Abstract

Third harmonic generation microscopy is used to make dynamical images of living systems for the first time. A 100 fs excitation pulse at 1.2 μm results in a 400 nm signal which is generated directly within the specimen. Chara plant rhizoids have been imaged, showing dynamic plant activity, and non-fading image characteristics even with continuous viewing, indicating prolonged viability under these THG-imaging conditions.

© 1998 Optical Society of America

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References

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  1. T. Y. F. Tsang, “Optical third-harmonic generation at interfaces,” Phys. Rev. A 52, 4116–4125 (1995).
    [Crossref] [PubMed]
  2. R. W. Boyd, Nonlinear Optics. (Academic Press, Boston, 1992).
  3. Y. Barad, H. Eisenberg, M. Horowitz, and Y. Silberberg, “Nonlinear scanning laser microscopy by third-harmonic generation,” Appl. Phys. Lett. 70, 922–924 (1997).
    [Crossref]
  4. M. Müller, J. Squier, K. R. Wilson, and G. J. Brakenhoff, “3D-microscopy of transparent objects using third-harmonic generation,” J. Microsc. 191, 266–274 (1998).
    [Crossref] [PubMed]
  5. J. Squier, M. Müller, K. R. Wilson, and G. J. Brakenhoff, “3D-Microscopy using third harmonic generation at interfaces in biological and nonbiological specimens,” to appear in proceedings of Ultrafast Phenomena XI, Garmisch-PartenKirchen/Germany, July 12–17, 1998.
  6. T. Tsang, “Reflected optical harmonics from dielectric mirrors,” Appl. Opt.,  33, 7720–7723, (1994).
    [Crossref] [PubMed]
  7. A. Sievers, B. Buchen, and D. Hodick, “Gravity sensing in tip-growing cells,” Trends in Plant Sci. 1, 273–279 (1996).
    [Crossref]
  8. M. Müller, J. Squier, R. Wolleschensky, U. Simon, and G. J. Brakenhoff, “Dispersion pre-compensation of 15 femtosecond optical pulses for high-numerical-aperture objectives,” J. Microsc. 191, 141–150 (1998).
    [Crossref] [PubMed]

1998 (3)

M. Müller, J. Squier, K. R. Wilson, and G. J. Brakenhoff, “3D-microscopy of transparent objects using third-harmonic generation,” J. Microsc. 191, 266–274 (1998).
[Crossref] [PubMed]

J. Squier, M. Müller, K. R. Wilson, and G. J. Brakenhoff, “3D-Microscopy using third harmonic generation at interfaces in biological and nonbiological specimens,” to appear in proceedings of Ultrafast Phenomena XI, Garmisch-PartenKirchen/Germany, July 12–17, 1998.

M. Müller, J. Squier, R. Wolleschensky, U. Simon, and G. J. Brakenhoff, “Dispersion pre-compensation of 15 femtosecond optical pulses for high-numerical-aperture objectives,” J. Microsc. 191, 141–150 (1998).
[Crossref] [PubMed]

1997 (1)

Y. Barad, H. Eisenberg, M. Horowitz, and Y. Silberberg, “Nonlinear scanning laser microscopy by third-harmonic generation,” Appl. Phys. Lett. 70, 922–924 (1997).
[Crossref]

1996 (1)

A. Sievers, B. Buchen, and D. Hodick, “Gravity sensing in tip-growing cells,” Trends in Plant Sci. 1, 273–279 (1996).
[Crossref]

1995 (1)

T. Y. F. Tsang, “Optical third-harmonic generation at interfaces,” Phys. Rev. A 52, 4116–4125 (1995).
[Crossref] [PubMed]

1994 (1)

Barad, Y.

Y. Barad, H. Eisenberg, M. Horowitz, and Y. Silberberg, “Nonlinear scanning laser microscopy by third-harmonic generation,” Appl. Phys. Lett. 70, 922–924 (1997).
[Crossref]

Boyd, R. W.

R. W. Boyd, Nonlinear Optics. (Academic Press, Boston, 1992).

Brakenhoff, G. J.

