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Virtual Journal for Biomedical Optics

| EXPLORING THE INTERFACE OF LIGHT AND BIOMEDICINE

  • Editor: Gregory W. Faris
  • Vol. 4, Iss. 11 — Oct. 21, 2009
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Three-dimensional combined photoacoustic and optical coherence microscopy for in vivo microcirculation studies

Li Li, Konstantin Maslov, Geng Ku, and Lihong V. Wang  »View Author Affiliations


Optics Express, Vol. 17, Issue 19, pp. 16450-16455 (2009)
http://dx.doi.org/10.1364/OE.17.016450


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Abstract

Photoacoustic microscopy is predominantly sensitive to optical absorption, while optical coherence tomography relies on optical backscattering. Integrating their complementary contrasts can provide comprehensive information about biological tissue. We have developed a dual-modality microscope that combines the two for studying microcirculation. Three-dimensional imaging of microvasculature and its local environment has been demonstrated at micrometer-order resolution using endogenous contrast in vivo.

© 2009 OSA

As a fast emerging, noninvasive imaging tool, photoacoustic microscopy (PAM) [1

1. H. F. Zhang, K. Maslov, G. Stoica, and L. V. Wang, “Functional photoacoustic microscopy for high-resolution and noninvasive in vivo imaging,” Nat. Biotechnol. 24(7), 848–851 (2006). [CrossRef] [PubMed]

3

3. G. Ku, K. Maslov, L. Li, and L. V. Wang, “Photoacoustic microscopy with 2-µm transverse resolution,” J. Biomed. Opt. under review. [PubMed]

] is currently capable of visualizing three-dimensional biological tissue in vivo at a resolution down to 2 µm. Compared to other optical microscopy technologies, PAM possesses a unique feature that it has a 100% relative sensitivity to optical absorption. A small fractional variation in the optical absorption coefficient causes the same amount of fractional change in the photoacoustic signal [4

4. L. V. Wang, “Tutorial on photoacoustic microscopy and computed tomography,” IEEE J. Sel. Top. Quantum Electron. 14(1), 171–179 (2008). [CrossRef]

]. Many physiologically important molecules, e.g., hemoglobin and melanin, have strong characteristic optical absorption. As a result, PAM is especially appropriate for studying absorbing structures, such as blood micro-vessels [1

1. H. F. Zhang, K. Maslov, G. Stoica, and L. V. Wang, “Functional photoacoustic microscopy for high-resolution and noninvasive in vivo imaging,” Nat. Biotechnol. 24(7), 848–851 (2006). [CrossRef] [PubMed]

3

3. G. Ku, K. Maslov, L. Li, and L. V. Wang, “Photoacoustic microscopy with 2-µm transverse resolution,” J. Biomed. Opt. under review. [PubMed]

] and melanoma [5

5. J. Oh, M. Li, H. F. Zhang, K. Maslov, G. Stoica, and L. V. Wang, “Three-dimensional imaging of skin melanoma in vivo by dual-wavelength photoacoustic microscopy,” J. Biomed. Opt. 11(3), 034032 (2006). [CrossRef]

]. However, without data about the surrounding tissues, the information carried in PAM images cannot be fully interpreted.

Recently, there exists an increasing trend in biomedical research to integrate multiple complementary microscopic imaging modalities into a single platform to provide comprehensive insights into physiology [6

6. C. Vinegoni, T. Ralston, W. Tan, W. Luo, D. L. Marks, and S. A. Boppart, “Integrated structural and functional optical imaging combining spectral-domain optical coherence and multiphoton microscopy,” Appl. Phys. Lett. 88(5), 053901 (2006). [CrossRef]

,7

7. H. T. Chen, H. F. Wang, M. N. Slipchenko, Y. K. Jung, Y. Z. Shi, J. B. Zhu, K. K. Buhman, and J. X. Cheng, “A multimodal platform for nonlinear optical microscopy and microspectroscopy,” Opt. Express 17(3), 1282–1290 (2009), http://www.opticsinfobase.org/abstract.cfm?URI=oe-17-3-1282. [CrossRef] [PubMed]

