Abstract

Development of a laser scanning microscope for simultaneous three-dimensional imaging in both a full-field laser scanning mode (FLSM) and a confocal laser scanning mode (CLSM) permits the direct comparison of axial resolution and out-of-focus background rejection as a function of sample thickness for both FLSM and CLSM with varying detector aperture (pinhole) radii. The sample-dependent detector aperture radii that optimize the signal-to-noise ratio (S/N) in the CLSM are experimentally determined.

The results verify earlier calculations [ Appl. Opt. 33, 603 ( 1994)]. Using these results, we discuss the practical and theoretical limits on the S/N in the CLSM and compare them with those of a full-field epifluorescence microscope (FEM) that is enhanced by image deconvolution. The specimen volume over which the FLSM exhibits imaging properties that are equivalent to a FEM is calculated in the appendices.

© 1995 Optical Society of America

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References

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  1. J. B. White, W. B. Amos, M. Fordham, “An evaluation of confocal versus conventional imaging of biological structures by fluorescence light microscopy.” J. Cell Biol. 105, 41–48 (1987).
    [CrossRef] [PubMed]
  2. M. Minsky, “Microscopy apparatus,” U.S. patent3,013,467 (19December1961).
  3. T. Wilson, C. J. R. Sheppard, Theory and Practice of Scanning Optical Microscopy (Academic, Boston, Mass., 1984).
  4. T. Wilson, Confocal Microscopy (Academic, Boston, Mass., 1990).
  5. S. Inoue, Video Microscopy (Plenum, New York, 1986), p. 21.
  6. I. J. Cox, C. J. R. Sheppard, “Information capacity and resolution in an optical system,” J. Opt. Soc. Am. A 3, 1152–1158 (1986).
    [CrossRef]
  7. D. R. Sandison, W. W. Webb, “Background rejection and signal-to-noise optimization in confocal and alternative fluorescence microscopes,” Appl. Opt. 33, 603–615 (1994).
    [CrossRef] [PubMed]
  8. T. Wilson, A. R. Carlini, “Three-dimensional imaging in confocal imaging systems with finite sized detectors,” J. Mi-crosc. 149, 51–66 (1988).
  9. S. Kimura, C. Munakata, “Calculation of three-dimensional optical transfer function for a confocal scanning fluorescent microscope,” J. Opt. Soc.Am.A 6, 1015–1019 (1989).
    [CrossRef]
  10. K. S. Wells, D. R. Sandison, J. Strickler, W. W. Webb, “Quantitative fluorescence imaging with laser scanning confocal microscopy,” in Handbook of Biological Confocal Microscopy, J. B. Pawley, ed. (Plenum, New York, 1990), Chap. 3.
    [CrossRef]
  11. D. R. Sandison, D. W. Piston, W W. Webb, “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, J. E. Trogadis, eds. (Academic, San Diego, Calif., 1994), Chap. 2, pp. 29–46.
  12. W. W. Webb, K. S. Wells, D. R. Sandison, J. Strickler, “Criteria for quantitative dynamical confocal fluorescence imaging,” in Optical Microscopy for Biology, B. Herman, K. Jacobson, eds. (Wiley, New York, 1990), Chap. 6, pp. 73–108.
  13. C. J. R. Sheppard, C. J. Cogswell, M. Gu, “Signal strength and noise in confocal microscopy: factors influencing selection of an optimum detector aperture,” Scanning 13, 233–240 (1991).
    [CrossRef]
  14. C. J. R. Sheppard, “Stray light and noise in confocal microscopy,” Micron Microsc. Acta 22, 239–243 (1991).
    [CrossRef]
  15. Optical units are dimensionless distances associated with the size of the PSF. We choose those units that are consistent with the sine condition of imaging (see Ref. 16). We modify the referenced definitions to account for the specimen's index of refraction n and a high objective-lens NA by replacing λ with λ/n and a/f with NA/n. Lateral υ and axial u optical units are then defined as follows: υ = k(NA)r and u = k(NA)2z/n, where r is the distance from the optical axis, z is the distance from the focal plane, and k = 2πλ is the vacuum wave number. In the detector aperture plane, υi = k(NA)ri/M, where ri is the image-plane distance from the optical axis and M is the total magnification of the optical path.
  16. M. Born, E. Wolf, Principles of Optics, 6th ed. (Pergamon, New York, 1980), p. 437.
  17. M. Born, E. Wolf, Principles of Optics, 6th ed. (Pergamon, New York, 1980).
  18. T. Wilson, “The role of the pinhole in confocal imaging systems,” in Handbook of Biological Confocal Microscopy, J. B. Pawley, ed. (Plenum, New York, 1990), Chap. 11.
    [CrossRef]
  19. G. Arfken, Mathematical Methods for Physicists, 3rd ed. (Academic, San Diego, Calif, 1985).
  20. Ref. 17, p. 443.
  21. C. J. R. Sheppard, H. J. Matthews, “Imaging in high-aperture optical systems,” J. Opt. Soc. Am. A 4, 1354–1360 (1987).
    [CrossRef]
  22. B. Richards, E. Wolf, “Electromagnetic diffraction in optical systems II. Structure of the image field in an aplanatic system,” Proc. R. Soc. London Ser. A 253, 358–379 (1959).
    [CrossRef]
  23. P. J. Shaw, D. J. Rawlins, “Three-dimensional fluorescence microscopy,” Prog. Biophys. Mol. Biol. 56, 187–213 (1991).
    [CrossRef] [PubMed]
  24. Y. Hiraoka, D. A. Agard, J. W. Sedat, “Temporal and spatial coordination of chromosome movement, spindle formation, and nuclear envelope breakdown during prometaphase in Drosophila melanogaster embryos,” J. Cell Biol. 111, 2815–2828 (1990).
    [CrossRef] [PubMed]
  25. M. Benveniste, J. Schlessinger, Z. Kam, “Characterization of internalization and endosome formation of epidermal growth factor in transfected nih-3t3 cells by computerized image-intensified three-dimensional fluorescence microscopy,” J. Cell Biol. 109, 2105–2115 (1989).
    [CrossRef] [PubMed]
  26. S. Inoue, Video Microscopy (Plenum, New York, 1986).
  27. Y. Hiraoka, J. W. Sedat, D. A. Agard, “Determination of three-dimensional imaging properties of a light microscope system,” Biophys. J. 57, 325–333 (1990).
    [CrossRef] [PubMed]
  28. M. E. Barnett, “Image formation in optical and electron transmission microscopy,” J. Microsc. 102, 1–28 (1974).
    [CrossRef]
  29. A. V. Crewe, J. Wall, “Contrast in a high-resolution scanning-transmission electron microscope,” Optik 30, 461–474 (1970).
  30. C. J. R. Sheppard, A. Choudhury“Image formation in the scanning microscope,” Opt. Acta 10, 1051–1073 (1977).
    [CrossRef]

