Abstract

We demonstrate that the Stimulated Emission Depletion (STED) concept, which is usually invoked for fluorescence, can be extended to photoacoustic effects. When two-nanosecond pulses of exciting and stimulating light are synchronized, 80% of the acoustic signal generated through excited state absorption (ESA) can be quenched. Regarding the cross-sections for stimulated emission and ESA, a model gives a good order of magnitude in the depletion efficiency. The transient molecular orientation, usually measured via the fluorescence anisotropy, can be accessed in photoacoustic when STED is implemented.

© 2019 Optical Society of America under the terms of the OSA Open Access Publishing Agreement

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References

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    [Crossref]
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    [Crossref]
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    [Crossref]
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    [Crossref]
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    [Crossref]
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    [Crossref]
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    [Crossref]

2018 (1)

2017 (1)

2016 (1)

W. R. Silva, C. T. Graefe, and R. R. Frontiera, “Toward Label-Free Super-Resolution Microscopy,” ACS Photonics 3(1), 79–86 (2016).
[Crossref]

2015 (1)

H. R. Zhang, Q. Wu, and M. Y. Berezin, “Fluorescence anisotropy (polarization): from drug screening to precision medicine,” Expert Opin. Drug Discovery 10(11), 1145–1161 (2015).
[Crossref]

2014 (2)

M. Frenette, M. Hatamimoslehabadi, S. Bellinger-Buckley, S. Laoui, J. La, S. Bag, S. Mallidi, T. Hasan, B. Bouma, C. Yeleswarapu, and J. Rochford, “Shining Light on the Dark Side of Imaging: Excited State Absorption Enhancement of a Bis-styryl BODIPY Photoacoustic Contrast Agent,” J. Am. Chem. Soc. 136(45), 15853–15856 (2014).
[Crossref]

T. Scheul, I. Wang, and J. C. Vial, “STED-SPIM made simple,” Opt. Express 22(25), 30852–30864 (2014).
[Crossref]

2013 (1)

2010 (1)

2008 (2)

2007 (1)

E. Rittweger, B. R. Rankin, V. Westphal, and S. W. Hell, “Fluorescence depletion mechanisms in super-resolving STED microscopy,” Chem. Phys. Lett. 442(4-6), 483–487 (2007).
[Crossref]

2006 (1)

S. Jerebtsov, A. Kolomenskii, M. Poudel, F. Zhu, and H. Schuessler, “Lifetime and anisotropy decay of excited Coumarin 30 measured by a femtosecond pump-probe technique,” J. Mod. Opt. 53(16-17), 2609–2617 (2006).
[Crossref]

2004 (1)

A. Ma and R. M. Stratt, “Multiphonon vibrational relaxation in liquids: Should it lead to an exponential-gap law?” J. Chem. Phys. 121(22), 11217–11226 (2004).
[Crossref]

1998 (2)

T. Gustavsson, L. Cassara, V. Gulbinas, G. Gurzadyan, J. C. Mialocq, S. Pommeret, M. Sorgius, and P. van der Meulen, “Femtosecond spectroscopic study of relaxation processes of three amino-substituted coumarin dyes in methanol and dimethyl sulfoxide,” J. Phys. Chem. A 102(23), 4229–4245 (1998).
[Crossref]

I. Gryczynski, Z. Gryczynski, and J. R. Lakowicz, “Fluorescence anisotropy controlled by light quenching,” Photochem. Photobiol. 67(6), 641–646 (1998).
[Crossref]

1997 (1)

S. A. Kovalenko, J. Ruthmann, and N. P. Ernsting, “Ultrafast Stokes shift and excited-state transient absorption of coumarin 153 in solution,” Chem. Phys. Lett. 271(1-3), 40–50 (1997).
[Crossref]

1995 (1)

M. Villagranmuniz, C. Garciasegundo, H. F. Raneasandoval, C. Gogorza, and G. M. Bilmes, “Photoacoustic Analysis of Stimulated-Emission in Pulsed Dye-Lasers,” Appl. Phys. B: Lasers Opt. 61(4), 361–366 (1995).
[Crossref]

1983 (3)

