In vivo dual-scale photoacoustic surveillance and assessment of burn healing.

Accurate diagnoses of superficial and deep dermal burns are difficult to make even by experienced investigators due to slight differences in dermis damage. Many imaging technologies have been developed to improve the burn depth assessment. But these imaging tools have limitations in deep imaging or resolving ability. Photoacoustic imaging is a hybrid modality combining optical and ultrasound imaging that remains high resolution in deep imaging depth. In this work, we used dual-scale photoacoustic imaging to noninvasively diagnose burn injury and monitor the burn healing. Real-time PACT provided cross-sectional and volumetric images of the burn region. High-resolution PAM allowed for imaging of angiogenesis on the hyperemic ring. A long-term surveillance was also performed to assess the difference between the two damage degrees of burn injuries. Our proposed method suggests an effective tool to diagnose and monitor burn injury.

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Animal preparation
All in vivo animal experimental procedures were approved by the Institutional Animal Care and Use Committee of Xiamen University. Male white mice (ICR, 20~22 g), bought from Xiamen University Laboratory Animal Center, were anesthetized using inhalation of 2% isoflurane to guarantee the entire procedure performed under anesthesia. The mouse hair in dorsal skin was firstly shaved and then depilated gently using hair removal creams. To avoid creams-induced chemical irritation on skin, the mice were raised normally for 24 h before burn modeling. Burn injuries were made on exposed dorsal skin by direct contact with an electric soldering iron thermostatically set to a temperature at 200°C, which is confirmed by a thermal temperature sensor (Ax5, FLIR). Mice were randomly divided into two groups and inflicted heating with 5 and 8 seconds duration, respectively, to model SDB and DDB injuries. To make different models, the burn areas were 10 × 6 mm 2 shaped in ellipse for PACT and 4 mm diameter round for PAM, respectively. Before imaging, ultrasonic gel was applied on skin and a customized water tank filled with deionized water was placed upon gel to optimize PA signals transmission efficiency. The bottom of water tank was a layer of transparent polyethylene membrane that allows laser beam and acoustic wave to travel through. While imaging, the mice limbs were fixed and kept still to avoid image artifacts brought by skin movement. A time point experiment was performed to investigate the PA signal changes of mouse skin healing after a burn injury. The healing process was completely natural without any ointments promotion.

PACT imaging
PACT images were collected using a commercial imaging system (Vevo LAZR-X, FUJIFILM VisualSonics Inc.) with a 40 MHz ultrasound array transducer (MX550D, FUJIFILM VisualSonics Inc.). When imaging, the ultrasound probe was immersed in water for acoustic coupling. The laser pulse was tunable in visible and near-infrared spectrum (680-970 nm and 1200-2000 nm), which was delivered through optical fiber bundles connected to both sides of ultrasound transducer and converged in front of transducer arrays. The operating laser wavelength was 680 nm in anatomical imaging for the reason that it was the shortest available wavelength with highest hemoglobin absorption. Meanwhile, 750/850 nm were selected in functional imaging because oxyhemoglobin (HbO 2 ) have higher absorption than deoxyhemoglobin (HbR) at 750 nm but HbR is dominant at 850 nm. The distinct molar extinction coefficients enable measuring the concentrations of HbO 2 and HbR to calculate sO 2 by the following equation [ . (1) The PACT imaging head was stabilized to a stepper motor to perform 3D volumetric imaging under a field of view (FOV) of 15 × 12 mm 2 with a step size of 0.1 mm, which generated about 150 frames images per scan. After that, multi-wavelength PA data were stored and reconstructed in VevoLab software (FUJIFILM VisualSonics Inc.) to display PA and sO 2 images. Here, every frame of 3D data presented cross-sectional view of skin tissue and could be processed individually. Burn region was outlined for quantification by circling PA burn region of interest (ROI) in every frame and fitting all circles to generate 3D burn volume and calculate signal intensities.

PAM imaging
We built one laboratory high-resolution PAM to image the partial burn injury. Our PAM was characterized to have a lateral resolution of 10 μm at depth of 0.7 mm, which was enough to satisfy our imaging requirements. The laser wavelength is 532 nm with 7 ns pulse width (AONano532-1-40-V, Advanced Optowave Corporation), where hemoglobin has high molar extinction coefficient. A collimated laser beam was focused by a 4 × objective lens (RMS4X, Thorlabs) on skin surface, traveling through an optical-acoustic combiner designed to separate laser beam and acoustic wave. The incident laser pulse energy focused on sample surface is well within the American National Standards Institute safety limits [19]. The laser induced PA signals were then acquired by a 50-MHz central frequency ultrasound transducer (V214-BB-RM, Olympus) and digitized by a 14-bit data acquisition card (CSE1422, GaGe Applied Science). A raster scanning was performed to acquire data. Two-dimensional maximum-amplitude-projection (MAP) images were reconstructed by projecting maximum PA amplitude of each one-dimensional depth-resolved data. The reconstructed gray PAM images were processed and pseudo colored to enhance image contrast.

Histological examination
The mice were sacrificed after experiments. Then skin tissues containing burn injury were biopsied and sectioned. The sections were then fixed and stained with hematoxylin and eosin (H&E).

Results and discussion
As depth measurement is important in burn diagnosis, we first investigated the depth changes of mice burn healing process in SDB and DDB groups (n = 3, respectively). To evaluate depth values, cross-sectional PA images were acquired at pre-burn and post-burn 1, 2, 3, 4, 5, 7, 10, 14, 21 days (Fig. 2). Here, burn depths at each time point were calculated by measuring the PA-signal distance from skin surface to underlying layer as indicated by white double arrows in Figs. 2(a) and 2(c). The quantitative burn depth changes were plotted in Fig. 2(e). Before burn damage, the whole normal skin depths were 0.71 ± 0.01 mm and 0.54 ± 0.03 mm in SDB and DDB, respectively. When thermal damage occurred, the burn depth rose sharply and stayed high in both SDB and DDB. The difference was, in the first three days, the mean burn values of DDB depth (lowest, 1.68 ± 0.10 mm) were deeper than 1.6 mm while SDB (highest, 1.55 ± 0.15 mm) were lower than 1.6 mm. Therefore, the depth value of 1.6 mm could be a critical point distinguishing SDB from DDB [13]. The signals pointed by the yellow arrows came from the eschar of the skin surface. The more severe burn led to stronger PA signal since the light absorption of the eschar was much higher than that of hemoglobin. This could explain why PA signal intensity of skin surface, compared to that in SDB, was much higher in DDB in Fig. 2(c). As burn wound recovering, the burn depth started to decrease gradually from day 4. Hence, the depth diagnosis should be performed as early as possible in the first three days in case of inaccurate results. Since post-burn day 14, burn depth had turned down to a level as low as normal skin.
As for cross-sectional sO 2 images in Figs. 2(b) and 2(d), the burn depths also changed along with burning process. But the regularity was not as continuous as PA images showed, because sO 2 was changeable owing to blood perfusion. Therefore, the depth of sO 2 is unreliable to assess burn depth. The more important point conveyed in sO 2 images was that central area of burn had more hypoxia compared against surrounding area in first week. This was mainly caused by dermis damage lacking blood perfusion, especially in DDB. High magnification (100-fold) H&E histology images of skin tissues in SDB and DDB were illustrated in indicate the b while reticula more severe th   how the 3D sO 2 us hypoxia area s in Fig. 2(d)