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.

Deep in vivo photoacoustic imaging of mammalian tissues using a tyrosinase-based genetic reporter

Subjects

Abstract

Photoacoustic imaging allows absorption-based high-resolution spectroscopic in vivo imaging at a depth beyond that of optical microscopy. Until recently, photoacoustic imaging has largely been restricted to visualizing the vasculature through endogenous haemoglobin contrast, with most non-vascularized tissues remaining invisible unless exogenous contrast agents are administered. Genetically encodable photoacoustic contrast is attractive as it allows selective labelling of cells, permitting studies of, for example, specific genetic expression, cell growth or more complex biological behaviours in vivo. In this study we report a novel photoacoustic imaging scanner and a tyrosinase-based reporter system that causes human cell lines to synthesize the absorbing pigment eumelanin, thus providing strong photoacoustic contrast. Detailed three-dimensional images of xenografts formed of tyrosinase-expressing cells implanted in mice are obtained in vivo to depths approaching 10 mm with a spatial resolution below 100 μm. This scheme is a powerful tool for studying cellular and genetic processes in deep mammalian tissues.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Figure 1: Photoacoustic (PA) scanner for imaging mice in vivo.
Figure 2: Construction and in vitro optical and PA characteristics of Tyr-expressing cells.
Figure 3: In vivo PA images of Tyr-expressing K562 cells after subcutaneous injection into the flank of a nude mouse (λex = 600 nm).
Figure 4: In vivo PA images of Tyr-expressing 293T cells acquired at different times post-innoculation, illustrating cell population growth (λex = 640 nm).
Figure 5: Deep tissue imaging of Tyr-expressing K562 cells (λex = 680 nm).

References

  1. 1

    Darne, C., Lu, Y. & Sevick-Muraca, E. M. Small animal fluorescence and bioluminescence tomography: a review of approaches, algorithms and technology update. Phys. Med. Biol. 59, R1–R64 (2014).

    ADS  Article  Google Scholar 

  2. 2

    Branchini, B. R. et al. Red-emitting luciferases for bioluminescence reporter and imaging applications. Anal. Biochem. 396, 290–297 (2010).

    Article  Google Scholar 

  3. 3

    Jathoul, A. P., Grounds, H., Anderson, J. C. & Pule, M. A. A dual-color far-red to near-infrared firefly luciferin analogue designed for multiparametric bioluminescence imaging. Angew. Chem. Int. Ed. 53, 13059–13063 (2014).

    Article  Google Scholar 

  4. 4

    Ntziachristos, V. Going deeper than microscopy: the optical imaging frontier in biology. Nature Methods 7, 603–614 (2010).

    Article  Google Scholar 

  5. 5

    Beard, P. Biomedical photoacoustic imaging. Interface Focus 1, 602–631 (2011).

    Article  Google Scholar 

  6. 6

    Wang, L. V. Multiscale photoacoustic microscopy and computed tomography. Nature Photon. 3, 503–509 (2009).

    ADS  Article  Google Scholar 

  7. 7

    Ntziachristos, V. & Razansky, D. Molecular imaging by means of multispectral optoacoustic tomography (MSOT). Chem. Rev. 110, 2783–2794 (2010).

    Article  Google Scholar 

  8. 8

    Oraevsky, A. A. & Karabutov, A. A. in Biomedical Photonics Handbook PM125 (ed. Vo-Dinh, T.) Ch. 34, 3401–3434 (CRC Press, 2003).

    Google Scholar 

  9. 9

    Zhang, E. Z., Laufer, J. G., Pedley, R. B. & Beard, P. C. In vivo high-resolution 3D photoacoustic imaging of superficial vascular anatomy. Phys. Med. Biol. 54, 1035–1046 (2009).

    Article  Google Scholar 

  10. 10

    Laufer, J. et al. In vivo preclinical photoacoustic imaging of tumor vasculature development and therapy. J. Biomed. Opt. 17, 056016 (2012).

    ADS  Article  Google Scholar 

  11. 11

    Laufer, J. et al. In vivo photoacoustic imaging of mouse embryos. J. Biomed. Opt. 17, 061220 (2012).

