1.

Introduction

Photoacoustic microscopy (PAM) has been demonstrated as an advanced biological or biomedical imaging technique capable of detecting the physiological specific endogenous chromophores based on the optical absorption properties of the investigated target.^1–3 This imaging modality utilizes the acoustic wave generated by the laser-induced thermal–elastic expansion of the target to construct an image. Based on its focusing mechanism, two types of PAM have been developed: acoustic-resolution PAM and optical-resolution PAM (OR-PAM).^4,5 In OR-PAM, the lateral resolution primarily depends on the optical focal spot size and the axial resolution is determined by the ultrasonic transducer. Nevertheless, in strong scattering tissue such as skin, breast, bioliquids, etc., the optical focusing capability degrades due to optical scattering. Meanwhile, in the visible and near infrared (NIR) wavelengths, scattering in tissues is higher than the absorption. In addition, the imaging depth of OR-PAM is limited by the optical transport mean free path and affected by both photon absorption and scattering.^6,7 In order to overcome these challenges, various physical and chemical methods have been deployed to enhance photoacoustic (PA) imaging depth, PA signal-to-noise ratio and imaging resolution such as careful choosing of the excitation laser wavelength and utilization of optical contrast agents (OCAs).^7,8–10

In recent years, the optical tissue clearing (OTC) technique has shown great potential in inducing optical clearing effects (OCEs) to reduce scattering in tissues using hyperosmotic and biocompatible chemicals agents.^11–14 The OCE induced by using OCA and the mechanisms of optical clearing (OC) are also discussed.^15–20 Recently, numerous investigations on OCA introduced effective OCAs, such as glucose solutions, dimethyl sulfoxide solution, glycerol, propylene glycol, etc., which have refractive index close to that of collagen and are applicable to alter the scattering properties of tissues.^{11,12,21–24} Studies done by Ku and Wang⁸ and Wang et al.⁹ demonstrated that the application of OCA into chicken breast muscle and animal brains can significantly reduce light scattering and thereby enhance the resolution and imaging depth of photoacoustic tomography (PAT). Liu et al.²⁵ also proved that biocompatible and rapid OC of skin can minimize light scattering and thus increase optical resolution and sensitivity of PA flow cytometry.

Among all the PA imaging technologies, laser-scanning optical-resolution photoacoustic microscopy (LSOR-PAM) is a relatively new one for imaging biological tissue at a microscopic scale. It has demonstrated capabilities to improve scanning speed using two-dimensional galvanometer²⁶ and perform approximately real-time microscopic scale three-dimensional imaging of biological tissues.^27–29 The contrast and depth of this imaging technology is still limited as they are based on the laser focusing capability, which degrades due to the high scattering of tissue as mentioned above. Glycerol is one of the most common and efficient OCA in skin OC in vitro and in vivo,^30,31 and it has been proved in medical applications such as tooth therapy and cosmetics study.³²

The OTC methods use high-refractive OCA mainly in order to reduce light scattering and thus enhance the light penetration depth in tissue. Although, a lot of research has been done to assess the OTC efficacy by various optical methods such as NIR reflectance spectroscopy and optical coherence tomography (OCT), none of the studies has investigated OCE with different quantities of OCA on LSOR-PAM imaging method. Meanwhile, optimum concentration of OCA is important in such applications as a low concentration would not generate sufficient OCE and a high concentration would damage the biological tissues, even obstruct the blood vessel or cause the blood flow to stop.^33,34 In this study, we demonstrate that both the imaging visibility and PA signals amplitude of LSOR-PAM can be enhanced by using glycerol–water solution of different concentrations, which is one of the most efficient used OCA for biomedical imaging.

2.

Materials and Methods

2.1.