]. Previously, ultrasonic imaging has been suggested to complement photoacoustic imaging in deeper tissue [8

8. S. Y. Emelianov, S. R. Aglyamov, A. B. Karpiouk, S. Mallidi, S. Park, S. Sethuraman, J. Shah, R. W. Smalling, J. M. Rubin, and W. G. Scott, “Synergy and applications of combined ultrasound, elasticity, and photoacoustic imaging,” 2006 IEEE Ultrasonics Symposium, 405–415 (2006).

,9

9. J. J. Niederhauser, M. Jaeger, R. Lemor, P. Weber, and M. Frenz, “Combined ultrasound and optoacoustic system for real-time high-contrast vascular imaging in vivo,” IEEE Trans. Med. Imaging 24(4), 436–440 (2005). [CrossRef] [PubMed]

]. However, it is difficult to resolve laterally beyond 10 µm while maintaining a reasonable penetration depth (~1 mm) in tissue using the ultrasonic method alone. In this letter, we demonstrate that a pure optical technique, optical coherence tomography (OCT), is a proper alternative. State-of-the-art OCT allows real-time, µm-resolution imaging of tissue up to a couple of millimeters deep in vivo [10

10. B. Cense, N. A. Nassif, T. C. Chen, M. C. Pierce, S. H. Yun, B. H. Park, B. E. Bouma, G. J. Tearney, and J. F. de Boer, “Ultrahigh-resolution high-speed retinal imaging using spectral-domain optical coherence tomography,” Opt. Express 12(11), 2435–2447 (2004), http://www.opticsinfobase.org/oe/abstract.cfm?URI=oe-12-11-2435. [CrossRef] [PubMed]

]. The combination of PAM and OCT has several advantages. First, it provides completely complementary imaging contrasts. OCT exploits the scattering properties of tissue, while PAM is predominantly sensitive to optical absorption. Second, when designed properly, OCT and PAM can have comparable imaging depths and spatial resolutions, which are suitable for microscopic imaging.

A particularly useful application of the combined photoacoustic and optical-coherence microscopy is in studying microcirculation. Microcirculation is vital to sustain human health, and disturbed microcirculation is involved in most pathological processes [11

11. B. Fagrell and M. Intaglietta, “Microcirculation: its significance in clinical and molecular medicine,” J. Intern. Med. 241(5), 349–362 (1997). [CrossRef] [PubMed]

]. Existing microscopic imaging techniques, although they have greatly facilitated our understanding of morphology, function and regulation of normal and pathological microcirculation, suffer from several limitations. Intravital microscopy [12

12. R. K. Jain, L. L. Munn, and D. Fukumura, “Dissecting tumour pathophysiology using intravital microscopy,” Nat. Rev. Cancer 2(4), 266–276 (2002). [CrossRef] [PubMed]

] and orthogonal polarization spectral imaging [13

13. R. G. Nadeau, W. Groner, J. W. Winkelman, A. G. Harris, C. Ince, G. J. Bouma, and K. Messmer, “Orthogonal polarization spectral imaging: a new method for study of the microcirculation,” Nat. Med. 5(10), 1209–1212 (1999). [CrossRef] [PubMed]

] lack depth resolution, and thus are unable to show three-dimensional morphology of microvasculature. Confocal and multiphoton microscopy [14

14. J. Seylaz, R. Charbonné, K. Nanri, D. Von Euw, J. Borredon, K. Kacem, P. Méric, and E. Pinard, “Dynamic in vivo measurement of erythrocyte velocity and flow in capillaries and of microvessel diameter in the rat brain by confocal laser microscopy,” J. Cereb. Blood Flow Metab. 19(8), 863–870 (1999). [CrossRef] [PubMed]

