Abstract

We have developed a novel, hybrid imaging modality, Transient Absorption Ultrasonic Microscopy (TAUM), which takes advantage of the optical nonlinearities afforded by transient absorption to achieve ultrahigh-resolution photoacoustic microscopy. The theoretical point spread function for TAUM is functionally equivalent to confocal and two-photon fluorescence microscopy, potentially enabling cellular/subcellular photoacoustic imaging. A prototype TAUM system was designed, built, and used to image a cross-section through several capillaries in the excised cheek pouch of a Syrian Hamster. The well-resolved capillaries in the TAUM image provided experimental evidence of the spatial resolution. These results suggest that TAUM has excellent potential for producing volumetric images with cellular/subcellular resolution in three dimensions deep inside living tissue.

© 2010 OSA

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References

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  1. K. Maslov, G. Stoica, and L. V. H. Wang, “In vivo dark-field reflection-mode photoacoustic microscopy,” Opt. Lett. 30(6), 625–627 (2005).
    [CrossRef] [PubMed]
  2. S. Hu and L. V. Wang, “Photoacoustic imaging and characterization of the microvasculature,” J. Biomed. Opt. 15(1), 011101 (2010).
    [CrossRef] [PubMed]
  3. J. T. Oh, M. L. Li, H. F. Zhang, K. Maslov, G. Stoica, and L. V. Wang, “Three-dimensional imaging of skin melanoma in vivo by dual-wavelength photoacoustic microscopy,” J. Biomed. Opt. 11(3), 034032 (2006).
    [CrossRef] [PubMed]
  4. Y. S. Yang, S. Vaithilingam, H. T. J. Ma, S. Salehi-Had, O. Oralkan, B. T. Khuri-Yakub, and S. Guccione, “Development of Nanoparticle-Based Gold Contrast Agent for Photoacoustic Tomography,” NSTI Nanotech 2008, Vol 1, Technical Proceedings 708–711, 1092 (2008).
  5. G. Kim, S. W. Huang, K. C. Day, M. O’Donnell, R. R. Agayan, M. A. Day, R. Kopelman, and S. Ashkenazi, “Indocyanine-green-embedded PEBBLEs as a contrast agent for photoacoustic imaging,” J. Biomed. Opt. 12(4), 044020 (2007).
    [CrossRef] [PubMed]
  6. K. Maslov, H. F. Zhang, S. Hu, and L. V. Wang, “Optical-resolution photoacoustic microscopy for in vivo imaging of single capillaries,” Opt. Lett. 33(9), 929–931 (2008).
    [CrossRef] [PubMed]
  7. K. Maslov, G. Ku, and L. V. Wang, “Photoacoustic microscopy with submicron resolution,” in (SPIE, 2010), 75640W.
  8. C. E. Crespo-Hernández, B. Cohen, and B. Kohler, “Base stacking controls excited-state dynamics in A.T DNA,” Nature 436(7054), 1141–1144 (2005).
    [CrossRef] [PubMed]
  9. B. E. Applegate and J. A. Izatt, “Molecular imaging of endogenous and exogenous chromophores using ground state recovery pump-probe optical coherence tomography,” Opt. Express 14(20), 9142–9155 (2006).
    [CrossRef] [PubMed]
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    [CrossRef] [PubMed]
  11. C. Y. Dong, P. T. C. So, C. Buehler, and E. Gratton, “Spatial resolution in scanning pump-probe fluorescence microscopy,” Optik (Stuttg.) 106, 7–14 (1997).
  12. C. J. R. Sheppard and M. Gu, “Image-Formation in 2-Photon Fluorescence Microscopy,” Optik (Stuttg.) 86, 104–106 (1990).
  13. C. M. W. Daft, G. A. D. Briggs, and W. D. O’Brien., “Frequency dependence of tissue attenuation measured by acoustic microscopy,” J. Acoust. Soc. Am. 85(5), 2194–2201 (1989).
    [CrossRef] [PubMed]
  14. J. Squier and M. Muller, “High resolution nonlinear microscopy: A review of sources and methods for achieving optimal imaging,” Rev. Sci. Instrum. 72(7), 2855–2867 (2001).
    [CrossRef]
  15. R. L. Shelton and B. E. Applegate, “Off-axis photoacoustic microscopy,” IEEE Trans. Biomed. Eng. 57(8), 1835–1838 (2010).
    [CrossRef] [PubMed]
  16. G. Ku, K. Maslov, L. Li, and L. V. Wang, “Photoacoustic microscopy with 2-microm transverse resolution,” J. Biomed. Opt. 15(2), 021302 (2010).
    [CrossRef] [PubMed]

