Abstract

Scanning confocal microscopy offers several potential advantages for light microscope studies of biological material. Foremost amongst these is the rejection of interfering signals from out-of-focus structures, which often seriously degrade images. The degradation in image quality with epifluorescence microscopy is particularly pronounced; an unfortunate situation, as this is one of the most commonly used techniques in biological research. Confocal imaging almost completely eliminates this problem and therefore promises to have a wide application in this area. We have developed a high-speed beam scanning confocal imaging system that can be used in conjunction with a conventional microscope, and have examined a variety of biological material using this system. In all cases we have found that confocal imaging gives a marked improvement in quality over conventional techniques. The improvement is particularly spectacular with thick specimens viewed with epifluorescence.

© 1987 Optical Society of America

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References

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  1. M. Minsky, U.S.Patent3,013,467.
  2. C. J. R. Sheppard, T. Wilson, “The Image of a Single Point in Microscopes of Large Numerical Aperture,” Proc. R. Soc. London Ser. A 379, 145 (1982).
    [Crossref]
  3. T. Wilson, C. J. R. Sheppard, Theory and Practice of Scanning Optical Microscopy (Academic, New York, 1984).
  4. G. W. Brakenhoff, H. T. M. van der Voort, E. A. van Sprousen, W. A. M. Linnemans, N. Nanninga, “Three-Dimensional Chromatin Distribution in Neuroblastoma Nuclei Shown by Confocal Scanning Laser Microscopy,” Nature 317, 748 (1985).
    [Crossref] [PubMed]
  5. J. G. White, W. B. Amos, M. Fordham, “An Evaluation of Confocal Versus Conventional Imaging of Biological Structures by Fluorescence Light Microscopy,” J.Cell Biol 105, 1 (1987).
    [Crossref]
  6. R. Y. Tsien, T. J. Rink, M. Poenie, “Measurement of Cytosolic Free Ca++ in Individual Small Cells Using Fluorescence Microscopy With Dual Excitation Wavelengths,” Cell Calcium 6, 145 (1985).
    [Crossref] [PubMed]
  7. A. M. Paradiso, R. Y. Tsien, T. E. Machen, “Digital Image Processing of Intracellular pH in Gastric Oxyntic and Chief Cells,” Nature 325, 447 (1987).
    [Crossref] [PubMed]
  8. W. N. Ross et al., “Changes in Absorption, Fluorescence, Dichroism and Birefringence in Stained Giant Axon: Optical Measurement of Membrane Potential,” J. Membr. Biol. 33, 141 (1977).
    [Crossref] [PubMed]
  9. M. Pétrǎn, M. Hadravski, M. D. Egger, R. Galambos, “Tandem Scanning and Reflected-Light Microscope,” J. Opt. Soc. Am. 58, 661 (1968).
    [Crossref]
  10. M. Pétrǎn, J. Hadravsky, J. Benes, R. Kucera, A. Boyd, “The Tandem Scanning Reflected Light Microscope; Part 1—The Principle and Its Design,” Proc. R. Micros. Soc. 20, 125 (1985).
  11. V. Wilke, “Optical Scanning Microscopy—The Laser Scan Microscope,” Scanning 7, 88 (1985).
    [Crossref]
  12. K. Carlsson, P. E. Danielsson, R. Lenz, A. Liljeborg, I. Majlof, N. Aslund, “Three-Dimensional Microscopy Using a Confocal Laser Scanning Microscope,” Opt. Lett. 10, 53 (1985).
    [Crossref] [PubMed]
  13. A. A. Hyman, J. G. White, “Determination of Cell Division Axes in the Early Embryogenesis of C. elegans,” J. Cell Biol. in press.
  14. S. Munro, H. R. B. Pelham, “A C-Terminal Signal Prevents Secretion of Luminal ER Proteins,” Cell 48, 899 (1987).
    [Crossref] [PubMed]
  15. D. A. Agard, J. W. Sedat, “Three-Dimensional Architecture of a Polytene Nucleus,” Nature 302, 676 (1983).
    [Crossref] [PubMed]
  16. S. F. Gull, J. Skilling, “Maximum Entropy Method in Image Processing,” IEE Proc. 131F, 646 (1984).

