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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Abstract

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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Materials

Basic Protocol 1: 3‐D Reconstruction of Neurons from Multichannel Confocal Laser Scanning Image Series

  Materials
  • 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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Figures

Videos

Literature Cited

  Avila, R., He, T., Hong, L., Kaufman, A., Pfister, H., Silva, C., Sobierajski, L., and Wang, S. 1994. VolVis: A Diversified Volume Visualization System. IEEE Visualization Proceedings, pp. 31‐38. IEEE Computer Society Press, Washington, D.C.
  Bates, M., Huang, B., and Zhuang, X. 2008. Super‐resolution microscopy by nanoscale localization of photo‐switchable fluorescent probes. Curr. Opin. Chem. Biol. 12:505‐514.
  Bertero, M., Boccacci, P., Brakenhoff, G.J., Malfanti, F., and van der Voort, H.T.M. 1990. Three‐dimensional image restoration and super‐resolution in fluorescence confocal microscopy. J. Microsc. 157:3‐20.
  Chen, H., Swedlow, J.R., Grote, M., Sedat, J.W., and Algard, D.A. 1996. The collection, processing and display of digital three‐dimensional images of biological specimens. In Handbook of Biological Confocal Microscopy (J.B. Pawley, ed.) pp. 197‐210. Plenum Press, New York.
  Ewers, H. 2012. Nano resolution optical imaging through localization microscopy. In Cellular Imaging Techniques for Neuroscience and Beyond (F.G. Wouterlood, ed.) pp. 82‐100. Academic Press, New York.
  Gaunt, W.A. and Gaunt, P.N. 1978. Three‐Dimensional Reconstruction in Biology. Pitman Medical Press, Kent, U.K.
  Hell, S.W. and Wichmann, J. 1994. Breaking the diffraction resolution limit by stimulated emission: Stimulated‐emission‐depletion fluorescence microscopy. Opt. Lett. 19:780‐782.
  Inoué, S. 1995. Foundations of confocal scanned imaging in light microscopy. In Handbook of Biological Confocal Microscopy (J.B. Pawley, ed.) pp. 1‐14. Plenum Press, New York.
  Kajiwara, R., Wouterlood, F.G., Sah, A., Boekel, A.J., Baks‐te Bulte, L.T.G., and Witter, M.P. 2008. Convergence of entorhinal and CA3 inputs onto pyramidal neurons and interneurons in hippocampal area CA1. An anatomical study in the rat. Hippocampus 18:266‐280.
  Kononenko, N.L. and Witter, M.P. 2011. Presubiculum layer III conveys retrosplenial input to the medial entorhinal cortex. Hippocampus 22:881‐895.
  Nägerl, UV. 2012. Beyond Abbe's resolution barrier: STED microscopy. In Cellular Imaging Techniques for Neuroscience and Beyond (F.G. Wouterlood, ed.) pp. 35‐54. Academic Press, New York.
  Peter, K. 1906. Die Methoden der Rekonstruktion, pp. 1‐140. Gustav Fischer Verlag, Jena, Germany.
  Platt, J.L. and Michael, A.F. 1983. Retardation of fading and enhancement of intensity of immunofluorescence by p‐phenylenediamine. J. Histochem. Cytochem. 31:840‐842.
  Schook, P. 1980. Morphogenetic Movements as Studied in the Embryonic Chick Eye: An Analysis with the Help of Light Microscopy, Three‐Dimensional Reconstruction, Transmission Electron Microscopy, Scanning Electron Microscopy. Thesis. Swets & Zeitlinger, Lisse, The Netherlands.
  Snyder, D.L., Schulz, T.J., and O'Sullivan, J.A. 1992. Deblurring subject to nonnegativity constraints. IEEE Trans. Sign. Proc. 40:1143‐1150.
  Staiger, J.F., Schubert, D., Zuschratter, W., Kotter, R., Luhmann, H.J., and Zilles, K. 2002. Innervation of interneurons immunoreactive for VIP by intrinsically bursting pyramidal cells and fast‐spiking interneurons in infragranular layers of juvenile rat neocortex. Eur. J. Neurosci. 16:11‐20.
  van der Voort, H.T.M. and Strasters, K.C. 1995. Restoration of confocal images for quantitative image analysis. J. Microsc. 158:43‐45.
  van Haeften, T., Baks‐te‐Bulte, L., Goede, P.H., Wouterlood, F.G., and Witter, M.P. 2003. Morphological and numerical analysis of synaptic interactions between neurons in deep and superficial layers of the entorhinal cortex of the rat. Hippocampus 13:943‐952.
  Vinkenoog, M., van den Oever, M., Uylings, H.B.M., and Wouterlood, F.G. 2004. Random or selective neuroanatomical connectivity. Study of the distribution of fibers over two populations of identified interneurons in cerebral cortex. Brain Res. Brain Res. Protoc. 14:67‐76.
  Wallace, W., Schaefer, L.H., and Swedlow, J.R. 2001. A working person's guide to deconvolution in light microscopy. Biotechniques 31:1076‐1097.
  Wouterlood, F.G., van Haeften T., Blijleven, N., Perez‐Templado, P., and Perez‐Templado, E. 2002. Double‐label confocal laserscanning microscopy, image restoration and real‐time 3D‐reconstruction to study axons in the CNS and their contacts with target neurons. Appl. Immunohistochem. Mol. Morphol. 10:85‐95.
  Wouterlood, F.G., Böckers, T., and Witter, M.P. 2003. Synaptic contacts between identified neurons visualized in the confocal laser scanning microscope. Neuroanatomical tracing combined with immunofluorescence detection of postsynaptic density proteins and target neuron‐markers. J. Neurosci. Meth. 128:129‐142.
  Wouterlood, F.G., van Haeften, T., Eijkhoudt, M., Baks‐te‐Bulte, L., Goede, P.H., and Witter, M.P. 2004. Input from the presubiculum to dendrites of layer‐V neurons of the medial entorhinal cortex of the rat. Brain Res. 1013:1‐12.
  Wouterlood, F.G., Canto, C.B., Aliane, V., Boekel, A.J., Grosche, J., Härtig, W., Beliën, J.A.M., and Witter, M.P. 2007. Co‐expression of vesicular glutamate transporters 1and 2, glutamic acid decarboxylase and calretinin in rat entorhinal cortex. Brain Struct. Funct. 212:303‐319.
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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