1994 (1)

1991 (3)

C. J. R. Sheppard, C. J. Cogswell, M. Gu, “Signal strength and noise in confocal microscopy: factors influencing selection of an optimum detector aperture,” Scanning 13, 233–240 (1991).
[CrossRef]

C. J. R. Sheppard, “Stray light and noise in confocal microscopy,” Micron Microsc. Acta 22, 239–243 (1991).
[CrossRef]

P. J. Shaw, D. J. Rawlins, “Three-dimensional fluorescence microscopy,” Prog. Biophys. Mol. Biol. 56, 187–213 (1991).
[CrossRef] [PubMed]

1990 (2)

Y. Hiraoka, D. A. Agard, J. W. Sedat, “Temporal and spatial coordination of chromosome movement, spindle formation, and nuclear envelope breakdown during prometaphase in Drosophila melanogaster embryos,” J. Cell Biol. 111, 2815–2828 (1990).
[CrossRef] [PubMed]

Y. Hiraoka, J. W. Sedat, D. A. Agard, “Determination of three-dimensional imaging properties of a light microscope system,” Biophys. J. 57, 325–333 (1990).
[CrossRef] [PubMed]

1989 (2)

M. Benveniste, J. Schlessinger, Z. Kam, “Characterization of internalization and endosome formation of epidermal growth factor in transfected nih-3t3 cells by computerized image-intensified three-dimensional fluorescence microscopy,” J. Cell Biol. 109, 2105–2115 (1989).
[CrossRef] [PubMed]

S. Kimura, C. Munakata, “Calculation of three-dimensional optical transfer function for a confocal scanning fluorescent microscope,” J. Opt. Soc.Am.A 6, 1015–1019 (1989).
[CrossRef]

1988 (1)

T. Wilson, A. R. Carlini, “Three-dimensional imaging in confocal imaging systems with finite sized detectors,” J. Mi-crosc. 149, 51–66 (1988).

