Abstract

Photobleaching of organic fluorescent labels is a ubiquitous problem in fluorescence microscopy, limiting imaging capabilities and presenting hurdles to quantitative biophysical measurements. We report here that a nonlinear optical signal from some organic fluorophores persists in the presence of photobleaching. Specifically, a four-wave mixing process that is enhanced by a two-photon absorption resonance in the target fluorophore, termed stimulated parametric emission (SPE), is essentially unaffected by the photobleaching of the fluorophore, for rhodamine 6G and other commercial green and red fluorophores. The stability of the SPE signal, and the ability to image weakly or nonfluorescent chromophores, should make this nonlinear microscopy useful for quantitative biophysical measurements.

© 2009 Optical Society of America

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    [CrossRef] [PubMed]

2008 (2)

F. Masia, W. Langbein, and P. Borri, Appl. Phys. Lett. 93, 021114 (2008).
[CrossRef]

A. Kachynski, A. Kuzmin, M. Nyk, I. Roy, and P. Prasad, J. Phys. Chem. C 112, 10721 (2008).
[CrossRef]

2006 (2)

2003 (2)

H. Kawano, Y. Nabekawa, A. Suda, Y. Oishi, H. Mizuno, A. Miyawaki, and K. Midorikawa, Biochem. Biophys. Res. Commun. 311, 592 (2003).
[CrossRef] [PubMed]

A. Lamprecht and J. P. Benoit, J. Chromatogr. B 787, 415 (2003).
[CrossRef]

2001 (2)

J. X. Cheng, L. D. Book, and X. S. Xie, Opt. Lett. 26, 1341 (2001).
[CrossRef]

P. Dittrich and P. Schwille, Appl. Phys. B 73, 829 (2001).
[CrossRef]

2000 (1)

1999 (1)

N. George, B. Aneeshkumar, P. Radhakrishnan, and C. Vallabhan, J. Phys. D 32, 1745 (1999).
[CrossRef]

1998 (1)

T. X. Wu, G. M. Liu, J. C. Zhao, H. Hidaka, and N. Serpone, J. Phys. Chem. B 102, 5845 (1998).
[CrossRef]

1996 (1)

W. Scheenen, L. Makings, L. Gross, T. Pozzon, and R. Y. Tsien, Chem. Biol. 3, 765 (1996).
[CrossRef] [PubMed]

1995 (1)

C. J. R. Sheppard, X. Gan, M. Gu, and M. Roy, in Handbook of Biological Confocal Microscopy, 2nd ed. (Springer, 1995), p. 363.

1994 (1)

M. Laurent, G. Johannin, N. Gilbert, L. Lucas, D. Cassio, P. Pettit, and A. Fleury, Biol. Cell 80, 229 (1994).
[CrossRef] [PubMed]

1992 (1)

R. W. Boyd, Nonlinear Optics, 1st ed. (Academic, 1992), p. 439.

1979 (1)

J.-L. Oudar, R. W. Smith, and Y.-R. Shen, Appl. Phys. Lett. 34, 758 (1979).
[CrossRef]

Aneeshkumar, B.

N. George, B. Aneeshkumar, P. Radhakrishnan, and C. Vallabhan, J. Phys. D 32, 1745 (1999).
[CrossRef]

Benoit, J. P.

A. Lamprecht and J. P. Benoit, J. Chromatogr. B 787, 415 (2003).
[CrossRef]

Book, L. D.

Borri, P.

F. Masia, W. Langbein, and P. Borri, Appl. Phys. Lett. 93, 021114 (2008).
[CrossRef]

Boyd, R. W.

R. W. Boyd, Nonlinear Optics, 1st ed. (Academic, 1992), p. 439.

Cassio, D.

M. Laurent, G. Johannin, N. Gilbert, L. Lucas, D. Cassio, P. Pettit, and A. Fleury, Biol. Cell 80, 229 (1994).
[CrossRef] [PubMed]

Cheng, J. X.

de Boeij, W.

Dittrich, P.

P. Dittrich and P. Schwille, Appl. Phys. B 73, 829 (2001).
[CrossRef]

Egami, C.

Fleury, A.

M. Laurent, G. Johannin, N. Gilbert, L. Lucas, D. Cassio, P. Pettit, and A. Fleury, Biol. Cell 80, 229 (1994).
[CrossRef] [PubMed]

Fukui, K.

Gan, X.

