Abstract

We have recently harnessed transient absorption, a resonant two-photon process, for ultrahigh resolution photoacoustic microscopy, achieving nearly an order of magnitude improvement in axial resolution. The axial resolution is optically constrained due to the two-photon process unlike traditional photoacoustic microscopy where the axial resolution is inversely proportional to the frequency bandwidth of the detector. As a resonant process, the arrival time of the two photons need not be instantaneous. Systematically recording the signal as a function of the delay between two pulses will result in the measurement of an exponential decay whose time constant is related to the molecular dynamics. This time constant, analogous to the fluorescence lifetime, but encompassing nonradiative decay as well, can be used to differentiate between molecular systems with overlapping absorption spectra. This is frequently the situation for closely related yet distinct molecules such as redox pairs. In order to enable the measure of the exponential decay, we have reconfigured our transient absorption ultrasonic microscopy (TAUM) system to incorporate two laser sources with precisely controlled pulse trains. The system was tested by measuring Rhodamine 6G, an efficient laser dye where the molecular dynamics are dominated by the fluorescence pathway. As expected, the measured exponential time constant or ground state recovery time, 3.3±0.7ns, was similar to the well-known fluorescence lifetime, 4.11±0.05ns. Oxy- and deoxy-hemoglobin are the quintessential pair whose relative concentration is related to the local blood oxygen saturation. We have measured the ground state recovery times of these two species in fully oxygenated and deoxygenated bovine whole blood to be 3.7±0.8ns and 7.9±1.0ns, respectively. Hence, even very closely related pairs of molecules may be differentiated with this technique.

© 2014 Optical Society of America

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References

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2013 (1)

Q. Shao, E. Morgounova, C. Jiang, J. Choi, J. Bischof, and S. Ashkenazi, J. Biomed. Opt. 18, 076019 (2013).
[CrossRef]

2012 (1)

L. V. Wang and S. Hu, Science 335, 1458 (2012).
[CrossRef]

2010 (2)

2009 (1)

2008 (2)

S. Ashkenazi, S. W. Huang, T. Horvath, Y. E. L. Koo, and R. Kopelman, J. Biomed. Opt. 13, 034023 (2008).
[CrossRef]

K. Maslov, H. F. Zhang, S. Hu, and L. V. Wang, Opt. Lett. 33, 929 (2008).
[CrossRef]

2007 (1)

D. Fu, T. Ye, T. E. Matthews, G. Yurtsever, and W. S. Warren, J. Biomed. Opt. 12, 054004 (2007).
[CrossRef]

2006 (1)

2001 (1)

Q. S. Hanley, V. Subramaniam, D. J. Arndt-Jovin, and T. M. Jovin, Cytometry 43, 248 (2001).
[CrossRef]

1982 (1)

R. F. Kubin and A. N. Fletcher, J. Lumin. 27, 455 (1982).
[CrossRef]

1961 (1)

K. Dalziel and J. R. P. O’brien, Biochem. J. 78, 236 (1961).

Applegate, B. E.

R. L. Shelton and B. E. Applegate, Biomed. Opt. Express 1, 676 (2010).
[CrossRef]

B. E. Applegate and J. A. Izatt, Opt. Express 14, 9142 (2006).
[CrossRef]

R. L. Shelton, S. P. Mattison, and B. E. Applegate, “Volumetric imaging of erythrocytes using label-free multiphoton photoacoustic microscopy,” J. Biophoton., doi:10.1002/jbio.201300059 (2013).
[CrossRef]

Arndt-Jovin, D. J.

Q. S. Hanley, V. Subramaniam, D. J. Arndt-Jovin, and T. M. Jovin, Cytometry 43, 248 (2001).
[CrossRef]

Ashkenazi, S.

Q. Shao, E. Morgounova, C. Jiang, J. Choi, J. Bischof, and S. Ashkenazi, J. Biomed. Opt. 18, 076019 (2013).
[CrossRef]

S. Ashkenazi, J. Biomed. Opt. 15, 040501 (2010).
[CrossRef]

S. W. Huang, J. F. Eary, C. X. Jia, L. Y. Huang, S. Ashkenazi, and M. O’Donnell, Opt. Lett. 34, 2393 (2009).
[CrossRef]

S. Ashkenazi, S. W. Huang, T. Horvath, Y. E. L. Koo, and R. Kopelman, J. Biomed. Opt. 13, 034023 (2008).
[CrossRef]

Bischof, J.

