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Seeing through the Skin: Photoacoustic Tomography of Skin Vasculature and Beyond

Open AccessPublished:June 24, 2021DOI:https://doi.org/10.1016/j.xjidi.2021.100039
      Skin diseases are the most common human diseases and manifest in distinct structural and functional changes to skin tissue components such as basal cells, vasculature, and pigmentation. Although biopsy is the standard practice for skin disease diagnosis, it is not sufficient to provide in vivo status of the skin and highly depends on the timing of diagnosis. Noninvasive imaging technologies that can provide structural and functional tissue information in real time would be invaluable for skin disease diagnosis and treatment evaluation. Among the modern medical imaging technologies, photoacoustic (PA) tomography (PAT) shows great promise as an emerging optical imaging modality with high spatial resolution, high imaging speed, deep penetration depth, rich contrast, and inherent sensitivity to functional and molecular information. Over the last decade, PAT has undergone an explosion in technical development and biomedical applications. Particularly, PAT has attracted increasing attention in skin disease diagnosis, providing structural, functional, metabolic, molecular, and histological information. In this concise review, we introduce the principles and imaging capability of various PA skin imaging technologies. We highlight the representative applications in the past decade with a focus on imaging skin vasculature and melanoma. We also envision the critical technical developments necessary to further accelerate the translation of PAT technologies to fundamental skin research and clinical impacts.

      Abbreviations:

      ACD (allergy contact dermatitis), AR-PAM (acoustic-resolution photoacoustic microscopy), CSC (cryogen spray cooling), CSVV (cutaneous small-vessel vasculitis), CTC (circulating tumor cell), FDA (Food and Drug Administration), NIR (near-infrared), OR-PAM (optical-resolution photoacoustic microscopy), PA (photoacoustic), PACT (photoacoustic computed tomography), PAM (photoacoustic microscopy), PAT (photoacoustic tomography), PWS (port-wine stain), RSOM (raster-scan optoacoustic mesoscopy), sO2 (oxygen saturation of hemoglobin), THb (total hemoglobin concentration)

      Introduction

      Skin is the largest organ in the human body and plays an important role in immunity and regulation (
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      ). The skin can be further subdivided into three major layers—the epidermis, the dermis, and the subcutaneous tissue (also known as the adipose layer)—each with its own unique structure and function. The epidermis serves to attenuate harmful UV light from the sunlight using melanin, a natural pigment capable of shielding nuclear DNA from ionizing UVR. The dermis contains hair roots, nerve endings, blood vessels, and sweat glands that regulate body temperature and remove waste products. The subcutaneous tissue provides body energy, reduces external impact, and protects deep tissue. Skin diseases are ranked as the fourth most common cause of global human health issues (
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      ). Currently, a skin biopsy is the standard practice for many skin disease diagnoses; however, this is far from sufficient in clinical practice. First, a skin biopsy cannot evaluate in vivo vascular functions. Increasing evidence has shown that skin diseases often lead to substantial functional changes in the affected blood vessels, resulting in abnormal delivery of oxygenated blood to tissues (
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      ), and these optical imaging technologies do not have sufficient penetration depths (Figure 1a). Although ultrasound imaging, another traditional imaging method, can achieve deep penetration, it lacks the sensitivity to visualize microvasculature without microbubbles. Thus, there is a clinical need for an imaging technology that can provide detailed deep tissue information for skin disease diagnosis and treatment monitoring.
      Figure thumbnail gr1
      Figure 1Photon propagation regimes in soft tissue and the penetration limits of representative high-resolution optical imaging modalities. (a) Photon propagation regimes in soft tissue and association with the penetration limits of high-resolution optical imaging modalities (
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      . AR-PAM, acoustic-resolution photoacoustic microscopy; CFM, confocal microscopy; OAC, optical‒acoustic combiner; OR-PAM, optical-resolution photoacoustic microscopy; PACT, photoacoustic computed tomography; PAT, photoacoustic tomography; TPM, two-photon microscopy; UTA, ultrasound transducer array.
      The aforementioned clinical need for skin imaging can be potentially addressed by photoacoustic (PA) tomography (PAT) (
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      ), ophthalmology (
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      ). Different from traditional optical imaging, PAT uses acoustic detection and thus can penetrate several millimeters into the skin with optically or ultrasonically determined resolutions (
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      • Stoica G.
      • Wang L.V.
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      ). By providing high-resolution images with unique optical contrast, PAT has been applied to many preclinical and clinical skin studies. Owing to the unprecedented sensitivity of PA imaging to hemoglobin and melanin, PAT has been commonly used for imaging the vascular system and melanoma. Melanin is abundant in most melanoma cells and provides high contrast images that show the location, shape, and depth of the tumor. PA vascular studies reveal vascular structure, oxygenation, and flow. In the state of disease, these parameters can provide information about the tumor microenvironment, such as angiogenesis and hypoxia. The unique sensitivity of vasculature also helps to detect nonmelanoma skin cancers. In addition, other skin diseases, such as psoriasis, dermatitis, and port-wine stains (PWSs), can be quantitatively detected and evaluated by PAT.
      In this concise review, we discuss the technical advances and (pre)clinical applications of PA skin imaging. We introduce the principle of PAT and describe various PA skin imaging systems as well as representative PA skin imaging studies. We conclude with a brief discussion of further improvements in PA skin imaging for future clinical translation. In the interest of brevity, only representative technologies and applications are highlighted in this review. Information about more PAT applications can be found in other comprehensive review articles (
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      ).