2010 (3)

S. Hu and L. V. Wang, “Photoacoustic imaging and characterization of the microvasculature,” J. Biomed. Opt. 15(1), 011101 (2010).
[CrossRef] [PubMed]

R. L. Shelton and B. E. Applegate, “Off-axis photoacoustic microscopy,” IEEE Trans. Biomed. Eng. 57(8), 1835–1838 (2010).
[CrossRef] [PubMed]

G. Ku, K. Maslov, L. Li, and L. V. Wang, “Photoacoustic microscopy with 2-microm transverse resolution,” J. Biomed. Opt. 15(2), 021302 (2010).
[CrossRef] [PubMed]

2008 (1)

2007 (1)

G. Kim, S. W. Huang, K. C. Day, M. O’Donnell, R. R. Agayan, M. A. Day, R. Kopelman, and S. Ashkenazi, “Indocyanine-green-embedded PEBBLEs as a contrast agent for photoacoustic imaging,” J. Biomed. Opt. 12(4), 044020 (2007).
[CrossRef] [PubMed]

2006 (2)

J. T. Oh, M. L. Li, H. F. Zhang, K. Maslov, G. Stoica, and L. V. Wang, “Three-dimensional imaging of skin melanoma in vivo by dual-wavelength photoacoustic microscopy,” J. Biomed. Opt. 11(3), 034032 (2006).
[CrossRef] [PubMed]

B. E. Applegate and J. A. Izatt, “Molecular imaging of endogenous and exogenous chromophores using ground state recovery pump-probe optical coherence tomography,” Opt. Express 14(20), 9142–9155 (2006).
[CrossRef] [PubMed]

2005 (3)

2001 (1)

J. Squier and M. Muller, “High resolution nonlinear microscopy: A review of sources and methods for achieving optimal imaging,” Rev. Sci. Instrum. 72(7), 2855–2867 (2001).
[CrossRef]

1997 (1)

C. Y. Dong, P. T. C. So, C. Buehler, and E. Gratton, “Spatial resolution in scanning pump-probe fluorescence microscopy,” Optik (Stuttg.) 106, 7–14 (1997).

1990 (1)

C. J. R. Sheppard and M. Gu, “Image-Formation in 2-Photon Fluorescence Microscopy,” Optik (Stuttg.) 86, 104–106 (1990).

1989 (1)

C. M. W. Daft, G. A. D. Briggs, and W. D. O’Brien., “Frequency dependence of tissue attenuation measured by acoustic microscopy,” J. Acoust. Soc. Am. 85(5), 2194–2201 (1989).
[CrossRef] [PubMed]

Agayan, R. R.

G. Kim, S. W. Huang, K. C. Day, M. O’Donnell, R. R. Agayan, M. A. Day, R. Kopelman, and S. Ashkenazi, “Indocyanine-green-embedded PEBBLEs as a contrast agent for photoacoustic imaging,” J. Biomed. Opt. 12(4), 044020 (2007).
[CrossRef] [PubMed]

Applegate, B. E.

Ashkenazi, S.

G. Kim, S. W. Huang, K. C. Day, M. O’Donnell, R. R. Agayan, M. A. Day, R. Kopelman, and S. Ashkenazi, “Indocyanine-green-embedded PEBBLEs as a contrast agent for photoacoustic imaging,” J. Biomed. Opt. 12(4), 044020 (2007).
[CrossRef] [PubMed]

Briggs, G. A. D.

C. M. W. Daft, G. A. D. Briggs, and W. D. O’Brien., “Frequency dependence of tissue attenuation measured by acoustic microscopy,” J. Acoust. Soc. Am. 85(5), 2194–2201 (1989).
[CrossRef] [PubMed]

Buehler, C.

