Live-cell imaging of dendritic spines by STED microscopy

TLDR

Time‑lapse fluorescence imaging is a key neurobiological tool, yet its diffraction‑limited resolution forces researchers to supplement live‑cell studies with electron microscopy. The study aims to demonstrate the first noninvasive, sub‑diffraction far‑field imaging of activity‑dependent dendritic spine plasticity. Using stimulated emission depletion (STED) microscopy, the authors performed time‑lapse imaging of YFP‑positive hippocampal neurons in organotypic slices. STED imaging outperforms confocal microscopy by revealing finer structural details and enabling more accurate quantification of spine neck width and head curvature, key parameters for synaptic function.

Abstract

Time lapse fluorescence imaging has become one of the most important approaches in neurobiological research. In particular, both confocal and two-photon microscopy have been used to study activity-dependent changes in synaptic morphology. However, the diffraction-limited resolution of light microscopy is often inadequate, forcing researchers to complement the live cell imaging strategy by EM. Here, we report on the first use of a far-field optical technique with subdiffraction resolution to noninvasively image activity-dependent morphological plasticity of dendritic spines. Specifically we show that time lapse stimulated emission depletion imaging of dendritic spines of YFP-positive hippocampal neurons in organotypic slices outperforms confocal microscopy in revealing important structural details. The technique substantially improves the quantification of morphological parameters, such as the neck width and the curvature of the heads of spines, which are thought to play critical roles for the function and plasticity of synaptic connections.