This article has been accepted for publication in a future issue of this journal, but has not been fully edited. Content may change prior to final publication. Citation information: DOI 10.1109/TMI.2016.2528284, IEEE Transactions on Medical Imaging
IEEE TMI-2015-1070
1
Skull optical clearing solution for enhancing ultrasonic and photoacoustic imaging Xiaoquan Yang¶, Member, IEEE, Yang Zhang¶, Kai Zhao, Yanjie Zhao, Yanyan Liu, Hui Gong, Qingming Luo*, and Dan Zhu*
Abstract—The performance of photoacoustic microscopy (PAM) degrades due to the turbidity of the skull that introduces attenuation and distortion of both laser and stimulated ultrasound. In this manuscript, we demonstrated that a newly developed skull optical clearing solution (SOCS) could enhance not only the transmittance of light, but also that of ultrasound in the skull in vitro. Thus the photoacoustic signal was effectively elevated, and the relative strength of the artifacts induced by the skull could be suppressed. Furthermore in vivo studies demonstrated that SOCS could drastically enhance the performance of photoacoustic microscopy for cerebral microvasculature imaging. Index Terms—Photoacoustic imaging, Skull optical clearing solution.
I. INTRODUCTION
H
emodynamic change in brain is tightly related to the brain
function. Many different methods have been used in mapping such dynamic changes, such as fMRI (functional magnetic resonance imaging), PET (positron emission tomography), and OI (optical imaging). OI can acquire the cerebral blood flow, oxy- and deoxy-hemoglobin of the brain. However, due to the high scattering of light in skull and brain, pure OI can hardly penetrated into the deep brain tissues with high spatial resolution [1]. Although using fluorescence imaging methods in second near infrared window or transcranial optical vascular imaging technique can get cerebral blood flow [2-4]. These methods depend on the repeated injection of contrast agent, This work was supported in part by National Major Scientific Research Program of China (No. 2011CB910401), in part by Science Fund for Creative Research Group of China (No. 61121004), National Natural Science Foundation of China (No. 81201067, 81171376, 91232710, 812111313) and in part by Director Fund of WNLO. ¶Authors contributed equally to this work. Asterisk indicates corresponding author. Xiaoquan Yang, Yang Zhang, Kai Zhao, Yanjie Zhao, Yanyan Liu, Hui Gong, Qingming Luo ([email protected]), and Dan Zhu ([email protected]) are with Britton Chance Center for Biomedical Photonics, Wuhan National Laboratory for Optoelectronics-Huazhong University of Science and Technology, Wuhan,430074, China, and Key Laboratory of Biomedical Photonics of Ministry of Education, Department of Biomedical Engineering, Huazhong University of Science and Technology, Wuhan, 430074, China
and they cannot provide the information of oxy- and deoxy-hemoglobin. Photoacoustic imaging is an emerging hybrid technique that can detect deep optical absorption contrast with unprecedented spatial resolution in optical imaging [5, 6]. It is facilitated by the rich endogenous and exogenous optical contrast agent in biological tissues [7, 8]. However, the performance of photoacoustic imaging in brain has been hindered by the presence of the skull [9]. Many researches prefer to remove the skull when PAM with a high-frequency ultrasound transducer is employed [10, 11]. The skull will attenuate the light due to its turbidity. It will also diminish the stimulated ultrasound due to the high attenuation coefficient of the skull and mismatch of the acoustic impendence between the skull and soft tissues [9]. Therefore, reducing the impact of the skull in photoacoustic imaging is highly desirable [5]. Optical clearing (OC) techniques, based on immersion of tissue into hyperosmotic and high refractive index agents, can be used to improve the delivery of light into deep tissues by reducing the optical scattering properties [11-14]. Previous works demonstrated that the optical clearing agents (OCA) could improve the sensitivity of photoacoustic flow cytometry in detection of single blood cell in subcutaneous blood vessels [15-17]. And the OCA can help the PAM to map the metastases in sentinel lymph nodes ex-vivo [18]. The following studies shown that the performance of both optical-resolution (OR) and acoustic-resolution (AR) PAM in imaging of subcutaneous blood vessels in the skin will be enhanced [19-21]. To the best of our knowledge, the previous researches are focused on the clearing of the soft tissue in the photoacoustic imaging. It is unknown that if the OC technique is suitable for clearing of the skull in the photoacoustic imaging, since the skin and skull are quite different in the aspect of optical and acoustic property. In this manuscript, a newly developed skull optical clearing solution (SOCS) was introduced in the AR-PAM for imaging cortical microvasculature with the skull intact. We investigated the difference of the ultrasound signal, optical signal, and photoacoustic signal through the skull before and after the treatment with SOCS in vitro. Additionally in vivo experiments were performed to evaluate the efficacy of SOCS on PAM. II. METHODS AND RESULTS The PAM used in this study was upgraded from the one
Copyright (c) 2015 IEEE. Personal use of this material is permitted. However, permission to use this material for any other purposes must be obtained from the IEEE by sending a request to [email protected].
0278-0062 (c) 2015 IEEE. Personal use is permitted, but republication/redistribution requires IEEE permission. See http://www.ieee.org/publications_standards/publications/rights/index.html for more information.
This article has been accepted for publication in a future issue of this journal, but has not been fully edited. Content may change prior to final publication. Citation information: DOI 10.1109/TMI.2016.2528284, IEEE Transactions on Medical Imaging
IEEE TMI-2015-1070
reported in our previous work [22, 23] by replacing the scanner with a more compact model (ML03K017, PI, Germany). The scheme of the system is shown in Fig.1.
2 applied to take the reflection images of the USAF target without the skull, with the untreated skull, and with the SOCS-treated skull. All data were processed by SPSS16.0 software. A one-factor analysis of variance (ANOVA) was applied to determine the significant difference of data. Changes were considered to be significant if P < 0.05 and extremely significant if P < 0.01. The typical reflection images of the USAF target are shown in Fig. 2. The central region of the target is clear which is shown in Fig. 2(a). When it was covered with intact skull samples, the image was completely blurred (Fig. 2(b)). After 25 minutes treatment by SOCS, the skull becomes transparent. And the target can be seen clearly through the treated skull again (Fig.2(c)).
Fig. 2 Reflection images of the USAF target (a) without skull, (b) with skull, (c) and with skull treated by SOCS. (d) optical transmittance with skull before and after SOCS treatment. Fig. 1 Scheme of the PAM used in this study. MF: multi-mode fiber, BS: beam splitter, CL: conical lens, UST: ultrasonic transducer, OC: optical condenser, WT: water tank, PD: photodiode.