      Principles of PAT

      Figure 2a illustrates the imaging principle of PAT. To summarize, as photons travel in tissue, some of them are absorbed by molecules, and their energy is partially or completely converted into heat. The heat then induces an initial pressure rise, which propagates as an acoustic wave. An ultrasonic transducer or transducer array detects the acoustic wave to form an image, which maps the original optical energy deposition in the tissue (
      • Wang L.V.
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      Biomedical optics: principles and imaging.
      ). It should be noted that conventional clinical ultrasound imaging systems often must be modified or optimized for PAT to avoid image reconstruction distortion and to improve the field of view (
      • Choi W.
      • Park E.Y.
      • Jeon S.
      • Kim C.
      Clinical photoacoustic imaging platforms.
      ). PAT has two major implementations on the basis of the image formation methods (
      • Yang J.M.
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      • Chen R.
      • Yao J.
      • Cai X.
      • Maslov K.
      • et al.
      Simultaneous functional photoacoustic and ultrasonic endoscopy of internal organs in vivo.
      ): reconstruction-based PA computed tomography (PACT) and acoustic lens‒based PA microscopy (PAM). Both implementations have been used for skin imaging. In PACT, the object is excited by a broadened laser beam. An array of ultrasonic transducers is then placed close to the object to simultaneously receive the emitted ultrasonic waves (Figure 2b). Various reconstruction methods, such as the universal back-projection method (
      • Xu M.
      • Wang L.V.
      Universal back-projection algorithm for photoacoustic computed tomography [published correction appears in Phys Rev E Stat Nonlin Soft Matter Phys 2007;75:059903].
      ) and iteration-based method (
      • Xu Y.
      • Wang L.V.
      Time reversal and its application to tomography with diffracting sources.
      ), can be used to map the laser-induced initial pressure rise distribution. Using near-infrared (NIR) light and low-frequency ultrasound detection, PACT can readily achieve a penetration depth of several centimeters, a length that extends deeper than any human skin. In PAM, instead of reconstructing the image digitally as PACT does, a focused ultrasound transducer is used for analog image reconstruction. On the basis of the spatial resolution and targeted imaging depth, PAM generally has two formats: optical-resolution PAM (OR-PAM) (Figure 2c) (
      • Maslov K.
      • Zhang H.F.
      • Hu S.
      • Wang L.V.
      Optical-resolution photoacoustic microscopy for in vivo imaging of single capillaries.
      ), which has a lateral resolution ranging from submicrometer to a few micrometers and an imaging depth <1.0 mm, and acoustic-resolution PAM (AR-PAM) (Figure 2d) (
      • Maslov K.
      • Stoica G.
      • Wang L.V.
      In vivo dark-field reflection-mode photoacoustic microscopy.
      ), which has a lateral resolution of tens of micrometers and an imaging depth of 1.0‒10 mm. The smallest vessels of the skin (known as papillary plexus) are mostly contained within the 1 mm depth of the dermis, and the larger vessels of the subcutis can reach up to 4 mm (Figure 3a); therefore, the papillary plexus can be imaged with single-vessel resolution using OR-PAM (Figure 3b), and the larger vessels of the subcutis can be imaged with deep penetration using AR-PAM (Figure 3c). To capture the highest degree of details and maximize the penetration depths, hybrid systems have been developed to incorporate both OR-PAM and AR-PAM (
      • Ma H.
      • Cheng Z.
      • Wang Z.
      • Zhang W.
      • Yang S.
      Switchable optical and acoustic resolution photoacoustic dermoscope dedicated into in vivo biopsy-like of human skin.
      ). Throughout the rest of this paper, we aim to simplify the engineering terminology for clinical readers using PAT in general for different implementations, if not otherwise stated. The readers are encouraged to refer to the corresponding literature for the technical details.
      Figure thumbnail gr2
      Figure 2Principles of PAT and major implementations. (a) Imaging principle of PAT. When a short laser pulse is fired at the biological tissue, some photons are absorbed by biomolecules, and their energy is converted into heat through the nonradiative relaxation of excited molecules. The local temperature rise induces a local pressure rise, which propagates as an acoustic wave through the tissue and reaches an UT or UTA. The received signals are used to form an image that maps the original optical energy deposition inside the tissue. (b) PACT system with a linear UTA (
      • Erpelding T.N.
      • Kim C.
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      • Maslov K.
      • Guo Z.J.
      • et al.
      Sentinel lymph nodes in the rat: noninvasive photoacoustic and US imaging with a clinical US system.
      ), which has been most commonly adapted for clinical studies (
      • Choi W.
      • Park E.Y.
      • Jeon S.
      • Kim C.
      Clinical photoacoustic imaging platforms.
      ). The excitation light is delivered through a fused-end, bifurcated fiber bundle that flanks both sides of the UTA. (c) Reflection-mode OR-PAM system with an optical‒acoustic combiner that transmits light but reflects sound (
      • Yao J.
      • Maslov K.I.
      • Zhang Y.
      • Xia Y.
      • Wang L.V.
      Label-free oxygen-metabolic photoacoustic microscopy in vivo.
      ). SOL sandwiched between two prisms. OR-PAM is mostly used for preclinical studies. (d) AR-PAM system with a dark-field illumination (
      • Zhang H.F.
      • Maslov K.
      • Stoica G.
      • Wang L.V.
      Functional photoacoustic microscopy for high-resolution and noninvasive in vivo imaging.
      ). The laser light is only weakly focused, with the UT in the dark cone. AR-PAM has also been used for the clinical study of skin vasculature disease (
      • Aguirre J.
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      • Omar M.
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      • Eyerich K.
      • et al.
      Precision assessment of label-free psoriasis biomarkers with ultra-broadband optoacoustic mesoscopy.
      ). Reprinted with permission from
      • Wang L.V.
      • Yao J.
      A practical guide to photoacoustic tomography in the life sciences.
      . AR-PAM, acoustic-resolution photoacoustic microscopy; NA, numerical aperture; OR-PAM, optical-resolution photoacoustic microscopy; PACT, photoacoustic computed tomography; PAT, photoacoustic tomography; SOL, silicone oil layer; UT, ultrasound transducer; UTA, ultrasound transducer array.
      Figure thumbnail gr3
      Figure 3Illustration of the penetration depths of OR-PAM and AR-PAM in the skin. (a) Schematic of the skin layers and the corresponding penetration depths of OR-PAM and AR-PAM. (b) Representative OR-PAM image of the mouse skin vasculature, showing a 1-mm penetration depth. (c) Representative AR-PAM of an ex vivo pig skin sample with blood preserved, showing a 5-mm pentation depth. Bar = 1 mm. AR-PAM, acoustic-resolution photoacoustic microscopy; OR-PAM, optical-resolution photoacoustic microscopy.
      To accommodate the rich optical absorption contrast in biological tissues, PAT has expanded its arsenal of optical excitation wavelengths to provide the most optimized imaging performance, such as detection sensitivity and contrast to noise ratio (
      • Upputuri P.K.
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      Recent advances in photoacoustic contrast agents for in vivo imaging.
      ;
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      Sensitivity of photoacoustic microscopy.
      ,
      • Yao J.
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      Photoacoustic microscopy.
      ). Hemoglobin in red blood cells is the major optical absorber that provides the high image contrast of blood vessels (
      • Hu S.
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      Photoacoustic imaging and characterization of the microvasculature.
      ). The visible light wavelength ranging from 500 nm to 650 nm is most often used for PAT of skin vasculature, balancing between the penetration depth and the signal to noise ratio. The distinct features in the optical absorption spectra of oxyhemoglobin and deoxyhemoglobin allow for multispectral unmixing of the two forms. Melanin has a broad and relatively featureless optical absorption spectrum ranging from visible light to NIR light and thus is often imaged by PAT at wavelengths longer than 750 nm to suppress the background signals from hemoglobin (
      • Park B.
      • Bang C.H.
      • Lee C.
      • Han J.H.
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      • Kim J.
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      3D wide-field multispectral photoacoustic imaging of human melanomas in vivo: a pilot study.
      ;
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      • Cui H.
      • Cohen M.S.
      • Yang X.
      Growth of melanoma brain tumors monitored by photoacoustic microscopy.
      ). DNA and RNA in the cell nuclei have strong optical absorption in UV light and can be imaged by PAT at wavelengths around 200‒300 nm (
      • Haven N.J.
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      Reflective objective-based ultraviolet photoacoustic remote sensing virtual histopathology.
      ;
      • Wong T.T.W.
      • Zhang R.
      • Hai P.
      • Zhang C.
      • Pleitez M.A.
      • Aft R.L.
      • et al.
      Fast label-free multilayered histology-like imaging of human breast cancer by photoacoustic microscopy.
      ,
      • Wong T.T.
      • Zhang R.
      • Zhang C.
      • Hsu H.C.
      • Maslov K.I.
      • Wang L.
      • et al.
      Label-free automated three-dimensional imaging of whole organs by microtomy-assisted photoacoustic microscopy.
      ;
      • Yao D.K.
      • Maslov K.
      • Shung K.K.
      • Zhou Q.
      • Wang L.V.
      In vivo label-free photoacoustic microscopy of cell nuclei by excitation of DNA and RNA.
      ). Water and lipids have relatively strong absorption in the far NIR or mid-infrared region and can be imaged by PAT at wavelengths >1,200 nm (
      • Pleitez M.A.
      • Khan A.A.
      • Soldà A.
      • Chmyrov A.
      • Reber J.
      • Gasparin F.
      • et al.
      Label-free metabolic imaging by mid-infrared optoacoustic microscopy in living cells.
      ;
      • Shi J.
      • Wong T.T.W.
      • He Y.
      • Li L.
      • Zhang R.
      • Yung C.S.
      • et al.
      High-resolution, high-contrast mid-infrared imaging of fresh biological samples with ultraviolet-localized photoacoustic microscopy.
      ).

