Tissue optical clearing technique has shown great potential for enhancing the imaging depth and contrast of optical imaging modalities. However, the mechanism of optical clearing is still in controversy. In this manuscript, we combined photoacoustic microscopy with ultrasonography to monitor the dermic changes induced by optical clearing agents at different immersion time points. The measured parameters were correlated with the optical clearing process, and could be used to assess the optical clearing effect. Both in vitro and in vivo results demonstrated that photoacoustic microscopy and ultrasonography can potentially be used as a powerful tool in screening optical clearing agents and exploring the mechanism of optical clearing.
© 2014 Optical Society of America
With the inherent advantages of rich contrast and high resolution, optical imaging has been widely used in biological research. However, the strong optical scattering of tissue hinders the penetration depth of optical imaging. Optical clearing technique using immersion of optical clearing agents (OCAs) [1, 2], is able to reduce the optical scattering and enhance the penetration depth. The enhancement of optical imaging has been validated in many optical imaging modalities, including laser speckle imaging , optical coherence tomography , confocal microscopy , two-photon microscopy  and even hybrid imaging modalities such as photoacoustic flow cytometry [7, 8] and photoacoustic microscopy (PAM) .
Research on the mechanism of optical clearing is necessary for selection and correct usage of OCAs. Although OCAs have been successfully applied to many imaging modalities, the mechanism of optical clearing is still not very clear. Different hypotheses have been suggested based on results from different measurement modalities. Tuchin et al measured the optical properties with Abbe refractometer and spectrophotometer. They proposed that matching of refractive indices between the cells and ground substance was a major mechanism . The dehydration was also proved to be an important mechanism based on the observation with optical coherence tomography, electron microscopy  and reflectance spectroscopy . Besides, the dissociation of collagen fibers , observed with second harmonic generation , may be an additional mechanism. However, many researches showed that the parameters associated with the hypotheses were not sufficient to explain the optical clearing and predict the optical clearing potential . Meanwhile, most previous studies were based on experiments ex vivo. As the structure and function of tissues in vivo are quite different from those ex vivo , the mechanism of optical clearing should be further investigated in vivo as well as ex vivo.
Ultrasonography is currently the most important clinical modality for non-invasive assessment of tissue structure . Ultrasound pulses are reflected and scattered within the tissues, the amplitude and echo pattern are correlated with the mechanical properties of tissue, such as density, elasticity, and speed of sound. However, the capability of resolving the vasculature for ultrasonography is limited. PAM is a hybrid imaging modality that detects laser induced ultrasonic wave . The imaging contrast comes from the optical absorption. Therefore, PAM is superior for monitoring the vasculature. With different imaging contrast of PAM and ultrasonography, the combination of PAM and ultrasonography can provide complementary information of the tissue during optical clearing. Zharov et al applied OCA to photoacoustic imaging and utilized ultrasonography to observe the structural changes after optical clearing . However, to the best of our knowledge, no work has demonstrated that the combined system could be used in the investigation of OCA. Multiple parameters can be acquired simultaneously by the combined system. And these parameters were correlated with each other, thus gave a bright view of the optical clearing.
In this manuscript, we combined PAM and ultrasonography to investigate the changes of skin during optical clearing, several parameters that measured by PAM and ultrasonography were correlated with the optical clearing process. The measurements ex vivo revealed the changes of different properties of skin during the diffusion process. Furthermore, dual-modality imaging was conducted in vivo. Ultrasonography illustrated the echogenicity and echoheterogeneity which were strongly correlated with the structure and constituents of skin. PAM monitored the changes of subcutaneous blood vessels that were determined by both optical properties and acoustic parameters of skin.
2. Materials and methods
2.1 Experimental apparatus
The experimental apparatus was implemented based on the acoustic-resolution PAM built by our group . As shown in Fig. 1(a), A Nd:YAG laser (Surelite I-30, Continuum, US) was utilized to induce the photoacoustic signal, which had a wavelength of 532 nm, pulse width of 6 ns and pulse repetition of 30 Hz. The laser was coupled into a multi-mode fiber. The laser from the distal end of the fiber was reshaped by a conical lens. Then it was weakly focused into the sample with a focal length of about 6 mm. A focused ultrasonic transducer (Center Frequency: 50 MHz, V30011, Olympus, US) was used to transmit ultrasonic wave and receive the reflected ultrasonic wave as well as the induced photoacoustic wave. A water tank with bottom sealed with membrane was used to couple the ultrasonic wave. A commercial pulser-receiver (5073PR, Olympus, US) was utilized to transmit and amplify the ultrasonic wave. The pulse generator (TFG2006, Shuying, P.R.C.) triggered the pulser-receiver to transmit pulsed ultrasonic wave. Then it also triggered Nd:YAG laser to induce photoacoustic wave with a delay of 6 μs. The oscilloscope (TDS5034B, Tektronix, US) acquired the reflected ultrasonic signal and photoacoustic signal from the pulser-receiver. Meanwhile, a photodiode (DET10A, Thorlabs, US) monitored the incident optical energy to compensate for the energy fluctuations of the laser pulses. B-mode scanning was implemented by a motorized stage (ML03K017, Physik Instrumente, Ger.) which was synchronized by the oscilloscope.
