Super-resolution imaging of neuronal dense-core vesicles

Bethe A. Scalettar, Daniel Shaver, Stefanie Kaech-Petrie, Janis E. Lochner

Research output: Contribution to journalArticle

3 Citations (Scopus)

Abstract

Detection of fluorescence provides the foundation for many widely utilized and rapidly advancing microscopy techniques employed in modern biological and medical applications. Strengths of fluorescence include its sensitivity, specificity, and compatibility with live imaging. Unfortunately, conventional forms of fluorescence microscopy suffer from one major weakness, diffraction-limited resolution in the imaging plane, which hampers studies of structures with dimensions smaller than ~250 nm. Recently, this limitation has been overcome with the introduction of super-resolution fluorescence microscopy techniques, such as photoactivated localization microscopy (PALM). Unlike its conventional counterparts, PALM can produce images with a lateral resolution of tens of nanometers. It is thus now possible to use fluorescence, with its myriad strengths, to elucidate a spectrum of previously inaccessible attributes of cellular structure and organization. Unfortunately, PALM is not trivial to implement, and successful strategies often must be tailored to the type of system under study. In this article, we show how to implement single-color PALM studies of vesicular structures in fixed, cultured neurons. PALM is ideally suited to the study of vesicles, which have dimensions that typically range from ~50-250 nm. Key steps in our approach include labeling neurons with photoconvertible (green to red) chimeras of vesicle cargo, collecting sparsely sampled raw images with a super-resolution microscopy system, and processing the raw images to produce a high-resolution PALM image. We also demonstrate the efficacy of our approach by presenting exceptionally well-resolved images of dense-core vesicles (DCVs) in cultured hippocampal neurons, which refute the hypothesis that extrasynaptic trafficking of DCVs is mediated largely by DCV clusters.

Original languageEnglish (US)
JournalJournal of visualized experiments : JoVE
Issue number89
DOIs
StatePublished - 2014

Fingerprint

Secretory Vesicles
Microscopy
Microscopic examination
Imaging techniques
Neurons
Fluorescence
Fluorescence microscopy
Fluorescence Microscopy
Medical applications
Cellular Structures
Labeling
Color
Diffraction
Sensitivity and Specificity
Processing

ASJC Scopus subject areas

  • Neuroscience(all)
  • Chemical Engineering(all)
  • Immunology and Microbiology(all)
  • Biochemistry, Genetics and Molecular Biology(all)

Cite this

Super-resolution imaging of neuronal dense-core vesicles. / Scalettar, Bethe A.; Shaver, Daniel; Kaech-Petrie, Stefanie; Lochner, Janis E.

In: Journal of visualized experiments : JoVE, No. 89, 2014.

Research output: Contribution to journalArticle

Scalettar, Bethe A. ; Shaver, Daniel ; Kaech-Petrie, Stefanie ; Lochner, Janis E. / Super-resolution imaging of neuronal dense-core vesicles. In: Journal of visualized experiments : JoVE. 2014 ; No. 89.
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