The light from an Nd: YAG laser (532nm, 30Hz, Surelite I-30, Continuum, U. S.) is focused into a multi-mode fiber. The output light from the fiber is reshaped by conical lens and an optical condenser is used to weakly focus the light into the sample. The pulse energy delivered to the sample is about 1mJ and the laser density at the surface of the sample is about 14mJ/cm2. The focused ultrasonic transducer (V30011, Olympus NDT, U. S.) with a central frequency of 46 MHz and -6 dB bandwidth of 35 MHz is placed at the center of the optical condenser to overlap the ultrasonic and optical focus. A photodiode is used to calibrate the power of each laser pulse. The whole system works under the control of a pulse generator. In ultrasonic imaging, the pulse generator triggers the pulser-receiver (5073PR, Olympus, U. S.) to transmit the pulse to the ultrasonic transducer. In photoacoustic imaging, the pulse generator also triggers the laser to output the laser pulse. The oscilloscope receives the reflected ultrasound or induced photoacoustic signal after the amplification with the pulser-receiver. Then the scanner, which is driven by a motion control PC, moves to the next location for sequential data acquisition. SOCS used in this work is composed of laurinol, dimethylsulfoxide, sorbitol, alcohol, glucose, and sodium dodecyl benzene sulfonate. More details of this kind of SOCS can be found in our previous research [15]. Fresh skull samples excised from Balb/c mice (male, 2 months old, 25±5 g, n=20) were cleaned with saline solution in order to remove the residual blood. The samples were put on a 1951 United States Air Force (USAF) resolution test target. A stereo microscope (SZ61TR, Olympus, Japan) and charge-coupled-device camera (Pixelfly USB, PCO Computer, Germany) were
In order to quantify the efficiency of the SOCS, the transmittance (533 nm) of the skull was measured in vitro with an optical power meter before and after topical SOCS treatment. As shown in Fig. 2(d), the mean transmittance of the skull almost tripled after SOCS treatment compared with the untreated, intact skull.
Fig. 3 Ultrasonic signal from the metal plate before (a) and after (b) SOCS treatment of the skull.
In vitro ultrasonic experiments were also performed to evaluate the change in the ultrasonic transmittance of the skull after SOCS treatment. A metal plate placed roughly 1mm beneath the untreated skull (n=6) was imaged with the AR-PAM system in the ultrasonic imaging mode. 20 frames of B-mode images were also acquired. The reflection signal of the metal plate could then be determined by taking the average of the maximum intensity projections of the metal plate in the averaged B-mode images. After treatment of the skulls with SOCS, the experiments were carried out again. In the experiments, the location of the transducer will be adjusted to promise the metal plate locating at the focus of the transducer. The results indicated that SOCS treatment increased the strength of the reflection signal by a factor of 1.58±0.20. Therefore SOCS can not only decrease the attenuation of laser light, but also enhance the transmission of ultrasound through skulls. The B-scan images of the ultrasonic signal from the metal plate before and after SOCS treatment can be seen in Fig. 3.
Copyright (c) 2015 IEEE. Personal use of this material is permitted. However, permission to use this material for any other purposes must be obtained from the IEEE by sending a request to [email protected].
0278-0062 (c) 2015 IEEE. Personal use is permitted, but republication/redistribution requires IEEE permission. See http://www.ieee.org/publications_standards/publications/rights/index.html for more information.
This article has been accepted for publication in a future issue of this journal, but has not been fully edited. Content may change prior to final publication. Citation information: DOI 10.1109/TMI.2016.2528284, IEEE Transactions on Medical Imaging
IEEE TMI-2015-1070
3 For the in vivo experiments, mice were anesthetized with a cocktail of 2% α-chloralose and 10% urethane (8 m1/kg) via intraperitoneal injection. Skin and fascia above the skull were removed carefully. A head-immobilization device including a custom built plate and a skull holder were used to reduce movement during imaging. Initial reflection images were taken by a stereo-microscope. After topical treatment of the skulls with SOCS for 25 minutes, reflection images were acquired again. Photoacoustic images were also acquired before and after the treatment. The procedures in this study were carried out according to the Institutional Animal Care and Use Committee of Hubei Province.
Fig. 4 B-mode images of carbon fiber, (a) without skull, (b) with untreated skull, (c) and with SOCS-treated skull. (a), (b), and (c) are central part of the each original images of carbon fiber. (d) A-line profile at the central position of (b) and (c), respectively. The position of the red curve is indicated in the (c) with red line, and the position of the blue curve is indicated in the (b) with blue line. S: signal, A: artifact, WTS: with treated skull, WUS: with untreated skull.
Furthermore we verified the changes in resolution and signal strength using PAM with untreated and treated skulls. The experimental procedure was identical to that used in the ultrasonic imaging studies described above. The resolution experiments were performed using a carbon fiber with a diameter of 6 µm. And the measured lateral and axial resolution of the system is 45µm and 15µm, respectively, using full width at half maximum of LSF. The B-mode images of the carbon fiber without skulls, with untreated skulls, and with treated skulls can be found in Fig. 4. Fig. 4(a) demonstrates the line spread function (LSF) of the PAM. The lateral resolution will be degraded to about 60µm by the skull due to disturbance of the ultrasound phase and reduction of the numerical aperture. In Fig. 4(c) we can see that the signal strength was greatly enhanced, and the relative width of LSF decreases to 0.98±0.28 times after SOCS treatment of the skull. No significant change in resolution was observed. However we found that artifacts induced by the skull were suppressed, as shown in fig. 4(d). The peaks of the signal strength for the treated and the untreated skulls are 1.48 and 0.64, respectively (Fig. 4(d)). Correspondingly, the peaks of artifact strength for the two groups in Fig. 4 (d) are 0.44 and 0.28, respectively. If we define the relative strength of artifact (RSA) as the ratio of the signal strength peak to the artifact, the RSA becomes 1.47 times higher after the skulls were treated with SOCS. The signal strength experiments were performed using a black tape. The results suggested that strength of the photoacoustic signal was 2.16±0.31 times higher after SOCS treatment of the skull. It is worth noting that the signal of the carbon fiber is slightly delayed after treatment, which is mainly due to the speed of sound in the SOCS being slower than that in the ultrasonic gel.