During the ex vivo experiments, we monitored both forward and backward photoacoustic signal. The diagrams of photoacoustic detection were depicted in Figs. 1(b) and 1(c). The forward photoacoustic detection was implemented by a contact transducer (Center Frequency: 50 MHz, V214, Olympus, US). The absorber was in contact with the transducer. And the stimulated forward photoacoustic wave was directly detected by the transducer.
2.2 Sample preparation
In our experiments, Spragure Dawley (SD) rats (about 150 g, Animal Biosafety Level 3 Laboratory, Wuhan, China) were used in both ex vivo and in vivo imaging. All the procedures in the experiments were carried out in accordance to the Institutional Animal Care and Use Committee of Hubei Province.
The ex vivo experiments were conducted with fresh dorsal skin excised. The stratum corneum and subcutaneous fat as well as blood vessels of the whole skin were removed for better penetration of OCAs. The skin samples were immersed with two layers of OCAs to guarantee the penetration of OCAs from both epidermis and dermis. Two sample holders were used to stretch the skin samples and keep the samples still. During ultrasonic measurements, a metal plate was inserted beneath the skin samples for reflecting the ultrasonic wave. During photoacoustic measurements, black tape was inserted beneath the skin samples for generation of photoacoustic wave. Each measurement contained 3 groups of 30 samples in total, including two experimental groups and a control group immersed by phosphate buffered solution (PBS).
Before each in vivo experiment, the rat was anesthetized by intraperitoneal administration of 1 g/kg urethane. And the dorsal hair was removed with depilatory cream (Veet, India). During the following experiment, the body temperature of the rat was kept to be 37 °C by a heating pad. Before the dual-modality imaging, a thin layer of ultrasonic gel was smeared on the dorsal skin and the dorsal skin was affixed to the membrane of the water tank. Each rat was first imaged without further processing to get the control images. Then the imaged area was immersed with OCAs for about 15 minutes to undergo another dual-modality imaging.
2.3 Chemical reagents
Two kinds of commonly used OCAs, glycerol and PEG-400, were selected to conduct the investigation . All the chemical reagents were purchased from Shenshi Chemical Instrument Corporation (Wuhan, China) with a purity of 99.9% and a concentration of 100%. Acoustic speed, impedance and absorption coefficient [Table 1] of the chemical reagents were measured by a flat ultrasonic transducer (V214, Olympus, US) following the procedures in Ref .
2.4 Investigation process
During ultrasonic measurements ex vivo, the transmitted ultrasonic wave passed through multiple OCA-skin interfaces and was reflected by the OCA-metal interface. Sequential B-mode scan was conducted at different samples. The scanning repeated for 20 times which covered about 1 hour. The amplitude of ultrasonic reflection at OCA-skin interface and OCA-metal interface was averaged within each B-scan image. The ultrasonic amplitude was further normalized to the base value.
During photoacoustic measurements ex vivo, the black tape absorbed the penetrated laser to generate photoacoustic wave. Sequential B-mode scan of the black tape was repeated for 1 hour. The amplitude of photoacoustic signal from the black tape was averaged within each B-scan image. Similarly, the photoacoustic amplitude was normalized to the base value. In addition, the arrival time of photoacoustic signal from the black tape was recorded and averaged within each B-scan image. The shift of arrival time was further calculated by subtracting the original arrival time.
During experiments in vivo, B-mode photoacoustic images and ultrasonic echo images were acquired simultaneously. Two sets of images were acquired before and after 15 minutes’ immersion with different OCAs. The photoacoustic images focused on the subcutaneous blood vessels, ultrasonic echo images focused on the structure of skin. Statistical analysis was conducted for the photoacoustic amplitude of subcutaneous blood vessels. The echoheterogeneity of ultrasonic echo images before and after the immersion was also compared. The echoheterogeneity was calculated as the standard deviation of a randomly chosen region in the skin range of the echo images . In order for quantitative comparison, 10 vessel pairs were selected randomly to conduct the statistical calculation. Each parameter was normalized to the control value and the 95% confidence intervals were fitted using normfit in MATLAB (R2010b).