,15

15. E. B. Brown, R. B. Campbell, Y. Tsuzuki, L. Xu, P. Carmeliet, D. Fukumura, and R. K. Jain, “In vivo measurement of gene expression, angiogenesis and physiological function in tumors using multiphoton laser scanning microscopy,” Nat. Med. 7(7), 864–868 (2001). [CrossRef] [PubMed]

], although they can image tissue in three dimensions, generally require invasive introduction of exogenous fluorescence contrast agents. Our new tool overcomes these problems and is capable of noninvasively imaging three-dimensional microvasculature and its local environment simultaneously, using only endogenous contrasts.

As the first demonstration of our dual-modality imaging strategy, we combined our optical-resolution PAM previously reported in [3

3. G. Ku, K. Maslov, L. Li, and L. V. Wang, “Photoacoustic microscopy with 2-µm transverse resolution,” J. Biomed. Opt. under review. [PubMed]

] with spectral-domain OCT [10

10. B. Cense, N. A. Nassif, T. C. Chen, M. C. Pierce, S. H. Yun, B. H. Park, B. E. Bouma, G. J. Tearney, and J. F. de Boer, “Ultrahigh-resolution high-speed retinal imaging using spectral-domain optical coherence tomography,” Opt. Express 12(11), 2435–2447 (2004), http://www.opticsinfobase.org/oe/abstract.cfm?URI=oe-12-11-2435. [CrossRef] [PubMed]

] in a single system. Figure 1
Fig. 1 Schematic of the combined photoacoustic and optical-coherence microscope. SLD: superluminescent diode. Solid lines represent single-mode optical fibers. Arrowhead solid lines show data flow. Arrowhead dashed lines show the flow of system control signals.
illustrates the experimental configuration of the dual-modality microscope. Both absorption- and scattering-contrast images were obtained from a unified platform, which resembled an inverted optical microscope. The object was illuminated from the bottom. The light used in both modalities was delivered to the platform through a single-mode optical fiber. The light coming out of the fiber tip was first shaped by an aspheric lens and then focused into the object by a microscope objective (Leica, E1, 4X/0.1). In both PAM and OCT images, the transverse resolution was determined by the optical focal size, and was estimated to be ~5 µm by imaging a USAF resolution target. During experiments, target objects were loaded on a cover slip using a custom-made holder, and were held stationary. The imaging platform was scanned using a fast two-dimensional precision linear stage. We were able to acquire x-z cross-sectional images across a 1-mm range within 0.4 seconds, which yielded a rate of 2.5 frames per second. A volumetric data set consisted of a series of x-z cross-sectional images taken at incremental y locations. A 2 µm lateral step size was typical for both PAM and OCT imaging.

The PAM subsystem measures the time-resolved acoustic wave generated from laser-induced thermoelastic expansion of absorbing structures, such as blood vessels. It was irradiated by a diode-pumped Nd:YVO4 laser (Elforlight, SPOT, 532 nm). Compared with other nanosecond-lasers currently used for photoacoustic imaging, this laser is compact, and can emit laser pulses as narrow as 1.2 ns at a pulse repetition rate as high as 20 KHz. To detect the generated photoacoustic wave, a spherically focused ultrasonic transducer (Olympus-NDT, V2022, center frequency: 75 MHz, focal length: 5 mm) was placed atop the object on the imaging platform. A drop of water was applied between the objective and the ultrasonic transducer for acoustic coupling. The photoacoustic axial resolution, mainly determined by the bandwidth of the ultrasonic detection, was estimated to be 14 µm [3

3. G. Ku, K. Maslov, L. Li, and L. V. Wang, “Photoacoustic microscopy with 2-µm transverse resolution,” J. Biomed. Opt. under review. [PubMed]

]. At each transverse location, we collected the photoacoustic signal for 1 µs, which translated to an axial range of 1.5 mm.