As shown in Fig. 5(a), the cortical blood vessels are hard to resolve though the turbid intact skull in the reflection images. But after 25 minutes of SOCS treatment, the microvessels in the cortex become clear through the transparent skull window (Fig. 5(b)). Fig. 5(c) and 5(d) show the photoacoustic images of cerebral vasculature through the untreated skull and treated skull, respectively, which correspond to the same area in Fig. 5(a) and 5(b). It can be seen that the photoacoustic signal amplitude of the same vessels in the cortex is significantly enhanced after topical treatment with SOCS. Concealed vessels in deep tissue can be observed clearly through the transparent skull window compared with the untreated, turbid skull. The diameters of the vessels are nearly the same. Fig. 5(e) shows the typical photoacoustic signal of a single vessel before and after SOCS treatment. The photoacoustic amplitude and diameter of 10 random vessels (indicated by the green arrows in Fig. 5 (c) and (d)) was recorded before and after SOCS–treatment. The results shown in Fig. 5(f) indicate that photoacoustic signal increased to 2.59 folds without affecting the diameter of the vessels. Interestingly the increase in photoacoustic amplitude of vessels in deep tissue was greater than that of surface vessels (3.20 fold vs. 2.19 fold). This may be due to the reduction of optical scattering properties of the skull which would guarantee that more photons reach deep tissue.
Fig. 5 Typical images of cortical blood vessels,(a) reflection images obtained from untreated skull, (b) reflection images obtained from the transparent skull after SOCS treatment for 25 min, (c) photoacoustic images obtained from intact skull, (d) photoacoustic images obtained from the transparent skull after SOCS treatment for 25 min, (e) typical photoacoustic signal of the same vessel denoted by green arrows in (c) and (d), (f) statistical analysis of amplitude of photoacoustic signal and diameter of vessels before and after SOCS treatment.
III. DISCUSSION In the ultrasonic experiments, the improvement of the
Copyright (c) 2015 IEEE. Personal use of this material is permitted. However, permission to use this material for any other purposes must be obtained from the IEEE by sending a request to [email protected].
0278-0062 (c) 2015 IEEE. Personal use is permitted, but republication/redistribution requires IEEE permission. See http://www.ieee.org/publications_standards/publications/rights/index.html for more information.
This article has been accepted for publication in a future issue of this journal, but has not been fully edited. Content may change prior to final publication. Citation information: DOI 10.1109/TMI.2016.2528284, IEEE Transactions on Medical Imaging
IEEE TMI-2015-1070
ultrasonic signal due to SOCS treatment is about 1.58 fold. However the parameters in the photoacoustic imaging are different, since there is only one-way transmission of ultrasound in the AR-PAM. Therefore, the improvement should be expected to be about 1.25 fold. The diffusion of SOCS into the skull reduces the mismatch of acoustic impendence between the skull and the ultrasonic coupler, thus the reflection at the interface of the skull/coupler can be reduced and further enhance the transmittance of ultrasound though the skull. The resolution of AR-PAM through the treated skull in our experiments hardly changed because the resolution of our system depends on the focus of ultrasound instead of laser light. Since SOCS greatly corrects the phase distortion, it may be useful in improving the spatial resolution in OR-PAM that relies on the focus of laser light [17]. The artifacts induced by the multiple reflection of ultrasound in skull are also suppressed after the usage of SOCS, as shown in Fig. 4. It seems that the improvement is moderate in the scanning PAM. And the improvement can only be seen in the B-mode image. However the SOCS may play an important part in systems requiring image reconstructions, since the negligible artifacts in the detection signals may introduce serious artifacts in the reconstructed images. Previous study indicated that, in OR-PAM, the usage of OCA can preserve the optical focal spot to improve the spatial resolution [17, 20]. But in AR-PAM the usage of OCA will enhance the transmission of the light to increase the amplitude of the photoacoustic signal [19]. And the OCA will also affect amplitude of the induced photoacoustic signal [23]. In this study, we find out that optical clearing of skin and the skull is similar regarding optics, yet quite different regarding acoustics. In the optical clearing of skin, the diffusion of OCAs and the dehydration will compete for the acoustic impendence of skin. The dehydration will eventually dominate and increase the mismatch of acoustic impendence, thus weakening the transmission of ultrasound [23]. But in the optical clearing of the skull, the dehydration is so weak that only the diffusion of the SOCS will dominate the impendence of the skull. Therefore the ultrasound signal in the SOCS-treated skull will be enhanced. The fundamental mechanism by which the skull optical clearing method improves the propagation of ultrasonic through the skull remains unclear. It is hypothesized that the dissociation of the collagen increases the homogeneity of the skull, leading to the matching of ultrasound propagation speed and suppressing the attenuation and distortion of stimulated ultrasound. Skull optical clearing technique can make the turbid skull transparent within a very short time, which leads to enhancement for the photoacoustic amplitude while retaining the spatial resolution in AR-PAM, especially for deep tissue vessels. Although this kind of SOCS is not designed for photoacoustic imaging, it still works in our AR-PAM. The results indicate that the main reason for improvement is the enhancement of the laser power through the skull. And the changing of ultrasound is still moderate both in the intensity and resolution. Better performance can be expected as SOCS is