3.1 Changes of forward photoacoustic amplitude
Previous research on optical clearing paid lots of attention to the optical transmittance . PAM is also able to monitor and quantify the optical transmittance. In order to validate the argument, we recorded the forward photoacoustic amplitude. As shown in Fig. 2(a), the black tape was in contact with the transducer, thus the forward photoacoustic signal was detected directly. The photoacoustic amplitude was proportional to the absorption coefficient multiplied by photon density. Since the absorption coefficient kept constant, the amplitude was only determined by the penetrated photon density. The changes of forward photoacoustic amplitude with OCAs were shown in Fig. 2(b). Glycerol showed continuously increased photon density while PEG-400 increased photon density within 20 minutes and kept nearly constant thereafter. The results revealed that glycerol showed greater optical clearing potential than PEG-400 ex vivo. The results were consistent with previous studies using other measurement apparatus .
3.2 Changes of ultrasonic reflection at different interfaces
During the ultrasonic measurements, the ultrasonic incident angle and detection angle kept constant. As a result, the reflection at the OCA-skin interface mainly depended on the difference of acoustic impedance between OCA and immersed skin. As illustrated in Fig. 3(a), both OCAs showed similar tendency. Reflection signal at OCA-skin interface decreased at first and increased thereafter. Considering the different acoustic impedance between OCAs [Table 1] and skin, we deduced that the flux in of OCAs reduced the acoustic impedance mismatch between the skin and OCAs, thereafter introduced a decrease in ultrasonic reflection. However, the continuous diffusion of glycerol and PEG-400 caused serious dehydration . The water loss in skin could lead to continuous increasing of acoustic impedance of skin and finally aggravated the mismatch of acoustic impedance. It is worth noting that, there were inflection points during the observation. This may suggest that there was competition between the diffusion of OCAs and the dehydration. The trends of the curves prompted that the diffusion of OCAs dominated the changes until the emerging of the inflection points. Then the dehydration would govern the trends of the curves. However, the ultrasonic reflection still did not recover to the base value by the end of the observation.
As metal had a much higher acoustic impedance than OCAs, ultrasonic reflection at OCA-metal interface could be regarded as 100%. However, the reflected ultrasonic wave had to pass through multiple skin-OCA interfaces as well as the skin. Along with the flux of OCAs and water, the ultrasonic reflection at skin-OCA interface changed as mentioned before. Meanwhile, the altered constituents of skin changed the absorption coefficient of skin. As a result, the changes of acoustic reflection at the OCA-metal interface [Fig. 3(b)] were determined by both the changes of ultrasonic reflection at the skin-OCA interface and the changes of acoustic absorption by the skin. PEG-400 and glycerol showed a decrease after a short-time rising, which was different from that at skin-OCA interface. Furthermore, the inflection points in the curves of PEG-400 and glycerol may suggest the competition between changes of ultrasonic reflection and absorption of the immersed skin. Before the inflection points, the changes of ultrasonic reflection dominated the trend of the curves, while the changes of ultrasonic absorption became the dominator after that. And the enhancement of absorption coefficient of both OCAs even lasted till the end of the observation. This made the reflection at OCA-metal interface hard to recover to the base value. It is worth noting that PBS also caused subtle changes of ultrasonic reflection. This may because that the physiological parameters of the compounded PBS were a little different from that of the excised skin.
3.3 Changes of backward photoacoustic amplitude
The dual-modality system detected backward photoacoustic signal. As shown in Fig. 4(a), the laser had to be scattered before penetrating into the black tape, which was the same as that in measurements of forward photoacoustic signal. However, the stimulated photoacoustic wave had to be reflected at the OCA-skin interface and absorbed by the skin. The photoacoustic amplitude was determined by the penetrated photon density and ultrasonic penetration efficiency. As a result, the changes of backward photoacoustic amplitude were summation of optical clearing and ultrasonic attenuation. The ultrasonic attenuation was referred to be the acoustic energy loss caused by both acoustic reflection and absorption . The changes of backward photoacoustic amplitude were illustrated in Fig. 4(b). Different from the curves in Fig. 2(b), glycerol showed a turning point at 25 minutes. PEG-400 caused decreased amplitude since 15 minutes. With more than 35 minutes’ immersion, PEG-400 even caused the skin to weaken the photoacoustic signal.