The OCT subsystem measures the depth-resolved backscattered light from the object. It was configured as a fiber-based Michelson interferometer, and was seeded by a broadband superluminescent diode (InPhenix, IPSDD0803, λ0 = 829 nm, Δλ = 36.4 nm). The axial resolution, determined by the source bandwidth, was estimated to be 8.4 μm in air or 5.9 μm in soft tissue. The interference between the backscattered light from the object and a reference mirror was recorded by a homemade spectrometer. It was designed to have a spectral resolution of 67.5 pm, which limited the axial imaging range of OCT to 2.5 mm in air or 1.8 mm in tissue.

Figure 2
Fig. 2 x-y projection images of a nude mouse ear obtained by (A) OCT and (B) PAM. (C) Overlay of (A) and (B). (D) Photograph taken through an optical microscope. SG: sebaceous gland; BV: blood vessel.
shows that the x-y projectional images of a nude mouse ear obtained in vivo by our dual-modality microscope correlate well with the photograph taken through an optical microscope with 4X magnification. OCT and PAM visualized different aspects of the tissue better than the traditional optical microscope. In the OCT image (Fig. 2A), the sebaceous glands are resolved with higher resolution, due to the better rejection of background scattered light. The PAM image (Fig. 2B) clearly maps the microvasculature at higher contrast in greater details than the photograph. The average contrast-to-background ratio between blood micro-vessels and surrounding tissue was estimated to ~25:1, which manifests the advantage of the absorption-based photoacoustic method in imaging microvascular morphology. In Fig. 2C, we co-registered the two images, with the blood vessels in red and avascular structures in gray.

Both PAM and OCT are intrinsically three-dimensional imaging modalities. Neither PAM nor OCT requires scanning to resolve along the axial direction. In order to obtain a volumetric data set, our dual-modality microscope only needs two-dimensional raster scanning, unlike confocal and multi-photon microscopes, which depend on three-dimensional scanning. The three-dimensional PAM image (Fig. 4A
Fig. 4 3D visualization of a mouse ear. (A) PAM image of the microvasculature only; (B) Dual-modality image showing the skin surface, a top view; (C) Dual-modality image showing the microvasculature in the ventral skin layer, a top view from 88 µm below the skin surface; (D) Dual-modality image showing the microvasculature in the dorsal skin layer, a top view from 148 µm below the skin surface. The bounding box has dimensions of 3.6 mm x 3.6 mm x 0.4 mm.
) reveals details of the shape, direction, branching and connectivity of blood micro-vessels. Figure 4B shows a top view of the volumetric rendering of the dual-modality data set, where the microscopic epidermal ridges are evident from the OCT contrast. The distinguishable vascular morphologies in the ventral and dorsal layers of the skin are also compared in Figs. 4C and 4D.

We want to point out that PAM and OCT cannot only visualize structural relations between microvasculature and its local environment, but also quantify many important complementary hemodynamic functions. By exploiting the difference between the absorption spectra of oxy-hemoglobin and deoxy-hemoglobin, we can calculate the oxygen saturation (SO2) and total concentration (THb) of hemoglobin locally in each single blood vessel using PAM images acquired with illumination at multiple wavelengths [20

20. H. F. Zhang, K. Maslov, M. Sivaramakrishnan, G. Stoica, and L. V. Wang, “Imaging of hemoglobin oxygen saturation variations in single vessels in vivo using photoacoustic microscopy,” Appl. Phys. Lett. 90(5), 053901 (2007). [CrossRef]

]. Moreover, OCT has been shown to be able to measure local blood flow as slow as 10 µm/s [21

21. Y. Zhao, Z. Chen, C. Saxer, S. Xiang, J. F. de Boer, and J. S. Nelson, “Phase-resolved optical coherence tomography and optical Doppler tomography for imaging blood flow in human skin with fast scanning speed and high velocity sensitivity,” Opt. Lett. 25(2), 114–116 (2000). [CrossRef]

]. Currently, we are working to add these functional imaging capabilities to our dual-modality systems. By quantifying local SO2, THb, and blood flow together, we expect to obtain additional functional indicators, such as the local oxygen metabolic rate, which may serve as a potential biomarker for diseases [22

22. L. V. Wang, “Prospects of photoacoustic tomography,” Med. Phys. 35(12), 5758–5767 (2008). [CrossRef]

].

