High‐Resolution Multi‐Photon Imaging of Morphological Structures of Caenorhabditis elegans

Gabriele M. Bixel1, Stephanie J.B. Fretham2, Michael Aschner3

1 Tissue Morphogenesis, Max Planck Institute for Molecular Biomedicine, and University of Muenster, Muenster, 2 Department of Biology, Luther College, Decorah, Iowa, 3 Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, New York
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 11.19
DOI:  10.1002/0471140856.tx1119s64
Online Posting Date:  May, 2015
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library


In this protocol, we combine two‐photon excitation fluorescence with nonlinear optical measurements to reconstruct the three‐dimensional architecture of the pharyngeal region and the muscular system of the anterior and mid‐body region of Caenorhabditis elegans (C. elegans). Femto‐second laser pulses excite second‐harmonic generation (SHG) and third‐harmonic generation (THG) signals, which show detailed structural information regarding the organization of myofibrils that are arranged around the central pharynx region. The combination of two‐photon excitation with SHG and THG imaging is a very powerful tool to study cell morphology, microarchitecture, and tissue arrangement in C. elegans. © 2015 by John Wiley & Sons, Inc.

Keywords: C. elegans; multi‐photon microscopy; label‐free imaging; SHG; THG

PDF or HTML at Wiley Online Library

Table of Contents

  • Introduction
  • Basic Protocol 1: C. Elegans Collection, Preparation, and Antibody Labeling
  • Basic Protocol 2: Imaging of C. Elegans with Multi‐Photon Microscopy
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
PDF or HTML at Wiley Online Library


Basic Protocol 1: C. Elegans Collection, Preparation, and Antibody Labeling

  • C. elegans worms on nematode growth media agar plates
  • Bouin's fixative (see recipe)
  • Methanol
  • 2‐mercaptoethanol
  • Liquid nitrogen
  • BT (see recipe)
  • BTB (see recipe)
  • ABA (see recipe)
  • ABA blocking (see recipe)
  • ABB (see recipe)
  • Mouse Anti‐GFP Antibody (Roche)
  • CyTM3‐conjugated anti‐mouse IgG (Jackson ImmunoResearch Laboratories)
  • Fluoromount‐G (SouthernBiotech)
  • Clear nail polish
  • 15‐ml conical tubes
  • 1.7‐ml SliptechTM Microcentrifuge Tubes (Denville Scientific)
  • Additional reagents and equipment required for growth and maintenance of C. elegans (Stiernagle, )

Basic Protocol 2: Imaging of C. Elegans with Multi‐Photon Microscopy

  • Single beam multi‐photon microscope setup (Fig. ; e.g., TriM Scope II, LaVision BioTec)
  • Olympus BX51 WI microscope stand
  • Titanium (Ti): Sapphire laser (e.g. Coherent Scientific Chameleon Ultra II), capable of 680 to 1080 nm emission, 120 fsec pulse width, 80 MHz repetition rate
  • IR light source (e.g., Coherent Chameleon Compact OPO), capable of automated 1000 to 1600 nm wavelength extension, 200 fsec pulse width, 80 MHz repetition rate
  • 20× IR objective lens (e.g., Olympus XLUMPlanFl 20×/1.0 W, working distance of 2.0 mm).
  • Two x–y galvanometric mirrors, for scanning the sample at a rate of up to 1200 lines/s
  • Dichroic mirrors and band‐pass filters: blue (395/11 nm, forward detection), green (525/50 nm, forward detection), and red (620/60 nm, backward detection)
  • Olympus U‐AAC condenser, for collecting emission signals in forward direction
  • High‐sensitivity non‐descanned photomultiplier tube (PMT) detectors: blue channel (e.g., Hamamatsu H67080‐01), green and red channels (e.g., Hamamatsu H67080‐20)
  • LaVision BioTec ImSpector Software, for 3D image acquisition
  • Additional reagents and equipment for optimization of laser sources (McConnell, )
PDF or HTML at Wiley Online Library