      Biomarkers for functional PAT

      Alongside its ability to penetrate deep into the skin to visualize vasculature, PAT is also powerful in its inherent sensitivity to a variety of functional and molecular biomarkers. In fact, as shown in Figure 4, PAT can be used for functional imaging of blood vessels surrounding a melanoma tumor by quantifying the vascular density, oxygen saturation of hemoglobin (sO2), and blood flow speed. In this example, the spatial resolution of PAT is sufficient to resolve single capillaries and even red blood cells (Figure 4a). PAT is also capable of quantitative analysis of tissue components with spectroscopic measurement, such as the melanin concentration in the melanoma tumor (Figure 4b). Because PAT exploits the absorption properties of biomolecules to generate contrast, functional information of the hemodynamics can be obtained without injecting any contrast agents. For example, sO2 is an important biomarker for skin cancer prognosis, treatment monitoring, and wound healing monitoring. PAT can provide high-resolution sO2 measurements using hemoglobin as the endogenous contrast (Figure 4c). In fact, sO2 has been successfully imaged by PAT at different length scales in a number of cases, including during cuffed occlusion of the arm (
      • Favazza C.
      • Maslov K.
      • Cornelius L.
      • Wang L.V.
      In vivo functional human imaging using photoacoustic microscopy: response to ischemic and thermal stimuli.
      ), controlled temperature changes (
      • Favazza C.
      • Maslov K.
      • Cornelius L.
      • Wang L.V.
      In vivo functional human imaging using photoacoustic microscopy: response to ischemic and thermal stimuli.
      ), and relatively homeostatic situations (
      • Deán-Ben X.L.
      • Razansky D.
      Hand-held optoacoustic probe for three-dimensional imaging of human morphology and function.
      ;
      • Favazza C.P.
      • Cornelius L.A.
      • Wang L.V.
      In vivo functional photoacoustic microscopy of cutaneous microvasculature in human skin.
      ;
      • Zhang H.F.
      • Maslov K.
      • Oh J.T.
      • Stoica G.
      • Wang L.V.
      Functional photoacoustic microscopy in vivo.
      • Zhang H.F.
      • Maslov K.
      • Sivaramakrishnan M.
      • Stoica G.
      • Wang L.H.V.
      Imaging of hemoglobin oxygen saturation variations in single vessels in vivo using photoacoustic microscopy.
      • Zhang H.F.
      • Maslov K.
      • Stoica G.
      • Wang L.V.
      Functional photoacoustic microscopy for high-resolution and noninvasive in vivo imaging.
      ). sO2 has even been resolved down to the single-cell level in a human cuticle (
      • Hsu H.C.
      • Wang L.D.
      • Wang L.V.
      In vivo photoacoustic microscopy of human cuticle microvasculature with single-cell resolution.
      ). The total hemoglobin concentration (THb) can also be calculated at an isosbestic wavelength, where deoxyhemoglobin and oxyhemoglobin have identical molar extinction coefficients (
      • Patrikeev I.
      • Brecht H.P.
      • Petrov Y.Y.
      • Petrova I.Y.
      • Prough D.S.
      • Esenaliev R.O.
      Wavelet differentiation of optoacoustic signals for monitoring of total hemoglobin concentration and oxygen saturation level in small blood vessels.
      ).
      Figure thumbnail gr4
      Figure 4Representative PA imaging of skin vasculature using endogenous contrast. (a) PA microvasculature imaging of a mouse ear bearing an implanted B16 melanoma tumor. Depth is coded by colors: blue (superficial) to red (deep). Bar = 1 mm. (b) Close-up PA image of the B16 melanoma tumor identified in the white box of a using spectroscopic measurements. (c) PA sO2 imaging of the principal arterial‒vein pair. Bar = 200 μm. (d) PA flow velocity imaging of the principal arterial‒vein pair. PA, photoacoustic; sO2, oxygen saturation of hemoglobin.
      Blood flow velocity is another critically important biomarker of vascular function and tissue viability. Although Doppler ultrasound techniques have proven useful for large veins and arteries, these techniques do not work well for smaller blood vessels and low flow speeds. In PAT, the Doppler effect can be utilized to measure flow speed.
      • Yao J.
      • Maslov K.I.
      • Shi Y.
      • Taber L.A.
      • Wang L.V.
      In vivo photoacoustic imaging of transverse blood flow by using Doppler broadening of bandwidth.
      showed that Doppler bandwidth broadening can be measured and can be related to the velocity of transverse blood flow. Once it was determined that flow rate could be measured with PA imaging, researchers sought to measure the metabolic rate of oxygen, another critical biomarker of tissue health. This parameter encompasses vessel geometry and weight, THb, oxygen saturation, and flow rate, all of which can be measured by PAT alone. Because the metabolic rate of oxygen indicates the rate at which tissue is consuming oxygen, it is valuable for cancer diagnosis and treatment evaluation (
      • Yao J.
      • Maslov K.I.
      • Zhang Y.
      • Xia Y.
      • Wang L.V.
      Label-free oxygen-metabolic photoacoustic microscopy in vivo.
      ). At clinically relevant depths, PAT can be potentially an invaluable tool to study skin disease and treatment.

      Applications of PAT for skin imaging

      Although each skin disease possesses its own unique properties, almost all skin diseases are characterized by neovascularization or changes in vascular function (
      • Miller A.J.
      • Mihm Jr., M.C.
      Melanoma.
      ). Since the first demonstration of PAT of human palm vasculature at an imaging depth of 4 mm (
      • Zhang E.Z.
      • Laufer J.G.
      • Pedley R.B.
      • Beard P.C.
      In vivo high-resolution 3D photoacoustic imaging of superficial vascular anatomy.
      ), PAT of the skin vasculature has become a valuable tool for detecting and characterizing various skin conditions. In fact, many vascular details, including thickness, diameter, and depth, can be obtained through the use of PA dermoscopy (
      • Xu D.
      • Yang S.
      • Wang Y.
      • Gu Y.
      • Xing D.
      Noninvasive and high-resolving photoacoustic dermoscopy of human skin.
      ). In this section, we will review the different ways through which PAT has been applied in preclinical and clinical studies of skin diseases on small animal models and patients.