C. Y. Dong, P. T. C. So, C. Buehler, and E. Gratton, “Spatial resolution in scanning pump-probe fluorescence microscopy,” Optik (Stuttg.) 106, 7–14 (1997).

Cohen, B.

C. E. Crespo-Hernández, B. Cohen, and B. Kohler, “Base stacking controls excited-state dynamics in A.T DNA,” Nature 436(7054), 1141–1144 (2005).
[CrossRef] [PubMed]

Crespo-Hernández, C. E.

C. E. Crespo-Hernández, B. Cohen, and B. Kohler, “Base stacking controls excited-state dynamics in A.T DNA,” Nature 436(7054), 1141–1144 (2005).
[CrossRef] [PubMed]

Daft, C. M. W.

C. M. W. Daft, G. A. D. Briggs, and W. D. O’Brien., “Frequency dependence of tissue attenuation measured by acoustic microscopy,” J. Acoust. Soc. Am. 85(5), 2194–2201 (1989).
[CrossRef] [PubMed]

Day, K. C.

G. Kim, S. W. Huang, K. C. Day, M. O’Donnell, R. R. Agayan, M. A. Day, R. Kopelman, and S. Ashkenazi, “Indocyanine-green-embedded PEBBLEs as a contrast agent for photoacoustic imaging,” J. Biomed. Opt. 12(4), 044020 (2007).
[CrossRef] [PubMed]

Day, M. A.

G. Kim, S. W. Huang, K. C. Day, M. O’Donnell, R. R. Agayan, M. A. Day, R. Kopelman, and S. Ashkenazi, “Indocyanine-green-embedded PEBBLEs as a contrast agent for photoacoustic imaging,” J. Biomed. Opt. 12(4), 044020 (2007).
[CrossRef] [PubMed]

Dong, C. Y.

C. Y. Dong, P. T. C. So, C. Buehler, and E. Gratton, “Spatial resolution in scanning pump-probe fluorescence microscopy,” Optik (Stuttg.) 106, 7–14 (1997).

Gratton, E.

C. Y. Dong, P. T. C. So, C. Buehler, and E. Gratton, “Spatial resolution in scanning pump-probe fluorescence microscopy,” Optik (Stuttg.) 106, 7–14 (1997).

Gu, M.

C. J. R. Sheppard and M. Gu, “Image-Formation in 2-Photon Fluorescence Microscopy,” Optik (Stuttg.) 86, 104–106 (1990).

Hu, S.

Huang, S. W.

G. Kim, S. W. Huang, K. C. Day, M. O’Donnell, R. R. Agayan, M. A. Day, R. Kopelman, and S. Ashkenazi, “Indocyanine-green-embedded PEBBLEs as a contrast agent for photoacoustic imaging,” J. Biomed. Opt. 12(4), 044020 (2007).
[CrossRef] [PubMed]

Izatt, J. A.

Kim, G.

G. Kim, S. W. Huang, K. C. Day, M. O’Donnell, R. R. Agayan, M. A. Day, R. Kopelman, and S. Ashkenazi, “Indocyanine-green-embedded PEBBLEs as a contrast agent for photoacoustic imaging,” J. Biomed. Opt. 12(4), 044020 (2007).
[CrossRef] [PubMed]

Kohler, B.

C. E. Crespo-Hernández, B. Cohen, and B. Kohler, “Base stacking controls excited-state dynamics in A.T DNA,” Nature 436(7054), 1141–1144 (2005).
[CrossRef] [PubMed]

Kopelman, R.

G. Kim, S. W. Huang, K. C. Day, M. O’Donnell, R. R. Agayan, M. A. Day, R. Kopelman, and S. Ashkenazi, “Indocyanine-green-embedded PEBBLEs as a contrast agent for photoacoustic imaging,” J. Biomed. Opt. 12(4), 044020 (2007).
[CrossRef] [PubMed]

Ku, G.

G. Ku, K. Maslov, L. Li, and L. V. Wang, “Photoacoustic microscopy with 2-microm transverse resolution,” J. Biomed. Opt. 15(2), 021302 (2010).
[CrossRef] [PubMed]

Li, L.

