Unit

UNIT 2.8 3-D Reconstruction of Neurons from Multichannel Confocal Laser Scanning Image Series

  1. Floris G. Wouterlood

Published Online: 1 AUG 2005

DOI: 10.1002/0471142301.ns0208s32

Current Protocols in Neuroscience

Current Protocols in Neuroscience

How to Cite

Wouterlood, F. G. 2005. 3-D Reconstruction of Neurons from Multichannel Confocal Laser Scanning Image Series. Current Protocols in Neuroscience. 32: 2.8:2.8.1–2.8.16.

Author Information

  1. Vrije Universiteit Medical Center, Amsterdam, The Netherlands

Publication History

  1. Published Online: 1 AUG 2005
  2. Published Print: JUL 2005

This is not the most recent version of the article. View current version (10 APR 2014)

Abstract

A confocal laser scanning microscope (CLSM) collects information from a thin, focal plane and ignores out-of-focus information. The operator configures separate channels (laser, filters, detector settings) for each fluorochrome used in a particular experiment. Then, 3-D reconstructions are made from Z-series of confocal images: one series per channel. Channel signal separation is extremely important and measures to avoid bleaching are vital. Post-acquisition deconvolution of the image series is then performed to increase resolution. In the 3-D reconstruction program 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 relationship of 3-D-reconstructed structures with respect to structures seen 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 wherever possible.

Keywords:

  • immunofluorescence;
  • neuroanatomical tracing;
  • fluorescence imaging;
  • multiple labeling;
  • visualization