Reflectance Confocal Microscopy Images Research Articles

Film-Forming, Moisturizing, and Sensory Properties of a Cosmetic Formulation Containing Tara Gum and Brazilian Berry Extracts.

The development of cosmetic formulations with moisturizing and film-forming properties has been very important to help keep skin physiology and protection. In this context, this study aimed to develop a cosmetic formulation containing Tara gum and Brazilian berry extract and evaluate its physical-mechanical, film-forming, and sensory properties. A gel formulation was developed based on Tara gum added to Plinia cauliflora extract and was characterized by its spreadability profile and sensory properties. A clinical study was carried out with ten participants to evaluate the skin microrelief, stratum corneum water content, transepidermal water loss (TEWL), and skin morphological characteristics by reflectance confocal microscopy (RCM) before and after 2h of application of the formulations. The formulation with Brazilian berry significantly decreased the work of shear parameter, which can be correlated with improved spreadability in the sensory analysis. The clinical study showed that both formulations improved skin hydration and reduced the TEWL. The RCM imaging analysis showed the visible film on the skin surface, a decrease in the size of furrows, an increase in the reflectance of the interkeratinocytes, and reflectance of the stratum corneum for both formulations. These results were more pronounced for the formulation containing Brazilian berry. The Tara gum in the gel formulation promoted the formation and visualization of a polymeric net on the stratum corneum surface, demonstrated by the images obtained from RCM. However, the formulation added with the Brazilian berry extract improved the skin microrelief, honeycomb pattern of the epidermis, and skin hydration in deeper layers of the epidermis.

A Prospective Double-Blinded Comparison of Reflectance Confocal Microscopy with Conventional Histopathology for In Vivo Assessment in Oral Cancer.

We investigated reflectance confocal microscopy (RCM) as a possible non-invasive approach for the diagnosis of cancer and real-time assessment of surgical margins. In a phase I study on 20 patients, we established the RCM imaging morphological features that distinguish OSCC from normal tissue with a newly developed intra-oral RCM probe. Our subsequent phase II prospective double-blinded study in 60 patients tested the diagnostic accuracy of RCM against histopathology. Five RCM videos from the tumor and five from normal surrounding mucosa were collected on each patient, followed by a 3-mm punch biopsy of the imaged area. An experienced RCM reader, who was blinded to biopsy location and histological diagnosis, examined the videos from both regions and classified each as "tumor" or "not-tumor" based on RCM features established in phase I. Hematoxylin and eosin slides from the biopsies were read by a pathologist who was blinded to RCM results. Using histology as the gold standard, we calculated the sensitivity and specificity of RCM. We report a high agreement between the blinded readers (95% for normal tissue and 81.7% for tumors), high specificity (98.3%) and negative predictive values (96.6%) for normal tissue identification, and high sensitivity (90%) and positive predictive values (88.2%) for tumor detection. RCM imaging is a promising technology for non-invasive in vivo diagnosis of OSCC and for real-time intraoperative evaluation of mucosal surgical margins. Its inherent constraint, however, stems from the diminished capability to evaluate structures located at more substantial depths within the tissue.

Abstract 4171: Utilizing biopsy-free virtual histology for improved clinical efficiency in the dermatologic surgical setting