4 improved, which could take into account both the transmittance and the phase correction of the light and ultrasound. IV. CONCLUSION In summary, the skull optical clearing technique could suppress the attenuation and distortion of both laser and stimulated ultrasound that is introduced by the turbid skull. The performance of photoacoustic microscopy for cerebral microvasculature imaging improves after treatment with innovative skull optical clearing solution in vivo. The relative strength of the artifacts induced by the skull could also be suppressed due to the treatment. This technique provides a simple way to solve the skull restriction in PA imaging which will be beneficial for neurovasculature research. ACKNOWLEDGMENT The authors appreciate Dr. Nathan Rudemillerin from Duke University for close reading of the manuscript. REFERENCES [1] [2]
[3] [4]
[5] [6]
[7] [8] [9]
[10]
[11]
[12] [13]
V. Ntziachristos, “Going Deeper Than Microscopy: The Optical Imaging Frontier in Biology,” Nat. Methods, vol. 7, no. 8, pp. 603−614. Aug. 2010. V. Kalchenko, D. Israeli, Y. Kuznetsov, I. Meglinski, and A. Harmelin, “A simple approach for non-invasive transcranial optical vascular imaging (nTOVI),” J. Biophoton., vol. 8, no. 11-12, pp. 897-901. Nov. 2015. V. Kalchenko, D. Israeli, Y. Kuznetsov, A. Harmelin, “Transcranial optical vascular imaging (TOVI) of cortical hemodynamics in mouse brain,” Sci. Rep., vol. 4, pp. 5839, Jul. 2014. G. Hong, S. Diao, J. Chang, A. L. Antaris, C. Chen, B. Zhang, S. Zhao, D. N. Atochin, P. L. Huang, K. I. Andreasson, C. J. Kuo, H. Dai, “Through-skull fluorescence imaging of the brain in a new near-infrared window,” Nat. Photonics., vol. 8, no. 9, pp. 723-730, Aug. 2014. L. V. Wang, and S. Hu, “Photoacoustic tomography: in vivo imaging from organelles to organs,” Science, vol. 335, no. 6075, pp. 1458-1462, Mar. 2004. X. Wang, Y. Pang, G. Ku, X. Xie, G. Stoica, and L. V. Wang, “Noninvasive laser-induced photoacoustic tomography for structural and functional in vivo imaging of the brain,” Nat. Biotechnol., vol. 21, no. 7, pp. 803-806, Jun. 2003. X. Yang, L. V. Wang, “Monkey brain cortex imaging by photoacoustic tomography,”J. Biomed. Opt., vol. 13, no. 4, 044009, Jul. 2008. M. Kneipp, J. Turner, H. Estrada, J. Rebling, S. Shoham, and, D. Razansky, “Effects of the murine skull in optoacoustic brain microscopy, ” J. Biophoton., accepted, 2015. S. Hu, P. Yan, K. Maslov, and L. V. Wang, “Intravital imaging of amyloid plaques in a transgenic mouse model using optical-resolution photoacoustic microscopy,” Opt. Lett., vol. 34, no. 24, pp. 3899-3901, Dec. 2009. H. Wang, X. Yang, Z. Wang, Z. Deng, H. Gong, and Q. Luo,“Early monitoring of cerebral hypoperfusion in rats by laser speckle imaging and functional photoacoustic microscopy,” J. Biomed. Opt., vol. 17, no. 6, 061207, Jun. 2012. V. V. Tuchin, I. L. Maksimova, D. A. Zimnyakov, I. L. Kon, A. H. Mavlyutov, and A. A. Mishin, “Light propagation in tissues with controlled optical properties,” J. Biomed. Opt., vol. 2, no. 4, pp. 401-417, Oct. 1997. D. Zhu, K. V. Larin, Q. Luo, and V. V. Tuchin, “ Recent progress in Tissue optical clearing,” Laser Photon. Rev., vol. 7, no. 5, pp. 732–757, Sep. 2013. J. Wang, Y. Zhang, P. Li, Q. Luo and D. Zhu, “Review: Tissue Optical Clearing Window for Blood Flow Monitoring Using Laser Speckle Contrast Imaging,” IEEE J. Sel. Top. Quantum Electron., vol. 20, no. 2, 6801112, Mar. 2014.
Copyright (c) 2015 IEEE. Personal use of this material is permitted. However, permission to use this material for any other purposes must be obtained from the IEEE by sending a request to [email protected].
0278-0062 (c) 2015 IEEE. Personal use is permitted, but republication/redistribution requires IEEE permission. See http://www.ieee.org/publications_standards/publications/rights/index.html for more information.
This article has been accepted for publication in a future issue of this journal, but has not been fully edited. Content may change prior to final publication. Citation information: DOI 10.1109/TMI.2016.2528284, IEEE Transactions on Medical Imaging
IEEE TMI-2015-1070 [14] J. Wang, Y. Zhang, T. Xu, Q. Luo, and D. Zhu, “An innovative transparent cranial window based on skull optical clearing,” Laser Phys. Lett., vol. 9, no. 6, pp. 469-473, Jun. 2012. [15] V. P. Zharov, E. I. Galanzha, Y. A. Menyaev, and V. V.Tuchin, “In vivo high-speed imaging of individual cells in fast blood flow,” J. Biomed. Opt.,Vol. 11, no.5, pp. 054034. Sep.-Oct .2006. [16] V. P. Zharov, E. I. Galanzha, E. V. Shashkov, N. G. Khlebtsov, and V. V. Tuchin, “In vivo photoacoustic flow cytometry for monitoring of circulating single cancer cells and contrast agents,” Opt. Lett., vol. 31, no. 24, pp. 3623-3625, Dec. 2006. [17] Y. A. Menyaev, D. A. Nedosekin, M. Sarimollaoglu, M. A. Juratli, E. I. Galanzha, V. V. Tuchin, and V. P. Zharov, “Optical clearing in photoacoustic flow cytometry,” Biomed. Opt. Express, vol. 4, no. 12, pp. 3030–3041, Nov. 2013. [18] E. I. Galanzha, M. S. Kokoska, E. V. Shashkov, J. Kim, V. V. Tuchin, and V. P. Zharov, “In vivo fiber-based multicolor photoacoustic detection and photothermal purging of metastasis in sentinel lymph
5
[19] [20] [21] [22] [23]
nodes targeted by nanoparticles,” J. Biophoton., vol. 2, no. 8-9, pp. 528-539. Sep. 2009. Y. Liu, X. Yang, D. Zhu, R. Shi, and Q. Luo, “Optical clearing agents improve photoacoustic imaging in the optical diffusive regime,” Opt. Lett., vol. 38, no. 20, pp. 4236-4239, Oct. 2013. Y. Zhou, J. Yao, L. V. Wang, “Optical clearing-aided photoacoustic microscopy with enhanced resolution and imaging depth,” Opt. Lett., vol. 38, no. 14, pp. 2592-2595, Jul. 2013. Z. Deng, L. Jing, N. Wu, P. Lv, X. Jiang, Q. Ren, and C. Li, “Viscous optical clearing agent for in vivo optical imaging”, J. Biomed. Opt., vol. 19, no. 7, pp. 076019, Jul. 2014. X. Yang, X. Cai, K. Maslov, L. V. Wang and Q. Luo, “High-resolution photoacoustic microscope for rat brain imaging in vivo,” Chin. Opt. Lett., vol. 8, no. 6, pp. 609-611, Jun. 2010. X. Yang, Y. Liu, D. Zhu, R. Shi, and Q. Luo, “Dynamic monitoring of optical clearing of skin using photoacoustic microscopy and ultrasonography,” Opt. Express, vol. 22, no. 1, 1094-1104, Jan. 2013.
Copyright (c) 2015 IEEE. Personal use of this material is permitted. However, permission to use this material for any other purposes must be obtained from the IEEE by sending a request to [email protected].
