Abstract

In order to achieve high-resolution deep-tissue imaging, multi-photon fluorescence microscopy and photoacoustic tomography had been proposed in the past two decades. However, combining the advantages of these two imaging systems to achieve optical-spatial resolution with an ultrasonic-penetration depth is still a field with challenges. In this paper, we investigate the detection of the two-photon photoacoustic ultrasound, and first demonstrate background-free two-photon photoacoustic imaging in a phantom sample. To generate the background-free two-photon photoacoustic signals, we used a high-repetition rate femtosecond laser to induce narrowband excitation. Combining a loss modulation technique, we successfully created a beating on the light intensity, which not only provides pure sinusoidal modulation, but also ensures the spectrum sensitivity and frequency selectivity. By using the lock-in detection, the power dependency experiment validates our methodology to frequency-select the source of the nonlinearity. This ensures our capability of measuring the background-free two-photon photoacoustic waves by detecting the 2nd order beating signal directly. Furthermore, by mixing the nanoparticles and fluorescence dyes as contrast agents, the two-photon photoacoustic signal was found to be enhanced and detected. In the end, we demonstrate subsurface two-photon photoacoustic bio-imaging based on the optical scanning mechanism inside phantom samples.

© 2014 Optical Society of America

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References

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  1. M. Rumi, J. W. Perry, “Two-photon absorption: an overview of measurements and principles,” Adv. Opt. Photonics 2(4), 451–518 (2010).
    [CrossRef]
  2. F. Helmchen, W. Denk, “Deep tissue two-photon microscopy,” Nat. Methods 2(12), 932–940 (2005).
    [CrossRef] [PubMed]
  3. L. V. Wang, S. Hu, “Photoacoustic tomography: In Vivo imaging from organelles to organs,” Science 335(6075), 1458–1462 (2012).
    [CrossRef] [PubMed]
  4. V. Ntziachristos, “Going deeper than microscopy: the optical imaging frontier in biology,” Nat. Methods 7(8), 603–614 (2010).
    [CrossRef] [PubMed]
  5. H. F. Zhang, K. Maslov, G. Stoica, L. V. Wang, “Functional photoacoustic microscopy for high-resolution and noninvasive in vivo imaging,” Nat. Biotechnol. 24(7), 848–851 (2006).
    [CrossRef] [PubMed]
  6. Y. Yamaoka, M. Nambu, T. Takamatsu, “Fine depth resolution of two-photon absorption-induced photoacoustic microscopy using low-frequency bandpass filtering,” Opt. Express 19(14), 13365–13377 (2011).
    [CrossRef] [PubMed]
  7. Y. Yamaoka, M. Nambub, T. Takamatsu, “Frequency-selective multiphoton-excitation-induced photoacoustic microscopy (MEPAM) to visualize the cross sections of dense objects,” Proc. SPIE7564, 1–9 (2010).
  8. N. Chandrasekharan, B. Gonzales, B. M. Cullum, “Non-resonant Multiphoton Photoacoustic Spectroscopy for Noninvasive Subsurface Chemical Diagnostics,” Appl. Spectrosc. 58(11), 1325–1333 (2004).
    [CrossRef] [PubMed]
  9. M.E. van Raaij, M. Lee, E. Cherin, B. Stefanovic, F.S. Foster, “Femtosecond photoacoustics: integrated two-photon fluorescence and photoacoustic microscopy,” Proc. SPIE7564, 1-6 (2010)
  10. G. Langer, K. D. Bouchal, H. Grün, P. Burgholzer, T. Berer, “Two-photon absorption-induced photoacoustic imaging of Rhodamine B dyed polyethylene spheres using a femtosecond laser,” Opt. Express 21(19), 22410–22422 (2013).
    [CrossRef] [PubMed]
  11. S. Dahal, J. B. Kiser, B. M. Cullum, “Depth and resolution characterization of two-photon photoacoustic spectroscopy for noninvasive subsurface chemical diagnostics,” Proc. SPIE8025, 1–7 (2011).
  12. W. R. Zipfel, R. M. Williams, W. W. Webb, “Nonlinear magic: multiphoton microscopy in the biosciences,” Nat. Biotechnol. 21(11), 1369–1377 (2003).
    [CrossRef] [PubMed]
  13. B. Huang, M. Bates, X. Zhuang, “Super-Resolution Fluorescence Microscopy,” Annu. Rev. Biochem. 78(1), 993–1016 (2009).
    [CrossRef] [PubMed]
  14. P. Tian, W. S. Warren, “Ultrafast measurement of two-photon absorption by loss modulation,” Opt. Lett. 27(18), 1634–1636 (2002).
    [CrossRef] [PubMed]
  15. R. L. Shelton, B. E. Applegate, “Ultrahigh resolution photoacoustic microscopy via transient absorption,” Biomed. Opt. Express 1(2), 676–686 (2010).
    [CrossRef] [PubMed]
  16. M. Xu, L. V. Wang, “Photoacoustic imaging in biomedicine,” Rev. Sci. Instrum. 77, 041101 (2006).
  17. L. V. Wang, “Tutorial on Photoacoustic Microscopy and Computed Tomography,” IEEE J. Sel. Top. Quantum Electron. 14(1), 171–179 (2008).
    [CrossRef]
  18. M. Pramanik, L. V. Wang, “Thermoacoustic and photoacoustic sensing of temperature,” J. Biomed. Opt. 14(5), 054024 (2009).
  19. A. Danielli, K. Maslov, J. Xia, L. V. Wang, “Wide range quantitative photoacoustic spectroscopy to measure non-linear optical absorption of hemoglobin,” Proc. SPIE8223, 1–6 (2012).
  20. Y. Yamaoka, T. Takamatsu, “Enhancement of multiphoton excitation-induced photoacoustic signals by using gold nanoparticles surrounded by fluorescent dyes,” Proc. SPIE7177, 1–9 (2009).
  21. M. Zhang, Z. Che, J. Chen, H. Zhao, L. Yang, Z. Zhong, J. Lu, “Experimental determination of thermal conductivity of water-agar gel at Different concentrations and temperatures,” J. Chem. Eng. Data 56(4), 859–864 (2011).
    [CrossRef]
  22. D. Haemmerich, D. J. Schutt, I. dos Santos, J. G. Webster, D. M. Mahvi, “Measurement of temperature-dependent specific heat of biological tissues,” Physiol. Meas. 26(1), 59–67 (2005).
    [CrossRef] [PubMed]
  23. Y. H. Lai, C. F. Chang, Y. H. Cheng, C. K. Sun, “Two-photon photoacoustics ultrasound measurement by a loss modulation technique,” Proc. SPIE8581, 1–8 (2013).