G. Ku, K. Maslov, L. Li, and L. V. Wang, “Photoacoustic microscopy with 2-microm transverse resolution,” J. Biomed. Opt. 15(2), 021302 (2010).
[CrossRef] [PubMed]

Li, M. L.

J. T. Oh, M. L. Li, H. F. Zhang, K. Maslov, G. Stoica, and L. V. Wang, “Three-dimensional imaging of skin melanoma in vivo by dual-wavelength photoacoustic microscopy,” J. Biomed. Opt. 11(3), 034032 (2006).
[CrossRef] [PubMed]

Maslov, K.

G. Ku, K. Maslov, L. Li, and L. V. Wang, “Photoacoustic microscopy with 2-microm transverse resolution,” J. Biomed. Opt. 15(2), 021302 (2010).
[CrossRef] [PubMed]

K. Maslov, H. F. Zhang, S. Hu, and L. V. Wang, “Optical-resolution photoacoustic microscopy for in vivo imaging of single capillaries,” Opt. Lett. 33(9), 929–931 (2008).
[CrossRef] [PubMed]

J. T. Oh, M. L. Li, H. F. Zhang, K. Maslov, G. Stoica, and L. V. Wang, “Three-dimensional imaging of skin melanoma in vivo by dual-wavelength photoacoustic microscopy,” J. Biomed. Opt. 11(3), 034032 (2006).
[CrossRef] [PubMed]

K. Maslov, G. Stoica, and L. V. H. Wang, “In vivo dark-field reflection-mode photoacoustic microscopy,” Opt. Lett. 30(6), 625–627 (2005).
[CrossRef] [PubMed]

Muller, M.

J. Squier and M. Muller, “High resolution nonlinear microscopy: A review of sources and methods for achieving optimal imaging,” Rev. Sci. Instrum. 72(7), 2855–2867 (2001).
[CrossRef]

O’Brien, W. D.

C. M. W. Daft, G. A. D. Briggs, and W. D. O’Brien., “Frequency dependence of tissue attenuation measured by acoustic microscopy,” J. Acoust. Soc. Am. 85(5), 2194–2201 (1989).
[CrossRef] [PubMed]

O’Donnell, M.

G. Kim, S. W. Huang, K. C. Day, M. O’Donnell, R. R. Agayan, M. A. Day, R. Kopelman, and S. Ashkenazi, “Indocyanine-green-embedded PEBBLEs as a contrast agent for photoacoustic imaging,” J. Biomed. Opt. 12(4), 044020 (2007).
[CrossRef] [PubMed]

Oh, J. T.

J. T. Oh, M. L. Li, H. F. Zhang, K. Maslov, G. Stoica, and L. V. Wang, “Three-dimensional imaging of skin melanoma in vivo by dual-wavelength photoacoustic microscopy,” J. Biomed. Opt. 11(3), 034032 (2006).
[CrossRef] [PubMed]

Shelton, R. L.

R. L. Shelton and B. E. Applegate, “Off-axis photoacoustic microscopy,” IEEE Trans. Biomed. Eng. 57(8), 1835–1838 (2010).
[CrossRef] [PubMed]

Sheppard, C. J. R.

C. J. R. Sheppard and M. Gu, “Image-Formation in 2-Photon Fluorescence Microscopy,” Optik (Stuttg.) 86, 104–106 (1990).

So, P. T. C.

C. Y. Dong, P. T. C. So, C. Buehler, and E. Gratton, “Spatial resolution in scanning pump-probe fluorescence microscopy,” Optik (Stuttg.) 106, 7–14 (1997).

Squier, J.

J. Squier and M. Muller, “High resolution nonlinear microscopy: A review of sources and methods for achieving optimal imaging,” Rev. Sci. Instrum. 72(7), 2855–2867 (2001).
[CrossRef]

Stoica, G.

J. T. Oh, M. L. Li, H. F. Zhang, K. Maslov, G. Stoica, and L. V. Wang, “Three-dimensional imaging of skin melanoma in vivo by dual-wavelength photoacoustic microscopy,” J. Biomed. Opt. 11(3), 034032 (2006).
[CrossRef] [PubMed]

K. Maslov, G. Stoica, and L. V. H. Wang, “In vivo dark-field reflection-mode photoacoustic microscopy,” Opt. Lett. 30(6), 625–627 (2005).
[CrossRef] [PubMed]

Wang, L. V.

