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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References

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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

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

1997

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

1995

1963

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.

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

J. Phys. Chem. A

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

Opt. Lett.

Phys. Rev. Lett.

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

Science

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

Other

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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Figures (4)

Fig. 1
Fig. 1

Schematic diagram of the optical setup: DM, dichroic mirror; PBC, polarizing beam cube; QWP, quarter-wave plate.

Fig. 2
Fig. 2

Two-dimensional images taken at different scan rates and excitation powers: (A) 950 pixels/s at 3 mW, (B) 4750 pixels/s at 15 mW.

Fig. 3
Fig. 3

Composite images of one-dimensional scans. The top row is raw data, and the bottom row contains the integrated intensity profiles of the raw data. The integrated images are offset for clarity.

Fig. 4
Fig. 4

Zero-dimensional time scans at different resolutions. (A) and (B) were binned at 128 time points/bin. B is the first 2 s of (B) binned at eight time points/bin. The arrows accompanying each time scan indicate the appropriate time and counts axes.

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