LSOR-PAM System

The schematic of our experimental LSOR-PAM system is shown in Fig. 1. A pulsed Nd:YAG laser (SPOT-10-100-532, Elforlight, Northampton, United Kingdom; Pulse width $<1.8\mathrm{ns}$) with a 532-nm wavelength and a repetition rate of 10 kHz is used in the experiment. The laser pulses are transferred into a fiber coupling and collimation system including two FiberPorts (PAF-X-11-A, Thorlabs, New Jersey) and a single-mode fiber, and then expanded twofold through two achromatic lenses with 30 mm (AC254-030-A1, Thorlabs) and 60-mm (AC254-060-A1, Thorlabs) focal length. The expanded laser pulses are directed to an $x–y$ galvanometer scanner (TSH8203M, Century Sunny Technology Co. Ltd., Beijing, China) and focused by a 30-mm focal length objective lens (AC254-030-A1, Thorlabs). The laser irradiation generation and $x–y$ galvanometer scanner are controlled and synchronized by PC1 in Fig. 1 using the scan controlling card (NI6221, National Instrument, Austin). An unfocused ultrasonic transducer (V356, Olympus NDT, Tokyo, Japan; center frequency: 30 MHz) is kept stationary to detect the PA signal. The signal is amplified by two amplifiers (ZFL-500, Mini-Circuits, New York; bandwidth: 0.1 to 500 MHz, gain: 28 dB) and then digitized and recorded by the acquisition card (CompuScope 14200, GAGE, Lockport; sampling rate: $200\mathrm{MS}/\mathrm{s}$) controlled by PC2 in Fig. 1. The fast photodiode (DET10A, Thorlabs) is used to detect the laser pulses and triggered the acquisition.

Fig. 1

Schematic of the experimental optical-resolution photoacoustic microscopy (OR-PAM) system. PD: photodiode; BS: Beam splitter; AL1-AL2: achromatic lenses; two-dimensional (2-D) GM: galvanometer; OL: objective lens; PC1-PC2: personal computer UT: ultrasonic transducer.

We measured the resolution of this experimental system through imaging of gold nanoparticles (diameter $<100\mathrm{nm}$) solution and calculation of the full-width half-maximum (FWHM) of the Gaussian fitting results.³⁵ The lateral and axial PA amplitude profiles in Fig. 2 are acquired from the PA response of one nanoparticle. The lateral and axial resolutions are estimated to be 5 and 50 μm according to the FWHM values. The laser pulse energy leaving objective lens is 40 nJ in both resolution measurement and the following experiments.

Fig. 2

Resolution of the imaging system by imaging of nanoparticles ($\text{diameter}<100\mathrm{nm}$). (a) Lateral cross-section profile of a nanoparticle; (b) axial cross-section profile of a nanoparticle.

2.2.

Materials and Measurements

Fresh abdominal porcine skin tissues are used as scattering biological tissues to cover a phantom light absorber. The phantom is the registered trademark sign [the ® sign in Fig. 3(a)] of a ruler. Each fresh skin is depilated and rinsed briefly with normal saline solution. The skin is cut into $\sim 1\times 1{\mathrm{cm}}^2$ squares samples with a mean thickness of 0.5 mm by microtome. Samples are stored in a refrigerator at $-70°\mathrm{C}$ for in vitro experiment. The anhydrous glycerol is purchased from Tianjin Damao Chemical Reagent Factory, China. Before experiments, glycerol is diluted to different concentrations, i.e., 20%, 40%, and 60% with distilled water. The solutions have a mean refractive index of 1.36, 1.39, and 1.41, respectively.³⁶ Accordingly, the experiments are divided into groups using 20%, 40%, and 60% glycerol solutions, respectively. Each concentration group consists of four independent experiments. The skin samples are defrosted in physiological saline at room temperature ($\sim 25°\mathrm{C}$) for $\sim 10\min$. The register trademark sign is covered by a piece of skin sample and submerged completely in the distilled water and imaged for 3 min to obtain a baseline value using LSOR-PAM. Then, the sign covered with a new skin sample is immersed in the glycerol solution of a certain concentration for 1 h. The PA signals and PAM images are obtained every 15 min during the period for each independent experiments of that concentration group. The procedure is repeated for 20%, 40%, and 60% glycerol solutions. Each skin sample is used only once.

