Confocal Microscopy

Peter Ekström1

1 University of Lund, Lund, Sweden
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 2.8
DOI:  10.1002/0471140856.tx0208s05
Online Posting Date:  May, 2001
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Abstract

Confocal microscopy allows visualization of optical sections of material labeled with fluorescence or reflecting probes. By excluding light from planes above and below the plane of focus it is possible to obtain sharp images of objects deep within sections. Sections can be combined to construct three‚Äźdimensional images. This unit provides an overview and introduction to confocal microscopy.

     
 
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Table of Contents

  • Image Capturing
  • Practical and Theoretical Limitations
  • Practical Guidelines
  • Acknowledgements
  • Figures
  • Tables
     
 
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Materials

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Figures

Videos

Literature Cited

Literature Cited
   Art, J. 1995. Photon detectors for confocal microscopy. In Handbook of Biological Confocal Microscopy, 2nd ed. (J.B. Pawley, ed.) pp. 183‐196. Plenum Press, New York.
   Baccalao, 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.B. Pawley, ed.) pp. 311‐325. Plenum Press, New York.
   Brandon, C. 1987. Cholinergic neurons in the rabbit retina: Dendritic branching and ultrastructural connectivity. Brain Res. 426:119‐130.
   Brismar, H. and Ulfhake, B. 1997. Fluorescence lifetime measurements in confocal microscopy of neurons labeled with multiple fluorophores. Nature Biotechnol. 15:373‐377.
   Carlsson, K. 1991. The influence of specimen refractive index, detector signal integration, and non‐uniform scan speed on the imaging properties in confocal microscopy. J. Microsc. 163:167‐178.
   Carlsson, K. 1993. Utraviolet‐excited fluorescence in confocal imaging. Neuroprotocols 2:141‐149.
   Carlsson, K., Åslund, N., Mossberg, K., and Philip, J. 1994. Simultaneous confocal recording of multiple fluorescent labels with improved channel separation. J. Microsc. 176:287‐299.
   Cogswell, C.J. and Larkin, K.G. 1995. The specimen illumination path and its effect on image quality. In Handbook of Biological Confocal Microscopy, 2nd ed. (J.B. Pawley, ed.) pp. 127‐137. Plenum Press, New York.
   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.B. Pawley, ed.) pp. 445‐458. Plenum Press, New York.
   Donaldson, J. 1998. Immunofluorescent staining. In Current Protocols in Cell Biology (J.S. Bonifacino, M. Dasso, J.B. Harford, J. Lippincott‐Schwartz, and K.M. Yamada, eds.) pp. 4.31‐4.36. John Wiley & Sons, New York.
   Florijn, R.J., Slats, J., Tanke, H.J., and Raap, A.K. 1995. Analysis of antifading reagents for fluorescence microscopy. Cytometry 19:177‐182.
   Gratton, E. and vande Ven, M.J. 1995. Laser sources for confocal microscopy. In Handbook of Biological Confocal Microscopy, 2nd ed. (J.B. Pawley, ed.) pp. 69‐97. Plenum Press, New York.
   Keller, H.E. 1995. Objective lenses for confocal microscopy. In Handbook of Biological Confocal Microscopy, 2nd ed. (J.B. Pawley, ed.) pp. 111‐126. Plenum Press, New York.
   Lindek, S., Stelzer, E.H.K., and Hell, S.W. 1995. Two new high‐resolution confocal fluorescence microscopies (4Pi, Theta) with one‐ and two‐photon excitation. In Handbook of Biological Confocal Microscopy, 2nd ed. (J.B. Pawley, ed.), pp. 417‐430. Plenum Press, New York.
   Pawley, J.B. 1994. Sources of noise in three‐dimensional microscopical data sets. In Three‐Dimensional Confocal Microscopy: Volume Investigation of Biological Specimens (J.K. Stevens, L.R. Mills, and J.E. Trogadis, eds.) pp. 47‐94. Academic Press, San Diego.
   Pawley, J.B. (ed.) 1995a. Handbook of Biological Confocal Microscopy, 2nd ed. Plenum Press, New York.
   Pawley, J.B. 1995b. Fundamental limits in confocal microscopy. In Handbook of Biological Confocal Microscopy, 2nd ed. (J.B. Pawley, ed.) pp. 19‐37. Plenum Press, New York.
   Russ, J.C. 1998. The Image Processing Handbook, 3rd ed. CRC Press, Boca Raton. Fla.
   Sandison, D.R., Piston, D.W., and Webb, W.W. 1994. Background rejection and optimization of signal to noise in confocal microscopy. In Three‐Dimensional Confocal Microscopy: Volume Investigation of Biological Specimens (J.K. Stevens, L.R. Mills. and J.E. Trogadis, eds.), pp. 29‐46, Academic Press, San Diego.
   Shaw, P.J. 1995. Comparison of wide‐field/deconvolution and confocal microscopy for imaging. In Handbook of Biological Confocal Microscopy, 2nd ed. (J.B. Pawley, ed.) pp. 373‐387, Plenum Press, New York.
