Correlative Video Light/Electron Microscopy

Roman S. Polishchuk1, Alexander A. Mironov1

1 Consorzio Mario Negri Sud, S. Maria Imbara (Chieti)
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 4.8
DOI:  10.1002/0471143030.cb0408s11
Online Posting Date:  August, 2001
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Abstract

This unit describes newly developed methods that allow the examination of living cells by time‐lapse analysis with the subsequent identification of the just‐observed organelle under an electron microscope. To understand how such cellular functions, such as intracellular traffic, cytokinesis, and cell migration, are organized and executed in vivo, it is most useful to observe living cells in real time with the spatial resolution afforded by electron microscopy (EM). Most suitable for this is a conceptually simple, yet powerful, method called correlative video light/electron microscopy (CVLEM), by which observations of the in vivo dynamics and the ultrastructure of intracellular objects can indeed be combined to achieve the above‐mentioned result. This unit describes this methodology, illustrates the type of questions that the CVLEM approach was designed to address, and discusses the expertise required for successful application of the technique.

     
 
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Table of Contents

  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1:

  Materials
  • Cells of interests
  • cDNA encoding an appropriate GFP fusion protein
  • Fixative A (see recipe)
  • Fixative B (see recipe)
  • PBS ( appendix 2A)
  • Blocking solution (see recipe)
  • Primary antibody to label structure of interest
  • Monovalent Fab fragments of secondary antibody conjugated with horseradish peroxidase (HRP; Rockland) or Nanogold (Nanoprobes)
  • 1% (w/v) glutaraldehyde in 0.2 M HEPES buffer, pH 7.3
  • Gold enhancement mixture (see recipe)
  • DAB solution (see recipe)
  • 2% (w/v) OsO 4 (Electron Microscopy Sciences) in water
  • 3% (w/v) potassium ferrocyanide in 0.2 M cacodylate buffer
  • 0.2 M cacodylate buffer, pH 7.4 (see recipe)
  • 50%, 70%, 90%, and 100% (v/v) ethanol
  • Epoxy resin (see recipe)
  • 8‐mm resin cylinder prepared in advance from a cylindrical mold
  • 35‐mm MatTek petri dishes with CELLocate coverslip and map of CELLocate grid (MatTek)
  • Inverted fluorescence microscope
  • Multiphoton microscope, laser‐scanning confocal microscope, or digitalized fluorescence inverted microscope capable of acquiring a time‐lapse series of images by computer
  • 60°C oven
  • Ultramicrotome with sample holder, glass knife, and diamond knife
  • Eyelashes
  • Pick‐up loop (Agar)
  • Adjustable‐angle laboratory clamps
  • Slot grids covered with carbon‐formvar supporting film (Electron Microscopy Sciences, Agar)
  • Electron microscope
  • Software for three‐dimensional reconstruction from serial sections
  • Additional reagents and equipment for transfection ( appendix 3A)
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Figures

Videos

Literature Cited

Literature Cited
   Brown, W.J. and Farquhar, M.G. 1989. Immunoperoxidase methods for the localization of antigens in cultured cells and tissue sections by electron microscopy. Methods Cell Biol. 31:553‐569.
   Burry, R.W., Vandre, D.D., and Hayes, D.M. 1992. Silver enhancement of gold antibody probes in pre‐embedding electron microscopic immunocytochemistry. J. Histochem. Cytochem. 40:1849‐1856.
  Deerinck, T.J., Martone, M.E., Lev‐Ram, V., Green, D.P., Tsien, R.Y., Spector, D.L., Huang, S., and Ellisman, M.H. 1994. Fluorescence photooxidation with eosin: A method for high resolution immunolocalization and in situ hybridization detection for light and electron microscopy. J. Cell Biol. 126:901‐910.
   Ellenberg, J., Lippincott‐Schwartz, J., and Presley, J.F. 1999. Dual‐colour imaging with GFP variants. Trends Cell Biol. 9:52‐56.
   Ladinsky, M.S., Kremer, J.R., Furcinitti, P.S., McIntosh, J.R., and Howell, K.E. 1994. HVEM tomography of the trans‐Golgi network: Structural insights and identification of a lace‐like vesicle coat. J. Cell Biol. 127:29‐38.
   Lippincott‐Schwartz, J. and Smith, C.L. 1997. Insights into secretory and endocytic membrane traffic using green fluorescent protein chimeras. Curr. Opin. Neurobiol. 7:631‐639.
   Mironov, A.A., Polishchuk, R.S., and Luini, A. 2000. Visualising membrane traffic in vivo by combined video fluorescence and 3‐D‐electron microscopy. Trends Cell Biol. 10:349‐353.
   Perez, F., Diamantopoulos, G.S., Stalder, R., and Kreis, T.E. 1999. CLIP‐170 highlights growing microtubule ends in vivo. Cell 96:517‐527.
   Polishchuk, R.S., Polishchuk, E.V., Marra, P., Buccione, R., Alberti, S., Luini, A., and Mironov, A.A. 2000. GFP‐based correlative light‐electron microscopy reveals the saccular‐tubular ultrastructure of carriers in transit from the Golgi apparatus to the plasma membrane. J. Cell Biol. 148:45‐58.
   Pollok, B.A. and Heim, R. 1999. Using GFP in FRET‐based applications. Trends Cell Biol. 9:57‐60.
   Powell, R.D., Halsey, C.M., and Hainfeld, J.F. 1998. Combined fluorescent and gold immunoprobes: Reagents and methods for correlative light and electron microscopy. Microsc. Res. Tech. 42:2‐12.
   Tokuyasu, K.T. and Maher, P.A. 1987. Immunocytochemical studies of cardiac myofibrillogenesis in early chick embryos. II. Generation of α‐actinin dots within titin spots at the time of the first myofibril formation. J. Cell Biol. 105:2795‐2801.
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