1987 (2)

J. B. White, W. B. Amos, M. Fordham, “An evaluation of confocal versus conventional imaging of biological structures by fluorescence light microscopy.” J. Cell Biol. 105, 41–48 (1987).
[CrossRef] [PubMed]

C. J. R. Sheppard, H. J. Matthews, “Imaging in high-aperture optical systems,” J. Opt. Soc. Am. A 4, 1354–1360 (1987).
[CrossRef]

1986 (1)

1977 (1)

C. J. R. Sheppard, A. Choudhury“Image formation in the scanning microscope,” Opt. Acta 10, 1051–1073 (1977).
[CrossRef]

1974 (1)

M. E. Barnett, “Image formation in optical and electron transmission microscopy,” J. Microsc. 102, 1–28 (1974).
[CrossRef]

1970 (1)

A. V. Crewe, J. Wall, “Contrast in a high-resolution scanning-transmission electron microscope,” Optik 30, 461–474 (1970).

1959 (1)

B. Richards, E. Wolf, “Electromagnetic diffraction in optical systems II. Structure of the image field in an aplanatic system,” Proc. R. Soc. London Ser. A 253, 358–379 (1959).
[CrossRef]

Agard, D. A.

Y. Hiraoka, D. A. Agard, J. W. Sedat, “Temporal and spatial coordination of chromosome movement, spindle formation, and nuclear envelope breakdown during prometaphase in Drosophila melanogaster embryos,” J. Cell Biol. 111, 2815–2828 (1990).
[CrossRef] [PubMed]

Y. Hiraoka, J. W. Sedat, D. A. Agard, “Determination of three-dimensional imaging properties of a light microscope system,” Biophys. J. 57, 325–333 (1990).
[CrossRef] [PubMed]

Amos, W. B.

J. B. White, W. B. Amos, M. Fordham, “An evaluation of confocal versus conventional imaging of biological structures by fluorescence light microscopy.” J. Cell Biol. 105, 41–48 (1987).
[CrossRef] [PubMed]

Arfken, G.

G. Arfken, Mathematical Methods for Physicists, 3rd ed. (Academic, San Diego, Calif, 1985).

Barnett, M. E.

M. E. Barnett, “Image formation in optical and electron transmission microscopy,” J. Microsc. 102, 1–28 (1974).
[CrossRef]

Benveniste, M.

M. Benveniste, J. Schlessinger, Z. Kam, “Characterization of internalization and endosome formation of epidermal growth factor in transfected nih-3t3 cells by computerized image-intensified three-dimensional fluorescence microscopy,” J. Cell Biol. 109, 2105–2115 (1989).
[CrossRef] [PubMed]

Born, M.

M. Born, E. Wolf, Principles of Optics, 6th ed. (Pergamon, New York, 1980).

M. Born, E. Wolf, Principles of Optics, 6th ed. (Pergamon, New York, 1980), p. 437.

Carlini, A. R.

T. Wilson, A. R. Carlini, “Three-dimensional imaging in confocal imaging systems with finite sized detectors,” J. Mi-crosc. 149, 51–66 (1988).

Choudhury, A.

C. J. R. Sheppard, A. Choudhury“Image formation in the scanning microscope,” Opt. Acta 10, 1051–1073 (1977).
[CrossRef]

Cogswell, C. J.

C. J. R. Sheppard, C. J. Cogswell, M. Gu, “Signal strength and noise in confocal microscopy: factors influencing selection of an optimum detector aperture,” Scanning 13, 233–240 (1991).
[CrossRef]

Cox, I. J.

Crewe, A. V.

A. V. Crewe, J. Wall, “Contrast in a high-resolution scanning-transmission electron microscope,” Optik 30, 461–474 (1970).

Fordham, M.

J. B. White, W. B. Amos, M. Fordham, “An evaluation of confocal versus conventional imaging of biological structures by fluorescence light microscopy.” J. Cell Biol. 105, 41–48 (1987).
[CrossRef] [PubMed]

Gu, M.

