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Waterproof Galvanometer Scanner-Based Handheld Photoacoustic Microscopy Probe for Wide-Field Vasculature Imaging In Vivo

Waterproof Galvanometer Scanner-Based Handheld Photoacoustic Microscopy Probe for Wide-Field... hv photonics Communication Waterproof Galvanometer Scanner-Based Handheld Photoacoustic Microscopy Probe for Wide-Field Vasculature Imaging In Vivo 1 , † 1 , 2 , † 1 , 3 1 1 1 Daewoon Seong , Sangyeob Han , Jaeyul Lee , Euimin Lee , Yoonseok Kim , Junsoo Lee , 1 , 1 Mansik Jeon * and Jeehyun Kim School of Electronic and Electrical Engineering, College of IT Engineering, Kyungpook National University, Daegu 41566, Korea; smc7095@knu.ac.kr (D.S.); syhan850224@knu.ac.kr (S.H.); jaeyul21@g.ucla.edu (J.L.); augustmini@knu.ac.kr (E.L.); otter0618@knu.ac.kr (Y.K.); jslee5399@knu.ac.kr (J.L.); jeehk@knu.ac.kr (J.K.) Institute of Biomedical Engineering, School of Medicine, Kyungpook National University, Daegu 41566, Korea Department of Bioengineering, University of California, Los Angeles, CA 90095, USA * Correspondence: msjeon@knu.ac.kr; Tel.: +82-53-950-7846 † These authors contributed equally to this work. Abstract: Photoacoustic imaging (PAI) is a hybrid non-invasive imaging technique used to merge high optical contrast and high acoustic resolution in deep tissue. PAI has been extensively developed by utilizing its advantages that include deep imaging depth, high resolution, and label-free imaging. As a representative implementation of PAI, photoacoustic microscopy (PAM) has been used in preclinical and clinical studies for its micron-scale spatial resolution capability with high optical absorption contrast. Several handheld and portable PAM systems have been developed that improve its applicability to several fields, making it versatile. In this study, we developed a laboratory- customized, two-axis, waterproof, galvanometer scanner-based handheld PAM (WP-GVS-HH-PAM), which provides an extended field of view (14.5  9 mm ) for wide-range imaging. The fully waterproof handheld probe enables free movement for imaging regardless of sample shape, and Citation: Seong, D.; Han, S.; Lee, J.; volume rate and scanning region are adjustable per experimental conditions. Results of WP-GVS-HH- Lee, E.; Kim, Y.; Lee, J.; Jeon, M.; Kim, J. Waterproof Galvanometer PAM-based phantom and in vivo imaging of mouse tissues (ear, iris, and brain) confirm the feasibility Scanner-Based Handheld and applicability of our system as an imaging modality for various biomedical applications. Photoacoustic Microscopy Probe for Wide-Field Vasculature Imaging In Keywords: photoacoustic microscopy; handheld probe; wide-field imaging; in vivo vasculature Vivo. Photonics 2021, 8, 305. https:// imaging; 3D imaging doi.org/10.3390/photonics8080305 Received: 29 June 2021 Accepted: 28 July 2021 1. Introduction Published: 30 July 2021 Photoacoustic imaging (PAI) is a label-free, non-invasive biomedical imaging tech- nique that has been extensively studied and developed [1,2]. PAI is based on the light- Publisher’s Note: MDPI stays neutral induced ultrasound (US) signal through the photoacoustic (PA) effect (i.e., thermal-elastic with regard to jurisdictional claims in expansion) [3]. The optical absorption contrast of target biological tissues determines the published maps and institutional affil- intensity of the broadband US waves (i.e., PA waves), which are converted into analog iations. electrical signals by US transducers [4]. Since the degree of scattering of PA signal in biological tissue is comparably less than that of the optical beam, PAI offers improved optical sensitivity with high US resolution compared to other optical imaging modalities in optically turbid media [5]. In addition, using the PA effect, PAI enables the non-invasive Copyright: © 2021 by the authors. characterization of biological and biomedical properties with endogenous and exogenous Licensee MDPI, Basel, Switzerland. agents such as metabolism, anatomy information, functional data, and molecular pro- This article is an open access article cesses [1,6]. Based on the aforementioned distinctive advantages, PAI has been functionally distributed under the terms and used in various applications including vasculature mapping, assessing hemoglobin oxygen conditions of the Creative Commons saturation, and blood flow [7–10]. Attribution (CC BY) license (https:// In particular, photoacoustic microscopy (PAM) is a major implementation of PAI, creativecommons.org/licenses/by/ which provides rich optical absorption with enhanced spatial resolution [11,12]. PAM is 4.0/). Photonics 2021, 8, 305. https://doi.org/10.3390/photonics8080305 https://www.mdpi.com/journal/photonics Photonics 2021, 8, 305 2 of 10 largely divided into two different types: optical resolution (OR) and acoustic resolution (AR) PAM, according to the focus type (i.e., optical and acoustic focus, respectively) [13]. OR-PAM provides a high spatial resolution of the subcellular level compared with AR-PAM by tight focusing of the optical excitation beam [14]. Since the ballistic photon regime and scattering limit the penetration depth of OR-PAM, it is required to optimize the spatial resolution and imaging depth because of the trade-off between spatial resolution and depth of focus [15]. With regard to AR-PAM, although the lateral resolution is inferior to that of optical imaging modalities, the penetration depth is enhanced by utilizing comparably weak acoustic scattering in biological tissue than the optical diffusion limit [16]. By utilizing the advantages of each imaging technique, PAM has been investigated for biomedical applications [17–20]. To expand the applicable conditions and enhance the versatility of PAM compared to the bench-top system, several miniaturized handheld PAM (HH-PAM) have been de- veloped [21–23]. To enable fast-scanning with the miniaturized probe, various imaging scanners-based HH-PAM systems were reported (e.g., micro-electro-mechanical system (MEMS) and galvanometer scanner (GVS)). To reduce the volume size of the HH-PAM probe, MEMS mirror has been widely employed following the rapid development of MEMS systems [24–26]. Because of the small size of a MEMS mirror, the dimension of the HH- PAM probe is able to be optimized according to the applications. However, the scanning region and scanning stability are relatively small and low compared to GVS. In addition, GVS has been developed as another representative imaging scanner for HH-PAM [27–30]. As an initial approach to use GVS for the composition of HH-PAM probe, an image-guided fiber bundle-based system was demonstrated [27]. Although the proposed system utilized two-axis GVS for real-time imaging, usage of the fiber bundle, which is costly to use, limits the field of view (FOV) and spatial resolution [27]. In addition, miniaturized HH-PAM probes with GVS and an adjustable light focus were developed for in vivo imaging [28–30]. However, limitations, including the small imaging range [28], low axial resolution due to the use of a cylindrically focused acoustic transducer, and relatively slow scanning speed [29,30] remained. In this study, we present a two-axis waterproof GVS-based HH-PAM (WP-GVS-HH- PAM) probe, which provides an extensive FOV for wide-range in vivo experiments. The proposed system is customized to enable portable PA imaging based on the previously reported bench-top type two-axis GVS PAM [31]. To improve the signal-to-noise ratio (SNR) and enable limitless movement of the probe, WP-GVS-HH-PAM was comprised of an opto-acoustic combiner (for coaxial and confocal alignment of the optical beam and acoustic signal) and a laboratory-made two-axis WP-GVS for wide FOV. The system performance was quantitatively evaluated by resolution (spatial and axial), penetration depth, scanning range, and SNR. In addition, the applicability of WP-GVS-HH-PAM for biomedical fields was demonstrated by phantom and mouse in vivo imaging (ear, iris, and brain). The obtained results present the feasibility of WP-GVS-HH-PAM as an imaging modality in various biomedical applications requiring a wide scanning region and high spatial resolution. 