Abstract

Two-photon fluorescence (TPF) is one of the most important discoveries for biological imaging. Although a cw laser is known to excite TPF, its application in TPF imaging has been very limited due to the perceived low efficiency of excitation. Here we directly excited fluorophores with an IR cw laser used for optical trapping and achieved single-molecule fluorescence sensitivity: discrete stepwise photobleaching of enhanced green fluorescent proteins was observed. The single-molecule fluorescence intensity analysis and on-time distribution strongly indicate that a cw laser can generate TPF detectable at the single-molecule level, and thus opens the door to single-molecule TPF imaging using cw lasers.

© 2011 Optical Society of America

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[CrossRef] [PubMed]

2010

P. D. Simonson, H. A. Deberg, P. Ge, J. K. Alexander, O. Jeyifous, W. N. Green, and P. R. Selvin, Biophys. J. 99, L81 (2010).
[CrossRef] [PubMed]

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M. Drobizhev, S. Tillo, N. S. Makarov, T. E. Hughes, and A. Rebane, J. Phys. Chem. B 113, 855 (2009).
[CrossRef] [PubMed]

2008

M. D. Cahalan and I. Parker, Annu. Rev. Immunol. 26, 585 (2008).
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[CrossRef]

2007

M. H. Ulbrich and E. Y. Isacoff, Nat. Methods 4, 319 (2007).
[PubMed]

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M. C. Leake, J. H. Chandler, G. H. Wadhams, F. Bai, R. M. Berry, and J. P. Armitage, Nature 443, 355 (2006).
[CrossRef] [PubMed]

K. Svoboda and R. Yasuda, Neuron 50, 823 (2006).
[CrossRef] [PubMed]

2005

F. Helmchen and W. Denk, Nat. Methods 2, 932 (2005).
[CrossRef] [PubMed]

N. J. Carter and R. A. Cross, Nature 435, 308 (2005).
[CrossRef] [PubMed]

N. C. Shaner, P. A. Steinbach, and R. Y. Tsien, Nat. Methods 2, 905 (2005).
[CrossRef] [PubMed]

P. J. Schuck, K. A. Willets, D. P. Fromm, R. J. Twieg, and W. E. Moerner, Chem. Phys. 318, 7 (2005).
[CrossRef]

2003

W. R. Zipfel, R. M. Williams, and W. W. Webb, Nat. Biotechnol. 21, 1369 (2003).
[CrossRef] [PubMed]

2001

A. M. van Oijen, R. Verberk, Y. Durand, J. Schmidt, J. N. J. van Lingen, A. A. Bol, and A. Meijerink, Appl. Phys. Lett. 79, 830 (2001).
[CrossRef]

2000

P. T. So, C. Y. Dong, B. R. Masters, and K. M. Berland, Annu. Rev. Biomed. Eng. 2, 399 (2000).
[CrossRef]

1999

D. W. Piston, Trends Cell Biol. 9, 66 (1999).
[CrossRef] [PubMed]

J. M. Squirrell, D. L. Wokosin, J. G. White, and B. D. Bavister, Nat. Biotechnol. 17, 763 (1999).
[CrossRef] [PubMed]

W. E. Moerner and M. Orrit, Science 283, 1670 (1999).
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E. J. Peterman, S. Brasselet, and W. E. Moerner, J. Phys. Chem. A 103, 10553 (1999).
[CrossRef]

1998

1997

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

1995

1990

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

1961

W. Kaiser and C. G. B. Garrett, Phys. Rev. Lett. 7, 229(1961).
[CrossRef]

Alexander, J. K.

P. D. Simonson, H. A. Deberg, P. Ge, J. K. Alexander, O. Jeyifous, W. N. Green, and P. R. Selvin, Biophys. J. 99, L81 (2010).
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M. C. Leake, J. H. Chandler, G. H. Wadhams, F. Bai, R. M. Berry, and J. P. Armitage, Nature 443, 355 (2006).
[CrossRef] [PubMed]

Arndt-Jovin, D. J.

Bai, F.

M. C. Leake, J. H. Chandler, G. H. Wadhams, F. Bai, R. M. Berry, and J. P. Armitage, Nature 443, 355 (2006).
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Bavister, B. D.

