Live-cell imaging of dendritic spines by STED microscopy

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.LTP ͉ nanoscopy ͉ spine neck diameter ͉ superresolution ͉ synaptic plasticity

Automated analysis of spine dynamics on live CA1 pyramidal cells

“…This illustrates a physical limitation of traditional diffraction-limited microscopy that can only be cured by reducing the size of the PSF, e.g. by STED microscopy (Nägerl et al, 2008;Takasaki et al, 2008;Testa et al, 2012). In the real 2PM images of the same dendritic section, recall was comparable, but several false-positive spines were detected in the background (low precision, Fig.…”

Fluorescence nanoscopy. Methods and applications

“…STED imaging can be performed on fusion constructs of fluorescent proteins that also span most of the visible spectrum: green (GFP [112]), yellow (YFP [113] and citrine [114]) and far-red emitting proteins (TagRFP657 [115] and the tetrametric E2-Crimson [116]). Several reversible photoswitchable proteins have also been demonstrated suitable for RESOLFT imaging, exploiting their reversible switching behaviour to deplete the periphery of the focal spot (Dronpa and its counterpart Padron derivative [15,22], rsEGFP2 [23] and Dreiklang [117]).…”

“…Super-resolution of fixed brain sections is also feasible: The morphological plasticity of dendritic spines in living brain slices has been described using YFP as a volume label with time-lapse STED microscopy [113].…”

Microscopic marvels: The glorious resolution

“…Simons's originally controversial theory, proposed in 1988 , held that certain lipids and proteins in a cell membrane form temporary clumps to, for example, facilitate the transmission of signals across the membrane 4 . But the proposed rafts would have been as small as 30 nanometres in diameter -invisible until STED microscopy came along.…”