2013 (1)

2012 (1)

L. V. Wang, S. Hu, “Photoacoustic tomography: In Vivo imaging from organelles to organs,” Science 335(6075), 1458–1462 (2012).
[CrossRef] [PubMed]

2011 (2)

Y. Yamaoka, M. Nambu, T. Takamatsu, “Fine depth resolution of two-photon absorption-induced photoacoustic microscopy using low-frequency bandpass filtering,” Opt. Express 19(14), 13365–13377 (2011).
[CrossRef] [PubMed]

M. Zhang, Z. Che, J. Chen, H. Zhao, L. Yang, Z. Zhong, J. Lu, “Experimental determination of thermal conductivity of water-agar gel at Different concentrations and temperatures,” J. Chem. Eng. Data 56(4), 859–864 (2011).
[CrossRef]

2010 (3)

R. L. Shelton, B. E. Applegate, “Ultrahigh resolution photoacoustic microscopy via transient absorption,” Biomed. Opt. Express 1(2), 676–686 (2010).
[CrossRef] [PubMed]

V. Ntziachristos, “Going deeper than microscopy: the optical imaging frontier in biology,” Nat. Methods 7(8), 603–614 (2010).
[CrossRef] [PubMed]

M. Rumi, J. W. Perry, “Two-photon absorption: an overview of measurements and principles,” Adv. Opt. Photonics 2(4), 451–518 (2010).
[CrossRef]

2009 (2)

B. Huang, M. Bates, X. Zhuang, “Super-Resolution Fluorescence Microscopy,” Annu. Rev. Biochem. 78(1), 993–1016 (2009).
[CrossRef] [PubMed]

M. Pramanik, L. V. Wang, “Thermoacoustic and photoacoustic sensing of temperature,” J. Biomed. Opt. 14(5), 054024 (2009).