    ADS  Article  Google Scholar 

  12. 12

    Brecht, H. P. et al. Whole-body three-dimensional optoacoustic tomography system for small animals. J. Biomed. Opt. 14, 064007 (2009).

    ADS  Article  Google Scholar 

  13. 13

    Kruger, R. A., Lam, R. B., Reinecke, D. R., Del Rio, S. P. & Doyle, R. P. Photoacoustic angiography of the breast. Med. Phys. 37, 6096–6100 (2010).

    Article  Google Scholar 

  14. 14

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

    Article  Google Scholar 

  15. 15

    De la Zerda, A., Kim, J. W., Galanzha, E. I., Gambhir, S. S. & Zharov, V. P. Advanced contrast nanoagents for photoacoustic molecular imaging, cytometry, blood test and photothermal theranostics. Contrast Media Mol. Imaging 6, 346–369 (2011).

    Article  Google Scholar 

  16. 16

    Luke, G. P., Yeager, D. & Emelianov, S. Y. Biomedical applications of photoacoustic imaging with exogenous contrast agents. Ann. Biomed. Eng. 40, 422–437 (2012).

    Article  Google Scholar 

  17. 17

    Kim, C., Favazza, C. & Wang, L. V. In vivo photoacoustic tomography of chemicals: high-resolution functional and molecular optical imaging at new depths. Chem. Rev. 110, 2756–2782 (2010).

    Article  Google Scholar 

  18. 18

    Shaner, N. C., Steinbach, P. A. & Tsien, R. Y. A guide to choosing fluorescent proteins. Nature Methods 2, 905–909 (2005).

    Article  Google Scholar 

  19. 19

    Razansky, D. et al. Multispectral opto-acoustic tomography of deep-seated fluorescent proteins in vivo. Nature Photon. 3, 412–417 (2009).

    ADS  Article  Google Scholar 

  20. 20

    Laufer, J., Jathoul, A., Pule, M. & Beard, P. In vitro characterization of genetically expressed absorbing proteins using photoacoustic spectroscopy. Biomed. Opt. Express 4, 2477–2490 (2013).

    Article  Google Scholar 

  21. 21

    Filonov, G. S. et al. Deep-tissue photoacoustic tomography of a genetically encoded near-infrared fluorescent probe. Angew. Chem. Int. Ed. 51, 1448–1451 (2012).

    Article  Google Scholar 

  22. 22

    Krumholz, A., Shcherbakova, D. M., Xia, J., Wang, L. V. & Verkhusha, V. V. Multicontrast photoacoustic in vivo imaging using near-infrared fluorescent proteins. Sci. Rep. 4, 3939 (2014).

    ADS  Article  Google Scholar 

  23. 23

    Li, L., Zhang, H. F., Zemp, R. J., Maslov, K. & Wang, L. Simultaneous imaging of a lacZ-marked tumor and microvasculature morphology in vivo by dual-wavelength photoacoustic microscopy. J. Innov. Opt. Health Sci. 1, 207–215 (2008).

    Article  Google Scholar 

  24. 24

    Raper, H. S. The tyrosinase-tyrosine reaction. Biochem. J. 21, 89–96 (1927).

    Article  Google Scholar 

  25. 25

    Paproski, R. J., Forbrich, A. E., Wachowicz, K., Hitt, M. M. & Zemp, R. J. Tyrosinase as a dual reporter gene for both photoacoustic and magnetic resonance imaging. Biomed. Opt. Express 2, 771–780 (2011).

    Article  Google Scholar 

  26. 26

    Krumholz, A. et al. Photoacoustic microscopy of tyrosinase reporter gene in vivo. J. Biomed. Opt. 16, 080503 (2011).

    ADS  Article  Google Scholar 

  27. 27

    Laufer, J. et al. in Proc. SPIE, Photons Plus Ultrasound: Imaging and Sensing 2012 (eds Oraevsky, A. A. & Wang, L. V.) 82230M–82230M-5 (2012).