2. Materials and Methods 2.1. System Configuration of WP-GVS-HH-PAM Figure 1 demonstrates the optical configuration and photograph of the developed WP- GVS-HH-PAM system. A Q-switched diode-pumped laser (SPOT-10-200-532, Elforlight Ltd., Daventry, UK), whose wavelength is 532 nm with tunable repetition rates from 1 to 50 kHz, was used for PA imaging (Figure 1a). L1 (AC254-075-A, Thorlabs Inc., Newton, NJ, USA) and L2 (AC254-030-A, Thorlabs Inc., USA) were used to reduce the size of beam diameter with collimation. In addition, the collimated beam was transmitted by C1 (TC12FC-532, Thorlabs Inc., USA) to multi-mode fiber (M64L02, Thorlabs Inc., USA) for beam delivery. At the end of the multi-mode fiber, C2 (TC25FC-532, Thorlabs Inc., USA) transferred the beam with an enlarged diameter than was larger than the incident beam of Photonics 2021, 8, 305 3 of 10 1 to 50 kHz, was used for PA imaging (Figure 1a). L1 (AC254-075-A, Thorlabs Inc., New- ton, NJ, USA) and L2 (AC254-030-A, Thorlabs Inc., USA) were used to reduce the size of beam diameter with collimation. In addition, the collimated beam was transmitted by C1 (TC12FC-532, Thorlabs Inc., USA) to multi-mode fiber (M64L02, Thorlabs Inc., USA) for Photonics 2021, 8, 305 3 of 10 beam delivery. At the end of the multi-mode fiber, C2 (TC25FC-532, Thorlabs Inc., USA) transferred the beam with an enlarged diameter than was larger than the incident beam of C1. The output beam from C2 was focused by an objective lens (AC254-100-AB, Thorlabs Inc., USA) and passed to a two-axis GVS through the laboratory-made opto- C1. The output beam from C2 was focused by an objective lens (AC254-100-AB, Thorlabs acoustic beam combiner. The custom opto-acoustic beam combiner consisted of an un- Inc., USA) and passed to a two-axis GVS through the laboratory-made opto-acoustic beam coated BK7 prism (PS910, Thorlabs Inc., USA), a dielectric-coated prism (MRA10-E02, combiner. The custom opto-acoustic beam combiner consisted of an uncoated BK7 prism Thorlabs Inc., USA), a correction lens with a 54 mm focal length (67–147, Edmund Optics (PS910, Thorlabs Inc., USA), a dielectric-coated prism (MRA10-E02, Thorlabs Inc., USA), Inc., Barringt a corr on, N ection J, USlens A), and with an a 54 acou mm stic focal lenslength with a(67–147, 27 mm aco Edmund ustic foc Optics al lengt Inc., h ( Barrington, 45– NJ, 384, Edmun USA), d Opti and cs Inc., an acoustic USA). To m lens inwith imize aat 27 temm nuatacoustic ion of the op focal tical b length eam (45–384, and acou Edmund stic Optics Inc., USA). To minimize attenuation of the optical beam and acoustic signal in the opto- signal in the opto-acoustic beam combiner, optical adhesive was applied to each junction of each component (37–322, Edm acoustic beam combiner und Opti , opticalcs Inc., adhesive USA) was . The correction l applied to eachejunction ns corrects opti of each -component (37–322, Edmund Optics Inc., USA). The correction lens corrects optical aberration caused cal aberration caused by the acoustic lens, and a dielectric coated film between two prisms by the acoustic lens, and a dielectric coated film between two prisms was used to reflect was used to reflect the optical beam and transmit the acoustic signal. To implement the the optical beam and transmit the acoustic signal. To implement the fully waterproof fully waterproof WP-GVS-HH-PAM probe, we modified the previously presented two- WP-GVS-HH-PAM probe, we modified the previously presented two-axis waterproof GVS axis waterproof GVS with a 3D printing-based customized probe [31]. This fully water- with a 3D printing-based customized probe [31]. This fully waterproof probe enables free proof probe enables free movement of WP-GVS-HH-PAM for wide-range scanning. The movement of WP-GVS-HH-PAM for wide-range scanning. The beam reflected by two-axis beam reflected by two-axis GVS (GVS102, Thorlabs Inc., USA) illuminates the sample GVS (GVS102, Thorlabs Inc., USA) illuminates the sample through a window, which is through a window, which is sealed with a polyethylene membrane for transmission of the sealed with a polyethylene membrane for transmission of the optical beam and PA signal. optical beam and PA signal. A photograph of the developed WP-GVS-HH-PAM probe is A photograph of the developed WP-GVS-HH-PAM probe is shown in Figure 1b. shown in Figure 1b. Figure 1. Schematic representation of WP-GVS-HH-PAM. (a) Optical configuration of the system. Figure 1. Schematic representation of WP-GVS-HH-PAM. (a) Optical configuration of the system. (b) A photograph of (b) A photograph of WP-GVS-HH-PAM probe. (c) utilizing WP-WGS-HH-PAM probe for PA im- WP-GVS-HH-PAM probe. (c) utilizing WP-WGS-HH-PAM probe for PA imaging. AL, acoustic lens; AMP, amplifier; C, aging. AL, acoustic lens; AMP, amplifier; C, collimator; CL, correction lens; L, lens; M, mirror; MMF, collimator; CL, correction lens; L, lens; M, mirror; MMF, multi-mode fiber; OL, objective lens; OUC, opto-acoustic combiner; multi-mode fiber; OL, objective lens; OUC, opto-acoustic combiner; TM, transparent membrane; UT, TM, transparent membrane; UT, ultrasound transducer; WT, water tank. ultrasound transducer; WT, water tank. As an aspect of PA signal processing, an ultrasound transducer (V214-BB-RM, Olym- pus NDT, Shinjuku, Tokyo, Japan) with a 50 MHz center frequency was utilized for con- verting the PA wave into electrical signals. The detected electrical signal was amplified by serially connecting two pre-amplifiers (ZFL-500LN+, Mini-Circuits, Brooklyn, NY, USA) with 52 dB gain. The amplified signal was transformed into a 12-bit digital signal by a digitizer (ATS9350, Alazar Technologies Inc., Pointe-Claire, QC, Canada) with a 500 MHz Photonics 2021, 8, 305 4 of 10 sampling rate and a 250 MHz full-power bandwidth. To precisely synchronize the scanning speed with data acquisition, a data acquisition board (DAQ, NI PCIe-6323, National In- strument Corporation, Austin, TX, USA) was implemented. At the rising edge of the main trigger from the DAQ, laser pulse generation, scanning of fast-axis scanner, and digitizer acquisition were simultaneously started, and PA imaging was successively conducted. In addition, the fast-axis of GVS was operated with a triangular waveform, which enhances the stability and durability of the scanner. Scanning was started according to the rising edge of the main trigger. 2.2. Animal Experiment Protocol For in-vivo animal experiments, all experimental procedures were performed fol- lowing the guidelines of the Institutional Animal Care and Use Committee (IACUC) of Kyungpook National University (No. KNU-2020-0025). The illumination laser beam for the tissues (mouse ear, iris, and brain) adhered to the American Standard Institute safety limit of 532 nm wavelength. A normal healthy Balb/c mouse (male, 12 weeks old) was used for in vivo study. We used an isoflurane machine to anesthetize the mouse with 1 L/min of oxygen and 0.75% isoflurane before PA imaging. An anesthetized mouse was placed on the imaging stage with an electronic heating pad to maintain the body temperature during the experiment and the state was checked by monitoring the movement of hands and feet. Ultrasound gel (Power sonic, Tamin Inc., Bloomfield, KY, USA) was applied to the region of interest (ROI) as an acoustic impedance-matching material between mouse and probe membrane. After the experiments, the mouse was sacrificed according to the approved techniques from the IACUC of KNU. 3. Results 3.1. Performance Evaluation of WP-GVS-HH-PAM To quantitatively evaluate the system performance of the proposed WP-GVS-HH- PAM, we obtained both lateral and axial resolutions (Figure 2a–d). To measure the spatial resolution, a sharp blade was used to obtain the PA maximum amplitude projection (MAP) image (Figure 2a). Based on this result, extracted PA intensities from the selected line, along with the x-direction, were used to calculate the lateral resolution. In accordance with the extracted intensity values from the white dashed line (a–b) in Figure 2a, the obtained spatial resolution was 11.5 m (Figure 2b), which was fitted using an edge-spread function and a line-spread function (LSF). To minimize the line-edge roughness of the sharp blade, we averaged 50 A-lines centered on line a–b. In addition, to measure the axial resolution, we imaged a carbon fiber of 7 m diameter (Carbon Fiber Yarn, Zhongfu shenying carbon fiber Co.,Ltd, Lianyungang city, China) and extracted the PA intensities along with the depth direction in the B-scan image (white dashed line of c–d in Figure 2c). According to the full width at half maximum of LSF fitting from the Gaussian profile in Figure 2d, the measured axial resolution was 31.3 m, which is close to the theoretical value of 33 m. To reduce the oscillation effect and so obtain the time-resolved A-line signal, we averaged 50 B-scan images for the selected center position of cross-section images. In addition, as an aspect of the envelope-fitting function to minimize the time oscillatory problem, we applied the Hilbert transform-based ultrasound envelope detection method in our PA signal acquisition software. The reason for the occurrent difference between the theoretical value and measured one is the reduced time variant effect from applied averaging. The theoretical value was