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Recent advances in photoacoustic endoscopy

Imaging based on photoacoustic effect relies on il-luminating with short light pulses absorbed by tissue absorbers, resulting in thermoelastic expansion, giving rise to ultrasonic waves. The ultrasonic waves are then detected by detectors placed around the sample. Photoacoustic endoscopy (PAE) is one of four major implementations of photoacoustic tomography that have been developed recently. The prototype PAE was based on scanning mirror system that deflected both the light and the ultrasound. A recently developed mini-probe was further miniaturized, and enabled simultaneous photoacoustic and ultrasound imaging. This PAE-endos-copic ultrasound (EUS) system can offer high-resolution vasculature information in the gastrointestinal (GI) tract and display differences between optical and mechanical contrast compared with single-mode EUS. However, PAE for endoscopic GI imaging is still at the preclinical stage. In this commentary, we describe the technological improvements in PAE for possible clinical application in endoscopic GI imaging. In addition, we discuss the technical details of the ultrasonic transducer incorporated into the photoacoustic endoscopic probe.


COMMENTARY ON HOT TOPICS
Photoacoustics is described as laser induced ultrasound [1] . Imaging based on photoacoustics uses short light pulses (nanosecond range) as the source. As pulsed light is absorbed by tissue absorbers, such as hemoglobin or melanin, a transient temperature increase is generated, resulting in local thermoelastic expansion, giving rise to ultrasonic waves [2] . These ultrasonic waves are then detected by ultrasonic detectors placed around the sample ( Figure 1). An important advantage of photoacoustic imaging is that the method can overcome the high degree of scattering of optical photons in biological tissue, resulting in high spatial resolution deep within tis-sue [3] . Although photoacoustic spectroscopy and simple imaging was developed in the 1970s, only recently has photoacoustic imaging become important in biomedical research [4] . A major photoacoustic imaging for biomedical applications is photoacoustic tomography (PAT). PAT is similar to conventional ultrasound imaging, because image information is provided by capturing the ultrasonic waves using mechanical scanning or by detection arrays [2] . However, while conventional ultrasound imaging measures only mechanical contrasts, PAT detects optical and thermoelastic contrasts [5] . Currently, PAT has four major implementations: raster-scan based photoacoustic microscopy (PAM), inverse-reconstruction based photoacoustic computed tomography (PACT), rotation-scan based photoacoustic endoscopy (PAE), and hybrid PAT systems with other imaging methods ( Figure 2) [6] .
Recently, Yang et al [7] showed photoacoustic images of the rat gastrointestinal tract ex vivo using a novel photoacoustic endoscope with a miniaturized imaging probe, which integrated a light-guiding optical fiber, ultrasonic sensor, and mechanical scanning unit for circumferential sector scanning. More recently, the same group [8] developed an integrated PAE and endoscopic ultrasound (EUS) imaging system for simultaneous photoacoustic and ultrasonic imaging of internal organs in vivo. In this commentary, we describe the technological improvements in PAE for possible clinical application in endoscopic gastrointestinal (GI) imaging. We also discuss the technical details of the ultrasonic transducer incorporated into the photoacoustic endoscopic probe.

PAT
PAT is cross-sectional or three-dimensional imaging using photoacoustic effect, an emerging optical imaging modality that can offer volumetric images of biological tissues in vivo with high spatial resolution and deep tissue optical contrast [5] . PAT is similar to ultrasound imaging in that both use detected ultrasonic waves to produce images [8] . However, PAT uses optical absorption-based contrast of tissue. PAT can provide high spatial resolution because ultrasonic scattering coefficients in tissue are two to three orders of magnitude less than optical scattering coefficients [5] . Additionally, unlike ultrasonography or optical coherence tomography, PAT produces specklefree images. As mentioned above, "PAT" includes PAM, PAE, PACT (Table 1). While PAM and PAE can image millimeters deep at microscopic resolution, PACT is available for microscopic and macroscopic imaging. In addition, PAT has been integrated into other imaging modalities, including ultrasound imaging [9] , optical coherence tomography (OCT) [10] , confocal microscopy [11] , twophoton microscopy [6] , and magnetic resonance imaging [12] .
Single-wavelength photoacoustic measurements of hemoglobin, a prominent light absorber in tissue, can provide images of blood vessels without exogenous contrasts. Deeper-seated vascular structures can be detected using a red or near infrared wavelength shift [2] . In addition, the technique can evaluate oxygen saturation inside blood vessels because oxyhemoglobin and deoxyhemoglobin have significantly different optical absorption spectra [13] . Other endogenous optical absorbers, such as melanin and other tissue chromophores, can contribute to photoacoustic signals. Sound reflectors such as calcification are useful in images of some tumors, including leiomyomas, leiomyosarcomas, or mucinous adenocarcinomas [2] .
Multispectral optoacoustic tomography (MSOT) with multiple illumination wavelengths can help differentiate extrinsic contrast agents (such as common fluorochromes, or photoabsorbing nanoparticles) from intrinsic contrasts (such as hemoglobin or melanin) by their unique spectral signatures [14] . This imaging modality can offer differentiation of physiological conditions with the combination of each image of different absorbers [2] . Using this method, Oh et al [15] reported three-dimensional images of subcutaneous melanomas and their surrounding vasculature in nude mice by dual-wavelength reflection-mode PAM, in which melanin distribution was imaged with a near-infrared light source and vascular system surrounding the melanoma with visible light. Extrinsically administered contrast agents for MSOT should have a sufficiently high optical absorption to be detected in tissues [3] . Such agents include near-infrared cyanine dyes, such as indocyanine green [16] , reporter gene products [17] , and light-absorbing nanoparticles, such as gold nanoparticles [18] and carbon nanotubes [19] . Several nanoparticles produce significantly stronger photoacoustic signals than organic dyes [2] . However, they also have limitations, including their larger size and safety concerns. MSOT can also detect activatable contrast agents, such as "smart probes" or molecular beacons, that are dark in their base state but produce fluorescence after target interaction [20] . MSOT can provide functional, genetic, and molecular imaging using these extrinsic contrast agents [5] .
In recent years, PAT has been used in a number of preclinical applications, including imaging of angiogenesis, the microcirculation, drug responses, brain func- Reproduced with permission from Yao et al [6] . PA: Photoacoustic.
tion, tumor microenvironments, biomarkers, and gene expression [5] . PAT is also in the early stages of clinical application including breast cancer diagnosis [21] , melanoma imaging [22] , prostate cancer treatment [23] , and non-invasive sentinel lymph node imaging [24] . Further developments in photoacoustic imaging techniques may provide better diagnosis of diseases and patient-management strategies.

