Live-cell imaging of dendritic spines by STED microscopy - PubMed (original) (raw)
Live-cell imaging of dendritic spines by STED microscopy
U Valentin Nägerl et al. Proc Natl Acad Sci U S A. 2008.
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.
Conflict of interest statement
The authors declare no conflict of interest.
Figures
Fig. 1.
STED microscopy of dendritic structures. (A) Schematic representation of the experimental setup. (B) Dendritic spines imaged either by confocal (Top) or STED (Bottom) microscopy. (Scale bar, 0.5 μm.) Note that pixel intensity representation is not saturated over the regions of interest, i.e., at the spine necks. (C) Normalized line profile of pixel intensity across the neck of a spine as imaged in confocal (gray curve) or STED mode (black curve); line width: four pixels, indicated by shaded bar in B. (D) Frequency histogram of FWHM values for spine necks imaged in confocal (gray bars) and STED (black bars) modes. (E) Plot of ratio of FWHM values in STED over confocal modes as a function of FWHM values of spine necks and dendritic shafts imaged in the STED mode.
Fig. 2.
High-resolution 3D STED imaging of dendritic structures. (A) Volume reconstruction of image stack data; the panels show individual image sections acquired at the z level indicated (Δz = 0.5 μm). (B) Another example of a reconstructed stretch of dendrite (Δz = 0.25 μm), the lateral pixel size was 29 nm/pixel in both cases. (Scale bars, 1 μm.)
Fig. 3.
Time lapse STED imaging of dendritic structures. (A and B) Series of image frames of YFP-labeled dendritic spines acquired by STED microscopy at 40 sec/frame (20 frames were acquired in total) under unstimulated conditions. Arrows indicate cup-like shapes of spine heads; STED pulse peak intensity 400 MW/cm2 (A) and 215 MW/cm2 (B). (Scale bars, 1 μm.) (C) Time course of fluorescence intensity in the heads of spines as a function of consecutively acquired image frames (20 sec/frame).
Fig. 4.
STED imaging of activity-dependent postsynaptic morphological plasticity. (A–D) Examples of structural changes of dendritic spines after plasticity-inducing stimulation using the chemical LTP protocol. The first frame was taken before the stimulation, while all subsequent ones at the times indicated (h:min). Arrows indicate sites of structural rearrangements. (Scale bars, 0.5 μm.)
Similar articles
- Stimulated emission depletion (STED) imaging of dendritic spines in living hippocampal slices.
Willig KI, Nägerl UV. Willig KI, et al. Cold Spring Harb Protoc. 2012 May 1;2012(5):pdb.prot069260. doi: 10.1101/pdb.prot069260. Cold Spring Harb Protoc. 2012. PMID: 22550296 - Stimulated emission depletion (STED) microscopy reveals nanoscale defects in the developmental trajectory of dendritic spine morphogenesis in a mouse model of fragile X syndrome.
Wijetunge LS, Angibaud J, Frick A, Kind PC, Nägerl UV. Wijetunge LS, et al. J Neurosci. 2014 Apr 30;34(18):6405-12. doi: 10.1523/JNEUROSCI.5302-13.2014. J Neurosci. 2014. PMID: 24790210 Free PMC article. - Considerations for Imaging and Analyzing Neural Structures by STED Microscopy.
Lenz MO, Tønnesen J. Lenz MO, et al. Methods Mol Biol. 2019;1941:29-46. doi: 10.1007/978-1-4939-9077-1_3. Methods Mol Biol. 2019. PMID: 30707425 - Super-resolution STED microscopy in live brain tissue.
Calovi S, Soria FN, Tønnesen J. Calovi S, et al. Neurobiol Dis. 2021 Aug;156:105420. doi: 10.1016/j.nbd.2021.105420. Epub 2021 Jun 5. Neurobiol Dis. 2021. PMID: 34102277 Review. - Dendritic spines and long-term plasticity.
Segal M. Segal M. Nat Rev Neurosci. 2005 Apr;6(4):277-84. doi: 10.1038/nrn1649. Nat Rev Neurosci. 2005. PMID: 15803159 Review.
Cited by
- Microglia either promote or restrain TRAIL-mediated excitotoxicity caused by Aβ1-42 oligomers.
Zou J, McNair E, DeCastro S, Lyons SP, Mordant A, Herring LE, Vetreno RP, Coleman LG Jr. Zou J, et al. J Neuroinflammation. 2024 Sep 1;21(1):215. doi: 10.1186/s12974-024-03208-2. J Neuroinflammation. 2024. PMID: 39218898 Free PMC article. - A modular framework for multi-scale tissue imaging and neuronal segmentation.
Cauzzo S, Bruno E, Boulet D, Nazac P, Basile M, Callara AL, Tozzi F, Ahluwalia A, Magliaro C, Danglot L, Vanello N. Cauzzo S, et al. Nat Commun. 2024 May 22;15(1):4102. doi: 10.1038/s41467-024-48146-y. Nat Commun. 2024. PMID: 38778027 Free PMC article. - Pushing the Resolution Limit of Stimulated Emission Depletion Optical Nanoscopy.
Jeong S, Koh D, Gwak E, Srambickal CV, Seo D, Widengren J, Lee JC. Jeong S, et al. Int J Mol Sci. 2023 Dec 19;25(1):26. doi: 10.3390/ijms25010026. Int J Mol Sci. 2023. PMID: 38203197 Free PMC article. Review. - Quantitative super-resolution microscopy reveals the differences in the nanoscale distribution of nuclear phosphatidylinositol 4,5-bisphosphate in human healthy skin and skin warts.
Hoboth P, Sztacho M, Quaas A, Akgül B, Hozák P. Hoboth P, et al. Front Cell Dev Biol. 2023 Jul 7;11:1217637. doi: 10.3389/fcell.2023.1217637. eCollection 2023. Front Cell Dev Biol. 2023. PMID: 37484912 Free PMC article. - Dense 4D nanoscale reconstruction of living brain tissue.
Velicky P, Miguel E, Michalska JM, Lyudchik J, Wei D, Lin Z, Watson JF, Troidl J, Beyer J, Ben-Simon Y, Sommer C, Jahr W, Cenameri A, Broichhagen J, Grant SGN, Jonas P, Novarino G, Pfister H, Bickel B, Danzl JG. Velicky P, et al. Nat Methods. 2023 Aug;20(8):1256-1265. doi: 10.1038/s41592-023-01936-6. Epub 2023 Jul 10. Nat Methods. 2023. PMID: 37429995 Free PMC article.
References
- Denk W, Strickler JH, Webb WW. 2-Photon Laser Scanning Fluorescence Microscopy. Science. 1990;248:73–76. - PubMed
- Yuste R, Bonhoeffer T. Morphological changes in dendritic spines associated with long-term synaptic plasticity. Annu Rev Neurosci. 2001;24:1071–1089. - PubMed
- Engert F, Bonhoeffer T. Dendritic spine changes associated with hippocampal long-term synaptic plasticity. Nature. 1999;399:66–70. - PubMed
- Maletic-Savatic M, Malinow R, Svoboda K. Rapid dendritic morphogenesis in CA1 hippocampal dendrites induced by synaptic activity. Science. 1999;283:1923–1927. - PubMed
MeSH terms
LinkOut - more resources
Full Text Sources
Other Literature Sources