Q. Shao, E. Morgounova, C. Jiang, J. Choi, J. Bischof, and S. Ashkenazi, J. Biomed. Opt. 18, 076019 (2013).
[CrossRef]

Choi, J.

Q. Shao, E. Morgounova, C. Jiang, J. Choi, J. Bischof, and S. Ashkenazi, J. Biomed. Opt. 18, 076019 (2013).
[CrossRef]

Dalziel, K.

K. Dalziel and J. R. P. O’brien, Biochem. J. 78, 236 (1961).

Eary, J. F.

Fletcher, A. N.

R. F. Kubin and A. N. Fletcher, J. Lumin. 27, 455 (1982).
[CrossRef]

Fu, D.

D. Fu, T. Ye, T. E. Matthews, G. Yurtsever, and W. S. Warren, J. Biomed. Opt. 12, 054004 (2007).
[CrossRef]

Hanley, Q. S.

Q. S. Hanley, V. Subramaniam, D. J. Arndt-Jovin, and T. M. Jovin, Cytometry 43, 248 (2001).
[CrossRef]

Horvath, T.

S. Ashkenazi, S. W. Huang, T. Horvath, Y. E. L. Koo, and R. Kopelman, J. Biomed. Opt. 13, 034023 (2008).
[CrossRef]

Hu, S.

Huang, L. Y.

Huang, S. W.

S. W. Huang, J. F. Eary, C. X. Jia, L. Y. Huang, S. Ashkenazi, and M. O’Donnell, Opt. Lett. 34, 2393 (2009).
[CrossRef]

S. Ashkenazi, S. W. Huang, T. Horvath, Y. E. L. Koo, and R. Kopelman, J. Biomed. Opt. 13, 034023 (2008).
[CrossRef]

Izatt, J. A.

Jia, C. X.

Jiang, C.

Q. Shao, E. Morgounova, C. Jiang, J. Choi, J. Bischof, and S. Ashkenazi, J. Biomed. Opt. 18, 076019 (2013).
[CrossRef]

Jovin, T. M.

Q. S. Hanley, V. Subramaniam, D. J. Arndt-Jovin, and T. M. Jovin, Cytometry 43, 248 (2001).
[CrossRef]

Koo, Y. E. L.

S. Ashkenazi, S. W. Huang, T. Horvath, Y. E. L. Koo, and R. Kopelman, J. Biomed. Opt. 13, 034023 (2008).
[CrossRef]

Kopelman, R.

S. Ashkenazi, S. W. Huang, T. Horvath, Y. E. L. Koo, and R. Kopelman, J. Biomed. Opt. 13, 034023 (2008).
[CrossRef]

Kubin, R. F.

R. F. Kubin and A. N. Fletcher, J. Lumin. 27, 455 (1982).
[CrossRef]

Maslov, K.

Matthews, T. E.

D. Fu, T. Ye, T. E. Matthews, G. Yurtsever, and W. S. Warren, J. Biomed. Opt. 12, 054004 (2007).
[CrossRef]

Mattison, S. P.

R. L. Shelton, S. P. Mattison, and B. E. Applegate, “Volumetric imaging of erythrocytes using label-free multiphoton photoacoustic microscopy,” J. Biophoton., doi:10.1002/jbio.201300059 (2013).
[CrossRef]

Morgounova, E.

Q. Shao, E. Morgounova, C. Jiang, J. Choi, J. Bischof, and S. Ashkenazi, J. Biomed. Opt. 18, 076019 (2013).
[CrossRef]

O’brien, J. R. P.

K. Dalziel and J. R. P. O’brien, Biochem. J. 78, 236 (1961).

O’Donnell, M.

Shao, Q.

Q. Shao, E. Morgounova, C. Jiang, J. Choi, J. Bischof, and S. Ashkenazi, J. Biomed. Opt. 18, 076019 (2013).
[CrossRef]

Shelton, R. L.

