Abstract

We describe a laser-scanning two-photon fluorescence microscope that is capable of observing single molecules with excellent temporal resolution and three-dimensional spatial resolution. To demonstrate the capabilities of the instrument we present single-molecule fluorescence data obtained in several different scanning modes. In addition, a polarization-sensitive detection scheme can provide detailed three-dimensional information about the orientations of molecules in any of these scanning modes.

© 1999 Optical Society of America

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  1. T. Basché, W. E. Moerner, M. Orrit, and U. Wild, eds., Single Molecule Optical Detection, Imaging and Spectroscopy (Verlag-Chemie, Weinheim, Germany, 1997).
  2. W. E. Moerner and M. Orrit, Science 283, 1670 (1999).
    [CrossRef] [PubMed]
  3. J. Mertz, C. Xu, and W. W. Webb, Opt. Lett. 20, 2532 (1995).
    [CrossRef]
  4. E. J. Sánchez, L. Novotny, G. R. Holtom, and X. S. Xie, J. Phys. Chem. A 101, 7019 (1997).
  5. M. K. Lewis, P. Wolanin, A. Gafni, and D. G. Steel, Opt. Lett. 23, 1111 (1998).
    [CrossRef]
  6. M. Sonnleitner, G. J. Schütz, and Th. Schmidt, Chem. Phys. Lett. 300, 221 (1999).
  7. W. L. Peticolas, J. P. Goldsborough, and K. E. Reickhoff, Phys. Rev. Lett. 10, 43 (1963).
    [CrossRef]
  8. In some confocal microscopes the collected fluorescence is reflected back off the scanning mirrors and thus can be focused to a constant position as the excitation position is scanned.?However, this scheme is somewhat lossy, and the angle-dependent depolarization of the scanning mirrors would preclude the use of the polarization-senstive detection system described here.
  9. P. T. C. So, T. French, W. M. Yu, K. M. Berland, C. Y. Dong, and E. Gratton, in Fluorescence Imaging Spectroscopy and Microscopy, X. F. Wang and B. Herman, eds. (Wiley, New York, 1996), pp. 351–374.

1999 (2)

W. E. Moerner and M. Orrit, Science 283, 1670 (1999).
[CrossRef] [PubMed]

M. Sonnleitner, G. J. Schütz, and Th. Schmidt, Chem. Phys. Lett. 300, 221 (1999).

1998 (1)

1997 (1)

E. J. Sánchez, L. Novotny, G. R. Holtom, and X. S. Xie, J. Phys. Chem. A 101, 7019 (1997).

1995 (1)

1963 (1)

W. L. Peticolas, J. P. Goldsborough, and K. E. Reickhoff, Phys. Rev. Lett. 10, 43 (1963).
[CrossRef]

Berland, K. M.

P. T. C. So, T. French, W. M. Yu, K. M. Berland, C. Y. Dong, and E. Gratton, in Fluorescence Imaging Spectroscopy and Microscopy, X. F. Wang and B. Herman, eds. (Wiley, New York, 1996), pp. 351–374.

Dong, C. Y.

P. T. C. So, T. French, W. M. Yu, K. M. Berland, C. Y. Dong, and E. Gratton, in Fluorescence Imaging Spectroscopy and Microscopy, X. F. Wang and B. Herman, eds. (Wiley, New York, 1996), pp. 351–374.

French, T.

P. T. C. So, T. French, W. M. Yu, K. M. Berland, C. Y. Dong, and E. Gratton, in Fluorescence Imaging Spectroscopy and Microscopy, X. F. Wang and B. Herman, eds. (Wiley, New York, 1996), pp. 351–374.

Gafni, A.

Goldsborough, J. P.

W. L. Peticolas, J. P. Goldsborough, and K. E. Reickhoff, Phys. Rev. Lett. 10, 43 (1963).
[CrossRef]

Gratton, E.

P. T. C. So, T. French, W. M. Yu, K. M. Berland, C. Y. Dong, and E. Gratton, in Fluorescence Imaging Spectroscopy and Microscopy, X. F. Wang and B. Herman, eds. (Wiley, New York, 1996), pp. 351–374.