0278-0062 (c) 2015 IEEE. Personal use is permitted, but republication/redistribution requires IEEE permission. See http://www.ieee.org/publications_standards/publications/rights/index.html for more information.
IEEE TMI-2015-1070
1
Skull optical clearing solution for enhancing ultrasonic and photoacoustic imaging Xiaoquan Yang¶, Member, IEEE, Yang Zhang¶, Kai Zhao, Yanjie Zhao, Yanyan Liu, Hui Gong, Qingming Luo*, and Dan Zhu*
Abstract—The performance of photoacoustic microscopy (PAM) degrades due to the turbidity of the skull that introduces attenuation and distortion of both laser and stimulated ultrasound. In this manuscript, we demonstrated that a newly developed skull optical clearing solution (SOCS) could enhance not only the transmittance of light, but also that of ultrasound in the skull in vitro. Thus the photoacoustic signal was effectively elevated, and the relative strength of the artifacts induced by the skull could be suppressed. Furthermore in vivo studies demonstrated that SOCS could drastically enhance the performance of photoacoustic microscopy for cerebral microvasculature imaging. Index Terms—Photoacoustic imaging, Skull optical clearing solution.
I. INTRODUCTION
H
emodynamic change in brain is tightly related to the brain
function. Many different methods have been used in mapping such dynamic changes, such as fMRI (functional magnetic resonance imaging), PET (positron emission tomography), and OI (optical imaging). OI can acquire the cerebral blood flow, oxy- and deoxy-hemoglobin of the brain. However, due to the high scattering of light in skull and brain, pure OI can hardly penetrated into the deep brain tissues with high spatial resolution [1]. Although using fluorescence imaging methods in second near infrared window or transcranial optical vascular imaging technique can get cerebral blood flow [2-4]. These methods depend on the repeated injection of contrast agent, This work was supported in part by National Major Scientific Research Program of China (No. 2011CB910401), in part by Science Fund for Creative Research Group of China (No. 61121004), National Natural Science Foundation of China (No. 81201067, 81171376, 91232710, 812111313) and in part by Director Fund of WNLO. ¶Authors contributed equally to this work. Asterisk indicates corresponding author. Xiaoquan Yang, Yang Zhang, Kai Zhao, Yanjie Zhao, Yanyan Liu, Hui Gong, Qingming Luo ([email protected]), and Dan Zhu ([email protected]) are with Britton Chance Center for Biomedical Photonics, Wuhan National Laboratory for Optoelectronics-Huazhong University of Science and Technology, Wuhan,430074, China, and Key Laboratory of Biomedical Photonics of Ministry of Education, Department of Biomedical Engineering, Huazhong University of Science and Technology, Wuhan, 430074, China
and they cannot provide the information of oxy- and deoxy-hemoglobin. Photoacoustic imaging is an emerging hybrid technique that can detect deep optical absorption contrast with unprecedented spatial resolution in optical imaging [5, 6]. It is facilitated by the rich endogenous and exogenous optical contrast agent in biological tissues [7, 8]. However, the performance of photoacoustic imaging in brain has been hindered by the presence of the skull [9]. Many researches prefer to remove the skull when PAM with a high-frequency ultrasound transducer is employed [10, 11]. The skull will attenuate the light due to its turbidity. It will also diminish the stimulated ultrasound due to the high attenuation coefficient of the skull and mismatch of the acoustic impendence between the skull and soft tissues [9]. Therefore, reducing the impact of the skull in photoacoustic imaging is highly desirable [5]. Optical clearing (OC) techniques, based on immersion of tissue into hyperosmotic and high refractive index agents, can be used to improve the delivery of light into deep tissues by reducing the optical scattering properties [11-14]. Previous works demonstrated that the optical clearing agents (OCA) could improve the sensitivity of photoacoustic flow cytometry in detection of single blood cell in subcutaneous blood vessels [15-17]. And the OCA can help the PAM to map the metastases in sentinel lymph nodes ex-vivo [18]. The following studies shown that the performance of both optical-resolution (OR) and acoustic-resolution (AR) PAM in imaging of subcutaneous blood vessels in the skin will be enhanced [19-21]. To the best of our knowledge, the previous researches are focused on the clearing of the soft tissue in the photoacoustic imaging. It is unknown that if the OC technique is suitable for clearing of the skull in the photoacoustic imaging, since the skin and skull are quite different in the aspect of optical and acoustic property. In this manuscript, a newly developed skull optical clearing solution (SOCS) was introduced in the AR-PAM for imaging cortical microvasculature with the skull intact. We investigated the difference of the ultrasound signal, optical signal, and photoacoustic signal through the skull before and after the treatment with SOCS in vitro. Additionally in vivo experiments were performed to evaluate the efficacy of SOCS on PAM. II. METHODS AND RESULTS The PAM used in this study was upgraded from the one
Copyright (c) 2015 IEEE. Personal use of this material is permitted. However, permission to use this material for any other purposes must be obtained from the IEEE by sending a request to [email protected].
0278-0062 (c) 2015 IEEE. Personal use is permitted, but republication/redistribution requires IEEE permission. See http://www.ieee.org/publications_standards/publications/rights/index.html for more information.
This article has been accepted for publication in a future issue of this journal, but has not been fully edited. Content may change prior to final publication. Citation information: DOI 10.1109/TMI.2016.2528284, IEEE Transactions on Medical Imaging
IEEE TMI-2015-1070
reported in our previous work [22, 23] by replacing the scanner with a more compact model (ML03K017, PI, Germany). The scheme of the system is shown in Fig.1.
2 applied to take the reflection images of the USAF target without the skull, with the untreated skull, and with the SOCS-treated skull. All data were processed by SPSS16.0 software. A one-factor analysis of variance (ANOVA) was applied to determine the significant difference of data. Changes were considered to be significant if P < 0.05 and extremely significant if P < 0.01. The typical reflection images of the USAF target are shown in Fig. 2. The central region of the target is clear which is shown in Fig. 2(a). When it was covered with intact skull samples, the image was completely blurred (Fig. 2(b)). After 25 minutes treatment by SOCS, the skull becomes transparent. And the target can be seen clearly through the treated skull again (Fig.2(c)).
Fig. 2 Reflection images of the USAF target (a) without skull, (b) with skull, (c) and with skull treated by SOCS. (d) optical transmittance with skull before and after SOCS treatment. Fig. 1 Scheme of the PAM used in this study. MF: multi-mode fiber, BS: beam splitter, CL: conical lens, UST: ultrasonic transducer, OC: optical condenser, WT: water tank, PD: photodiode.