C. J. R. Sheppard, C. J. Cogswell, M. Gu, “Signal strength and noise in confocal microscopy: factors influencing selection of an optimum detector aperture,” Scanning 13, 233–240 (1991).
[CrossRef]

Hiraoka, Y.

Y. Hiraoka, D. A. Agard, J. W. Sedat, “Temporal and spatial coordination of chromosome movement, spindle formation, and nuclear envelope breakdown during prometaphase in Drosophila melanogaster embryos,” J. Cell Biol. 111, 2815–2828 (1990).
[CrossRef] [PubMed]

Y. Hiraoka, J. W. Sedat, D. A. Agard, “Determination of three-dimensional imaging properties of a light microscope system,” Biophys. J. 57, 325–333 (1990).
[CrossRef] [PubMed]

Inoue, S.

S. Inoue, Video Microscopy (Plenum, New York, 1986).

S. Inoue, Video Microscopy (Plenum, New York, 1986), p. 21.

Kam, Z.

M. Benveniste, J. Schlessinger, Z. Kam, “Characterization of internalization and endosome formation of epidermal growth factor in transfected nih-3t3 cells by computerized image-intensified three-dimensional fluorescence microscopy,” J. Cell Biol. 109, 2105–2115 (1989).
[CrossRef] [PubMed]

Kimura, S.

S. Kimura, C. Munakata, “Calculation of three-dimensional optical transfer function for a confocal scanning fluorescent microscope,” J. Opt. Soc.Am.A 6, 1015–1019 (1989).
[CrossRef]

Matthews, H. J.

Minsky, M.

M. Minsky, “Microscopy apparatus,” U.S. patent3,013,467 (19December1961).

Munakata, C.

S. Kimura, C. Munakata, “Calculation of three-dimensional optical transfer function for a confocal scanning fluorescent microscope,” J. Opt. Soc.Am.A 6, 1015–1019 (1989).
[CrossRef]

Piston, D. W.

D. R. Sandison, D. W. Piston, W W. Webb, “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, J. E. Trogadis, eds. (Academic, San Diego, Calif., 1994), Chap. 2, pp. 29–46.

Rawlins, D. J.

P. J. Shaw, D. J. Rawlins, “Three-dimensional fluorescence microscopy,” Prog. Biophys. Mol. Biol. 56, 187–213 (1991).
[CrossRef] [PubMed]

Richards, B.

B. Richards, E. Wolf, “Electromagnetic diffraction in optical systems II. Structure of the image field in an aplanatic system,” Proc. R. Soc. London Ser. A 253, 358–379 (1959).
[CrossRef]

Sandison, D. R.

D. R. Sandison, W. W. Webb, “Background rejection and signal-to-noise optimization in confocal and alternative fluorescence microscopes,” Appl. Opt. 33, 603–615 (1994).
[CrossRef] [PubMed]

D. R. Sandison, D. W. Piston, W W. Webb, “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, J. E. Trogadis, eds. (Academic, San Diego, Calif., 1994), Chap. 2, pp. 29–46.

W. W. Webb, K. S. Wells, D. R. Sandison, J. Strickler, “Criteria for quantitative dynamical confocal fluorescence imaging,” in Optical Microscopy for Biology, B. Herman, K. Jacobson, eds. (Wiley, New York, 1990), Chap. 6, pp. 73–108.

K. S. Wells, D. R. Sandison, J. Strickler, W. W. Webb, “Quantitative fluorescence imaging with laser scanning confocal microscopy,” in Handbook of Biological Confocal Microscopy, J. B. Pawley, ed. (Plenum, New York, 1990), Chap. 3.
[CrossRef]

Schlessinger, J.

M. Benveniste, J. Schlessinger, Z. Kam, “Characterization of internalization and endosome formation of epidermal growth factor in transfected nih-3t3 cells by computerized image-intensified three-dimensional fluorescence microscopy,” J. Cell Biol. 109, 2105–2115 (1989).
[CrossRef] [PubMed]

Sedat, J. W.

Y. Hiraoka, J. W. Sedat, D. A. Agard, “Determination of three-dimensional imaging properties of a light microscope system,” Biophys. J. 57, 325–333 (1990).
[CrossRef] [PubMed]

Y. Hiraoka, D. A. Agard, J. W. Sedat, “Temporal and spatial coordination of chromosome movement, spindle formation, and nuclear envelope breakdown during prometaphase in Drosophila melanogaster embryos,” J. Cell Biol. 111, 2815–2828 (1990).
[CrossRef] [PubMed]

Shaw, P. J.

