Functional photoacoustic microscopy for high-resolution and noninvasive in vivo imaging


Although optical absorption is strongly associated with the physiological status of biological tissue, existing high-resolution optical imaging modalities, including confocal microscopy1,2, two-photon microscopy3,4 and optical coherence tomography5, do not sense optical absorption directly. Furthermore, optical scattering prevents these methods from imaging deeper than 1 mm below the tissue surface. Here we report functional photoacoustic microscopy (fPAM), which provides multiwavelength imaging of optical absorption and permits high spatial resolution beyond this depth limit with a ratio of maximum imaging depth to depth resolution greater than 100. Reflection mode, rather than orthogonal or transmission mode, is adopted because it is applicable to more anatomical sites than the others. fPAM is demonstrated with in vivo imaging of angiogenesis, melanoma, hemoglobin oxygen saturation (sO2) of single vessels in animals and total hemoglobin concentration in humans.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Figure 1: Experimental fPAM system.
Figure 2: In vivo imaging of a subcutaneously inoculated B16-melanoma in an immunocompromised nude mouse using fPAM at 584 nm and 764 nm.
Figure 3: Functional imaging of sO2 by fPAM in vivo in a 150-g Sprague-Dawley rat.
Figure 4: In vivo imaging of the total hemoglobin concentration in subcutaneous vasculature of the palm of a human hand by fPAM at 584 nm.


  1. 1

    Wilson, T. & Sheppard, C. Theory and Practice of Scanning Optical Microscopy (Academic Press, London, 1984).

    Google Scholar 

  2. 2

    Sipkins, D.A. et al. In vivo imaging of specialized bone marrow endothelial microdomains for tumour engraftment. Nature 435, 969–973 (2005).

    CAS  Article  Google Scholar 

  3. 3

    Denk, W., Strickler, J.H. & Webb, W.W. Two-photon laser scanning fluorescence microscopy. Science 248, 73–76 (1990).

    CAS  Article  Google Scholar 

  4. 4

    So, P.T.C., Dong, C.Y., Masters, B.R. & Berland, K.M. Two-photon excitation fluorescence microcopy. Annu. Rev. Biomed. Eng. 2, 399–429 (2000).

    CAS  Article  Google Scholar 

  5. 5

    Huang, D. et al. Optical coherence tomography. Science 254, 1178–1181 (1991).

    CAS  Article  Google Scholar 

  6. 6

    Sun, T. & Diebold, G.J. Generation of ultrasonic waves from a layered photoacoustic source. Nature 355, 806–808 (1992).

    CAS  Article  Google Scholar 

  7. 7

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

  8. 8

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

    CAS  Article  Google Scholar 

  9. 9

    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).

    CAS  Article  Google Scholar 

  10. 10

    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 

  11. 11

    Briggs, G.A.D. Acoustic Microscopy, p. 27 (Clarendon, Oxford, 1992).

    Google Scholar 

  12. 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. 13

    Carmeliet, P. & Jain, R.K. Angiogenesis in cancer and other diseases. Nature 407, 249–257 (2000).

    CAS  Article  Google Scholar 

  14. 14

    Jobsis, F.F. Noninvasive, infrared monitoring of cerebral and myocardial oxygen sufficiency and circulatory parameters. Science 198, 1264–1267 (1977).

    CAS  Article  Google Scholar 

  15. 15

    Chance, B. et al. Comparison of time-resolved and -unresolved measurements of deoxyhemoglobin in brain. Proc. Natl. Acad. Sci. USA 85, 4971–4975 (1988).

    CAS  Article  Google Scholar 

  16. 16

    Tsao, M.U., Sethna, S.S., Sloan, C.H. & Wyngarden, L.J. Spectrophotometric determination of the oxygen saturation of whole blood. J. Biol. Chem. 217, 479–488 (1955).

    CAS  PubMed  Google Scholar 

  17. 17

    Vanzetta, I. & Grinvald, A. Increased cortical oxidative metabolism due to sensory stimulation: implications for functional brain imaging. Science 286, 1555–1558 (1999).

    CAS  Article  Google Scholar 

  18. 18

    Levasseur, J., Wei, E., Raper, A., Kontos, H. & Patterson, J. Detailed description of a cranial window technique for acute and chronic experiments. Stroke 6, 308–317 (1975).

    CAS  Article  Google Scholar 

  19. 19

    Hall, D.G. & Stoica, G. Characterization of brain and bone-metastasizing clones selected from an ethylnitrosourea-induced rat mammary carcinoma. Clin. Exp. Metastasis 12, 283–295 (1994).

    CAS  Article  Google Scholar 

  20. 20

    Ambach, G. & Palkovits, M. Blood supply of the rat hypothalamus I. nucleus supraopticus. Acta Morphol. Acad. Sci. Hung. 22, 291–310 (1974).

    CAS  PubMed  Google Scholar 

Download references


We thank O. Craciun, J. Oh, G. Ku, M.L. Li and G. Lungu for experimental assistance. This work was sponsored by National Institutes of Health grants R01 EB000712 and R01 NS46214.

Author information



Corresponding author

Correspondence to Lihong V Wang.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

An illustrative example showing the penetration depth of fPAM in tissue. (PDF 69 kb)

Supplementary Fig. 2

Images of vasculature in a rat acquired in vivo by fPAM at the isosbestic optical wavelength of 584 nm before, two days post, and five days post subcutaneous inoculation of BR7C5 tumor cells. (PDF 76 kb)

Supplementary Table 1

Comparison among the modern high-resolution microscopic imaging techniques, whose depth-to-resolution ratios are all greater than 100. (PDF 15 kb)

Supplementary Video 1

(1.7 MB) Movie for the composite volumetric visualization of a melanoma in the skin acquired in vivo. (AVI 1774 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Zhang, H., Maslov, K., Stoica, G. et al. Functional photoacoustic microscopy for high-resolution and noninvasive in vivo imaging. Nat Biotechnol 24, 848–851 (2006).

Download citation

Further reading


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