Line-field confocal optical coherence tomography for in vivo visualization of morphological characteristics of squamous cell carcinoma in a murine model.

OBJECTIVES: Non-invasive imaging with line-field confocal optical coherence tomography (LC-OCT) can support the diagnosis of squamous cell carcinoma (SCC) through visualization of morphological characteristics specific to skin cancer. We aimed to visualize prominent morphological characteristics of SCC using LC-OCT in a well-established murine SCC model. MATERIALS AND METHODS: Nine hairless mice were exposed to ultraviolet radiation three times weekly for 9 months to induce SCC development. Visible SCC tumors (n = 9) were imaged with LC-OCT and the presence of 10 well-described morphological characteristics of SCC were evaluated in the scans by two physicians with adjudication by a third. RESULTS: Overall, murine morphological characteristics resembled corresponding features previously reported in human SCCs. Interrupted dermal-epidermal junction occurred in 100% of tumors. In epidermis, the most frequently observed characteristics were severe epidermal dysplasia (100%) and tumor budding (89%). Common dermal characteristics included broad strands (100%) and collagen alterations (78%). CONCLUSION: LC-OCT imaging can be used to non-invasively visualize morphological characteristics specific to SCC in an in vivo preclinical model.

Dermatologic Scar Assessment With Stereoscopic Imaging and Digital Three-Dimensional Models: A Validation Study.

BACKGROUND AND OBJECTIVE: We evaluated a new handheld stereoscopic imaging system capable of visualizing scars with digital three-dimensional (3D) models and providing automated morphometric estimates. The objective was to validate the repeatability and accuracy of intra- and inter-investigator scan results. STUDY DESIGN/MATERIALS AND METHODS: Engineered metal plates with depressed and elevated model scars (n = 72) were scanned six times by one investigator. In vivo hypertrophic and atrophic scars (n = 15) were scanned once by three investigators. The repeatability of morphometric estimates was assessed using coefficients of variation (CVs) to compare the variation among multiple scan results for both models and in vivo scars, with 0% reflecting a perfect match. Scar estimates from digital 3D reconstructions were compared with the known dimensions of physical model scars and with ruler measurements of in vivo scars. RESULTS: A total of 48 model scars and 12 in vivo scars were eligible for automated analyses with the imaging system's proprietary software. Intra-investigator scan results for the model scars were repeatable, with low variance for all parameters: volume, area, length, and depth/height (CV: 1.8-3.1%). By comparison, inter-investigator scans of real in vivo scars resulted in slightly higher median CVs (4.4-7.3%; P < 0.05). 3D model scar estimates correlated well with the known physical dimensions of model scars for all parameters (P < 0.001) and accurately reflected the measurements of in vivo scars (P < 0.001). The six in vivo scars situated on the chest and abdomen showed the highest inter-investigator variation, due to respiratory movement artifacts. CONCLUSION: Stereoscopic imaging of scars generates accurate and repeatable measurement estimates that show little intra- and inter-investigator-based assessment variation. The best results are achieved by minimizing subject movement. Lasers Surg. Med. © 2020 Wiley Periodicals LLC.

Electronic Pneumatic Injection-Assisted Dermal Drug Delivery Visualized by Ex Vivo Confocal Microscopy.

BACKGROUND AND OBJECTIVES: Electronic pneumatic injection (EPI) is a technique for dermal drug delivery, which is increasingly being used in clinical practice. However, only few studies have been reported on cutaneous drug distribution and related clinical endpoints. We aimed to visualize the immediate cutaneous drug distribution, changes in skin architecture, and related clinical endpoint of EPI. STUDY DESIGN/MATERIALS AND METHODS: Acridine orange (AO) solution was administered to ex vivo porcine skin by EPI at pressure levels from 4 to 6 bar with a fixed injection volume of 50 µl and nozzle size of 200 µm. Immediate cutaneous distribution was visualized using ex vivo confocal microscopy (EVCM). Changes in skin architecture were visualized using both EVCM and hematoxylin and eosin-stained cryosections. RESULTS: The defined immediate endpoint was a clinically visible papule formation on the skin. The pressure threshold to consistently induce a papule was 4 bar, achieving delivery of AO to the deep dermis (2319 µm axial and 5944 µm lateral distribution). Increasing the pressure level to 6 bar did not lead to significant differences in axial and lateral dispersion (P = 0.842, P = 0.905; respectively). A distinctively hemispherical distribution pattern was identified. Disruption of skin architecture occurred independently of pressure level, and consisted of subepidermal clefts, dermal vacuoles, and fragmented collagen. CONCLUSIONS: This is the first study to relate a reproducible clinical endpoint to EPI-assisted immediate drug delivery using EVCM. An EPI-induced skin papule indicates dermal drug delivery throughout all layers of the dermis, independent of pressure level settings. Lasers Surg. Med. © 2020 The Authors. Lasers in Surgery and Medicine published by Wiley Periodicals LLC.

