Skin Imaging Using Ultrasound Imaging, Optical Coherence Tomography, Confocal Microscopy, and Two-Photon Microscopy in Cutaneous Oncology

With the recognition of dermoscopy as a new medical technology and its available fee assessment in Korea comes an increased interest in imaging-based dermatological diagnosis. For the dermatologist, who treats benign tumors and malignant skin cancers, imaging-based evaluations can assist with determining the surgical method and future follow-up plans. The identification of the tumor's location and the existence of blood vessels can guide safe treatment and enable the use of minimal incisions. The recent development of high-resolution microscopy based on laser reflection has enabled observation of the skin at the cellular level. Despite the limitation of a shallow imaging depth, non-invasive light-based histopathologic examinations are being investigated as a rapid and pain-free process that would be appreciated by patients and feature reduced time from consultation to treatment. In the United States, the current procedural terminology billing code was established for reflectance confocal microscopy in 2016 and has been used for the skin cancer diagnosis ever since. In this review, we introduce the basic concepts and images of ultrasound imaging, optical coherence tomography, confocal microscopy, and two-photon microscopy and discuss how they can be utilized in the field of dermatological oncology.


INTRODUCTION
Efforts to diagnose skin cancer without skin biopsy are ongoing. The diagnoses of patients with suspected skin cancer are confirmed by punch biopsy followed by histopathological examination, which involve the collection of a small portion of the entire lesion to diagnose skin cancer (1). In this case, since only vertical information of a specific region is acquired, dermoscopy can supplement horizontal information of the entire lesion to identify the most suitable biopsy site. However, dermoscopy has an inherent depth limit confined to the upper dermis ( Table 1). To observe lesions deep to the upper dermis, the maximum depth that can be observed with dermoscopy, non-invasive techniques, such as confocal microscopy, multiphoton microscopy, optical coherence tomography, and ultrasound must be used. Although each operation principle is different, they all use the reflection characteristic as if it is mirrored, and the skin's depth and resolution differ among device types ( Table 2). Here we briefly discuss each available device and its clinical use in the dermatology field.

ULTRASOUND IMAGING
Ultrasound imaging uses high-frequency sound waves that cannot be heard by the human ear. When it is sent inside the human body, the degree of absorption and reflection is cut off depending on the constituents and the reflected sound waves are sensed and imaged (2). Therefore, the probe that sends and detects the sound waves forms the core equipment for ultrasound technology. Higher-frequency (MHz) sound waves enable highresolution observation of the skin surface, but the observable depth decreases. In the field of dermatology, ultrasound is mainly used to identify benign tumor type and extent ( Table 3). Before surgery, it can provide information about tumor type and size, locate the existence of surrounding vessels, identify the best location for the incision, and set the range while viewing the ultrasound screen in real time with the patient. It can also help the clinician evaluate whether the tumor was completely removed after surgery (Figure 1).
In the case of epidermoid cysts, one of the most common benign tumors, it is often seen as a well-defined ovoidshaped heterogeneous hypoechoic lesion in the subcutaneous layer with strong posterior acoustic enhancement (Figure 2). Ultrasonographic findings corresponding to epidermal cyst rupture include pericystic changes, increased vascularity, deep abscess formation, and others (9). Trichilemmal cyst, a benign Abbreviations: OCT, optical coherence tomography; TPM, two-photon microscopy; EMPD, extramammary Paget's disease; SHG, second harmonic generation; MPM, multi-photon microscopy; BCC, basal cell carcinoma; SCC, squamous cell carcinoma; AK, actinic keratosis. appendage lesion derived from the outer root sheath of the hair follicle, is often seen as a well-defined hypoechoic lesion with internal calcification and posterior sound enhancement (Figure 3) (8). Identifying these sites just prior to surgery and optimizing the incision site and approach can improve the success rate and reduce recurrence rates. Pilomatricoma, a benign superficial tumor of the hair follicle, is often seen as a well-defined mass with inner echogenic foci and a peripheral hypoechoic rim or a completely echogenic mass with strong posterior acoustic shadowing in the subcutaneous layer on ultrasonography (Figure 4) (7). Pilomatricoma often shows angiographic findings and may be difficult to differentiate from hemangioma.
A lipoma appears as a well-defined hypoechoic mass with multiple echogenic strands on ultrasound ( Figure 5). If the encapsulation is well-formed, it is easier to remove. Ultrasonography is especially useful for diagnosing and treating lipoma in the forehead. A lipoma occurring in the forehead is often located under the frontalis muscles, and it is important to confirm its precise position using preoperative ultrasonography. It typically has a semispherical shape when located under the muscles and an ovoid shape when it is located in the subcutaneous fat layer (Figure 6) (11). However, this is not always the case, so a comprehensive judgment should be made by checking whether it is close to the periosteum or using a special technique that uses the angulation of the probe to point out the lateral borders of the lesion (12).
There are no obvious criteria that can diagnose malignant cutaneous tumors using ultrasound imaging. However, tumor size >5 cm, infiltrated margins, rapid clinical growth, moderate to severe intratumoral hypervascularity (Figure 7), and an absence of the typical features of benign tumors are highly suggestive of malignancy (13,14). High-definition ultrasound with transducers up to 70 MHz, which can observe more detail, has been used to diagnose cutaneous angiosarcoma of the breast and is expected to be useful for the identification of malignant skin cancers (15).