Abstract The ability to accurately define tumor margins may enhance tissue sparing and increase efficiency in the dermatologic surgery process, but no device exists that serves this role. Reflectance Confocal Microscopy (RCM) provides non-invasive cellular resolution of the skin. The only clinically-approved RCM device is bulky, non-portable, and requires a tissue cap which makes mapping of the underlying tissue impossible. We recently combined “virtual histology”, a machine learning algorithm with RCM images from this standard RCM device to generate biopsy-free histology to overcome these limitations. Whether virtual histology can be used with a portable, handheld RCM device to scan for residual tumor and tumor margins is currently unknown. We hypothesize that combining a handheld RCM device with virtual histology could provide accurate tumor margin assessment. We determined whether our established virtual histology algorithm could be applied to images from a portable RCM device and whether these pseudo-stained virtual histology images correlated with histology from skin specimens. The study was conducted as a prospective, consecutive non-randomized trial at a Veterans Affairs Medical Center dermatologic surgery clinic. All patients greater than 18 years of age with previously biopsied BCC, SCC, or SCCis were included. Successive confocal images from the epidermis to the dermis were obtained 1.5 microns apart from the handheld RCM device to detect residual skin cancer. The handheld, in-vivo RCM images were processed through a conditional generative adversarial network-based machine learning algorithm to digitally convert the images into H&E pseudo-stained virtual histology images. Virtual histology of in-vivo RCM images from unbiopsied skin captured with the portable RCM device were similar to those obtained with the standard RCM device and virtual histology applied to portable RCM images correctly correlated with frozen section histology. Residual tumors detected with virtual histology generated from the portable RCM images accurately corresponded with residual tumors shown in the frozen surgical tissue specimen. Residual tumor was also not detected when excised tissue was clear of tumor following surgical procedure. Thus, the combination of virtual histology with portable RCM may provide accurate histology-quality data for evaluation of residual skin cancer prior to surgery. Combining machine learning-based virtual histology with handheld RCM images demonstrates promise in providing insights into tumor characteristics and has the potential to assist the surgeon and better guide practice decisions to more efficiently serve patients, leading to decreased layers and appointment times. Future work is needed to provide real time virtual histology, convert horizontal/confocal sections into vertical or 3D sections, and to perform clinical studies to map tumors in tissue. Citation Format: Rachel Wahhab, Nelly Kokikian, Jingxi Li, Jason Garfinkel, Stephanie Martin, David Beynet, Aydogan Ozcan, Philip Scumpia. Utilizing biopsy-free virtual histology for improved clinical efficiency in the dermatologic surgical setting [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4171.

Features of tattoo-associated cutaneous lymphoid hyperplasia on reflectance confocal microscopy and line-field confocal optical coherence tomography.

The popularity of tattoos has led to an increase in associated skin reactions, including complications such as infection, allergic reactions and rare conditions such as tattoo-induced cutaneous lymphoid hyperplasia (CLH). CLH is a benign lymphoproliferative reaction with clinical features resembling malignant cutaneous lymphomas. Non-invasive diagnostic tools like reflectance confocal microscopy (RCM) and the new line-field confocal optical coherence tomography (LC-OCT) are being studied in dermatology better to understand the morphological patterns of many dermatological diseases. Between September 2021 and May 2023, patients with suspicious lesions for tattoo-related CLH were analysed using RCM and LC-OCT before confirming the diagnosis of CLH through skin biopsy and histopathological examination. The study included five cases of CLH. It focused on the analysis of high-quality LC-OCT images/videos and RCM images to investigate the features of CLH in tattooed individuals. Most (80%) cases exhibited a mixed T and B lymphocyte infiltration subtype, while 20% showed a predominant T infiltration subtype. RCM and LC-OCT revealed characteristic features, including architectural disarray, fibrosis, lymphoid infiltrates, and pigment deposits in the epidermis and dermis. Non-invasive tools such as RCM and LC-OCT are valuable in diagnosing tattoo-related CLH. While skin biopsy remains the current standard for diagnosis, RCM and LC-OCT can serve as helpful adjuncts in identifying the most representative area for biopsy. They may potentially become alternative diagnostic options in the future, offering benefits in terms of cost, diagnostic efficiency, aesthetics and patient satisfaction as the prevalence of tattoo-related adverse reactions continues to rise.