      PAT of melanoma

      Melanoma is the deadliest form of skin cancer projected to claim the lives of 7,180 Americans diagnosed in 2021 alone, according to the 2021 melanoma skin cancer statistics by the American Cancer Society. Characterized by the dysregulated division of melanocytes due to an accumulation of mutations in genes responsible for cell division, melanoma can be lethal if allowed to metastasize (
      • Davis L.E.
      • Shalin S.C.
      • Tackett A.J.
      Current state of melanoma diagnosis and treatment.
      ). Thus, the best way to reduce mortality is through the detection of early-stage melanoma before metastasis. In fact, according to the Melanoma Research Alliance, patients with localized melanoma (stages 0, I, and II) have a 5-year survival rate of 98.4% in comparison with the 5-year survival rate of patients with metastatic melanoma who have a survival rate of only 22.5%. In clinical practice, a biopsy may be performed to identify malignant lesions, but the diagnostic accuracy of a given biopsy depends largely on the skill and experience of the doctor. In fact, the diagnostic certainty of an early stage in situ melanoma is only 73% for excisional biopsy, 75% for deep shave, 44% for punch, and 42% for superficial shave (
      • Hieken T.J.
      • Hernández-Irizarry R.
      • Boll J.M.
      • Jones Coleman J.E.
      Accuracy of diagnostic biopsy for cutaneous melanoma: implications for surgical oncologists.
      ). Given the large margin of diagnostic error in biopsies, there is clinical value in looking for diagnostic alternatives.
      PAT presents itself as a viable diagnostic alternative to biopsy. PAT is extremely sensitive to melanin’s strong light absorption over the NIR wavelength spectrum, and it is capable of penetrating the dermis, making it an ideal method for longitudinal monitoring of melanoma. It is also worth noting that other pigmented lesions can be characterized by PAT, including seborrheic keratosis and dysplastic nevi, both of which are benign and are commonly mistaken for melanoma. In fact, PAT has shown success in delineating the lower boundary of pigmented lesions, revealing the lesions propensity for further invasion (Figure 5a and b) (
      • Breathnach A.
      • Concannon L.
      • Aalto L.
      • Dorairaj J.
      • Subhash H.M.
      • Kelly J.
      • et al.
      Assessment of cutaneous melanoma and pigmented skin lesions with photoacoustic imaging.
      ;
      • Schwarz M.
      • Aguirre J.
      • Buehler A.
      • Omar M.
      • Ntziachristos V.
      Visualization of the microcirculatory network in skin by high frequency optoacoustic mesoscopy.
      ).
      • Zhang H.F.
      • Maslov K.
      • Oh J.T.
      • Stoica G.
      • Wang L.V.
      Functional photoacoustic microscopy in vivo.
      were among the first to use dual-wavelength PAT to perform functional imaging on mouse models of melanoma (
      • Oh J.T.
      • Li M.L.
      • Zhang H.F.
      • Maslov K.
      • Stoica G.
      • Wang L.V.
      Three-dimensional imaging of skin melanoma in vivo by dual-wavelength photoacoustic microscopy.
      ;
      • Zhang H.F.
      • Maslov K.
      • Stoica G.
      • Wang L.V.
      Functional photoacoustic microscopy for high-resolution and noninvasive in vivo imaging.
      ).
      • Zhou Y.
      • Li G.
      • Zhu L.
      • Li C.
      • Cornelius L.A.
      • Wang L.V.
      Handheld photoacoustic probe to detect both melanoma depth and volume at high speed in vivo.
      showed the volumetric melanoma growth rate over the course of tumor progression in nude mice in vivo, which was extremely useful for diagnosis, staging, and treatment evaluation.
      Figure thumbnail gr5
      Figure 5PAT of melanoma and pigmented lesion. (a) Photograph of the melanoma in a nude mouse in vivo. Bar = 1 mm. (b) Handheld PAM of melanoma clearly shows both the top and bottom boundaries. The dashed line indicates 3.6 mm. Bar = 1 mm. (c) Photograph of handheld PAM imaging a red mole on a volunteer’s leg. (d) A handheld PAM image of the mole reveals an irregular blood vessel pattern. The inset shows the photograph of the mole. Bar = 1 mm. Reprinted with permission from
      • Lin L.
      • Zhang P.
      • Xu S.
      • Shi J.
      • Li L.
      • Yao J.
      • et al.
      Handheld optical-resolution photoacoustic microscopy.
      and
      • Zhou Y.
      • Li G.
      • Zhu L.
      • Li C.
      • Cornelius L.A.
      • Wang L.V.
      Handheld photoacoustic probe to detect both melanoma depth and volume at high speed in vivo.
      . PAM, photoacoustic microscopy; PAT, photoacoustic tomography.
      In addition to elevated melanocyte activity, melanoma is also characterized by neovascularization, with linear irregular vessels as the most common vascular structure (33.3%), followed by punctate vessels and polymorphic/atypical vessels (
      • Argenziano G.
      • Zalaudek I.
      • Corona R.
      • Sera F.
      • Cicale L.
      • Petrillo G.
      • et al.
      Vascular structures in skin tumors: a dermoscopy study.
      ). In fact,
      • Omar M.
      • Schwarz M.
      • Soliman D.
      • Symvoulidis P.
      • Ntziachristos V.
      Pushing the optical imaging limits of cancer with multi-frequency-band raster-scan optoacoustic mesoscopy (RSOM).
      used AR-PAM to study the vascular network supporting the growth of melanoma through several millimeters of tumor depth. The principles of PA imaging of melanoma vasculature can also be extended to other skin carcinomas. For example,
      • Zhou W.T.
      • Chen Z.
      • Yang S.
      • Xing D.
      Optical biopsy approach to basal cell carcinoma and melanoma based on all-optically integrated photoacoustic and optical coherence tomography.
      successfully conducted all-optical integrated PA optical coherence tomography to gather in vivo imaging of basal cell carcinoma and analyze its vascular morphology.
      Success in preclinical melanoma research by PAT has since seen applications in the clinic.
      • Chuah S.Y.
      • Attia A.B.
      • Long V.
      • Ho C.J.
      • Malempati P.
      • Fu C.Y.
      • et al.
      Structural and functional 3D mapping of skin tumours with non-invasive multispectral optoacoustic tomography.
      showed a high histological agreement of melanoma depth measurements with multispectral PAT in three human patients.
      • Kim J.
      • Kim Y.H.
      • Park B.
      • Seo H.M.
      • Bang C.H.
      • Park G.S.
      • et al.
      Multispectral ex vivo photoacoustic imaging of cutaneous melanoma for better selection of the excision margin.
      later showed the utility of PAT in assessing the margins of human tumor resections ex vivo. Dual-wavelength imaging has also been performed on humans using the commercial PAT system Vevo Lazr (
      • Breathnach A.
      • Concannon L.
      • Aalto L.
      • Dorairaj J.
      • Subhash H.M.
      • Kelly J.
      • et al.
      Assessment of cutaneous melanoma and pigmented skin lesions with photoacoustic imaging.
      ).
      • Park B.
      • Bang C.H.
      • Lee C.
      • Han J.H.
      • Choi W.
      • Kim J.
      • et al.
      3D wide-field multispectral photoacoustic imaging of human melanomas in vivo: a pilot study.
      showed success in identifying the depths of in situ and nodular melanoma as well as surrounding neovascularization in six patients in vivo using a three-dimensional multispectral PA imaging system. As of late, melanoma diagnosis has even seen further advancement through the development of handheld PAT systems (Figure 5c and d) (
      • Chuah S.Y.
      • Attia A.B.
      • Long V.
      • Ho C.J.
      • Malempati P.
      • Fu C.Y.
      • et al.
      Structural and functional 3D mapping of skin tumours with non-invasive multispectral optoacoustic tomography.
      ;
      • Lee C.
      • Choi W.
      • Kim J.
      • Kim C.
      Three-dimensional clinical handheld photoacoustic/ultrasound scanner.
      ;
      • Zhou Y.
      • Li G.
      • Zhu L.
      • Li C.
      • Cornelius L.A.
      • Wang L.V.
      Handheld photoacoustic probe to detect both melanoma depth and volume at high speed in vivo.
      ). In addition, PAT has been applied to melanoma detection in conjunction with other imaging modalities, such as optical coherence tomography (
      • Rahlves M.
      • Varkentin A.
      • Stritzel J.
      • Blumenrother E.
      • Mazurenka M.
      • Wollweber M.
      • et al.
      Towards multimodal detection of melanoma thickness based on Optical Coherence Tomography and Optoacoustics.
      ) and ultrasonography (
      • Wang Y.
      • Xu D.
      • Yang S.
      • Xing D.
      Toward in vivo biopsy of melanoma based on photoacoustic and ultrasound dual imaging with an integrated detector.
      ). Acknowledging its function and flexibility within the expansive field of diagnostic imaging technologies, it is likely that PA systems will soon find a place in the field of melanoma diagnosis.