2008 (1)

L. V. Wang, “Tutorial on Photoacoustic Microscopy and Computed Tomography,” IEEE J. Sel. Top. Quantum Electron. 14(1), 171–179 (2008).
[CrossRef]

2006 (2)

M. Xu, L. V. Wang, “Photoacoustic imaging in biomedicine,” Rev. Sci. Instrum. 77, 041101 (2006).

H. F. Zhang, K. Maslov, G. Stoica, L. V. Wang, “Functional photoacoustic microscopy for high-resolution and noninvasive in vivo imaging,” Nat. Biotechnol. 24(7), 848–851 (2006).
[CrossRef] [PubMed]

2005 (2)

F. Helmchen, W. Denk, “Deep tissue two-photon microscopy,” Nat. Methods 2(12), 932–940 (2005).
[CrossRef] [PubMed]

D. Haemmerich, D. J. Schutt, I. dos Santos, J. G. Webster, D. M. Mahvi, “Measurement of temperature-dependent specific heat of biological tissues,” Physiol. Meas. 26(1), 59–67 (2005).
[CrossRef] [PubMed]

2004 (1)

2003 (1)

W. R. Zipfel, R. M. Williams, W. W. Webb, “Nonlinear magic: multiphoton microscopy in the biosciences,” Nat. Biotechnol. 21(11), 1369–1377 (2003).
[CrossRef] [PubMed]

2002 (1)

Applegate, B. E.

Bates, M.

B. Huang, M. Bates, X. Zhuang, “Super-Resolution Fluorescence Microscopy,” Annu. Rev. Biochem. 78(1), 993–1016 (2009).
[CrossRef] [PubMed]

Berer, T.

Bouchal, K. D.

Burgholzer, P.

Chandrasekharan, N.

Chang, C. F.

Y. H. Lai, C. F. Chang, Y. H. Cheng, C. K. Sun, “Two-photon photoacoustics ultrasound measurement by a loss modulation technique,” Proc. SPIE8581, 1–8 (2013).

Che, Z.

M. Zhang, Z. Che, J. Chen, H. Zhao, L. Yang, Z. Zhong, J. Lu, “Experimental determination of thermal conductivity of water-agar gel at Different concentrations and temperatures,” J. Chem. Eng. Data 56(4), 859–864 (2011).
[CrossRef]

Chen, J.

M. Zhang, Z. Che, J. Chen, H. Zhao, L. Yang, Z. Zhong, J. Lu, “Experimental determination of thermal conductivity of water-agar gel at Different concentrations and temperatures,” J. Chem. Eng. Data 56(4), 859–864 (2011).
[CrossRef]

Cheng, Y. H.

Y. H. Lai, C. F. Chang, Y. H. Cheng, C. K. Sun, “Two-photon photoacoustics ultrasound measurement by a loss modulation technique,” Proc. SPIE8581, 1–8 (2013).

Cherin, E.

M.E. van Raaij, M. Lee, E. Cherin, B. Stefanovic, F.S. Foster, “Femtosecond photoacoustics: integrated two-photon fluorescence and photoacoustic microscopy,” Proc. SPIE7564, 1-6 (2010)

Cullum, B. M.

N. Chandrasekharan, B. Gonzales, B. M. Cullum, “Non-resonant Multiphoton Photoacoustic Spectroscopy for Noninvasive Subsurface Chemical Diagnostics,” Appl. Spectrosc. 58(11), 1325–1333 (2004).
[CrossRef] [PubMed]

S. Dahal, J. B. Kiser, B. M. Cullum, “Depth and resolution characterization of two-photon photoacoustic spectroscopy for noninvasive subsurface chemical diagnostics,” Proc. SPIE8025, 1–7 (2011).

Dahal, S.

S. Dahal, J. B. Kiser, B. M. Cullum, “Depth and resolution characterization of two-photon photoacoustic spectroscopy for noninvasive subsurface chemical diagnostics,” Proc. SPIE8025, 1–7 (2011).

Danielli, A.

A. Danielli, K. Maslov, J. Xia, L. V. Wang, “Wide range quantitative photoacoustic spectroscopy to measure non-linear optical absorption of hemoglobin,” Proc. SPIE8223, 1–6 (2012).

