Abstract

Photoacoustic microscopy (PAM) is a hybrid imaging modality that combines optical illumination with ultrasonic detection to achieve absorption contrast imaging of endogenous and exogenous chromophores. Optical resolution PAM achieves high lateral-resolution by tightly focusing the excitation light; however the axial resolution is still dependent upon the bandwidth of the ultrasonic transducer. As a result, PAM images have highly asymmetric voxels with submicron lateral resolution and axial resolution typically limited to tens of microns. We have previously reported on a resonant multiphoton approach to PAM called transient absorption ultrasonic microscopy (TAUM), which enables high axial resolution by frequency encoding the photoacoustic signal at the overlap of a pump and a probe beam. This approach enables photoacoustic imaging with subcellular resolution on par with other multiphoton microscopy techniques. Here, we report on an innovation that enables TAUM imaging with a much less sophisticated optical system than previously reported. If we allow the time delay between the pump and probe to collapse to zero, the pump and probe optical paths can be combined. An amplitude modulator in the single beam path is sufficient to encode the TAUM signal at the second harmonic of the modulation frequency. The resulting system is essentially a standard optical resolution PAM system that incorporates an amplitude modulator and utilizes a Fourier post processing algorithm to improve the axial resolution by approximately an order of magnitude. A prototype system based on this approach has been assembled and tested on fixed bovine erythrocytes.

© 2014 Optical Society of America

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  1. K. Maslov, G. Stoica, and L. V. Wang, Opt. Lett. 30, 625 (2005).
    [CrossRef]
  2. C. Zhang, Y.-J. Cheng, J. Chen, S. Wickline, and L. V. Wang, J. Biomed. Opt. 17, 060506 (2012).
    [CrossRef]
  3. K. Maslov, H. F. Zhang, S. Hu, and L. V. Wang, Opt. Lett. 33, 929 (2008).
    [CrossRef]
  4. R. L. Shelton, S. P. Mattison, and B. E. Applegate, J. Biophoton. (2013).
    [CrossRef]
  5. R. L. Shelton, S. P. Mattison, and B. E. Applegate, Opt. Lett. 39, 3102 (2014).
    [CrossRef]
  6. R. L. Shelton and B. E. Applegate, Biomed. Opt. Express 1, 676 (2010).
    [CrossRef]
  7. R. L. Shelton and B. E. Applegate, IEEE Trans. Biomed. Eng. 57, 1835 (2010).
    [CrossRef]
  8. H. F. Zhang, K. Maslov, G. Stoica, and L. V. Wang, Nat. Biotechnol. 24, 848 (2006).
    [CrossRef]

2014 (1)

2012 (1)

C. Zhang, Y.-J. Cheng, J. Chen, S. Wickline, and L. V. Wang, J. Biomed. Opt. 17, 060506 (2012).
[CrossRef]

2010 (2)

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

R. L. Shelton and B. E. Applegate, IEEE Trans. Biomed. Eng. 57, 1835 (2010).
[CrossRef]

2008 (1)

2006 (1)

H. F. Zhang, K. Maslov, G. Stoica, and L. V. Wang, Nat. Biotechnol. 24, 848 (2006).
[CrossRef]

2005 (1)

Applegate, B. E.

R. L. Shelton, S. P. Mattison, and B. E. Applegate, Opt. Lett. 39, 3102 (2014).
[CrossRef]

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

R. L. Shelton and B. E. Applegate, IEEE Trans. Biomed. Eng. 57, 1835 (2010).
[CrossRef]

R. L. Shelton, S. P. Mattison, and B. E. Applegate, J. Biophoton. (2013).
[CrossRef]

Chen, J.

C. Zhang, Y.-J. Cheng, J. Chen, S. Wickline, and L. V. Wang, J. Biomed. Opt. 17, 060506 (2012).
[CrossRef]

Cheng, Y.-J.

C. Zhang, Y.-J. Cheng, J. Chen, S. Wickline, and L. V. Wang, J. Biomed. Opt. 17, 060506 (2012).
[CrossRef]

Hu, S.

Maslov, K.

Mattison, S. P.

R. L. Shelton, S. P. Mattison, and B. E. Applegate, Opt. Lett. 39, 3102 (2014).
[CrossRef]

R. L. Shelton, S. P. Mattison, and B. E. Applegate, J. Biophoton. (2013).
[CrossRef]

Shelton, R. L.