Fig. 3

Photograph of the ruler used and its registered trademark sign (a) and the corresponding 2-D OR-PAM maximum amplitude projection (MAP) image obtained 15 min after application of the 60% glycerol solution. (b) The red solid square in (a) is the registered trademark sign to be imaged. The photoacoustic (PA) signals from the red dotted square area in (b) are statistically analyzed.

The OCE of each glycerol solution at time intervals 15, 30, 45, and 60 min is evaluated by calculating the enhancement in PA as

Eq. (1)

$$\mathrm{SEC}=\frac{T_{\text{treated}}-T_{\text{control}}}{T_{\text{control}}},$$

where $T_{\text{control}}$ is the sum of PA signal amplitude in the red dotted square area in Fig. 3(b) obtained at 0 min and $T_{\text{treated}}$ is the sum obtained at time interval 15, 30, 45, and 60 min.

3.

Results and Discussion

In order to research the OCE of glycerol solutions with different concentrations on the LSOR-PAM imaging modality, the maximum amplitude projection (MAP) images of the registered trademark sign of a ruler covered with the porcine skin tissue are obtained at different time stages. Figure 4 shows photographs of a registered trademark sign covered with a porcine skin tissue under different concentrations of glycerol solution 15 min after the glycerol application, respectively. In Fig. 4, the different OCE is reflected by the visibility of the sign without glycerol as a control, (a) 20% glycerol, (b) 40% glycerol, (c) 60% glycerol, and (d). Visibility differences are mainly due to the hyperosmotic glycerol resulting in refractive index matching between cellular tissue components: cell membrane, cell nucleus, cell organelles, melanin granules, and the extracellular fluid.^11,14 Visibility differences in Figs. 4(b)–4(d) demonstrate that with the increasing of concentration of glycerol, the OCE of skin is dramatically improved. 60% glycerol resulted in the greatest increase of light transmittance and the sign integrality, indicating that more light may be through the skin tissue and delivered into the sign by applying 60% glycerol than others concentrations. The results obtained were similar to these of Mao et al.,³⁷ who have demonstrated larger optical skin clearing improvement than that with low-concentration glycerol injected into the dorsal dermas of Sprague Dawley (SD) rats from reflectance spectra. Also, Zhong et al.³⁸ also have proved that the high-concentration solutions induce more transparency of skin than the surroundings tissue. Figure 5 shows the change of LSOR-PAM MAP images of the sign covered with a porcine skin tissue at different time (0, 15, 30, 45, and 60 min) immersed in 20%, 40%, and 60% glycerol solution. Visibility of the sign is improved with the increasing of time and concentration of glycerol in all groups. Still, comparing Figs. 5(a)–5(c), there is a striking difference between each two different concentration groups. The sign becomes slightly clearer after being immersed in 20% glycerol group, but there is still no clearly visible and complete image during the whole process. Meanwhile, the sign is always visible in 40% glycerol group. Moreover, the improvement of sign visibility is extremely prominent in 60% glycerol group as the MAP images are quite clear and complete. The PA signal amplitude changes of different glycerol concentration solutions at time intervals 15, 30, 45, and 60 min are shown in Fig. 6. The better MAP image quality reflects that PA signal amplitude in higher glycerol concentration groups is stronger as can be seen from Fig. 6. The PA signal amplitude can be caused by light penetration differences or ultrasound transmitting change. As higher concentration glycerol solution has higher acoustic attenuation, stronger PA signal amplitude in higher glycerol concentration groups is due to the improved OCE of higher concentration glycerol, which leads to more light penetrating through the skin tissue. A further analysis of the increases of PA signal amplitude is presented in Fig. 7. The result, in Figs. 6(a)–6(h), corresponds to the Fig. 5 results described above. Figure 6 demonstrates that PA signal amplitude increases with the increasing of glycerol concentration at time intervals 15, 30, 45, and 60 min, respectively. From the Fig. 7, we especially observed that PA signal amplitudes of the sign show significant enhancement after applying glycerol solutions with different concentrations gradient. For the low concentration of 20% glycerol, the greatest increase in PA signal amplitude is only 8.1% at 15 min. For the moderate concentration of 40% glycerol, the greatest increase in PA signal amplitude is 76% at 15 min. However, for the high concentration of 60% glycerol, the greatest increase in PA signal amplitude remarkably increases to 165.5% at 30 min, which is 20.4-fold and 2.2-fold to that of 20% and 40% glycerol at 15 min, respectively. As can be seen from Fig. 5, for both 20% and 40% glycerol groups, the visibility of the sign is best at 15 min, whereas for 60% glycerol group, the best is at 30 min. This result corresponds to the Fig. 7 showed above. The difference in the tendency of PA signal amplitude change possibly reflects that high concentrated glycerol solution’s diffusion is lower than solutions with a lower concentration. This phenomenon is consistent with previous studies on the relationship of the concentration of aqueous sucrose solution and its diffusion through semi-permeable membranes.^39,40 We believe that the enhancements of PA signal amplitude were mainly caused by the enhancement of the optical penetration due to the reduced scattering coefficient imparted by application of the OCAs and that higher glycerol concentration may result in stronger dehydration effect.⁴¹ The steady-state thermodynamics of water permeation could provide an additional explanation to the behavior of the permeability observed in the study.³⁹ It is mainly due to that the biological tissues are mostly hypertonic to the applied glycerol–water solution and the water molecules would then spill out of the tissue to reach equilibrium with the concentration gradient between the intercellular space and cell membranes.⁴² When the concentration difference is small, such as a dilute solution, the impedance caused by water’s flow is not significant and does not greatly affect the flow of glucose.⁴³

