3‐D Reconstruction of Neurons from Multichannel Confocal Laser Scanning Image Series

Floris G. Wouterlood1

1 Department of Anatomy and Neurosciences, Vrije Universiteit Medical Center, Amsterdam
Publication Name:  Current Protocols in Neuroscience
Unit Number:  Unit 2.8
DOI:  10.1002/0471142301.ns0208s67
Online Posting Date:  April, 2014
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A confocal laser scanning microscope (CLSM) collects information from a thin, focal plane and ignores out‐of‐focus information. Scanning of a specimen, with stepwise axial (Z‐) movement of the stage in between each scan, produces Z‐series of confocal images of a tissue volume, which then can be used to 3‐D reconstruct structures of interest. The operator first configures separate channels (e.g., laser, filters, and detector settings) for each applied fluorochrome and then acquires Z‐series of confocal images: one series per channel. Channel signal separation is extremely important. Measures to avoid bleaching are vital. Post‐acquisition deconvolution of the image series is often performed to increase resolution before 3‐D reconstruction takes place. In the 3‐D reconstruction programs described in this unit, reconstructions can be inspected in real time from any viewing angle. By altering viewing angles and by switching channels off and on, the spatial relationships of 3‐D‐reconstructed structures with respect to structures visualized in other channels can be studied. Since each brand of CLSM, computer program, and 3‐D reconstruction package has its own proprietary set of procedures, a general approach is provided in this protocol wherever possible. Curr. Protoc. Neurosci 67:2.8.1‐2.8.18. © 2014 by John Wiley & Sons, Inc.

Keywords: immunofluorescence; neuroanatomical tracing; fluorescence imaging; multiple labeling; visualization

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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: 3‐D Reconstruction of Neurons from Multichannel Confocal Laser Scanning Image Series
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: 3‐D Reconstruction of Neurons from Multichannel Confocal Laser Scanning Image Series

  • Fluorochromes (e.g., carbocyanine dyes, Cy‐dyes, Amersham Biosciences and Jackson Immunoresearch; or the Alexa Fluor series of fluorochromes, Invitrogen‐Molecular Probes; see Table 2.8.1)
  • Streptavidin conjugates: Alexa Fluor 488, 546, 594, and 633
  • Histological sections (see Strategic Planning)
  • Histological controls:
    • Standard preparation containing intentionally double‐ or triple‐labeled fibers for the purpose of instrument calibration—use before acquisition scan
    • Set of sections single‐stained with one of each of the fluorochromes being used—use to test channel crosstalk
  • TetraSpeck microspheres kit (Invitrogen‐Molecular Probes)
  • Confocal laser scanning microscope (CLSM; e.g., Leica Microsystems, Zeiss, Olympus, Nikon)
  • Computer for viewing high‐resolution graphics: A “gaming” PC with a large amount of working memory and a high‐end graphical card will perform the jobs of deconvolution and 3‐D rendering in a fast, efficient, and cost‐effective way (best results according to our experience are obtained on a fast platform in a Linux operating system environment)
  • Software (see Strategic Planning):
    • Deconvolution software: Huygens II Professional deconvolution software (Scientific Volume Imaging, http://www.svi.nl)—versions of this package are available that run in Linux‐ and Microsoft Windows environments (Huygens Essentials for Windows)
    • 3‐D rendering software: FluVR module of Huygens‐II (Scientific Volume Imaging, http://www.svi.nl) for volume rendering, or with Amira (http://www.vsg3d.com/amira) or Imaris (http://www.bitplane.com) for volume and surface rendering—Amira is available in Linux‐ and Windows versions; Imaris is available for Windows and Macintosh platforms (all software may run on the same platform as the deconvolution software)
Table 2.8.1   MaterialsExcitation Maximum and Laser Wavelengths for Fluorochromes Used in Confocal Microscopes

Fluorochrome Excitation maximum (nm) a Illuminate with laser wavelength(s) (nm)
Cy2 489 488
Cy3 554 543, 568
Cy5 649 633, 647
Alexa Fluor 488 491 488
Alexa Fluor 546 556 543
Alexa Fluor 556 577 568
Alexa Fluor 594 590 594
Alexa Fluor 633 632 633, 647
Alexa Fluor 647 650 633, 647
Texas Red 595 594

 aExcitation maximum provided by the manufacturer.
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Literature Cited

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Key References
  Pawley, J.B. 2006. Handbook of Biological Confocal Microscopy, 5th ed. Plenum Press, New York.
  First and foremost acknowledged work covering all aspects of confocal imaging.
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