The light from an Nd: YAG laser (532nm, 30Hz, Surelite I-30, Continuum, U. S.) is focused into a multi-mode fiber. The output light from the fiber is reshaped by conical lens and an optical condenser is used to weakly focus the light into the sample. The pulse energy delivered to the sample is about 1mJ and the laser density at the surface of the sample is about 14mJ/cm2. The focused ultrasonic transducer (V30011, Olympus NDT, U. S.) with a central frequency of 46 MHz and -6 dB bandwidth of 35 MHz is placed at the center of the optical condenser to overlap the ultrasonic and optical focus. A photodiode is used to calibrate the power of each laser pulse. The whole system works under the control of a pulse generator. In ultrasonic imaging, the pulse generator triggers the pulser-receiver (5073PR, Olympus, U. S.) to transmit the pulse to the ultrasonic transducer. In photoacoustic imaging, the pulse generator also triggers the laser to output the laser pulse. The oscilloscope receives the reflected ultrasound or induced photoacoustic signal after the amplification with the pulser-receiver. Then the scanner, which is driven by a motion control PC, moves to the next location for sequential data acquisition. SOCS used in this work is composed of laurinol, dimethylsulfoxide, sorbitol, alcohol, glucose, and sodium dodecyl benzene sulfonate. More details of this kind of SOCS can be found in our previous research [15]. Fresh skull samples excised from Balb/c mice (male, 2 months old, 25±5 g, n=20) were cleaned with saline solution in order to remove the residual blood. The samples were put on a 1951 United States Air Force (USAF) resolution test target. A stereo microscope (SZ61TR, Olympus, Japan) and charge-coupled-device camera (Pixelfly USB, PCO Computer, Germany) were
In order to quantify the efficiency of the SOCS, the transmittance (533 nm) of the skull was measured in vitro with an optical power meter before and after topical SOCS treatment. As shown in Fig. 2(d), the mean transmittance of the skull almost tripled after SOCS treatment compared with the untreated, intact skull.
Fig. 3 Ultrasonic signal from the metal plate before (a) and after (b) SOCS treatment of the skull.
In vitro ultrasonic experiments were also performed to evaluate the change in the ultrasonic transmittance of the skull after SOCS treatment. A metal plate placed roughly 1mm beneath the untreated skull (n=6) was imaged with the AR-PAM system in the ultrasonic imaging mode. 20 frames of B-mode images were also acquired. The reflection signal of the metal plate could then be determined by taking the average of the maximum intensity projections of the metal plate in the averaged B-mode images. After treatment of the skulls with SOCS, the experiments were carried out again. In the experiments, the location of the transducer will be adjusted to promise the metal plate locating at the focus of the transducer. The results indicated that SOCS treatment increased the strength of the reflection signal by a factor of 1.58±0.20. Therefore SOCS can not only decrease the attenuation of laser light, but also enhance the transmission of ultrasound through skulls. The B-scan images of the ultrasonic signal from the metal plate before and after SOCS treatment can be seen in Fig. 3.
Copyright (c) 2015 IEEE. Personal use of this material is permitted. However, permission to use this material for any other purposes must be obtained from the IEEE by sending a request to [email protected].
0278-0062 (c) 2015 IEEE. Personal use is permitted, but republication/redistribution requires IEEE permission. See http://www.ieee.org/publications_standards/publications/rights/index.html for more information.
This article has been accepted for publication in a future issue of this journal, but has not been fully edited. Content may change prior to final publication. Citation information: DOI 10.1109/TMI.2016.2528284, IEEE Transactions on Medical Imaging
IEEE TMI-2015-1070
3 For the in vivo experiments, mice were anesthetized with a cocktail of 2% α-chloralose and 10% urethane (8 m1/kg) via intraperitoneal injection. Skin and fascia above the skull were removed carefully. A head-immobilization device including a custom built plate and a skull holder were used to reduce movement during imaging. Initial reflection images were taken by a stereo-microscope. After topical treatment of the skulls with SOCS for 25 minutes, reflection images were acquired again. Photoacoustic images were also acquired before and after the treatment. The procedures in this study were carried out according to the Institutional Animal Care and Use Committee of Hubei Province.
Fig. 4 B-mode images of carbon fiber, (a) without skull, (b) with untreated skull, (c) and with SOCS-treated skull. (a), (b), and (c) are central part of the each original images of carbon fiber. (d) A-line profile at the central position of (b) and (c), respectively. The position of the red curve is indicated in the (c) with red line, and the position of the blue curve is indicated in the (b) with blue line. S: signal, A: artifact, WTS: with treated skull, WUS: with untreated skull.
Furthermore we verified the changes in resolution and signal strength using PAM with untreated and treated skulls. The experimental procedure was identical to that used in the ultrasonic imaging studies described above. The resolution experiments were performed using a carbon fiber with a diameter of 6 µm. And the measured lateral and axial resolution of the system is 45µm and 15µm, respectively, using full width at half maximum of LSF. The B-mode images of the carbon fiber without skulls, with untreated skulls, and with treated skulls can be found in Fig. 4. Fig. 4(a) demonstrates the line spread function (LSF) of the PAM. The lateral resolution will be degraded to about 60µm by the skull due to disturbance of the ultrasound phase and reduction of the numerical aperture. In Fig. 4(c) we can see that the signal strength was greatly enhanced, and the relative width of LSF decreases to 0.98±0.28 times after SOCS treatment of the skull. No significant change in resolution was observed. However we found that artifacts induced by the skull were suppressed, as shown in fig. 4(d). The peaks of the signal strength for the treated and the untreated skulls are 1.48 and 0.64, respectively (Fig. 4(d)). Correspondingly, the peaks of artifact strength for the two groups in Fig. 4 (d) are 0.44 and 0.28, respectively. If we define the relative strength of artifact (RSA) as the ratio of the signal strength peak to the artifact, the RSA becomes 1.47 times higher after the skulls were treated with SOCS. The signal strength experiments were performed using a black tape. The results suggested that strength of the photoacoustic signal was 2.16±0.31 times higher after SOCS treatment of the skull. It is worth noting that the signal of the carbon fiber is slightly delayed after treatment, which is mainly due to the speed of sound in the SOCS being slower than that in the ultrasonic gel.