      PAT of other skin disorders

      Psoriasis is a chronic inflammatory skin disease that presents in the majority of affected individuals as irritated patches of red skin covered in white scales or plaques. It is typically characterized by dysfunction of the immune system, which causes sustained inflammation and subsequent dysregulated division of keratinocytes. One of the main concerns with psoriasis is the progression of the disease to psoriatic arthritis, which impacts up to 40% of patients with psoriasis and causes pain and swelling of the joints (
      • Rendon A.
      • Schäkel K.
      Psoriasis pathogenesis and treatment.
      ). Although there is no known cure for psoriasis, several viable treatments include topical steroid creams, phototherapy, retinoids, and immunosuppressant prescription drugs (
      • Kim W.B.
      • Jerome D.
      • Yeung J.
      Diagnosis and management of psoriasis.
      ). Because psoriasis diagnosis is primarily clinical, subtle changes indicating the onset of psoriatic lesions can go unnoticed. In fact, epidermal hyperplasia in patients with psoriasis is preceded by extensive angiogenesis in the superficial dermis (
      • Heidenreich R.
      • Röcken M.
      • Ghoreschi K.
      Angiogenesis drives psoriasis pathogenesis.
      ). These changes in vasculature can be distinguished from nondiseased skin. In fact, electron microscopy was used to identify capillary loops that curl around the papillary vessel and are closely enveloped by the epidermis as a strong indicator of early psoriatic angiogenesis (
      • Braverman I.M.
      • Yen A.
      Ultrastructure of the capillary loops in the dermal papillae of psoriasis.
      ;
      • Ryan T.J.
      Microcirculation in psoriasis: blood vessels, lymphatics and tissue fluid.
      ). Because PAT can successfully visualize vascular structures at large depths in tissue, it has shown great potential for application in early psoriasis diagnosis. Whereas low-sensitivity PA systems appear to be outperformed by other imaging alternatives such as confocal microscopy (
      • Ossadnik K.
      • Philipp S.
      • Bost W.
      • Fournelle M.
      • Richter H.
      • Lademann J.
      Application of photoacoustic methods and confocal microscopy for monitoring of therapeutic response in plaque psoriasis.
      ), advances in ultrasound transducers (
      • Wang Z.
      • Yang F.
      • Ma H.
      • Cheng Z.
      • Yang S.
      Photoacoustic and ultrasound (PAUS) dermoscope with high sensitivity and penetration depth by using a bimorph transducer.
      ) demonstrate that high-sensitivity PA imaging modalities can be used to study psoriasis-derived changes in vasculature. In fact,
      • Aguirre J.
      • Schwarz M.
      • Garzorz N.
      • Omar M.
      • Buehler A.
      • Eyerich K.
      • et al.
      Precision assessment of label-free psoriasis biomarkers with ultra-broadband optoacoustic mesoscopy.
      used a portable raster-scan optoacoustic mesoscopy (RSOM) system to provide quantitative vascular analysis of psoriasis progress, including increased vessel density and capillary bending in the upper dermis, with high sensitivity and high resolution (Figure 6).
      Figure thumbnail gr6
      Figure 6AR-PAM of healthy skin versus that of adjacent psoriatic skin and validation by histology. (a) Cross-sectional AR-PAM image of psoriatic skin showing the top part of elongated capillary loops (cyan arrow) that almost climbed to the skin surface through elongated rete ridges. Bar = 200 μm. (b) Cross-sectional AR-PAM image of adjacent healthy skin showing a layered EP structure with clearly resolved vessels in the DR. Bar = 200 μm. (c) Histology image (left) of the psoriatic skin and the corresponding AR-PAM cross-sectional image (right). The histology image shows the acanthosis, the elongated capillary loops through the rete ridge, and the increased vascularization of the DR. Bar = 300 μm. (d) Histology image (left) of healthy skin and the corresponding AR-PAM cross-sectional image (right). Bar = 300 μm. Reprinted with permission from
      • Aguirre J.
      • Schwarz M.
      • Garzorz N.
      • Omar M.
      • Buehler A.
      • Eyerich K.
      • et al.
      Precision assessment of label-free psoriasis biomarkers with ultra-broadband optoacoustic mesoscopy.
      . AR-PAM, acoustic-resolution photoacoustic microscopy; DR, dermis; EP, epidermal.
      Dermatitis describes a class of skin diseases that involve irritation of the skin manifesting in rashes as a result of inflammation. Allergy contact dermatitis (ACD), a subset of dermatitis cases, affects around 15‒20% of the general population (
      • Thyssen J.P.
      • Linneberg A.
      • Menné T.
      • Johansen J.D.
      The epidemiology of contact allergy in the general population--prevalence and main findings.
      ) and is typically triggered by the innate and adaptive immune responses to the infiltration of the epidermal layer by an exogenous irritant (
      • Martin S.F.
      Contact dermatitis: from pathomechanisms to immunotoxicology.
      ). In diagnosing ACD, physicians typically employ patch testing to identify the specific allergens associated with flare-ups. Unfortunately, there is a large margin of error in patch testing because test results are subjectively determined by a physician’s visual assessment, and skin irritation may be the result of the patch test itself rather than that of the allergen being tested. In fact, positive tests were only reproducible in 86% of cases, and the reproducibility was far lower for mild reactions (
      • Lazzarini R.
      • Duarte I.
      • Ferreira A.L.
      Patch tests.
      ). However, using RSOM,
      • Hindelang B.
      • Aguirre J.
      • Berezhnoi A.
      • He H.
      • Eyerich K.
      • Ntziachristos V.
      • et al.
      Optoacoustic mesoscopy shows potential to increase accuracy of allergy patch testing.
      were able to distinguish between mild (+) and strong (++) reactions to patch-tested allergens using vessel fragmentation and the ratio of low- to high-frequency content as biomarkers. Although
      • Hindelang B.
      • Aguirre J.
      • Berezhnoi A.
      • He H.
      • Eyerich K.
      • Ntziachristos V.
      • et al.
      Optoacoustic mesoscopy shows potential to increase accuracy of allergy patch testing.
      are one of the first to explore PAT in patch testing, their results are promising for future applications of PAT to diagnose this disease. In fact, preliminary studies of RSOM in atopic dermatitis (eczema) have already shown greater strength than the studies of optical coherence tomography in identifying disease severity (
      • Yew Y.W.
      • Unnimadhava Kurup Soudamini Amma D.
      • Kuan A.H.Y.
      • Li X.
      • Dev K.
      • Ebrahim Attia A.B.
      • et al.
      Raster-scanning optoacoustic mesoscopy imaging as an objective disease severity tool in atopic dermatitis patients.
      ).
      PA imaging has also been applied to the study of PWS because it is associated with many malformed venules concentrated in the superficial dermis of the affected area. PWSs are reddish birthmarks that can appear anywhere on the body and are due to excessive vascularization of the capillaries. The most common form of treatment is laser therapy to coagulate the unnecessary capillaries causing the redness. This is often paired with simultaneous cryogen spray cooling (CSC) of the epidermis to protect it from overheating. However, to optimize treatment parameters and guide the laser to the optimal focal points, accurate depth and morphology of PWS vascularization are necessary. In a series of studies,
      • Viator J.A.
      • Kolkman R.G.M.
      • Steenbergen W.
      Photoacoustic depth determination and imaging of port wine stain birthmarks.
      ,
      • Viator J.A.
      • Aguilar G.
      • Jacques S.L.
      • Nelson J.S.
      Optimization of cryogen spray cooling for port wine stain laser therapy using photoacoustic measurement of epidermal melanin.
      ,
      • Viator J.A.
      • Au G.
      • Choi B.
      • Nelson J.S.
      Design limitations of a photoacoustic probe for port wine stain depth determination.
      ,
      • Viator J.A.
      • Au G.
      • Paltauf G.
      • Jacques S.L.
      • Prahl S.A.
      • Ren H.W.
      • et al.
      Clinical testing of a photoacoustic probe for port wine stain depth determination.
      developed a PA imaging system to study the excessive blood vessels and capillary malformation in PWS and to quantify the PWS thickness for laser treatment planning. Using PAT, CSC and laser therapy can be adjusted appropriately to remove as much of the PWS as possible while still preserving the epidermis.