Denk, W.

F. Helmchen, W. Denk, “Deep tissue two-photon microscopy,” Nat. Methods 2(12), 932–940 (2005).
[CrossRef] [PubMed]

dos Santos, I.

D. Haemmerich, D. J. Schutt, I. dos Santos, J. G. Webster, D. M. Mahvi, “Measurement of temperature-dependent specific heat of biological tissues,” Physiol. Meas. 26(1), 59–67 (2005).
[CrossRef] [PubMed]

Foster, F.S.

M.E. van Raaij, M. Lee, E. Cherin, B. Stefanovic, F.S. Foster, “Femtosecond photoacoustics: integrated two-photon fluorescence and photoacoustic microscopy,” Proc. SPIE7564, 1-6 (2010)

Gonzales, B.

Grün, H.

Haemmerich, D.

D. Haemmerich, D. J. Schutt, I. dos Santos, J. G. Webster, D. M. Mahvi, “Measurement of temperature-dependent specific heat of biological tissues,” Physiol. Meas. 26(1), 59–67 (2005).
[CrossRef] [PubMed]

Helmchen, F.

F. Helmchen, W. Denk, “Deep tissue two-photon microscopy,” Nat. Methods 2(12), 932–940 (2005).
[CrossRef] [PubMed]

Hu, S.

L. V. Wang, S. Hu, “Photoacoustic tomography: In Vivo imaging from organelles to organs,” Science 335(6075), 1458–1462 (2012).
[CrossRef] [PubMed]

Huang, B.

B. Huang, M. Bates, X. Zhuang, “Super-Resolution Fluorescence Microscopy,” Annu. Rev. Biochem. 78(1), 993–1016 (2009).
[CrossRef] [PubMed]

Kiser, J. B.

S. Dahal, J. B. Kiser, B. M. Cullum, “Depth and resolution characterization of two-photon photoacoustic spectroscopy for noninvasive subsurface chemical diagnostics,” Proc. SPIE8025, 1–7 (2011).

Lai, Y. H.

Y. H. Lai, C. F. Chang, Y. H. Cheng, C. K. Sun, “Two-photon photoacoustics ultrasound measurement by a loss modulation technique,” Proc. SPIE8581, 1–8 (2013).

Langer, G.

Lee, M.

M.E. van Raaij, M. Lee, E. Cherin, B. Stefanovic, F.S. Foster, “Femtosecond photoacoustics: integrated two-photon fluorescence and photoacoustic microscopy,” Proc. SPIE7564, 1-6 (2010)

Lu, J.

M. Zhang, Z. Che, J. Chen, H. Zhao, L. Yang, Z. Zhong, J. Lu, “Experimental determination of thermal conductivity of water-agar gel at Different concentrations and temperatures,” J. Chem. Eng. Data 56(4), 859–864 (2011).
[CrossRef]

Mahvi, D. M.

D. Haemmerich, D. J. Schutt, I. dos Santos, J. G. Webster, D. M. Mahvi, “Measurement of temperature-dependent specific heat of biological tissues,” Physiol. Meas. 26(1), 59–67 (2005).
[CrossRef] [PubMed]

Maslov, K.

H. F. Zhang, K. Maslov, G. Stoica, L. V. Wang, “Functional photoacoustic microscopy for high-resolution and noninvasive in vivo imaging,” Nat. Biotechnol. 24(7), 848–851 (2006).
[CrossRef] [PubMed]

A. Danielli, K. Maslov, J. Xia, L. V. Wang, “Wide range quantitative photoacoustic spectroscopy to measure non-linear optical absorption of hemoglobin,” Proc. SPIE8223, 1–6 (2012).

Nambu, M.

Nambub, M.

Y. Yamaoka, M. Nambub, T. Takamatsu, “Frequency-selective multiphoton-excitation-induced photoacoustic microscopy (MEPAM) to visualize the cross sections of dense objects,” Proc. SPIE7564, 1–9 (2010).

Ntziachristos, V.

V. Ntziachristos, “Going deeper than microscopy: the optical imaging frontier in biology,” Nat. Methods 7(8), 603–614 (2010).
[CrossRef] [PubMed]

Perry, J. W.

