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Photoacoustic Imaging Lab, Department of Biomedical Engineering, Duke University, Durham, North Carolina, USADepartment of Biomedical Engineering, School of Engineering, Vanderbilt University, Nashville, Tennessee, USA
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.
). 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 (
). Both neoplastic and inflammatory cutaneous diseases, such as skin cancer, diabetes, and vasculitis, manifest structural and functional changes in the skin, especially in the skin vasculature (
). 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 (
). Moreover, cutaneous pathology patterns have shown to be highly sensitive to the timing of the biopsy and the age of the lesion. In fact, histology late in the course of skin disease is difficult to interpret by dermatologists because active inflammation often cannot be distinguished from late disease progress. Because of the varied pathogenic mechanisms and the specific organs involved in different skin diseases, a skin biopsy cannot stand by itself; it must be correlated with clinical history, physical and laboratory findings, and skin morphology and function. Therefore, a noninvasive imaging device that can scan skin tissue in vivo would be preferred for skin disease diagnosis. Thus far, clinical skin imaging has been performed with traditional optical imaging methods, including dermoscopy, laser Doppler, optical coherence tomography, and confocal and two-photon microscopy (
), 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 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 (
). The four regimes are divided at photon propagation depths of approximately 0.1 mm (aberration limit), 1 mm (diffusion limit), 10 cm (dissipation limit), and 1 m (absorption limit), with an optical absorption coefficient of 0.1 cm–1, optical scattering coefficient of 100 cm–1, and anisotropy of 0.9. The classification holds in optical scattering dominant media. Note that the penetration limits shown in this image are order-of-magnitude approximations. (b) Signal generation and detection in CFM, TPM, and PAT, with different penetration limits in scattering tissue. Note that the penetration limits shown in the figure are drawn on a logarithmic scale. The colors of the excitation light do not represent the true optical wavelengths. Reprinted with permission from
). In PAT, excitation light is absorbed by tissue and is converted into pressure waves to form an image. PAT capitalizes on low acoustic scattering of tissue to achieve high spatial resolution at the depths of up to several centimeters (Figure 1b) (
Integrating photoacoustic ophthalmoscopy with scanning laser ophthalmoscopy, optical coherence tomography, and fluorescein angiography for a multimodal retinal imaging platform.
). Different from traditional optical imaging, PAT uses acoustic detection and thus can penetrate several millimeters into the skin with optically or ultrasonically determined resolutions (
). 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 (
Calibration-free absolute quantification of optical absorption coefficients using acoustic spectra in 3D photoacoustic microscopy of biological tissue.
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 (
). 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 (
): 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 (
Universal back-projection algorithm for photoacoustic computed tomography [published correction appears in Phys Rev E Stat Nonlin Soft Matter Phys 2007;75:059903].
Phys Rev E Stat Nonlin Soft Matter Phys.2005; 71: 016706
), 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) (
), 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) (
), 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 (
). 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 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 (
). 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 (
). 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 (
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 (
). 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 (
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 (
in: Photons plus ultrasound: imaging and sensing 2006: the Seventh Conference on Biomedical Thermoacoustics, Optoacoustics, and Acousto-Optics, proceedings. vol. 6086. SPIE,
Wellingham, WA2006
). The total hemoglobin concentration (THb) can also be calculated at an isosbestic wavelength, where deoxyhemoglobin and oxyhemoglobin have identical molar extinction coefficients (
Wavelet differentiation of optoacoustic signals for monitoring of total hemoglobin concentration and oxygen saturation level in small blood vessels.
in: Photons plus ultrasound: imaging and sensing 2007: the Eighth Conference on Biomedical Thermoacoustics, Optoacoustics, and Acousto-Optics, proceedings. vol. 6437. SPIE,
Bellingham, WA2007643717
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.
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 (
). 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 (
), 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 (
). 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 (
). 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 (
). 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) (
in: Photons plus ultrasound: imaging and sensing 2006: the Seventh Conference on Biomedical Thermoacoustics, Optoacoustics, and Acousto-Optics, proceedings. vol. 6086. SPIE,
Wellingham, WA2006
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 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
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 (
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,
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.
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 (
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) (
). 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 (
). Although there is no known cure for psoriasis, several viable treatments include topical steroid creams, phototherapy, retinoids, and immunosuppressant prescription drugs (
). 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 (
). 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 (
). 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 (
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 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
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 (
). 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 (
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
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 (
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,
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 (
). 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 (
). 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) (
High-resolution burn imaging in pig skin by photoacoustic microscopy.
in: Photons plus ultrasound: imaging and sensing 2007: the Eight Conference on Biomedical Thermoacousitcs, Optoacoustics, and Acousto-Optics, proceedings. vol. 6437. SPIE,
Wellingham, WA2007
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.
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 (
). 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.
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,
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.
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 (
). 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 (
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
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 (
). 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 (
). 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 (
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,
showed single-melanoma CTC detection in vivo, followed by immediate laser ablation of the CTCs (Figure 8).
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
Photoacoustic and photothermal cytometry using photoswitchable proteins and nanoparticles with ultrasharp resonances [published correction appears in J Biophotonics 2015;8:687].
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.
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 (
In vivo flow cytometry and time-resolved near-IR angiography and lymphography.
in: Saratov Fall Meeting 2006: Optical technologies in biophysics and medicine VIII. vol. 6535. SPIE—The International Society for Optical Engineering,
Bellingham, WA200765350K
). 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 (
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 (
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 (
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 (
). 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 (
). The laser system is another important safety concern. Although studies have been performed to elucidate the necessary energy requirements for high-quality skin imaging (
), 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 (
). 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 (
), 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 (
). 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 (
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.
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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In vivo flow cytometry and time-resolved near-IR angiography and lymphography.
in: Saratov Fall Meeting 2006: Optical technologies in biophysics and medicine VIII. vol. 6535. SPIE—The International Society for Optical Engineering,
Bellingham, WA200765350K
Calibration-free absolute quantification of optical absorption coefficients using acoustic spectra in 3D photoacoustic microscopy of biological tissue.
Wavelet differentiation of optoacoustic signals for monitoring of total hemoglobin concentration and oxygen saturation level in small blood vessels.
in: Photons plus ultrasound: imaging and sensing 2007: the Eighth Conference on Biomedical Thermoacoustics, Optoacoustics, and Acousto-Optics, proceedings. vol. 6437. SPIE,
Bellingham, WA2007643717
Integrating photoacoustic ophthalmoscopy with scanning laser ophthalmoscopy, optical coherence tomography, and fluorescein angiography for a multimodal retinal imaging platform.
Clinicopathologic correlation of 282 leukocytoclastic vasculitis cases in a tertiary hospital: a focus on direct immunofluorescence findings at the blood vessel wall.
Universal back-projection algorithm for photoacoustic computed tomography [published correction appears in Phys Rev E Stat Nonlin Soft Matter Phys 2007;75:059903].
Phys Rev E Stat Nonlin Soft Matter Phys.2005; 71: 016706
in: Photons plus ultrasound: imaging and sensing 2006: the Seventh Conference on Biomedical Thermoacoustics, Optoacoustics, and Acousto-Optics, proceedings. vol. 6086. SPIE,
Wellingham, WA2006 (60861G)
High-resolution burn imaging in pig skin by photoacoustic microscopy.
in: Photons plus ultrasound: imaging and sensing 2007: the Eight Conference on Biomedical Thermoacousitcs, Optoacoustics, and Acousto-Optics, proceedings. vol. 6437. SPIE,
Wellingham, WA2007
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