Vamping: Stereology-based automated quantification of fluorescent puncta size and density

Dani Dumitriu, Seth I. Berger, Carine Hamo, Yuko Hara, Megan Bailey, Amarelle Hamo, Yael S. Grossman, William G. Janssen, John Morrison

Research output: Contribution to journalArticlepeer-review

13 Scopus citations

Abstract

The size of dendritic spines and postsynaptic densities (PSDs) is well known to be correlated with molecular and functional characteristics of the synapse. Thus, the development of microscopy methods that allow high throughput quantification and measurement of PSDs is a contemporary need in the field of neurobiology. While the gold standard for measurement of sub-micrometer structures remains electron microscopy (EM), this method is exceedingly laborious and therefore not always feasible. Immunohistochemistry (IHC) is a much faster technique for identifying biological structures such as PSDs, but the fluorescent images resulting from it have traditionally been harder to interpret and quantify. Here, we report on two new image analysis tools that result in accurate size and density measurements of fluorescent puncta. Anti-PSD-95 staining was used to target synapses. The new technique of vamping, using Volume Assisted Measurement of Puncta in 2 and 3 Dimensions (VAMP2D and VAMP3D) respectively, is based on stereological principles. The fully automated image analysis tool was tested on the same subjects for whom we had previously obtained EM measurements of PSD size and/or density. Based on highly consistent results between data obtained by each of these methods, vamping offers an expedient alternative to EM that can nonetheless deliver a high level of accuracy in measuring sub-cellular structures.

Original languageEnglish (US)
Pages (from-to)97-105
Number of pages9
JournalJournal of Neuroscience Methods
Volume209
Issue number1
DOIs
StatePublished - Jul 30 2012
Externally publishedYes

Keywords

  • Confocal microscopy
  • Electron microscopy
  • Image analysis
  • Postsynaptic densities
  • PSD-95
  • Stereology

ASJC Scopus subject areas

  • Neuroscience(all)

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