J. M. Squirrell, D. L. Wokosin, J. G. White, and B. D. Bavister, Nat. Biotechnol. 17, 763 (1999).
[CrossRef] [PubMed]

Bell, S. C.

S. C. Kohout, M. H. Ulbrich, S. C. Bell, and E. Y. Isacoff, Nat. Struct. Mol. Biol. 15, 106 (2008).
[CrossRef]

Berland, K. M.

P. T. So, C. Y. Dong, B. R. Masters, and K. M. Berland, Annu. Rev. Biomed. Eng. 2, 399 (2000).
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Berns, M. W.

Berry, R. M.

M. C. Leake, J. H. Chandler, G. H. Wadhams, F. Bai, R. M. Berry, and J. P. Armitage, Nature 443, 355 (2006).
[CrossRef] [PubMed]

Bol, A. A.

A. M. van Oijen, R. Verberk, Y. Durand, J. Schmidt, J. N. J. van Lingen, A. A. Bol, and A. Meijerink, Appl. Phys. Lett. 79, 830 (2001).
[CrossRef]

Booth, M.

Brasselet, S.

E. J. Peterman, S. Brasselet, and W. E. Moerner, J. Phys. Chem. A 103, 10553 (1999).
[CrossRef]

Cahalan, M. D.

M. D. Cahalan and I. Parker, Annu. Rev. Immunol. 26, 585 (2008).
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N. J. Carter and R. A. Cross, Nature 435, 308 (2005).
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M. C. Leake, J. H. Chandler, G. H. Wadhams, F. Bai, R. M. Berry, and J. P. Armitage, Nature 443, 355 (2006).
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W. Ji, P. Xu, Z. Li, J. Lu, L. Liu, Y. Zhan, Y. Chen, B. Hille, T. Xu, and L. Chen, Proc. Natl. Acad. Sci. USA 105, 13668 (2008).
[CrossRef] [PubMed]

Chen, Y.

W. Ji, P. Xu, Z. Li, J. Lu, L. Liu, Y. Zhan, Y. Chen, B. Hille, T. Xu, and L. Chen, Proc. Natl. Acad. Sci. USA 105, 13668 (2008).
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Cheng, W.

Cross, R. A.

N. J. Carter and R. A. Cross, Nature 435, 308 (2005).
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Deberg, H. A.

P. D. Simonson, H. A. Deberg, P. Ge, J. K. Alexander, O. Jeyifous, W. N. Green, and P. R. Selvin, Biophys. J. 99, L81 (2010).
[CrossRef] [PubMed]

Denk, W.

F. Helmchen and W. Denk, Nat. Methods 2, 932 (2005).
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W. Denk, J. H. Strickler, and W. W. Webb, Science 248, 73 (1990).
[CrossRef] [PubMed]

Dong, C. Y.

P. T. So, C. Y. Dong, B. R. Masters, and K. M. Berland, Annu. Rev. Biomed. Eng. 2, 399 (2000).
[CrossRef]

Drobizhev, M.

M. Drobizhev, N. S. Makarov, S. E. Tillo, T. E. Hughes, and A. Rebane, Nat. Methods 8, 393 (2011).
[CrossRef] [PubMed]

M. Drobizhev, S. Tillo, N. S. Makarov, T. E. Hughes, and A. Rebane, J. Phys. Chem. B 113, 855 (2009).
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A. M. van Oijen, R. Verberk, Y. Durand, J. Schmidt, J. N. J. van Lingen, A. A. Bol, and A. Meijerink, Appl. Phys. Lett. 79, 830 (2001).
[CrossRef]

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P. J. Schuck, K. A. Willets, D. P. Fromm, R. J. Twieg, and W. E. Moerner, Chem. Phys. 318, 7 (2005).
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W. Kaiser and C. G. B. Garrett, Phys. Rev. Lett. 7, 229(1961).
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P. D. Simonson, H. A. Deberg, P. Ge, J. K. Alexander, O. Jeyifous, W. N. Green, and P. R. Selvin, Biophys. J. 99, L81 (2010).
[CrossRef] [PubMed]

Green, W. N.

P. D. Simonson, H. A. Deberg, P. Ge, J. K. Alexander, O. Jeyifous, W. N. Green, and P. R. Selvin, Biophys. J. 99, L81 (2010).
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Hell, S. W.

Helmchen, F.