M. Rumi, J. W. Perry, “Two-photon absorption: an overview of measurements and principles,” Adv. Opt. Photonics 2(4), 451–518 (2010).
[CrossRef]

Pramanik, M.

M. Pramanik, L. V. Wang, “Thermoacoustic and photoacoustic sensing of temperature,” J. Biomed. Opt. 14(5), 054024 (2009).

Rumi, M.

M. Rumi, J. W. Perry, “Two-photon absorption: an overview of measurements and principles,” Adv. Opt. Photonics 2(4), 451–518 (2010).
[CrossRef]

Schutt, D. J.

D. Haemmerich, D. J. Schutt, I. dos Santos, J. G. Webster, D. M. Mahvi, “Measurement of temperature-dependent specific heat of biological tissues,” Physiol. Meas. 26(1), 59–67 (2005).
[CrossRef] [PubMed]

Shelton, R. L.

Stefanovic, B.

M.E. van Raaij, M. Lee, E. Cherin, B. Stefanovic, F.S. Foster, “Femtosecond photoacoustics: integrated two-photon fluorescence and photoacoustic microscopy,” Proc. SPIE7564, 1-6 (2010)

Stoica, G.

H. F. Zhang, K. Maslov, G. Stoica, L. V. Wang, “Functional photoacoustic microscopy for high-resolution and noninvasive in vivo imaging,” Nat. Biotechnol. 24(7), 848–851 (2006).
[CrossRef] [PubMed]

Sun, C. K.

Y. H. Lai, C. F. Chang, Y. H. Cheng, C. K. Sun, “Two-photon photoacoustics ultrasound measurement by a loss modulation technique,” Proc. SPIE8581, 1–8 (2013).

Takamatsu, T.

Y. Yamaoka, M. Nambu, T. Takamatsu, “Fine depth resolution of two-photon absorption-induced photoacoustic microscopy using low-frequency bandpass filtering,” Opt. Express 19(14), 13365–13377 (2011).
[CrossRef] [PubMed]

Y. Yamaoka, M. Nambub, T. Takamatsu, “Frequency-selective multiphoton-excitation-induced photoacoustic microscopy (MEPAM) to visualize the cross sections of dense objects,” Proc. SPIE7564, 1–9 (2010).

Y. Yamaoka, T. Takamatsu, “Enhancement of multiphoton excitation-induced photoacoustic signals by using gold nanoparticles surrounded by fluorescent dyes,” Proc. SPIE7177, 1–9 (2009).

Tian, P.

van Raaij, M.E.

M.E. van Raaij, M. Lee, E. Cherin, B. Stefanovic, F.S. Foster, “Femtosecond photoacoustics: integrated two-photon fluorescence and photoacoustic microscopy,” Proc. SPIE7564, 1-6 (2010)

Wang, L. V.

L. V. Wang, S. Hu, “Photoacoustic tomography: In Vivo imaging from organelles to organs,” Science 335(6075), 1458–1462 (2012).
[CrossRef] [PubMed]

M. Pramanik, L. V. Wang, “Thermoacoustic and photoacoustic sensing of temperature,” J. Biomed. Opt. 14(5), 054024 (2009).

L. V. Wang, “Tutorial on Photoacoustic Microscopy and Computed Tomography,” IEEE J. Sel. Top. Quantum Electron. 14(1), 171–179 (2008).
[CrossRef]

H. F. Zhang, K. Maslov, G. Stoica, L. V. Wang, “Functional photoacoustic microscopy for high-resolution and noninvasive in vivo imaging,” Nat. Biotechnol. 24(7), 848–851 (2006).
[CrossRef] [PubMed]

M. Xu, L. V. Wang, “Photoacoustic imaging in biomedicine,” Rev. Sci. Instrum. 77, 041101 (2006).

A. Danielli, K. Maslov, J. Xia, L. V. Wang, “Wide range quantitative photoacoustic spectroscopy to measure non-linear optical absorption of hemoglobin,” Proc. SPIE8223, 1–6 (2012).

Warren, W. S.

Webb, W. W.

W. R. Zipfel, R. M. Williams, W. W. Webb, “Nonlinear magic: multiphoton microscopy in the biosciences,” Nat. Biotechnol. 21(11), 1369–1377 (2003).
[CrossRef] [PubMed]

Webster, J. G.