Pigmented contact dermatitis: An updated review

Pigmented contact dermatitis (PCD) is a disorder brought on by repeated exposure to low-intensity allergens, usually presenting as blotchy or reticulate slate-gray pigmentation affecting Fitzpatrick skin type IV-VI. The pathogenesis remains unclear; however, type IV hypersensitivity reactions due to allergic sensitization, genetics, ultraviolet exposure, and autoimmunity are to blame. Clinical examination, dermoscopy, patch/photo patch testing, histopathology, and recently, a novel reflectance confocal microscopy and multimodality skin imaging system aid in the diagnosis. Several contact allergens have been linked to PCD, but from an Indian perspective, Kumkum and Paraphenylenediamine are the incriminating agents. Patch testing plays an immense role whenever PCD is diagnosed, primarily due to contact allergens. Devastating psychological impacts can result from PCD-related deformity on social acceptance, mental health, and self-esteem. Avoiding allergens, wearing broad-spectrum sunscreen, and engaging in sun-protective behavior are general measures for treating the condition.

Confocal Assessment of Pigmented-Mucosal Lesions: A Monocentric, Retrospective Evaluation of Lip and Genital Area.

Pigmentation of lip and/or genitalia is mainly due to the development of benign melanotic macules, with a less occurrence of melanocytic and other non-melanocytic lesions. Mucosal melanoma has worse prognosis compared with cutaneous counterpart, hence identification of atypical features for an early diagnosis is crucial. The aim of this study was to report further data of confocal features characterizing pigmented mucosal lesions of genital area and of the lips and test the diagnostic role of the reflectance confocal microscopy (RCM)lip score. Clinical, dermoscopic and RCM images of histologically proven pigmented lesions, involving the genital area (vulva or glans penis) and lip, were retrospectively reviewed. RCM images were evaluated for malignant criteria, and statistical analysis was conducted for categorical variables. Seventy pigmented lesions were included in the study and divided into two groups based on the body area location: lip (17) and genital area (53). Architectural disarray (P = 0.002), dendritic (P = 0.031) and roundish cells in epidermis (P < 0.0001), interpapillary dendritic cells (P = 0.039) and junctional atypical cells (P = 0.002) were associated to genital melanoma. Melanoma involving the lip was characterized by roundish cells in epidermis, a criterion found in one labial benign lesion, only (P = 0.005). Main limitations of the study are the inclusion of low melanomas and the presence of epidermal dendritic cells in melanosis and melanoma, as a confusing factor in imaging. Dermatologists should consider confocal microscopy as an adjunctive tool to dermoscopy in the differential diagnosis of pigmented mucosal lesions, especially in presence of clinical and dermoscopic findings suspicious for malignancy.

Preliminary Evaluation of in vivo Reflectance Confocal Microscopy Features of Neonatal Lupus Erythematosus

Introduction: Neonatal lupus erythematosus (NLE) is a rare autoimmune disease, which needs to be distinguished from eczema, congenital syphilis, and tinea corporis in newborns. Reflectance confocal microscopy (RCM) could be a helpful noninvasive diagnostic tool, which has been used to evaluate several inflammatory skin conditions. The aim of this study was to describe the RCM characteristics of NLE. Methods: Eleven NLE patients were included in the study, and all patients were evaluated clinically with RCM. We also evaluated RCM images from 11 eczema patients as controls. Results: Some major key diagnostic features of NLE can be observed by RCM: an enlarged honeycomb pattern (9/11, 81.8%), round-to-oval cyst-like structures were present (6/11, 54.5%), the normal ring-like structures were totally or partially obliterated (11/11, 100%) at the level of the dermo-epidermal junction, medium refractivity collagen fibers that were disorganized (10/11, 90.9%), numerous high refractivity round cells (11/11, 100%) in the dermis. Conclusion: RCM allows the visualization of major key diagnostic features of NLE and serves as a complementary diagnostic tool for NLE.

The combination of dermoscopy and reflectance confocal microscopy increases the diagnostic confidence of amelanotic/hypomelanotic lentigo maligna.