OPTICAL COHERENCE TOMOGRAPHY
Optical coherence tomography (OCT), a three-dimensional (3D) imaging technique based on low coherence interferometry, OCT non-invasively provides skin images similar to the B mode of ultrasound to a depth of 1-2 mm and a resolution of 2-10 µm with high imaging speed. Functional OCT techniques that can provide additional information, such as polarization and vasculature were recently developed and applied for the detection of abnormal vasculature of a port-wine stain or skin cancer (16)(17)(18). Our research group developed a device that matches an OCT image with that obtained by dermoscopic imaging and provides more information than dermoscopy alone (19). Through this, we expect to be able to assess the extent of scar treatment (Figure 8). It is expected that a stage of nevus flammeus will be established, and treatment feasibility and degree will be evaluated (Figure 9). The limitations of OCT are limited depth of examination and lack of resolution to observe cancer cell morphology. Line-field confocal OCT, which can reveal comprehensive structural mapping of the skin at the cellular level with an isotropic spatial resolution of ∼1 µm to a depth of ∼500 µm, was recently reported to correlate with conventional histopathological images of skin tumors (20). Key articles comparing OCT and histopathology are summarized in Table 4.

CONFOCAL MICROSCOPY
Confocal microscopy is based on the existence of one focal point when a laser, used as a light source, is reflected off a subject. The "out of focus" signal is blocked by a pinhole, and contrast is generated by reflections at the interfaces of tissue and cellular structures due to variations of the index of refraction. Since image acquisition is not possible with a single signal point, imaging occurs by scanning across several pinholes. Imaging up to a depth of 100-200 µm at a 1-µm resolution is possible. Confocal microscopy is capable of providing rapid bedside pathological analysis by producing images with subcellular resolution without skin biopsy and physical sectioning (24)(25)(26). There are two ways to use this approach for Mohs surgery. One is used in vivo and can help the identification of the surgical margins in a perioperative setting (27). It is also possible to check the remaining lesion using intraoperative images in vivo after removing the main skin cancer mass (28). The other is for ex vivo use, in which the surgical margins are removed and confocal microscopy is used to confirm whether the tumor remains within it (29). However, when used for detection in Mohs surgery, the grayscale confocal image was difficult to interpret by the surgeons. To improve this, each frozen specimen was stained with acridine orange    (pH 6.0) and eosin (pH 6.0) and then scanned with confocal mosaicking microscopy to imitate hematoxylin and eosin-stained Mohs frozen sections. This approach and physician training can improve the accuracy of the non-melanoma skin cancer diagnosis (30). Key articles comparing confocal microscopy and histopathology are summarized in Table 5. Confocal microscopy has also been applied to diagnose mammary and extramammary Paget's disease (EMPD) (37), frequently showing Paget cells predominantly within the epidermis (38). However, due to the limited depth of imaging (100-200 µm) when applied non-invasively, the invasion site is difficult to determine. A major limitation of this technique is that it can only provide morphological information and does not reflect the tissue's internal structure or functional state.