3.4 Shifting of photoacoustic arrival time
The flux in of OCAs, flux out of water may change the constituents and thickness of the skin. As depicted in Fig. 5(a), the photoacoustic signal from the black tape had to go through the OCA layer with a length of L-L1, and also the skin with a length of L1. Altered constituents led to changed speed of sound in skin, dehydration caused thinner skin. Both cases shifted the arrival time of photoacoustic signal. In Fig. 5(b), the arrival time of the black tape both negatively shifted for glycerol and PEG-400. Glycerol, with the maximum speed of sound, caused notable time shift. The slope of the delay time also revealed the diffusion rate of OCAs. PEG-400 showed a fast diffusion rate only during the first 12 minutes, while glycerol lasted for about 45 minutes. We speculated that glycerol may penetrate more deeply compared with PEG-400 ex vivo, without any penetration enhancers. The accuracy of shift time could be estimated according to the sampling rate and axial resolution. The sampling rate of 250 MHz provided a temporal resolution of 4 ns. The axial resolution (15 μm) may blur the time stamp of arrival, but the subtraction significantly weakened the influence. As a result, the accuracy should be no worse than 10 ns.
3.5 Dermal changes during optical clearing in vivo
The OCAs were then applied to rat dorsal skin in vivo. PAM and Ultrasonography monitored dermal changes from different perspectives simultaneously. As shown in Fig. 6, PEG-400 caused subtle enhanced photoacoustic amplitude of subcutaneous blood vessels, with a factor of 1.67 ± 0.28, indicating mild increasing of photon density at the blood vessel layer as a result of optical clearing. The blood vessels were slightly closer to the skin surface along with the dehydration. Ultrasonic echo images in Figs. 6(c) and 6(d) showed shallow imaging depth but more speckles in skin. The echoheterogeneity was 1.22 ± 0.11 times after application of PEG-400, showing slightly densely packed collagen fibers. The fused images in Figs. 6(e) and 6(f) gave an intuitive display of the changes of skin, with the subcutaneous blood vessels and dermal structure shown together. As we expected, the result of the PEG-400 was not good as previous research. This was because it was hard for PEG-400 to penetrate into the dermis without penetration enhancer, such as thiazone .
Dual-modality images before and after immersion with glycerol were showcased in Fig. 7. Glycerol induced similar dehydration compared with the PEG-400. Photoacoustic signal of subcutaneous blood vessels was enhanced with a factor of 2.31 ± 0.28, indicating moderate optical clearing effect. The vessels were also closer to the skin surface along with the dehydration. Ultrasonic echo images were similar to that immersed with the PEG-400. The echoheterogeneity was 1.27 ± 0.10 times after optical clearing, indicating densely packed collagen fibers. The fused images in Figs. 7(e) and 7(f) were registered well within the two modalities, further proving the capability of assessing dermal changes during optical clearing. It is worth noting that the skin surface enhancement in photoacoustic images was side effects of optical clearing. The surface enhancement weakened the enhancement of subcutaneous blood vessels. And the surface signal should be removed before volumetric visualization. Here we just presented the raw images to show the optical clearing effects.
Optical clearing of tissue is a complex process during which flux of reagents and molecular interactions  may happen together. Multi-parameter monitoring provides multiple perspectives, promoting a better understanding of the mechanism of optical clearing. In this manuscript, PAM and ultrasonography were combined to monitor the changes of photoacoustic and ultrasonic echo signal. The ultrasonic amplitude in echo images, recognized as echogenicity, indicated the capability of reflecting ultrasound of skin. The echo speckle, recognized as echoheterogenity, indicated the capability of scattering ultrasound. The former corresponded to the constituents of skin, while the latter corresponded to collagen distribution and pattern . Photoacoustic images focused on the subcutaneous blood vessels, which can reveal the optical transmittance and gave an estimation of interactions between optical penetration and acoustic attenuation.
The key results were summarized in Table 2. The forward photoacoustic measurement was consistent with data acquired by spectrophotometer , where glycerol and PEG-400 caused a relative transmittance of 2 and 1.6, respectively. While the backward photoacoustic measurement supplied additional information about the dehydration. Combined with the ultrasonic echo and photoacoustic amplitude measurements, we concluded the temporal relationship between optical clearing and dehydration quantitatively, which was hard to derive using other modalities. As ultrasonography is sensitive to depth, the measurement of arrival time was accurate, thus gave a quite reasonable estimation of diffusion rate. The results were similar to that measured by Raman spectroscopy , where the diffusion rate showed a trend from ascent to descent and reached the maximum at about 30 minutes. Previous research on configuration of fibers had to take advantage of skin chamber, while ultrasonography can give us the estimation with skin intact. The echoheterogeneity measurement met the results by second harmonic generation , where 27.3% fineness of collagen fibers was observed.