P. J. Shaw, D. J. Rawlins, “Three-dimensional fluorescence microscopy,” Prog. Biophys. Mol. Biol. 56, 187–213 (1991).
[CrossRef] [PubMed]

Sheppard, C. J. R.

C. J. R. Sheppard, “Stray light and noise in confocal microscopy,” Micron Microsc. Acta 22, 239–243 (1991).
[CrossRef]

C. J. R. Sheppard, C. J. Cogswell, M. Gu, “Signal strength and noise in confocal microscopy: factors influencing selection of an optimum detector aperture,” Scanning 13, 233–240 (1991).
[CrossRef]

C. J. R. Sheppard, H. J. Matthews, “Imaging in high-aperture optical systems,” J. Opt. Soc. Am. A 4, 1354–1360 (1987).
[CrossRef]

I. J. Cox, C. J. R. Sheppard, “Information capacity and resolution in an optical system,” J. Opt. Soc. Am. A 3, 1152–1158 (1986).
[CrossRef]

C. J. R. Sheppard, A. Choudhury“Image formation in the scanning microscope,” Opt. Acta 10, 1051–1073 (1977).
[CrossRef]

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

Strickler, J.

W. W. Webb, K. S. Wells, D. R. Sandison, J. Strickler, “Criteria for quantitative dynamical confocal fluorescence imaging,” in Optical Microscopy for Biology, B. Herman, K. Jacobson, eds. (Wiley, New York, 1990), Chap. 6, pp. 73–108.

K. S. Wells, D. R. Sandison, J. Strickler, W. W. Webb, “Quantitative fluorescence imaging with laser scanning confocal microscopy,” in Handbook of Biological Confocal Microscopy, J. B. Pawley, ed. (Plenum, New York, 1990), Chap. 3.
[CrossRef]

Wall, J.

A. V. Crewe, J. Wall, “Contrast in a high-resolution scanning-transmission electron microscope,” Optik 30, 461–474 (1970).

Webb, W W.

D. R. Sandison, D. W. Piston, W W. Webb, “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, J. E. Trogadis, eds. (Academic, San Diego, Calif., 1994), Chap. 2, pp. 29–46.

Webb, W. W.

D. R. Sandison, W. W. Webb, “Background rejection and signal-to-noise optimization in confocal and alternative fluorescence microscopes,” Appl. Opt. 33, 603–615 (1994).
[CrossRef] [PubMed]

K. S. Wells, D. R. Sandison, J. Strickler, W. W. Webb, “Quantitative fluorescence imaging with laser scanning confocal microscopy,” in Handbook of Biological Confocal Microscopy, J. B. Pawley, ed. (Plenum, New York, 1990), Chap. 3.
[CrossRef]

W. W. Webb, K. S. Wells, D. R. Sandison, J. Strickler, “Criteria for quantitative dynamical confocal fluorescence imaging,” in Optical Microscopy for Biology, B. Herman, K. Jacobson, eds. (Wiley, New York, 1990), Chap. 6, pp. 73–108.

Wells, K. S.

W. W. Webb, K. S. Wells, D. R. Sandison, J. Strickler, “Criteria for quantitative dynamical confocal fluorescence imaging,” in Optical Microscopy for Biology, B. Herman, K. Jacobson, eds. (Wiley, New York, 1990), Chap. 6, pp. 73–108.

K. S. Wells, D. R. Sandison, J. Strickler, W. W. Webb, “Quantitative fluorescence imaging with laser scanning confocal microscopy,” in Handbook of Biological Confocal Microscopy, J. B. Pawley, ed. (Plenum, New York, 1990), Chap. 3.
[CrossRef]

White, J. B.

J. B. White, W. B. Amos, M. Fordham, “An evaluation of confocal versus conventional imaging of biological structures by fluorescence light microscopy.” J. Cell Biol. 105, 41–48 (1987).
[CrossRef] [PubMed]

Wilson, T.

T. Wilson, A. R. Carlini, “Three-dimensional imaging in confocal imaging systems with finite sized detectors,” J. Mi-crosc. 149, 51–66 (1988).