L. J. Rothberg, M. Bernstein, and K. S. Peters, “Time Resolved Photo-Acoustic Spectroscopy Applied to Properties of Picosecond Transients,” J. Chem. Phys. 79(6), 2569–2576 (1983).
[Crossref]

R. F. Kubin and A. N. Fletcher, “The Effect of Oxygen on the Fluorescence Quantum Yields of Some Coumarin Dyes in Ethanol,” Chem. Phys. Lett. 99(1), 49–52 (1983).
[Crossref]

J. M. Heritier, “Electrostrictive Limit and Focusing Effects in Pulsed Photo-Acoustic Detection,” Opt. Commun. 44(4), 267–272 (1983).
[Crossref]

1982 (1)

R. Buisson, R. Chicault, F. Madeore, M. Poirier, and J. C. Vial, “Direct Detection of Phonons Created by a Quenching Process in Laf3-Pr3+,” Solid State Commun. 42(3), 157–160 (1982).
[Crossref]

1981 (1)

J. Wiedmann and A. Penzkofer, “Excited-State Absorption Cross-Sections in Rhodamine Dyes Determined after Molecular-Reorientation,” Nuovo Cimento B 63(1), 459–469 (1981).
[Crossref]

1953 (1)

G. Weber, “Rotational Brownian motion and polarization of the fluorescence of solutions,” Adv. Protein Chem. 8, 415–459 (1953).
[Crossref]

Bag, S.

M. Frenette, M. Hatamimoslehabadi, S. Bellinger-Buckley, S. Laoui, J. La, S. Bag, S. Mallidi, T. Hasan, B. Bouma, C. Yeleswarapu, and J. Rochford, “Shining Light on the Dark Side of Imaging: Excited State Absorption Enhancement of a Bis-styryl BODIPY Photoacoustic Contrast Agent,” J. Am. Chem. Soc. 136(45), 15853–15856 (2014).
[Crossref]

Bellinger-Buckley, S.

M. Frenette, M. Hatamimoslehabadi, S. Bellinger-Buckley, S. Laoui, J. La, S. Bag, S. Mallidi, T. Hasan, B. Bouma, C. Yeleswarapu, and J. Rochford, “Shining Light on the Dark Side of Imaging: Excited State Absorption Enhancement of a Bis-styryl BODIPY Photoacoustic Contrast Agent,” J. Am. Chem. Soc. 136(45), 15853–15856 (2014).
[Crossref]

Berezin, M. Y.

H. R. Zhang, Q. Wu, and M. Y. Berezin, “Fluorescence anisotropy (polarization): from drug screening to precision medicine,” Expert Opin. Drug Discovery 10(11), 1145–1161 (2015).
[Crossref]

Bernstein, M.

L. J. Rothberg, M. Bernstein, and K. S. Peters, “Time Resolved Photo-Acoustic Spectroscopy Applied to Properties of Picosecond Transients,” J. Chem. Phys. 79(6), 2569–2576 (1983).
[Crossref]

Bilmes, G. M.

M. Villagranmuniz, C. Garciasegundo, H. F. Raneasandoval, C. Gogorza, and G. M. Bilmes, “Photoacoustic Analysis of Stimulated-Emission in Pulsed Dye-Lasers,” Appl. Phys. B: Lasers Opt. 61(4), 361–366 (1995).
[Crossref]

Bouma, B.

M. Frenette, M. Hatamimoslehabadi, S. Bellinger-Buckley, S. Laoui, J. La, S. Bag, S. Mallidi, T. Hasan, B. Bouma, C. Yeleswarapu, and J. Rochford, “Shining Light on the Dark Side of Imaging: Excited State Absorption Enhancement of a Bis-styryl BODIPY Photoacoustic Contrast Agent,” J. Am. Chem. Soc. 136(45), 15853–15856 (2014).
[Crossref]

Buisson, R.

R. Buisson, R. Chicault, F. Madeore, M. Poirier, and J. C. Vial, “Direct Detection of Phonons Created by a Quenching Process in Laf3-Pr3+,” Solid State Commun. 42(3), 157–160 (1982).
[Crossref]

Cassara, L.

