Abstract

We demonstrate the acquisition of high-quality two-photon fluorescence microscopy images using an all-solid-state self-mode-locked Cr:LiSAF laser. We contrast the performance of the two-photon technique with single-photon confocal fluorescence microscopy images taken with an argon-ion laser. Examples of improved depth penetration and reduced dye bleaching are presented.

© 1997 Optical Society of America

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References

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  1. W. Denk, J. H. Stickler, W. W. Webb, “2-Photon laser scanning fluorescence microscopy,” Science 248, 73–76 (1990).
    [CrossRef] [PubMed]
  2. C. J. R. Sheppard, “Scanning optical microscopy,” in Advances in Optical and Electrical Microscopy, R. Barer, V. E. Cosselett, eds. (Academic, London, 1987), Vol. 10, pp. 1–98.
  3. S. Hell, E. H. K. Stelzer, “Fundamental improvement of resolution with a 4 pi - confocal fluorescence microscope using 2-photon excitation,” Opt. Commun. 93, 277–282 (1992).
    [CrossRef]
  4. O. Nakamura, “3-dimensional imaging characteristics of laser scan fluorescence microscopy - 2-photon excitation vs. single-photon excitation,” Optik 93, 39–42 (1993).
  5. M. J. P. Dymott, A. Ferguson, “Self-mode-locked diode pumped Cr:LiSAF laser producing 34 fs pulses at 42 mW average power,” Opt. Lett. 20, 1–3 (1995).

1995 (1)

1993 (1)

O. Nakamura, “3-dimensional imaging characteristics of laser scan fluorescence microscopy - 2-photon excitation vs. single-photon excitation,” Optik 93, 39–42 (1993).

1992 (1)

S. Hell, E. H. K. Stelzer, “Fundamental improvement of resolution with a 4 pi - confocal fluorescence microscope using 2-photon excitation,” Opt. Commun. 93, 277–282 (1992).
[CrossRef]

1990 (1)

W. Denk, J. H. Stickler, W. W. Webb, “2-Photon laser scanning fluorescence microscopy,” Science 248, 73–76 (1990).
[CrossRef] [PubMed]

Denk, W.

W. Denk, J. H. Stickler, W. W. Webb, “2-Photon laser scanning fluorescence microscopy,” Science 248, 73–76 (1990).
[CrossRef] [PubMed]

Dymott, M. J. P.

Ferguson, A.

Hell, S.

S. Hell, E. H. K. Stelzer, “Fundamental improvement of resolution with a 4 pi - confocal fluorescence microscope using 2-photon excitation,” Opt. Commun. 93, 277–282 (1992).
[CrossRef]

Nakamura, O.

O. Nakamura, “3-dimensional imaging characteristics of laser scan fluorescence microscopy - 2-photon excitation vs. single-photon excitation,” Optik 93, 39–42 (1993).

Sheppard, C. J. R.

C. J. R. Sheppard, “Scanning optical microscopy,” in Advances in Optical and Electrical Microscopy, R. Barer, V. E. Cosselett, eds. (Academic, London, 1987), Vol. 10, pp. 1–98.

Stelzer, E. H. K.

S. Hell, E. H. K. Stelzer, “Fundamental improvement of resolution with a 4 pi - confocal fluorescence microscope using 2-photon excitation,” Opt. Commun. 93, 277–282 (1992).
[CrossRef]

Stickler, J. H.

W. Denk, J. H. Stickler, W. W. Webb, “2-Photon laser scanning fluorescence microscopy,” Science 248, 73–76 (1990).
[CrossRef] [PubMed]

Webb, W. W.

W. Denk, J. H. Stickler, W. W. Webb, “2-Photon laser scanning fluorescence microscopy,” Science 248, 73–76 (1990).
[CrossRef] [PubMed]

Opt. Commun. (1)

S. Hell, E. H. K. Stelzer, “Fundamental improvement of resolution with a 4 pi - confocal fluorescence microscope using 2-photon excitation,” Opt. Commun. 93, 277–282 (1992).
[CrossRef]

Opt. Lett. (1)

Optik (1)

O. Nakamura, “3-dimensional imaging characteristics of laser scan fluorescence microscopy - 2-photon excitation vs. single-photon excitation,” Optik 93, 39–42 (1993).

Science (1)

W. Denk, J. H. Stickler, W. W. Webb, “2-Photon laser scanning fluorescence microscopy,” Science 248, 73–76 (1990).
[CrossRef] [PubMed]

Other (1)

C. J. R. Sheppard, “Scanning optical microscopy,” in Advances in Optical and Electrical Microscopy, R. Barer, V. E. Cosselett, eds. (Academic, London, 1987), Vol. 10, pp. 1–98.

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Figures (4)

Fig. 1
Fig. 1

Schematic diagram of the Cr:LiSAF laser: L1–L4, lenses; L1, L4, spherical lenses of focal length 6.5 and 90 mm, respectively; L2, L3, cylindrical lenses of focal lengths 5 and 100 mm, respectively.

Fig. 2
Fig. 2

Schematic diagram of the microscope: P1, P2, photomultiplier tubes; D1, D2, dichroic beam splitters; SM1, SM2, scanning mirrors.

Fig. 3
Fig. 3

Sample of testis stained with Haemotoxylin and Eosin: (a) single-photon fluorescence image, (b) two-photon fluorescence image taken with the Cr:LiSAF laser, showing improved depth penetration.

Fig. 4
Fig. 4

Stained plastic sample exposed to a parked beam from the Cr:LiSAF laser. The image was formed by scanning in the xz plane to form a depth sample.

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