Abstract

We present a transmission-mode confocal laser scanning microscope system based on the use of second-harmonic generation (SHG) for signal detection. Our method exploits the quadratic intensity dependence of SHG to preferentially reveal unscattered signal light and reject out-of-focus scattered background. The SHG crystal acts as a virtual pinhole that remains self-aligned without the need for descanning.

© 2003 Optical Society of America

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References

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  1. M. Minsky, “Microscopy Apparatus,” U.S. patent3,013,467 (November7, 1957).
  2. T. Wilson and C. Sheppard, Theory and Practice of Scanning Optical Microscopy (Academic, London, 1984).
  3. A. E. Dixon, S. Damaskinos, and M. R. Atkinson, Nature 351, 551 (1991).
    [CrossRef]
  4. G. J. Brakenhoff, J. Microsc. 117, 233 (1979).
    [CrossRef]
  5. M. Born and E. Wolf, Principles of Optics, 7th ed. (Cambridge U. Press, Cambridge, 1999).
    [CrossRef]
  6. C. Bohren and D. Huffman, Absorption and Scattering of Light by Small Particles (Wiley-Interscience, New York, 1983).
  7. W. Denk, J. H. Strickler, and W. W. Webb, Science 248, 73 (1990).
    [CrossRef] [PubMed]

1991

A. E. Dixon, S. Damaskinos, and M. R. Atkinson, Nature 351, 551 (1991).
[CrossRef]

1990

W. Denk, J. H. Strickler, and W. W. Webb, Science 248, 73 (1990).
[CrossRef] [PubMed]

1979

G. J. Brakenhoff, J. Microsc. 117, 233 (1979).
[CrossRef]

Atkinson, M. R.

A. E. Dixon, S. Damaskinos, and M. R. Atkinson, Nature 351, 551 (1991).
[CrossRef]

Bohren, C.

C. Bohren and D. Huffman, Absorption and Scattering of Light by Small Particles (Wiley-Interscience, New York, 1983).

Born, M.

M. Born and E. Wolf, Principles of Optics, 7th ed. (Cambridge U. Press, Cambridge, 1999).
[CrossRef]

Brakenhoff, G. J.

G. J. Brakenhoff, J. Microsc. 117, 233 (1979).
[CrossRef]

Damaskinos, S.

A. E. Dixon, S. Damaskinos, and M. R. Atkinson, Nature 351, 551 (1991).
[CrossRef]

Denk, W.

W. Denk, J. H. Strickler, and W. W. Webb, Science 248, 73 (1990).
[CrossRef] [PubMed]

Dixon, A. E.

A. E. Dixon, S. Damaskinos, and M. R. Atkinson, Nature 351, 551 (1991).
[CrossRef]

Huffman, D.

C. Bohren and D. Huffman, Absorption and Scattering of Light by Small Particles (Wiley-Interscience, New York, 1983).

Minsky, M.

M. Minsky, “Microscopy Apparatus,” U.S. patent3,013,467 (November7, 1957).

Sheppard, C.

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

Strickler, J. H.

W. Denk, J. H. Strickler, and W. W. Webb, Science 248, 73 (1990).
[CrossRef] [PubMed]

Webb, W. W.

W. Denk, J. H. Strickler, and W. W. Webb, Science 248, 73 (1990).
[CrossRef] [PubMed]

Wilson, T.

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

Wolf, E.

M. Born and E. Wolf, Principles of Optics, 7th ed. (Cambridge U. Press, Cambridge, 1999).
[CrossRef]

J. Microsc.

G. J. Brakenhoff, J. Microsc. 117, 233 (1979).
[CrossRef]

Nature

A. E. Dixon, S. Damaskinos, and M. R. Atkinson, Nature 351, 551 (1991).
[CrossRef]

Science

W. Denk, J. H. Strickler, and W. W. Webb, Science 248, 73 (1990).
[CrossRef] [PubMed]

Other

M. Born and E. Wolf, Principles of Optics, 7th ed. (Cambridge U. Press, Cambridge, 1999).
[CrossRef]

C. Bohren and D. Huffman, Absorption and Scattering of Light by Small Particles (Wiley-Interscience, New York, 1983).

M. Minsky, “Microscopy Apparatus,” U.S. patent3,013,467 (November7, 1957).

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

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Figures (4)

Fig. 1
Fig. 1

Experimental layout: MO’s, microscope objectives; PMT, photomultiplier tube. The filter transmits only SHG light.

Fig. 2
Fig. 2

Measured SHG/SHG0 and P2/P02 for a Z scan of a 530-nm latex bead (averaged over five scans). Traces are derived from a Gaussian approximation model. (a) Nonaveraged SHG and (b) direct P2 xy scans of the bead. Scale bars, 5 µm.

Fig. 3
Fig. 3

Measured SHG and P2 for a Z scan of a 170µm-thick agarose slab of 1µm latex beads, and a theoretical trace derived from a Gaussian approximation, normalized to arbitrary units.

Fig. 4
Fig. 4

xy images of an onion slice beneath a 200µm agarose slab of 1µm latex beads obtained left, by SHG detection and right, by direct P2 detection. Scale bars, 100 µm.

Equations (5)

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Ir,z=P01-zW0r+zηWSr,z,
SHGSHG0=1-z2+2z1-zηA00A0S+z2η2A00ASS.
SHGSHG0 z=zslab-L/2zslab+L/21-2z1-ηzA00A0S+δz21-2ηzA00A0S+ηz2A00ASS,
SHGSHG0exp-2Nσzslab-L/2zslab+L/21-ηzA00A0S-σ2Uzδz,
P2P02exp-2Nσzslab-L/2zslab+L/21-ηzδz.

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