   Shaw, P.J. and Rawlins, D.J. 1991. Three‐dimensional fluorescence microscopy. Prog. Biophys. Molec. Biol. 56:187‐213.
   Sheppard, C.J.R. and Gu, M. 1992. 3‐D transfer functions in confocal scanning microscopy. In Visualization in Biomedical Microscopies. 3‐D Imaging and Computer Applications (A. Kriete, ed.) pp. 251‐282. VCH Publishers, Weinheim, Germany.
   Shotton, D.M. 1995. Electronic light microscopy: Present capabilities and future prospects. Histochem. Cell Biol. 104:97‐137.
   Tekola, P., Zhu, Q., and Baak, J.P.A. 1994. Confocal laser microscopy and image processing for three‐dimensional microscopy. Technical principles and an application to breast cancer. Prog. Pathol. 25:12‐21.
   Tsien, R.Y. and Waggoner, A. 1995. Fluorophores for confocal microscopy. Photophysics and photochemistry. In Handbook of Biological Confocal Microscopy, 2nd ed. (J.B. Pawley, ed.) pp. 267‐279. Plenum Press, New York.
   Wallén, P., Carlsson, K., and Mossberg, K. 1992. Confocal laser scanning microscopy as a tool for studying the 3D‐morphology of nerve cells. In Visualization in Biomedical Microscopies. 3‐D Imaging and Computer Applications (A. Kriete, ed.) pp. 109‐143. VCH Publishers, Weinheim, Germany
   Webb, R.H. and Dorey, C.K. 1995. The pixilated image. In Handbook of Biological Confocal Microscopy, 2nd ed. (J.B. Pawley, ed.) pp. 55‐67. Plenum Press, New York.
   Wells, S. and Johnson, I. 1994. Fluorescent labels for confocal microscopy. In Three‐Dimensional Confocal Microscopy: Volume Investigation of Biological Specimens (J.K. Stevens, L.R. Mills, and J.E. Trogadis, eds.) pp. 101‐129. Academic Press, San Diego.
   Wessendorf, M.W. and Brelje, T.C. 1993. Multicolor fluorescence microscopy using the laser‐scanning confocal microscope. Neuroprotocols 2:121‐140.
   White, N.S. 1995. Visualization systems for multidimensional CLSM images. In Handbook of Biological Confocal Microscopy, 2nd ed. (J.B. Pawley, ed.) pp. 211‐254. Plenum Press, New York.
   Wilson, T. 1993. Image formation in confocal microscopy. In Electronic Light Microscopy (D.M. Shotton, ed.) pp. 231‐246. John Wiley & Sons, New York.
   Wilson, T. 1995. The role of the pinhole in confocal imaging systems. In Handbook of Biological Confocal Microscopy, 2nd ed. (J.B. Pawley, ed.) pp. 167‐182. Plenum Press, New York.
Key References
   Inoue, S. 1997. Video Microscopy. The Fundamentals. 2nd ed. Plenum Press, New York.
   Important source of information about light microscopy and video microscopy.
   Kriete, A. (ed.). 1992. Visualization in Biomedical Microscopies. 3‐D Imaging and Computer Applications. VCH Publishing, Weinheim, Germany.
   Detailed considerations of computer‐assisted image analysis in various microscopy techniques.
   Matsumoto, B. (ed.). 1993. Cell Biological Applications of Confocal Microscopy. Methods in Cell Biology, Vol. 38. Academic Press, San Diego.
   Useful source of practical information.
   Pawley 1995a. See above.
   Excellent source of information on theoretical and technical aspects of confocal microscopy.
   Russ 1998. See above.
   Excellent source of information about digital image processing.
   Shotton 1995. See above.
   Concise discussion of many aspects of digital imaging in light and confocal microscopy.
   Stevens, J.K., Mills, L.R., and Trogadis, J.E. (eds.). 1994. Three‐Dimensional Confocal Microscopy: Volume Investigation of Biological Specimens. Academic Press, San Diego.
   Covers basic theoretical considerations, gives examples of practical applications, and discusses alternative methods.
   Wilson, T. (ed.). 1990. Confocal Microscopy. Academic Press, London.
   Very useful source of theoretical information on confocal microscopy.
Internet Resources
   http://optics.jct.ac.il/aryeh/Spectra
   Excitation/emission spectra for common fluorophores.
   http://rsb.info.nih.gov/nih-image/
   Use to obtain NIH Image, a powerful image analysis program for Macintosh computers developed by W. Rasband (Research Services Branch, National Institute of Mental Health, NIH).
   http://www.bioimage.org/misc/companies.html
   List of commercial suppliers in optical and electron microscopy.
   http://corn.eng.buffalo.edu/
   Links, literature, and courses.
   http://www.cs.ubc.ca/spider/ladic/confocal.html
   General information and links about confocal microscopy.
   http://www.probes.com/
   Molecular Probes, supplier of fluorescent probes and optical filters. Information about fluorescence characteristics, and numerous useful links.
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