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

Floris G. Wouterlood1

1Vrije Universiteit Medical Center, Amsterdam, The Netherlands

Publication Name: 
Current Protocols in Neuroscience
Unit Number: 
UNIT  2.8
DOI: 
10.1002/0471142301.ns0208s32
Online Posting Date: 
August, 2005
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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

     
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Table of Contents

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

Basic Protocol: 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 Alexa Fluor series of fluorochromes, Molecular Probes; see Table 2.8.1)
  • Histological sections (see Strategic Planning)
  • TetraSpeck microspheres kit (Molecular Probes)
  • Confocal laser scanning microscope (CLSM, e.g., Leica Microsystems, Zeiss, BioRad, Olympus, and Nikon)
  • Computer for viewing high-resolution graphics: SGI Octane workstation (Silicon Graphics, http://www.sgi.com; processor R-10000/175 MHz, 2 Gb working memory) running the Irix Unix-type operating system
  • 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, and with Amira, http://www.amiravis.de/). Amira is available in Linux- and Windows versions. Both FluVR and Amira can run on the same Silicon Graphics workstation as the deconvolution software. FluVR is a so-called volume-rendering program whereas Amira is a surface-rendering program.
       
      Table  2.8.1 Excitation Maximum and Laser Wavelengths for Fluorochromes Used in Confocal Microscopes
      FluorochromeExcitation maximum (nm)aIlluminate with laser wavelength(s) (nm)
      Cy2489488
      Cy3554543, 568
      Cy5649633, 647
      Alexa Fluor 488491488
      Alexa Fluor 546556543
      Alexa Fluor 556577568
      Alexa Fluor 594590594
      Alexa Fluor 633632633, 647
      Alexa Fluor 647650633, 647
      Texas Red595594
       aExcitation maximum provided by the manufacturer.
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Figures

  • Figure 2.8.1
    Confocal images of a 50-µm thick rat entorhinal cortex section containing three fluorochromes. Projection images in single channels (A,B,C) and a composite image (D) of the labeled structures after scanning in sequential mode. (A) Channel #1 (488 nm excitation, fluorochrome Alexa Fluor 488): fibers and boutons labeled with biotinylated dextran amine neuroanatomical tracer (injected in presubiculum). (B) Channel #2 (543 nm excitation, fluorochrome Cy3): neurons and processes containing calretinin (CR). (C) Channel #3 (647 nm excitation, fluorochrome Cy5): neurons and processes containing parvalbumin (PV). (D) A composite image (overlaid projection image) showing all three markers.

    View Image
  • Figure 2.8.2
    (A) Combined radial (XY), and axial (XZ, YZ) composite confocal images (at time of acquisition) of a 100-nm fluorescent bead scanned with the 594-nm laser, 63× glycerin objective, NA 1.3, electronic zoom 8. While in the XY image the bead appears a perfect sphere, the image is distorted in the XZ and YZ directions, illustrating that the axial component of the PSFi is different from the radial component. (B) 3-D reconstruction of this bead. (C) 4-µm multifluorescent beads scanned in two channels: 488 nm and 633 nm. Whereas in the radial image (XY) there is no observable pixel shift, an axial pixel shift occurs. The result of the axial pixel shift is that the projection of the 488-nm image is slightly axially displaced compared with the 633-nm image (inset right lower corner). (D) The same appears after 3-D reconstruction of these beads (no deconvolution) with representation of the image in channel #1 with a wire frame, and representation of channel #2 in solid color. Inset shows the XY view (dimensions compressed for the sake of illustration).

    View Image
  • Figure 2.8.3
    Excitation crosstalk and its removal. (A) Excitation spectra of the fluorochromes Cy3 and Cy5 (source: Bio-Rad). The asterisk indicates an overlap of the Cy5 curve with the Cy3 curve. This overlap causes illumination of double-stained sections with the 568-nm laser to excite Cy5 to ~15% of maximal intensity. (B) Image obtained after scanning with the 568-nm laser of sections containing Cy3 and Cy5 (same sections as in Fig. 2.8.1; Alexa Fluor 488 does not suffer from excitation crosstalk in this channel). The PV neuron and its processes (labeled with Cy5) are clearly visible (arrows). (C) Dye separation is a post-acquisition computer processing step that removes Cy5 crosstalk based on its excitation-emission spectrum. (D) Result of the post-acquisition dye separation: the Cy5 crosstalk is completely eliminated from this 568-nm channel.

    View Image
  • Figure 2.8.4
    The deconvolution program, Huygens-II, is used to statistically improve images while reducing background. (A) Acquired Z-series from a 488-nm channel (projection image, see Fig. 2.8.1; fibers labeled with neuroanatomical tracer). There is considerable background, and the fibers and boutons appear blurred. Panel C is an enlarged portion of panel A. (B) The same Z-series after deconvolution with an improvement to the signal-to-noise ratio, blur is removed and the fibers and boutons appear sharper. Details in the background become visible (e.g., in the encircled area).

    View Image
  • Figure 2.8.5
    3-D reconstructions with the Amira surface-rendering program after image acquisition (Fig. 2.8.1), dye separation (Fig. 2.8.3), and deconvolution (Fig. 2.8.4). Triple staining. (A) Reconstruction of fibers (488-nm channel; 488 Z-series) (green). (B) Reconstruction of calretinin cells (543-nm channel; 543 Z-series) (red). (C) Reconstruction of parvalbumin cells (633-nm channel; 633 Z-series) (blue). (D) Final 3-D reconstruction: merge of the images of the three individual color channels.

    View Image
  • Figure 2.8.6
    High-magnification 3-D reconstructions after deconvolution. (A) Fibers were labeled with a neuroanatomical tracer (633-nm fluorochrome, channel #1), and (B) dendrites were labeled via pericellular injection with neurobiotin (488-nm fluorochrome, channel #2). (C) Final surface-rendered 3-D reconstruction after merge of channels #1 and #2. B1-B4 are axon terminals; D1 and D2 are dendrites. Rotation (in real time) of the reconstruction and inspection reveals that several boutons are in direct contact with the dendrites whereas others are not. Panel D shows two frames of a volume rendering, using the same images as for panels A through C. Each frame is a separate 3-D rendering, calculated at a different angle of view. Reconstruction made with FluVR.

    View Image

Literature Cited

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.
    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.
    Gaunt, W.A. and Gaunt, P.N. 1978. Three-Dimensional Reconstruction in Biology. Pitman Medical Press, Kent, U.K.
    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.
    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. Immunohisto. M. M. 10:85-102.
    Wouterlood, F.G., Böckers, T., and Witter, M.P. 2003. Synaptic contacts between identified neurons visualized in the confocal laserscanning microscope. Neuroanatomical tracing combined with immunofluorescence detection of postsynaptic density proteins and target neuron-markers. J. Neurosci. Methods 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.
 Key References
    Pawley, J.B. 1995. Handbook of Biological Confocal Microscopy, 2nd ed. Plenum Press, New York.
     
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