T. Gustavsson, L. Cassara, V. Gulbinas, G. Gurzadyan, J. C. Mialocq, S. Pommeret, M. Sorgius, and P. van der Meulen, “Femtosecond spectroscopic study of relaxation processes of three amino-substituted coumarin dyes in methanol and dimethyl sulfoxide,” J. Phys. Chem. A 102(23), 4229–4245 (1998).
[Crossref]

Chicault, R.

R. Buisson, R. Chicault, F. Madeore, M. Poirier, and J. C. Vial, “Direct Detection of Phonons Created by a Quenching Process in Laf3-Pr3+,” Solid State Commun. 42(3), 157–160 (1982).
[Crossref]

Ernsting, N. P.

S. A. Kovalenko, J. Ruthmann, and N. P. Ernsting, “Ultrafast Stokes shift and excited-state transient absorption of coumarin 153 in solution,” Chem. Phys. Lett. 271(1-3), 40–50 (1997).
[Crossref]

Fletcher, A. N.

R. F. Kubin and A. N. Fletcher, “The Effect of Oxygen on the Fluorescence Quantum Yields of Some Coumarin Dyes in Ethanol,” Chem. Phys. Lett. 99(1), 49–52 (1983).
[Crossref]

Frenette, M.

M. Frenette, M. Hatamimoslehabadi, S. Bellinger-Buckley, S. Laoui, J. La, S. Bag, S. Mallidi, T. Hasan, B. Bouma, C. Yeleswarapu, and J. Rochford, “Shining Light on the Dark Side of Imaging: Excited State Absorption Enhancement of a Bis-styryl BODIPY Photoacoustic Contrast Agent,” J. Am. Chem. Soc. 136(45), 15853–15856 (2014).
[Crossref]

Frontiera, R. R.

W. R. Silva, C. T. Graefe, and R. R. Frontiera, “Toward Label-Free Super-Resolution Microscopy,” ACS Photonics 3(1), 79–86 (2016).
[Crossref]

Garciasegundo, C.

M. Villagranmuniz, C. Garciasegundo, H. F. Raneasandoval, C. Gogorza, and G. M. Bilmes, “Photoacoustic Analysis of Stimulated-Emission in Pulsed Dye-Lasers,” Appl. Phys. B: Lasers Opt. 61(4), 361–366 (1995).
[Crossref]

Gogorza, C.

M. Villagranmuniz, C. Garciasegundo, H. F. Raneasandoval, C. Gogorza, and G. M. Bilmes, “Photoacoustic Analysis of Stimulated-Emission in Pulsed Dye-Lasers,” Appl. Phys. B: Lasers Opt. 61(4), 361–366 (1995).
[Crossref]

Graefe, C. T.

W. R. Silva, C. T. Graefe, and R. R. Frontiera, “Toward Label-Free Super-Resolution Microscopy,” ACS Photonics 3(1), 79–86 (2016).
[Crossref]

Gryczynski, I.

I. Gryczynski, Z. Gryczynski, and J. R. Lakowicz, “Fluorescence anisotropy controlled by light quenching,” Photochem. Photobiol. 67(6), 641–646 (1998).
[Crossref]

Gryczynski, Z.

I. Gryczynski, Z. Gryczynski, and J. R. Lakowicz, “Fluorescence anisotropy controlled by light quenching,” Photochem. Photobiol. 67(6), 641–646 (1998).
[Crossref]

Gulbinas, V.

T. Gustavsson, L. Cassara, V. Gulbinas, G. Gurzadyan, J. C. Mialocq, S. Pommeret, M. Sorgius, and P. van der Meulen, “Femtosecond spectroscopic study of relaxation processes of three amino-substituted coumarin dyes in methanol and dimethyl sulfoxide,” J. Phys. Chem. A 102(23), 4229–4245 (1998).
[Crossref]

Gurzadyan, G.

T. Gustavsson, L. Cassara, V. Gulbinas, G. Gurzadyan, J. C. Mialocq, S. Pommeret, M. Sorgius, and P. van der Meulen, “Femtosecond spectroscopic study of relaxation processes of three amino-substituted coumarin dyes in methanol and dimethyl sulfoxide,” J. Phys. Chem. A 102(23), 4229–4245 (1998).
[Crossref]

Gustavsson, T.