primarily determined by the specification of the ultrasound transducer, whose 6 dB bandwidth was 40.63 MHz with a 50 MHz center frequency, while the velocity of sound was 1540 m/s. The obtained lateral and axial resolutions were sufficient for vascular mapping in small animals in vivo, whose diameter varied from 4 to 50 m according to the type of vessel [32,33]. Photonics 2021, 8, 305 5 of 10 the ultrasound transducer, whose −6 dB bandwidth was 40.63 MHz with a 50 MHz center frequency, while the velocity of sound was 1540 m/s. The obtained lateral and axial reso- Photonics 2021, 8, 305 5 of 10 lutions were sufficient for vascular mapping in small animals in vivo, whose diameter varied from 4 to 50 μm according to the type of vessel [32,33]. Figure 2. Quantitative evaluations of WP-GVS-HH-PAM performance. (a) PA MAP image of a sharp Figure 2. Quantitative evaluations of WP-GVS-HH-PAM performance. (a) PA MAP image of a sharp blade. (b) ESF and LSF blade. (b) ESF and LSF fitting graph of the PA MAP data across the line a–b in (a). (c) PA B-scan fitting graph of the PA MAP data across the line a–b in (a). (c) PA B-scan image of a 7 m diameter carbon fiber. (d) LSF fitting image of a 7 μm diameter carbon fiber. (d) LSF fitting graph with Gaussian profiling of the PA data graph with Gaussian profiling of the PA data across the line cd in (c). (e) Photograph of the prepared chicken breast tissue across the line c−d in (c). (e) Photograph of the prepared chicken breast tissue inserted with the inserted with the black need for measuring an imaging depth. (f) PA B-scan image for imaging depth measurement obtained black need for measuring an imaging depth. (f) PA B-scan image for imaging depth measurement with the prepared sample in (e). ESF, edge spread function; LSF, line spread function; MAP, maximum amplitude projection. obtained with the prepared sample in (e). ESF, edge spread function; LSF, line spread function; MAP, maximum amplitude projection. To measure the effective imaging depth of WP-GVS-HH-PAM in the biological tissue, we prepared a black needle inserted at chicken breast tissue (Figure 2e). The black needle To measure the effective imaging depth of WP-GVS-HH-PAM in the biological tis- was diagonally inserted to generate the PA signal. The PA signals were detected well sue, we prepared a black needle inserted at chicken breast tissue (Figure 2e). The black regardless of the depth of the needle from the surface of the chicken breast tissue (Figure 2f). needle was diagonally inserted to generate the PA signal. The PA signals were detected The measured imaging depth in biological tissue was 788 m, which was in the range of well regardless of the depth of the needle from the surface of the chicken breast tissue 128 pixels in the depth direction (6.16 m per single pixel). Because a two-axis GVS was (Figure 2f). The measured imaging depth in biological tissue was 788 μm, which was in implemented in the probe, the scanning range varied from micro-scale (142 m at 0.1 V the range of 128 pixels in the depth direction (6.16 μm per single pixel). Because a two- of galvanometer scanner) to 14.5  9 mm (fast axis  slow axis) according to the voltage axis GVS was implemented in the probe, the scanning range varied from micro-scale (142 value applied to the scanners. Therefore, the possible maximum scanning volume was μm at 0.1 V of galvanometer scanner) to 14.5 × 9 mm (fast axis × slow axis) according to 14.5  9  0.78 mm (x  y  z). The upper limit in the depth direction is determined the voltage value applied to the scanners. Therefore, the possible maximum scanning vol- by the acoustic signal transmission time and acquisition rate of the digitizer. Moreover, ume was 14.5 × 9 × 0.78 mm (x × y × z). The upper limit in the depth direction is deter- the axial range was selected by the depth of focus range. In addition, with regard to the mined by the acoustic signal transmission time and acquisition rate of the digitizer. More- scanning speed, the B-scan (800 (fast-axis)  512 (depth-direction)) and volumetric images over, the axial range was selected by the depth of focus range. In addition, with regard to (800 (fast-axis)  800 (slow-axis)  512 (depth-direction)) were obtained in 0.04 s and 32 s, respectively. Moreover, the SNR of our system was measured using carbon fiber for axial resolution measurement. The obtained averaged SNR is 37.8 dB and this value is sufficient to image a single red blood cell [34]. Photonics 2021, 8, 305 6 of 10 the scanning speed, the B-scan (800 (fast-axis) × 512 (depth-direction)) and volumetric im- ages (800 (fast-axis) × 800 (slow-axis) × 512 (depth-direction)) were obtained in 0.04 s and 32 s, respectively. Moreover, the SNR of our system was measured using carbon fiber for Photonics 2021, 8, 305 6 of 10 axial resolution measurement. The obtained averaged SNR is 37.8 dB and this value is sufficient to image a single red blood cell [34]. 3.2. Phantom Images Using WP-GVS-HH-PAM 3.2. Phantom Images Using WP-GVS-HH-PAM To verify the applicability of the developed system to in vivo imaging of microvas- To verify the applicability of the developed system to in vivo imaging of microvascu- culature, we imaged carbon-fiber networks and a leaf skeleton target as vessel-mimicking lature, we imaged carbon-fiber networks and a leaf skeleton target as vessel-mimicking phantom samples (Figure 3). Each phantom sample was used to demonstrate the versatil- phantom samples (Figure 3). Each phantom sample was used to demonstrate the versatility ity for imaging a narrow area with high-resolution (carbon fiber networks) and scanning for imaging a narrow area with high-resolution (carbon fiber networks) and scanning a a wide region (leaf skeleton target), respectively. We minimized the laser power enough wide region (leaf skeleton target), respectively. We minimized the laser power enough to measur to meae sure the structure since both pha the structure since both phantom ntom sa samples mple easily s easily burn burn due due to the hig to the high absorption h absorp- rate. tion ra Similar te. Sim to ila that r to tha done t done wi with carbon th carbon f fiber networks, iber netwo to rks, to prepar prepare a bunch e a bunch o of fibers f fibers (7 (7 m diameter), we randomly placed them on the slide glass with distilled water and covered μm diameter), we randomly placed them on the slide glass with distilled water and cov- them ered them w with a cover ith a cover glass. glass. The The role of ro the le o cover f the cover glass gl was ass maintai was mning aintathe ininsample g the saposition mple po- for sitithe on for micr th oscope e microscope and PAM and PA imaging. M ima Although ging. Alsignal though si attenuation gnal attenua through tion through cover cover glass is occurred, we nonetheless used it for exact matching of the sample position. The scanning glass is occurred, we nonetheless used it for exact matching of the sample position. The range for the carbon fiber network was 1.7  1.4 mm for the ROI (red square in Figure 3a; scanning range for the carbon fiber network was 1.7 × 1.4 mm for the ROI (red square in this is a photograph obtained using a microscope). Figure 3c is the measured PA MAP Figure 3a; this is a photograph obtained using a microscope). Figure 3c is the measured image of ROI, which clearly identifies not only single fibers but also overlapped regions. PA MAP image of ROI, which clearly identifies not only single fibers but also overlapped In addition, we captured the PA MAP image of the leaf phantom shown in Figure 3d regions. In addition, we captured the PA MAP image of the leaf phantom shown in Figure for the ROI (red square in Figure 3b), wherein the scanning range was 11.6  6.1 mm to 3d for the ROI (red square in Figure 3b), wherein the scanning range was 11.6 × 6.1 mm to cover the wide region. PA MAP yielded structural information identical to the photograph cover the wide region. PA MAP yielded structural information identical to the photo- (Figure 3b) regardless of the thickness of the leaf veins. Based on the imaging results of a graph (Figure 3b) regardless of the thickness of the leaf veins. Based on the imaging results vessel-mimicking sample, the system was capable of in vivo imaging of microvasculature of a vessel-mimicking sample, the system was capable of in vivo imaging of microvascu- in a small animal. lature in a small animal. Figure 3. Photographs and PA MAP images of carbon fiber networks and a leaf skeleton target. (a) Figure 3. Photographs and PA MAP images of carbon fiber networks and a leaf skeleton target. Magnified photograph of carbon fiber networks. (b) Magnified photograph of leaf skeleton target. (a) Magnified photograph of carbon fiber networks. (b) Magnified photograph of leaf skeleton target. (c,d) PA MAP images of the region in (a,b), respectively. MAP, maximum amplitude projection. (c,d) PA MAP images of the region in (a,b), respectively. MAP, maximum amplitude projection. 