1987 (3)

J. G. White, W. B. Amos, M. Fordham, “An Evaluation of Confocal Versus Conventional Imaging of Biological Structures by Fluorescence Light Microscopy,” J.Cell Biol 105, 1 (1987).
[Crossref]

A. M. Paradiso, R. Y. Tsien, T. E. Machen, “Digital Image Processing of Intracellular pH in Gastric Oxyntic and Chief Cells,” Nature 325, 447 (1987).
[Crossref] [PubMed]

S. Munro, H. R. B. Pelham, “A C-Terminal Signal Prevents Secretion of Luminal ER Proteins,” Cell 48, 899 (1987).
[Crossref] [PubMed]

1985 (5)

M. Pétrǎn, J. Hadravsky, J. Benes, R. Kucera, A. Boyd, “The Tandem Scanning Reflected Light Microscope; Part 1—The Principle and Its Design,” Proc. R. Micros. Soc. 20, 125 (1985).

V. Wilke, “Optical Scanning Microscopy—The Laser Scan Microscope,” Scanning 7, 88 (1985).
[Crossref]

K. Carlsson, P. E. Danielsson, R. Lenz, A. Liljeborg, I. Majlof, N. Aslund, “Three-Dimensional Microscopy Using a Confocal Laser Scanning Microscope,” Opt. Lett. 10, 53 (1985).
[Crossref] [PubMed]

R. Y. Tsien, T. J. Rink, M. Poenie, “Measurement of Cytosolic Free Ca++ in Individual Small Cells Using Fluorescence Microscopy With Dual Excitation Wavelengths,” Cell Calcium 6, 145 (1985).
[Crossref] [PubMed]

G. W. Brakenhoff, H. T. M. van der Voort, E. A. van Sprousen, W. A. M. Linnemans, N. Nanninga, “Three-Dimensional Chromatin Distribution in Neuroblastoma Nuclei Shown by Confocal Scanning Laser Microscopy,” Nature 317, 748 (1985).
[Crossref] [PubMed]

1984 (1)

S. F. Gull, J. Skilling, “Maximum Entropy Method in Image Processing,” IEE Proc. 131F, 646 (1984).

1983 (1)

D. A. Agard, J. W. Sedat, “Three-Dimensional Architecture of a Polytene Nucleus,” Nature 302, 676 (1983).
[Crossref] [PubMed]

1982 (1)

C. J. R. Sheppard, T. Wilson, “The Image of a Single Point in Microscopes of Large Numerical Aperture,” Proc. R. Soc. London Ser. A 379, 145 (1982).
[Crossref]

1977 (1)

W. N. Ross et al., “Changes in Absorption, Fluorescence, Dichroism and Birefringence in Stained Giant Axon: Optical Measurement of Membrane Potential,” J. Membr. Biol. 33, 141 (1977).
[Crossref] [PubMed]

1968 (1)

M. Pétrǎn, M. Hadravski, M. D. Egger, R. Galambos, “Tandem Scanning and Reflected-Light Microscope,” J. Opt. Soc. Am. 58, 661 (1968).
[Crossref]

Agard, D. A.

D. A. Agard, J. W. Sedat, “Three-Dimensional Architecture of a Polytene Nucleus,” Nature 302, 676 (1983).
[Crossref] [PubMed]

Amos, W. B.

J. G. White, W. B. Amos, M. Fordham, “An Evaluation of Confocal Versus Conventional Imaging of Biological Structures by Fluorescence Light Microscopy,” J.Cell Biol 105, 1 (1987).
[Crossref]

Aslund, N.

Benes, J.

M. Pétrǎn, J. Hadravsky, J. Benes, R. Kucera, A. Boyd, “The Tandem Scanning Reflected Light Microscope; Part 1—The Principle and Its Design,” Proc. R. Micros. Soc. 20, 125 (1985).

Boyd, A.

M. Pétrǎn, J. Hadravsky, J. Benes, R. Kucera, A. Boyd, “The Tandem Scanning Reflected Light Microscope; Part 1—The Principle and Its Design,” Proc. R. Micros. Soc. 20, 125 (1985).

Brakenhoff, G. W.

G. W. Brakenhoff, H. T. M. van der Voort, E. A. van Sprousen, W. A. M. Linnemans, N. Nanninga, “Three-Dimensional Chromatin Distribution in Neuroblastoma Nuclei Shown by Confocal Scanning Laser Microscopy,” Nature 317, 748 (1985).
[Crossref] [PubMed]

Carlsson, K.

Danielsson, P. E.

Egger, M. D.

M. Pétrǎn, M. Hadravski, M. D. Egger, R. Galambos, “Tandem Scanning and Reflected-Light Microscope,” J. Opt. Soc. Am. 58, 661 (1968).
[Crossref]

Fordham, M.