Last, it is also important to note that the presented microscope is just a paradigm of the strategy to combine photoacoustic imaging with optical coherence tomography. The concept itself is versatile in nature. The dual-modality microscopy can work fully in the reflection mode to allow access to various anatomical locations of interest by adopting alternative designs, for example using a light-ultrasound splitting component similar to the one used in Ref [2

2. K. Maslov, H. F. Zhang, S. Hu, and L. V. Wang, “Optical-resolution photoacoustic microscopy for in vivo imaging of single capillaries,” Opt. Lett. 33(9), 929–931 (2008). [CrossRef] [PubMed]

]. Also, its applications will not be limited to studying cutaneous microcirculation. With proper alternative designs, we expect that variant systems following the same concept can be potentially applied in studying other subjects, such as cerebral microcirculation and ophthalmological diseases.

Acknowledgements

This work was supported in part by National Institutes of Health grants R01 CA092415, R01 NS46214 (BRP), R01 EB000712, R01 EB008085, and U54 CA136398 (NTR). We also thank Jim Ballard and Christopher Favazza for proofreading the manuscript. L.W. has a financial interest in Microphotoacoustics, Inc. and Endra, Inc., which, however, did not support this work.

References and links

1.

H. F. Zhang, K. Maslov, G. Stoica, and L. V. Wang, “Functional photoacoustic microscopy for high-resolution and noninvasive in vivo imaging,” Nat. Biotechnol. 24(7), 848–851 (2006). [CrossRef] [PubMed]

2.

K. Maslov, H. F. Zhang, S. Hu, and L. V. Wang, “Optical-resolution photoacoustic microscopy for in vivo imaging of single capillaries,” Opt. Lett. 33(9), 929–931 (2008). [CrossRef] [PubMed]

3.

G. Ku, K. Maslov, L. Li, and L. V. Wang, “Photoacoustic microscopy with 2-µm transverse resolution,” J. Biomed. Opt. under review. [PubMed]

4.

L. V. Wang, “Tutorial on photoacoustic microscopy and computed tomography,” IEEE J. Sel. Top. Quantum Electron. 14(1), 171–179 (2008). [CrossRef]

5.

J. Oh, M. Li, H. F. Zhang, K. Maslov, G. Stoica, and L. V. Wang, “Three-dimensional imaging of skin melanoma in vivo by dual-wavelength photoacoustic microscopy,” J. Biomed. Opt. 11(3), 034032 (2006). [CrossRef]

6.

C. Vinegoni, T. Ralston, W. Tan, W. Luo, D. L. Marks, and S. A. Boppart, “Integrated structural and functional optical imaging combining spectral-domain optical coherence and multiphoton microscopy,” Appl. Phys. Lett. 88(5), 053901 (2006). [CrossRef]

7.

H. T. Chen, H. F. Wang, M. N. Slipchenko, Y. K. Jung, Y. Z. Shi, J. B. Zhu, K. K. Buhman, and J. X. Cheng, “A multimodal platform for nonlinear optical microscopy and microspectroscopy,” Opt. Express 17(3), 1282–1290 (2009), http://www.opticsinfobase.org/abstract.cfm?URI=oe-17-3-1282. [CrossRef] [PubMed]

8.

S. Y. Emelianov, S. R. Aglyamov, A. B. Karpiouk, S. Mallidi, S. Park, S. Sethuraman, J. Shah, R. W. Smalling, J. M. Rubin, and W. G. Scott, “Synergy and applications of combined ultrasound, elasticity, and photoacoustic imaging,” 2006 IEEE Ultrasonics Symposium, 405–415 (2006).

