Abstract

Fluorescence laser scanning microscopy (LSM) offers many advantages over conventional fluorescence microscopy. Very strong excitation light can be concentrated on small spots (0.5 μm) of the specimen, enabling the detection of low concentrations of fluorescent substances. The low levels of autofluorescence generated in the microscope objective and in the immersion oil in LSM provide images of great contrast, even with weakly fluorescent specimens. Confocal LSM permits the visualization of multiple focal layers of the specimen and 3-D image reconstructions. Combination of images stored in computer memory allow the comparison of phase contrast and fluorescence images of the same area of the specimen enabling multiparameter analysis of cells.

© 1987 Optical Society of America

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References

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  1. C. Cremer, T. Cremer, “Considerations on a Laser-Scanning Microscope with High Resolution and Depth of Field,” Microsc. Acta 81, 31 (1978).
    [PubMed]
  2. M. Achats, R. Beck, C. Seger, G. von Sengbusch, “Laser-scanning Technologie zur Bildanalyse vpon Zellen,” Acta Histochem. Suppl. 21, 147 (1980).
  3. P. H. Bartels et al., “Ultrafast Laser Scanner Microscope,” Anal. Quant. Cytol. 3, 55 (1981).
    [PubMed]
  4. W. Deinet, M. Linke, R. Mueller, I. Sander, “Laser-Scanning-Mikroskop Mit Automatischer Fokussierung,” Microsc. Acta 87, 129 (1983).
  5. R. L. Shoemaker, P. H. Bartels, D. W. Hillman, J. Jonas, D. Kessler, R. V. Shack, D. Vukobratovich, “An Ultrafast Laser Scanner Microscope for Digital Image Analysis,” IEEE Trans. Biomed. Eng. BME-29, 82 (1982).
    [Crossref]
  6. V. Wilke, “Laser Scanning in Microscopy,” Proc. R. Microsc. Soc. 17, 21 (1982).
  7. V. Wilke, “Laser Scanning in Microscopy,” Proc. Soc. Photo-Opt. Instrum. Eng. 396, 164 (1983).
  8. R. Mueller, “Scanning Laser Microscope for Inspection of Microelectronic Devices,” Siemens Forsch. Entwicklungsber. 13, 9 (1984).
  9. V. Wilke, “Optical Scanning Microscopy—The Laser Scan Microscope,” Scanning 7, 88 (1985).
    [Crossref]
  10. G. J. Brakenhoff, P. Blom, P. J. Barends, “Confocal Scanning Light Microscopy with High Aperture Immersion Lenses,” J. Microsc. 117, 219(1979).
    [Crossref]
  11. H. T. M. van der Voort, G. J. Brakenhoff, J. A. C. Valkenburg, N. Nanninga, “Design and Use of a Computer Controlled Confocal Microscope for Biological Applications,” Scanning 6, 66 (1985).
    [Crossref]
  12. C. J. R. Sheppard, A. Choudhury, “Image Formation in the Scanning Microscope,” Opt. Acta 24, 1051 (1977).
    [Crossref]
  13. C. J. R. Sheppard, T. Wilson, “Image Formation in the Scanning Microscope,” Opt. Acta 25, 315 (1978).
    [Crossref]
  14. T. Wilson, C. Sheppard, Theory and Practice of Scanning Optical Microscopy (Academic, London, 1984).
  15. J. S. Ploem, “Organic and Biological Surfaces: Fluorescence Microscopy,” in Analysis of Organic and Biological Surfaces, P. Echlin, Ed. (Wiley, New York, 1984), pp. 609–628.
  16. W. E. Kalish, T. Whitmore, A. Siegel, “Laser Scanning Microscopy of Surface Spread Polytene Chromosomes,” J. Microsc 117, 217 (1985).
    [Crossref]
  17. J. S. Ploem, “Automated Methods in Immunofluorescence Studies,” in Immunofluorescence Technology, R. A. Wick et al., Eds. (Elsevier Biomedical, New York, 1982), pp. 73–94.
  18. G. T. Reynolds, D. L. Taylor, “Image Intensification Applied to Light Microscopy,” BioScience 30, 586 (1980).
    [Crossref]
  19. R. D. Allen, N. S. Allen, “Video-Enhanced Microscopy With a Computer Frame Memory,” J. Microsc. 129, 3 (1983).
    [Crossref] [PubMed]

1985 (3)

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

H. T. M. van der Voort, G. J. Brakenhoff, J. A. C. Valkenburg, N. Nanninga, “Design and Use of a Computer Controlled Confocal Microscope for Biological Applications,” Scanning 6, 66 (1985).
[Crossref]

W. E. Kalish, T. Whitmore, A. Siegel, “Laser Scanning Microscopy of Surface Spread Polytene Chromosomes,” J. Microsc 117, 217 (1985).
[Crossref]

1984 (1)

R. Mueller, “Scanning Laser Microscope for Inspection of Microelectronic Devices,” Siemens Forsch. Entwicklungsber. 13, 9 (1984).