J. G. White, W. B. Amos, M. Fordham, “An Evaluation of Confocal Versus Conventional Imaging of Biological Structures by Fluorescence Light Microscopy,” J.Cell Biol 105, 1 (1987).
[Crossref]

Galambos, R.

M. Pétrǎn, M. Hadravski, M. D. Egger, R. Galambos, “Tandem Scanning and Reflected-Light Microscope,” J. Opt. Soc. Am. 58, 661 (1968).
[Crossref]

Gull, S. F.

S. F. Gull, J. Skilling, “Maximum Entropy Method in Image Processing,” IEE Proc. 131F, 646 (1984).

Hadravski, M.

M. Pétrǎn, M. Hadravski, M. D. Egger, R. Galambos, “Tandem Scanning and Reflected-Light Microscope,” J. Opt. Soc. Am. 58, 661 (1968).
[Crossref]

Hadravsky, J.

M. Pétrǎn, J. Hadravsky, J. Benes, R. Kucera, A. Boyd, “The Tandem Scanning Reflected Light Microscope; Part 1—The Principle and Its Design,” Proc. R. Micros. Soc. 20, 125 (1985).

Hyman, A. A.

A. A. Hyman, J. G. White, “Determination of Cell Division Axes in the Early Embryogenesis of C. elegans,” J. Cell Biol. in press.

Kucera, R.

M. Pétrǎn, J. Hadravsky, J. Benes, R. Kucera, A. Boyd, “The Tandem Scanning Reflected Light Microscope; Part 1—The Principle and Its Design,” Proc. R. Micros. Soc. 20, 125 (1985).

Lenz, R.

Liljeborg, A.

Linnemans, W. A. M.

G. W. Brakenhoff, H. T. M. van der Voort, E. A. van Sprousen, W. A. M. Linnemans, N. Nanninga, “Three-Dimensional Chromatin Distribution in Neuroblastoma Nuclei Shown by Confocal Scanning Laser Microscopy,” Nature 317, 748 (1985).
[Crossref] [PubMed]

Machen, T. E.

A. M. Paradiso, R. Y. Tsien, T. E. Machen, “Digital Image Processing of Intracellular pH in Gastric Oxyntic and Chief Cells,” Nature 325, 447 (1987).
[Crossref] [PubMed]

Majlof, I.

Minsky, M.

M. Minsky, U.S.Patent3,013,467.

Munro, S.

S. Munro, H. R. B. Pelham, “A C-Terminal Signal Prevents Secretion of Luminal ER Proteins,” Cell 48, 899 (1987).
[Crossref] [PubMed]

Nanninga, N.

G. W. Brakenhoff, H. T. M. van der Voort, E. A. van Sprousen, W. A. M. Linnemans, N. Nanninga, “Three-Dimensional Chromatin Distribution in Neuroblastoma Nuclei Shown by Confocal Scanning Laser Microscopy,” Nature 317, 748 (1985).
[Crossref] [PubMed]

Paradiso, A. M.

A. M. Paradiso, R. Y. Tsien, T. E. Machen, “Digital Image Processing of Intracellular pH in Gastric Oxyntic and Chief Cells,” Nature 325, 447 (1987).
[Crossref] [PubMed]

Pelham, H. R. B.

S. Munro, H. R. B. Pelham, “A C-Terminal Signal Prevents Secretion of Luminal ER Proteins,” Cell 48, 899 (1987).
[Crossref] [PubMed]

Pétran, M.

M. Pétrǎn, J. Hadravsky, J. Benes, R. Kucera, A. Boyd, “The Tandem Scanning Reflected Light Microscope; Part 1—The Principle and Its Design,” Proc. R. Micros. Soc. 20, 125 (1985).

M. Pétrǎn, M. Hadravski, M. D. Egger, R. Galambos, “Tandem Scanning and Reflected-Light Microscope,” J. Opt. Soc. Am. 58, 661 (1968).
[Crossref]

Poenie, M.

R. Y. Tsien, T. J. Rink, M. Poenie, “Measurement of Cytosolic Free Ca++ in Individual Small Cells Using Fluorescence Microscopy With Dual Excitation Wavelengths,” Cell Calcium 6, 145 (1985).
[Crossref] [PubMed]

Rink, T. J.

R. Y. Tsien, T. J. Rink, M. Poenie, “Measurement of Cytosolic Free Ca++ in Individual Small Cells Using Fluorescence Microscopy With Dual Excitation Wavelengths,” Cell Calcium 6, 145 (1985).
[Crossref] [PubMed]

Ross, W. N.