9.

J. J. Niederhauser, M. Jaeger, R. Lemor, P. Weber, and M. Frenz, “Combined ultrasound and optoacoustic system for real-time high-contrast vascular imaging in vivo,” IEEE Trans. Med. Imaging 24(4), 436–440 (2005). [CrossRef] [PubMed]

10.

B. Cense, N. A. Nassif, T. C. Chen, M. C. Pierce, S. H. Yun, B. H. Park, B. E. Bouma, G. J. Tearney, and J. F. de Boer, “Ultrahigh-resolution high-speed retinal imaging using spectral-domain optical coherence tomography,” Opt. Express 12(11), 2435–2447 (2004), http://www.opticsinfobase.org/oe/abstract.cfm?URI=oe-12-11-2435. [CrossRef] [PubMed]

11.

B. Fagrell and M. Intaglietta, “Microcirculation: its significance in clinical and molecular medicine,” J. Intern. Med. 241(5), 349–362 (1997). [CrossRef] [PubMed]

12.

R. K. Jain, L. L. Munn, and D. Fukumura, “Dissecting tumour pathophysiology using intravital microscopy,” Nat. Rev. Cancer 2(4), 266–276 (2002). [CrossRef] [PubMed]

13.

R. G. Nadeau, W. Groner, J. W. Winkelman, A. G. Harris, C. Ince, G. J. Bouma, and K. Messmer, “Orthogonal polarization spectral imaging: a new method for study of the microcirculation,” Nat. Med. 5(10), 1209–1212 (1999). [CrossRef] [PubMed]

14.

J. Seylaz, R. Charbonné, K. Nanri, D. Von Euw, J. Borredon, K. Kacem, P. Méric, and E. Pinard, “Dynamic in vivo measurement of erythrocyte velocity and flow in capillaries and of microvessel diameter in the rat brain by confocal laser microscopy,” J. Cereb. Blood Flow Metab. 19(8), 863–870 (1999). [CrossRef] [PubMed]

15.

E. B. Brown, R. B. Campbell, Y. Tsuzuki, L. Xu, P. Carmeliet, D. Fukumura, and R. K. Jain, “In vivo measurement of gene expression, angiogenesis and physiological function in tumors using multiphoton laser scanning microscopy,” Nat. Med. 7(7), 864–868 (2001). [CrossRef] [PubMed]

16.

E. Eriksson, J. V. Boykin, and R. N. Pittman, “Method for in vivo microscopy of the cutaneous microcirculation of the hairless mouse ear,” Microvasc. Res. 19(3), 374–379 (1980). [CrossRef] [PubMed]

17.

H. F. Zhang, K. Maslov, and L. V. Wang, “In vivo imaging of subcutaneous structures using functional photoacoustic microscopy,” Nat. Protocols 2(4), 797–804 (2007). [CrossRef]

18.

J. Ai and L. V. Wang, “Synchronous self-elimination of autocorrelation interference in Fourier-domain optical coherence tomography,” Opt. Lett. 30(21), 2939–2941 (2005). [CrossRef] [PubMed]

19.

European mutant mouse pathology database (Pathbase), “Scanning electron microscopy of the mouse ear,” (University of Cambridge, 08/2009). http://eulep.pdn.cam.ac.uk/~skinbase/Anatomic_hair_types/EAR_ ANNOTAT ED.jpg

20.

H. F. Zhang, K. Maslov, M. Sivaramakrishnan, G. Stoica, and L. V. Wang, “Imaging of hemoglobin oxygen saturation variations in single vessels in vivo using photoacoustic microscopy,” Appl. Phys. Lett. 90(5), 053901 (2007). [CrossRef]

21.

Y. Zhao, Z. Chen, C. Saxer, S. Xiang, J. F. de Boer, and J. S. Nelson, “Phase-resolved optical coherence tomography and optical Doppler tomography for imaging blood flow in human skin with fast scanning speed and high velocity sensitivity,” Opt. Lett. 25(2), 114–116 (2000). [CrossRef]

22.

