Automatic Dendritic Spine Quantification from Confocal Data with Neurolucida 360

Dara L. Dickstein1, Daniel R. Dickstein2, William G. M. Janssen2, Patrick R. Hof1, Jacob R. Glaser3, Alfredo Rodriguez3, Nate O'Connor3, Paul Angstman3, Susan J. Tappan3

1 Computational Neurobiology and Imaging Center, Icahn School of Medicine at Mount Sinai, New York, New York, 2 Friedman Brain Institute, Icahn School of Medicine at Mount Sinai, New York, New York, 3 MBF Bioscience, Williston, Vermont
Publication Name:  Current Protocols in Neuroscience
Unit Number:  Unit 1.27
DOI:  10.1002/cpns.16
Online Posting Date:  October, 2016
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Determining the density and morphology of dendritic spines is of high biological significance given the role of spines in synaptic plasticity and in neurodegenerative and neuropsychiatric disorders. Precise quantification of spines in three dimensions (3D) is essential for understanding the structural determinants of normal and pathological neuronal function. However, this quantification has been restricted to time‐ and labor‐intensive methods such as electron microscopy and manual counting, which have limited throughput and are impractical for studies of large samples. While there have been some automated software packages that quantify spine number, they are limited in terms of their characterization of spine structure. This unit presents methods for objective dendritic spine morphometric analysis by providing image acquisition parameters needed to ensure optimal data series for proper spine detection, characterization, and quantification with Neurolucida 360. These protocols will be a valuable reference for scientists working towards quantifying and characterizing spines. © 2016 by John Wiley & Sons, Inc.

Keywords: dendritic spines; neurons; confocal microscopy; automated quantification

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Table of Contents

  • Introduction
  • Basic Protocol 1: Imaging of Fluorescently Labeled Dendritic Segments
  • Support Protocol 1: Post‐Processing Deconvolution
  • Basic Protocol 2: Dendritic Spine Modeling and Reconstruction with Neurolucida 360
  • Alternate Protocol 1: Spine Classification
  • Basic Protocol 3: Imaging Complete Neurons
  • Basic Protocol 4: Neuron Reconstruction Using Neurolucida 360
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Imaging of Fluorescently Labeled Dendritic Segments

  • Fluorescently labeled tissue, mounted onto subbed glass slides and coverslipped using anti‐fade fluorescence mounting media
  • Immersion media appropriate for the specifications of the system
  • Confocal microscope equipped with low and high NA objective lenses
  • Image capture software

Support Protocol 1: Post‐Processing Deconvolution

  • Confocal images (see protocol 1)
  • AutoQuant Image Deconvolution Software (Media Cybernetics)

Basic Protocol 2: Dendritic Spine Modeling and Reconstruction with Neurolucida 360

  • Image data with known scaling either embedded within the file or written in your laboratory notebook (see protocol 2Support Protocol)
  • Neurolucida 360 v2.7 or later (MBF Bioscience)
  • Neurolucida 360 Explorer (MBF Bioscience)

Alternate Protocol 1: Spine Classification

  • Data file from Neurolucida 360 with modeled dendritic spines
  • Neurolucida 360 v2.7 or later (MBF Bioscience)
  • Neurolucida 360 Explorer (MBF Bioscience)

Basic Protocol 3: Imaging Complete Neurons

  • Neuron to be imaged
  • Confocal microscope with 10× objective and 63× or 100× oil‐immersion objective
  • Image capture software

Basic Protocol 4: Neuron Reconstruction Using Neurolucida 360

  • Image data with known scaling either embedded within the file or written in your laboratory notebook
  • Neurolucida 360 v2.7 or later (MBF Bioscience)
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Internet Resources
  Drs. Dickstein and Tappan hosted a webinar on this topic based on a previous version of Neurolucida 360. The recorded version of the webinar is available for viewing here.‐software/zen‐lite.html
  Zen Black image acquisition software described in the protocol. Free versions or free trials are available from the vendor.
  AutoQuant image deconvolution software (Media Cybernetics) described in the protocol. Free versions or free tria4sls are available from the vendor.
  Neurolucida 360 automated neuron reconstruction software (MBF Bioscience) described in the protocol. Free versions or free trials are available from the vendor.
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