Abstract

We report the observation of two-photon fluorescence excitation in a continuous-wave (cw) single-beam gradient force optical trap and demonstrate its use as an in situ probe to study the physiological state of an optically confined sample. In particular, a cw Nd:YAG (1064-nm) laser is used simultaneously to confine, and excite visible fluorescence from submicrometer regions of, cell specimens. Two-photon fluorescence emission spectra are presented for motile human sperm cells and immotile Chinese hamster ovary cells that have been labeled with nucleic acid (Propidium Iodide) and pH-sensitive (Snarf) fluorescent probes. The resulting spectra are correlated to light-induced changes in the physiological state experienced by the trapped cells. This spectral technique should prove extremely useful for monitoring cellular activity and the effects of confinement by optical tweezers.

© 1995 Optical Society of America

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References

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  1. A. Ashkin, J. M. Dziedzic, J. E. Bjorkholm, S. Chu, Opt. Lett. 11, 288 (1986).
    [CrossRef] [PubMed]
  2. K. Svoboda, S. M. Block, Annu. Rev. Biophys. Biomol. Struct. 23, 247 (1994).
    [CrossRef] [PubMed]
  3. Y. Liu, D. K. Cheng, G. J. Sonek, M. W. Berns, C. F. Chapman, B. J. Tromberg, Biophys. J. 68, 2137 (1995).
    [CrossRef] [PubMed]
  4. K. Visscher, “Optical manipulation and confocal microscopy,” Ph.D. dissertation (University of Amsterdam, Amsterdam, 1993), pp. 73–77.
  5. P. E. Hanninen, E. Soini, S. W. Hell, J. Microsc. (Oxford)176, 222 (1994).
    [CrossRef]
  6. S. P. Jiang, Prog. React. Kinet. 15, 77 (1989).
  7. W. Denk, J. H. Strickler, W. W. Webb, Science 248, 73 (1990).
    [CrossRef] [PubMed]
  8. D. W. Piston, M. S. Kirby, H. Cheng, W. J. Lederer, W. Webb, Appl. Opt. 33, 662 (1994).
    [CrossRef] [PubMed]
  9. W. Denk, Proc. Natl. Acad. Sci. (USA) 91, 6629 (1994).
    [CrossRef] [PubMed]
  10. R. G. Haugland, Handbook of Fluorescence Probes and Research Chemicals (Molecular Probes, Eugene, Ore., 1995).
  11. Y. Tadir, W. H. Wright, O. Vafa, T. Ord, R. H. Asch, M. W. Berns, Fertil. Steril. 53, 944 (1990).
    [PubMed]

1995 (1)

Y. Liu, D. K. Cheng, G. J. Sonek, M. W. Berns, C. F. Chapman, B. J. Tromberg, Biophys. J. 68, 2137 (1995).
[CrossRef] [PubMed]

1994 (3)

K. Svoboda, S. M. Block, Annu. Rev. Biophys. Biomol. Struct. 23, 247 (1994).
[CrossRef] [PubMed]

D. W. Piston, M. S. Kirby, H. Cheng, W. J. Lederer, W. Webb, Appl. Opt. 33, 662 (1994).
[CrossRef] [PubMed]

W. Denk, Proc. Natl. Acad. Sci. (USA) 91, 6629 (1994).
[CrossRef] [PubMed]

1990 (2)

Y. Tadir, W. H. Wright, O. Vafa, T. Ord, R. H. Asch, M. W. Berns, Fertil. Steril. 53, 944 (1990).
[PubMed]

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

1989 (1)

S. P. Jiang, Prog. React. Kinet. 15, 77 (1989).

1986 (1)

Asch, R. H.

Y. Tadir, W. H. Wright, O. Vafa, T. Ord, R. H. Asch, M. W. Berns, Fertil. Steril. 53, 944 (1990).
[PubMed]

Ashkin, A.

Berns, M. W.

Y. Liu, D. K. Cheng, G. J. Sonek, M. W. Berns, C. F. Chapman, B. J. Tromberg, Biophys. J. 68, 2137 (1995).
[CrossRef] [PubMed]

Y. Tadir, W. H. Wright, O. Vafa, T. Ord, R. H. Asch, M. W. Berns, Fertil. Steril. 53, 944 (1990).
[PubMed]

Bjorkholm, J. E.

Block, S. M.

K. Svoboda, S. M. Block, Annu. Rev. Biophys. Biomol. Struct. 23, 247 (1994).
[CrossRef] [PubMed]

Chapman, C. F.

Y. Liu, D. K. Cheng, G. J. Sonek, M. W. Berns, C. F. Chapman, B. J. Tromberg, Biophys. J. 68, 2137 (1995).
[CrossRef] [PubMed]

Cheng, D. K.

Y. Liu, D. K. Cheng, G. J. Sonek, M. W. Berns, C. F. Chapman, B. J. Tromberg, Biophys. J. 68, 2137 (1995).
[CrossRef] [PubMed]

Cheng, H.

Chu, S.

Denk, W.

W. Denk, Proc. Natl. Acad. Sci. (USA) 91, 6629 (1994).
[CrossRef] [PubMed]

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

Dziedzic, J. M.