As shown in Fig. 5(a), the cortical blood vessels are hard to resolve though the turbid intact skull in the reflection images. But after 25 minutes of SOCS treatment, the microvessels in the cortex become clear through the transparent skull window (Fig. 5(b)). Fig. 5(c) and 5(d) show the photoacoustic images of cerebral vasculature through the untreated skull and treated skull, respectively, which correspond to the same area in Fig. 5(a) and 5(b). It can be seen that the photoacoustic signal amplitude of the same vessels in the cortex is significantly enhanced after topical treatment with SOCS. Concealed vessels in deep tissue can be observed clearly through the transparent skull window compared with the untreated, turbid skull. The diameters of the vessels are nearly the same. Fig. 5(e) shows the typical photoacoustic signal of a single vessel before and after SOCS treatment. The photoacoustic amplitude and diameter of 10 random vessels (indicated by the green arrows in Fig. 5 (c) and (d)) was recorded before and after SOCS–treatment. The results shown in Fig. 5(f) indicate that photoacoustic signal increased to 2.59 folds without affecting the diameter of the vessels. Interestingly the increase in photoacoustic amplitude of vessels in deep tissue was greater than that of surface vessels (3.20 fold vs. 2.19 fold). This may be due to the reduction of optical scattering properties of the skull which would guarantee that more photons reach deep tissue.
Fig. 5 Typical images of cortical blood vessels,(a) reflection images obtained from untreated skull, (b) reflection images obtained from the transparent skull after SOCS treatment for 25 min, (c) photoacoustic images obtained from intact skull, (d) photoacoustic images obtained from the transparent skull after SOCS treatment for 25 min, (e) typical photoacoustic signal of the same vessel denoted by green arrows in (c) and (d), (f) statistical analysis of amplitude of photoacoustic signal and diameter of vessels before and after SOCS treatment.
III. DISCUSSION In the ultrasonic experiments, the improvement of the
Copyright (c) 2015 IEEE. Personal use of this material is permitted. However, permission to use this material for any other purposes must be obtained from the IEEE by sending a request to [email protected].
0278-0062 (c) 2015 IEEE. Personal use is permitted, but republication/redistribution requires IEEE permission. See http://www.ieee.org/publications_standards/publications/rights/index.html for more information.
This article has been accepted for publication in a future issue of this journal, but has not been fully edited. Content may change prior to final publication. Citation information: DOI 10.1109/TMI.2016.2528284, IEEE Transactions on Medical Imaging
IEEE TMI-2015-1070
ultrasonic signal due to SOCS treatment is about 1.58 fold. However the parameters in the photoacoustic imaging are different, since there is only one-way transmission of ultrasound in the AR-PAM. Therefore, the improvement should be expected to be about 1.25 fold. The diffusion of SOCS into the skull reduces the mismatch of acoustic impendence between the skull and the ultrasonic coupler, thus the reflection at the interface of the skull/coupler can be reduced and further enhance the transmittance of ultrasound though the skull. The resolution of AR-PAM through the treated skull in our experiments hardly changed because the resolution of our system depends on the focus of ultrasound instead of laser light. Since SOCS greatly corrects the phase distortion, it may be useful in improving the spatial resolution in OR-PAM that relies on the focus of laser light [17]. The artifacts induced by the multiple reflection of ultrasound in skull are also suppressed after the usage of SOCS, as shown in Fig. 4. It seems that the improvement is moderate in the scanning PAM. And the improvement can only be seen in the B-mode image. However the SOCS may play an important part in systems requiring image reconstructions, since the negligible artifacts in the detection signals may introduce serious artifacts in the reconstructed images. Previous study indicated that, in OR-PAM, the usage of OCA can preserve the optical focal spot to improve the spatial resolution [17, 20]. But in AR-PAM the usage of OCA will enhance the transmission of the light to increase the amplitude of the photoacoustic signal [19]. And the OCA will also affect amplitude of the induced photoacoustic signal [23]. In this study, we find out that optical clearing of skin and the skull is similar regarding optics, yet quite different regarding acoustics. In the optical clearing of skin, the diffusion of OCAs and the dehydration will compete for the acoustic impendence of skin. The dehydration will eventually dominate and increase the mismatch of acoustic impendence, thus weakening the transmission of ultrasound [23]. But in the optical clearing of the skull, the dehydration is so weak that only the diffusion of the SOCS will dominate the impendence of the skull. Therefore the ultrasound signal in the SOCS-treated skull will be enhanced. The fundamental mechanism by which the skull optical clearing method improves the propagation of ultrasonic through the skull remains unclear. It is hypothesized that the dissociation of the collagen increases the homogeneity of the skull, leading to the matching of ultrasound propagation speed and suppressing the attenuation and distortion of stimulated ultrasound. Skull optical clearing technique can make the turbid skull transparent within a very short time, which leads to enhancement for the photoacoustic amplitude while retaining the spatial resolution in AR-PAM, especially for deep tissue vessels. Although this kind of SOCS is not designed for photoacoustic imaging, it still works in our AR-PAM. The results indicate that the main reason for improvement is the enhancement of the laser power through the skull. And the changing of ultrasound is still moderate both in the intensity and resolution. Better performance can be expected as SOCS is