      PAT of burn injury and wound healing

      Evaluating burn injury and monitoring its healing process are other important medical applications of PAT. Burn injury is commonly induced by excessive heat exposure, and severe burn is life threatening. In the United States only, >400,000 burn injuries need medical treatment annually, leading to >3,000 deaths (
      • Crowe C.S.
      • Massenburg B.B.
      • Morrison S.D.
      • Naghavi M.
      • Pham T.N.
      • Gibran N.S.
      Trends of burn injury in the United States: 1990 to 2016.
      ). Surface area and depth of the burn are the two most important indicators for the prognosis of a patient with a burn. Whereas the burn surface area is relatively simple to measure by visual examination, the burn depth is much more challenging to precisely evaluate. An accurate burn depth is a valuable information to decide whether skin grafts are necessary and guide the clearance of necrotic tissue.
      Using only hemoglobin and water as the endogenous contrast, PAT has the potential to characterize the pathological features of healthy skin, superficial dermal burns, deep dermal burns, and deep burns (
      • Sato S.
      • Yamazaki M.
      • Saitoh D.
      • Tsuda H.
      • Okada Y.
      • Obara M.
      • et al.
      Photoacoustic diagnosis of burns in rats.
      ,
      • Sato S.
      • Shimada T.
      • Arai T.
      • Kikuchi M.
      • Obara M.
      • Ashida H.
      Measurement of photoacoustic signals from skin: potential application to burn depth estimation.
      ;
      • Yamazaki M.
      • Sato S.
      • Ashida H.
      • Saito D.
      • Okada Y.
      • Obara M.
      Measurement of burn depths in rats using multiwavelength photoacoustic depth profiling.
      ,
      • Yamazaki M.
      • Sato S.
      • Saito D.
      • Okada Y.
      • Kurita A.
      • Kikuchi M.
      • et al.
      Photoacoustic diagnosis of burns in rats: two-dimensional photoacoustic imaging of burned tissue.
      ,
      • Yamazaki M.
      • Sato S.
      • Saito D.
      • Fujita M.
      • Okada Y.
      • Kikuchi M.
      • et al.
      Photoacoustic signal measurement for burned skins in the spectral range of 500–650nm: experiment with rat burn models.
      ). There are several recent studies using PAT for burn injuries, which have majorly focused on establishing the technical feasibility, enhancing the system performance, and collecting preliminary data to support future clinical trials. For acute burns, PAT can clearly delineate the hyperemic bowl, the boundary between the edemic coagulated burned tissue and the healthy perfused tissue (Figure 7a and b) (
      • Zhang H.F.
      • Maslov K.
      • Stoica G.
      • Wang L.V.
      High-resolution burn imaging in pig skin by photoacoustic microscopy.
      ,
      • Zhang H.F.
      • Maslov K.
      • Stoica G.
      • Wang L.V.
      Imaging acute thermal burns by photoacoustic microscopy.
      ).
      • Ida T.
      • Iwazaki H.
      • Kawaguchi Y.
      • Kawauchi S.
      • Ohkura T.
      • Iwaya K.
      • et al.
      Burn depth assessments by photoacoustic imaging and laser Doppler imaging.
      recently compared the PAT measurement of burn depth with laser Doppler measurement and have found that PAT is more accurate, especially in burns induced by high temperature.
      • Talbert R.J.
      • Holan S.H.
      • Viator J.A.
      Photoacoustic discrimination of viable and thermally coagulated blood using a two-wavelength method for burn injury monitoring.
      reported that PAT can distinguish coagulated blood from normal blood using spectral measurements. The coagulated blood is often brownish in color, characteristic of burn tissue (
      • Talbert R.J.
      • Holan S.H.
      • Viator J.A.
      Photoacoustic discrimination of viable and thermally coagulated blood using a two-wavelength method for burn injury monitoring.
      ). Edema is another biomarker of thermal damage to the skin because increased blood vessel permeability at the burn site leads to abnormal accumulation of water.
      • Yoshida K.
      • Sato S.
      • Hatanaka K.
      • Saitoh D.
      • Ashida H.
      • Sakamoto T.
      • et al.
      Photoacoustic diagnosis of edema in rat burned skin.
      have used PAT to successfully characterize edema in burned rat skin, allowing better evaluation of the risks for multiple organ dysfunction syndrome and systemic inflammatory response syndrome. In this study, 1,430 nm excitation light was used, at which water absorbs strongly. Moreover, PAT can be used to trace albumin, the most abundant protein in the blood, to study changes in the permeability of burned skin. For example,
      • Tsunoi Y.
      • Sato S.
      • Kawauchi S.
      • Ashida H.
      • Saitoh D.
      • Terakawa M.
      In vivo photoacoustic molecular imaging of the distribution of serum albumin in rat burned skin.
      used PAT to image the extravascular leakage of Evans blue dye as the exogenous contrast that binds albumin with a high affinity. In addition to providing diagnostic assistance, PAT can also be used to monitor the healing process of burned or other skin wounds.
      • Später T.
      • Körbel C.
      • Frueh F.S.
      • Nickels R.M.
      • Menger M.D.
      • Laschke M.W.
      Seeding density is a crucial determinant for the in vivo vascularisation capacity of adipose tissue-derived microvascular fragments.
      used PAT to study the lipid-derived microvascular debris density required to reconstruct the host blood vessels. In addition, different tissue conditions such as angiogenesis and healing can be evaluated using PAT in a periodic manner, taking advantage of its noninvasive nonionizing nature (
      • Aizawa K.
      • Sato S.
      • Saitoh D.
      • Ashida H.
      • Obara M.
      Photoacoustic monitoring of burn healing process in rats.
      ). For laser microsurgery, PAT can be used to monitor the vasculature injury and healing process (
      • Hu S.
      • Maslov K.I.
      • Wang L.V.
      Monitoring the healing process of laser-induced microvascular lesions using optical-resolution photoacoustic microscopy.
      ). For treating burn tissue infection by antibacterial photodynamic therapy, PAT can be used to map the photosensitizer distribution in the skin to ensure adequate diffusion over the entire infected area (
      • Hirao A.
      • Sato S.
      • Saitoh D.
      • Shinomiya N.
      • Ashida H.
      • Obara M.
      In vivo photoacoustic monitoring of photosensitizer distribution in burned skin for antibacterial photodynamic therapy.
      ;
      • Tsunoi Y.
      • Sato S.
      • Ashida H.
      • Terakawa M.
      Photoacoustic imaging of intravenously injected photosensitizer in rat burn models for efficient antibacterial photodynamic therapy.
      ).
      Figure thumbnail gr7
      Figure 7PAT of burn injury. (a) Photograph and (b) AR-PAM image of an acute skin burn induced by 175 °C heat exposure for 20 seconds, showing the characteristic hyperemic ring. Bar = 1mm. Reprinted with permission from
      • Zhang H.F.
      • Maslov K.
      • Stoica G.
      • Wang L.V.
      Imaging acute thermal burns by photoacoustic microscopy.
      . AR-PAM, acoustic-resolution photoacoustic microscopy; PAT, photoacoustic tomography.