Holtom, G. R.

E. J. Sánchez, L. Novotny, G. R. Holtom, and X. S. Xie, J. Phys. Chem. A 101, 7019 (1997).

Lewis, M. K.

Mertz, J.

Moerner, W. E.

W. E. Moerner and M. Orrit, Science 283, 1670 (1999).
[CrossRef] [PubMed]

Novotny, L.

E. J. Sánchez, L. Novotny, G. R. Holtom, and X. S. Xie, J. Phys. Chem. A 101, 7019 (1997).

Orrit, M.

W. E. Moerner and M. Orrit, Science 283, 1670 (1999).
[CrossRef] [PubMed]

Peticolas, W. L.

W. L. Peticolas, J. P. Goldsborough, and K. E. Reickhoff, Phys. Rev. Lett. 10, 43 (1963).
[CrossRef]

Reickhoff, K. E.

W. L. Peticolas, J. P. Goldsborough, and K. E. Reickhoff, Phys. Rev. Lett. 10, 43 (1963).
[CrossRef]

Sánchez, E. J.

E. J. Sánchez, L. Novotny, G. R. Holtom, and X. S. Xie, J. Phys. Chem. A 101, 7019 (1997).

Schmidt, Th.

M. Sonnleitner, G. J. Schütz, and Th. Schmidt, Chem. Phys. Lett. 300, 221 (1999).

Schütz, G. J.

M. Sonnleitner, G. J. Schütz, and Th. Schmidt, Chem. Phys. Lett. 300, 221 (1999).

So, P. T. C.

P. T. C. So, T. French, W. M. Yu, K. M. Berland, C. Y. Dong, and E. Gratton, in Fluorescence Imaging Spectroscopy and Microscopy, X. F. Wang and B. Herman, eds. (Wiley, New York, 1996), pp. 351–374.

Sonnleitner, M.

M. Sonnleitner, G. J. Schütz, and Th. Schmidt, Chem. Phys. Lett. 300, 221 (1999).

Steel, D. G.

Webb, W. W.

Wolanin, P.

Xie, X. S.

E. J. Sánchez, L. Novotny, G. R. Holtom, and X. S. Xie, J. Phys. Chem. A 101, 7019 (1997).

Xu, C.

Yu, W. M.

P. T. C. So, T. French, W. M. Yu, K. M. Berland, C. Y. Dong, and E. Gratton, in Fluorescence Imaging Spectroscopy and Microscopy, X. F. Wang and B. Herman, eds. (Wiley, New York, 1996), pp. 351–374.

Chem. Phys. Lett. (1)

M. Sonnleitner, G. J. Schütz, and Th. Schmidt, Chem. Phys. Lett. 300, 221 (1999).

J. Phys. Chem. A (1)

E. J. Sánchez, L. Novotny, G. R. Holtom, and X. S. Xie, J. Phys. Chem. A 101, 7019 (1997).

Opt. Lett. (2)

Phys. Rev. Lett. (1)

W. L. Peticolas, J. P. Goldsborough, and K. E. Reickhoff, Phys. Rev. Lett. 10, 43 (1963).
[CrossRef]

Science (1)

W. E. Moerner and M. Orrit, Science 283, 1670 (1999).
[CrossRef] [PubMed]

Other (3)

T. Basché, W. E. Moerner, M. Orrit, and U. Wild, eds., Single Molecule Optical Detection, Imaging and Spectroscopy (Verlag-Chemie, Weinheim, Germany, 1997).

In some confocal microscopes the collected fluorescence is reflected back off the scanning mirrors and thus can be focused to a constant position as the excitation position is scanned.?However, this scheme is somewhat lossy, and the angle-dependent depolarization of the scanning mirrors would preclude the use of the polarization-senstive detection system described here.

P. T. C. So, T. French, W. M. Yu, K. M. Berland, C. Y. Dong, and E. Gratton, in Fluorescence Imaging Spectroscopy and Microscopy, X. F. Wang and B. Herman, eds. (Wiley, New York, 1996), pp. 351–374.

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