Basic Confocal Microscopy

Carolyn L. Smith1

1 National Institute of Neurological Disorders and Stroke, Bethesda, Maryland
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 2C.1
DOI:  10.1002/9780471729259.mc02c01s01
Online Posting Date:  June, 2006
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

This unit introduces the reader to the basic principles of confocal microscopy and the design and capabilities of current confocal microscopes. The advantages and disadvantages of confocal microscopy as compared to other techniques for fluorescence imaging are described. There are also practical guidelines for sample preparation and optimizing imaging parameters and examples of some of the applications of confocal microscopy.

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Introduction
  • Basis of Optical Sectioning
  • Configuration of an LSCM
  • Practical Guidelines
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

   Ando, R., Hama, H., Yamamoto‐Hino, M., Mizuno, H., and Miyawaki, A. 2002. An optical marker based on the UV‐induced green‐to‐red photoconversion of a fluorescent protein. Proc. Natl. Acad. Sci. U.S.A. 99:12651‐12656.
   Art, J. 1995. Photon detectors for confocal microscopy. In Handbook of Biological Confocal Microscopy, 2nd ed. (J. Pawley, ed.) pp. 183‐196. Plenum, New York.
   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.
   Benham, G. 2002. Practical aspects of objective lens selection for confocal and multiphoton digital imaging techniques. In Cell Biological Applications of Confocal Microscopy, 2nd ed. (B. Matsumoto, ed.) pp. 247‐300. Academic Press, San Diego, Calif.
   Boccacci, P. and Bertero, M. 2002. Image‐restoration methods: Basics and algorithms. In Confocal and Two‐Photon Microscopy: Foundations, Applications, and Advances (A. Diaspro, A., ed.), pp. 253‐269. John Wiley & Sons, Hoboken, N.J.
   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, Calif.
   Brelje, T.C., Wessendorf, M.W., and Sorenson, R.L. 2002. Multicolor laser scanning confocal immunofluorescence microscopy: Practical applications and limitations. In Cell Biological Applications of Confocal Microscopy, 2nd ed. (B. Matsumoto, ed.), pp. 166‐244. Academic Press, San Diego, Calif.
   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.
   Daims, H., Lucker, S., and Wagner, M. 2006. Daime, a novel image analysis program for microbial ecology and biofilm research. Environ. Microbiol. 8:200‐213.
   Diaspro, A. (ed.) 2002. Confocal and Two Photon Microscopy: Foundations, Applications and Advances. Wiley‐Liss, New York.
   Dickinson, M.E., Bearman, G., Tille, S., Lansford, R., and Fraser, S.E. 2001. Multi‐spectral imaging and llinear unmixing add a whole new dimension to laser scanning fluorescence microscopy. Biotechniques 31:1274‐1278.
   Drenkard, E. and Ausubel, F.M. 2002. Pseudomonas biofilm formation and antibiotic resistance are linked to phenotypic variation. Nature 416:740‐743.
   Elphick, G.F., Querbes, W., Jordan, J.A. Gee, G.V., Eash, S., Manley, K., Dugan, A., Stanifer, M., Bhatnagar, A., Koreze, W.K., Roth, B.L., and Atwood, J.W. 2004. The Human Polyomavirus, JCV, uses serotonin receptors to infect cells. Science 306:1380‐1383.
   Ferrari, A., Pellegrini, V., Arcangeli, C., Fittipaldi, A., Giacca, M., and Beltram, F. 2003. Caveolae‐mediated internalization of extracellular HIV‐1 Tat fusion proteins visualized in real time. Mol. Ther. 8:284‐294.
   Forest, T., Barnard, S., and Baines, J.D. 2005. Active intranuclear movement of herpes virus capsids. Nat. Cell Biol. 7:429‐431.
   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.
   Glushakova, S., Yin, D., Li, T., and Zimmerberg, J. 2005. Membrane transformation during malaria parasite release from human red blood cells. Curr. Biol. 15:1645‐1650.
   Inoué, S. and Inoué, T. 2002. Direct‐View High‐Speed Confocal Scanner: The CSU‐10. In Cell Biological Applications of Confocal Microscopy, 2nd ed. (B. Matsumoto, ed.) pp. 88‐128. Academic Press, San Diego, Calif.
   Inoué, S. and Spring, K.R. 1997. Video Microscopy: The Fundamentals, 2nd ed. Plenum, New York.
   Jares‐Erijman, E.A. and Jovin, T.M. 2003. FRET imaging. Nat. Biotechnol. 21:1387‐1395.
   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 Dev. 12:246‐260.
   Karbowski, M., Arnoult, D., Chen, H., Chan, D.C., Smith, C.L., and Youle, R.J. 2004. Quantitation of mitochondrial dynamics by photolabeling of individual organelles shows that mitochondrial fusion is blocked during the Bax activation phase of apoptosis. J. Cell Biol. 164:493‐499.