D. Haemmerich, D. J. Schutt, I. dos Santos, J. G. Webster, D. M. Mahvi, “Measurement of temperature-dependent specific heat of biological tissues,” Physiol. Meas. 26(1), 59–67 (2005).
[CrossRef] [PubMed]

Williams, R. M.

W. R. Zipfel, R. M. Williams, W. W. Webb, “Nonlinear magic: multiphoton microscopy in the biosciences,” Nat. Biotechnol. 21(11), 1369–1377 (2003).
[CrossRef] [PubMed]

Xia, J.

A. Danielli, K. Maslov, J. Xia, L. V. Wang, “Wide range quantitative photoacoustic spectroscopy to measure non-linear optical absorption of hemoglobin,” Proc. SPIE8223, 1–6 (2012).

Xu, M.

M. Xu, L. V. Wang, “Photoacoustic imaging in biomedicine,” Rev. Sci. Instrum. 77, 041101 (2006).

Yamaoka, Y.

Y. Yamaoka, M. Nambu, T. Takamatsu, “Fine depth resolution of two-photon absorption-induced photoacoustic microscopy using low-frequency bandpass filtering,” Opt. Express 19(14), 13365–13377 (2011).
[CrossRef] [PubMed]

Y. Yamaoka, M. Nambub, T. Takamatsu, “Frequency-selective multiphoton-excitation-induced photoacoustic microscopy (MEPAM) to visualize the cross sections of dense objects,” Proc. SPIE7564, 1–9 (2010).

Y. Yamaoka, T. Takamatsu, “Enhancement of multiphoton excitation-induced photoacoustic signals by using gold nanoparticles surrounded by fluorescent dyes,” Proc. SPIE7177, 1–9 (2009).

Yang, L.

M. Zhang, Z. Che, J. Chen, H. Zhao, L. Yang, Z. Zhong, J. Lu, “Experimental determination of thermal conductivity of water-agar gel at Different concentrations and temperatures,” J. Chem. Eng. Data 56(4), 859–864 (2011).
[CrossRef]

Zhang, H. F.

H. F. Zhang, K. Maslov, G. Stoica, L. V. Wang, “Functional photoacoustic microscopy for high-resolution and noninvasive in vivo imaging,” Nat. Biotechnol. 24(7), 848–851 (2006).
[CrossRef] [PubMed]

Zhang, M.

M. Zhang, Z. Che, J. Chen, H. Zhao, L. Yang, Z. Zhong, J. Lu, “Experimental determination of thermal conductivity of water-agar gel at Different concentrations and temperatures,” J. Chem. Eng. Data 56(4), 859–864 (2011).
[CrossRef]

Zhao, H.

M. Zhang, Z. Che, J. Chen, H. Zhao, L. Yang, Z. Zhong, J. Lu, “Experimental determination of thermal conductivity of water-agar gel at Different concentrations and temperatures,” J. Chem. Eng. Data 56(4), 859–864 (2011).
[CrossRef]

Zhong, Z.

M. Zhang, Z. Che, J. Chen, H. Zhao, L. Yang, Z. Zhong, J. Lu, “Experimental determination of thermal conductivity of water-agar gel at Different concentrations and temperatures,” J. Chem. Eng. Data 56(4), 859–864 (2011).
[CrossRef]

Zhuang, X.

B. Huang, M. Bates, X. Zhuang, “Super-Resolution Fluorescence Microscopy,” Annu. Rev. Biochem. 78(1), 993–1016 (2009).
[CrossRef] [PubMed]

Zipfel, W. R.