R. L. Shelton and B. E. Applegate, Biomed. Opt. Express 1, 676 (2010).
[CrossRef]

R. L. Shelton, S. P. Mattison, and B. E. Applegate, “Volumetric imaging of erythrocytes using label-free multiphoton photoacoustic microscopy,” J. Biophoton., doi:10.1002/jbio.201300059 (2013).
[CrossRef]

Subramaniam, V.

Q. S. Hanley, V. Subramaniam, D. J. Arndt-Jovin, and T. M. Jovin, Cytometry 43, 248 (2001).
[CrossRef]

Wang, L. V.

Warren, W. S.

D. Fu, T. Ye, T. E. Matthews, G. Yurtsever, and W. S. Warren, J. Biomed. Opt. 12, 054004 (2007).
[CrossRef]

Ye, T.

D. Fu, T. Ye, T. E. Matthews, G. Yurtsever, and W. S. Warren, J. Biomed. Opt. 12, 054004 (2007).
[CrossRef]

Yurtsever, G.

D. Fu, T. Ye, T. E. Matthews, G. Yurtsever, and W. S. Warren, J. Biomed. Opt. 12, 054004 (2007).
[CrossRef]

Zhang, H. F.

Biochem. J. (1)

K. Dalziel and J. R. P. O’brien, Biochem. J. 78, 236 (1961).

Biomed. Opt. Express (1)

Cytometry (1)

Q. S. Hanley, V. Subramaniam, D. J. Arndt-Jovin, and T. M. Jovin, Cytometry 43, 248 (2001).
[CrossRef]

J. Biomed. Opt. (4)

S. Ashkenazi, S. W. Huang, T. Horvath, Y. E. L. Koo, and R. Kopelman, J. Biomed. Opt. 13, 034023 (2008).
[CrossRef]

S. Ashkenazi, J. Biomed. Opt. 15, 040501 (2010).
[CrossRef]

Q. Shao, E. Morgounova, C. Jiang, J. Choi, J. Bischof, and S. Ashkenazi, J. Biomed. Opt. 18, 076019 (2013).
[CrossRef]

D. Fu, T. Ye, T. E. Matthews, G. Yurtsever, and W. S. Warren, J. Biomed. Opt. 12, 054004 (2007).
[CrossRef]

J. Lumin. (1)

R. F. Kubin and A. N. Fletcher, J. Lumin. 27, 455 (1982).
[CrossRef]

Opt. Express (1)

Opt. Lett. (2)

Science (1)

L. V. Wang and S. Hu, Science 335, 1458 (2012).
[CrossRef]

Other (1)

R. L. Shelton, S. P. Mattison, and B. E. Applegate, “Volumetric imaging of erythrocytes using label-free multiphoton photoacoustic microscopy,” J. Biophoton., doi:10.1002/jbio.201300059 (2013).
[CrossRef]

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Figures (3)

Fig. 1.
Fig. 1.

Schematic of the TAUM system modified for ground state recovery measurements. A, Isometric view of the modified TAUM microscope. B, Top-down view of the microscope with components labeled. BS, 50 / 50 beam splitter; DFC, dual-frequency optical chopper; G, galvanometer scanning mirror pair; SL, scan lens, TL, tube lens; MH, microscope head.

Fig. 2.
Fig. 2.

Ground state recovery time plots measured using TAUM. A, Rhodamine 6G recovery time data with a single exponential decay plotted with a decay constant of 1 / 3.5 ns 1 . B, Oxygenated and deoxygenated blood recovery time data with single exponential decays plotted with decay constants of 1 / 3.5 and 1 / 6.5 ns 1 , respectively. C, Histogram showing the distribution of measured ground state recovery times in R6G ( N = 44 ), oxygenated blood ( N = 50 ), and deoxygenated blood ( N = 44 ). The histogram shows a clear distinction between oxygenated and deoxygenated states of whole blood.

Fig. 3.
Fig. 3.

Film strip panel of TAUM images of a 100 μm capillary tube filled with R6G and imaged at varying interpulse delay values. The drop in signal crosses the 1 / e value between panels 2 and 3, which is consistent with the measured recovery time for R6G reported above. Scalebar = 40 μm .

Equations (1)

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Δ p ( r , t ) = σ λ p u F p u h c σ N 1 0 F p r κ ( r , t ) Σ i exp ( t d τ i ) ,

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