Source: http://aoot.osa.org/boe/abstract.cfm?uri=boe-5-12-4235
Timestamp: 2019-04-25 04:26:21+00:00

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We developed a reflection-mode subwavelength-resolution photoacoustic microscopy system capable of imaging optical absorption contrast in vivo. The simultaneous high-resolution and reflection-mode imaging capacity of the system was enabled by delicately configuring a miniature high-frequency ultrasonic transducer tightly under a water-immersion objective with numerical aperture of 1.0. At 532-nm laser illumination, the lateral resolution of the system was measured to be ~320 nm. With this system, subcellular structures of red blood cells and B16 melanoma cells were resolved ex vivo; microvessels, including individual capillaries, in a mouse ear were clearly imaged label-freely in vivo, using the intrinsic optical absorption from hemoglobin. The current study suggests that, the optical-absorption contrast, subwavelength resolution, and reflection-mode ability of the developed photoacoustic microscopy may empower a wide range of biomedical studies for visualizing cellular and/or subcellular structures.
L. V. Wang and S. Hu, “Photoacoustic tomography: in vivo imaging from organelles to organs,” Science 335(6075), 1458–1462 (2012).
S. Hu and L. V. Wang, “Optical-resolution photoacoustic microscopy: auscultation of biological systems at the cellular level,” Biophys. J. 105(4), 841–847 (2013).
C. Zhang, K. Maslov, and L. V. Wang, “Subwavelength-resolution label-free photoacoustic microscopy of optical absorption in vivo,” Opt. Lett. 35(19), 3195–3197 (2010).
J. Chen, R. Lin, H. Wang, J. Meng, H. Zheng, and L. Song, “Blind-deconvolution optical-resolution photoacoustic microscopy in vivo,” Opt. Express 21(6), 7316–7327 (2013).
Y. Yuan, S. Yang, and D. Xing, “Optical-resolution photoacoustic microscopy based on two-dimensional scanning galvanometer,” Appl. Phys. Lett. 100(2), 023702 (2012).
C. Zhang, K. Maslov, S. Hu, R. Chen, Q. Zhou, K. K. Shung, and L. V. Wang, “Reflection-mode submicron-resolution in vivo photoacoustic microscopy,” J. Biomed. Opt. 17(2), 020501 (2012).
Y. Wang, S. Hu, K. Maslov, Y. Zhang, Y. Xia, and L. V. Wang, “In vivo integrated photoacoustic and confocal microscopy of hemoglobin oxygen saturation and oxygen partial pressure,” Opt. Lett. 36(7), 1029–1031 (2011).
S. Oladipupo, S. Hu, J. Kovalski, J. Yao, A. C. Santeford, R. E. Sohn, R. V. Shohet, K. Maslov, L. V. Wang, and J. M. Arbeit, “VEGF is essential for hypoxia-inducible factor-mediated neovascularization but dispensable for endothelial sprouting,” Proc. Natl. Acad. Sci. U.S.A. 108(32), 13264–13269 (2011).
T. P. Matthews, C. Zhang, D. K. Yao, K. Maslov, and L. V. Wang, “Label-free photoacoustic microscopy of peripheral nerves,” J. Biomed. Opt. 19(1), 016004 (2014).
S. Jiao, M. Jiang, J. Hu, A. Fawzi, Q. Zhou, K. K. Shung, C. A. Puliafito, and H. F. Zhang, “Photoacoustic ophthalmoscopy for in vivo retinal imaging,” Opt. Express 18(4), 3967–3972 (2010).
H. Wang, X. Yang, Y. Liu, B. Jiang, and Q. Luo, “Reflection-mode optical-resolution photoacoustic microscopy based on a reflective objective,” Opt. Express 21(20), 24210–24218 (2013).
G. M. Lerman and U. Levy, “Effect of radial polarization and apodization on spot size under tight focusing conditions,” Opt. Express 16(7), 4567–4581 (2008).
W. Gong, K. Si, and C. J. Sheppard, “Optimization of axial resolution in a confocal microscope with D-shaped apertures,” Appl. Opt. 48(20), 3998–4002 (2009).
C. J. Sheppard and A. Choudhury, “Annular pupils, radial polarization, and superresolution,” Appl. Opt. 43(22), 4322–4327 (2004).
W. Song, Q. Wei, T. Liu, D. Kuai, J. M. Burke, S. Jiao, and H. F. Zhang, “Integrating photoacoustic ophthalmoscopy with scanning laser ophthalmoscopy, optical coherence tomography, and fluorescein angiography for a multimodal retinal imaging platform,” J. Biomed. Opt. 17(6), 061206 (2012).
Z. Xie, S. Jiao, H. F. Zhang, and C. A. Puliafito, “Laser-scanning optical-resolution photoacoustic microscopy,” Opt. Lett. 34(12), 1771–1773 (2009).
L. Wang, K. Maslov, and L. V. Wang, “Single-cell label-free photoacoustic flowoxigraphy in vivo,” Proc. Natl. Acad. Sci. U.S.A. 110(15), 5759–5764 (2013).
L. Li, C. Yeh, S. Hu, L. Wang, B. T. Soetikno, R. Chen, Q. Zhou, K. K. Shung, K. I. Maslov, and L. V. Wang, “Fully motorized optical-resolution photoacoustic microscopy,” Opt. Lett. 39(7), 2117–2120 (2014).
R. Weissleder and M. J. Pittet, “Imaging in the era of molecular oncology,” Nature 452(7187), 580–589 (2008).
Fig. 1 (a) Schematic diagram of the reflection-mode subwavelength-resolution photoacoustic microscopy system. (b) Photograph of the configured imaging probe consisting of a high-NA objective and a miniature high-frequency ultrasonic transducer. GM: galvanometer scanner; L1: scan lens; L2: tube lens.
Fig. 2 (a) Theoretical optical diffraction profiles with and without the ultrasonic transducer (UT) under the objective; (b) Subwavelength-resolution PAM image of graphite nanoparticles of a mean diameter of ~80 nm; (c) Lateral resolution of subwavelength-resolution PAM estimated by Gaussian fitting of the photoacoustic amplitude along the red dot-line in Fig. 2(b); (d) Subwavelength-resolution PAM imaging of a copper net with holes of 95 µm in diameter and bars of ~30 μm in width.
Fig. 3 Subwavelength-resolution PAM imaging of individual RBCs and B16 melanoma cells ex vivo. (a) and (b) show the subwavelength-resolution PAM and OM images of RBCs, respectively; (c) Photoacoustic amplitude profile of one RBC along the marked dotted line; (d) and (e) show the subwavelength-resolution PAM and OM images of B16 melanoma cells, respectively.
Fig. 4 In vivo subwavelength-resolution PAM of a mouse ear. (a) Maximum amplitude projection (MAP) subwavelength-resolution PAM image of the mouse ear microvasculature; (b) Close-up of the white dashed square area in Fig. 4(a) where individual RBCs can be identified; (c) Representative cross-sectional images at different depths; (d) Volumetric rendering of the data set (Media 1).

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