T. Wilson, “The role of the pinhole in confocal imaging systems,” in Handbook of Biological Confocal Microscopy, J. B. Pawley, ed. (Plenum, New York, 1990), Chap. 11.
[CrossRef]

T. Wilson, Confocal Microscopy (Academic, Boston, Mass., 1990).

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

Wolf, E.

B. Richards, E. Wolf, “Electromagnetic diffraction in optical systems II. Structure of the image field in an aplanatic system,” Proc. R. Soc. London Ser. A 253, 358–379 (1959).
[CrossRef]

M. Born, E. Wolf, Principles of Optics, 6th ed. (Pergamon, New York, 1980), p. 437.

M. Born, E. Wolf, Principles of Optics, 6th ed. (Pergamon, New York, 1980).

Appl. Opt. (1)

Biophys. J. (1)

Y. Hiraoka, J. W. Sedat, D. A. Agard, “Determination of three-dimensional imaging properties of a light microscope system,” Biophys. J. 57, 325–333 (1990).
[CrossRef] [PubMed]

J. Cell Biol. (3)

Y. Hiraoka, D. A. Agard, J. W. Sedat, “Temporal and spatial coordination of chromosome movement, spindle formation, and nuclear envelope breakdown during prometaphase in Drosophila melanogaster embryos,” J. Cell Biol. 111, 2815–2828 (1990).
[CrossRef] [PubMed]

M. Benveniste, J. Schlessinger, Z. Kam, “Characterization of internalization and endosome formation of epidermal growth factor in transfected nih-3t3 cells by computerized image-intensified three-dimensional fluorescence microscopy,” J. Cell Biol. 109, 2105–2115 (1989).
[CrossRef] [PubMed]

J. B. White, W. B. Amos, M. Fordham, “An evaluation of confocal versus conventional imaging of biological structures by fluorescence light microscopy.” J. Cell Biol. 105, 41–48 (1987).
[CrossRef] [PubMed]

J. Mi-crosc. (1)

T. Wilson, A. R. Carlini, “Three-dimensional imaging in confocal imaging systems with finite sized detectors,” J. Mi-crosc. 149, 51–66 (1988).

J. Microsc. (1)

M. E. Barnett, “Image formation in optical and electron transmission microscopy,” J. Microsc. 102, 1–28 (1974).
[CrossRef]

J. Opt. Soc. Am. A (2)

J. Opt. Soc.Am.A (1)

S. Kimura, C. Munakata, “Calculation of three-dimensional optical transfer function for a confocal scanning fluorescent microscope,” J. Opt. Soc.Am.A 6, 1015–1019 (1989).
[CrossRef]

Micron Microsc. Acta (1)

C. J. R. Sheppard, “Stray light and noise in confocal microscopy,” Micron Microsc. Acta 22, 239–243 (1991).
[CrossRef]

Opt. Acta (1)

C. J. R. Sheppard, A. Choudhury“Image formation in the scanning microscope,” Opt. Acta 10, 1051–1073 (1977).
[CrossRef]

Optik (1)

A. V. Crewe, J. Wall, “Contrast in a high-resolution scanning-transmission electron microscope,” Optik 30, 461–474 (1970).

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

B. Richards, E. Wolf, “Electromagnetic diffraction in optical systems II. Structure of the image field in an aplanatic system,” Proc. R. Soc. London Ser. A 253, 358–379 (1959).
[CrossRef]

Prog. Biophys. Mol. Biol. (1)

P. J. Shaw, D. J. Rawlins, “Three-dimensional fluorescence microscopy,” Prog. Biophys. Mol. Biol. 56, 187–213 (1991).
[CrossRef] [PubMed]

Scanning (1)

C. J. R. Sheppard, C. J. Cogswell, M. Gu, “Signal strength and noise in confocal microscopy: factors influencing selection of an optimum detector aperture,” Scanning 13, 233–240 (1991).
[CrossRef]

Other (14)

S. Inoue, Video Microscopy (Plenum, New York, 1986).

Optical units are dimensionless distances associated with the size of the PSF. We choose those units that are consistent with the sine condition of imaging (see Ref. 16). We modify the referenced definitions to account for the specimen's index of refraction n and a high objective-lens NA by replacing λ with λ/n and a/f with NA/n. Lateral υ and axial u optical units are then defined as follows: υ = k(NA)r and u = k(NA)2z/n, where r is the distance from the optical axis, z is the distance from the focal plane, and k = 2πλ is the vacuum wave number. In the detector aperture plane, υi = k(NA)ri/M, where ri is the image-plane distance from the optical axis and M is the total magnification of the optical path.