3.3. WP-GVS-HH-PAM for In Vivo Experiments with a Mouse 3.3. WP-GVS-HH-PAM for In Vivo Experiments with a Mouse As an in vivo experiment, the microvasculature of mouse ear, iris, and brain were As an in vivo experiment, the microvasculature of mouse ear, iris, and brain were imaged using WP-GVS-HH-PAM. The intensity-based PA MAP images captured for each imaged using WP-GVS-HH-PAM. The intensity-based PA MAP images captured for each target tissue are shown in Figure 4. The obtained image of the vasculature map in the mouse ear (Figure 4a) demonstrates the micro-vessel networks including single capillaries, an artery, and a vein. The FOV for imaging a mouse ear was 9.0  8.2 mm, which was sufficient to obtain complete structural information of the ear with a single scan. In addition, we captured the PA MAP image of the iris (Figure 4b) with a FOV of 3.1  2.6 mm. The resulting image showed the microvasculature of the iris; we have adjusted the focus by Photonics 2021, 8, 305 7 of 10 target tissue are shown in Figure 4. The obtained image of the vasculature map in the mouse ear (Figure 4a) demonstrates the micro-vessel networks including single capillar- ies, an artery, and a vein. The FOV for imaging a mouse ear was 9.0 × 8.2 mm, which was Photonics 2021, 8, 305 7 of 10 sufficient to obtain complete structural information of the ear with a single scan. In addi- tion, we captured the PA MAP image of the iris (Figure 4b) with a FOV of 3.1 × 2.6 mm. The resulting image showed the microvasculature of the iris; we have adjusted the focus regulating the position of the objective lens to image the lateral region for this image. The by regulating the position of the objective lens to image the lateral region for this image. PA MAP image of the mouse brain under an intact skull (Figure 4c) was also obtained. The The PA MAP image of the mouse brain under an intact skull (Figure 4c) was also obtained. FOV was 10.5  7.0 mm, and the distinctive blood vascular features, which are indicated The FOV was 10.5 × 7.0 mm, and the distinctive blood vascular features, which are indi- as white arrow (e.g., sagittal sinus and coronal suture) were visualized. In addition, as an cated as white arrow (e.g., sagittal sinus and coronal suture) were visualized. In addition, aspect of depth-encoded PA MAP images to provide 3D volume data-based quantitative as an aspect of depth-encoded PA MAP images to provide 3D volume data-based quanti- information, the depth profile along with the z-axis direction was applied to in vivo PA tative information, the depth profile along with the z-axis direction was applied to in vivo images, as shown in Figure 4d–f. By using the wide FOV of WP-GVS-HH-PAM, we were PA images, as shown in Figure 4d–f. By using the wide FOV of WP-GVS-HH-PAM, we able to measure the whole range of each target tissue without applying an additional were able to measure the whole range of each target tissue without applying an additional imaging technique, such as a linear stage, to overcome the limited scanning range. imaging technique, such as a linear stage, to overcome the limited scanning range. Figure 4. In vivo PA MAP images of micro-vascular maps using WP-GVS-HH-PAM in mouse ear, Figure 4. In vivo PA MAP images of micro-vascular maps using WP-GVS-HH-PAM in mouse ear, iris, and brain, respectively. (a–c) PA signal intensity-based MAP images. (d–f) Depth-encoded PA iris, and brain, respectively. (a–c) PA signal intensity-based MAP images. (d–f) Depth-encoded PA MAP images. MAP images. 4. Discussions Here, we presented a HH-PAM probe with an extended FOV for wide-range scanning regardless of sample shape. The previously presented GVS-based HH-PAM probe has a limited FOV, caused by the clear aperture or usable region of other optical components Photonics 2021, 8, 305 8 of 10 used after GVS (e.g., fiber bundles and objective lens) [27,28]. In contrast, the scanning range of the proposed system was relatively enlarged, given that the two-axis WP-GVS was located at the terminal end of the HH probe. Furthermore, the presented system provides comparably improved axial resolution, from 90 m to 31.3 m, compared to cylindrically- focused acoustic transducer-based systems [29,30]. In addition, although the MEMS-based HH-PAM system provides an enhanced scanning rate compared to GVS and miniaturized probe volume size, the FOV was limited because of the small size of the MEMS mirror. WP- GVS-HH-PAM, however, can capture a wide area in a single scan, owing to the relatively large scanning angle of the GVS mirror (25 ) compared to the MEMS scanner (18 for fast-axis and 11 for slow-axis) [21]. In addition, through triangular wave signal-based GVS scanning, the durability of the developed scanner is enhanced, and a high sensitivity for the scanning position was maintained even at high-speed scanning. WP-GVS-HH-PAM advances the previously reported system [31] as an aspect of the movement of the imaging probe, which enhances the applicability of PA imaging systems. By applying fiber coupling through the implementation of the multimode fiber and modified, 3D modeling-based probe construction, WP-GVS-HH-PAM provides unrestricted movement of the probe motion and enhances the convenience of PA imaging. With regard to resolution, the measured lateral resolution of the proposed system was 11.5 m. Although the obtained value was sufficient for in vivo microvascular mapping of small animals, the lateral resolution of the system can be further improved by adding a lens pair (i.e., beam expander) before the objective lens. In addition, the scanning time for volumetric imaging using WP-GVS-HH-PAM was 32 s for an 800  800  512 pixel image. This time can be adjusted by the number of pixels according to the experimental requirements. The scanning speed and the sensitivity of WP-GVS-HH-PAM for in vivo imaging can be improved by using another laser source with an enhanced pulse-repetition rate and high-power light. Moreover, the FOV can also be adjusted following the ROI from a micrometer scale to up to 14.5  9 mm , which is sufficient to measure the whole region of the mouse brain. 5. Conclusions In conclusion, we demonstrated the WP-GVS-HH-PAM with a laboratory customized two-axis WP-GVS that provides an extensive scanning range for a wide range in vivo 3D imaging. The developed HH-probe consisted of a two-axis WP-GVS for wide scanning and an opto-acoustic combiner for coaxial and confocal alignment of the optical beam and acoustic signal. Compared to the conventional HH-PAM systems, our proposed WP-GVS- HH-PAM provides an extended FOV (from micro-scale (142 m at 0.1 V of galvanometer scanner) to 14.5  9 mm ) for in vivo imaging. The spatial and axial resolutions were 11.5 m and 31.3 m, respectively, which are sufficient for vascular mapping. We quan- titatively evaluated the system performance using vessel-mimicking phantom samples (carbon fiber networks and leaf skeleton) and in vivo imaging in a small animal (mouse ear, iris, and brain). The obtained results of the implemented WP-GVS-HH-PAM demon- strate the feasibility of capturing a microvascular map of several animal tissues. Hence, the proposed system shows promising results encouraging the applications of HH-PAM to various biomedical applications including preclinical and clinical research with small animals in vivo. Author Contributions: Conceptualization, M.J. and J.K.; methodology, D.S. and S.H.; software, S.H.; validation, D.S., J.L. (Jaeyul Lee) and Y.K.; formal analysis, D.S. and S.H; investigation, Y.K., E.L. and J.L. (Junsoo Lee); resources, M.J. and J.K.; data curation, D.S., S.H., Y.K. and E.L.; writing—original draft preparation, D.S.; writing—review and editing, S.H. and J.L. (Jaeyul Lee); visualization, D.S., S.H. and E.L.; supervision, M.J. and J.K.; project administration, M.J.; funding acquisition, M.J. and J.K. All authors have read and agreed to the published version of the manuscript. Funding: This work was supported by LG Yonam Foundation (of Korea) and also supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (2020R1I1A1A01072399), and supported by Basic Science Research Program Photonics 2021, 8, 305 9 of 10 through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (2018R1D1A1B07043340). Institutional Review Board Statement: The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Institutional Review Board of the Institutional Animal and Human Care and Use Committee of Kyungpook National University (No. KNU-2020-0025). Informed Consent Statement: Not applicable. 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Waterproof Galvanometer Scanner-Based Handheld Photoacoustic Microscopy Probe for Wide-Field Vasculature Imaging In Vivo

