Basic Confocal Microscopy

Carolyn L. Smith1

1 National Institute of Neurological Disorders and Stroke, Bethesda, Maryland
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 4.5
DOI:  10.1002/0471143030.cb0405s01
Online Posting Date:  May, 2001
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Abstract

This comprehensive overview unit introduces the reader to confocal microscopy from the basic principles of imaging and optical sectioning, to selection of laser, practical guidelines for fixation, choice of fluorophore, control samples, and mounting the sample. There are also suggestions for optimizing the imaging parameters.

     
 
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Table of Contents

  • Basis of Optical Sectioning
  • Types of Confocal Microscopes
  • Practical Guidelines
  • Figures
  • Tables
     
 
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Materials

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Figures

Literature Cited

Literature Cited
   Amos, W.B., 1995. Appendix 1: Optical units. In Handbook of Biological Confocal Microscopy, 2nd ed. (J. Pawley ed.) pp. 579‐580. Plenum, New York.
   Art, J.J. and Goodman, M.B. 1993. Rapid scanning confocal microscopy. In Cell Biological Applications of Confocal Microscopy (B. Matsumoto, ed.) pp. 47‐78. Academic Press, San Diego.
   Bacallao, R., Kiai, K., and Jesaitis, L. 1995. Guiding principles of specimen preservation for confocal fluorescence microscopy. In Handbook of Biological Confocal Microscopy, 2nd ed. (J. Pawley, ed.) pp. 311‐326. Plenum New York.
   Brelje, T.C., Wessendorf, M.W., and Sorenson, R.L. 1993. Multicolor laser scanning confocal immunofluorescence microscopy: Practical applications and limitations. In Cell Biological Applications of Confocal Microscopy (B. Matsumoto, ed.) pp. 98‐182. Academic Press, San Diego.
   Bridgman, P.C. and Reese, T. 1984. The structure of cytoplasm in directly frozen cultured cells. 1. Filamentous meshworks and the cytoplasmic ground substance. J. Cell. Biol. 99: 1655‐1668.
   Callaway, E.M. and Katz, L.C. 1993. Photostimulation using caged glutamate reveals functional circuitry in living brain slices. Proc. Natl. Acad. Sci. U.S.A. 90: 7661‐7665.
   Centonze, V. and Pawley, J., 1995. Tutorial on practical confocal microscopy and use of the confocal test specimen. In Handbook of Biological Confocal Microscopy, 2nd ed. (J. Pawley, ed.) pp. 549‐570. Plenum, New York.
   Cole, N., Smith, C., Sciaky, N., Terasaki, M., Edidin, M., and Lippincott‐Schwartz, J. 1996. Diffusional mobility of Golgi proteins in membranes of living cells. Science 237: 797‐801.
   Denk, W., Piston, D.W., and Webb, W.W. 1995. Two‐photon molecular excitation in laser‐scanning microscopy. In Handbook of Biological Confocal Microscopy, 2nd ed. (J. Pawley, ed.) pp. 445‐458. Plenum, New York.
   Ellenberg, J., Siggia, E.D., Moreira, J.E., Smith, C.L., Presley, J.F., Worman, J.J., and Lippincott‐Schwartz, J. 1997. Nuclear membrane dynamics and reassembly in living cells: Targeting of an inner nuclear membrane protein in interphase and mitosis. J. Cell Biol. 138: 1193‐1206.
   Giloh, H. and Sedat, J.W. 1982. Fluorescence microscopy: Reduced photobleaching of rhodamine and fluorescein protein conjugates by n‐propyl gallate. Science 217: 1252‐1255.
  Inoué, S. and Spring, K.R. 1997. Video Microscopy: The Fundamentals, 2nd ed. Plenum, New York.
   Johnson, G.D., Davidson, R.S., McNamee, K.C., Russell, G., Goodwin, D., and Holborow, E.J. 1982. Fading of immunofluorescence during microscopy: A study of the phenomenon and its remedy. J. Immunol. Methods. 55:231‐242.
   Kamabadur, R., Koizumi, K., Stivers, C., Nagle, J., Poole, S., and Odenwald, W. 1998. Regulation of POU genes by castor and hunchback establishes layered compartments in the Drosophila CNS. Genes & Devel. 12: 246‐260.
   Keller, E. 1995. Objective lenses for confocal microscopy. In Handbook of Biological Confocal Microscopy, 2nd ed. (J. Pawley, ed.) pp. 111‐126. Plenum, New York.
   Kino, G.S. 1995. Intermediate optics in Nipkow disk microscopes. In Handbook of Biological Confocal Microscopy, 2nd ed. (J. Pawley, ed.) pp. 155‐166. Plenum, New York.
   Majilof, L. and Forsgren, P. 1993. Confocal microscopy: Important considerations for accurate imaging. In Cell Biological Applications of Confocal Microscopy (B. Matsumoto, ed.) pp. 79‐97. Academic Press, San Diego.
  Matsumoto, B. (ed.) 1993. Cell Biological Applications of Confocal Microscopy. Academic Press, London.
   Sandison, D.R., Williams, R.M., Wells, K.S., Strickler, J., and Webb, W.W. 1995. Quantitative fluorescence confocal laser scanning microscopy. In Handbook of Biological Confocal Microscopy, 2nd ed. (J. Pawley, ed.) pp. 39‐54. Plenum, New York.
   Shotton, D.M. 1993. Electronic acquisition of light microscope images. In Electronic Light Microscopy (D.M. Shotton, ed.) pp. 1‐38. Wiley‐Liss, New York.
   Svobodoa, K., Tank, D.W., and Denk, W. 1996. Direct measurement of coupling between dendritic spines and shaft. Science 272: 716‐719.
   Terasaki, M. and Dailey, M.E. 1995. Confocal microscopy of living cells. In Handbook of Biological Confocal Microscopy, 2nd ed. (J. Pawley, ed.) pp. 327‐346. Plenum, New York.
   Tsien, R.Y. and Bacskai, B.J., 1995. Video‐rate confocal microscopy. In Handbook of Biological Confocal Microscopy, 2nd ed. (J. Pawley, ed.) pp. 459‐478. Plenum, New York.
   Tsien, R.Y. and Waggoner, A. 1995. Fluorophores for confocal microscopy. In Handbook of Biological Confocal Microscopy, 2nd ed. (J. Pawley, ed.) pp. 267‐280. Plenum, New York.
   Waterman‐Storer, C.M., Sanger, J.W., and Sanger, J.M. 1993. Dynamics of organelles in the mitotic spindles of living cells: Membrane and microtubule interactions. Cell Motil. Cytoskel. 26: 19‐39.
   Webb, R.H. and Dorey, C.K. 1995. The pixilated image. In Handbook of Biological Confocal Microscopy, 2nd ed. (J. Pawley, ed.) pp. 55‐68. Plenum New York.
   Wedekind, P., Kubitscheck, U., Heinrich, O., and Peters, R. 1996. Line‐scanning microphotolysis for diffraction‐limited measurements of lateral diffusion. Biophys. J. 71: 1621‐1632.
   Wessendorf, M.W. and Brelje, T.C. 1993. Multicolor fluorescence microscopy using the laser‐scanning confocal microscope. Neuroprotocols 2: 121‐140.
  Wilson, T. (ed.) 1990. Confocal Microscopy. Academic Press, London.
   Wilson, T. 1995. The role of the pinhole in confocal imaging system. In Handbook of Biological Confocal Microscopy, 2nd ed. (J. Pawley, ed.) pp. 167‐182. Plenum, New York.
Key References
   Inoué and Spring. 1997. See above.
  Covers basics of light microscopy, video microscopy, and much more.
   Matsumoto. 1993. See above.
  Good source of practical information.
  Pawley, J. (ed.) 1995. Handbook of Biological Confocal Microscopy, 2nd ed. Plenum, New York.
  Excellent source of theoretical and technical information.
  Russ, J. 1995. The Image Processing Handbook. CRC Press, Boca Raton, Fla.
  Excellent source of information about digital image processing.
  Shotton, D.M. (ed.) 1993. Electronic Light Microscopy. John Wiley and Sons, New York.
  Covers many aspects of light microscopy, including confocal microscopy.
   Wilson, 1990. See above.
  Good source of theoretical background information.
Internet Resources
  zippy.nimh.nih.gov
  Use to obtain NIH Image via FTP.
  http://optics.jct.ac.il/∼aryeh/Spectra
  Source of excitation and emission spectra for common fluorophores.
  http://rsb.info.nih.gov/nih‐image/
  Use to obtain NIH Image.
  http://www.probes.com
  Molecular Probes web site, including product listings and much more.
  http://www.mwrn
  Microworld Resources, a comprehensive list of microscopy products and vendors.
  listserv@ubvm.cc.buffalo.edu
  Confocal e‐mail listserv network.
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