W. N. Ross et al., “Changes in Absorption, Fluorescence, Dichroism and Birefringence in Stained Giant Axon: Optical Measurement of Membrane Potential,” J. Membr. Biol. 33, 141 (1977).
[Crossref] [PubMed]

Sedat, J. W.

D. A. Agard, J. W. Sedat, “Three-Dimensional Architecture of a Polytene Nucleus,” Nature 302, 676 (1983).
[Crossref] [PubMed]

Sheppard, C. J. R.

C. J. R. Sheppard, T. Wilson, “The Image of a Single Point in Microscopes of Large Numerical Aperture,” Proc. R. Soc. London Ser. A 379, 145 (1982).
[Crossref]

T. Wilson, C. J. R. Sheppard, Theory and Practice of Scanning Optical Microscopy (Academic, New York, 1984).

Skilling, J.

S. F. Gull, J. Skilling, “Maximum Entropy Method in Image Processing,” IEE Proc. 131F, 646 (1984).

Tsien, R. Y.

A. M. Paradiso, R. Y. Tsien, T. E. Machen, “Digital Image Processing of Intracellular pH in Gastric Oxyntic and Chief Cells,” Nature 325, 447 (1987).
[Crossref] [PubMed]

R. Y. Tsien, T. J. Rink, M. Poenie, “Measurement of Cytosolic Free Ca++ in Individual Small Cells Using Fluorescence Microscopy With Dual Excitation Wavelengths,” Cell Calcium 6, 145 (1985).
[Crossref] [PubMed]

van der Voort, H. T. M.

G. W. Brakenhoff, H. T. M. van der Voort, E. A. van Sprousen, W. A. M. Linnemans, N. Nanninga, “Three-Dimensional Chromatin Distribution in Neuroblastoma Nuclei Shown by Confocal Scanning Laser Microscopy,” Nature 317, 748 (1985).
[Crossref] [PubMed]

van Sprousen, E. A.

G. W. Brakenhoff, H. T. M. van der Voort, E. A. van Sprousen, W. A. M. Linnemans, N. Nanninga, “Three-Dimensional Chromatin Distribution in Neuroblastoma Nuclei Shown by Confocal Scanning Laser Microscopy,” Nature 317, 748 (1985).
[Crossref] [PubMed]

White, J. G.

J. G. White, W. B. Amos, M. Fordham, “An Evaluation of Confocal Versus Conventional Imaging of Biological Structures by Fluorescence Light Microscopy,” J.Cell Biol 105, 1 (1987).
[Crossref]

A. A. Hyman, J. G. White, “Determination of Cell Division Axes in the Early Embryogenesis of C. elegans,” J. Cell Biol. in press.

Wilke, V.

V. Wilke, “Optical Scanning Microscopy—The Laser Scan Microscope,” Scanning 7, 88 (1985).
[Crossref]

Wilson, T.

C. J. R. Sheppard, T. Wilson, “The Image of a Single Point in Microscopes of Large Numerical Aperture,” Proc. R. Soc. London Ser. A 379, 145 (1982).
[Crossref]

T. Wilson, C. J. R. Sheppard, Theory and Practice of Scanning Optical Microscopy (Academic, New York, 1984).

Cell (1)

S. Munro, H. R. B. Pelham, “A C-Terminal Signal Prevents Secretion of Luminal ER Proteins,” Cell 48, 899 (1987).
[Crossref] [PubMed]

Cell Calcium (1)

R. Y. Tsien, T. J. Rink, M. Poenie, “Measurement of Cytosolic Free Ca++ in Individual Small Cells Using Fluorescence Microscopy With Dual Excitation Wavelengths,” Cell Calcium 6, 145 (1985).
[Crossref] [PubMed]

IEE Proc. (1)

S. F. Gull, J. Skilling, “Maximum Entropy Method in Image Processing,” IEE Proc. 131F, 646 (1984).

J. Membr. Biol. (1)

W. N. Ross et al., “Changes in Absorption, Fluorescence, Dichroism and Birefringence in Stained Giant Axon: Optical Measurement of Membrane Potential,” J. Membr. Biol. 33, 141 (1977).
[Crossref] [PubMed]

J. Opt. Soc. Am (1)

M. Pétrǎn, M. Hadravski, M. D. Egger, R. Galambos, “Tandem Scanning and Reflected-Light Microscope,” J. Opt. Soc. Am. 58, 661 (1968).
[Crossref]