The dermoscopic diagnosis of amelanotic/hypomelanotic lentigo maligna/lentigo maligna melanoma (AHLM/LMM) may be very difficult in its early stages because of lack of pigment. Reflectance confocal microscopy (RCM) is an imaging technique that is especially helpful for the diagnosis of lentigo maligna. To determine the diagnostic performances of dermoscopy and RCM in the diagnosis of AHLM/LMMs we evaluated dermoscopic and RCM images of consecutive cases of histopathologically confirmed AHLM/LMMs, amelanotic/hypomelanotic basal cell carcinoma and squamous cell carcinoma (AHBCCs/AHSCCs), amelanotic/hypomelanotic benign lesions (AHBLs), and actinic keratoses (AKs) from five participating centers. Sensitivity, specificity, accuracy, predictive values, and level of diagnosis confidence were calculated for both diagnostic procedures. Both dermoscopy and RCM showed diagnostic performance >97% in the diagnosis of AHLM/LMMs versus AHBCC/AHSCCs and their combination slightly improved diagnostic performance, with accuracy increasing from 98.0% to 99.1%. Similarly, RCM in combination with dermoscopy showed a tiny increase in the diagnostic performance in the diagnosis of AHLM/LMMs versus AHBLs (accuracy increased from 87.2% to 88.8%) and versus AKs (accuracy increased from 91.4% to 93.4%). Although the increase in diagnostic performance due to RCM was modest, the combination of dermoscopy and RCM greatly increased the level of confidence; high confidence in the diagnosis of AHLM/LMMs versus AHBLs increased from 36.2% with dermoscopy alone to 76.6% with dermoscopy plus RMC. Based on our results, dermoscopy and RCM should be complementary to improve not only diagnostic accuracy but also the level of diagnostic certainty in the diagnosis of AHLM/LMMs.

Dermatoscopy and reflectance confocal microscopy for basal cell carcinoma diagnosis and diagnosis prediction score: A prospective and multicenter study on 1005 lesions

BackgroundBasal cell carcinoma is usually diagnosed by clinical and dermoscopy examination, but diagnostic accuracy may be suboptimal. Reflectance confocal microscopy (RCM) imaging increases skin cancer diagnostic accuracy. ObjectiveTo evaluate additional benefit in diagnostic accuracy of handheld RCM in a prospective controlled clinical setting MethodsA prospective, multicenter study in 3 skin cancer reference centers in Italy enrolling consecutive lesions with clinical-dermoscopic suspicion of basal cell carcinoma. Clinicaltrials.gov; NCT04789421 ResultsA total of 1005 lesions were included, of which 474 histopathologically confirmed versus 531 diagnosed by clinical-dermoscopic-RCM correlation, confirmed with 2 years of follow up. Specifically, 740 were confirmed basal cell carcinomas. Sensitivity and specificity for dermoscopy alone was 93.2% (CI 95% 91.2 - 94.9) and 51.7% (CI 95% 45.5 - 57.9); PPV was 84.4 (CI 95% 81.7 – 86.8) and NPV 73.3 (66.3 - 79.5). Adjunctive reflectance confocal microscopy reported higher rates: 97.8 (CI 95% 96.5 - 98.8) sensitivity and 86.8 (CI 95% 82.1 - 90.6) specificity, with PPV of 95.4 (CI 95% 93.6 – 96.8) and NPV 93.5 (89.7 - 96.2). LimitationsStudy conducted in one single Country ConclusionAdjunctive handheld RCM assessment of lesions clinically suspicious for basal cell carcinoma permits higher diagnostic accuracy with minimal false negative lesions.

Concordance between reflectance confocal microscopy and histopathology for the diagnosis of acral lentiginous melanoma.

