Going deeper than microscopy: the optical imaging frontier in biology


Optical microscopy has been a fundamental tool of biological discovery for more than three centuries, but its in vivo tissue imaging ability has been restricted by light scattering to superficial investigations, even when confocal or multiphoton methods are used. Recent advances in optical and optoacoustic (photoacoustic) imaging now allow imaging at depths and resolutions unprecedented for optical methods. These abilities are increasingly important to understand the dynamic interactions of cellular processes at different systems levels, a major challenge of postgenome biology. This Review discusses promising photonic methods that have the ability to visualize cellular and subcellular components in tissues across different penetration scales. The methods are classified into microscopic, mesoscopic and macroscopic approaches, according to the tissue depth at which they operate. Key characteristics associated with different imaging implementations are described and the potential of these technologies in biological applications is discussed.

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Figure 1: Simplified metrics of photon propagation in tissue.
Figure 2: The penetration depth and resolution of modern photonic imaging techniques is depicted.
Figure 3: Principle of operation of SPIM.
Figure 4: Hybrid optical tomography using priors.
Figure 5: Principle of operation of volumetric optoacoustic tomography.
Figure 6: Examples of mesoscopic imaging.


  1. 1

    Beauvoit, B., Evans, S.M., Jenkins, T.W., Miller, E.E. & Chance, B. Correlation between the light-scattering and the mitochondrial content of normal-tissues and transplantable rodent tumors. Anal. Biochem. 226, 167–174 (1995).

    CAS  PubMed  Google Scholar 

  2. 2

    Webb, R.H. Theoretical basis of confocal microscopy. Methods Enzymol. 307, 3–20 (1999).A concise description of confocal microscopy technology and performance metrics.

    CAS  PubMed  Google Scholar 

  3. 3

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

    CAS  Google Scholar 

  4. 4

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

    CAS  Article  Google Scholar 

  5. 5

    Tsien, R.Y. Building and breeding molecules to spy on cells and tumors. FEBS Lett. 579, 927–932 (2005).A concise review of fluorescence reporters and probes for in vivo imaging.

    CAS  PubMed  Google Scholar 

  6. 6

    Weissleder, R. & Pittet, M. Imaging in the era of molecular oncology. Nature 452, 580–589 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  7. 7

    Stephens, D.J. & Allan, V.J. Light microscopy techniques for live cell imaging. Science 300, 82–86 (2003).

    CAS  Google Scholar 

  8. 8

    Zipfel, W.R., Williams, R.M. & Webb, W.W. Nonlinear magic: multiphoton microscopy in the biosciences. Nat. Biotechnol. 21, 1368–1376 (2003).

    Google Scholar 

  9. 9

    Jain, R.K. Normalization of tumor vasculature: An emerging concept in antiangiogenic therapy. Science 307, 58–62 (2005).

    CAS  Google Scholar 

  10. 10

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

    CAS  PubMed  PubMed Central  Google Scholar 

  11. 11

    Walls, J.R., Sled, J.G., Sharpe, J. & Henkelman, R.M. Resolution improvement in emission optical projection tomography. Phys. Med. Biol. 52, 2775–2790 (2007).

    PubMed  Google Scholar 

  12. 12

    Alanentalo, T. et al. High-resolution three-dimensional imaging of islet-infiltrate interactions based on optical projection tomography assessments of the intact adult mouse pancreas. J. Biomed. Opt. 13, 054070 (2008).

    PubMed  Google Scholar 

  13. 13

    Kerwin, J. et al. 3 dimensional modelling of early human brain development using optical projection tomography. BMC Neurosci. 5, 27 (2004).

    PubMed  PubMed Central  Google Scholar 

  14. 14

    Boot, M.J. et al. In vitro whole-organ imaging: 4D quantification of growing mouse limb buds. Nat. Methods 5, 609–612 (2008).

    CAS  PubMed  Google Scholar 

  15. 15

    Huisken, J., Swoger, J., Del Bene, F., Wittbrodt, J. & Stelzer, E.H.K. Optical sectioning deep inside live embryos by selective plane illumination microscopy. Science 305, 1007–1009 (2004).An introduction of selective plane illumination microscopy.

    CAS  Google Scholar 

  16. 16

    Huisken, J. & Stainier, D.Y. Even fluorescence excitation by multidirectional selective plane illumination microscopy (mSPIM). Opt. Lett. 32, 2608–2610 (2007).

    PubMed  Google Scholar 

  17. 17

    Verveer, P.J. et al. High-resolution three-dimensional imaging of large specimens with light sheet-based microscopy. Nat. Methods 4, 311–313 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  18. 18

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

    CAS  PubMed  PubMed Central  Google Scholar 

  19. 19

    Ermolayev, V. et al. Ultramicroscopy reveals axonal transport impairments in cortical motor neurons at prion disease. Biophys. J. 96, 3390–3398 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  20. 20

    Andreev, V.G., Karabutov, A.A. & Oraevsky, A.A. Detection of ultrawide-band ultrasound pulses in optoacoustic tomography. IEEE Trans. Ultrason. Ferroelectr. Freq. Control 50, 1383–1390 (2003).

    PubMed  Google Scholar 

  21. 21

    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).A demonstration of blood-vessel imaging using optoacoustic (photoacoustic) tomography.

    CAS  PubMed  Google Scholar 

  22. 22

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

    CAS  PubMed  PubMed Central  Google Scholar 

  23. 23

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

    CAS  PubMed  Google Scholar 

  24. 24

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

    CAS  PubMed  Google Scholar 

  25. 25

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

    PubMed  Google Scholar 

  26. 26

    Maslov, K., Zhang, H., Hu, S. & Wang, L. Optical-resolution photoacoustic microscopy for in vivo imaging of single capillaries. Opt. Lett. 33, 929–931 (2008).

    PubMed  Google Scholar 

  27. 27

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

    CAS  PubMed  PubMed Central  Google Scholar 

  28. 28

    Tearney, G.J. et al. In vivo endoscopic optical biopsy with optical coherence tomography. Science 276, 2037–2039 (1997).

    CAS  PubMed  Google Scholar 

  29. 29

    Bredfeldt, J.S., Vinegoni, C., Marks, D.L. & Boppart, S.A. Molecularly sensitive optical coherence tomography. Opt. Lett. 30, 495–497 (2005).

    CAS  PubMed  Google Scholar 

  30. 30

    Skala, M.C., Crow, M.J., Wax, A. & Izatt, J.A. Photothermal optical coherence tomography of epidermal growth factor receptor in live cells using immunotargeted gold nanospheres. Nano Lett. 8, 3461–3467 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  31. 31

    Sarunic, M.V., Applegate, B.E. & Izatt, J.A. Spectral domain second-harmonic optical coherence tomography. Opt. Lett. 30, 2391–2393 (2005).

    PubMed  Google Scholar 

  32. 32

    Drexler, W. & Fujimoto, J.G. State-of-the-art retinal optical coherence tomography. Prog. Retin. Eye Res. 27, 45–88 (2008).

    PubMed  Google Scholar 

  33. 33

    Chamberland, D. et al. Photoacoustic tomography of joints aided by an Etanercept-conjugated gold nanoparticle contrast agent—an ex vivo preliminary rat study. Nanotechnology 19, 095101 (2008).

    PubMed  Google Scholar 

  34. 34

    Tolentino, T.P. et al. Measuring diffusion and binding kinetics by contact area FRAP. Biophys. J. 95, 920–930 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  35. 35

    McNally, J.G. Quantitative FRAP in analysis of molecular binding dynamics in vivo. Methods Cell Biol. 85, 329–351 (2008).

    CAS  PubMed  Google Scholar 

  36. 36

    Mavrakis, M., Rikhy, R., Lilly, M. & Lippincott-Schwartz, J. Fluorescence imaging techniques for studying Drosophila embryo development. Curr. Protoc. Cell Biol. 4, 18 (2008).

    PubMed  Google Scholar 

  37. 37

    Sprague, B.L., Pego, R.L., Stavreva, D.A. & McNally, J.G. Analysis of binding reactions by fluorescence recovery after photobleaching. Biophys. J. 86, 3473–3495 (2004).

    CAS  PubMed  PubMed Central  Google Scholar 

  38. 38

    Provenzano, P.P., Eliceiri, K.W. & Keely, P.J. Multiphoton microscopy and fluorescence lifetime imaging microscopy (FLIM) to monitor metastasis and the tumor microenvironment. Clin. Exp. Metastasis 26, 357–370 (2008).

    PubMed  Google Scholar 

  39. 39

    Hallworth, R., Currall, B., Nichols, M.G., Wu, X. & Zuo, J. Studying inner ear protein-protein interactions using FRET and FLIM. Brain Res. 1091, 122–131 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  40. 40

    Chen, Y., Mills, J.D. & Periasamy, A. Protein localization in living cells and tissues using FRET and FLIM. Differentiation 71, 528–541 (2003).

    CAS  PubMed  PubMed Central  Google Scholar 

  41. 41

    Tadrous, P.J. Methods for imaging the structure and function of living tissues and cells: 2. fluorescence lifetime imaging. J. Pathol. 191, 229–234 (2000).

    CAS  PubMed  Google Scholar 

  42. 42

    Bastiaens, P.I. & Squire, A. Fluorescence lifetime imaging microscopy: spatial resolution of biochemical processes in the cell. Trends Cell Biol. 9, 48–52 (1999).

    CAS  PubMed  Google Scholar 

  43. 43

    Rodriguez, L.G., Lockett, S.J. & Holtom, G.R. Coherent anti-stokes Raman scattering microscopy: a biological review. Cytometry A 69, 779–791 (2006).

    PubMed  Google Scholar 

  44. 44

    Rinia, H.A., Wurpel, G.W. & Muller, M. Measuring molecular order and orientation using coherent anti-stokes Raman scattering microscopy. Methods Mol. Biol. 400, 45–61 (2007).

    CAS  PubMed  Google Scholar 

  45. 45

    Cheng, J.X. Coherent anti-Stokes Raman scattering microscopy. Appl. Spectrosc. 61, 197–208 (2007).

    PubMed  PubMed Central  Google Scholar 

  46. 46

    Imanishi, Y., Lodowski, K.H. & Koutalos, Y. Two-photon microscopy: shedding light on the chemistry of vision. Biochemistry 46, 9674–9684 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  47. 47

    Botvinick, E.L. & Shah, J.V. Laser-based measurements in cell biology. Methods Cell Biol. 82, 81–109 (2007).

    CAS  PubMed  Google Scholar 

  48. 48

    Campagnola, P.J. & Loew, L.M. Second-harmonic imaging microscopy for visualizing biomolecular arrays in cells, tissues and organisms. Nat. Biotechnol. 21, 1356–1360 (2003).

    CAS  PubMed  PubMed Central  Google Scholar 

  49. 49

    Patterson, M.S., Chance, B. & Wilson, B.C. Time resolved reflectance and transmittance for the noninvasive measurement of tissue optical-properties. Appl. Opt. 28, 2331–2336 (1989).

    CAS  PubMed  Google Scholar 

  50. 50

    Arridge, S.R. Optical tomography in medical imaging. Inverse Probl. 15, R41–R93 (1999).

    Google Scholar 

  51. 51

    Shu, X. et al. Mammalian expression of infrared fluorescent proteins engineered from a bacterial phytochrome. Science 324, 804–807 (2009).

    PubMed  PubMed Central  Google Scholar 

  52. 52

    Shashkov, E., Everts, M., Galanzha, E. & Zharov, V. Quantum dots as multimodal photoacoustic and photothermal contrast agents. Nano Lett. 8, 3953–3958 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  53. 53

    De La Zerda et al. Carbon nano-tubes as photoacoustic molecular imaging agents in living mice. Nat. Nanotechnol. 3, 557–562 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  54. 54

    Weissleder, R. & Ntziachristos, V. Shedding light onto live molecular targets. Nat. Med. 9, 123–128 (2003).

    CAS  PubMed  Google Scholar 

  55. 55

    Ntziachristos, V., Tung, C.H., Bremer, C. & Weissleder, R. Fluorescence molecular tomography resolves protease activity in vivo. Nat. Med. 8, 757–760 (2002).An introduction of fluorescence molecular tomography.

    CAS  PubMed  Google Scholar 

  56. 56

    Ntziachristos, V. & Weissleder, R. Experimental three-dimensional fluorescence reconstruction of diffuse media using a normalized Born approximation. Opt. Lett. 26, 893–895 (2001).

    CAS  PubMed  Google Scholar 

  57. 57

    Schwaiger, M., Ziegler, S. & Nekolla, S. PET/CT: challenge for nuclear cardiology. J. Nucl. Med. 46, 1664–1678 (2005).

    PubMed  Google Scholar 

  58. 58

    Judenhofer, M.S. et al. Simultaneous PET-MRI: a new approach for functional and morphological imaging. Nat. Med. 14, 459–465 (2008).

    CAS  Google Scholar 

  59. 59

    Barbour, R. et al. MRI-guided optical tomography: prospects and computation for a new imaging method. IEEE Comput. Sci. Eng. 2, 63–77 (1995).

    Google Scholar 

  60. 60

    Ntziachristos, V., Yodh, A.G., Schnall, M. & Chance, B. Concurrent MRI and diffuse optical tomography of breast after indocyanine green enhancement. Proc. Natl. Acad. Sci. USA 97, 2767–2772 (2000).