1983 (3)

R. D. Allen, N. S. Allen, “Video-Enhanced Microscopy With a Computer Frame Memory,” J. Microsc. 129, 3 (1983).
[Crossref] [PubMed]

V. Wilke, “Laser Scanning in Microscopy,” Proc. Soc. Photo-Opt. Instrum. Eng. 396, 164 (1983).

W. Deinet, M. Linke, R. Mueller, I. Sander, “Laser-Scanning-Mikroskop Mit Automatischer Fokussierung,” Microsc. Acta 87, 129 (1983).

1982 (2)

R. L. Shoemaker, P. H. Bartels, D. W. Hillman, J. Jonas, D. Kessler, R. V. Shack, D. Vukobratovich, “An Ultrafast Laser Scanner Microscope for Digital Image Analysis,” IEEE Trans. Biomed. Eng. BME-29, 82 (1982).
[Crossref]

V. Wilke, “Laser Scanning in Microscopy,” Proc. R. Microsc. Soc. 17, 21 (1982).

1981 (1)

P. H. Bartels et al., “Ultrafast Laser Scanner Microscope,” Anal. Quant. Cytol. 3, 55 (1981).
[PubMed]

1980 (2)

M. Achats, R. Beck, C. Seger, G. von Sengbusch, “Laser-scanning Technologie zur Bildanalyse vpon Zellen,” Acta Histochem. Suppl. 21, 147 (1980).

G. T. Reynolds, D. L. Taylor, “Image Intensification Applied to Light Microscopy,” BioScience 30, 586 (1980).
[Crossref]

1979 (1)

G. J. Brakenhoff, P. Blom, P. J. Barends, “Confocal Scanning Light Microscopy with High Aperture Immersion Lenses,” J. Microsc. 117, 219(1979).
[Crossref]

1978 (2)

C. Cremer, T. Cremer, “Considerations on a Laser-Scanning Microscope with High Resolution and Depth of Field,” Microsc. Acta 81, 31 (1978).
[PubMed]

C. J. R. Sheppard, T. Wilson, “Image Formation in the Scanning Microscope,” Opt. Acta 25, 315 (1978).
[Crossref]

1977 (1)

C. J. R. Sheppard, A. Choudhury, “Image Formation in the Scanning Microscope,” Opt. Acta 24, 1051 (1977).
[Crossref]

Achats, M.

M. Achats, R. Beck, C. Seger, G. von Sengbusch, “Laser-scanning Technologie zur Bildanalyse vpon Zellen,” Acta Histochem. Suppl. 21, 147 (1980).

Allen, N. S.

R. D. Allen, N. S. Allen, “Video-Enhanced Microscopy With a Computer Frame Memory,” J. Microsc. 129, 3 (1983).
[Crossref] [PubMed]

Allen, R. D.

R. D. Allen, N. S. Allen, “Video-Enhanced Microscopy With a Computer Frame Memory,” J. Microsc. 129, 3 (1983).
[Crossref] [PubMed]

Barends, P. J.

G. J. Brakenhoff, P. Blom, P. J. Barends, “Confocal Scanning Light Microscopy with High Aperture Immersion Lenses,” J. Microsc. 117, 219(1979).
[Crossref]

Bartels, P. H.

R. L. Shoemaker, P. H. Bartels, D. W. Hillman, J. Jonas, D. Kessler, R. V. Shack, D. Vukobratovich, “An Ultrafast Laser Scanner Microscope for Digital Image Analysis,” IEEE Trans. Biomed. Eng. BME-29, 82 (1982).
[Crossref]

P. H. Bartels et al., “Ultrafast Laser Scanner Microscope,” Anal. Quant. Cytol. 3, 55 (1981).
[PubMed]

Beck, R.