F. Helmchen and W. Denk, Nat. Methods 2, 932 (2005).
[CrossRef] [PubMed]

Hille, B.

W. Ji, P. Xu, Z. Li, J. Lu, L. Liu, Y. Zhan, Y. Chen, B. Hille, T. Xu, and L. Chen, Proc. Natl. Acad. Sci. USA 105, 13668 (2008).
[CrossRef] [PubMed]

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

Hou, X.

Hughes, T. E.

M. Drobizhev, N. S. Makarov, S. E. Tillo, T. E. Hughes, and A. Rebane, Nat. Methods 8, 393 (2011).
[CrossRef] [PubMed]

M. Drobizhev, S. Tillo, N. S. Makarov, T. E. Hughes, and A. Rebane, J. Phys. Chem. B 113, 855 (2009).
[CrossRef] [PubMed]

Isacoff, E. Y.

S. C. Kohout, M. H. Ulbrich, S. C. Bell, and E. Y. Isacoff, Nat. Struct. Mol. Biol. 15, 106 (2008).
[CrossRef]

M. H. Ulbrich and E. Y. Isacoff, Nat. Methods 4, 319 (2007).
[PubMed]

Jauffred, L.

L. Jauffred and L. B. Oddershede, Nano Lett. 10, 1927(2010).
[CrossRef] [PubMed]

Jeyifous, O.

P. D. Simonson, H. A. Deberg, P. Ge, J. K. Alexander, O. Jeyifous, W. N. Green, and P. R. Selvin, Biophys. J. 99, L81 (2010).
[CrossRef] [PubMed]

Ji, W.

W. Ji, P. Xu, Z. Li, J. Lu, L. Liu, Y. Zhan, Y. Chen, B. Hille, T. Xu, and L. Chen, Proc. Natl. Acad. Sci. USA 105, 13668 (2008).
[CrossRef] [PubMed]

Jovin, T. M.

Kaiser, W.

W. Kaiser and C. G. B. Garrett, Phys. Rev. Lett. 7, 229(1961).
[CrossRef]

Kirsch, A. K.

Kohout, S. C.

S. C. Kohout, M. H. Ulbrich, S. C. Bell, and E. Y. Isacoff, Nat. Struct. Mol. Biol. 15, 106 (2008).
[CrossRef]

Konig, K.

Leake, M. C.

M. C. Leake, J. H. Chandler, G. H. Wadhams, F. Bai, R. M. Berry, and J. P. Armitage, Nature 443, 355 (2006).
[CrossRef] [PubMed]

Li, Z.

W. Ji, P. Xu, Z. Li, J. Lu, L. Liu, Y. Zhan, Y. Chen, B. Hille, T. Xu, and L. Chen, Proc. Natl. Acad. Sci. USA 105, 13668 (2008).
[CrossRef] [PubMed]

Liu, L.

W. Ji, P. Xu, Z. Li, J. Lu, L. Liu, Y. Zhan, Y. Chen, B. Hille, T. Xu, and L. Chen, Proc. Natl. Acad. Sci. USA 105, 13668 (2008).
[CrossRef] [PubMed]

Liu, Y.

Lu, H. P.

H. P. Lu, L. Xun, and X. S. Xie, Science 282, 1877 (1998).
[CrossRef] [PubMed]

Lu, J.

W. Ji, P. Xu, Z. Li, J. Lu, L. Liu, Y. Zhan, Y. Chen, B. Hille, T. Xu, and L. Chen, Proc. Natl. Acad. Sci. USA 105, 13668 (2008).
[CrossRef] [PubMed]

Makarov, N. S.

M. Drobizhev, N. S. Makarov, S. E. Tillo, T. E. Hughes, and A. Rebane, Nat. Methods 8, 393 (2011).
[CrossRef] [PubMed]

M. Drobizhev, S. Tillo, N. S. Makarov, T. E. Hughes, and A. Rebane, J. Phys. Chem. B 113, 855 (2009).
[CrossRef] [PubMed]

Masters, B. R.

P. T. So, C. Y. Dong, B. R. Masters, and K. M. Berland, Annu. Rev. Biomed. Eng. 2, 399 (2000).
[CrossRef]

Meijerink, A.

A. M. van Oijen, R. Verberk, Y. Durand, J. Schmidt, J. N. J. van Lingen, A. A. Bol, and A. Meijerink, Appl. Phys. Lett. 79, 830 (2001).
[CrossRef]

Moerner, W. E.