Acral lentiginous melanoma (ALM) is a highly malignant and invasive type of melanoma with unique locations of onset. Its incidence is increasing and early diagnosis is challenging. Reflectance confocal microscopy (RCM) is a non-invasive technique that provides an accurate image of tissue pathology. There are few reports on the use of RCM for the assessment of ALM. In this retrospective study, data from 31 patients with a clinical diagnosis of ALM were collected. RCM image features were compared with histopathological findings to determine the concordance between the two methods. The sensitivity, specificity, positive predictive value, and negative predictive value of RCM for the diagnosis of ALM were evaluated. RCM and histopathology findings were concordant in 29 of 31 patients (93.5%). There were no false-negative results, although there were two false positives in RCM diagnosis. The sensitivity of RCM for diagnosing ALM was 100%, specificity was 50%, positive predictive value was 93.1%, and negative predictive value was 100%. RCM showed substantial concordance with histopathology in the diagnosis of ALM. It is a reliable and valuable non-invasive diagnostic tool that holds promise for the early diagnosis of ALM.

In-depth study of Wood's lamp examination combined with reflective confocal laser scanning microscopy for the guidance of vitiligo staging and treatment.

Both Wood's lamp and reflective confidential laser scanning microcopy are helpful for the diagnosis and treatment of vitiligo. However, there is few research that contains large samples and consistent observations. To analyze the characteristics of Wood's lamp images and reflectance confocal microscopy (RCM) images of vitiligo lesions and to evaluate their significance in vitiligo staging. We analyzed the characteristics of RCM images, Wood's lamp images, the vitiligo disease activity (VIDA) score, and clinical features to guide vitiligo staging and treatment. The expert consensus based on the clinical features, VIDA score, Wood's lamp findings, and isomorphic response was consistent with the Wood's lamp findings (χ2 = 3.63, p > 0.05) and RCM findings (χ2 = 3.60, p > 0.05) in diagnosing vitiligo and assessing the disease stage. There was a correlation between the three lesion grades based on the Wood's lamp findings and the stage of vitiligo (p < 0.01). Lesions that appeared porcelain white under the Wood's lamp were in the slowly progressive stage; lesions that appeared gray-white or trichromatic under the Wood's lamp were in the rapidly progressive stage; lesions with clear borders under the Wood's lamp needed further analysis by RCM for the stage to be determined; lesions with blurred borders under the Wood's lamp were in the rapidly progressive stage; lesions that were visible under the naked eye and under the Wood's lamp were in the rapidly progressive stage. The study demonstrates a reliable correlation between the findings of RCM (a sophisticated expensive tool) and Wood's lamp examination (a simple, readily available, inexpensive tool) in the assessment of the disease activity of vitiligo lesions.

Continuing Medical Education-accredited reflectance confocal microscopy course in the USA and its impact on learning and clinical integration: report from a single centre.

Reflectance confocal microscopy (RCM) images skin lesions noninvasively at quasi-histological resolution. RCM has improved the diagnosis of skin neoplasms, and recently acquired billing codes in the USA.1 However, reading RCM images requires training, imposing a major barrier to its widespread adoption.2 To date, RCM learning has not been standardized and is primarily based on ‘mentorship’ and ‘on-site’ learning. There is a paucity of CME-accredited RCM courses in the USA and across the globe, as well as a lack of reports on CME’s clinical impact. This study aimed to evaluate the impact of a short-duration RCM course on learning and clinical integration.

Characterization and Efficacy of Essential Oil-Based Cosmetic Formulations for Acne-Prone Skin

This randomized, placebo-controlled, double-blind clinical in vivo study aimed to evaluate the clinical efficacy of a cosmetic formulation for non-inflammatory acne using essential oils. Fifty-three male participants were divided into four groups: a formulation containing a mixture of four essential oils (4EO), Melaleuca alternifolia (M.a.), nanoemulsion of M. alternifolia (Nanoem.), and a placebo group. The participants applied the formulation daily for 90 days and non-invasive skin imaging techniques were employed to assess the outcomes. Skin microrelief images and reflectance confocal microscopy images were captured in the malar region, and Raman spectroscopy was used to analyze the terpene composition of the essential oils, oil mixture, and nanoemulsion. The results indicated that the nanoemulsion, M.a. essential oil, and 4EO formulation effectively reduced the overall number of comedone and improved follicular hyperkeratinization. The nanoemulsion of M.a. demonstrated the most promising outcomes in reducing comedone areas, especially in the infundibular region. This effect could be attributed to the presence of terpinene-4-ol in the essential oil and the enhanced penetration provided by the nanoemulsion formulation. These findings suggest that cosmetic formulations containing essential oils, particularly in nanoemulsion form, have potential against mild acne. This study contributes to our understanding of the relationship between terpene composition and clinical activity, highlighting the importance of innovative delivery systems.