Literature Cited

Literature Cited
  Andresen, V., Alexander, S., Heupel, W.M., Hirschberg, M., Hoffman, R.M., and Friedl, P. 2009. Infrared multiphoton microscopy: Subcellular‐resolved deep tissue imaging. Curr. Opin. Biotechnol. 20:54‐62.
  Brenner, S. 1974. The genetics of Caenorhabditis elegans. Genetics 77:71‐94.
  Campagnola, P.J., Millard, A.C., Terasaki, M., Hoppe, P.E., Malone, C.J., and Mohler, W.A. 2002. Three‐dimensional high‐resolution second‐harmonic generation imaging of endogenous structural proteins in biological tissues. Biophys. J. 82:493‐508.
  Denk, W., Strickler, J.H., and Webb, W.W. 1990. Two‐photon laser scanning fluorescence microscopy. Science 248:73‐76.
  Duerr, J.S. 2006. Immunohistochemistry (June 19, 2006). WormBook. ed. The C. elegans Research Community, WormBook, doi/10.1895/wormbook.1.105.1, http://www.wormbook.org.
  Filippidis, G., Gualda, E.J., Mari, M., Troulinaki, K., Fotakis, C., and Tavernarakis, N. 2009. In vivo imaging of cell morphology and cellular processes in Caenorhabditis elegans, using non‐linear phenomena. Micron 40:876‐880.
  Friedl, P. 2004. Dynamic imaging of cellular interactions with extracellular matrix. Histochem. Cell Biol. 122:183‐190.
  Friedl, P., Wolf, K., von Andrian, U.H., and Harms, G. 2007. Biological second and third harmonic generation microscopy. Curr. Protoc. Cell Biol. 34:4.15:4.15.1‐4.15.21.
  Gualda, E.J., Filippidis, G., Voglis, G., Mari, M., Fotakis, C., and Tavernarakis, N. 2008. In vivo imaging of cellular structures in Caenorhabditis elegans by combined TPEF, SHG and THG microscopy. J. Microsc. 229(Pt 1):141‐150.
  Helmchen, F. and Denk, W. 2005. Deep tissue two‐photon microscopy. Nat. Methods 2:932‐940.
  McConnell, G. 2006. Optimizing laser source operation for confocal and multiphoton laser scanning microscopy. Curr. Protoc. Cytom. 38:2.13:2.13.1‐2.13.7.
  Nonet, M.L., Staunton, J.E., Kilgard, M.P., Fergestad, T., Hartwieg, E., Horvitz, H.R., Jorgensen, E.M., and Meyer, B.J. 1997. Caenorhabditis elegans rab‐3 mutant synapses exhibit impaired function and are partially depleted of vesicles. J. Neurosci. 17:8061‐8073.
  Oheim, M., Beaurepaire, E., Chaigneau, E., Mertz, J., and Charpak, S. 2001. Two‐photon microscopy in brain tissue: Parameters influencing the imaging depth. J. Neurosci. Methods 111:29‐37.
  Rehberg, M., Krombach, F., Pohl, U., and Dietzel, S. 2011. Label‐free 3D visualization of cellular and tissue structures in intact muscle with second and third harmonic generation microscopy. PLoS One 6:e28237.
  Stiernagle, T. 2006. Maintenance of C. elegans (February 11, 2006). WormBook. ed. The C. elegans Research Community, WormBook, doi/10.1895/wormbook.1.101.1, http://www.wormbook.org.
  Weigelin, B., Bakker, G.‐J., and Friedl, P. 2012. Intravital third harmonic generation microscopy of collective melanoma cell invasion. IntraVital 1:32‐43.
  Zipfel, W.R., Williams, R.M., and Webb, W.W. 2003. Nonlinear magic: Multiphoton microscopy in the biosciences. Nat. Biotechnol. 21:1369‐1377.
PDF or HTML at Wiley Online Library