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‐dimensional images. This unit provides an overview and introduction to confocal microscopy.

     
 
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Table of Contents

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

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Figures

  •  FigureFigure 2.8.1 (A) Schematic drawing of confocal laser scanning microscope and light path. Laser light passes an excitation filter, by which the appropriate excitation wavelength is selected. The light beam is reflected by a dichroic mirror to the scanning device, into the objective, and is focused onto the specimen. The specimen is scanned line by line in a raster system. Fluorescence from the specimen travels back through the objective, via the scanning device, through the dichroic mirror to the pinhole aperture. An emission filter selects the appropriate emission wavelength range, and an ocular focuses light from the focal plane in the specimen on the small pinhole aperture. Light passing through the pinhole is captured by a photomultiplier tube. Simultaneous two‐wavelength scanning requires that the dichroic mirror reflects both excitation wavelengths and lets both emission wavelengths through. Also, the two emission wavelengths passing through the pinhole are separated by a beamsplitter, and captured by two photomultipliers. (B) The confocal principle. The parallel light of the laser beam is focused in the specimen. Light from the focal plane is focused on the pinhole aperture, whereas light from planes above or below focus either in front of, or behind, the pinhole, thus contributing only low photon flux through the pinhole. (C) The numerical aperture (NA) of an objective is given by the formula NA = ηsinα, where η is the refractive index of the immersion medium between the objective and the specimen, and α is half the opening angle of the objective. (D) Chromatic aberration. Light of different wavelengths have different focal lengths with a given lens. Unless this is corrected for (e.g., by using high‐quality planapochromat lenses), scanning at different wavelengths will give incorrect optical sectioning. (Adapted from Shotton, 1995.)
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  •  FigureFigure 2.8.2 Intensity distribution and diffraction pattern of (A) laser light and (B) normal light. Due to a high degree of parallelism, the diameter of the diffraction‐limited laser light spot is smaller than that obtained with normal light. This results in better lateral resolution with the CLSM. The full width half maximum (FWHM), i.e., the full width of the irradiance distribution at 50% irradiance, is often referred to as the optical section thickness. (Adapted from Tekola et al., 1994.)
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  •  FigureFigure 2.8.3 The excitation (dashed lines) and emission (solid lines) spectra of three fluorophores, FITC, LRSC, and Cy‐5, that are excellently suited to the emission lines of the mixed gas Ar/Kr ion laser (arrows). Also, the spectra of a UV‐excited fluorophore, AMCA, is included. Above, the emission lines of some commonly used laser types are shown. (Adapted from Wessendorf and Brelje, 1993 and Tsien and Waggoner, 1995.)
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  •  FigureFigure 2.8.4 (A‐C) Autofocus projections of a three‐dimensional Gaussian filtered stack comprising 50 confocal sections, showing photoreceptor cells immunoreactive for serotonin (A, LRSC fluorescence), the phototransduction protein arrestin (B, FITC fluorescence), and the photopigment apoprotein opsin (C, Cy5 fluorescence), in the pineal organ of the salmon. The scanning was performed with a MultiProbe 2001 CLSM, using the excitation wavelengths 488 nm (B), 568 nm (A) and 647 nm of the Ar/Kr laser. (D) Confocal section, after three‐dimensional Gaussian filtering, of a neuron that exhibits immunoreactivity for acetylated α‐tubulin (mouse monoclonal clone 6‐11B‐1), in the salmon pineal organ. (E‐H) Stereo pairs of (E, F) an autofocus projection, and (G, H) a surface rendering projection of 100 confocal sections of the same neuron as in (D). Scale bars represent 10 µm in A‐C, and 5 µm in D‐H.
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  •  FigureFigure 2.8.5 Confocal section (0°) of a whole‐mounted salmon pineal organ, showing photosensory cells exhibiting immunoreactivity for the phototransduction protein arrestin (mouse monoclonal clone 5C6‐47; courtesy of Dr. L. Donoso). The section was chosen out of a stack of 80 confocal sections. The line indicated by the arrow shows the plane of section through the stack shown below (−90°); the thickness of this digital section corresponds to a 0.5‐µm‐thick physical section. Note that the resolution and fluorescence intensity decreases dramatically with depth. The scanning was performed with a MultiProbe 2001 CLSM, using the excitation wavelength 488 nm (FITC fluorescence).
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  •  FigureFigure 2.8.6 Autofocus projections of 125 confocal sections of a region in the salmon pineal organ that contains three neurons that exhibit immunoreactivity for acetylated α tubulin (mouse monoclonal clone 6‐11B‐1). Tilting the volume around the x axis (−45° or −90° lat.), and around the y axis (−45° or −90° long.) allows inspection of the volume from different angles. Presentation of such views as stereo pairs (not shown here) are of great help in determining spatial relationships between cells. When viewing the volume from a 90° angle, it is apparent that thin structures, like axons, appear broader than at 0°. This is because axial resolution is worse than lateral resolution. The scanning was performed with a MultiProbe 2001 CLSM, using the excitation wavelength 488 nm (FITC fluorescence).
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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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