Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Protocol
  • Published:

In vivo imaging of subcutaneous structures using functional photoacoustic microscopy

Abstract

Functional photoacoustic microscopy (fPAM) is a hybrid technology that permits noninvasive imaging of the optical absorption contrast in subcutaneous biological tissues. fPAM uses a focused ultrasonic transducer to detect high-frequency photoacoustic (PA) signals. Volumetric images of biological tissues can be formed by two-dimensional raster scanning, and functional parameters can be further extracted from spectral measurements. fPAM is safe and applicable to animals as well as humans. This protocol provides guidelines for parameter selection, system alignment, imaging operation, laser safety and data processing for in vivo fPAM. It currently takes 100 min to carry out this protocol, including 50 min for data acquisition using a 10-Hz pulse-repetition-rate laser system. The data acquisition time, however, can be significantly reduced by using a laser system with a higher pulse repetition rate.

This is a preview of subscription content, access via your institution

Access options

Buy this article

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Instrument of fPAM.
Figure 2: Image acquisition by fPAM.
Figure 3
Figure 4: B-scan image of the cross-section of a carbon fiber (diameter: 6 μm) immersed in an optically scattering medium.

Similar content being viewed by others

References

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

    Article  Google Scholar 

  2. Wang, X. et al. Noninvasive laser-induced photoacoustic tomography for structural and functional imaging of the brain in vivo. Nat. Biotechnol. 21, 803–806 (2003).

    Article  CAS  Google Scholar 

  3. Hoelen, C.G.A., de Mul, F.F.M., Pongers, R. & Dekker, A. Three-dimensional photoacoustic imaging of blood vessels in tissue. Opt. Lett. 23, 648–650 (1998).

    Article  CAS  Google Scholar 

  4. Oraevsky, A.A. & Karabutov, A.A. Optoacoustic Tomography. in Biomedical Photonics Handbook, Vol. PM125 (ed. Vo-Dinh, T.) (CRC Press, Boca Raton, Florida, 2003).

    Google Scholar 

  5. Kruger, R.A., Liu, P., Fang, Y.R. & Appledorn, C.R. Photoacoustic ultrasound (PAUS)—reconstruction tomography. Med. Phys. 22, 1605–1609 (1995).

    Article  CAS  Google Scholar 

  6. Bell, A.G. On the production and reproduction of sound by light. Am. J. Sci. 20, 305–324 (1880).

    Article  Google Scholar 

  7. Diebold, G.J., Khan, M.I. & Park, S.M. Photoacoustic “signature” of particulate matter: optical production of acoustic monopole radiation. Science 250, 101–104 (1990).

    Article  CAS  Google Scholar 

  8. Tam, A.C. Applications of photoacoustic sensing techniques. Rev. Mod. Phys. 58, 381–431 (1986).

    Article  CAS  Google Scholar 

  9. Oleary, M.A., Boas, D.A., Chance, B. & Yodh, A.G. Experimental images of heterogeneous turbid media by frequency-domain diffuse-photon tomography. Opt. Lett. 20, 426–428 (1995).

    Article  CAS  Google Scholar 

  10. Boas, D.A. et al. Imaging the body with diffuse optical tomography. IEEE Signal Processing Mag. 18, 57–75 (2001).

    Article  Google Scholar 

  11. Duck, F.A. Physical Properties of Tissue (Academy Press, London, 1990).

    Google Scholar 

  12. Maslov, K., Stoica, G. & Wang, L.V. In vivo dark-field reflection-mode photoacoustic microscopy. Opt. Lett. 30, 625–627 (2005).

    Article  Google Scholar 

  13. Zhang, H.F., Maslov, K., Stoica, G. & Wang, L.V. Functional photoacoustic microscopy for high-resolution and noninvasive in vivo imaging. Nat. Biotechnol. 24, 848–851 (2006).