   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.
   Lippincott‐Schwartz, J., Altan‐Bonnet, N., and Patterson, G.H. 2003. Photobleaching and photoactivation: Following protein dynamics in living cells. Nat. Cell Biol. Suppl:S7‐S14.
   Majlof, L. and Forsgren, P. 2002. Confocal microscopy: Important considerations for accurate imaging. In Cell Biological Applications of Confocal Microscopy 2nd ed. (B. Matsumoto, ed.) pp. 149‐164. Academic Press, San Diego, Calif.
   Matsumoto, B. (ed.) 2002. Cell Biological Applications of Confocal Microscopy. 2nd ed. Academic Press, London.
   McNally, J., Karpova, T., Cooper, J., and Conchello, J. 1999. Three‐dimensional imaging by deconvolution microscopy. Methods 19:373‐385.
   McNally, J.G. and Smith, C.L. 2002. Photobleaching by confocal microscopy. In Confocal and Two Photon Microscopy: Foundations, Applications and Advances (A. Diaspro, ed.) pp. 525‐538. Wiley‐Liss, New York.
   Nerbonne, J.M. 1996. Caged compounds: Tools for illuminating neuronal responses and connections. Curr. Opin. Neurobiol. 6:379‐386.
   Patterson, G.H. and Lippincott‐Schwartz, J. 2002. A photoactivatable GFP for selective photolabeling of proteins and cells. Science 297:1873‐1877.
   Perrin, A.J., Jiang, X., Birmingham, C.L., So, N.S., Burmell, J.H. 2004. Recognition of bacteria in the cytosol of mammalian cells by the ubiquitin system. Curr. Biol. 14:806‐811.
   Rani, S.A., Pitts, B., and Stewart, P.S. 2005. Rapid diffusion of fluorescent tracers into Staphylococcus epidermis biofilms visualized by time lapse microscopy. Antimicrob. Agents Chemother. 49:728‐732.
   Roux, P., Munter, S., Frischknecht, F., Herbomel, P., and Shorte, P. 2004. Focusing light on infection in four dimensions. Cell. Microbiol. 6:333‐343.
   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.
   Satpute‐Krishnan, P., DeGiorgis, J.A., and Bearer, E.L. 2003. Fast anterograde transport of Herpes Simplex Virus: Role for the amyloid precursor protein of Alzheimer's disease. Aging Cell 2:305‐318.
   Shaner, N.C., Campbell, R.E., Steinbach, P.A., Giepmans, B.N., Palmer, A.E., and Tsien, R.Y. 2004. Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. Red fluorescent protein. Nat. Biotechnol. 22:1567‐1572.
   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.
   Sprague, B.L. and McNally, J.G. 2005. FRAP analysis of binding: Proper and fitting. Trends Cell Biol. 15:84‐91.
   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 Waggoner, A. 1995. Fluorophores for confocal microscopy. In Handbook of Biological Confocal Microscopy, 2nd ed. (J. Pawley, ed.) pp. 267‐280. Plenum, New York.
   Viachou, D., Zimmermann, T., Cantera, R., Janse, C.J., Waters, A.P., and Kafatos, F.C. 2004. Real‐time, in vivo analysis of malaria ookinete locomotion and mosquito midgut invasion. Cell Microbiol. 6:671‐685.
   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. Cytoskeleton 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.
   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.
   Wouterlood, F.G. 2005. 3‐D reconstruction of neurons from multichannel confocal laser scanning image series. In Current Protocols in Neuroscience (J.N. Crawley, C.R. Gerfen, M.A. Rogawski, D.R. Sibley, P. Skolnick, and S. Wray, eds) pp. 2.8.1‐2.2.8.16. John Wiley & Sons, Hoboken, N.J.
   Wouters, F.S., and Bastiaens, P.I.H. 2000. Imaging protein‐protein interactions by fluorescence resonance energy transfer (FRET) microscopy. In Current Protocols in Cell Biology (J.S. Bonifacino, M. Dasso, J.B. Harford, J. Lippincott‐Schwartz, and K.M. Yamada, eds.) pp. 17.1.1‐17.1.15. John Wiley & Sons, Hoboken, N.J.
   Zhang, J., Campbell, R.E, Ting, A., and Tsien, R.Y. 2002. Creating new fluorescent probes for cell biology. Nature Rev. Mol. Cell Biol. 3:906‐918.
Key References
   Inoué and Spring, 1997. See above.
  Covers the basics of light microscopy, video microscopy, and much more.
   Matsumoto, 2002. See above.
  Good source of practical information about confocal imaging.
   Pawley, J. (ed.) 1995. See above.
  Comprehensive reference book on confocal microscopy.
   Russ, J. 2002. The Image Processing Handbook. 4th edition. CRC Press, Boca Raton, Fla.
  Guide to digital image processing.
Internet Resources
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library