      PAT of single cells in the skin

      High-resolution imaging of single cells and cell organelles is crucial in early disease diagnosis. For example, circulating tumor cells (CTCs) are cancerous cells that enter the blood circulation from primary or metastatic lesions and play an important role in tumor recurrence and metastasis (
      • Karakousis G.
      • Yang R.
      • Xu X.
      Circulating melanoma cells as a predictive biomarker.
      ). Detection of CTCs has high clinical value for cancer staging. OR-PAM, with submicrometer to micrometer resolution, is well-suited for imaging single-melanoma cell morphology and functions (
      • Strohm E.M.
      • Moore M.J.
      • Kolios M.C.
      Single cell photoacoustic microscopy: a review.
      ). Owing to the rare occurrence of melanoma CTCs in early-stage cancer, blood test‒based ex vivo detection of CTCs is incredibly challenging with a small volume of blood sample. Alternatively, on-site PA cytometry can be used to detect the melanoma CTCs without drawing blood (
      • Galanzha E.I.
      • Shashkov E.V.
      • Kelly T.
      • Kim J.W.
      • Yang L.
      • Zharov V.P.
      In vivo magnetic enrichment and multiplex photoacoustic detection of circulating tumour cells.
      ).
      • O'Brien C.M.
      • Rood K.D.
      • Bhattacharyya K.
      • DeSouza T.
      • Sengupta S.
      • Gupta S.K.
      • et al.
      Capture of circulating tumor cells using photoacoustic flowmetry and two phase flow.
      used microfluidic two-phase flow in vitro to separate circulating melanoma tumor cells from the bloodstream on PA detection of melanin.
      • Juratli M.A.
      • Galanzha E.I.
      • Sarimollaoglu M.
      • Nedosekin D.A.
      • Suen J.Y.
      • Zharov V.P.
      In vivo detection of circulating tumor cells during tumor manipulation.
      discovered that using PA flow cytometry, head and neck melanomas release extra tumor cells into the bloodstream on disturbance by palpation or biopsy. More recently,
      • He Y.
      • Wang L.
      • Shi J.
      • Yao J.
      • Li L.
      • Zhang R.
      • et al.
      In vivo label-free photoacoustic flow cytography and on-the-spot laser killing of single circulating melanoma cells.
      showed single-melanoma CTC detection in vivo, followed by immediate laser ablation of the CTCs (Figure 8).
      Figure thumbnail gr8
      Figure 8PAT of melanoma CTC in vivo. Three fused OR-PAM images spanning ∼3 s show a single melanoma CTC traveling in the artery and then returning in the vein. The yellow boxes indicate the single CTC acquired by OR-PAM at 1,064 nm. Bar = 200 μm. Reprinted with permission from
      • He Y.
      • Wang L.
      • Shi J.
      • Yao J.
      • Li L.
      • Zhang R.
      • et al.
      In vivo label-free photoacoustic flow cytography and on-the-spot laser killing of single circulating melanoma cells.
      . CTC, circulating tumor cell; Norm., normal; OR-PAM, optical-resolution photoacoustic microscopy; PA, photoacoustic; PAT, photoacoustic tomography; s, second.
      In addition to pigmented CTCs, PAT has also been used to detect nonpigmented circulating cells (
      • Bhattacharyya K.
      • Goldschmidt B.S.
      • Viator J.A.
      Detection and capture of breast cancer cells with photoacoustic flow cytometry.
      ;
      • Cai C.
      • Carey K.A.
      • Nedosekin D.A.
      • Menyaev Y.A.
      • Sarimollaoglu M.
      • Galanzha E.I.
      • et al.
      In vivo photoacoustic flow cytometry for early malaria diagnosis.
      ;
      • Galanzha E.I.
      • Nedosekin D.A.
      • Sarimollaoglu M.
      • Orza A.I.
      • Biris A.S.
      • Verkhusha V.V.
      • et al.
      Photoacoustic and photothermal cytometry using photoswitchable proteins and nanoparticles with ultrasharp resonances [published correction appears in J Biophotonics 2015;8:687].
      ).
      • de la Zerda A.
      • Kim J.W.
      • Galanzha E.I.
      • Gambhir S.S.
      • Zharov V.P.
      Advanced contrast nanoagents for photoacoustic molecular imaging, cytometry, blood test and photothermal theranostics.
      have shown PA flowmetry of circulating breast cancer cells using targeted gold nanoparticles. PA flow cytometry has also been applied in the human cuticle for visualization of single red blood cells, which are indicative of overall blood perfusion and vascular health.
      • Hsu H.C.
      • Wang L.D.
      • Wang L.V.
      In vivo photoacoustic microscopy of human cuticle microvasculature with single-cell resolution.
      tracked the oxygen saturation and flow speed of each red blood cells as they traveled through the capillary beds. PA and photothermal flow cytometry of the blood and lymph has also been shown for tracking particles besides melanin and hemoglobin (
      • Galanzha E.I.
      • Tuchin V.V.
      • Brock R.W.
      • Zharov V.P.
      In vivo flow cytometry and time-resolved near-IR angiography and lymphography.
      ;
      • Tuchin V.V.
      In vivo optical flow cytometry and cell imaging.
      ). Moreover, for skin cell morphology, OR-PAM is capable of performing single-cell histology without staining by imaging DNA and RNA within the cell nuclei and cytochromes within the cytoplasm. For cell functions, PA flowmetry is capable of label-free imaging of oxygen unloading in single red blood cells in vivo (
      • Wang L.D.
      • Maslov K.
      • Wang L.V.
      Single-cell label-free photoacoustic flowoxigraphy in vivo.
      ). OR-PAM can image the mitosis of living mesenchymal stem cells, labeled with gold nanoparticles, over 7 days (
      • Zhang Y.S.
      • Wang Y.
      • Wang L.
      • Cai X.
      • Zhang C.
      • Wang L.V.
      • et al.
      Labeling human mesenchymal stem cells with gold nanocages for in vitro and in vivo tracking by two-photon microscopy and photoacoustic microscopy.
      ). Using the temperature dependence of the PA signal, OR-PAM can measure the temperature of living cells loaded with iron oxide nanoparticles (
      • Gao L.
      • Wang L.
      • Li C.
      • Liu Y.
      • Ke H.
      • Zhang C.
      • et al.
      Single-cell photoacoustic thermometry.
      ,
      • Gao L.
      • Zhang C.
      • Li C.
      • Wang L.V.
      Intracellular temperature mapping with fluorescence-assisted photoacoustic-thermometry.
      ).