J.Cell Biol (1)

J. G. White, W. B. Amos, M. Fordham, “An Evaluation of Confocal Versus Conventional Imaging of Biological Structures by Fluorescence Light Microscopy,” J.Cell Biol 105, 1 (1987).
[Crossref]

Nature (3)

D. A. Agard, J. W. Sedat, “Three-Dimensional Architecture of a Polytene Nucleus,” Nature 302, 676 (1983).
[Crossref] [PubMed]

A. M. Paradiso, R. Y. Tsien, T. E. Machen, “Digital Image Processing of Intracellular pH in Gastric Oxyntic and Chief Cells,” Nature 325, 447 (1987).
[Crossref] [PubMed]

G. W. Brakenhoff, H. T. M. van der Voort, E. A. van Sprousen, W. A. M. Linnemans, N. Nanninga, “Three-Dimensional Chromatin Distribution in Neuroblastoma Nuclei Shown by Confocal Scanning Laser Microscopy,” Nature 317, 748 (1985).
[Crossref] [PubMed]

Opt. Lett. (1)

Proc. R. Micros. Soc. (1)

M. Pétrǎn, J. Hadravsky, J. Benes, R. Kucera, A. Boyd, “The Tandem Scanning Reflected Light Microscope; Part 1—The Principle and Its Design,” Proc. R. Micros. Soc. 20, 125 (1985).

Proc. R. Soc. London Ser. A (1)

C. J. R. Sheppard, T. Wilson, “The Image of a Single Point in Microscopes of Large Numerical Aperture,” Proc. R. Soc. London Ser. A 379, 145 (1982).
[Crossref]

Scanning (1)

V. Wilke, “Optical Scanning Microscopy—The Laser Scan Microscope,” Scanning 7, 88 (1985).
[Crossref]

Other (3)

T. Wilson, C. J. R. Sheppard, Theory and Practice of Scanning Optical Microscopy (Academic, New York, 1984).

M. Minsky, U.S.Patent3,013,467.

A. A. Hyman, J. G. White, “Determination of Cell Division Axes in the Early Embryogenesis of C. elegans,” J. Cell Biol. in press.

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Figures (7)

Fig. 1
Fig. 1

Schematic diagram of the confocal microscope. Light passes from the laser into a reflector, which is a chromatic reflector for fluorescence microscopy or a half-silvered mirror for reflection imaging. The optical scanning system directs a parallel beam of light into the eyepiece of a conventional microscope. The beam is focused to a diffraction-limited spot in the specimen and light reflected or emitted by the specimen passes back along the same path, being descanned and separated from the incident light at the reflector.

Fig. 2
Fig. 2

Images showing the organization of microtubules within an intact nematode embryo. The microtubules have been stained with fluorescein-labeled antibodies. The microscope has been focused on a plane near the bottom of the embryo, so that the light path travels through about 20 μm of tissue. As can be clearly seen there is a considerable amount of detail present in the confocal image (a) which is not present in the conventional image (b). Objective ×100 1.3 N.A., excitation 488 nm, scale bar 20 μm.

Fig. 3
Fig. 3

Images showing an optical section through a whole mount of Daphnia. Specimen is unstained, the fluorescence signal is probably derived from autofluorescence of the specimen and the mounting medium. Again there is a striking difference between the conventional and the confocal images (lower and upper half, respectively). Objective ×16 0.4 N.A., excitation 415 nm, scale bar 100 μm.

Fig. 4
Fig. 4

Human chromosome spread stained with quinacrine. This is a very thin (<0.4 μm) specimen and so there is a negligible amount of signal from out-of-focus elements. The conventional image (lower half) compares favorably with the confocal image (upper half) in this case, although there does seem to be a slight improvement in the confocal image. Objective ×100 1.3 N.A., excitation 488 nm, scale bar 4 μm.

Fig. 5
Fig. 5

Reflection image of the diatom Pleurosigma angulatum showing the hexagonal (400-nm spacing) markings on its shell. Objective ×100 1.3 N.A., excitation 488 nm.

Fig. 6
Fig. 6

Reflection interference contrast image of live human buccal epithelial cells. High-contrast detail maybe seen in these unstained cells. Objective ×6.3 0.2 N.A., scale bar 100 μm.

Fig. 7
Fig. 7

Fluorescence image of chloroplasts within the live cells of a blade of grass (upper half). The conventional image completely fails to show the chloroplasts (lower half). Objective ×40 N.A. 0.75, excitation 415 nm, scale bar 40 μm.

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