Handheld cross-polarized microscope for imaging individual pigmented cells in human skin in vivo.

We present the development of a simple, handheld cross-polarized microscope (CPM) and demonstration of imaging individual pigmented cells in human skin in vivo. In the CPM device, the cross-polarized detection approach is used to reduce the specular reflection from the skin surface and preferentially detect multiply-scattered light. The multiply-scattered light works as back illumination from within the tissue towards the skin surface, and superficial pigment such as intraepidermal melanin absorbs some spectral bands of the multiply-scattered light and cast colored shadows. Since the light that interacted with the superficial pigment only needs to travel a short distance before it exits the skin surface, microscopic details of the pigment can be preserved. The CPM device uses a water-immersion objective lens with a high numerical aperture to image the microscopic details with minimal spherical aberrations and a small depth of focus. Preliminary results from a pilot study of imaging skin lesions in vivo showed that the CPM device could reveal three-dimensional distribution of pigmented cells and intracellular distribution of pigment. Co-registered CPM and reflectance confocal microcopy images showed good correspondence between dark, brown cells in CPM images and bright, melanin-containing cells in reflectance confocal microscopy images. Lay Description: We present the development of a simple, handheld cross-polarized microscope (CPM) and demonstration of imaging individual pigmented cells in human skin in vivo. In the CPM device, the cross-polarized detection approach is used to reduce the specular reflection from the skin surface and preferentially detect multiply-scattered light. The multiply-scattered light works as back illumination from within the tissue towards the skin surface, and superficial pigment such as intraepidermal melanin absorbs some spectral bands of the multiply-scattered light and cast colored shadows. Since the light that interacted with the superficial pigment only needs to travel a short distance before it exits the skin surface, microscopic details of the pigment can be preserved. Preliminary results from a pilot study of imaging skin lesions in vivo showed that the CPM device could reveal three-dimensional distribution of pigmented cells and intracellular distribution of pigment. Co-registered CPM and reflectance confocal microcopy images showed good correspondence between dark, brown cells in CPM images and bright, melanin-containing cells in reflectance confocal microscopy images. This article is protected by copyright. All rights reserved.

Perifollicular linear projections: A dermatoscopic criterion for the diagnosis of lentigo maligna on the face

Lentigo maligna (LM) can mimic benign flat pigmented lesions and can be challenging to diagnose. To describe a new dermoscopic feature termed 'perifollicular linear projections (PLP)' as a diagnostic criterion for LM on the face. Retrospective study on reflectance confocal microscopy (RCM) and dermoscopy images of flat facial pigmented lesions originating from 2 databases. PLP were defined as short, linear, pigmented projections emanating from hair follicles. Dermoscopy readers were blinded to the final histopathologic diagnosis. From 83 consecutive LMs, 21/83 (25.3%) displayed 'bulging of hair follicles' on RCM and 18 these 21 (85.7%), displayed PLP on dermoscopy. From a database of 2,873 consecutively imaged and biopsied lesions, 252 flat pigmented facial lesions were included. PLP was seen in 47/76 melanomas (61.8%), compared with 7/176 lesions (3.9%) with other diagnosis (p<0.001). The sensitivity was 61.8% (95% CI 49.9 - 72.7%), specificity 96.0% (95% CI 92.9 - 98.4%). PLP was independently associated with LM diagnosis on multivariate analysis (OR 26.1 [95% CI 9.6 - 71.0]). Retrospective study. PLP is a newly described dermoscopic criterion that may add specificity and sensitivity to the early diagnosis of LM located on the face. We postulate that PLP constitutes an intermediary step in the LM progression model.