C. J. R. Sheppard, X. Gan, M. Gu, and M. Roy, in Handbook of Biological Confocal Microscopy, 2nd ed. (Springer, 1995), p. 363.

George, N.

N. George, B. Aneeshkumar, P. Radhakrishnan, and C. Vallabhan, J. Phys. D 32, 1745 (1999).
[CrossRef]

Gilbert, N.

M. Laurent, G. Johannin, N. Gilbert, L. Lucas, D. Cassio, P. Pettit, and A. Fleury, Biol. Cell 80, 229 (1994).
[CrossRef] [PubMed]

Gross, L.

W. Scheenen, L. Makings, L. Gross, T. Pozzon, and R. Y. Tsien, Chem. Biol. 3, 765 (1996).
[CrossRef] [PubMed]

Gu, M.

C. J. R. Sheppard, X. Gan, M. Gu, and M. Roy, in Handbook of Biological Confocal Microscopy, 2nd ed. (Springer, 1995), p. 363.

Hidaka, H.

T. X. Wu, G. M. Liu, J. C. Zhao, H. Hidaka, and N. Serpone, J. Phys. Chem. B 102, 5845 (1998).
[CrossRef]

Higashi, T.

Isobe, K.

Itoh, K.

Johannin, G.

M. Laurent, G. Johannin, N. Gilbert, L. Lucas, D. Cassio, P. Pettit, and A. Fleury, Biol. Cell 80, 229 (1994).
[CrossRef] [PubMed]

Kachynski, A.

A. Kachynski, A. Kuzmin, M. Nyk, I. Roy, and P. Prasad, J. Phys. Chem. C 112, 10721 (2008).
[CrossRef]

Kataoka, S.

Kawakami, S.

Kawano, H.

H. Kawano, Y. Nabekawa, A. Suda, Y. Oishi, H. Mizuno, A. Miyawaki, and K. Midorikawa, Biochem. Biophys. Res. Commun. 311, 592 (2003).
[CrossRef] [PubMed]

Kawata, Y.

Kobayashi, N.

Kuzmin, A.

A. Kachynski, A. Kuzmin, M. Nyk, I. Roy, and P. Prasad, J. Phys. Chem. C 112, 10721 (2008).
[CrossRef]

Lamprecht, A.

A. Lamprecht and J. P. Benoit, J. Chromatogr. B 787, 415 (2003).
[CrossRef]

Langbein, W.

F. Masia, W. Langbein, and P. Borri, Appl. Phys. Lett. 93, 021114 (2008).
[CrossRef]

Laurent, M.

M. Laurent, G. Johannin, N. Gilbert, L. Lucas, D. Cassio, P. Pettit, and A. Fleury, Biol. Cell 80, 229 (1994).
[CrossRef] [PubMed]

Liu, G. M.

T. X. Wu, G. M. Liu, J. C. Zhao, H. Hidaka, and N. Serpone, J. Phys. Chem. B 102, 5845 (1998).
[CrossRef]

Lucas, L.

M. Laurent, G. Johannin, N. Gilbert, L. Lucas, D. Cassio, P. Pettit, and A. Fleury, Biol. Cell 80, 229 (1994).
[CrossRef] [PubMed]

Makings, L.

W. Scheenen, L. Makings, L. Gross, T. Pozzon, and R. Y. Tsien, Chem. Biol. 3, 765 (1996).
[CrossRef] [PubMed]

Masaki, M.

Masia, F.

F. Masia, W. Langbein, and P. Borri, Appl. Phys. Lett. 93, 021114 (2008).
[CrossRef]

Matsunaga, S.

Midorikawa, K.

H. Kawano, Y. Nabekawa, A. Suda, Y. Oishi, H. Mizuno, A. Miyawaki, and K. Midorikawa, Biochem. Biophys. Res. Commun. 311, 592 (2003).
[CrossRef] [PubMed]

Miyawaki, A.

H. Kawano, Y. Nabekawa, A. Suda, Y. Oishi, H. Mizuno, A. Miyawaki, and K. Midorikawa, Biochem. Biophys. Res. Commun. 311, 592 (2003).
[CrossRef] [PubMed]

Mizuno, H.

H. Kawano, Y. Nabekawa, A. Suda, Y. Oishi, H. Mizuno, A. Miyawaki, and K. Midorikawa, Biochem. Biophys. Res. Commun. 311, 592 (2003).
[CrossRef] [PubMed]

Murase, R.

Nabekawa, Y.