L. V. Wang, “Prospects of photoacoustic tomography,” Med. Phys. 35(12), 5758–5767 (2008). [CrossRef]

OCIS Codes
(170.1870) Medical optics and biotechnology : Dermatology
(170.4470) Medical optics and biotechnology : Ophthalmology
(170.4500) Medical optics and biotechnology : Optical coherence tomography
(170.5120) Medical optics and biotechnology : Photoacoustic imaging
(170.6900) Medical optics and biotechnology : Three-dimensional microscopy

ToC Category:
Medical Optics and Biotechnology

History
Original Manuscript: July 2, 2009
Revised Manuscript: August 11, 2009
Manuscript Accepted: August 13, 2009
Published: August 31, 2009

Virtual Issues
Vol. 4, Iss. 11 Virtual Journal for Biomedical Optics

Citation
Li Li, Konstantin Maslov, Geng Ku, and Lihong V. Wang, "Three-dimensional combined photoacoustic and optical coherence microscopy for in vivo microcirculation studies," Opt. Express 17, 16450-16455 (2009)
http://www.opticsinfobase.org/vjbo/abstract.cfm?URI=oe-17-19-16450


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References

  1. H. F. Zhang, K. Maslov, G. Stoica, and L. V. Wang, “Functional photoacoustic microscopy for high-resolution and noninvasive in vivo imaging,” Nat. Biotechnol. 24(7), 848–851 (2006). [CrossRef] [PubMed]
  2. K. Maslov, H. F. Zhang, S. Hu, and L. V. Wang, “Optical-resolution photoacoustic microscopy for in vivo imaging of single capillaries,” Opt. Lett. 33(9), 929–931 (2008). [CrossRef] [PubMed]
  3. G. Ku, K. Maslov, L. Li, and L. V. Wang, “Photoacoustic microscopy with 2-µm transverse resolution,” J. Biomed. Opt. under review. [PubMed]
  4. L. V. Wang, “Tutorial on photoacoustic microscopy and computed tomography,” IEEE J. Sel. Top. Quantum Electron. 14(1), 171–179 (2008). [CrossRef]
  5. J. Oh, M. Li, H. F. Zhang, K. Maslov, G. Stoica, and L. V. Wang, “Three-dimensional imaging of skin melanoma in vivo by dual-wavelength photoacoustic microscopy,” J. Biomed. Opt. 11(3), 034032 (2006). [CrossRef]
  6. C. Vinegoni, T. Ralston, W. Tan, W. Luo, D. L. Marks, and S. A. Boppart, “Integrated structural and functional optical imaging combining spectral-domain optical coherence and multiphoton microscopy,” Appl. Phys. Lett. 88(5), 053901 (2006). [CrossRef]
  7. H. T. Chen, H. F. Wang, M. N. Slipchenko, Y. K. Jung, Y. Z. Shi, J. B. Zhu, K. K. Buhman, and J. X. Cheng, “A multimodal platform for nonlinear optical microscopy and microspectroscopy,” Opt. Express 17(3), 1282–1290 (2009), http://www.opticsinfobase.org/abstract.cfm?URI=oe-17-3-1282 . [CrossRef] [PubMed]
  8. S. Y. Emelianov, S. R. Aglyamov, A. B. Karpiouk, S. Mallidi, S. Park, S. Sethuraman, J. Shah, R. W. Smalling, J. M. Rubin, and W. G. Scott, “Synergy and applications of combined ultrasound, elasticity, and photoacoustic imaging,” 2006 IEEE Ultrasonics Symposium, 405–415 (2006).
  9. J. J. Niederhauser, M. Jaeger, R. Lemor, P. Weber, and M. Frenz, “Combined ultrasound and optoacoustic system for real-time high-contrast vascular imaging in vivo,” IEEE Trans. Med. Imaging 24(4), 436–440 (2005). [CrossRef] [PubMed]