T. Gustavsson, L. Cassara, V. Gulbinas, G. Gurzadyan, J. C. Mialocq, S. Pommeret, M. Sorgius, and P. van der Meulen, “Femtosecond spectroscopic study of relaxation processes of three amino-substituted coumarin dyes in methanol and dimethyl sulfoxide,” J. Phys. Chem. A 102(23), 4229–4245 (1998).
[Crossref]

Harke, B.

Hasan, T.

M. Frenette, M. Hatamimoslehabadi, S. Bellinger-Buckley, S. Laoui, J. La, S. Bag, S. Mallidi, T. Hasan, B. Bouma, C. Yeleswarapu, and J. Rochford, “Shining Light on the Dark Side of Imaging: Excited State Absorption Enhancement of a Bis-styryl BODIPY Photoacoustic Contrast Agent,” J. Am. Chem. Soc. 136(45), 15853–15856 (2014).
[Crossref]

Hatamimoslehabadi, M.

M. Frenette, M. Hatamimoslehabadi, S. Bellinger-Buckley, S. Laoui, J. La, S. Bag, S. Mallidi, T. Hasan, B. Bouma, C. Yeleswarapu, and J. Rochford, “Shining Light on the Dark Side of Imaging: Excited State Absorption Enhancement of a Bis-styryl BODIPY Photoacoustic Contrast Agent,” J. Am. Chem. Soc. 136(45), 15853–15856 (2014).
[Crossref]

Hell, S. W.

B. Harke, J. Keller, C. K. Ullal, V. Westphal, A. Schoenle, and S. W. Hell, “Resolution scaling in STED microscopy,” Opt. Express 16(6), 4154–4162 (2008).
[Crossref]

E. Rittweger, B. R. Rankin, V. Westphal, and S. W. Hell, “Fluorescence depletion mechanisms in super-resolving STED microscopy,” Chem. Phys. Lett. 442(4-6), 483–487 (2007).
[Crossref]

Heritier, J. M.

J. M. Heritier, “Electrostrictive Limit and Focusing Effects in Pulsed Photo-Acoustic Detection,” Opt. Commun. 44(4), 267–272 (1983).
[Crossref]

Hrelescu, C.

Hu, S.

Jacak, J.

Jerebtsov, S.

S. Jerebtsov, A. Kolomenskii, M. Poudel, F. Zhu, and H. Schuessler, “Lifetime and anisotropy decay of excited Coumarin 30 measured by a femtosecond pump-probe technique,” J. Mod. Opt. 53(16-17), 2609–2617 (2006).
[Crossref]

Katzmann, J.

Keller, J.

Klar, T. A.

Kolomenskii, A.

S. Jerebtsov, A. Kolomenskii, M. Poudel, F. Zhu, and H. Schuessler, “Lifetime and anisotropy decay of excited Coumarin 30 measured by a femtosecond pump-probe technique,” J. Mod. Opt. 53(16-17), 2609–2617 (2006).
[Crossref]

Kovalenko, S. A.

S. A. Kovalenko, J. Ruthmann, and N. P. Ernsting, “Ultrafast Stokes shift and excited-state transient absorption of coumarin 153 in solution,” Chem. Phys. Lett. 271(1-3), 40–50 (1997).
[Crossref]

Kubin, R. F.

R. F. Kubin and A. N. Fletcher, “The Effect of Oxygen on the Fluorescence Quantum Yields of Some Coumarin Dyes in Ethanol,” Chem. Phys. Lett. 99(1), 49–52 (1983).
[Crossref]

Kyhm, K.

La, J.

M. Frenette, M. Hatamimoslehabadi, S. Bellinger-Buckley, S. Laoui, J. La, S. Bag, S. Mallidi, T. Hasan, B. Bouma, C. Yeleswarapu, and J. Rochford, “Shining Light on the Dark Side of Imaging: Excited State Absorption Enhancement of a Bis-styryl BODIPY Photoacoustic Contrast Agent,” J. Am. Chem. Soc. 136(45), 15853–15856 (2014).
[Crossref]

Lakowicz, J. R.

