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The limited sampling of biopsy and histopathology can lead to incomplete and/or inaccurate assessment of basal cell carcinomas (BCCs), subtypes and depth, which can affect diagnosis and treatment outcome. Reflectance confocal microscopy (RCM) combined with optical coherence tomography (OCT) can help achieve comprehensive 3-dimensional sampling in vivo, which may improve the diagnostic accuracy and margin assessment of BCCs. In a clinical study, we tested a combined RCM-OCT probe on 85 patients, with either clinically-suspicious (n=60, in intact skin) or biopsy-proven BCCs (n=25, in scarred skin). We correlated BCC features in RCM and OCT images with histopathology, calculated diagnostic accuracy and correlated depth predicted by OCT with histopathologically measured depth. The main features were small tumors extending from the basal cell layer at the dermal-epidermal junction; small and large tumor nests; in dermis; dark silhouettes; dilated blood vessels; horn cyst and bright peritumoral stroma. Deeper features such as necrosis and intratumoral mucin pools were correlated on OCT and histology. Higher sensitivity and negative predictive value (100%) and comparable specificity (48% vs 56% on RCM) and positive predictive value (82.19 vs 84.59 % on RCM) were observed for the combined RCM-OCT device for diagnosis of all lesions (n=85). Relatively higher specificity (94.1%) and positive predictive value (75%) were observed in the clinically suspicious lesions (n=60, in intact skin). High correlation was observed (R=0.86) between the OCT predicted depth and histopathologically measured depth. Therefore, RCM-OCT imaging may be prospectively used to comprehensively diagnose suspicious BCC lesions, determine subtype and triage for treatment.

In this report we describe a computer vision based pipeline to convert in-vivo reflectance confocal microscopy (RCM) videos collected with a handheld system into large field of view (FOV) mosaics. For many applications such as imaging of hard to access lesions, intraoperative assessment of MOHS margins, or delineation of lesion margins beyond clinical borders, raster scan based mosaicing techniques have clinically significant limitations. In such cases, clinicians often capture RCM videos by freely moving a handheld microscope over the area of interest, but the resulting videos lose large-scale spatial relationships. Videomosaicking is a standard computational imaging technique to register, and stitch together consecutive frames of videos into large FOV high resolution mosaics. However, mosaicing RCM videos collected in-vivo has unique challenges: (i) tissue may deform or warp due to physical contact with the microscope objective lens, (ii) discontinuities or “jumps” between consecutive images and motion blur artifacts may occur, due to manual operation of the microscope, and (iii) optical sectioning and resolution may vary between consecutive images due to scattering and aberrations induced by changes in imaging depth and tissue morphology. We addressed these challenges by adapting or developing new algorithmic methods for videomosaicking, specifically by modeling non-rigid deformations, followed by automatically detecting discontinuities (cut locations) and, finally, applying a data-driven image stitching approach that fully preserves resolution and tissue morphologic detail without imposing arbitrary pre-defined boundaries. We will present example mosaics obtained by clinical imaging of both melanoma and non-melanoma skin cancers. The ability to combine freehand mosaicing for handheld microscopes with preserved cellular resolution will have high impact application in diverse clinical settings, including low-resource healthcare systems.

Reflectance confocal microscopy (RCM) imaging shows promise for guiding surgical treatment of skin cancers. Recent technological advancements such as the introduction of the handheld version of the reflectance confocal microscope, video acquisition and video-mosaicing have improved RCM as an emerging tool to evaluate cancer margins during routine surgical skin procedures such as Mohs micrographic surgery (MMS). Detection of residual non-melanoma skin cancer (NMSC) tumor during MMS is feasible, as demonstrated by the introduction of real-time perioperative imaging on patients in the surgical setting. Our study is currently testing the feasibility of a new mosaicing algorithm for perioperative RCM imaging of NMSC cancer margins on patients during MMS. We report progress toward imaging and image analysis on forty-five patients, who presented for MMS at the MSKCC Dermatology service. The first 10 patients were used as a training set to establish an RCM imaging algorithm, which was implemented on the remaining test set of 35 patients. RCM imaging, using 35% AlCl3 for nuclear contrast, was performed pre- and intra-operatively with the Vivascope 3000 (Caliber ID). Imaging was performed in quadrants in the wound, to simulate the Mohs surgeon’s examination of pathology. Videos were taken at the epidermal and deep dermal margins. Our Mohs surgeons assessed all videos and video-mosaics for quality and correlation to histology. Overall, our RCM video-mosaicing algorithm is feasible. RCM videos and video-mosaics of the epidermal and dermal margins were found to be of clinically acceptable quality. Assessment of cancer margins was affected by type of NMSC, size and location. Among the test set of 35 patients, 83% showed acceptable imaging quality, resolution and contrast. Visualization of nuclear and cellular morphology of residual BCC/SCC tumor and normal skin features could be detected in the peripheral and deep dermal margins. We observed correlation between the RCM videos/video-mosaics and the corresponding histology in 32 lesions. Peri-operative RCM imaging shows promise for improved and faster detection of cancer margins and guiding MMS in the surgical setting.

Laser ablation offers precise and fast removal of superficial and early nodular types of basal cell carcinomas (BCCs). Nevertheless, the lack of histological confirmation has been a limitation. Reflectance confocal microscopy (RCM) imaging combined with a contrast agent can offer cellular-level histology-like feedback to detect the presence (or absence) of residual BCC directly on the patient. We conducted an ex vivo bench-top study to provide a set of effective ablation parameters (fluence, number of passes) to remove superficial BCCs while also controlling thermal coagulation post-ablation to allow uptake of contrast agent. The results for an Er:YAG laser (2.9 um and pulse duration 250us) show that with 6 passes of 25 J/cm 2 , thermal coagulation can be effectively controlled, to allow both the uptake of acetic acid (contrast agent) and detection of residual (or absence) BCCs. Confirmation was provided with histological examination. An initial in vivo study on 35 patients shows that the uptake of contrast agent aluminum chloride) and imaging quality is similar to that observed in the ex vivo study. The detection of the presence of residual tumor or complete clearance was confirmed in 10 wounds with (additional) histology and in 25 lesions with follow-up imaging. Our results indicate that resolution is sufficient but further development and use of appropriate contrast agent are necessary to improve sensitivity and specificity. Advances in RCM technology for imaging of lateral and deep margins directly on the patient may provide less invasive, faster and less expensive image-guided approaches for treatment of BCCs.

When used for intra-operative imaging of residual basal cell carcinomas (BCCs), reflectance confocal microscopy (RCM) is limited to detection of relatively large tumors. Small tumors remain hidden in the surrounding bright dermis. Fluorescence confocal microscopy (FCM) may improve the sensitivity for detecting small tumors. Fluorescein enhances cell cytoplasm contrast in fluorescence confocal images, but has had limited clinical impact on imaging BCCs in vivo because there is a lack of a well-defined protocol (concentration and application time) that can be effectively used for intraoperative imaging. We conducted an ex vivo study, using discarded tissue from Mohs surgery and a benchtop FCM with 488nm wavelength for excitation and 521nm detection for imaging Concentrations of 6, 0.6 and 0.6 mM with immersion times of 5, 15, 30, and 60 seconds were repeatedly tested (total of 76 specimens).. The 0.6 mM and immersion time of 60 seconds showed that cellular cytoplasm can be labeled with controlled saturation and without leaving the yellow color on the surface of the tissue. Initial results show that, fluorescein may enhance cellular structures contrast relative to other normal dermal structures, improving the detection of small BCCs. This study provide an optimized set of parameters for subsequently testing of topical application in vivo for intraopertive imaging of BCCs.