During the measurements ex vivo, PAM based on the detection of forward photoacoustic signal can be used to assess the optical transmittance. As visualized in Fig. 2, glycerol and PEG-400 showed great optical clearing effect. However, PAM based on the detection of backward photoacoustic signal [Fig. 4] showed moderate optical clearing effect, especially after a long time immersion. The ultrasonic measurements in Fig. 3 provided a reasonable explanation. Ultrasonic reflection and absorption changed during the optical clearing process, introducing decreased ultrasonic penetration after long time immersion. Although the optical transmittance was enhanced after immersion, the generated photoacoustic wave attenuated more through the skin. Finally the optical clearing effect was weakened. The diffusion rate was monitored through shifting of arrival time. The permeability of OCAs is less than that of water , leading to thinner thickness and higher density of skin. The speed of sound in skin can be considered to be determined only by the density under constant temperature . As a result, the diffusion of OCAs induced higher speed of sound in skin and finally negatively shifted the arrival time of photoacoustic signal. Higher diffusion rate caused rapid changes of density and thickness, leading to rapid shift of arrival time. As shown in Fig. 5, by the end of the observation, the diffusion of OCAs and water was in dynamic equilibrium. It is worth noting that after immersion of 60 minutes, the ultrasonic and photoacoustic signal were still changing. Although the diffusion process was completed after 60 minutes , the molecular interaction  was still continued, which may alter the optical and ultrasonic parameters for a long time.
The parameters monitored ex vivo were correlated with each other. All parameters reflected the diffusion process, but with different quantitative relationship. The enhancement of forward photoacoustic signal for glycerol was sustained throughout the observation. While that for PEG-400 was only sustained during the first 20 minutes. The shifting of photoacoustic arrival time revealed that the diffusion process for glycerol was about 45 minutes, while for PEG-400 it was only 12 minutes. It can be deduced that the optical clearing was maintained during and shortly after the diffusion process. The shorter diffusion process for PEG-400 caused earlier appearance of inflection point in curve of ultrasonic reflection at skin-OCA interface. The diffusion process brought serious dehydration and increased absorption of ultrasound. The dehydration quickly governed the trend of ultrasonic reflection at skin-OCA interface. As the ultrasonic amplitude after absorption was exponentially decayed, the increased ultrasonic absorption pulled down the ultrasonic reflection at OCA-metal interface severely. Finally, the backward photoacoustic signal was summation of optical transmittance and ultrasonic attenuation. The continuously increased ultrasonic attenuation quickly governed the trend of backward photoacoustic signal. However, different test conditions and different affecting factors during the diffusion caused complex relationship among the measured parameters. We can observe the interaction trends of different parameters, but a quantitative relationship among the parameters was hard to obtain.
The capability of assessing dermal changes during optical clearing was further validated in vivo. The PEG-400 was proved to be with mild optical clearing effect in vivo, along with the dehydration. The dual-modality images provided an intuitive visualization of enhanced subcutaneous blood vessels and closer distribution from the skin surface. What is more, the echo pattern provided an estimation of enhanced echoheterogeneity. We suggested that this phenomenon proved the hypothesis that more densely packed collagen fibers will come along with the dehydration. The observation of skin immersed with glycerol was similar to that with PEG-400. It has to be noted that some trials did not show optical clearing effect with glycerol, and dehydration was not found during such trials. It can be concluded that the optical clearing effect with glycerol came with dehydration, and the dual-modality system can give a reasonable prediction of optical clearing. It was strange that we did not observe the changes of ultrasonic echo pattern ex vivo. As the speckles mainly came from the collagen fibers, we suspected that the excision and processing of skin may destroy the fiber structure.
Combined system of PAM and ultrasonography was validated in the investigation of dermal changes during optical clearing. Multiple parameters were monitored during optical clearing, and revealed the changes of constituents and structure of skin. The capability was further validated during in vivo experiments. Therefore, the dual-modality system can be potentially used in investigating the optical clearing effect and helping us to screen appropriate OCAs. On the other hand, the capability was not limited to skin optical clearing. Optical clearing on other tissues such as skull  and muscle  can also be investigated by the dual-modality system.
This work was supported by National Major Scientific Research Program of China (Grant No. 2011CB910401), Science Fund for Creative Research Group of China (Grant No. 61121004), and National Natural Science Foundation of China (Grants No. 81201067, 81171376).
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