M. Born, E. Wolf, Principles of Optics, 6th ed. (Pergamon, New York, 1980), p. 437.

M. Born, E. Wolf, Principles of Optics, 6th ed. (Pergamon, New York, 1980).

T. Wilson, “The role of the pinhole in confocal imaging systems,” in Handbook of Biological Confocal Microscopy, J. B. Pawley, ed. (Plenum, New York, 1990), Chap. 11.
[CrossRef]

G. Arfken, Mathematical Methods for Physicists, 3rd ed. (Academic, San Diego, Calif, 1985).

Ref. 17, p. 443.

K. S. Wells, D. R. Sandison, J. Strickler, W. W. Webb, “Quantitative fluorescence imaging with laser scanning confocal microscopy,” in Handbook of Biological Confocal Microscopy, J. B. Pawley, ed. (Plenum, New York, 1990), Chap. 3.
[CrossRef]

D. R. Sandison, D. W. Piston, W W. Webb, “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, J. E. Trogadis, eds. (Academic, San Diego, Calif., 1994), Chap. 2, pp. 29–46.

W. W. Webb, K. S. Wells, D. R. Sandison, J. Strickler, “Criteria for quantitative dynamical confocal fluorescence imaging,” in Optical Microscopy for Biology, B. Herman, K. Jacobson, eds. (Wiley, New York, 1990), Chap. 6, pp. 73–108.

M. Minsky, “Microscopy apparatus,” U.S. patent3,013,467 (19December1961).

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

T. Wilson, Confocal Microscopy (Academic, Boston, Mass., 1990).

S. Inoue, Video Microscopy (Plenum, New York, 1986), p. 21.

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Figures (13)

Fig. 1
Fig. 1

Microtubule networks that are dissociating after cytokinesis in fixed sea urchin (Lytechinus pictus) embryos are immunofluores-cently labeled with Rhodamine. Images are taken with a 40×/1.3-numerical-aperture (NA) plan-neofluar oil-immersion objective and show three different levels of background rejection: (a) no background rejection, (b) a detector aperture 6 times the width of the Airy pattern [23 optical units (ou)], and (c) with a detector aperture 0.9 times the width of the Airy pattern 3.3 ou). Total bleaching during the acquisition of all three images was less than 3%. The scale bar in (b) is 50 μm. Sea urchin samples were generously provided by R. G. Summers of the State University of New York-Buffalo medical school.

Fig. 2
Fig. 2

Calculated image-intensity profiles of 5000 signal photons from a point source in the presence of background and shot noise. (a) An isolated point source with a uniform average background intensity 100 times the point source peak intensity (S/B = 0.01); the point source is lost in the background-associated shot noise, (b) The same point source with a confocal detector aperture to reduce the background (S/B = 1); the point source is now resolvable.

Fig. 3
Fig. 3

Schematic diagram of the simultaneous CLSM–FLSM laser scanning microscope. Sample fluorescence collected by the objective lens is split by DM2. The transmitted portion passes through the usual MRC600 CLSM optics. The reflected portion is collected by the external FLSM optics inside the broken-border box. AOM, acousto-optic modulator; BF, barrier filter; D, detector aperture plane; DF, discriminating filter; DM, dichroic mirror; EP, eyepiece; I, intermediate image plane; L, lens; OBJ, objective lens; PMT, photomultiplier tube.

Fig. 4
Fig. 4

Measured images of a point-source estimate for the xz sections through the PSF for three detector aperture radii: (a) υD = 10 ou, (b) υD = 4.2 ou, and (c) υD = 1.4 ou. The illumination intensity increases with decreasing aperture size. The beads are immersed in oil with an index of refraction of 1.518 to avoid artifacts in the axial distances measurements that would be due to a refractive-index mismatch between the cover slip and the specimen.17 The scale bar in (c) is 0.5 μm.