P. J. Schuck, K. A. Willets, D. P. Fromm, R. J. Twieg, and W. E. Moerner, Chem. Phys. 318, 7 (2005).
[CrossRef]

E. J. Peterman, S. Brasselet, and W. E. Moerner, J. Phys. Chem. A 103, 10553 (1999).
[CrossRef]

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

Novotny, L.

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

Oddershede, L. B.

L. Jauffred and L. B. Oddershede, Nano Lett. 10, 1927(2010).
[CrossRef] [PubMed]

Orrit, M.

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

Parker, I.

M. D. Cahalan and I. Parker, Annu. Rev. Immunol. 26, 585 (2008).
[CrossRef] [PubMed]

Peterman, E. J.

E. J. Peterman, S. Brasselet, and W. E. Moerner, J. Phys. Chem. A 103, 10553 (1999).
[CrossRef]

Piston, D. W.

D. W. Piston, Trends Cell Biol. 9, 66 (1999).
[CrossRef] [PubMed]

Rebane, A.

M. Drobizhev, N. S. Makarov, S. E. Tillo, T. E. Hughes, and A. Rebane, Nat. Methods 8, 393 (2011).
[CrossRef] [PubMed]

M. Drobizhev, S. Tillo, N. S. Makarov, T. E. Hughes, and A. Rebane, J. Phys. Chem. B 113, 855 (2009).
[CrossRef] [PubMed]

Sanchez, E. J.

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

Schmidt, J.

A. M. van Oijen, R. Verberk, Y. Durand, J. Schmidt, J. N. J. van Lingen, A. A. Bol, and A. Meijerink, Appl. Phys. Lett. 79, 830 (2001).
[CrossRef]

Schnetter, C. M.

Schuck, P. J.

P. J. Schuck, K. A. Willets, D. P. Fromm, R. J. Twieg, and W. E. Moerner, Chem. Phys. 318, 7 (2005).
[CrossRef]

Selvin, P. R.

P. D. Simonson, H. A. Deberg, P. Ge, J. K. Alexander, O. Jeyifous, W. N. Green, and P. R. Selvin, Biophys. J. 99, L81 (2010).
[CrossRef] [PubMed]

Shaner, N. C.

N. C. Shaner, P. A. Steinbach, and R. Y. Tsien, Nat. Methods 2, 905 (2005).
[CrossRef] [PubMed]

Simonson, P. D.

P. D. Simonson, H. A. Deberg, P. Ge, J. K. Alexander, O. Jeyifous, W. N. Green, and P. R. Selvin, Biophys. J. 99, L81 (2010).
[CrossRef] [PubMed]

So, P. T.

P. T. So, C. Y. Dong, B. R. Masters, and K. M. Berland, Annu. Rev. Biomed. Eng. 2, 399 (2000).
[CrossRef]

Sonek, G. J.

Squirrell, J. M.

J. M. Squirrell, D. L. Wokosin, J. G. White, and B. D. Bavister, Nat. Biotechnol. 17, 763 (1999).
[CrossRef] [PubMed]

Steinbach, P. A.

N. C. Shaner, P. A. Steinbach, and R. Y. Tsien, Nat. Methods 2, 905 (2005).
[CrossRef] [PubMed]

Strickler, J. H.

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

Svoboda, K.

K. Svoboda and R. Yasuda, Neuron 50, 823 (2006).
[CrossRef] [PubMed]

Thromberg, B. J.

Tillo, S.

M. Drobizhev, S. Tillo, N. S. Makarov, T. E. Hughes, and A. Rebane, J. Phys. Chem. B 113, 855 (2009).
[CrossRef] [PubMed]

Tillo, S. E.

M. Drobizhev, N. S. Makarov, S. E. Tillo, T. E. Hughes, and A. Rebane, Nat. Methods 8, 393 (2011).
[CrossRef] [PubMed]

Tsien, R. Y.

N. C. Shaner, P. A. Steinbach, and R. Y. Tsien, Nat. Methods 2, 905 (2005).
[CrossRef] [PubMed]

Twieg, R. J.

P. J. Schuck, K. A. Willets, D. P. Fromm, R. J. Twieg, and W. E. Moerner, Chem. Phys. 318, 7 (2005).
[CrossRef]

Ulbrich, M. H.