S. Hu and L. V. Wang, “Photoacoustic imaging and characterization of the microvasculature,” J. Biomed. Opt. 15(1), 011101 (2010).
[CrossRef] [PubMed]

G. Ku, K. Maslov, L. Li, and L. V. Wang, “Photoacoustic microscopy with 2-microm transverse resolution,” J. Biomed. Opt. 15(2), 021302 (2010).
[CrossRef] [PubMed]

K. Maslov, H. F. Zhang, S. Hu, and L. V. Wang, “Optical-resolution photoacoustic microscopy for in vivo imaging of single capillaries,” Opt. Lett. 33(9), 929–931 (2008).
[CrossRef] [PubMed]

J. T. Oh, M. L. Li, H. F. Zhang, K. Maslov, G. Stoica, and L. V. Wang, “Three-dimensional imaging of skin melanoma in vivo by dual-wavelength photoacoustic microscopy,” J. Biomed. Opt. 11(3), 034032 (2006).
[CrossRef] [PubMed]

Wang, L. V. H.

Yang, C.

Zhang, H. F.

K. Maslov, H. F. Zhang, S. Hu, and L. V. Wang, “Optical-resolution photoacoustic microscopy for in vivo imaging of single capillaries,” Opt. Lett. 33(9), 929–931 (2008).
[CrossRef] [PubMed]

J. T. Oh, M. L. Li, H. F. Zhang, K. Maslov, G. Stoica, and L. V. Wang, “Three-dimensional imaging of skin melanoma in vivo by dual-wavelength photoacoustic microscopy,” J. Biomed. Opt. 11(3), 034032 (2006).
[CrossRef] [PubMed]

IEEE Trans. Biomed. Eng. (1)

R. L. Shelton and B. E. Applegate, “Off-axis photoacoustic microscopy,” IEEE Trans. Biomed. Eng. 57(8), 1835–1838 (2010).
[CrossRef] [PubMed]

J. Acoust. Soc. Am. (1)

C. M. W. Daft, G. A. D. Briggs, and W. D. O’Brien., “Frequency dependence of tissue attenuation measured by acoustic microscopy,” J. Acoust. Soc. Am. 85(5), 2194–2201 (1989).
[CrossRef] [PubMed]

J. Biomed. Opt. (4)

G. Ku, K. Maslov, L. Li, and L. V. Wang, “Photoacoustic microscopy with 2-microm transverse resolution,” J. Biomed. Opt. 15(2), 021302 (2010).
[CrossRef] [PubMed]

S. Hu and L. V. Wang, “Photoacoustic imaging and characterization of the microvasculature,” J. Biomed. Opt. 15(1), 011101 (2010).
[CrossRef] [PubMed]

J. T. Oh, M. L. Li, H. F. Zhang, K. Maslov, G. Stoica, and L. V. Wang, “Three-dimensional imaging of skin melanoma in vivo by dual-wavelength photoacoustic microscopy,” J. Biomed. Opt. 11(3), 034032 (2006).
[CrossRef] [PubMed]

G. Kim, S. W. Huang, K. C. Day, M. O’Donnell, R. R. Agayan, M. A. Day, R. Kopelman, and S. Ashkenazi, “Indocyanine-green-embedded PEBBLEs as a contrast agent for photoacoustic imaging,” J. Biomed. Opt. 12(4), 044020 (2007).
[CrossRef] [PubMed]

Nature (1)

C. E. Crespo-Hernández, B. Cohen, and B. Kohler, “Base stacking controls excited-state dynamics in A.T DNA,” Nature 436(7054), 1141–1144 (2005).
[CrossRef] [PubMed]

Opt. Express (2)

Opt. Lett. (2)

Optik (Stuttg.) (2)

C. Y. Dong, P. T. C. So, C. Buehler, and E. Gratton, “Spatial resolution in scanning pump-probe fluorescence microscopy,” Optik (Stuttg.) 106, 7–14 (1997).