I. Gryczynski, Z. Gryczynski, and J. R. Lakowicz, “Fluorescence anisotropy controlled by light quenching,” Photochem. Photobiol. 67(6), 641–646 (1998).
[Crossref]

Laoui, S.

M. Frenette, M. Hatamimoslehabadi, S. Bellinger-Buckley, S. Laoui, J. La, S. Bag, S. Mallidi, T. Hasan, B. Bouma, C. Yeleswarapu, and J. Rochford, “Shining Light on the Dark Side of Imaging: Excited State Absorption Enhancement of a Bis-styryl BODIPY Photoacoustic Contrast Agent,” J. Am. Chem. Soc. 136(45), 15853–15856 (2014).
[Crossref]

Liu, L. M.

H. Qin and L. M. Liu, “High-Efficiency FRET-Enhanced Photoacoustic Probes for in vivo tumor Imaging,” in International Conference on Innovative Optical Health Science10245, 1024503 (2017).
[Crossref]

Ma, A.

A. Ma and R. M. Stratt, “Multiphonon vibrational relaxation in liquids: Should it lead to an exponential-gap law?” J. Chem. Phys. 121(22), 11217–11226 (2004).
[Crossref]

Madeore, F.

R. Buisson, R. Chicault, F. Madeore, M. Poirier, and J. C. Vial, “Direct Detection of Phonons Created by a Quenching Process in Laf3-Pr3+,” Solid State Commun. 42(3), 157–160 (1982).
[Crossref]

Mallidi, S.

M. Frenette, M. Hatamimoslehabadi, S. Bellinger-Buckley, S. Laoui, J. La, S. Bag, S. Mallidi, T. Hasan, B. Bouma, C. Yeleswarapu, and J. Rochford, “Shining Light on the Dark Side of Imaging: Excited State Absorption Enhancement of a Bis-styryl BODIPY Photoacoustic Contrast Agent,” J. Am. Chem. Soc. 136(45), 15853–15856 (2014).
[Crossref]

Maslov, K.

Mialocq, J. C.

T. Gustavsson, L. Cassara, V. Gulbinas, G. Gurzadyan, J. C. Mialocq, S. Pommeret, M. Sorgius, and P. van der Meulen, “Femtosecond spectroscopic study of relaxation processes of three amino-substituted coumarin dyes in methanol and dimethyl sulfoxide,” J. Phys. Chem. A 102(23), 4229–4245 (1998).
[Crossref]

Park, S.

Penzkofer, A.

J. Wiedmann and A. Penzkofer, “Excited-State Absorption Cross-Sections in Rhodamine Dyes Determined after Molecular-Reorientation,” Nuovo Cimento B 63(1), 459–469 (1981).
[Crossref]

Peters, K. S.

L. J. Rothberg, M. Bernstein, and K. S. Peters, “Time Resolved Photo-Acoustic Spectroscopy Applied to Properties of Picosecond Transients,” J. Chem. Phys. 79(6), 2569–2576 (1983).
[Crossref]

Poirier, M.

R. Buisson, R. Chicault, F. Madeore, M. Poirier, and J. C. Vial, “Direct Detection of Phonons Created by a Quenching Process in Laf3-Pr3+,” Solid State Commun. 42(3), 157–160 (1982).
[Crossref]

Pommeret, S.

T. Gustavsson, L. Cassara, V. Gulbinas, G. Gurzadyan, J. C. Mialocq, S. Pommeret, M. Sorgius, and P. van der Meulen, “Femtosecond spectroscopic study of relaxation processes of three amino-substituted coumarin dyes in methanol and dimethyl sulfoxide,” J. Phys. Chem. A 102(23), 4229–4245 (1998).
[Crossref]

Poudel, M.

S. Jerebtsov, A. Kolomenskii, M. Poudel, F. Zhu, and H. Schuessler, “Lifetime and anisotropy decay of excited Coumarin 30 measured by a femtosecond pump-probe technique,” J. Mod. Opt. 53(16-17), 2609–2617 (2006).
[Crossref]

Qin, H.