Two-photon microscopy (TPM) is a technique that uses the fluorescence released after excitation from simultaneously
Frontiers in Medicine | www.frontiersin.org   absorbing two photons with long wavelengths and low energy. TPM allows observation of vital phenomena in cells and in vivo at the molecular level. In particular, it has the advantage of being able to identify the distribution of collagen within the dermis using the second harmonic generation (SHG) produced when two photons simultaneously interfere. Non-invasive in vivo 2009

Reflectance confocal microscopy (in vivo)
All 38 cases displayed an atypical honeycomb and/or disarranged pattern of the spinous-granular layer of the epidermis; round nucleated cells were seen in 20 SCCs (65%) and 1 AK (14%) Round blood vessels were seen in the superficial dermis in 28 SCCs (90%) and 5 AKs (72%) Round nucleated cells at the spinous-granular layer correspond to atypical keratinocytes or dyskeratotic cells    multi-photon microscopy (MPM) imaging also reportedly provides label-free contrast and reveals several characteristic features of basal cell carcinoma lesions (39). This feature correlates well with histopathological examination, findings, and SHG in particular shows collagen and elastin bundles around the tumor (Figure 10) ( Table 6). However, since TPM and MPM utilize weak endogenous fluorescence in tissue, there is a need for high excitation laser power and extension of pixel duration (44,45). To overcome this limitation and reduce photodamage, moxifloxacin, an FDAapproved antibiotic, has been reported as a cell-labeling agent for MPM (46). Moxifloxacin has bright intrinsic multi-photon fluorescence, good tissue penetration, and high intracellular concentration. In addition, moxifloxacin-based MPM imaging is 10 times faster than imaging based on endogenous fluorescence (Figure 11) (46).
Although imaging depth remains a limitation, various methods to achieve a clear and high-resolution image are being developed. It is also expected that the diagnosis rate can be increased by tumor marker labeling. A recent report stated that in patients with EMPD, a subclinical extension can be assessed by MPM using whole-mount immunostaining with anti-cytokeratin 7 antibody to label Paget cells (35). These trials will be used in the ex vivo skin tissue to find the tumor's margins, and it is anticipated that it may replace frozen sections in the future. For more generalized clinical applications, the cost of the equipment is the greatest hinderance. MPM equipment is expensive because it uses a femtosecond laser (36).

CONCLUSION
In addition to ultrasonic devices that can closely observe the skin and deep structures, the development of dermatological equipment that unites laser and optical technology has shown visible progress. The principle of these devices is to analyze signals reflected or scattered from the skin, and there is a fundamental limitation that it is evaluated by looking into the mirror. These limitations are expected to improve in the near future by the development of fluorescent probes targeting tumors or diseases and will be used more actively for the diagnosis and treatment of skin lesions.
For dermatologists, this is a good opportunity to strengthen the specialty of dermatology. We are already familiar with laser equipment and have demonstrated a correlation between clinical and histopathological findings. When we use imaging equipment to further investigate a patient's skin and present objectively explainable data by linking "clinical imaging-histopathological findings, " a more robust doctor-patient relationship can be established.

AUTHOR CONTRIBUTIONS
BO conceived the concept and wrote the manuscript. KK co-conceived the concept and drafted the figures and tables. KC co-conceived the concept and edited and improved the manuscript.