W. R. Zipfel, R. M. Williams, W. W. Webb, “Nonlinear magic: multiphoton microscopy in the biosciences,” Nat. Biotechnol. 21(11), 1369–1377 (2003).
[CrossRef] [PubMed]

Adv. Opt. Photonics (1)

M. Rumi, J. W. Perry, “Two-photon absorption: an overview of measurements and principles,” Adv. Opt. Photonics 2(4), 451–518 (2010).
[CrossRef]

Annu. Rev. Biochem. (1)

B. Huang, M. Bates, X. Zhuang, “Super-Resolution Fluorescence Microscopy,” Annu. Rev. Biochem. 78(1), 993–1016 (2009).
[CrossRef] [PubMed]

Appl. Spectrosc. (1)

Biomed. Opt. Express (1)

IEEE J. Sel. Top. Quantum Electron. (1)

L. V. Wang, “Tutorial on Photoacoustic Microscopy and Computed Tomography,” IEEE J. Sel. Top. Quantum Electron. 14(1), 171–179 (2008).
[CrossRef]

J. Biomed. Opt (1)

M. Pramanik, L. V. Wang, “Thermoacoustic and photoacoustic sensing of temperature,” J. Biomed. Opt. 14(5), 054024 (2009).

J. Chem. Eng. Data (1)

M. Zhang, Z. Che, J. Chen, H. Zhao, L. Yang, Z. Zhong, J. Lu, “Experimental determination of thermal conductivity of water-agar gel at Different concentrations and temperatures,” J. Chem. Eng. Data 56(4), 859–864 (2011).
[CrossRef]

Nat. Biotechnol. (2)

W. R. Zipfel, R. M. Williams, W. W. Webb, “Nonlinear magic: multiphoton microscopy in the biosciences,” Nat. Biotechnol. 21(11), 1369–1377 (2003).
[CrossRef] [PubMed]

H. F. Zhang, K. Maslov, G. Stoica, L. V. Wang, “Functional photoacoustic microscopy for high-resolution and noninvasive in vivo imaging,” Nat. Biotechnol. 24(7), 848–851 (2006).
[CrossRef] [PubMed]

Nat. Methods (2)

F. Helmchen, W. Denk, “Deep tissue two-photon microscopy,” Nat. Methods 2(12), 932–940 (2005).
[CrossRef] [PubMed]

V. Ntziachristos, “Going deeper than microscopy: the optical imaging frontier in biology,” Nat. Methods 7(8), 603–614 (2010).
[CrossRef] [PubMed]

Opt. Express (2)

Opt. Lett. (1)

Physiol. Meas. (1)

D. Haemmerich, D. J. Schutt, I. dos Santos, J. G. Webster, D. M. Mahvi, “Measurement of temperature-dependent specific heat of biological tissues,” Physiol. Meas. 26(1), 59–67 (2005).
[CrossRef] [PubMed]

Rev. Sci. Instrum. (1)

M. Xu, L. V. Wang, “Photoacoustic imaging in biomedicine,” Rev. Sci. Instrum. 77, 041101 (2006).

Science (1)

L. V. Wang, S. Hu, “Photoacoustic tomography: In Vivo imaging from organelles to organs,” Science 335(6075), 1458–1462 (2012).
[CrossRef] [PubMed]

Other (6)

Y. Yamaoka, M. Nambub, T. Takamatsu, “Frequency-selective multiphoton-excitation-induced photoacoustic microscopy (MEPAM) to visualize the cross sections of dense objects,” Proc. SPIE7564, 1–9 (2010).

S. Dahal, J. B. Kiser, B. M. Cullum, “Depth and resolution characterization of two-photon photoacoustic spectroscopy for noninvasive subsurface chemical diagnostics,” Proc. SPIE8025, 1–7 (2011).

M.E. van Raaij, M. Lee, E. Cherin, B. Stefanovic, F.S. Foster, “Femtosecond photoacoustics: integrated two-photon fluorescence and photoacoustic microscopy,” Proc. SPIE7564, 1-6 (2010)

A. Danielli, K. Maslov, J. Xia, L. V. Wang, “Wide range quantitative photoacoustic spectroscopy to measure non-linear optical absorption of hemoglobin,” Proc. SPIE8223, 1–6 (2012).

Y. Yamaoka, T. Takamatsu, “Enhancement of multiphoton excitation-induced photoacoustic signals by using gold nanoparticles surrounded by fluorescent dyes,” Proc. SPIE7177, 1–9 (2009).

Y. H. Lai, C. F. Chang, Y. H. Cheng, C. K. Sun, “Two-photon photoacoustics ultrasound measurement by a loss modulation technique,” Proc. SPIE8581, 1–8 (2013).

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Figures (12)

Fig. 1
Fig. 1

The experimental design for testing the photo-acoustic phenomenon.