M. Müller, J. Squier, K. R. Wilson, and G. J. Brakenhoff, “3D-microscopy of transparent objects using third-harmonic generation,” J. Microsc. 191, 266–274 (1998).
[Crossref] [PubMed]

J. Squier, M. Müller, K. R. Wilson, and G. J. Brakenhoff, “3D-Microscopy using third harmonic generation at interfaces in biological and nonbiological specimens,” to appear in proceedings of Ultrafast Phenomena XI, Garmisch-PartenKirchen/Germany, July 12–17, 1998.

M. Müller, J. Squier, R. Wolleschensky, U. Simon, and G. J. Brakenhoff, “Dispersion pre-compensation of 15 femtosecond optical pulses for high-numerical-aperture objectives,” J. Microsc. 191, 141–150 (1998).
[Crossref] [PubMed]

Buchen, B.

A. Sievers, B. Buchen, and D. Hodick, “Gravity sensing in tip-growing cells,” Trends in Plant Sci. 1, 273–279 (1996).
[Crossref]

Eisenberg, H.

Y. Barad, H. Eisenberg, M. Horowitz, and Y. Silberberg, “Nonlinear scanning laser microscopy by third-harmonic generation,” Appl. Phys. Lett. 70, 922–924 (1997).
[Crossref]

Hodick, D.

A. Sievers, B. Buchen, and D. Hodick, “Gravity sensing in tip-growing cells,” Trends in Plant Sci. 1, 273–279 (1996).
[Crossref]

Horowitz, M.

Y. Barad, H. Eisenberg, M. Horowitz, and Y. Silberberg, “Nonlinear scanning laser microscopy by third-harmonic generation,” Appl. Phys. Lett. 70, 922–924 (1997).
[Crossref]

Müller, M.

M. Müller, J. Squier, K. R. Wilson, and G. J. Brakenhoff, “3D-microscopy of transparent objects using third-harmonic generation,” J. Microsc. 191, 266–274 (1998).
[Crossref] [PubMed]

M. Müller, J. Squier, R. Wolleschensky, U. Simon, and G. J. Brakenhoff, “Dispersion pre-compensation of 15 femtosecond optical pulses for high-numerical-aperture objectives,” J. Microsc. 191, 141–150 (1998).
[Crossref] [PubMed]

J. Squier, M. Müller, K. R. Wilson, and G. J. Brakenhoff, “3D-Microscopy using third harmonic generation at interfaces in biological and nonbiological specimens,” to appear in proceedings of Ultrafast Phenomena XI, Garmisch-PartenKirchen/Germany, July 12–17, 1998.

Sievers, A.

A. Sievers, B. Buchen, and D. Hodick, “Gravity sensing in tip-growing cells,” Trends in Plant Sci. 1, 273–279 (1996).
[Crossref]

Silberberg, Y.

Y. Barad, H. Eisenberg, M. Horowitz, and Y. Silberberg, “Nonlinear scanning laser microscopy by third-harmonic generation,” Appl. Phys. Lett. 70, 922–924 (1997).
[Crossref]

Simon, U.

M. Müller, J. Squier, R. Wolleschensky, U. Simon, and G. J. Brakenhoff, “Dispersion pre-compensation of 15 femtosecond optical pulses for high-numerical-aperture objectives,” J. Microsc. 191, 141–150 (1998).
[Crossref] [PubMed]

Squier, J.

M. Müller, J. Squier, K. R. Wilson, and G. J. Brakenhoff, “3D-microscopy of transparent objects using third-harmonic generation,” J. Microsc. 191, 266–274 (1998).
[Crossref] [PubMed]

J. Squier, M. Müller, K. R. Wilson, and G. J. Brakenhoff, “3D-Microscopy using third harmonic generation at interfaces in biological and nonbiological specimens,” to appear in proceedings of Ultrafast Phenomena XI, Garmisch-PartenKirchen/Germany, July 12–17, 1998.

M. Müller, J. Squier, R. Wolleschensky, U. Simon, and G. J. Brakenhoff, “Dispersion pre-compensation of 15 femtosecond optical pulses for high-numerical-aperture objectives,” J. Microsc. 191, 141–150 (1998).
[Crossref] [PubMed]

Tsang, T.

Tsang, T. Y. F.

