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Multispectral opto-acoustic tomography of deep-seated fluorescent proteins in vivo


Fluorescent proteins have become essential reporter molecules for studying life at the cellular and sub-cellular level, re-defining the ways in which we investigate biology. However, because of intense light scattering, most organisms and tissues remain inaccessible to current fluorescence microscopy techniques at depths beyond several hundred micrometres. We describe a multispectral opto-acoustic tomography technique capable of high-resolution visualization of fluorescent proteins deep within highly light-scattering living organisms. The method uses multiwavelength illumination over multiple projections combined with selective-plane opto-acoustic detection for artifact-free data collection. Accurate image reconstruction is enabled by making use of wavelength-dependent light propagation models in tissue. By performing whole-body imaging of two biologically important and optically diffuse model organisms, Drosophila melanogaster pupae and adult zebrafish, we demonstrate the facility to resolve tissue-specific expression of eGFP and mCherrry fluorescent proteins for precise morphological and functional observations in vivo.

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Figure 1: Experimental setup of multispectral opto-acoustic tomography.
Figure 2: Multispectral opto-acoustic imaging of tissue-mimicking phantom containing DsRed-expressing HeLa cells.
Figure 3: Imaging of eGFP distribution in Drosophila melanogaster pupa.
Figure 4: Imaging of mCherry distribution in the vertebral column of an adult zebrafish.
Figure 5: Three-dimensional in vivo imaging through the brain of an adult (six-month-old) mCherry-expressing transgenic zebrafish.

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  1. Giepmans, B. N. G., Adams, S. R., Ellisman, M. H. & Tsien, R. Y. The fluorescent toolbox for assessing protein location and function. Science 312, 217–224 (2006).

    Article  ADS  Google Scholar 

  2. Lichtman, J. W. & Conchello, J. A. Fluorescence microscopy. Nature Methods 2, 910–919 (2005).

    Article  Google Scholar 

  3. Conchello J. A. & Lichtman, J. W. Optical sectioning microscopy. Nature Methods 2, 920–931 (2005).

    Article  Google Scholar 

  4. Bahlmann, K. et al. Multifocal multiphoton microscopy (MMM) at a frame rate beyond 600 Hz. Opt. Express 15, 10991–10998 (2007).

    Article  ADS  Google Scholar 

  5. Minsky, M. Microscopy apparatus. US patent 3,013,467 (1961).

  6. Denk, W., Strickler, J. H. & Webb, W. W. 2-photon laser scanning fluorescence microscopy. Science 248, 73–76 (1990).

    Article  ADS  Google Scholar 

  7. Helmchen, F. & Denk, W. Deep tissue two-photon microscopy. Nature Methods 2, 932–940 (2005).

    Article  Google Scholar 

  8. Ntziachristos, V., Ripoll, J., Wang, L. H. V. & Weissleder, R. Looking and listening to light: the evolution of whole-body photonic imaging. Nature Biotechnol. 23, 313–320 (2005).

    Article  Google Scholar 

  9. Hove, J. R. et al. Intracardiac hemodynamics are an essential epigenetic factor for embryonic cardiogenesis. Nature 421, 172–177 (2003).

    Article  ADS  Google Scholar 

  10. Jain, R. K., Munn, L. L. & Fukumura, D. Dissecting tumor pathophysiology using intravital microscopy. Nature Rev. Cancer 2, 266–276 (2002).

    Article  Google Scholar 

  11. Sharpe, J. et al. Optical projection tomography as a tool for 3D microscopy and gene expression studies. Science 296, 541–545 (2002).

    Article  ADS  Google Scholar 

  12. Huisken, J. et al. Optical sectioning deep inside live embryos by selective plane illumination microscopy. Science 305, 1007–1009 (2004).

    Article  ADS  Google Scholar 

  13. Dodt, H. U. et al. Ultramicroscopy: three-dimensional visualization of neuronal networks in the whole mouse brain. Nature Methods 4, 331–336 (2007).

    Article  Google Scholar 

  14. Editorial. Geneticist seeks engineer: must like flies and worms. Nature Methods 4, 463 (2007).

  15. Schroeder, T. Imaging stem-cell-driven regeneration in mammals. Nature 453, 345–351 (2008).

    Article  ADS  Google Scholar 

  16. Gusev, V. E. & Karabutov, A. A. Laser Optoacoustics (American Institute of Physics, 1993).

    Google Scholar 

  17. 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 

  18. Lao, Y., Xing, D., Yang, S. & Xiang, L. Noninvasive photoacoustic imaging of the developing vasculature during early tumor growth. Phys. Med. Biol. 53, 4203–4212 (2008).

    Article  Google Scholar 

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

    Article  Google Scholar 

  20. 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 

  21. Razansky, D., Vinegoni, C. & Ntziachristos, V. Multispectral photoacoustic imaging of fluorochromes in small animals. Opt. Lett. 32, 2891–2893 (2007).

    Article  ADS  Google Scholar 

  22. Li, L., Zemp, R. J., Lungu, G., Stoica, G. & Wang, L. V. Photoacoustic imaging of lacZ gene expression in vivo. J. Biomed. Opt. 12, 020504 (2007).

    Article  ADS  Google Scholar 

  23. De La Zerda, A. et al. Carbon nanotubes as photoacoustic molecular imaging agents in living mice. Nature Nanotechnol. 3, 557–562 (2008).

    Article  ADS  Google Scholar 

  24. Razansky, D., Baeten, J. & Ntziachristos, V. Sensitivity of molecular target detection by multispectral optoacoustic tomography (MSOT). Med. Phys. 36, 2891–2893 (2009).

    Article  Google Scholar 

  25. Vinegoni, C., Pitsouli, C., Razansky, D., Perrimon, N. & Ntziachristos, V. In vivo imaging of Drospophila melanogaster pupae with mesoscopic fluorescence tomography. Nature Methods 5, 45–47 (2008).

    Article  Google Scholar 

  26. Ntziachristos, V., Tung, C.-H., Bremer, C. & Weissleder, R. Fluorescence molecular tomography resolves protease activity in vivo. Nature Med. 8, 757–760 (2002).

  27. Xu, M. & Wang, L. V. Universal back-projection algorithm for photoacoustic computed tomography. Phys. Rev. E 71, 016706 (2005).

    Article  ADS  Google Scholar 

  28. Razansky, D. & Ntziachristos, V. Hybrid photoacoustic fluorescence molecular tomography using finite-element-based inversion. Med. Phys. 34, 4293–4301 (2007).

    Article  Google Scholar 

  29. Cox, B. T., Arridge, S. R., Kostli, K. P. & Beard, P. C. 2D quantitative photoacoustic image reconstruction of absorption distributions in scattering media using a simple iterative method. Appl. Opt. 45, 1866–1875 (2006).

    Article  ADS  Google Scholar 

  30. Jetzfellner, T. et al. Iterative optoacoustic image normalization in non-uniform illumination configurations. Appl. Phys. Lett. 95, (2009) (in the press).

  31. Laufer, J. G., Delpy, D. T., Elwell, C. E. & Beard, P. C. 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  Google Scholar 

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D.R. acknowledges financial support by the Deutsche Forschungsgemeinschaft (DFG) research grant RA 1848/1-1. M.D. is a fellow of the Studienstiftung des deutschen Volkes. R.W.K. is supported by a BioFuture Award Grant (0311889) of the German Ministry for Education and Research (BMBF). We thank R. Jagasia for providing Hela mitodsRed cells.

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Correspondence to Daniel Razansky or Vasilis Ntziachristos.

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

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