Fig. 4

Photographs of the registered trademark sign covered with a porcine skin tissue under 0%, (a) 20%, (b) 40%, (c) 60%, and (d) glycerol at 15 min, respectively. The black circle is the location of the treatment site of the imaging sign.

Fig. 5

The OR-PAM MAP images of the sign covered with the skin immersed in glycerol solutions imaged at different time intervals. (a) MAP images with the 20% glycerol (b) MAP images with 40% glycerol. (c) MAP images with 60% glycerol.

Fig. 6

The normalized PA signal curves and amplitudes changes of the phantom immersed in 20%, 40%, and 60% glycerol at 15 min (a and e), 30 min (b and f), 45 min (c and g), and 60 min (d and h), respectively.

Fig. 7

Dynamic changes of the PA amplitude enhancement after application of 20%, 40%, and 60% glycerol at 0, 15, 30, 45, and 60 min, respectively.

From the analysis of the aforementioned results, we could conclude that the PAM imaging can be optimized through employing OCA like glycerol. And our results also demonstrate the importance of concentration of OCA to achieve desired OCE on the PAM imaging. In order to provide a more realistic guide for PAM imaging optimization through OTC technology, we would further investigate the OCE of the different kinds and different concentrations of OCA on in vivo OR-PAM imaging using animal models. And, it is possible that optical time for PA maximal amplitude is $<15\min$ for the low glycerol concentration, so more researches on studying the real time mutual function mechanism on OR-PAM imaging with OTC technology are necessary. Nonetheless, we for the first time demonstrated that the optical penetration ability and LSOR-PAM imaging depth and contrast could be enhanced by OTC technology. With this technology PAM imaging will have a greater potential for applications in clinical diagnosis and therapy in dermatology.

4.

Conclusions

In this study, we investigated the OCE of different concentrations of glycerol solutions in biological tissue on LSOR-PAM imaging. The time-varying and concentration-dependent properties of PA signal amplitude were determined. We have demonstrated a significant improvement of LSOR-PAM imaging performance using tissue OC technique. The PA signal amplitudes and the visibility of a phantom light absorber covered by skin tissues were found to be increasing with the concentration of the glycerol solution. The OCE of the skin tissue caused by OCA could improve the imaging depth and contrast of LSOR-PAM imaging. This result agrees well with studies of the deeply penetrating PAT of biological tissues enhanced with an OCA. Results presented here suggest that this tissue OC method has the potential to become a useful tool for the enhancement of the PAM imaging of biological tissues. And this study may provide a guideline regarding to the use of glycerol for optimal diagnostic and therapeutic effects in dermatology.