M. Achats, R. Beck, C. Seger, G. von Sengbusch, “Laser-scanning Technologie zur Bildanalyse vpon Zellen,” Acta Histochem. Suppl. 21, 147 (1980).

Blom, P.

G. J. Brakenhoff, P. Blom, P. J. Barends, “Confocal Scanning Light Microscopy with High Aperture Immersion Lenses,” J. Microsc. 117, 219(1979).
[Crossref]

Brakenhoff, G. J.

H. T. M. van der Voort, G. J. Brakenhoff, J. A. C. Valkenburg, N. Nanninga, “Design and Use of a Computer Controlled Confocal Microscope for Biological Applications,” Scanning 6, 66 (1985).
[Crossref]

G. J. Brakenhoff, P. Blom, P. J. Barends, “Confocal Scanning Light Microscopy with High Aperture Immersion Lenses,” J. Microsc. 117, 219(1979).
[Crossref]

Choudhury, A.

C. J. R. Sheppard, A. Choudhury, “Image Formation in the Scanning Microscope,” Opt. Acta 24, 1051 (1977).
[Crossref]

Cremer, C.

C. Cremer, T. Cremer, “Considerations on a Laser-Scanning Microscope with High Resolution and Depth of Field,” Microsc. Acta 81, 31 (1978).
[PubMed]

Cremer, T.

C. Cremer, T. Cremer, “Considerations on a Laser-Scanning Microscope with High Resolution and Depth of Field,” Microsc. Acta 81, 31 (1978).
[PubMed]

Deinet, W.

W. Deinet, M. Linke, R. Mueller, I. Sander, “Laser-Scanning-Mikroskop Mit Automatischer Fokussierung,” Microsc. Acta 87, 129 (1983).

Hillman, D. W.

R. L. Shoemaker, P. H. Bartels, D. W. Hillman, J. Jonas, D. Kessler, R. V. Shack, D. Vukobratovich, “An Ultrafast Laser Scanner Microscope for Digital Image Analysis,” IEEE Trans. Biomed. Eng. BME-29, 82 (1982).
[Crossref]

Jonas, J.

R. L. Shoemaker, P. H. Bartels, D. W. Hillman, J. Jonas, D. Kessler, R. V. Shack, D. Vukobratovich, “An Ultrafast Laser Scanner Microscope for Digital Image Analysis,” IEEE Trans. Biomed. Eng. BME-29, 82 (1982).
[Crossref]

Kalish, W. E.

W. E. Kalish, T. Whitmore, A. Siegel, “Laser Scanning Microscopy of Surface Spread Polytene Chromosomes,” J. Microsc 117, 217 (1985).
[Crossref]

Kessler, D.

R. L. Shoemaker, P. H. Bartels, D. W. Hillman, J. Jonas, D. Kessler, R. V. Shack, D. Vukobratovich, “An Ultrafast Laser Scanner Microscope for Digital Image Analysis,” IEEE Trans. Biomed. Eng. BME-29, 82 (1982).
[Crossref]

Linke, M.

W. Deinet, M. Linke, R. Mueller, I. Sander, “Laser-Scanning-Mikroskop Mit Automatischer Fokussierung,” Microsc. Acta 87, 129 (1983).

Mueller, R.

R. Mueller, “Scanning Laser Microscope for Inspection of Microelectronic Devices,” Siemens Forsch. Entwicklungsber. 13, 9 (1984).

W. Deinet, M. Linke, R. Mueller, I. Sander, “Laser-Scanning-Mikroskop Mit Automatischer Fokussierung,” Microsc. Acta 87, 129 (1983).

Nanninga, N.

H. T. M. van der Voort, G. J. Brakenhoff, J. A. C. Valkenburg, N. Nanninga, “Design and Use of a Computer Controlled Confocal Microscope for Biological Applications,” Scanning 6, 66 (1985).
[Crossref]

Ploem, J. S.

J. S. Ploem, “Organic and Biological Surfaces: Fluorescence Microscopy,” in Analysis of Organic and Biological Surfaces, P. Echlin, Ed. (Wiley, New York, 1984), pp. 609–628.

J. S. Ploem, “Automated Methods in Immunofluorescence Studies,” in Immunofluorescence Technology, R. A. Wick et al., Eds. (Elsevier Biomedical, New York, 1982), pp. 73–94.

Reynolds, G. T.