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hv photonics Communication Waterproof Galvanometer Scanner-Based Handheld Photoacoustic Microscopy Probe for Wide-Field Vasculature Imaging In Vivo 1 , † 1 , 2 , † 1 , 3 1 1 1 Daewoon Seong , Sangyeob Han , Jaeyul Lee , Euimin Lee , Yoonseok Kim , Junsoo Lee , 1 , 1 Mansik Jeon * and Jeehyun Kim School of Electronic and Electrical Engineering, College of IT Engineering, Kyungpook National University, Daegu 41566, Korea; smc7095@knu.ac.kr (D.S.); syhan850224@knu.ac.kr (S.H.); jaeyul21@g.ucla.edu (J.L.); augustmini@knu.ac.kr (E.L.); otter0618@knu.ac.kr (Y.K.); jslee5399@knu.ac.kr (J.L.); jeehk@knu.ac.kr (J.K.) Institute of Biomedical Engineering, School of Medicine, Kyungpook National University, Daegu 41566, Korea Department of Bioengineering, University of California, Los Angeles, CA 90095, USA * Correspondence: msjeon@knu.ac.kr; Tel.: +82-53-950-7846 † These authors contributed equally to this work. Abstract: Photoacoustic imaging (PAI) is a hybrid non-invasive imaging technique used to merge high optical contrast and high acoustic resolution in deep tissue. PAI has been extensively developed by utilizing its advantages that include deep imaging depth, high resolution, and label-free imaging. As a representative implementation of PAI, photoacoustic microscopy (PAM) has been used in preclinical and clinical studies for its micron-scale spatial resolution capability with high optical absorption contrast. Several handheld and portable PAM systems have been developed that improve its applicability to several fields, making it versatile. In this study, we developed a laboratory- customized, two-axis, waterproof, galvanometer scanner-based handheld PAM (WP-GVS-HH-PAM), which provides an extended field of view (14.5  9 mm ) for wide-range imaging. The fully waterproof handheld probe enables free movement for imaging regardless of sample shape, and Citation: Seong, D.; Han, S.; Lee, J.; volume rate and scanning region are adjustable per experimental conditions. Results of WP-GVS-HH- Lee, E.; Kim, Y.; Lee, J.; Jeon, M.; Kim, J. Waterproof Galvanometer PAM-based phantom and in vivo imaging of mouse tissues (ear, iris, and brain) confirm the feasibility Scanner-Based Handheld and applicability of our system as an imaging modality for various biomedical applications. Photoacoustic Microscopy Probe for Wide-Field Vasculature Imaging In Keywords: photoacoustic microscopy; handheld probe; wide-field imaging; in vivo vasculature Vivo. Photonics 2021, 8, 305. https:// imaging; 3D imaging doi.org/10.3390/photonics8080305 Received: 29 June 2021 Accepted: 28 July 2021 1. Introduction Published: 30 July 2021 Photoacoustic imaging (PAI) is a label-free, non-invasive biomedical imaging tech- nique that has been extensively studied and developed [1,2]. PAI is based on the light- Publisher’s Note: MDPI stays neutral induced ultrasound (US) signal through the photoacoustic (PA) effect (i.e., thermal-elastic with regard to jurisdictional claims in expansion) [3]. The optical absorption contrast of target biological tissues determines the published maps and institutional affil- intensity of the broadband US waves (i.e., PA waves), which are converted into analog iations. electrical signals by US transducers [4]. Since the degree of scattering of PA signal in biological tissue is comparably less than that of the optical beam, PAI offers improved optical sensitivity with high US resolution compared to other optical imaging modalities in optically turbid media [5]. In addition, using the PA effect, PAI enables the non-invasive Copyright: © 2021 by the authors. characterization of biological and biomedical properties with endogenous and exogenous Licensee MDPI, Basel, Switzerland. agents such as metabolism, anatomy information, functional data, and molecular pro- This article is an open access article cesses [1,6]. Based on the aforementioned distinctive advantages, PAI has been functionally distributed under the terms and used in various applications including vasculature mapping, assessing hemoglobin oxygen conditions of the Creative Commons saturation, and blood flow [7–10]. Attribution (CC BY) license (https:// In particular, photoacoustic microscopy (PAM) is a major implementation of PAI, creativecommons.org/licenses/by/ which provides rich optical absorption with enhanced spatial resolution [11,12]. PAM is 4.0/). Photonics 2021, 8, 305. https://doi.org/10.3390/photonics8080305 https://www.mdpi.com/journal/photonics Photonics 2021, 8, 305 2 of 10 largely divided into two different types: optical resolution (OR) and acoustic resolution (AR) PAM, according to the focus type (i.e., optical and acoustic focus, respectively) [13]. OR-PAM provides a high spatial resolution of the subcellular level compared with AR-PAM by tight focusing of the optical excitation beam [14]. Since the ballistic photon regime and scattering limit the penetration depth of OR-PAM, it is required to optimize the spatial resolution and imaging depth because of the trade-off between spatial resolution and depth of focus [15]. With regard to AR-PAM, although the lateral resolution is inferior to that of optical imaging modalities, the penetration depth is enhanced by utilizing comparably weak acoustic scattering in biological tissue than the optical diffusion limit [16]. By utilizing the advantages of each imaging technique, PAM has been investigated for biomedical applications [17–20]. To expand the applicable conditions and enhance the versatility of PAM compared to the bench-top system, several miniaturized handheld PAM (HH-PAM) have been de- veloped [21–23]. To enable fast-scanning with the miniaturized probe, various imaging scanners-based HH-PAM systems were reported (e.g., micro-electro-mechanical system (MEMS) and galvanometer scanner (GVS)). To reduce the volume size of the HH-PAM probe, MEMS mirror has been widely employed following the rapid development of MEMS systems [24–26]. Because of the small size of a MEMS mirror, the dimension of the HH- PAM probe is able to be optimized according to the applications. However, the scanning region and scanning stability are relatively small and low compared to GVS. In addition, GVS has been developed as another representative imaging scanner for HH-PAM [27–30]. As an initial approach to use GVS for the composition of HH-PAM probe, an image-guided fiber bundle-based system was demonstrated [27]. Although the proposed system utilized two-axis GVS for real-time imaging, usage of the fiber bundle, which is costly to use, limits the field of view (FOV) and spatial resolution [27]. In addition, miniaturized HH-PAM probes with GVS and an adjustable light focus were developed for in vivo imaging [28–30]. However, limitations, including the small imaging range [28], low axial resolution due to the use of a cylindrically focused acoustic transducer, and relatively slow scanning speed [29,30] remained. In this study, we present a two-axis waterproof GVS-based HH-PAM (WP-GVS-HH- PAM) probe, which provides an extensive FOV for wide-range in vivo experiments. The proposed system is customized to enable portable PA imaging based on the previously reported bench-top type two-axis GVS PAM [31]. To improve the signal-to-noise ratio (SNR) and enable limitless movement of the probe, WP-GVS-HH-PAM was comprised of an opto-acoustic combiner (for coaxial and confocal alignment of the optical beam and acoustic signal) and a laboratory-made two-axis WP-GVS for wide FOV. The system performance was quantitatively evaluated by resolution (spatial and axial), penetration depth, scanning range, and SNR. In addition, the applicability of WP-GVS-HH-PAM for biomedical fields was demonstrated by phantom and mouse in vivo imaging (ear, iris, and brain). The obtained results present the feasibility of WP-GVS-HH-PAM as an imaging modality in various biomedical applications requiring a wide scanning region and high spatial resolution. 