    Google Scholar 

  28. 28

    Stritzker, J. et al. Vaccinia virus-mediated melanin production allows MR and optoacoustic deep tissue imaging and laser-induced thermotherapy of cancer. Proc. Natl Acad. Sci. USA 110, 3316–3320 (2013).

    ADS  Article  Google Scholar 

  29. 29

    Qin, C. et al. Tyrosinase as a multifunctional reporter gene for photoacoustic/MRI/PET triple modality molecular imaging. Sci. Rep. 3, 1490 (2013).

    Article  Google Scholar 

  30. 30

    Paproski, R. J., Heinmiller, A., Wachowicz, K. & Zemp, R. J. Multi-wavelength photoacoustic imaging of inducible tyrosinase reporter gene expression in xenograft tumors. Sci. Rep. 4, 5329 (2014).

    ADS  Article  Google Scholar 

  31. 31

    Zhang, E., Laufer, J. & Beard, P. Backward-mode multiwavelength photoacoustic scanner using a planar Fabry–Perot polymer film ultrasound sensor for high-resolution three-dimensional imaging of biological tissues. Appl. Opt. 47, 561–577 (2008).

    ADS  Article  Google Scholar 

  32. 32

    Treeby, B. E., Zhang, E. Z. & Cox, B. T. Photoacoustic tomography in absorbing acoustic media using time reversal. Inv. Prob. 26, 115003 (2010).

    MathSciNet  Article  Google Scholar 

  33. 33

    Treeby, B. E. & Cox, B. T. k-Wave: MATLAB toolbox for the simulation and reconstruction of photoacoustic wave fields. J. Biomed. Opt. 15, 021314 (2010).

    ADS  Article  Google Scholar 

  34. 34

    Treeby, B. E. Acoustic attenuation compensation in photoacoustic tomography using time-variant filtering. J. Biomed. Opt. 18, 036008 (2013).

    ADS  Article  Google Scholar 

  35. 35

    Treeby, B. E., Varslot, T. K., Zhang, E. Z., Laufer, J. G. & Beard, P. C. Automatic sound speed selection in photoacoustic image reconstruction using an autofocus approach. J. Biomed. Opt. 16, 090501 (2011).

    ADS  Article  Google Scholar 

  36. 36

    Koestli, K. P., Frenz, M., Bebie, H., Weber, H. P. & Köstli, K. P. Temporal backward projection of optoacoustic pressure transients using Fourier transform methods. Phys. Med. Biol. 46, 1863–1872 (2001).

    Article  Google Scholar 

  37. 37

    British standard safety of laser products (BS EN 60825-1) edn 1.2, August (2001).

  38. 38

    Calvo, P. A., Frank, D. W., Bieler, B. M., Berson, J. F. & Marks, M. S. A cytoplasmic sequence in human tyrosinase defines a second class of di-leucine-based sorting signals for late endosomal and lysosomal delivery. J. Biol. Chem. 274, 12780–12789 (1999).

    Article  Google Scholar 

  39. 39

    Berson, J. F., Harper, D. C., Tenza, D., Raposo, G. & Marks, M. S. Pmel17 initiates premelanosome morphogenesis within multivesicular bodies. Mol. Biol. Cell 12, 3451–3464 (2001).

    Article  Google Scholar 

  40. 40

    Tachibana, M. et al. Ectopic expression of MITF, a gene for Waardenburg syndrome type 2, converts fibroblasts to cells with melanocyte characteristics. Nature Genet. 14, 50–54 (1996).

    Article  Google Scholar 

  41. 41

    Kuzumaki, T., Matsuda, A., Wakamatsu, K., Ito, S. & Ishikawa, K. Eumelanin biosynthesis is regulated by coordinate expression of tyrosinase and tyrosinase-related protein-1 genes. Exp. Cell Res. 207, 33–40 (1993).

    Article  Google Scholar 

  42. 42

    Raposo, G. & Marks, M. S. Melanosomes-dark organelles enlighten endosomal membrane transport. Nature Rev. Mol. Cell Biol. 8, 786–797 (2007).