G. T. Reynolds, D. L. Taylor, “Image Intensification Applied to Light Microscopy,” BioScience 30, 586 (1980).
[Crossref]

Sander, I.

W. Deinet, M. Linke, R. Mueller, I. Sander, “Laser-Scanning-Mikroskop Mit Automatischer Fokussierung,” Microsc. Acta 87, 129 (1983).

Seger, C.

M. Achats, R. Beck, C. Seger, G. von Sengbusch, “Laser-scanning Technologie zur Bildanalyse vpon Zellen,” Acta Histochem. Suppl. 21, 147 (1980).

Shack, R. V.

R. L. Shoemaker, P. H. Bartels, D. W. Hillman, J. Jonas, D. Kessler, R. V. Shack, D. Vukobratovich, “An Ultrafast Laser Scanner Microscope for Digital Image Analysis,” IEEE Trans. Biomed. Eng. BME-29, 82 (1982).
[Crossref]

Sheppard, C.

T. Wilson, C. Sheppard, Theory and Practice of Scanning Optical Microscopy (Academic, London, 1984).

Sheppard, C. J. R.

C. J. R. Sheppard, T. Wilson, “Image Formation in the Scanning Microscope,” Opt. Acta 25, 315 (1978).
[Crossref]

C. J. R. Sheppard, A. Choudhury, “Image Formation in the Scanning Microscope,” Opt. Acta 24, 1051 (1977).
[Crossref]

Shoemaker, R. L.

R. L. Shoemaker, P. H. Bartels, D. W. Hillman, J. Jonas, D. Kessler, R. V. Shack, D. Vukobratovich, “An Ultrafast Laser Scanner Microscope for Digital Image Analysis,” IEEE Trans. Biomed. Eng. BME-29, 82 (1982).
[Crossref]

Siegel, A.

W. E. Kalish, T. Whitmore, A. Siegel, “Laser Scanning Microscopy of Surface Spread Polytene Chromosomes,” J. Microsc 117, 217 (1985).
[Crossref]

Taylor, D. L.

G. T. Reynolds, D. L. Taylor, “Image Intensification Applied to Light Microscopy,” BioScience 30, 586 (1980).
[Crossref]

Valkenburg, J. A. C.

H. T. M. van der Voort, G. J. Brakenhoff, J. A. C. Valkenburg, N. Nanninga, “Design and Use of a Computer Controlled Confocal Microscope for Biological Applications,” Scanning 6, 66 (1985).
[Crossref]

van der Voort, H. T. M.

H. T. M. van der Voort, G. J. Brakenhoff, J. A. C. Valkenburg, N. Nanninga, “Design and Use of a Computer Controlled Confocal Microscope for Biological Applications,” Scanning 6, 66 (1985).
[Crossref]

von Sengbusch, G.

M. Achats, R. Beck, C. Seger, G. von Sengbusch, “Laser-scanning Technologie zur Bildanalyse vpon Zellen,” Acta Histochem. Suppl. 21, 147 (1980).

Vukobratovich, D.

R. L. Shoemaker, P. H. Bartels, D. W. Hillman, J. Jonas, D. Kessler, R. V. Shack, D. Vukobratovich, “An Ultrafast Laser Scanner Microscope for Digital Image Analysis,” IEEE Trans. Biomed. Eng. BME-29, 82 (1982).
[Crossref]

Whitmore, T.

W. E. Kalish, T. Whitmore, A. Siegel, “Laser Scanning Microscopy of Surface Spread Polytene Chromosomes,” J. Microsc 117, 217 (1985).
[Crossref]

Wilke, V.

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

V. Wilke, “Laser Scanning in Microscopy,” Proc. Soc. Photo-Opt. Instrum. Eng. 396, 164 (1983).

V. Wilke, “Laser Scanning in Microscopy,” Proc. R. Microsc. Soc. 17, 21 (1982).

Wilson, T.

C. J. R. Sheppard, T. Wilson, “Image Formation in the Scanning Microscope,” Opt. Acta 25, 315 (1978).
[Crossref]

T. Wilson, C. Sheppard, Theory and Practice of Scanning Optical Microscopy (Academic, London, 1984).

Acta Histochem. Suppl. (1)

M. Achats, R. Beck, C. Seger, G. von Sengbusch, “Laser-scanning Technologie zur Bildanalyse vpon Zellen,” Acta Histochem. Suppl. 21, 147 (1980).