2. Materials and Methods 2.1. System Configuration of WP-GVS-HH-PAM Figure 1 demonstrates the optical configuration and photograph of the developed WP- GVS-HH-PAM system. A Q-switched diode-pumped laser (SPOT-10-200-532, Elforlight Ltd., Daventry, UK), whose wavelength is 532 nm with tunable repetition rates from 1 to 50 kHz, was used for PA imaging (Figure 1a). L1 (AC254-075-A, Thorlabs Inc., Newton, NJ, USA) and L2 (AC254-030-A, Thorlabs Inc., USA) were used to reduce the size of beam diameter with collimation. In addition, the collimated beam was transmitted by C1 (TC12FC-532, Thorlabs Inc., USA) to multi-mode fiber (M64L02, Thorlabs Inc., USA) for beam delivery. At the end of the multi-mode fiber, C2 (TC25FC-532, Thorlabs Inc., USA) transferred the beam with an enlarged diameter than was larger than the incident beam of Photonics 2021, 8, 305 3 of 10 1 to 50 kHz, was used for PA imaging (Figure 1a). L1 (AC254-075-A, Thorlabs Inc., New- ton, NJ, USA) and L2 (AC254-030-A, Thorlabs Inc., USA) were used to reduce the size of beam diameter with collimation. In addition, the collimated beam was transmitted by C1 (TC12FC-532, Thorlabs Inc., USA) to multi-mode fiber (M64L02, Thorlabs Inc., USA) for Photonics 2021, 8, 305 3 of 10 beam delivery. At the end of the multi-mode fiber, C2 (TC25FC-532, Thorlabs Inc., USA) transferred the beam with an enlarged diameter than was larger than the incident beam of C1. The output beam from C2 was focused by an objective lens (AC254-100-AB, Thorlabs Inc., USA) and passed to a two-axis GVS through the laboratory-made opto- C1. The output beam from C2 was focused by an objective lens (AC254-100-AB, Thorlabs acoustic beam combiner. The custom opto-acoustic beam combiner consisted of an un- Inc., USA) and passed to a two-axis GVS through the laboratory-made opto-acoustic beam coated BK7 prism (PS910, Thorlabs Inc., USA), a dielectric-coated prism (MRA10-E02, combiner. The custom opto-acoustic beam combiner consisted of an uncoated BK7 prism Thorlabs Inc., USA), a correction lens with a 54 mm focal length (67–147, Edmund Optics (PS910, Thorlabs Inc., USA), a dielectric-coated prism (MRA10-E02, Thorlabs Inc., USA), Inc., Barringt a corr on, N ection J, USlens A), and with an a 54 acou mm stic focal lenslength with a(67–147, 27 mm aco Edmund ustic foc Optics al lengt Inc., h ( Barrington, 45– NJ, 384, Edmun USA), d Opti and cs Inc., an acoustic USA). To m lens inwith imize aat 27 temm nuatacoustic ion of the op focal tical b length eam (45–384, and acou Edmund stic Optics Inc., USA). To minimize attenuation of the optical beam and acoustic signal in the opto- signal in the opto-acoustic beam combiner, optical adhesive was applied to each junction of each component (37–322, Edm acoustic beam combiner und Opti , opticalcs Inc., adhesive USA) was . The correction l applied to eachejunction ns corrects opti of each -component (37–322, Edmund Optics Inc., USA). The correction lens corrects optical aberration caused cal aberration caused by the acoustic lens, and a dielectric coated film between two prisms by the acoustic lens, and a dielectric coated film between two prisms was used to reflect was used to reflect the optical beam and transmit the acoustic signal. To implement the the optical beam and transmit the acoustic signal. To implement the fully waterproof fully waterproof WP-GVS-HH-PAM probe, we modified the previously presented two- WP-GVS-HH-PAM probe, we modified the previously presented two-axis waterproof GVS axis waterproof GVS with a 3D printing-based customized probe [31]. This fully water- with a 3D printing-based customized probe [31]. This fully waterproof probe enables free proof probe enables free movement of WP-GVS-HH-PAM for wide-range scanning. The movement of WP-GVS-HH-PAM for wide-range scanning. The beam reflected by two-axis beam reflected by two-axis GVS (GVS102, Thorlabs Inc., USA) illuminates the sample GVS (GVS102, Thorlabs Inc., USA) illuminates the sample through a window, which is through a window, which is sealed with a polyethylene membrane for transmission of the sealed with a polyethylene membrane for transmission of the optical beam and PA signal. optical beam and PA signal. A photograph of the developed WP-GVS-HH-PAM probe is A photograph of the developed WP-GVS-HH-PAM probe is shown in Figure 1b. shown in Figure 1b. Figure 1. Schematic representation of WP-GVS-HH-PAM. (a) Optical configuration of the system. Figure 1. Schematic representation of WP-GVS-HH-PAM. (a) Optical configuration of the system. (b) A photograph of (b) A photograph of WP-GVS-HH-PAM probe. (c) utilizing WP-WGS-HH-PAM probe for PA im- WP-GVS-HH-PAM probe. (c) utilizing WP-WGS-HH-PAM probe for PA imaging. AL, acoustic lens; AMP, amplifier; C, aging. AL, acoustic lens; AMP, amplifier; C, collimator; CL, correction lens; L, lens; M, mirror; MMF, collimator; CL, correction lens; L, lens; M, mirror; MMF, multi-mode fiber; OL, objective lens; OUC, opto-acoustic combiner; multi-mode fiber; OL, objective lens; OUC, opto-acoustic combiner; TM, transparent membrane; UT, TM, transparent membrane; UT, ultrasound transducer; WT, water tank. ultrasound transducer; WT, water tank. As an aspect of PA signal processing, an ultrasound transducer (V214-BB-RM, Olym- pus NDT, Shinjuku, Tokyo, Japan) with a 50 MHz center frequency was utilized for con- verting the PA wave into electrical signals. The detected electrical signal was amplified by serially connecting two pre-amplifiers (ZFL-500LN+, Mini-Circuits, Brooklyn, NY, USA) with 52 dB gain. The amplified signal was transformed into a 12-bit digital signal by a digitizer (ATS9350, Alazar Technologies Inc., Pointe-Claire, QC, Canada) with a 500 MHz Photonics 2021, 8, 305 4 of 10 sampling rate and a 250 MHz full-power bandwidth. To precisely synchronize the scanning speed with data acquisition, a data acquisition board (DAQ, NI PCIe-6323, National In- strument Corporation, Austin, TX, USA) was implemented. At the rising edge of the main trigger from the DAQ, laser pulse generation, scanning of fast-axis scanner, and digitizer acquisition were simultaneously started, and PA imaging was successively conducted. In addition, the fast-axis of GVS was operated with a triangular waveform, which enhances the stability and durability of the scanner. Scanning was started according to the rising edge of the main trigger. 2.2. Animal Experiment Protocol For in-vivo animal experiments, all experimental procedures were performed fol- lowing the guidelines of the Institutional Animal Care and Use Committee (IACUC) of Kyungpook National University (No. KNU-2020-0025). The illumination laser beam for the tissues (mouse ear, iris, and brain) adhered to the American Standard Institute safety limit of 532 nm wavelength. A normal healthy Balb/c mouse (male, 12 weeks old) was used for in vivo study. We used an isoflurane machine to anesthetize the mouse with 1 L/min of oxygen and 0.75% isoflurane before PA imaging. An anesthetized mouse was placed on the imaging stage with an electronic heating pad to maintain the body temperature during the experiment and the state was checked by monitoring the movement of hands and feet. Ultrasound gel (Power sonic, Tamin Inc., Bloomfield, KY, USA) was applied to the region of interest (ROI) as an acoustic impedance-matching material between mouse and probe membrane. After the experiments, the mouse was sacrificed according to the approved techniques from the IACUC of KNU. 3. Results 3.1. Performance Evaluation of WP-GVS-HH-PAM To quantitatively evaluate the system performance of the proposed WP-GVS-HH- PAM, we obtained both lateral and axial resolutions (Figure 2a–d). To measure the spatial resolution, a sharp blade was used to obtain the PA maximum amplitude projection (MAP) image (Figure 2a). Based on this result, extracted PA intensities from the selected line, along with the x-direction, were used to calculate the lateral resolution. In accordance with the extracted intensity values from the white dashed line (a–b) in Figure 2a, the obtained spatial resolution was 11.5 m (Figure 2b), which was fitted using an edge-spread function and a line-spread function (LSF). To minimize the line-edge roughness of the sharp blade, we averaged 50 A-lines centered on line a–b. In addition, to measure the axial resolution, we imaged a carbon fiber of 7 m diameter (Carbon Fiber Yarn, Zhongfu shenying carbon fiber Co.,Ltd, Lianyungang city, China) and extracted the PA intensities along with the depth direction in the B-scan image (white dashed line of c–d in Figure 2c). According to the full width at half maximum of LSF fitting from the Gaussian profile in Figure 2d, the measured axial resolution was 31.3 m, which is close to the theoretical value of 33 m. To reduce the oscillation effect and so obtain the time-resolved A-line signal, we averaged 50 B-scan images for the selected center position of cross-section images. In addition, as an aspect of the envelope-fitting function to minimize the time oscillatory problem, we applied the Hilbert transform-based ultrasound envelope detection method in our PA signal acquisition software. The reason for the occurrent difference between the theoretical value and measured one is the reduced time variant effect from applied averaging. The theoretical value was primarily determined by the specification