Morphometric Optical Imaging of Microporated Nail Tissue: An Investigation of Intermethod Agreement, Reliability, and Technical Limitations.

BACKGROUND AND OBJECTIVES: While optical imaging is a useful technique to quantitate morphological differences and treatment effects, comparative investigations of the various techniques are lacking. This study aimed at evaluating intermethod agreement, reliability, and technical limitations of wide-field microscopy (WFM), reflectance confocal microscopy (RCM), and optical coherence tomography (OCT) for morphometry by assessing fractionally ablated nail tissue. STUDY DESIGN/MATERIALS AND METHODS: Fifty healthy nail clippings were processed with a fractionated CO2 -laser (20 mJ/microbeam, density 15%), measured with calipers, and imaged using WFM, OCT, and RCM. Images were assessed for nail plate thickness, micropore dimensions, degree of poration, and artifacts. Repeated measurements (2-5) were taken to evaluate method repeatability using Cronbach's α and coefficients of variation (CoV), and estimate the intermethod correlation through linear correlation assessment (Pearson correlation coefficient [PCC]), ranked correlation (Kendall's tau; tau-c), and intraclass correlation (Shrout-Fleiss reliability coefficient; ICC). RESULTS: The repeatability varied substantially between methods and target measurements. The level of intermethod agreement for thickness measurements performed with calipers, WFM, and OCT was high (tau-c ≥ 0.7; ICC ≥ 0.8; PCC ≥ 0.9). RCM could only image 28 out of 50 samples due to its limited penetration depth. OCT demonstrated the highest repeatability of all imaging techniques (CoV 4-7%) and nail thickness showed the highest measurement reliability (α = 0.92). Micropore dimensions correlated strongest between OCT and RCM (tau-c/ICC/PCC ≥ 0.5). All modalities were prone to artifacts, which may have adversely affected measurement variation and intermethod agreement. CONCLUSION: Intermethod agreement and reliability appear to be highly dependent on the specific modality and target measurement. To reap the benefits of each technique while mitigating their limitations, an integrated approach to optical imaging is recommended. Lasers Surg. Med. © 2020 Wiley Periodicals LLC.

The ablative fractional coagulation zone influences skin fluorescence intensities of topically applied test molecules-An in vitro study with fluorescence microscopy and fluorescence confocal microscopy.

BACKGROUND: Ablative fractional laser (AFL) increases uptake of topically applied skin agents. The coagulation zone (CZ) surrounding vertically ablated channels may influence uptake of drugs. OBJECTIVES: To investigate impact of CZ thickness on skin fluorescence intensities (FI) of a hydrophilic molecule by means of fluorescence microscopy (FM) and fluorescence confocal microscopy (FCM). Second, to compare FI of hydrophilic and lipophilic test molecules by FCM. STUDY DESIGN/METHODS AND MATERIALS: Microchannels with CZ thicknesses of 0, 20, and 80 µm were generated by microneedles or AFL (10,600 nm). Channels were 700 µm deep and number of channels kept constant per skin area. After 4 hours of incubation, FI induced by sodium fluorescein (NAF, hydrophilic, logarithmic partition-coefficient (logP) = -1.52, MW = 376.26) were quantified in both CZ and surrounding skin by FM (0-1,500 µm) and FCM (0-90 µm). FI of NAF and carboxyfluorescein (CAF, lipophilic, logP = 2.9, MW = 376.32) were compared by FCM. RESULTS: By FM, NAF-induced FI were higher in CZ than in surrounding skin (P ≤ 0.001). Highest NAF-FI were induced in skin pretreated with a thin CZ (CZ-20 µm), assessed by both FM and FCM and in particular, FI were higher than in skin pretreated with no CZ (CZ-0 µm) (FM P ≤ 0.041, FCM P < 0.012). Skin FI remained constant to a depth of 500 µm, which corresponded to approximate depth of microchannels (CZ-0 µm, CZ-20 µm, CZ-80 µm: 0-500 µm P ≥ 0.107). In accordance with FM data, FCM showed higher FI within CZ than in surrounding skin, but gradually decreased to zero at a depth of 90 µm. NAF-FI were higher than CAF-FI (P ≤ 0.036), and highest CAF-FI were induced by CZ-0 µm and CZ-20 µm compared to CZ-80 µm (P ≤ 0.009). CONCLUSIONS: The influence of the CZ thickness on skin FI differs between small hydrophilic and lipophilic test molecules. Results may have clinical relevance for laser-assisted drug delivery. Lasers Surg. Med. 51:68-78, 2019. © 2018 Wiley Periodicals, Inc.