H. Qin and L. M. Liu, “High-Efficiency FRET-Enhanced Photoacoustic Probes for in vivo tumor Imaging,” in International Conference on Innovative Optical Health Science10245, 1024503 (2017).
[Crossref]

Raneasandoval, H. F.

M. Villagranmuniz, C. Garciasegundo, H. F. Raneasandoval, C. Gogorza, and G. M. Bilmes, “Photoacoustic Analysis of Stimulated-Emission in Pulsed Dye-Lasers,” Appl. Phys. B: Lasers Opt. 61(4), 361–366 (1995).
[Crossref]

Rankin, B. R.

E. Rittweger, B. R. Rankin, V. Westphal, and S. W. Hell, “Fluorescence depletion mechanisms in super-resolving STED microscopy,” Chem. Phys. Lett. 442(4-6), 483–487 (2007).
[Crossref]

Rittweger, E.

E. Rittweger, B. R. Rankin, V. Westphal, and S. W. Hell, “Fluorescence depletion mechanisms in super-resolving STED microscopy,” Chem. Phys. Lett. 442(4-6), 483–487 (2007).
[Crossref]

Rochford, J.

M. Frenette, M. Hatamimoslehabadi, S. Bellinger-Buckley, S. Laoui, J. La, S. Bag, S. Mallidi, T. Hasan, B. Bouma, C. Yeleswarapu, and J. Rochford, “Shining Light on the Dark Side of Imaging: Excited State Absorption Enhancement of a Bis-styryl BODIPY Photoacoustic Contrast Agent,” J. Am. Chem. Soc. 136(45), 15853–15856 (2014).
[Crossref]

Rothberg, L. J.

L. J. Rothberg, M. Bernstein, and K. S. Peters, “Time Resolved Photo-Acoustic Spectroscopy Applied to Properties of Picosecond Transients,” J. Chem. Phys. 79(6), 2569–2576 (1983).
[Crossref]

Ruthmann, J.

S. A. Kovalenko, J. Ruthmann, and N. P. Ernsting, “Ultrafast Stokes shift and excited-state transient absorption of coumarin 153 in solution,” Chem. Phys. Lett. 271(1-3), 40–50 (1997).
[Crossref]

Scheul, T.

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G. Weber, “Rotational Brownian motion and polarization of the fluorescence of solutions,” Adv. Protein Chem. 8, 415–459 (1953).
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Appl. Phys. B: Lasers Opt. (1)

M. Villagranmuniz, C. Garciasegundo, H. F. Raneasandoval, C. Gogorza, and G. M. Bilmes, “Photoacoustic Analysis of Stimulated-Emission in Pulsed Dye-Lasers,” Appl. Phys. B: Lasers Opt. 61(4), 361–366 (1995).
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Expert Opin. Drug Discovery (1)

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J. Am. Chem. Soc. (1)

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J. Chem. Phys. (2)

A. Ma and R. M. Stratt, “Multiphonon vibrational relaxation in liquids: Should it lead to an exponential-gap law?” J. Chem. Phys. 121(22), 11217–11226 (2004).
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S. Jerebtsov, A. Kolomenskii, M. Poudel, F. Zhu, and H. Schuessler, “Lifetime and anisotropy decay of excited Coumarin 30 measured by a femtosecond pump-probe technique,” J. Mod. Opt. 53(16-17), 2609–2617 (2006).
[Crossref]

J. Phys. Chem. A (1)

T. Gustavsson, L. Cassara, V. Gulbinas, G. Gurzadyan, J. C. Mialocq, S. Pommeret, M. Sorgius, and P. van der Meulen, “Femtosecond spectroscopic study of relaxation processes of three amino-substituted coumarin dyes in methanol and dimethyl sulfoxide,” J. Phys. Chem. A 102(23), 4229–4245 (1998).
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Nuovo Cimento B (1)

J. Wiedmann and A. Penzkofer, “Excited-State Absorption Cross-Sections in Rhodamine Dyes Determined after Molecular-Reorientation,” Nuovo Cimento B 63(1), 459–469 (1981).
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Solid State Commun. (1)