PAE
Conventional white light endoscopic imaging of GI tract allows direct visualization of morphological changes and lesions, and subsequent histological analysis of tissue is the gold standard for final diagnosis. However, this method is limited by human vision and the lack of sensitivity to subsurface activity [2] . Recent advances in optics and digital imaging techniques have been introduced in GI endoscopy. Several methods, including narrow-band imaging, autofluorescence imaging, confocal endomicroscopy, OCT, and two-photon microscopy, have been developed and are under investigation. Some of these methods have been used in clinical practice; however, their diagnostic accuracy and efficacy need to be confirmed in large-scale clinical trials. Additionally, these imaging methods cannot achieve greater penetration depth [25] . EUS-based imaging can penetrate for several millimeters to centimeters in tissue. However, its limitations include poor contrast and difficult interpretation of data [2] . In addition, the mechanical contrast in EUS images often does not provide the required sensitivity and specificity [26] . PAE may be useful as a new, minimally invasive diagnostic imaging tool because it provides functional optical contrast with high spatial resolution and maintains the benefits of traditional ultrasound endoscopy [7] . Although the penetration depth of PAT can provide images that are centimeters deep, internal organs, such as the gastrointestinal tract and cardiovascular system, are not reachable [6] . The photoacoustic probe must be positioned close to the area of interest by means of endoscopy in hollow organs [7] . Viator et al [1] first developed a photoacoustic endoscopic probe for 1D sensing. Sethuraman et al [27] demonstrated photoacoustic images of rabbit blood vessels ex vivo using a high-frequency intravascular ultrasound imaging catheter. However, the system was not truly endoscopic because it used external illumination.
PAE has been investigated intensively as a tool of GI tract imaging. A prototype PAE system with a miniatur-
ized imaging probe integrates a light-guiding optical fiber, an ultrasonic sensor, and a mechanical scanning unit into one small unit placed at the distal end of the endoscope [7] . This probe used a scanning mirror system instead of conventional flexible shaft-based mechanical scanning, enabling circumferential sector scanning without moving other illumination optics or the ultrasonic detector. The large intestinal tract of a rat was imaged ex vivo with this probe. However, probe diameter was 4.2 mm due to the larger transducer size. One recently developed probe is 3.8 mm in diameter and approximate 38 mm in length, enabling simultaneous photoacoustic and ultrasound imaging using a single device [8] . In this endoscopic system, a focused ultrasonic transducer detects one-dimensional, depth-resolved signals (or the A-line). Additionally, crosssectional images (or B-scan) can be achieved by constant rotation of a scanning mirror that directs both optical and acoustic waves. This system records and shows a set of dual wavelength photoacoustic to differentiate oxyand deoxyhemoglobin, two of the dominant absorbers of visible light in soft biological tissues, and ultrasonic B-scan images in real time. It provides anatomical information about a rabbit esophagus and organs surrounding the esophagus, covering an approximately 14-cm long and 18-mm diameter volume ( Figure 3). Volume rendering enabled three-dimensional visualization of the morphology and configuration of tissues and proximal organs surrounding the esophagus. Also, simultaneous, coregistered PAE-EUS colonoscopic pseudo-color images of the rat colon in vivo, and images of the lymphovascular system near the rat colon, could be achieved using the same scanning parameters as imaging of the esophagus. Thus, PAE-EUS system can provide high-resolution information on the GI tract vasculature and display differences between optical and mechanical contrast compared with single-mode EUS. However, the probe was too large to fit in the working channel (usually approximate 2.8or 3.7-mm diameter) of a standard endoscope. More recently, a newer generation probe was further miniaturized, with probe diameter of 2.5 mm and a approximate 35 mm rigid length [28] . This mini-probe may be inserted into the working channel of a standard endoscope and be used with endoscopic guidance.
In conclusion, PAE is an emerging modality, and provides image information of optical contrast or functional properties by detecting ultrasonic waves. The major advantage of PAE is the greater pentration depth, of millimeters to centimeters, in tissue. It has great potential for in vivo endoscopic applications, such as early-stage tumor detection, accurate diagnosis of submucosal lesions, and in situ characterization of diseased tissues. Targeted contrast agents may improve the capabilities of endoscopic imaging, resulting in the earlier and more accurate detection of malignant and premalignant lesions, and further extend PAE to molecular imaging. Several technical challenges regarding the use of PAE in biomedical applications must be overcome. High-repetition lasers with fast wavelength tuning at each scan position are required for high-speed multicontrast PAE. Additionally, further miniaturization of the PAE probe is essential so that it can be inserted into the working channel of a standard endoscope. Although PAE for GI endoscopic imaging is at the preclinical stage, it would become an important imaging modality with further technological improvements.