    Article  CAS  Google Scholar 

  14. Zhang, H.F., Maslov, K., Li, M.-L., Stoica, G. & Wang, L.V. In vivo volumetric imaging of subcutaneous microvasculature by photoacoustic microscopy. Opt. Express. 14, 9317–9323 (2006).

    Article  Google Scholar 

  15. Zhang, H.F., Maslov, K., Sivaramakrishnan, M., Stoica, G. & Wang, L.V. Imaging of hemoglobin oxygen saturation variations in single vessels in vivo using photoacoustic microscopy. Appl. Phys. Lett. 90, 052901 (2007).

    Article  Google Scholar 

  16. Oh, J.-T., Li, M.-L., Zhang, H.F., Maslov, K., Stoica, G. & Wang, L.V. Three-dimensional imaging of skin melanoma in vivo by dual-wavelength photoacoustic microscopy. J. Biomed. Opt. 11, 034032 (2006).

    Article  Google Scholar 

  17. Zhang, H.F., Maslov, K., Stoica, G. & Wang, L.V. Imaging acute thermal burns by photoacoustic microscopy. J. Biomed. Opt. 11, 054033 (2006).

    Article  Google Scholar 

  18. Li, L., Zemp, R.J., Lungu, G., Stoica, G. & Wang, L.V. Imaging of gene expression in vivo with photoacoustic tomography. Proc. SPIE 6086, 62–67 (2006).

    Google Scholar 

  19. Maslov, K., Sivaramakrishnan, M., Zhang, H.F., Stoica, G. & Wang, L.V. Technical considerations in quantitative blood oxygenation measurement using photoacoustic microscopy in vivo . Proc. SPIE 6086, 215–225 (2006).

    Google Scholar 

  20. Briggs, G.A.D. Acoustic Microscopy (Clarendon, Oxford, 1992).

    Google Scholar 

  21. Laser Institute of America, American National Standard for Safe Use of Lasers ANSI Z136.1-2000, (American National Standards Institute Inc., New York, NY, 2000).

  22. Li, M.-L., Zhang, H.F., Maslov, K., Stoica, G. & Wang, L.V. Improved in vivo photoacoustic microscopy based on a virtual-detector concept. Opt. Lett. 31, 474–476 (2006).

    Article  Google Scholar 

  23. Brown, D.G. & Riederer, S.J. Contrast-to-noise ratio in maximum intensity projection images. Magn. Reson. Med. 23, 130–137 (1992).

    Article  CAS  Google Scholar 

  24. Anderson, C.M., Saloner, D., Tsuruda, J.S., Shapeero, L.G. & Lee, R.E. Artifacts in maximum-intensity-projection display of MR angiograms. Am. J. Roentgenol. 154, 623–629 (1990).

    Article  CAS  Google Scholar 

  25. Laufer, J., Deply, D., Elwell, C. & Beard, P. Quantitative spatially resolved measurement of tissue chromophore concentrations using photoacoustic spectroscopy: application to the measurement of blood oxygenation and haemoglobin concentration. Phys. Med. Biol. 52, 141–168 (2007).

    Article  CAS  Google Scholar 

  26. Wilson, B.C. & Jacques, S.L. Optical reflectance and transmittance of tissues: principles and applications. IEEE J. Quant. Electron. 26, 2186–2199 (1990).

    Article  Google Scholar 

Download references

Acknowledgements

We thank G. Stoica, O. Craciun, J. Oh, G. Ku, M.-L. Li, X. Xie, G. Lungu, R. Zemp and M. Sivaramakrishnan for experimental assistance. This work was sponsored by grants from the National Institutes of Health (R01 EB000712 and R01 NS46214 to L.V.W.).

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Lihong V Wang.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Zhang, H., Maslov, K. & Wang, L. In vivo imaging of subcutaneous structures using functional photoacoustic microscopy. Nat Protoc 2, 797–804 (2007). https://doi.org/10.1038/nprot.2007.108

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nprot.2007.108

This article is cited by

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing