Abstract

Optical absorption provides essential biological functional information but cannot be sensed by mainstream optical microscopy technologies directly, which detect fluorescence or scattering and may require undesirable labeling. Here we developed in vivo subwavelength-resolution photoacoustic microscopy (SW-PAM) that provides exquisitely high optical-absorption contrast due to nonfluorescent, or fluorescent, endogenous pigments. Having approached the ultimate diffraction-limited optical resolution, SW-PAM can resolve subcellular organelles. Vasculature and early-stage melanoma were imaged with 12:1 and 17:1 contrasts, respectively, without labeling. SW-PAM along with the scaled-up macroscopy, as the only technology that measures the same contrast origin over such a wide length scale, can potentially accelerate translation from microscopic research to clinical practice.

© 2010 Optical Society of America

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2010

C. Kim, C. Favazza, and L. V. Wang, Chem. Rev. 110, 2756 (2010).
[CrossRef] [PubMed]

M. P. Fronheiser, S. A. Ermilov, H. Brecht, A. Conjusteau, R. Su, K. Mehta, and A. A. Oraevsky, J. Biomed. Opt. 15, 021305 (2010).
[CrossRef] [PubMed]

2009

W. Verkruysse, L. O. Svaasand, and W. Franco, J. Biomed. Opt. 14, 014005 (2009).
[CrossRef] [PubMed]

L. V. Wang, Nat. Photonics 3, 503 (2009).
[CrossRef]

C. Li and L. V. Wang, Phys. Med. Biol. 54, R59 (2009).
[CrossRef] [PubMed]

J. Laufer, E. Zhang, G. Raivich, and P. Beard, Appl. Opt. 48, D299 (2009).
[CrossRef] [PubMed]

2008

K. Maslov, H. F. Zhang, S. Hu, and L. V. Wang, Opt. Lett. 33, 929 (2008).
[CrossRef] [PubMed]

L. V. Wang, IEEE J. Sel. Top. Quantum Electron. 14, 171 (2008).
[CrossRef]

K. H. Song, E. W. Stein, J. A. Margenthaler, and L. V. Wang, J. Biomed. Opt. 13, 054033 (2008).
[CrossRef] [PubMed]

2007

L. Li, R. J. Zemp, G. Lungu, G. Stoica, and L. V. Wang, J. Biomed. Opt. 12, 020504 (2007).
[CrossRef] [PubMed]

V. Gray-Schopher, C. Wellbrock, and R. Marais, Nature 445, 851 (2007).
[CrossRef]

2006

H. F. Zhang, K. Maslov, G. Stoica, and L. V. Wang, Nat. Biotech. 24, 848 (2006).
[CrossRef]

2003

D. M. McDonald and P. L. Choyke, Nat. Med. 9, 713 (2003).
[CrossRef] [PubMed]

X. Wang, Y. Pang, G. Ku, X. Xie, G. Stoica, and L. V. Wang, Nat. Biotech. 21, 803 (2003).
[CrossRef]

1990

K. Kietzmann, D. Lubach, and H.-J. Heeren, Lab. Animal 24, 321 (1990).
[CrossRef]

Beard, P.

Brecht, H.

M. P. Fronheiser, S. A. Ermilov, H. Brecht, A. Conjusteau, R. Su, K. Mehta, and A. A. Oraevsky, J. Biomed. Opt. 15, 021305 (2010).
[CrossRef] [PubMed]

Choyke, P. L.

D. M. McDonald and P. L. Choyke, Nat. Med. 9, 713 (2003).
[CrossRef] [PubMed]

Conjusteau, A.

M. P. Fronheiser, S. A. Ermilov, H. Brecht, A. Conjusteau, R. Su, K. Mehta, and A. A. Oraevsky, J. Biomed. Opt. 15, 021305 (2010).
[CrossRef] [PubMed]

Ermilov, S. A.

M. P. Fronheiser, S. A. Ermilov, H. Brecht, A. Conjusteau, R. Su, K. Mehta, and A. A. Oraevsky, J. Biomed. Opt. 15, 021305 (2010).
[CrossRef] [PubMed]

Favazza, C.

C. Kim, C. Favazza, and L. V. Wang, Chem. Rev. 110, 2756 (2010).
[CrossRef] [PubMed]

Franco, W.

W. Verkruysse, L. O. Svaasand, and W. Franco, J. Biomed. Opt. 14, 014005 (2009).
[CrossRef] [PubMed]

Fronheiser, M. P.

M. P. Fronheiser, S. A. Ermilov, H. Brecht, A. Conjusteau, R. Su, K. Mehta, and A. A. Oraevsky, J. Biomed. Opt. 15, 021305 (2010).
[CrossRef] [PubMed]

Gray-Schopher, V.

V. Gray-Schopher, C. Wellbrock, and R. Marais, Nature 445, 851 (2007).
[CrossRef]

Heeren, H.-J.

K. Kietzmann, D. Lubach, and H.-J. Heeren, Lab. Animal 24, 321 (1990).
[CrossRef]

Hu, S.

Kietzmann, K.

K. Kietzmann, D. Lubach, and H.-J. Heeren, Lab. Animal 24, 321 (1990).
[CrossRef]

Kim, C.

C. Kim, C. Favazza, and L. V. Wang, Chem. Rev. 110, 2756 (2010).
[CrossRef] [PubMed]

Ku, G.

X. Wang, Y. Pang, G. Ku, X. Xie, G. Stoica, and L. V. Wang, Nat. Biotech. 21, 803 (2003).
[CrossRef]

Laufer, J.

Li, C.

C. Li and L. V. Wang, Phys. Med. Biol. 54, R59 (2009).
[CrossRef] [PubMed]

Li, L.

L. Li, R. J. Zemp, G. Lungu, G. Stoica, and L. V. Wang, J. Biomed. Opt. 12, 020504 (2007).
[CrossRef] [PubMed]

Lubach, D.

K. Kietzmann, D. Lubach, and H.-J. Heeren, Lab. Animal 24, 321 (1990).
[CrossRef]

Lungu, G.

L. Li, R. J. Zemp, G. Lungu, G. Stoica, and L. V. Wang, J. Biomed. Opt. 12, 020504 (2007).
[CrossRef] [PubMed]

Marais, R.

V. Gray-Schopher, C. Wellbrock, and R. Marais, Nature 445, 851 (2007).
[CrossRef]

Margenthaler, J. A.

K. H. Song, E. W. Stein, J. A. Margenthaler, and L. V. Wang, J. Biomed. Opt. 13, 054033 (2008).
[CrossRef] [PubMed]

Maslov, K.

K. Maslov, H. F. Zhang, S. Hu, and L. V. Wang, Opt. Lett. 33, 929 (2008).
[CrossRef] [PubMed]

H. F. Zhang, K. Maslov, G. Stoica, and L. V. Wang, Nat. Biotech. 24, 848 (2006).
[CrossRef]

McDonald, D. M.

D. M. McDonald and P. L. Choyke, Nat. Med. 9, 713 (2003).
[CrossRef] [PubMed]

Mehta, K.

M. P. Fronheiser, S. A. Ermilov, H. Brecht, A. Conjusteau, R. Su, K. Mehta, and A. A. Oraevsky, J. Biomed. Opt. 15, 021305 (2010).
[CrossRef] [PubMed]

Oraevsky, A. A.

M. P. Fronheiser, S. A. Ermilov, H. Brecht, A. Conjusteau, R. Su, K. Mehta, and A. A. Oraevsky, J. Biomed. Opt. 15, 021305 (2010).
[CrossRef] [PubMed]

Pang, Y.

X. Wang, Y. Pang, G. Ku, X. Xie, G. Stoica, and L. V. Wang, Nat. Biotech. 21, 803 (2003).
[CrossRef]

Raivich, G.

Song, K. H.

K. H. Song, E. W. Stein, J. A. Margenthaler, and L. V. Wang, J. Biomed. Opt. 13, 054033 (2008).
[CrossRef] [PubMed]

Stein, E. W.

K. H. Song, E. W. Stein, J. A. Margenthaler, and L. V. Wang, J. Biomed. Opt. 13, 054033 (2008).
[CrossRef] [PubMed]

Stoica, G.

L. Li, R. J. Zemp, G. Lungu, G. Stoica, and L. V. Wang, J. Biomed. Opt. 12, 020504 (2007).
[CrossRef] [PubMed]

H. F. Zhang, K. Maslov, G. Stoica, and L. V. Wang, Nat. Biotech. 24, 848 (2006).
[CrossRef]

X. Wang, Y. Pang, G. Ku, X. Xie, G. Stoica, and L. V. Wang, Nat. Biotech. 21, 803 (2003).
[CrossRef]

Su, R.

M. P. Fronheiser, S. A. Ermilov, H. Brecht, A. Conjusteau, R. Su, K. Mehta, and A. A. Oraevsky, J. Biomed. Opt. 15, 021305 (2010).
[CrossRef] [PubMed]

Svaasand, L. O.

W. Verkruysse, L. O. Svaasand, and W. Franco, J. Biomed. Opt. 14, 014005 (2009).
[CrossRef] [PubMed]

Verkruysse, W.

W. Verkruysse, L. O. Svaasand, and W. Franco, J. Biomed. Opt. 14, 014005 (2009).
[CrossRef] [PubMed]

Wang, L. V.

C. Kim, C. Favazza, and L. V. Wang, Chem. Rev. 110, 2756 (2010).
[CrossRef] [PubMed]

C. Li and L. V. Wang, Phys. Med. Biol. 54, R59 (2009).
[CrossRef] [PubMed]

L. V. Wang, Nat. Photonics 3, 503 (2009).
[CrossRef]

K. H. Song, E. W. Stein, J. A. Margenthaler, and L. V. Wang, J. Biomed. Opt. 13, 054033 (2008).
[CrossRef] [PubMed]

L. V. Wang, IEEE J. Sel. Top. Quantum Electron. 14, 171 (2008).
[CrossRef]

K. Maslov, H. F. Zhang, S. Hu, and L. V. Wang, Opt. Lett. 33, 929 (2008).
[CrossRef] [PubMed]

L. Li, R. J. Zemp, G. Lungu, G. Stoica, and L. V. Wang, J. Biomed. Opt. 12, 020504 (2007).
[CrossRef] [PubMed]

H. F. Zhang, K. Maslov, G. Stoica, and L. V. Wang, Nat. Biotech. 24, 848 (2006).
[CrossRef]

X. Wang, Y. Pang, G. Ku, X. Xie, G. Stoica, and L. V. Wang, Nat. Biotech. 21, 803 (2003).
[CrossRef]

L. V. Wang and H. Wu, Biomedical Optics: Principles and Imaging (Wiley, 2007).

Wang, X.

X. Wang, Y. Pang, G. Ku, X. Xie, G. Stoica, and L. V. Wang, Nat. Biotech. 21, 803 (2003).
[CrossRef]

Wellbrock, C.

V. Gray-Schopher, C. Wellbrock, and R. Marais, Nature 445, 851 (2007).
[CrossRef]

Wu, H.

L. V. Wang and H. Wu, Biomedical Optics: Principles and Imaging (Wiley, 2007).

Xie, X.

X. Wang, Y. Pang, G. Ku, X. Xie, G. Stoica, and L. V. Wang, Nat. Biotech. 21, 803 (2003).
[CrossRef]

Zemp, R. J.

L. Li, R. J. Zemp, G. Lungu, G. Stoica, and L. V. Wang, J. Biomed. Opt. 12, 020504 (2007).
[CrossRef] [PubMed]

Zhang, E.

Zhang, H. F.

K. Maslov, H. F. Zhang, S. Hu, and L. V. Wang, Opt. Lett. 33, 929 (2008).
[CrossRef] [PubMed]

H. F. Zhang, K. Maslov, G. Stoica, and L. V. Wang, Nat. Biotech. 24, 848 (2006).
[CrossRef]

Appl. Opt.

Chem. Rev.

C. Kim, C. Favazza, and L. V. Wang, Chem. Rev. 110, 2756 (2010).
[CrossRef] [PubMed]

IEEE J. Sel. Top. Quantum Electron.

L. V. Wang, IEEE J. Sel. Top. Quantum Electron. 14, 171 (2008).
[CrossRef]

J. Biomed. Opt.

W. Verkruysse, L. O. Svaasand, and W. Franco, J. Biomed. Opt. 14, 014005 (2009).
[CrossRef] [PubMed]

M. P. Fronheiser, S. A. Ermilov, H. Brecht, A. Conjusteau, R. Su, K. Mehta, and A. A. Oraevsky, J. Biomed. Opt. 15, 021305 (2010).
[CrossRef] [PubMed]

L. Li, R. J. Zemp, G. Lungu, G. Stoica, and L. V. Wang, J. Biomed. Opt. 12, 020504 (2007).
[CrossRef] [PubMed]

K. H. Song, E. W. Stein, J. A. Margenthaler, and L. V. Wang, J. Biomed. Opt. 13, 054033 (2008).
[CrossRef] [PubMed]

Lab. Animal

K. Kietzmann, D. Lubach, and H.-J. Heeren, Lab. Animal 24, 321 (1990).
[CrossRef]

Nat. Biotech.

X. Wang, Y. Pang, G. Ku, X. Xie, G. Stoica, and L. V. Wang, Nat. Biotech. 21, 803 (2003).
[CrossRef]

H. F. Zhang, K. Maslov, G. Stoica, and L. V. Wang, Nat. Biotech. 24, 848 (2006).
[CrossRef]

Nat. Med.

D. M. McDonald and P. L. Choyke, Nat. Med. 9, 713 (2003).
[CrossRef] [PubMed]

Nat. Photonics

L. V. Wang, Nat. Photonics 3, 503 (2009).
[CrossRef]

Nature

V. Gray-Schopher, C. Wellbrock, and R. Marais, Nature 445, 851 (2007).
[CrossRef]

Opt. Lett.

Phys. Med. Biol.

C. Li and L. V. Wang, Phys. Med. Biol. 54, R59 (2009).
[CrossRef] [PubMed]

Other

L. V. Wang and H. Wu, Biomedical Optics: Principles and Imaging (Wiley, 2007).

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Figures (4)

Fig. 1
Fig. 1

SW-PAM: (a) schematic diagram, (b) close-up diagram showing the confocal structure of the optical objective and the ultrasonic transducer, and (c) point spread function of the system to measure the transverse spatial resolution (red circle, the averaged pixel value and blue line, the theoretical Bessel-form function).

Fig. 2
Fig. 2

Ex vivo images of cells. (a) Melanoma cells grown on a cover glass and fixed. From left to right: PAM image, OM image (0.55 NA), and a composite of the PAM image and the fluorescence OM image of the stained nuclei (blue). In the PAM images, the strong signals come mainly from melanin, and the white dots are melanosomes: CN, cell nucleus. (b) PAM and OM (1.0 NA) images of red blood cells. The strong signals in the PAM image come mainly from hemoglobin.

Fig. 3
Fig. 3

PAM images of a black mouse ear, showing the distribution of melanin. (a) Image focusing at 10 μm deep (the close-up image indicates melanosomes). (b) Image focusing at 30 μm deep: SG, sebaceous gland.

Fig. 4
Fig. 4

Monitoring of melanoma growing on a nude mouse ear. (a) PAM image of blood vessels in the ear acquired before injection of melanoma cells. (b) PAM image where in vivo red blood cells (RBCs) can be identified (the close-up image indicates the biconcave structure of RBCs). (c) PAM image of blood vessels and melanoma taken four days after injecting melanoma cells. The melanoma is extracted by taking the difference of the two PAM images [(a) and (c)] and is plotted in gray: MT, melanoma tumor. (d) OM image (0.057 NA) of the same area in (a) and (c).

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