S. C. Kohout, M. H. Ulbrich, S. C. Bell, and E. Y. Isacoff, Nat. Struct. Mol. Biol. 15, 106 (2008).
[CrossRef]

M. H. Ulbrich and E. Y. Isacoff, Nat. Methods 4, 319 (2007).
[PubMed]

van Lingen, J. N. J.

A. M. van Oijen, R. Verberk, Y. Durand, J. Schmidt, J. N. J. van Lingen, A. A. Bol, and A. Meijerink, Appl. Phys. Lett. 79, 830 (2001).
[CrossRef]

van Oijen, A. M.

A. M. van Oijen, R. Verberk, Y. Durand, J. Schmidt, J. N. J. van Lingen, A. A. Bol, and A. Meijerink, Appl. Phys. Lett. 79, 830 (2001).
[CrossRef]

Verberk, R.

A. M. van Oijen, R. Verberk, Y. Durand, J. Schmidt, J. N. J. van Lingen, A. A. Bol, and A. Meijerink, Appl. Phys. Lett. 79, 830 (2001).
[CrossRef]

Wadhams, G. H.

M. C. Leake, J. H. Chandler, G. H. Wadhams, F. Bai, R. M. Berry, and J. P. Armitage, Nature 443, 355 (2006).
[CrossRef] [PubMed]

Webb, W. W.

W. R. Zipfel, R. M. Williams, and W. W. Webb, Nat. Biotechnol. 21, 1369 (2003).
[CrossRef] [PubMed]

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

White, J. G.

J. M. Squirrell, D. L. Wokosin, J. G. White, and B. D. Bavister, Nat. Biotechnol. 17, 763 (1999).
[CrossRef] [PubMed]

Willets, K. A.

P. J. Schuck, K. A. Willets, D. P. Fromm, R. J. Twieg, and W. E. Moerner, Chem. Phys. 318, 7 (2005).
[CrossRef]

Williams, R. M.

W. R. Zipfel, R. M. Williams, and W. W. Webb, Nat. Biotechnol. 21, 1369 (2003).
[CrossRef] [PubMed]

Wilms, S.

Wokosin, D. L.

J. M. Squirrell, D. L. Wokosin, J. G. White, and B. D. Bavister, Nat. Biotechnol. 17, 763 (1999).
[CrossRef] [PubMed]

Xie, X. S.

H. P. Lu, L. Xun, and X. S. Xie, Science 282, 1877 (1998).
[CrossRef] [PubMed]

E. J. Sanchez, L. Novotny, G. R. Holtom, and X. S. Xie, J. Phys. Chem. A 101, 7019 (1997).
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Figures (4)

Fig. 1
Fig. 1

Experimental setup for single-molecule TPF imaging. Inner surface of the microfluidic chamber is sparsely coated with EGFP. The laser focus is placed precisely at the inner surface. Lateral scanning of the chamber driven by a 3D motion stage (ESP300, Newport) allows searching of EGFP molecules on the surface.

Fig. 2
Fig. 2

Fluorescence time courses of EGFP molecules immobilized on glass coverslips. Gray traces are fluorescence intensity data collected with 1 s exposure time for each data point. Red lines show the stepwise photobleaching of EGFP fluorescence detected and simulated by a custom-written Matlab program.

Fig. 3
Fig. 3

Statistics of single EGFP fluorescence. (a) Histogram of fluorescence intensity (bin size of 5 a . u . ). The top x axis represents the number of photon counts per second. The black curve is fit by a double Gaussian function. (b) Histogram of fluorescence on-time before photobleaching (bin size of 10 s ). The black curve is fit by a single exponential decay. The histograms were constructed based on the analysis of 176 fluorescence time traces represented by traces in Figs. 2a, 2b.

Fig. 4
Fig. 4

EGFP fluorescence intensity as a function of laser power. (a) At the single-molecule level, fluorescence intensity increases with increasing laser power and then bleaches in a stepwise manner. (b) Inset shows the TPF image of an EGFP-coated polystyrene bead ( 1.3 μm ) immobilized on top of a micropipette. Different symbols represent measurements from different beads. Straight lines in different colors with different symbols show linear fits in a double logarithmic scale, which give an average slope of 1.89 ± 0.07 .

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