    Article  Google Scholar 

  43. 43

    Bouchard, B., Fuller, B. B., Vijayasaradhi, S. & Houghton, A. N. Induction of pigmentation in mouse fibroblasts by expression of human tyrosinase cDNA. J. Exp. Med. 169, 2029–2042 (1989).

    Article  Google Scholar 

  44. 44

    Fehse, B. et al. CD34 splice variant: an attractive marker for selection of gene-modified cells. Mol. Ther. 1, 448–456 (2000).

    Article  Google Scholar 

  45. 45

    Shaner, N. C. et al. Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein. Nature Biotechnol. 22, 1567–1572 (2004).

    Article  Google Scholar 

  46. 46

    Wang, L., Jackson, W. C., Steinbach, P. A. & Tsien, R. Y. Evolution of new nonantibody proteins via iterative somatic hypermutation. Proc. Natl Acad. Sci. USA 101, 16745–16749 (2004).

    ADS  Article  Google Scholar 

  47. 47

    Hafler, D. A. et al. Oligoclonal T lymphocytes in the cerebrospinal fluid of patients with multiple sclerosis. J. Exp. Med. 167, 1313–1322 (1988).

    Article  Google Scholar 

Download references

Acknowledgements

This work was funded by the UK Biotechnology Research Council (BBSRC) grant no. BB/I014357/1. Additional funding was provided by the gene-therapy division of the UK NIHR University College London Hospital Biomedical Research Centre. This work was also supported by King's College London and University College London Comprehensive Cancer Imaging Centre, Cancer Research UK and the Engineering and Physical Sciences Research Council (EPSRC), in association with the Medical Research Council and Department of Health, UK, and European Union project FAMOS (FP7 ICT, contract no. 317744). P.B. is funded by an EPSRC Leadership Fellowship and J.L. is funded by an ERC starting grant (281356). The authors thank J. Paterson (UCL Advanced Diagnostics) for assistance with immunohistochemistry, K. Venner for assistance with transmission electron microscopy (TEM) and C. Futter for assistance in interpreting the electron micrographs. H. Dortay (TU Berlin) is thanked for helpful comments on the manuscript and P. Varga (AO Research Institute Davos, Switzerland) for assistance with the use of Amira.

Author information

Affiliations

Authors

Contributions

A.J. carried out molecular cloning, cell preparation, maintenance and analysis, animal work, the design of experiments, in vitro characterizations and in vivo photoacoustic imaging, and assisted with preparation of the manuscript. J.L. undertook the photoacoustic spectroscopy and imaging studies, the reconstruction, processing and analysis of the in vivo images, and assisted with preparation of the manuscript. O.O. contributed to tissue phantom experiments and implemented the cell detection limit study. B.T. and B.C. developed the signal processing, image reconstruction and visualization methods. E.Z. designed and constructed the photoacoustic scanner. P.J. provided cell lines, and helped with in vitro and in vivo imaging and histological analyses. A.P. helped with analysis of cells by flow cytometry and with the general experimental design. RBP carried out the production of virus, and helped with cellular analyses and the use of different iterations of his novel marker gene. T.M. performed immunohistochemistry. M.L. was responsible for invocation of the project, and contributed to experimental planning, motivation, the use of facilities and equipment, experimental focus and editing of the manuscript. R.B. provided cell lines, mice, microscopy and the use of the Home Office Project Licence. M.P. provided gene and vector designs, codon optimization, experimental designs, and directed the overall focus of the work and writing of the manuscript. P.B. directed the photoacoustic imaging component of the project and organized and co-wrote the manuscript.

Corresponding author

Correspondence to Paul Beard.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary information

Supplementary information (PDF 7169 kb)

Supplementary Movie 1

Supplementary Movie 1 (MPG 9698 kb)

Supplementary Movie 2

Supplementary Movie 2 (WMV 15380 kb)

Supplementary Movie 3

Supplementary Movie 3 (MPG 14003 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Jathoul, A., Laufer, J., Ogunlade, O. et al. Deep in vivo photoacoustic imaging of mammalian tissues using a tyrosinase-based genetic reporter. Nature Photon 9, 239–246 (2015). https://doi.org/10.1038/nphoton.2015.22

Download citation

Further reading

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