H. Kawano, Y. Nabekawa, A. Suda, Y. Oishi, H. Mizuno, A. Miyawaki, and K. Midorikawa, Biochem. Biophys. Res. Commun. 311, 592 (2003).
[CrossRef] [PubMed]

Nyk, M.

A. Kachynski, A. Kuzmin, M. Nyk, I. Roy, and P. Prasad, J. Phys. Chem. C 112, 10721 (2008).
[CrossRef]

Oishi, Y.

H. Kawano, Y. Nabekawa, A. Suda, Y. Oishi, H. Mizuno, A. Miyawaki, and K. Midorikawa, Biochem. Biophys. Res. Commun. 311, 592 (2003).
[CrossRef] [PubMed]

Oudar, J.-L.

J.-L. Oudar, R. W. Smith, and Y.-R. Shen, Appl. Phys. Lett. 34, 758 (1979).
[CrossRef]

Pettit, P.

M. Laurent, G. Johannin, N. Gilbert, L. Lucas, D. Cassio, P. Pettit, and A. Fleury, Biol. Cell 80, 229 (1994).
[CrossRef] [PubMed]

Potma, E.

Pozzon, T.

W. Scheenen, L. Makings, L. Gross, T. Pozzon, and R. Y. Tsien, Chem. Biol. 3, 765 (1996).
[CrossRef] [PubMed]

Prasad, P.

A. Kachynski, A. Kuzmin, M. Nyk, I. Roy, and P. Prasad, J. Phys. Chem. C 112, 10721 (2008).
[CrossRef]

Radhakrishnan, P.

N. George, B. Aneeshkumar, P. Radhakrishnan, and C. Vallabhan, J. Phys. D 32, 1745 (1999).
[CrossRef]

Roy, I.

A. Kachynski, A. Kuzmin, M. Nyk, I. Roy, and P. Prasad, J. Phys. Chem. C 112, 10721 (2008).
[CrossRef]

Roy, M.

C. J. R. Sheppard, X. Gan, M. Gu, and M. Roy, in Handbook of Biological Confocal Microscopy, 2nd ed. (Springer, 1995), p. 363.

Scheenen, W.

W. Scheenen, L. Makings, L. Gross, T. Pozzon, and R. Y. Tsien, Chem. Biol. 3, 765 (1996).
[CrossRef] [PubMed]

Schwille, P.

P. Dittrich and P. Schwille, Appl. Phys. B 73, 829 (2001).
[CrossRef]

Serpone, N.

T. X. Wu, G. M. Liu, J. C. Zhao, H. Hidaka, and N. Serpone, J. Phys. Chem. B 102, 5845 (1998).
[CrossRef]

Shen, Y.-R.

J.-L. Oudar, R. W. Smith, and Y.-R. Shen, Appl. Phys. Lett. 34, 758 (1979).
[CrossRef]

Sheppard, C. J. R.

C. J. R. Sheppard, X. Gan, M. Gu, and M. Roy, in Handbook of Biological Confocal Microscopy, 2nd ed. (Springer, 1995), p. 363.

Smith, R. W.

J.-L. Oudar, R. W. Smith, and Y.-R. Shen, Appl. Phys. Lett. 34, 758 (1979).
[CrossRef]

Suda, A.

H. Kawano, Y. Nabekawa, A. Suda, Y. Oishi, H. Mizuno, A. Miyawaki, and K. Midorikawa, Biochem. Biophys. Res. Commun. 311, 592 (2003).
[CrossRef] [PubMed]

Tsien, R. Y.

W. Scheenen, L. Makings, L. Gross, T. Pozzon, and R. Y. Tsien, Chem. Biol. 3, 765 (1996).
[CrossRef] [PubMed]

Vallabhan, C.

N. George, B. Aneeshkumar, P. Radhakrishnan, and C. Vallabhan, J. Phys. D 32, 1745 (1999).
[CrossRef]

Watanabe, W.

Wiersma, D.

Wu, T. X.

T. X. Wu, G. M. Liu, J. C. Zhao, H. Hidaka, and N. Serpone, J. Phys. Chem. B 102, 5845 (1998).
[CrossRef]

Xie, X. S.

Zhao, J. C.