R. L. Shelton, S. P. Mattison, and B. E. Applegate, Opt. Lett. 39, 3102 (2014).
[CrossRef]

R. L. Shelton and B. E. Applegate, IEEE Trans. Biomed. Eng. 57, 1835 (2010).
[CrossRef]

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

R. L. Shelton, S. P. Mattison, and B. E. Applegate, J. Biophoton. (2013).
[CrossRef]

Stoica, G.

H. F. Zhang, K. Maslov, G. Stoica, and L. V. Wang, Nat. Biotechnol. 24, 848 (2006).
[CrossRef]

K. Maslov, G. Stoica, and L. V. Wang, Opt. Lett. 30, 625 (2005).
[CrossRef]

Wang, L. V.

C. Zhang, Y.-J. Cheng, J. Chen, S. Wickline, and L. V. Wang, J. Biomed. Opt. 17, 060506 (2012).
[CrossRef]

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

H. F. Zhang, K. Maslov, G. Stoica, and L. V. Wang, Nat. Biotechnol. 24, 848 (2006).
[CrossRef]

K. Maslov, G. Stoica, and L. V. Wang, Opt. Lett. 30, 625 (2005).
[CrossRef]

Wickline, S.

C. Zhang, Y.-J. Cheng, J. Chen, S. Wickline, and L. V. Wang, J. Biomed. Opt. 17, 060506 (2012).
[CrossRef]

Zhang, C.

C. Zhang, Y.-J. Cheng, J. Chen, S. Wickline, and L. V. Wang, J. Biomed. Opt. 17, 060506 (2012).
[CrossRef]

Zhang, H. F.

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

H. F. Zhang, K. Maslov, G. Stoica, and L. V. Wang, Nat. Biotechnol. 24, 848 (2006).
[CrossRef]

Biomed. Opt. Express (1)

IEEE Trans. Biomed. Eng. (1)

R. L. Shelton and B. E. Applegate, IEEE Trans. Biomed. Eng. 57, 1835 (2010).
[CrossRef]

J. Biomed. Opt. (1)

C. Zhang, Y.-J. Cheng, J. Chen, S. Wickline, and L. V. Wang, J. Biomed. Opt. 17, 060506 (2012).
[CrossRef]

Nat. Biotechnol. (1)

H. F. Zhang, K. Maslov, G. Stoica, and L. V. Wang, Nat. Biotechnol. 24, 848 (2006).
[CrossRef]

Opt. Lett. (3)

Other (1)

R. L. Shelton, S. P. Mattison, and B. E. Applegate, J. Biophoton. (2013).
[CrossRef]

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Figures (4)

Fig. 1.
Fig. 1.

Frequency response of TAUM system when ωrep is 50 kHz, ωpu and ωp are 2 kHz, and ωpr is 1.42 kHz. The modulation frequencies of the beam are shifted by the carrier frequency generated during digitization of discretely acquired photoacoustic signals. The dotted blue line shows the response of the system with two beam paths, while the solid red lines represent the signal in the simplified design of a single optical pathway.

Fig. 2.
Fig. 2.

(a) Schematic of the updated TAUM system, where OC is an optical chopper, G is an xy galvanometer scanning pair, SL and TL are a relay lens pair, and MH is the microscope head. (b) Inset view of the microscope head. Obj is the objective, UT is an ultrasonic transducer, and W is a tank for water immersion of the transducer objective pair. Without the optical chopper, this is simply the schematic of an off-axis PAM system.

Fig. 3.
Fig. 3.

(a) Axial scan through fixed erythrocytes on a coverslip captured using TAUM with a 5 MHz ultrasonic transducer. The left side of the figure shows two overlapping erythrocytes. The vertical scale bar is 10 um. (b) A single depth scan through the center of an erythrocyte, the red line is a Gaussian fit of the axial line (FWHM=1μm).

Fig. 4.
Fig. 4.

Volumetric rendering of erythrocytes fixed on a coverslip captured using TAUM with a 5 MHz ultrasonic transducer. Field of view is 60 by 60 by 30 μm.

Equations (6)

Equations on this page are rendered with MathJax. Learn more.

Fpr=Fpr,0[1(μa+Δμa/2+Δμa/2cos(ωput))l],
Δμa=nμa,netd/τnμa,blnetd/τn,
Fpr(t)=Fpr,0Δμa/2cos(ωput)l.
Fpr(t)=Fpr,0Δμa/4cos((ωpu±ωpr)t)l.
Δμa=nμa,nμa,bl
Fpr(t)=Fpr,0Δμa/4cos(2ωpt)l.

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