    CAS  PubMed  Google Scholar 

  61. 61

    Brooksby, B. et al. Imaging breast adipose and fibroglandular tissue molecular signatures by using hybrid MRI-guided near-infrared spectral tomography. Proc. Natl. Acad. Sci. USA 103, 8828–8833 (2006).

    CAS  PubMed  Google Scholar 

  62. 62

    Davis, S.C. et al. Magnetic resonance-coupled fluorescence tomography scanner for molecular imaging of tissue. Rev. Sci. Instrum. 79, 064302 (2008).

    PubMed  PubMed Central  Google Scholar 

  63. 63

    Fang, Q. et al. Combined optical imaging and mammography of the healthy breast: optical contrast derived from breast structure and compression. IEEE Trans. Med. Imaging 28, 30–42 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  64. 64

    Schulz, R. et al. Hybrid system for simultaneous fluorescence and X-ray computed tomography. IEEE Trans. Med. Imaging 29, 465–473 (2010).

    PubMed  Google Scholar 

  65. 65

    Hyde, D. et al. Hybrid FMT-CT imaging of amyloid-beta plaques in a murine Alzheimer's disease model. Neuroimage 44, 1304–1311 (2009).

    PubMed  Google Scholar 

  66. 66

    Lin, Y., Gao, H., Nalcioglu, O. & Gulsen, G. Fluorescence diffuse optical tomography with functional and anatomical a priori information: feasibility study. Phys. Med. Biol. 52, 5569–5585 (2007).

    CAS  PubMed  Google Scholar 

  67. 67

    Guven, M., Yazici, B., Intes, X. & Chance, B. Diffuse optical tomography with a priori anatomical information. Phys. Med. Biol. 50, 2837–2858 (2005).

    PubMed  Google Scholar 

  68. 68

    Hintersteiner, M. et al. In vivo detection of amyloid-beta deposits by near-infrared imaging using an oxazine-derivative probe. Nat. Biotechnol. 23, 577–583 (2005).

    CAS  PubMed  Google Scholar 

  69. 69

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

    PubMed  Google Scholar 

  70. 70

    Rosenthal, A., Razansky, D. & Ntziachristos, V. Fast semi-analytical model-based acoustic inversion for quantitative optoacoustic tomography. IEEE Trans. Med. Imaging 29, 1275–1285 (2010).

    PubMed  Google Scholar 

  71. 71

    Bowen, T. Radiation-induced thermoacoustic soft-tissue imaging. Proc. IEEE Ultrason. Symp. 817–822 (1981).

  72. 72

    Zemp, R.J. et al. Photoacoustic imaging of the microvasculature with a high-frequency ultrasound array transducer. J. Biomed. Opt. 12, 010501 (2007).

    PubMed  Google Scholar 

  73. 73

    Allen, T.J. & Beard, P.C. Pulsed near-infrared laser diode excitation system for biomedical photoacoustic imaging. Opt. Lett. 31, 3462–3464 (2006).

    PubMed  Google Scholar 

  74. 74

    Kolkman, R.G.M. et al. Photoacoustic determination of blood vessel diameter. Phys. Med. Biol. 49, 4745–4756 (2004).

    PubMed  Google Scholar 

  75. 75

    Eghtedari, M. et al. High sensitivity of in vivo detection of gold nanorods using a laser optoacoustic imaging system. Nano Lett. 7, 1914–1918 (2007).

    CAS  PubMed  Google Scholar 

  76. 76

    Cox, B., Arridge, S. & Beard, P. Estimating chromophore distributions from multiwavelength photoacoustic images. J. Opt. Soc. Am. A Opt. Image Sci. Vis. 26, 443–455 (2009).

    CAS  PubMed  Google Scholar 

  77. 77

    Jetzfellner, T., Rozenthal, A., Englmeier, K., Razansky, D. & Ntziachristos, V. Multispectral optoacoustic tomography by means of normalized spectral ratio. Opt. Lett. (in the press).

  78. 78

    Li, M. et al. Simultaneous molecular and hypoxia imaging of brain tumors in vivo using spectroscopic photoacoustic tomography. Proc. IEEE 96, 481–489 (2008).

    CAS  Google Scholar 

  79. 79

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

  80. 80

    Kruger, R.A, Kiser, W. Jr., Reinecke, D., Kruger, G. & Miller, K. Thermoacoustic optical molecular imaging of small animals. Mol. Imaging 2, 113–123 (2003).

    PubMed  Google Scholar 

  81. 81

    Rayavarapu, R. et al. Synthesis and bioconjugation of gold nanoparticles as potential molecular probes for light-based imaging techniques. Int. J. Biomed. Imaging 2007 29817 (2007).