Fig. 5
Fig. 5

Calculated lateral and axial CLSM resolution as a function of the detector aperture radius. The curves represent the lateral (lower curve) and axial (upper curve) FWHM resolution for a point source calculated with a paraxial PSF. The symbols mark the detector aperture radii that correspond to the images in Figs. 4(a) (triangle), 4(b) (circle), and 4(c) (square).

Fig. 6
Fig. 6

Measured xz images that display the axial resolution for a fluorescent plane. The same horizontal fluorescent plane with a 33-um hole photobleached at its center was imaged for each part, as follows: (a) FLSM with υD = ∞, (b) CLSM with υD = 16 ou, and (c) CLSM with υD = 2.3 ou.

Fig. 7
Fig. 7

Measured intensity profiles from the FLSM image of a planar sample [Fig. 6(a)]. Intensity as a function of the lateral distance from the optic axis is extracted at z = 0 μm (top), z = 20 μm (middle), and z = 40 μm (bottom). The square well seen clearly in the top plot is blurred by defocus until only the zero spatial frequency remains.

Fig. 8
Fig. 8

Calculated OTF's. The lateral OTF is calculated as a function of defocus for three levels of background rejection: (a) the FLSM, (b) the CLSM with υD = 16 ou, and (c) the ideal CLSM with υD = 0. The CLSM background rejection reduces the s = 0 component with defocus so that a uniform planar specimen is axially resolved.

Fig. 9
Fig. 9

Signal from 93-nm fluorescence beads as a function of the detector aperture radius for three objective lenses: 100×/0.8-NA (squares), 100×/1.25-NA (triangles), and 60×/1.4-NA (circles). The signal is calculated with a paraxial PSF for the on-axis pixel (dashed curve) and for the integrated image (solid curve). Paraxial calculations are not accurate for the 1.4-NA objective.

Fig. 10
Fig. 10

Measured background from a uniform bulk source as a function of sample thickness and detector aperture radius. The laser illumination was focused at the cover-slip-fluorophore interface. The size of a square indicates its relative aperture radius, and the dashed curves are splines fitted to the data points to aid the eye. The background is calculated for υD = 10 ou (solid curve) with a paraxial PSF.

Fig. 11
Fig. 11

S/B determined as a function of specimen thickness and detector aperture radius by use of a measured high-NA signal (Fig. 9) and background (Fig. 10). The size of a square indicates its relative aperture radius, and the dashed curves are splines fitted to the data points to aid the eye.

Fig. 12
Fig. 12

Measured S/N as a function of the detector aperture radius at two background florophore concentrations: 5 μM (circles) and 20 μM (squares). Dashed curves are splines fitted to the data points to aid the eye. Note that the S/N is optimized at υD ∼ 3.3 ou.

Fig. 13
Fig. 13

Three-dimensional Köhler illumination profile measured in the xz plane. The illumination profile is cylindrically symmetric about the z axis, and its in-focus diameter (here 120 μm) is determined by the field aperture. The solid lines form the boundary of the volume of a double-sided cone over which the illumination intensity varies by less than 15%. The scale bar (lower right-hand side) is 20 μm.

Equations (7)

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S / N = S / ( S + B ) 1 / 2 ,
S FEM ( S / B ) FEM = S CLSM ( S / B ) CLSM .
I [ z 0 , x 0 , y 0 ] ( illuminated area ) d x S d y S PSF { z 0 , [ ( x S x 0 ) 2 + ( y S y 0 ) 2 ] 1 / 2 } .
I [ z 0 , x 0 , y 0 , t ] 1 T 0 T d t PSF { z 0 , [ ( x S [ t ] x 0 ) 2 + ( y S [ t ] y 0 ) 2 ] 1 / 2 } ,
I [ z 0 , x 0 , y 0 ] ( illuminated area ) d x S d y S PSF { z 0 , [ ( x S x 0 ) 2 + ( y S y 0 ) 2 ] 1 / 2 } .
I ( x I , y I ) PSF { z 0 , [ ( x I M x 0 ) 2 + ( y I M y 0 ) 2 ] 1 / 2 } × I [ z 0 , x 0 , y 0 ] ,
I [ x I , y I ] PSF { z 0 , [ ( x I M x 0 ) 2 + ( y I M y 0 ) 2 ] 1 / 2 } × ( detector aperture ) d x d d y d PSF { z 0 , [ ( x d M x 0 ) 2 + ( y d M y 0 ) 2 ] 1 / 2 } .

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