boutons, indicating a continuous vesicle pool traversing axons, and the work demonstrates real‑time nanoscale investigation of intracellular physiology by optical microscopy.

Abstract

We present video-rate (28 frames per second) far-field optical imaging with a focal spot size of 62 nanometers in living cells. Fluorescently labeled synaptic vesicles inside the axons of cultured neurons were recorded with stimulated emission depletion (STED) microscopy in a 2.5-micrometer by 1.8-micrometer field of view. By reducing the cross-sectional area of the focal spot by about a factor of 18 below the diffraction limit (260 nanometers), STED allowed us to map and describe the vesicle mobility within the highly confined space of synaptic boutons. Although restricted within boutons, the vesicle movement was substantially faster in nonbouton areas, consistent with the observation that a sizable vesicle pool continuously transits through the axons. Our study demonstrates the emerging ability of optical microscopy to investigate intracellular physiological processes on the nanoscale in real time.