of the ultrasound transducer, whose 6 dB bandwidth was 40.63 MHz with a 50 MHz center frequency, while the velocity of sound was 1540 m/s. The obtained lateral and axial resolutions were sufficient for vascular mapping in small animals in vivo, whose diameter varied from 4 to 50 m according to the type of vessel [32,33]. Photonics 2021, 8, 305 5 of 10 the ultrasound transducer, whose −6 dB bandwidth was 40.63 MHz with a 50 MHz center frequency, while the velocity of sound was 1540 m/s. The obtained lateral and axial reso- Photonics 2021, 8, 305 5 of 10 lutions were sufficient for vascular mapping in small animals in vivo, whose diameter varied from 4 to 50 μm according to the type of vessel [32,33]. Figure 2. Quantitative evaluations of WP-GVS-HH-PAM performance. (a) PA MAP image of a sharp Figure 2. Quantitative evaluations of WP-GVS-HH-PAM performance. (a) PA MAP image of a sharp blade. (b) ESF and LSF blade. (b) ESF and LSF fitting graph of the PA MAP data across the line a–b in (a). (c) PA B-scan fitting graph of the PA MAP data across the line a–b in (a). (c) PA B-scan image of a 7 m diameter carbon fiber. (d) LSF fitting image of a 7 μm diameter carbon fiber. (d) LSF fitting graph with Gaussian profiling of the PA data graph with Gaussian profiling of the PA data across the line cd in (c). (e) Photograph of the prepared chicken breast tissue across the line c−d in (c). (e) Photograph of the prepared chicken breast tissue inserted with the inserted with the black need for measuring an imaging depth. (f) PA B-scan image for imaging depth measurement obtained black need for measuring an imaging depth. (f) PA B-scan image for imaging depth measurement with the prepared sample in (e). ESF, edge spread function; LSF, line spread function; MAP, maximum amplitude projection. obtained with the prepared sample in (e). ESF, edge spread function; LSF, line spread function; MAP, maximum amplitude projection. To measure the effective imaging depth of WP-GVS-HH-PAM in the biological tissue, we prepared a black needle inserted at chicken breast tissue (Figure 2e). The black needle To measure the effective imaging depth of WP-GVS-HH-PAM in the biological tis- was diagonally inserted to generate the PA signal. The PA signals were detected well sue, we prepared a black needle inserted at chicken breast tissue (Figure 2e). The black regardless of the depth of the needle from the surface of the chicken breast tissue (Figure 2f). needle was diagonally inserted to generate the PA signal. The PA signals were detected The measured imaging depth in biological tissue was 788 m, which was in the range of well regardless of the depth of the needle from the surface of the chicken breast tissue 128 pixels in the depth direction (6.16 m per single pixel). Because a two-axis GVS was (Figure 2f). The measured imaging depth in biological tissue was 788 μm, which was in implemented in the probe, the scanning range varied from micro-scale (142 m at 0.1 V the range of 128 pixels in the depth direction (6.16 μm per single pixel). Because a two- of galvanometer scanner) to 14.5  9 mm (fast axis  slow axis) according to the voltage axis GVS was implemented in the probe, the scanning range varied from micro-scale (142 value applied to the scanners. Therefore, the possible maximum scanning volume was μm at 0.1 V of galvanometer scanner) to 14.5 × 9 mm (fast axis × slow axis) according to 14.5  9  0.78 mm (x  y  z). The upper limit in the depth direction is determined the voltage value applied to the scanners. Therefore, the possible maximum scanning vol- by the acoustic signal transmission time and acquisition rate of the digitizer. Moreover, ume was 14.5 × 9 × 0.78 mm (x × y × z). The upper limit in the depth direction is deter- the axial range was selected by the depth of focus range. In addition, with regard to the mined by the acoustic signal transmission time and acquisition rate of the digitizer. More- scanning speed, the B-scan (800 (fast-axis)  512 (depth-direction)) and volumetric images over, the axial range was selected by the depth of focus range. In addition, with regard to (800 (fast-axis)  800 (slow-axis)  512 (depth-direction)) were obtained in 0.04 s and 32 s, respectively. Moreover, the SNR of our system was measured using carbon fiber for axial resolution measurement. The obtained averaged SNR is 37.8 dB and this value is sufficient to image a single red blood cell [34]. Photonics 2021, 8, 305 6 of 10 the scanning speed, the B-scan (800 (fast-axis) × 512 (depth-direction)) and volumetric im- ages (800 (fast-axis) × 800 (slow-axis) × 512 (depth-direction)) were obtained in 0.04 s and 32 s, respectively. Moreover, the SNR of our system was measured using carbon fiber for Photonics 2021, 8, 305 6 of 10 axial resolution measurement. The obtained averaged SNR is 37.8 dB and this value is sufficient to image a single red blood cell [34]. 3.2. Phantom Images Using WP-GVS-HH-PAM 3.2. Phantom Images Using WP-GVS-HH-PAM To verify the applicability of the developed system to in vivo imaging of microvas- To verify the applicability of the developed system to in vivo imaging of microvascu- culature, we imaged carbon-fiber networks and a leaf skeleton target as vessel-mimicking lature, we imaged carbon-fiber networks and a leaf skeleton target as vessel-mimicking phantom samples (Figure 3). Each phantom sample was used to demonstrate the versatil- phantom samples (Figure 3). Each phantom sample was used to demonstrate the versatility ity for imaging a narrow area with high-resolution (carbon fiber networks) and scanning for imaging a narrow area with high-resolution (carbon fiber networks) and scanning a a wide region (leaf skeleton target), respectively. We minimized the laser power enough wide region (leaf skeleton target), respectively. We minimized the laser power enough to measur to meae sure the structure since both pha the structure since both phantom ntom sa samples mple easily s easily burn burn due due to the hig to the high absorption h absorp- rate. tion ra Similar te. Sim to ila that r to tha done t done wi with carbon th carbon f fiber networks, iber netwo to rks, to prepar prepare a bunch e a bunch o of fibers f fibers (7 (7 m diameter), we randomly placed them on the slide glass with distilled water and covered μm diameter), we randomly placed them on the slide glass with distilled water and cov- them ered them w with a cover ith a cover glass. glass. The The role of ro the le o cover f the cover glass gl was ass maintai was mning aintathe ininsample g the saposition mple po- for sitithe on for micr th oscope e microscope and PAM and PA imaging. M ima Although ging. Alsignal though si attenuation gnal attenua through tion through cover cover glass is occurred, we nonetheless used it for exact matching of the sample position. The scanning glass is occurred, we nonetheless used it for exact matching of the sample position. The range for the carbon fiber network was 1.7  1.4 mm for the ROI (red square in Figure 3a; scanning range for the carbon fiber network was 1.7 × 1.4 mm for the ROI (red square in this is a photograph obtained using a microscope). Figure 3c is the measured PA MAP Figure 3a; this is a photograph obtained using a microscope). Figure 3c is the measured image of ROI, which clearly identifies not only single fibers but also overlapped regions. PA MAP image of ROI, which clearly identifies not only single fibers but also overlapped In addition, we captured the PA MAP image of the leaf phantom shown in Figure 3d regions. In addition, we captured the PA MAP image of the leaf phantom shown in Figure for the ROI (red square in Figure 3b), wherein the scanning range was 11.6  6.1 mm to 3d for the ROI (red square in Figure 3b), wherein the scanning range was 11.6 × 6.1 mm to cover the wide region. PA MAP yielded structural information identical to the photograph cover the wide region. PA MAP yielded structural information identical to the photo- (Figure 3b) regardless of the thickness of the leaf veins. Based on the imaging results of a graph (Figure 3b) regardless of the thickness of the leaf veins. Based on the imaging results vessel-mimicking sample, the system was capable of in vivo imaging of microvasculature of a vessel-mimicking sample, the system was capable of in vivo imaging of microvascu- in a small animal. lature in a small animal. Figure 3. Photographs and PA MAP images of carbon fiber networks and a leaf skeleton target. (a) Figure 3. Photographs and PA MAP images of carbon fiber networks and a leaf skeleton target. Magnified photograph of carbon fiber networks. (b) Magnified photograph of leaf skeleton target. (a) Magnified photograph of carbon fiber networks. (b) Magnified photograph of leaf skeleton target. (c,d) PA MAP images of the region in (a,b), respectively. MAP, maximum amplitude projection. (c,d) PA MAP images of the region in (a,b), respectively. MAP, maximum amplitude projection. 