C. J. R. Sheppard and M. Gu, “Image-Formation in 2-Photon Fluorescence Microscopy,” Optik (Stuttg.) 86, 104–106 (1990).

Rev. Sci. Instrum. (1)

J. Squier and M. Muller, “High resolution nonlinear microscopy: A review of sources and methods for achieving optimal imaging,” Rev. Sci. Instrum. 72(7), 2855–2867 (2001).
[CrossRef]

Other (2)

K. Maslov, G. Ku, and L. V. Wang, “Photoacoustic microscopy with submicron resolution,” in (SPIE, 2010), 75640W.

Y. S. Yang, S. Vaithilingam, H. T. J. Ma, S. Salehi-Had, O. Oralkan, B. T. Khuri-Yakub, and S. Guccione, “Development of Nanoparticle-Based Gold Contrast Agent for Photoacoustic Tomography,” NSTI Nanotech 2008, Vol 1, Technical Proceedings 708–711, 1092 (2008).

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Figures (4)

Fig. 1
Fig. 1

Schematic diagram of the prototype TAUM system. f1 and f2 are the modulation frequency of the pump and probe, respectively, as induced by a dual frequency optical chopper. L1 and L2 form a beam expander to fill the aperture of the objective (b). The sample is placed in a water bath (c) for acoustic coupling. A 6 or 25 MHz center frequency ultrasonic transducer serves as the detector (d).

Fig. 2
Fig. 2

Simulation of P(r 0,ω) with n = 1, Eq. (7). Panel A shows the resulting frequency band due to single photon photoacoustic emission (PA) at the laser repetition rate of 10 kHz (ωl ). Panel B includes the amplitude modulation of the pump (Pu) and probe (Pr) fluence, which adds beat frequencies of the pump and probe at 1 kHz ( ± ω pu) and 0.7 kHz ( ± ω pr), respectively to the 10 kHz band. All bands are due to single photon photoacoustic emission. Panel C includes the pump-probe interaction (coefficient D, Eq. (7), which induces sidebands at the sum (PPs), 1.7 kHz ( ± (ω pu + ω pr)) and difference (PPd), 0.3 kHz ( ± (ω pu- ω pr)) of the pump and probe modulation.

Fig. 3
Fig. 3

Axial scans through the thrombus sample. Cartoon of sample to left. A) Photoacoustic microscopy A-line. Nominal axial resolution is 300 μm. B) Integrated photoacoustic signal, analogous to single photon fluorescence microscopy. No axial sectioning ability. C) TAUM axial line, analogous to multiphoton microscopy. Nominal axial resolution is 7 μm (twice objective Rayleigh range).

Fig. 4
Fig. 4

, Ex vivo images of capillaries in the cheek pouch of a Syrian hamster. A) Photoacoustic B-scan (cross-section) of capillaries. B) 300 µm inset of B-scan to display 1:1 ratio with TAUM image. C) TAUM image of capillaries (cross-section) showing 3 capillaries of varying orientations. The scale bar is 20 μm.

Equations (7)

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p ( r , t ) = σ N 1 F κ ( r , t ) ,
N 1 = N 1 0 ( 1 σ λ p u F p u h c exp ( t d τ ) ) ,
Δ p ( r , t ) = σ N 1 0 ( σ λ p u F p u h c exp ( t d τ ) ) F p r κ ( r , t ) .
P S F T A U M = I ( r , z ) I ( r , z ) ,
I ( r , z ) = | 2 0 1 J 0 ( k r sin α , ρ ) exp ( 1 2 i 4 k z sin 2 ( α 2 ) ρ 2 ) d ρ | 2 ,
p ( r , t ) = σ N 1 0 F p u ( 1 + cos ( ω p u t ) 2 ) ( 1 exp ( t d τ ) ) κ ( r , t ) + σ N 1 0 ( 1 σ λ p u F p u ( 1 + cos ( ω p u t ) 2 ) h c exp ( t d τ ) ) F p r ( 1 + cos ( ω p r t ) 2 ) κ ( r , t ) ,
P ( r 0 , ω ) = Κ ( r 0 , ω ) n = 0 ( A δ ( n ω l ) + B δ ( n ω l ± ω p u ) + C δ ( n ω l ± ω p r ) + D δ ( n ω l ± ( ω p u ± ω p r ) ) ) ,

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