R. Buisson, R. Chicault, F. Madeore, M. Poirier, and J. C. Vial, “Direct Detection of Phonons Created by a Quenching Process in Laf3-Pr3+,” Solid State Commun. 42(3), 157–160 (1982).
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H. Qin and L. M. Liu, “High-Efficiency FRET-Enhanced Photoacoustic Probes for in vivo tumor Imaging,” in International Conference on Innovative Optical Health Science10245, 1024503 (2017).
[Crossref]

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Figures (6)

Fig. 1.
Fig. 1. (a) Jablonski diagram with excitation, vibronic/non-radiative relaxation, fluorescence, and stimulated emission. (b) Absorbance and fluorescence spectrum of Coumarin-490 in ethanol, where arrows indicate the two wavelengths of excitation (355 nm) and stimulated emission (532 nm).
Fig. 2.
Fig. 2. (a) Experimental setup of S-STED in Coumarin 490. While two beams of 532 nm (SHG) and 352 nm (SFG) with a ns pulse duration are generated from the fundamental beam of 1064 nm, they are self-synchronized and self-aligned. The two beams are focused by an achromatic lens into a cuvette, which is filled with an alcoholic solution of Coumarin 490. While 355 nm is used for excitation, 532 nm induces a stimulated emission in Coumarin 490. Photoacoustic signal is collected by a 75 MHz bandwidth piezoelectric transducer attached directly to the cuvette, and the amplified signal is sent to a fast oscilloscope. A camera is also placed near the cuvette to image fluorescence simultaneously. (b) For S-STED in Rhodamine 6G, 532 nm (SHG) beam is used not only for excitation, but also for generating a stimulating pulse of 600 nm through Rhodamine 101. Note that the stimulating beam of 600 nm is delayed by ∼ 100 ps with a short pulse duration compared to 1 ns pulse duration of the excitation pulse (532 nm). (c) A typical photoacoustic signal collected by a 75 MHz bandwidth piezoelectric transducer.
Fig. 3.
Fig. 3. (a) When Coumarin 490 in ethanol (∼millimolar concentration) is excited by only 355 nm pulse with 0.4 µJ (black curve), strong photoacoustic signals appear. However, the signal becomes suppressed significantly when a stimulating 532 nm beam is added (red curve). When only the stimulation beam of 532 nm is used (blue), no photoacoustic signal is observed. The laser beam waist at the entrance of the dye is 1.5 µm. (b) With 532 nm excitation, photoacoustic signal of Rhodamine 6G dye in ethanol (500 µmol/L) is observed with (red) and without (black) 600 nm stimulating beam, where other experimental conditions are similar to those for Coumarin 490. In both the cases, the polarizations of excitation and stimulation beams are parallel. The time difference between Fig. 3(a) and 3(b) is irrelevant to sample properties but due to different delay line settings of ultrasonic transducers.
Fig. 4.
Fig. 4. (a) Ratio of the photoacoustic signal of Coumarin 490 in ethanol (millimolar concentration) with and without STED is plotted as a function of stimulating energy at 532 nm. The excitation energy at 355 nm is 0.4 µJ, and the laser beam waist at the entrance of the dye solution is 1.5 µm. (b) Excitation (355 nm) laser energy dependence of photoacoustic signal for Coumarin 490 in ethanol is plotted as a function of the square of excitation laser energy. The linear scale excitation energy is also shown in inset. The beam waist at the entrance of the dye solution is 2.2 µm. The polarizations of excitation and stimulation beams are parallel
Fig. 5.
Fig. 5. Given three optical configurations for the linear polarization of excitation (355 nm) and stimulation (532 nm) beams (a), photoacoustic signals are observed from Coumarin 490 in ethanol. The energy of the stimulation pulse is 0.9 µJ and the beam waist at the cuvette entrance is 1.5 µm.
Fig. 6.
Fig. 6. Absorption (black) and fluorescence spectra of Coumarin 490 in ethanol. The length of arrows for absorption and ESA are the same.

Equations (2)

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R = Δ Δ + δ = h ν e x h ν f l h ν e x h ν f l . Q E
r = D D D + 2 D