T. Y. F. Tsang, “Optical third-harmonic generation at interfaces,” Phys. Rev. A 52, 4116–4125 (1995).
[Crossref] [PubMed]

Wilson, K. R.

M. Müller, J. Squier, K. R. Wilson, and G. J. Brakenhoff, “3D-microscopy of transparent objects using third-harmonic generation,” J. Microsc. 191, 266–274 (1998).
[Crossref] [PubMed]

J. Squier, M. Müller, K. R. Wilson, and G. J. Brakenhoff, “3D-Microscopy using third harmonic generation at interfaces in biological and nonbiological specimens,” to appear in proceedings of Ultrafast Phenomena XI, Garmisch-PartenKirchen/Germany, July 12–17, 1998.

Wolleschensky, R.

M. Müller, J. Squier, R. Wolleschensky, U. Simon, and G. J. Brakenhoff, “Dispersion pre-compensation of 15 femtosecond optical pulses for high-numerical-aperture objectives,” J. Microsc. 191, 141–150 (1998).
[Crossref] [PubMed]

Appl. Opt. (1)

Appl. Phys. Lett. (1)

Y. Barad, H. Eisenberg, M. Horowitz, and Y. Silberberg, “Nonlinear scanning laser microscopy by third-harmonic generation,” Appl. Phys. Lett. 70, 922–924 (1997).
[Crossref]

J. Microsc. (2)

M. Müller, J. Squier, K. R. Wilson, and G. J. Brakenhoff, “3D-microscopy of transparent objects using third-harmonic generation,” J. Microsc. 191, 266–274 (1998).
[Crossref] [PubMed]

M. Müller, J. Squier, R. Wolleschensky, U. Simon, and G. J. Brakenhoff, “Dispersion pre-compensation of 15 femtosecond optical pulses for high-numerical-aperture objectives,” J. Microsc. 191, 141–150 (1998).
[Crossref] [PubMed]

Phys. Rev. A (1)

T. Y. F. Tsang, “Optical third-harmonic generation at interfaces,” Phys. Rev. A 52, 4116–4125 (1995).
[Crossref] [PubMed]

Trends in Plant Sci. (1)

A. Sievers, B. Buchen, and D. Hodick, “Gravity sensing in tip-growing cells,” Trends in Plant Sci. 1, 273–279 (1996).
[Crossref]

Other (2)

R. W. Boyd, Nonlinear Optics. (Academic Press, Boston, 1992).

J. Squier, M. Müller, K. R. Wilson, and G. J. Brakenhoff, “3D-Microscopy using third harmonic generation at interfaces in biological and nonbiological specimens,” to appear in proceedings of Ultrafast Phenomena XI, Garmisch-PartenKirchen/Germany, July 12–17, 1998.

Supplementary Material (5)

» Media 1: MOV (672 KB)     
» Media 2: MOV (1327 KB)     
» Media 3: MOV (1054 KB)     
» Media 4: MOV (644 KB)     
» Media 5: MOV (669 KB)     

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Figures (8)

Fig. 1.
Fig. 1.

Microscope showing the input beam and collection beam paths.

Fig. 2.
Fig. 2.

Upper graph is a measure of the third order power dependence of the signal. The slope is 2.9. Lower graph is a measure of the axial sectioning through a glass/air interface. The excitation objective was 100x/ 1.25 NA, and the collection objective was a 20x/0.6 NA.

Fig. 3.
Fig. 3.

THG image series through a 250 μm diameter glass sphere. Note all images are raw data - no image processing has been performed, only an artificial color look up table has been used to assign a value to the image intensity. [Media 1]

Fig. 4.
Fig. 4.

THG image series of Chara rhizoid. [Media 2]

Fig. 5.
Fig. 5.

THG image series showing cytoplasmic streaming within the rhizoid. [Media 3]

Fig. 6.
Fig. 6.

THG image series at root tip showing motion of statoliths. [Media 4]

Fig. 7.
Fig. 7.

High NA series of rhizoid tip. The sections proceed in increments of 1.5 microns, the final image (4) stopping at the outer cell membrane.

Fig. 8.
Fig. 8.

THG series showing small organisms rapidly moving across and through the image plane. The sample is a drop of pond water. [Media 5]

Metrics