  10. B. Cense, N. A. Nassif, T. C. Chen, M. C. Pierce, S. H. Yun, B. H. Park, B. E. Bouma, G. J. Tearney, and J. F. de Boer, “Ultrahigh-resolution high-speed retinal imaging using spectral-domain optical coherence tomography,” Opt. Express 12(11), 2435–2447 (2004), http://www.opticsinfobase.org/oe/abstract.cfm?URI=oe-12-11-2435 . [CrossRef] [PubMed]
  11. B. Fagrell and M. Intaglietta, “Microcirculation: its significance in clinical and molecular medicine,” J. Intern. Med. 241(5), 349–362 (1997). [CrossRef] [PubMed]
  12. R. K. Jain, L. L. Munn, and D. Fukumura, “Dissecting tumour pathophysiology using intravital microscopy,” Nat. Rev. Cancer 2(4), 266–276 (2002). [CrossRef] [PubMed]
  13. R. G. Nadeau, W. Groner, J. W. Winkelman, A. G. Harris, C. Ince, G. J. Bouma, and K. Messmer, “Orthogonal polarization spectral imaging: a new method for study of the microcirculation,” Nat. Med. 5(10), 1209–1212 (1999). [CrossRef] [PubMed]
  14. J. Seylaz, R. Charbonné, K. Nanri, D. Von Euw, J. Borredon, K. Kacem, P. Méric, and E. Pinard, “Dynamic in vivo measurement of erythrocyte velocity and flow in capillaries and of microvessel diameter in the rat brain by confocal laser microscopy,” J. Cereb. Blood Flow Metab. 19(8), 863–870 (1999). [CrossRef] [PubMed]
  15. E. B. Brown, R. B. Campbell, Y. Tsuzuki, L. Xu, P. Carmeliet, D. Fukumura, and R. K. Jain, “In vivo measurement of gene expression, angiogenesis and physiological function in tumors using multiphoton laser scanning microscopy,” Nat. Med. 7(7), 864–868 (2001). [CrossRef] [PubMed]
  16. E. Eriksson, J. V. Boykin, and R. N. Pittman, “Method for in vivo microscopy of the cutaneous microcirculation of the hairless mouse ear,” Microvasc. Res. 19(3), 374–379 (1980). [CrossRef] [PubMed]
  17. H. F. Zhang, K. Maslov, and L. V. Wang, “In vivo imaging of subcutaneous structures using functional photoacoustic microscopy,” Nat. Protocols 2(4), 797–804 (2007). [CrossRef]
  18. J. Ai and L. V. Wang, “Synchronous self-elimination of autocorrelation interference in Fourier-domain optical coherence tomography,” Opt. Lett. 30(21), 2939–2941 (2005). [CrossRef] [PubMed]
  19. European mutant mouse pathology database (Pathbase), “Scanning electron microscopy of the mouse ear,” (University of Cambridge, 08/2009). http://eulep.pdn.cam.ac.uk/~skinbase/Anatomic_hair_types/EAR_
ANNOTAT ED.jpg
  20. H. F. Zhang, K. Maslov, M. Sivaramakrishnan, G. Stoica, and L. V. Wang, “Imaging of hemoglobin oxygen saturation variations in single vessels in vivo using photoacoustic microscopy,” Appl. Phys. Lett. 90(5), 053901 (2007). [CrossRef]
  21. Y. Zhao, Z. Chen, C. Saxer, S. Xiang, J. F. de Boer, and J. S. Nelson, “Phase-resolved optical coherence tomography and optical Doppler tomography for imaging blood flow in human skin with fast scanning speed and high velocity sensitivity,” Opt. Lett. 25(2), 114–116 (2000). [CrossRef]
  22. L. V. Wang, “Prospects of photoacoustic tomography,” Med. Phys. 35(12), 5758–5767 (2008). [CrossRef]

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