Lentigo maligna and lentigo maligna melanoma in-vivo differentiation with dermoscopy and reflectance confocal microscopy: a retrospective, multi-center study.

Dermoscopic predictors of lentigo maligna (LM) and lentigo maligna melanoma (LMM) have been recently reported, but these have not been reported in reflectance confocal microscopy (RCM). (i) To validate dermoscopic predictors for LM/LMM, (ii) to identify RCM patterns in LM and LMM and (iii) correlations between dermoscopic and RCM features in LM and LMM. A retrospective, multicenter study of consecutive lesions with histologically proven LM or LMM subtypes of the head and face, with complete sets of dermoscopic and RCM images. A total of 180 lesions were included (n=40 LMM). Previously reported differential dermoscopic features for LM subtypes were confirmed. Other features significantly associated with LMM diagnosis included irregular hyperpigmented areas, shiny white streaks, atypical vessels, and light brown color at dermoscopy and medusa head-like structures, dermal nests and nucleated cells within the papillae at RCM (p<0.05). Correlations among LM lesions between dermoscopic and RCM features included brown to-gray dots and atypical cells (epidermis), grey color and inflammation and obliterated follicles and medusa head-like structures. Among LMM lesions, significant correlations included obliterated follicles with folliculotropism, both irregular hyperpigmented areas and irregular blotches with widespread atypical cell distribution (epidermis), dermal nests and nucleated cells within the papillae (dermis). Irregular blotches were also associated with medusa head-like structures (dermal epidermal junction [DEJ]). Dermoscopic and RCM features can assist in the in-vivo identification of LM histopathological subtypes and many are correlated. RCM 3-dimensional analysis of skin layers allows the identification of invasive components in the DEJ and dermis.

Photodynamic Therapy is an Effective Treatment of Facial Pigmented Actinic Keratosis.

Actinic keratosis (AK), or solar keratosis, is a precancerous condition of the skin, mainly caused by excessive and chronic exposure to ultraviolet radiation. Pigmented AK (pAK) is a rare variant of AK. Photodynamic therapy (PDT) is widely used to treat the classical variant of AK, but very limited data are available on the use of PDT in patients with pAK. The objective of this study was to assess the usefulness of PDT in the treatment of pAK. The study included 16 patients with 20 pAK lesions treated with PDT. All skin lesions were clinically and dermatoscopically assessed for typical features characteristic of pAK. Reflectance confocal microscopy (RCM) was also used to assess keratinocyte atypia, confirm pAK diagnosis, and rule out other disease entities. After three PDT sessions, the complete resolution of all clinical features of pAK was observed in 80% of the studied lesions. Dermatoscopically, 65% of the lesions achieved 100% response and no cellular atypia was seen in the follow-up RCM images of 85% of lesions. Photodynamic therapy is an effective treatment modality for pAK in fair-skinned individuals.

Age-dependent changes in epidermal architecture explored using an automated image analysis algorithm on in vivo reflectance confocal microscopy images.