      Discussion and outlook

      As the largest organ of the human body, the skin is the most accessible organ for PA imaging. PAT is capable of multiscale imaging with consistent optical absorption contrast, providing anatomical, functional, and molecular information in scattering living tissue. This capability has permitted biomedical studies that require high-resolution images at depths beyond the human skin and filled a void between the traditional microscopic and macroscopic imaging methods used in dermatology. The noninvasiveness of PAT has further allowed longitudinal imaging of morphological and functional changes in skin diseases, enabling analysis of the experimental observations and clinical examination in a larger temporal context. In turn, skin imaging is also one of the most promising paths for translation of PA imaging from a benchtop laboratory technology to a true clinical tool of choice. The strong momentum of PAT development is fueled by its increasingly popular applications in skin imaging. In fact, a great effort has been made to develop real-time, modifiable PA imaging systems that can be easily tailored to a given patient’s condition in a clinical setting (
      • Kim J.
      • Park E.Y.
      • Park B.
      • Choi W.
      • Lee K.J.
      • Kim C.
      Towards clinical photoacoustic and ultrasound imaging: probe improvement and real-time graphical user interface.
      ).
      The advancements in PAT technologies signify great potential for clinical or research-based skin imaging. The preclinical and clinical applications reviewed in this paper are highly representative of the current state of PA skin imaging technologies. Many technological challenges still remain, but few are beyond reach. Each PAT implementation has specific strengths toward particular clinical and research applications. As PA imaging becomes more translational, the safety and cost considerations of the system will play a greater role in its continued development. Currently, the most significant barrier to PAT’s commercial entry is Food and Drug Administration (FDA) regulation and clinical approval, determined by the quality control and safety measures. To translate PAT to clinical skin imaging, the imaging quality in different patient groups (e.g., skin colors, ages, sex) needs to be consistent, providing clinically robust diagnosis information. Developing a rigorous and well-accepted testing system (e.g., skin phantoms, resolution targets, molecular probes) to evaluate the imaging technologies is also crucial before moving on to human studies. In one such effort, a stable phantom was developed from polyvinyl chloride plastisol to evaluate the precision of different PAT instruments (
      • Bohndiek S.E.
      • Bodapati S.
      • Van De Sompel D.
      • Kothapalli S.R.
      • Gambhir S.S.
      Development and application of stable phantoms for the evaluation of photoacoustic imaging instruments.
      ).
      The safety of PAT systems for skin imaging is another area of concern. Endogenous contrasts, such as hemoglobin, melanin, and lipids, are by nature biologically safe (
      • Mehrmohammadi M.
      • Yoon S.J.
      • Yeager D.
      • Emelianov S.Y.
      Photoacoustic imaging for cancer detection and staging.
      ). Successful PA applications should take advantage of endogenous contrasts over exogenous ones to accelerate the regulatory process. However, if exogenous contrasts must be employed, PAT systems have also shown great success in monitoring microneedle delivery of nanoparticles (
      • Moothanchery M.
      • Seeni R.Z.
      • Xu C.
      • Pramanik M.
      In vivo studies of transdermal nanoparticle delivery with microneedles using photoacoustic microscopy.
      ). The laser system is another important safety concern. Although studies have been performed to elucidate the necessary energy requirements for high-quality skin imaging (
      • Schwarz M.
      • Soliman D.
      • Omar M.
      • Buehler A.
      • Ovsepian S.V.
      • Aguirre J.
      • et al.
      Optoacoustic dermoscopy of the human skin: tuning excitation energy for optimal detection bandwidth with fast and deep imaging in vivo.
      ), the output is ultimately limited by the American National Standards Institute, which specifies a maximum exposure limit of 26.4 mJ/cm2 on the skin surface at 760 nm (
      Laser Institute of America
      American National Standards Institute - Z136.1: safe use of lasers.
      ). For dark-skinned patients, the relatively high concentration of melanin poses a challenge for PAT because the skin of these patients is more optically absorbing and thus might be prone to photothermal damage. Potential solutions include the application of transparent coolant to the skin surface while performing PA imaging or clearing the skin surface using a NIR laser. One recent safety advancement in PA skin imaging is the use of more cost-effective and compact excitation sources such as diode lasers (
      • Heres H.M.
      • Arabul M.U.
      • Rutten M.C.
      • Van de Vosse F.N.
      • Lopata R.G.
      Visualization of vasculature using a hand-held photoacoustic probe: phantom and in vivo validation.
      ;
      • Zeng L.
      • Piao Z.
      • Huang S.
      • Jia W.
      • Chen Z.
      Label-free optical-resolution photoacoustic microscopy of superficial microvasculature using a compact visible laser diode excitation.
      ) and class I lasers (
      • Bost W.
      • Lemor R.
      • Fournelle M.
      Optoacoustic imaging of subcutaneous microvasculature with a class one laser.
      ), which have much lower energy levels than traditional classes III and IV lasers. Translational efforts have even seen the modification of other components of PA systems such as the creation of human skin‒adapted probes (
      • Ma H.
      • Yang S.
      • Cheng Z.
      • Xing D.
      Photoacoustic confocal dermoscope with a waterless coupling and impedance matching opto-sono probe.
      ). The cost and safety advantages of low-power light sources will likely be extremely advantageous during clinical translation. As the developers of PAT continue focusing on quality control, efficient light delivery, multimodality integration, and safe contrast agents, PAT will further advance toward clinical approval and adoption by physicians and scientists for broad applications on skins. With more commercially available PAT systems tailored for clinical skin imaging, the user base will also experience a fast expansion in the next several years, resulting in a large number of published clinical studies. Of particular importance is the first FDA-approved PAT system by Seno Medical Instruments, Inc (San Antonio, TX), which has paved the way for more commercial PAT systems to receive regulatory clearance.
      Meanwhile, it is critically important to identify killer applications in skin imaging that can pilot the technology throughout the translational process. Many skin diseases are essentially blood vessel diseases, and PAT can be used to inform diagnostic and treatment decisions on the basis of the skin vessel conditions. One of the most promising candidates, cutaneous small-vessel vasculitis (CSVV), is a common skin vascular disease. In CSVV, the diseased skin shows red blood cell extravasation, fibrin deposition, and disruption of the vessel wall (
      • Takatu C.M.
      • Heringer A.P.R.
      • Aoki V.
      • Valente N.Y.S.
      • de Faria Sanchez P.C.
      • de Carvalho J.F.
      • et al.
      Clinicopathologic correlation of 282 leukocytoclastic vasculitis cases in a tertiary hospital: a focus on direct immunofluorescence findings at the blood vessel wall.
      ). CSVV can occur at anatomical locations that might be difficult to access, such as the neck, arm, and face. Imaging the cutaneous vasculature functions using PAT, in combination with a skin biopsy, will allow dermatologists to better evaluate the severity of the cutaneous disease and discover any accompanying systemic small vessel involvement. Understanding the dynamics of vascular functions in various skin diseases, such as microvessel density, blood flow, and oxygenation, which cannot be biopsied, will provide knowledge that is crucial for monitoring disease progression and response to therapy. The knowledge gained from PAT may also be applicable to severe vasculitic or vasculopathic disease models, such as dermatomyositis or scleroderma, in which biopsy of the affected area (in both cases, the tips of the fingers) may not be feasible. We envision that PAT of skin tissues will serve as a valuable surrogate to predict and correlate with vascular disruption in other internal organs such as kidneys and lungs that are difficult to assess.
      All in all, with its unique imaging capability, PAT has been advancing quickly toward practical impact in healthcare, both in the technology development and in the clinical translation. We expect that PAT will become a technology of choice for diagnosing skin diseases in the near future.

      Author Contributions

      Conceptualization: JY; Writing - Original Draft Preparation: DL, LH, EV, TV, JY; Writing - Review and Editing: DL, LH, JY

      Acknowledgments

      This work was partially supported by National Institutes of Health grants (R01 EB028143, R01 NS111039, RF1 NS115581, R01GM134036, R21 EB027304, R21EB027981, R43 CA243822, R43 CA239830, R44 HL138185), Duke Institute of Brain Science Incubator Award, American Heart Association Collaborative Sciences Award (18CSA34080277), and Chan Zuckerberg Initiative Grant 2020-226178 by Silicon Valley Community Foundation . We thank Caroline Connor for editing the manuscript.

      Conflict of Interest

      The authors state no conflict of interest.

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