3.3. WP-GVS-HH-PAM for In Vivo Experiments with a Mouse 3.3. WP-GVS-HH-PAM for In Vivo Experiments with a Mouse As an in vivo experiment, the microvasculature of mouse ear, iris, and brain were As an in vivo experiment, the microvasculature of mouse ear, iris, and brain were imaged using WP-GVS-HH-PAM. The intensity-based PA MAP images captured for each imaged using WP-GVS-HH-PAM. The intensity-based PA MAP images captured for each target tissue are shown in Figure 4. The obtained image of the vasculature map in the mouse ear (Figure 4a) demonstrates the micro-vessel networks including single capillaries, an artery, and a vein. The FOV for imaging a mouse ear was 9.0  8.2 mm, which was sufficient to obtain complete structural information of the ear with a single scan. In addition, we captured the PA MAP image of the iris (Figure 4b) with a FOV of 3.1  2.6 mm. The resulting image showed the microvasculature of the iris; we have adjusted the focus by Photonics 2021, 8, 305 7 of 10 target tissue are shown in Figure 4. The obtained image of the vasculature map in the mouse ear (Figure 4a) demonstrates the micro-vessel networks including single capillar- ies, an artery, and a vein. The FOV for imaging a mouse ear was 9.0 × 8.2 mm, which was Photonics 2021, 8, 305 7 of 10 sufficient to obtain complete structural information of the ear with a single scan. In addi- tion, we captured the PA MAP image of the iris (Figure 4b) with a FOV of 3.1 × 2.6 mm. The resulting image showed the microvasculature of the iris; we have adjusted the focus regulating the position of the objective lens to image the lateral region for this image. The by regulating the position of the objective lens to image the lateral region for this image. PA MAP image of the mouse brain under an intact skull (Figure 4c) was also obtained. The The PA MAP image of the mouse brain under an intact skull (Figure 4c) was also obtained. FOV was 10.5  7.0 mm, and the distinctive blood vascular features, which are indicated The FOV was 10.5 × 7.0 mm, and the distinctive blood vascular features, which are indi- as white arrow (e.g., sagittal sinus and coronal suture) were visualized. In addition, as an cated as white arrow (e.g., sagittal sinus and coronal suture) were visualized. In addition, aspect of depth-encoded PA MAP images to provide 3D volume data-based quantitative as an aspect of depth-encoded PA MAP images to provide 3D volume data-based quanti- information, the depth profile along with the z-axis direction was applied to in vivo PA tative information, the depth profile along with the z-axis direction was applied to in vivo images, as shown in Figure 4d–f. By using the wide FOV of WP-GVS-HH-PAM, we were PA images, as shown in Figure 4d–f. By using the wide FOV of WP-GVS-HH-PAM, we able to measure the whole range of each target tissue without applying an additional were able to measure the whole range of each target tissue without applying an additional imaging technique, such as a linear stage, to overcome the limited scanning range. imaging technique, such as a linear stage, to overcome the limited scanning range. Figure 4. In vivo PA MAP images of micro-vascular maps using WP-GVS-HH-PAM in mouse ear, Figure 4. In vivo PA MAP images of micro-vascular maps using WP-GVS-HH-PAM in mouse ear, iris, and brain, respectively. (a–c) PA signal intensity-based MAP images. (d–f) Depth-encoded PA iris, and brain, respectively. (a–c) PA signal intensity-based MAP images. (d–f) Depth-encoded PA MAP images. MAP images. 4. Discussions Here, we presented a HH-PAM probe with an extended FOV for wide-range scanning regardless of sample shape. The previously presented GVS-based HH-PAM probe has a limited FOV, caused by the clear aperture or usable region of other optical components Photonics 2021, 8, 305 8 of 10 used after GVS (e.g., fiber bundles and objective lens) [27,28]. In contrast, the scanning range of the proposed system was relatively enlarged, given that the two-axis WP-GVS was located at the terminal end of the HH probe. Furthermore, the presented system provides comparably improved axial resolution, from 90 m to 31.3 m, compared to cylindrically- focused acoustic transducer-based systems [29,30]. In addition, although the MEMS-based HH-PAM system provides an enhanced scanning rate compared to GVS and miniaturized probe volume size, the FOV was limited because of the small size of the MEMS mirror. WP- GVS-HH-PAM, however, can capture a wide area in a single scan, owing to the relatively large scanning angle of the GVS mirror (25 ) compared to the MEMS scanner (18 for fast-axis and 11 for slow-axis) [21]. In addition, through triangular wave signal-based GVS scanning, the durability of the developed scanner is enhanced, and a high sensitivity for the scanning position was maintained even at high-speed scanning. WP-GVS-HH-PAM advances the previously reported system [31] as an aspect of the movement of the imaging probe, which enhances the applicability of PA imaging systems. By applying fiber coupling through the implementation of the multimode fiber and modified, 3D modeling-based probe construction, WP-GVS-HH-PAM provides unrestricted movement of the probe motion and enhances the convenience of PA imaging. With regard to resolution, the measured lateral resolution of the proposed system was 11.5 m. Although the obtained value was sufficient for in vivo microvascular mapping of small animals, the lateral resolution of the system can be further improved by adding a lens pair (i.e., beam expander) before the objective lens. In addition, the scanning time for volumetric imaging using WP-GVS-HH-PAM was 32 s for an 800  800  512 pixel image. This time can be adjusted by the number of pixels according to the experimental requirements. The scanning speed and the sensitivity of WP-GVS-HH-PAM for in vivo imaging can be improved by using another laser source with an enhanced pulse-repetition rate and high-power light. Moreover, the FOV can also be adjusted following the ROI from a micrometer scale to up to 14.5  9 mm , which is sufficient to measure the whole region of the mouse brain. 5. Conclusions In conclusion, we demonstrated the WP-GVS-HH-PAM with a laboratory customized two-axis WP-GVS that provides an extensive scanning range for a wide range in vivo 3D imaging. The developed HH-probe consisted of a two-axis WP-GVS for wide scanning and an opto-acoustic combiner for coaxial and confocal alignment of the optical beam and acoustic signal. Compared to the conventional HH-PAM systems, our proposed WP-GVS- HH-PAM provides an extended FOV (from micro-scale (142 m at 0.1 V of galvanometer scanner) to 14.5  9 mm ) for in vivo imaging. The spatial and axial resolutions were 11.5 m and 31.3 m, respectively, which are sufficient for vascular mapping. We quan- titatively evaluated the system performance using vessel-mimicking phantom samples (carbon fiber networks and leaf skeleton) and in vivo imaging in a small animal (mouse ear, iris, and brain). The obtained results of the implemented WP-GVS-HH-PAM demon- strate the feasibility of capturing a microvascular map of several animal tissues. Hence, the proposed system shows promising results encouraging the applications of HH-PAM to various biomedical applications including preclinical and clinical research with small animals in vivo. Author Contributions: Conceptualization, M.J. and J.K.; methodology, D.S. and S.H.; software, S.H.; validation, D.S., J.L. (Jaeyul Lee) and Y.K.; formal analysis, D.S. and S.H; investigation, Y.K., E.L. and J.L. (Junsoo Lee); resources, M.J. and J.K.; data curation, D.S., S.H., Y.K. and E.L.; writing—original draft preparation, D.S.; writing—review and editing, S.H. and J.L. (Jaeyul Lee); visualization, D.S., S.H. and E.L.; supervision, M.J. and J.K.; project administration, M.J.; funding acquisition, M.J. and J.K. All authors have read and agreed to the published version of the manuscript. Funding: This work was supported by LG Yonam Foundation (of Korea) and also supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (2020R1I1A1A01072399), and supported by Basic Science Research Program Photonics 2021, 8, 305 9 of 10 through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (2018R1D1A1B07043340). 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Journal

PhotonicsMultidisciplinary Digital Publishing Institute

Published: Jul 30, 2021

Keywords: photoacoustic microscopy; handheld probe; wide-field imaging; in vivo vasculature imaging; 3D imaging

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