  82. 82

    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).

    PubMed  Google Scholar 

  83. 83

    Galanzha, E. et al. In vivo magnetic enrichment and multiplex photoacoustic detection of circulating tumour cells. Nat. Nanotechnol. 4, 855–860 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  84. 84

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

    CAS  PubMed  Google Scholar 

  85. 85

    Razansky, D. et al. Imaging of mesoscopic targets using selective-plane optoacoustic tomography. Nat. Photonics 3, 412–417 (2009).A demonstration of visualizing optical reporter molecules in vivo using mesoscopic multispectral optoacoustic tomography.

    CAS  Google Scholar 

  86. 86

    Jain, R.K., Munn, L.L. & Fukumura, D. Dissecting tumour pathophysiology using intravital microscopy. Nat. Rev. Cancer 2, 266–276 (2002).A description of in vivo applications of intravital microscopy in cancer.

    CAS  PubMed  PubMed Central  Google Scholar 

  87. 87

    Wang, T.D., Contag, C.H., Mandella, M.J., Chan, N.Y. & Kino, G.S. Confocal fluorescence microscope with dual-axis architecture and biaxial postobjective scanning. J. Biomed. Opt. 9, 735–742 (2004).

    PubMed  PubMed Central  Google Scholar 

  88. 88

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

    CAS  PubMed  PubMed Central  Google Scholar 

  89. 89

    Kleinfeld, D., Mitra, P.P., Helmchen, F. & Denk, W. Fluctuations and stimulus-induced changes in blood flow observed in individual capillaries in layers 2 through 4 of rat neocortex. Proc. Natl. Acad. Sci. USA 95, 15741–15746 (1998).

    CAS  PubMed  Google Scholar 

  90. 90

    Majewska, A.K., Newton, J.R. & Sur, M. Remodeling of synaptic structure in sensory cortical areas in vivo. J. Neurosci. 26, 3021–3029 (2006).

    CAS  PubMed  Google Scholar 

  91. 91

    Germain, R.N. et al. An extended vision for dynamic high-resolution intravital immune imaging. Semin. Immunol. 17, 431–441 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

  92. 92

    Molitoris, B.A. & Sandoval, R.M. Intravital multiphoton microscopy of dynamic renal processes. Am. J. Physiol. Renal Physiol. 288, F1084–F1089 (2005).

    CAS  PubMed  Google Scholar 

  93. 93

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

    CAS  PubMed  Google Scholar 

  94. 94

    Boas, D.A., Oleary, M.A., Chance, B. & Yodh, A.G. Scattering of diffuse photon fensity eaves ny dpherical inhomogeneities within turbid media—analytic solution and applications. Proc. Natl. Acad. Sci. USA 91, 4887–4891 (1994).

    CAS  PubMed  Google Scholar 

  95. 95

    Schotland, J.C. Continuous-wave diffusion imaging. J. Opt. Soc. Am. A Opt. Image Sci. Vis. 14, 275–279 (1997).

    Google Scholar 

  96. 96

    Cheong, W., Prahl, S. & Welch, A. A review of the optical-properties of biological tissues. IEEE J. Quantum Electron. 26, 2166–2185 (1990).

    Google Scholar 

  97. 97

    Beek, J.F., van Staveren, H.J., Posthumus, P., Sterenborg, H.J.C.M. & van Gemert, M.J.C. The optical properties of lung as a function of respiration. Phys. Med. Biol. 42, 2263–2272 (1997).

    CAS  PubMed  Google Scholar 

  98. 98

    Pogue, B.W. et al. Characterization of hemoglobin, water, and NIR scattering in breast tissue: analysis of intersubject variability and menstrual cycle changes. J. Biomed. Opt. 9, 541–552 (2004).

    CAS  PubMed  Google Scholar 

  99. 99

    Niedre, M., Turner, G. & Ntziachristos, V. Time-resolved imaging of optical coefficients through murine chest cavities. J. Biomed. Opt. 11, 064017–064011–064017 (2006).

    Google Scholar 

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I acknowledge investigators and students for their multiple contributions in understanding optical and optoacoustic performance; J. Ripoll for the contribution of Figure 1b; and support from a European Research Council Senior Investigator Award, the German Federal Ministry of Education and Research and the Institute for Biological and Medical Imaging

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Ntziachristos, V. Going deeper than microscopy: the optical imaging frontier in biology. Nat Methods 7, 603–614 (2010). https://doi.org/10.1038/nmeth.1483

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