Reflectance confocal microscopy (RCM) allows for real-time in vivo visualization of the epidermis at the cellular level noninvasively. Parameters relating to tissue architecture can be extracted from RCM images, however, analysis of such images requires manual identification of cells to derive these parameters, which can be time-consuming and subject to human error, highlighting the need for an automated cell identification method. First, the region-of-interest (ROI) containing cells needs to be identified, followed by the identification of individual cells within the ROI. To perform this task, we use successive applications of Sato and Gabor filters. The final step is post-processing improvement of cell detection and removal of size outliers. The proposed algorithm is evaluated on manually annotated real data. It is then applied to 5345 images to study the evolution of epidermal architecture in children and adults. The images were acquired on the volar forearm of healthy children (3 months to 10 years) and women (25-80 years), and on the volar forearm and cheek of women (40-80 years). Following the identification of cell locations, parameters such as cell area, cell perimeter, and cell density are calculated, as well as the probability distribution of the number of nearest neighbors per cell. The thicknesses of the Stratum Corneum and supra-papillary epidermis are also calculated using a hybrid deep-learning method. Epidermal keratinocytes are significantly larger (area and perimeter) in the granular layer than in the spinous layer and they get progressively larger with a child's age. Skin continues to mature dynamically during adulthood, as keratinocyte size continues to increase with age on both the cheeks and volar forearm, but the topology and cell aspect ratio remain unchanged across different epidermal layers, body sites, and age. Stratum Corneum and supra-papillary epidermis thicknesses increase with age, at a faster rate in children than in adults. The proposed methodology can be applied to large datasets to automate image analysis and the calculation of parameters relevant to skin physiology. These data validate the dynamic nature of skin maturation during childhood and skin aging in adulthood.

Automatic granular and spinous epidermal cell identification and analysis on in vivo reflectance confocal microscopy images using cell morphological features.

Reflectance confocal microscopy (RCM) allows for real-time in vivo visualization of the skin at the cellular level. The study of RCM images provides information on the structural properties of the epidermis. These may change in each layer of the epidermis, depending on the subject's age and the presence of certain dermatological conditions. Studying RCM images requires manual identification of cells to derive these properties, which is time consuming and subject to human error, highlighting the need for an automated cell identification method. We aim to design an automated pipeline for the analysis of the structure of the epidermis from RCM images of the Stratum granulosum and Stratum spinosum. We identified the region of interest containing the epidermal cells and the individual cells in the segmented tissue area using tubeness filters to highlight membranes. We used prior biological knowledge on cell size to process the resulting detected cells, removing cells that were too small and reapplying the used filters locally on detected regions that were too big to be considered a single cell. The proposed full image analysis pipeline (FIAP) was compared with machine learning-based approaches (cell cutter, different U-Net configurations, and loss functions). All methods were evaluated both on simulated data (four images) and on manually annotated RCM data (seven images). Accuracy was measured using recall and precision metrics. Both accuracy metrics were higher in the proposed FIAP for both real ( , ) and synthetic images ( , ). The tested machine learning methods failed to identify and segment keratinocytes on RCM images with a satisfactory accuracy. We showed that automatic cell segmentation can be achieved using a pipeline based on membrane detection, with an accuracy that matches expert manual cell identification. To our knowledge, this is the first method based on membrane detection to study healthy skin using RCM images evaluated against manually identified cell positions.

High-speed reflectance confocal microscopy of human skin at 1251-1342 nm.

Reflectance confocal microscopy (RCM) is an imaging method that can noninvasively visualize microscopic features of the human skin. The utility of RCM can be further improved by increasing imaging speed. In this paper, we report high-speed RCM imaging of human skin with a frame rate that is over 10 times faster and an area imaging rate that is 6-9 times faster than those of commercially available RCM devices. The higher imaging speed was achieved using a high-speed RCM technique, termed spectrally encoded confocal microscopy (SECM). SECM uses a diffraction grating and a high-speed, wavelength-swept source to conduct confocal imaging at a very high rate. We developed a handheld SECM probe using a scanned-grating approach. The SECM probe was used in conjunction with a wavelength-swept source with a spectral band of 1251-1342 nm. The SECM probe achieved high lateral resolution of 1.3-1.6 µm and an axial resolution of 3.5 µm. SECM images of the human skin (image size = 439 × 439 µm2 ) obtained at 100 frames/s clearly show previously reported RCM features of the human skin in vivo with adequate image quality. The fast imaging speed allowed for the rapid acquisiton of volumetric SECM image data (200 frames covering a depth range of 200 µm) within 2 s. The use of 1251-1342 nm provided sufficient signal level and contrast required to visualize key cellular morphologic features. These preliminary results demonstrate that high-speed SECM imaging of the human skin at 1251-1342 nm is feasible.