4 improved, which could take into account both the transmittance and the phase correction of the light and ultrasound. IV. CONCLUSION In summary, the skull optical clearing technique could suppress the attenuation and distortion of both laser and stimulated ultrasound that is introduced by the turbid skull. The performance of photoacoustic microscopy for cerebral microvasculature imaging improves after treatment with innovative skull optical clearing solution in vivo. The relative strength of the artifacts induced by the skull could also be suppressed due to the treatment. This technique provides a simple way to solve the skull restriction in PA imaging which will be beneficial for neurovasculature research. ACKNOWLEDGMENT The authors appreciate Dr. Nathan Rudemillerin from Duke University for close reading of the manuscript. REFERENCES [1] [2]
[3] [4]
[5] [6]
[7] [8] [9]
[10]
[11]
[12] [13]
V. Ntziachristos, “Going Deeper Than Microscopy: The Optical Imaging Frontier in Biology,” Nat. Methods, vol. 7, no. 8, pp. 603−614. Aug. 2010. V. Kalchenko, D. Israeli, Y. Kuznetsov, I. Meglinski, and A. Harmelin, “A simple approach for non-invasive transcranial optical vascular imaging (nTOVI),” J. Biophoton., vol. 8, no. 11-12, pp. 897-901. Nov. 2015. V. Kalchenko, D. Israeli, Y. Kuznetsov, A. Harmelin, “Transcranial optical vascular imaging (TOVI) of cortical hemodynamics in mouse brain,” Sci. Rep., vol. 4, pp. 5839, Jul. 2014. G. Hong, S. Diao, J. Chang, A. L. Antaris, C. Chen, B. Zhang, S. Zhao, D. N. Atochin, P. L. Huang, K. I. Andreasson, C. J. Kuo, H. Dai, “Through-skull fluorescence imaging of the brain in a new near-infrared window,” Nat. Photonics., vol. 8, no. 9, pp. 723-730, Aug. 2014. L. V. Wang, and S. Hu, “Photoacoustic tomography: in vivo imaging from organelles to organs,” Science, vol. 335, no. 6075, pp. 1458-1462, Mar. 2004. X. Wang, Y. Pang, G. Ku, X. Xie, G. Stoica, and L. V. Wang, “Noninvasive laser-induced photoacoustic tomography for structural and functional in vivo imaging of the brain,” Nat. Biotechnol., vol. 21, no. 7, pp. 803-806, Jun. 2003. X. Yang, L. V. Wang, “Monkey brain cortex imaging by photoacoustic tomography,”J. Biomed. Opt., vol. 13, no. 4, 044009, Jul. 2008. M. Kneipp, J. Turner, H. Estrada, J. Rebling, S. Shoham, and, D. Razansky, “Effects of the murine skull in optoacoustic brain microscopy, ” J. Biophoton., accepted, 2015. S. Hu, P. Yan, K. Maslov, and L. V. Wang, “Intravital imaging of amyloid plaques in a transgenic mouse model using optical-resolution photoacoustic microscopy,” Opt. Lett., vol. 34, no. 24, pp. 3899-3901, Dec. 2009. H. Wang, X. Yang, Z. Wang, Z. Deng, H. Gong, and Q. Luo,“Early monitoring of cerebral hypoperfusion in rats by laser speckle imaging and functional photoacoustic microscopy,” J. Biomed. Opt., vol. 17, no. 6, 061207, Jun. 2012. V. V. Tuchin, I. L. Maksimova, D. A. Zimnyakov, I. L. Kon, A. H. Mavlyutov, and A. A. Mishin, “Light propagation in tissues with controlled optical properties,” J. Biomed. Opt., vol. 2, no. 4, pp. 401-417, Oct. 1997. D. Zhu, K. V. Larin, Q. Luo, and V. V. Tuchin, “ Recent progress in Tissue optical clearing,” Laser Photon. Rev., vol. 7, no. 5, pp. 732–757, Sep. 2013. J. Wang, Y. Zhang, P. Li, Q. Luo and D. Zhu, “Review: Tissue Optical Clearing Window for Blood Flow Monitoring Using Laser Speckle Contrast Imaging,” IEEE J. Sel. Top. Quantum Electron., vol. 20, no. 2, 6801112, Mar. 2014.
Copyright (c) 2015 IEEE. Personal use of this material is permitted. However, permission to use this material for any other purposes must be obtained from the IEEE by sending a request to [email protected].
0278-0062 (c) 2015 IEEE. Personal use is permitted, but republication/redistribution requires IEEE permission. See http://www.ieee.org/publications_standards/publications/rights/index.html for more information.
This article has been accepted for publication in a future issue of this journal, but has not been fully edited. Content may change prior to final publication. Citation information: DOI 10.1109/TMI.2016.2528284, IEEE Transactions on Medical Imaging
IEEE TMI-2015-1070 [14] J. Wang, Y. Zhang, T. Xu, Q. Luo, and D. Zhu, “An innovative transparent cranial window based on skull optical clearing,” Laser Phys. Lett., vol. 9, no. 6, pp. 469-473, Jun. 2012. [15] V. P. Zharov, E. I. Galanzha, Y. A. Menyaev, and V. V.Tuchin, “In vivo high-speed imaging of individual cells in fast blood flow,” J. Biomed. Opt.,Vol. 11, no.5, pp. 054034. Sep.-Oct .2006. [16] V. P. Zharov, E. I. Galanzha, E. V. Shashkov, N. G. Khlebtsov, and V. V. Tuchin, “In vivo photoacoustic flow cytometry for monitoring of circulating single cancer cells and contrast agents,” Opt. Lett., vol. 31, no. 24, pp. 3623-3625, Dec. 2006. [17] Y. A. Menyaev, D. A. Nedosekin, M. Sarimollaoglu, M. A. Juratli, E. I. Galanzha, V. V. Tuchin, and V. P. Zharov, “Optical clearing in photoacoustic flow cytometry,” Biomed. Opt. Express, vol. 4, no. 12, pp. 3030–3041, Nov. 2013. [18] E. I. Galanzha, M. S. Kokoska, E. V. Shashkov, J. Kim, V. V. Tuchin, and V. P. Zharov, “In vivo fiber-based multicolor photoacoustic detection and photothermal purging of metastasis in sentinel lymph
5
[19] [20] [21] [22] [23]
nodes targeted by nanoparticles,” J. Biophoton., vol. 2, no. 8-9, pp. 528-539. Sep. 2009. Y. Liu, X. Yang, D. Zhu, R. Shi, and Q. Luo, “Optical clearing agents improve photoacoustic imaging in the optical diffusive regime,” Opt. Lett., vol. 38, no. 20, pp. 4236-4239, Oct. 2013. Y. Zhou, J. Yao, L. V. Wang, “Optical clearing-aided photoacoustic microscopy with enhanced resolution and imaging depth,” Opt. Lett., vol. 38, no. 14, pp. 2592-2595, Jul. 2013. Z. Deng, L. Jing, N. Wu, P. Lv, X. Jiang, Q. Ren, and C. Li, “Viscous optical clearing agent for in vivo optical imaging”, J. Biomed. Opt., vol. 19, no. 7, pp. 076019, Jul. 2014. X. Yang, X. Cai, K. Maslov, L. V. Wang and Q. Luo, “High-resolution photoacoustic microscope for rat brain imaging in vivo,” Chin. Opt. Lett., vol. 8, no. 6, pp. 609-611, Jun. 2010. X. Yang, Y. Liu, D. Zhu, R. Shi, and Q. Luo, “Dynamic monitoring of optical clearing of skin using photoacoustic microscopy and ultrasonography,” Opt. Express, vol. 22, no. 1, 1094-1104, Jan. 2013.
Copyright (c) 2015 IEEE. Personal use of this material is permitted. However, permission to use this material for any other purposes must be obtained from the IEEE by sending a request to [email protected].
0278-0062 (c) 2015 IEEE. Personal use is permitted, but republication/redistribution requires IEEE permission. See http://www.ieee.org/publications_standards/publications/rights/index.html for more information.