Fig. 2
Fig. 2

(a) The power dependency experiment for different kinds of nano-particles and fluorescence dyes. Note that the fluorescence dyes had strong nonlinear absorption. The pulse energy was 2.06nJ/pulse. (b) The imperfect temporal response of the AOMs to the external sinusoidal amplitude modulation. The high harmonics existed and caused the linear background. The HFR was −30dB, and the power dependency of the high harmonics was linear. (−20dB/OD)

Fig. 3
Fig. 3

Schematic diagram showing the basic experiment setup to test the loss modulation technique. For the infrared light experiment, the BBO crystal was removed. For the blue light experiment, we used color filters to remove the infrared light.

Fig. 4
Fig. 4

(a) The collinear NIR light beats with itself if its components have different frequency shifts provided by AOMs. (b) When the collinear NIR light passes through the BBO crystal, the collinear light creates sum frequency generation (SFG) and second harmonic generation (SHG). The blue light creates 1st order beating and 2nd order beating on itself.

Fig. 5
Fig. 5

(a) The original repetition rate of the femtosecond laser (b) The first order beating spectrum (c) The second order beating spectrum (d) The low frequency spectrum of the collinear NIR light (λR = 800nm) (e) The blue light generated by BBO (λB = 400nm). Note that the frequency separation is related to the diffraction order of the beam, the modulation frequency of AOMs, and the laser repetition rate. Moreover, the 2nd order beating peak originates from the nonlinearity of the sample or of the detector.

Fig. 6
Fig. 6

(a) The NIR light (λR = 800nm) power dependency with 0.5mW incident power. The two-photon absorption is quadratically dependent on the incident power. (b) The blue light (λB = 400nm) power dependency with 0.2mW incident power. Note that both the 1st and 2nd beating signals are generated by BBO, so they have the same power dependency. (ND: Neutral Density filter)

Fig. 7
Fig. 7

Lasers with different repetition rate induce different system responses. (a) The low repetition rate laser excites the system with observable transient response, which is equivalent to the broad band response in the frequency domain. (b) The high repetition rate laser excites the system by forced resonance, so only the frequencies equal to the multiple of the repetition rate can be excited. The response spectrum becomes discrete, which is equivalent to the narrow band response if we detect the response at a specific frequency.

Fig. 8
Fig. 8

The enhancement mechanism of the two-photon photoacoustic signal by mixing carbon particles and fluorescence dyes. The energy transfers from 2PF into 2PA with the help of carbon particles. This phenomenon is similar to fluorescence resonance energy transfer (FRET).

Fig. 9
Fig. 9

(a) The experiment setup. (b) The design of the optical cavity. Since the concentration of the solution is very high, the light cannot penetrate. Therefore, we prevent the direct incidence of the laser beam on the ultrasonic probe.

Fig. 10
Fig. 10

(a) The existence of the 2nd order beating signal in the dye-carbon mixture. This peak vanishes if we only use pure dilute carbon solution, shown as the red curve in the enlarged spectrum. (b) The power dependency of the 2nd order beating (2PA US) is quadratic. The laser pulse energy is 9.34nJ/pulse with a repetition rate of 80.305MHz.

Fig. 11
Fig. 11

(a) The cavity, the pattern and the phantom design (3mm agar pattern with 7mm thick agar layer) (b) The image of the pattern under the microscope (c) The single-photon photoacoustic image. The large background signal reduces the contrast ratio of the image, so the edge is blurred. (d) The two-photon photoacoustic image (depth z = 250μm). The signal only exists with the presence of the contrast agent, so the edge of the pattern is sharp. The pulse energy is 5.6nJ/pulse.

Fig. 12
Fig. 12

(a) The image of the leaf tissue under the microscope. (b) The two-photon photoacoustic image of the leaf tissue underneath 1mm-thick phantom. The image shows the application of the two-photon photoacoustic imaging on a biological tissue. The strong scattering in the agar phantom degraded the lateral resolution to approximately 10μm. The pulse energy is 2.8nJ/pulse.

Equations (1)

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Ω( f 1 , f 2 , f R )=min(| n 1 f 1 n 2 f 2 +n f R |) ( f 1 ~ f 2 ~ f R >>Ω, n 1 , n 2 ,n)

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