Anal. Quant. Cytol. (1)

P. H. Bartels et al., “Ultrafast Laser Scanner Microscope,” Anal. Quant. Cytol. 3, 55 (1981).
[PubMed]

BioScience (1)

G. T. Reynolds, D. L. Taylor, “Image Intensification Applied to Light Microscopy,” BioScience 30, 586 (1980).
[Crossref]

IEEE Trans. Biomed. Eng. (1)

R. L. Shoemaker, P. H. Bartels, D. W. Hillman, J. Jonas, D. Kessler, R. V. Shack, D. Vukobratovich, “An Ultrafast Laser Scanner Microscope for Digital Image Analysis,” IEEE Trans. Biomed. Eng. BME-29, 82 (1982).
[Crossref]

J. Microsc (1)

W. E. Kalish, T. Whitmore, A. Siegel, “Laser Scanning Microscopy of Surface Spread Polytene Chromosomes,” J. Microsc 117, 217 (1985).
[Crossref]

J. Microsc. (2)

G. J. Brakenhoff, P. Blom, P. J. Barends, “Confocal Scanning Light Microscopy with High Aperture Immersion Lenses,” J. Microsc. 117, 219(1979).
[Crossref]

R. D. Allen, N. S. Allen, “Video-Enhanced Microscopy With a Computer Frame Memory,” J. Microsc. 129, 3 (1983).
[Crossref] [PubMed]

Microsc. Acta (2)

W. Deinet, M. Linke, R. Mueller, I. Sander, “Laser-Scanning-Mikroskop Mit Automatischer Fokussierung,” Microsc. Acta 87, 129 (1983).

C. Cremer, T. Cremer, “Considerations on a Laser-Scanning Microscope with High Resolution and Depth of Field,” Microsc. Acta 81, 31 (1978).
[PubMed]

Opt. Acta (2)

C. J. R. Sheppard, A. Choudhury, “Image Formation in the Scanning Microscope,” Opt. Acta 24, 1051 (1977).
[Crossref]

C. J. R. Sheppard, T. Wilson, “Image Formation in the Scanning Microscope,” Opt. Acta 25, 315 (1978).
[Crossref]

Proc. R. Microsc. Soc. (1)

V. Wilke, “Laser Scanning in Microscopy,” Proc. R. Microsc. Soc. 17, 21 (1982).

Proc. Soc. Photo-Opt. Instrum. Eng. (1)

V. Wilke, “Laser Scanning in Microscopy,” Proc. Soc. Photo-Opt. Instrum. Eng. 396, 164 (1983).

Scanning (2)

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

H. T. M. van der Voort, G. J. Brakenhoff, J. A. C. Valkenburg, N. Nanninga, “Design and Use of a Computer Controlled Confocal Microscope for Biological Applications,” Scanning 6, 66 (1985).
[Crossref]

Siemens Forsch. Entwicklungsber. (1)

R. Mueller, “Scanning Laser Microscope for Inspection of Microelectronic Devices,” Siemens Forsch. Entwicklungsber. 13, 9 (1984).

Other (3)

J. S. Ploem, “Automated Methods in Immunofluorescence Studies,” in Immunofluorescence Technology, R. A. Wick et al., Eds. (Elsevier Biomedical, New York, 1982), pp. 73–94.

T. Wilson, C. Sheppard, Theory and Practice of Scanning Optical Microscopy (Academic, London, 1984).

J. S. Ploem, “Organic and Biological Surfaces: Fluorescence Microscopy,” in Analysis of Organic and Biological Surfaces, P. Echlin, Ed. (Wiley, New York, 1984), pp. 609–628.

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Figures (5)

Fig. 1
Fig. 1

Cyclic GMP reactivity in the hippocampus of the rat. The brain of the rat was fixed with paraformaldehyde. Subsequently 35-μm sections were treated with antibodies to cGMP. Magnification 2.5×. Different magnifications are shown in Figs. 25.

Fig. 2
Fig. 2

Hippocampus (brain) of the rat. Antibody to cyclic GNP. 4× objective.

Fig. 3
Fig. 3

Hippocampus (brain) of the rat. Antibody to cyclic GNP. 10× objective.

Fig. 4
Fig. 4

Hippocampus (brain) of the rat. Antibody to cyclic GNP. 16× objective.

Fig. 5
Fig. 5

Hippocampus (brain) of the rat. Antibody to cyclic GNP. 40× objective.

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