T. X. Wu, G. M. Liu, J. C. Zhao, H. Hidaka, and N. Serpone, J. Phys. Chem. B 102, 5845 (1998).
[CrossRef]

Appl. Phys. B (1)

P. Dittrich and P. Schwille, Appl. Phys. B 73, 829 (2001).
[CrossRef]

Appl. Phys. Lett. (2)

F. Masia, W. Langbein, and P. Borri, Appl. Phys. Lett. 93, 021114 (2008).
[CrossRef]

J.-L. Oudar, R. W. Smith, and Y.-R. Shen, Appl. Phys. Lett. 34, 758 (1979).
[CrossRef]

Biochem. Biophys. Res. Commun. (1)

H. Kawano, Y. Nabekawa, A. Suda, Y. Oishi, H. Mizuno, A. Miyawaki, and K. Midorikawa, Biochem. Biophys. Res. Commun. 311, 592 (2003).
[CrossRef] [PubMed]

Biol. Cell (1)

M. Laurent, G. Johannin, N. Gilbert, L. Lucas, D. Cassio, P. Pettit, and A. Fleury, Biol. Cell 80, 229 (1994).
[CrossRef] [PubMed]

Chem. Biol. (1)

W. Scheenen, L. Makings, L. Gross, T. Pozzon, and R. Y. Tsien, Chem. Biol. 3, 765 (1996).
[CrossRef] [PubMed]

J. Chromatogr. B (1)

A. Lamprecht and J. P. Benoit, J. Chromatogr. B 787, 415 (2003).
[CrossRef]

J. Opt. Soc. Am. B (1)

J. Phys. Chem. B (1)

T. X. Wu, G. M. Liu, J. C. Zhao, H. Hidaka, and N. Serpone, J. Phys. Chem. B 102, 5845 (1998).
[CrossRef]

J. Phys. Chem. C (1)

A. Kachynski, A. Kuzmin, M. Nyk, I. Roy, and P. Prasad, J. Phys. Chem. C 112, 10721 (2008).
[CrossRef]

J. Phys. D (1)

N. George, B. Aneeshkumar, P. Radhakrishnan, and C. Vallabhan, J. Phys. D 32, 1745 (1999).
[CrossRef]

Opt. Express (1)

Opt. Lett. (2)

Other (2)

R. W. Boyd, Nonlinear Optics, 1st ed. (Academic, 1992), p. 439.

C. J. R. Sheppard, X. Gan, M. Gu, and M. Roy, in Handbook of Biological Confocal Microscopy, 2nd ed. (Springer, 1995), p. 363.

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Figures (5)

Fig. 1
Fig. 1

Energy level diagrams for four-wave mixing microscopies. Left, CARS is enhanced by a resonance between the difference frequency of two input beams and a vibrational level v. However, a chromatically indistinguishable optical output may arise in a process termed SPE (center), which may be enhanced by a two-photon resonance. The longer-wavelength SPE at 2 ω 2 ω 1 can have no CARS contribution (right).

Fig. 2
Fig. 2

Polarization geometry of the input fields E 1 ( λ = 790 nm ) and E 2 ( λ = 1030 nm ) and the nonlinear SPE signals. The signal E NR for an NR nonlinearity in an isotropic material fulfilling the Kleinman symmetry is linearly polarized in the direction marked ‖. The addition of a (nearly) resonant dopant gives an elliptically polarized signal E Tot , which has a component perpendicular to E NR . A polarizer/analyzer with its pass direction along ⊥ ( α + 90 ° ) will block the NR background and allow observation of the resonant signal.

Fig. 3
Fig. 3

SPE microscope. A mode-locked Ti:sapphire laser synchronously pumps an OPO, providing illumination at 790 and 1030 nm .

Fig. 4
Fig. 4

IR-SPE (top) and TPF images (bottom) of mixed dyed and blank beads, taken as simultaneous pairs. The top left image shows the IR-SPE signal with the polarizer/analyzer oriented along the ‖ direction. All beads are visible in the SPE, while only the dye-doped beads are seen in the two-photon fluorescence image (bottom left). The middle pair of images shows the same field with the polarizer/analyzer oriented in the ⊥ direction. Only the dye-doped beads are clearly visible in the resonant SPE image. After the second pair of images was taken, the beads were intentionally photobleached. The fluorescence was greatly diminished (bottom right), but the SPE along ⊥ (top right) was unchanged.

Fig. 5
Fig. 5

Simultaneous TPF and IR-SPE measured from a rhodamine 6G-doped PMMA film. The IR-SPE was measured with the polarizer/analyzer in the ⊥ direction. The two-photon fluorescence is rapidly photobleached, but the IR-SPE signal is stable. Inset, similar IR-SPE signal stability (solid curve) was observed as beads doped with a green dye were photobleached (dashed curve).

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