Hanninen, P. E.

P. E. Hanninen, E. Soini, S. W. Hell, J. Microsc. (Oxford)176, 222 (1994).
[CrossRef]

Haugland, R. G.

R. G. Haugland, Handbook of Fluorescence Probes and Research Chemicals (Molecular Probes, Eugene, Ore., 1995).

Hell, S. W.

P. E. Hanninen, E. Soini, S. W. Hell, J. Microsc. (Oxford)176, 222 (1994).
[CrossRef]

Jiang, S. P.

S. P. Jiang, Prog. React. Kinet. 15, 77 (1989).

Kirby, M. S.

Lederer, W. J.

Liu, Y.

Y. Liu, D. K. Cheng, G. J. Sonek, M. W. Berns, C. F. Chapman, B. J. Tromberg, Biophys. J. 68, 2137 (1995).
[CrossRef] [PubMed]

Ord, T.

Y. Tadir, W. H. Wright, O. Vafa, T. Ord, R. H. Asch, M. W. Berns, Fertil. Steril. 53, 944 (1990).
[PubMed]

Piston, D. W.

Soini, E.

P. E. Hanninen, E. Soini, S. W. Hell, J. Microsc. (Oxford)176, 222 (1994).
[CrossRef]

Sonek, G. J.

Y. Liu, D. K. Cheng, G. J. Sonek, M. W. Berns, C. F. Chapman, B. J. Tromberg, Biophys. J. 68, 2137 (1995).
[CrossRef] [PubMed]

Strickler, J. H.

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

Svoboda, K.

K. Svoboda, S. M. Block, Annu. Rev. Biophys. Biomol. Struct. 23, 247 (1994).
[CrossRef] [PubMed]

Tadir, Y.

Y. Tadir, W. H. Wright, O. Vafa, T. Ord, R. H. Asch, M. W. Berns, Fertil. Steril. 53, 944 (1990).
[PubMed]

Tromberg, B. J.

Y. Liu, D. K. Cheng, G. J. Sonek, M. W. Berns, C. F. Chapman, B. J. Tromberg, Biophys. J. 68, 2137 (1995).
[CrossRef] [PubMed]

Vafa, O.

Y. Tadir, W. H. Wright, O. Vafa, T. Ord, R. H. Asch, M. W. Berns, Fertil. Steril. 53, 944 (1990).
[PubMed]

Visscher, K.

K. Visscher, “Optical manipulation and confocal microscopy,” Ph.D. dissertation (University of Amsterdam, Amsterdam, 1993), pp. 73–77.

Webb, W.

Webb, W. W.

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

Wright, W. H.

Y. Tadir, W. H. Wright, O. Vafa, T. Ord, R. H. Asch, M. W. Berns, Fertil. Steril. 53, 944 (1990).
[PubMed]

Annu. Rev. Biophys. Biomol. Struct. (1)

K. Svoboda, S. M. Block, Annu. Rev. Biophys. Biomol. Struct. 23, 247 (1994).
[CrossRef] [PubMed]

Appl. Opt. (1)

Biophys. J. (1)

Y. Liu, D. K. Cheng, G. J. Sonek, M. W. Berns, C. F. Chapman, B. J. Tromberg, Biophys. J. 68, 2137 (1995).
[CrossRef] [PubMed]

Fertil. Steril. (1)

Y. Tadir, W. H. Wright, O. Vafa, T. Ord, R. H. Asch, M. W. Berns, Fertil. Steril. 53, 944 (1990).
[PubMed]

Opt. Lett. (1)

Proc. Natl. Acad. Sci. (1)

W. Denk, Proc. Natl. Acad. Sci. (USA) 91, 6629 (1994).
[CrossRef] [PubMed]

Prog. React. Kinet. (1)

S. P. Jiang, Prog. React. Kinet. 15, 77 (1989).

Science (1)

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

Other (3)

R. G. Haugland, Handbook of Fluorescence Probes and Research Chemicals (Molecular Probes, Eugene, Ore., 1995).

K. Visscher, “Optical manipulation and confocal microscopy,” Ph.D. dissertation (University of Amsterdam, Amsterdam, 1993), pp. 73–77.

P. E. Hanninen, E. Soini, S. W. Hell, J. Microsc. (Oxford)176, 222 (1994).
[CrossRef]

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Figures (4)

Fig. 1
Fig. 1

Measured absorption and emission spectra for the fluorescent dye PI bound to DNA. Fluorescence emission is excited in a dye-tagged human sperm cell by a two-photon absorption process while the sample is held in 300-mW, 1064-nm cw optical tweezers.

Fig. 2
Fig. 2

Dependence of PI fluorescence intensity (IFLUOR) on trapping (pump) laser power. The intensity varies with nearly a square-law dependence on incident laser power (IFLUOR ~ P1.9). The CCD integration time was 5 s.

Fig. 3
Fig. 3

Time dependence of fluorescence intensity, measured for an optically trapped human sperm cell that is held stationary for 7 min in a 300-mW optical trap.

Fig. 4
Fig. 4

Two-photon fluorescence emission spectra for two different CHO’s that have been tagged with PI and Snarf.

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