Fast, large area multiphoton exoscope (FLAME) for macroscopic imaging with microscopic resolution of human skin.

We introduce a compact, fast large area multiphoton exoscope (FLAME) system with enhanced molecular contrast for macroscopic imaging of human skin with microscopic resolution. A versatile imaging platform, FLAME combines optical and mechanical scanning mechanisms with deep learning image restoration to produce depth-resolved images that encompass sub-mm2 to cm2 scale areas of tissue within minutes and provide means for a comprehensive analysis of live or resected thick human skin tissue. The FLAME imaging platform, which expands on a design recently introduced by our group, also features time-resolved single photon counting detection to uniquely allow fast discrimination and 3D virtual staining of melanin. We demonstrate its performance and utility by fast ex vivo and in vivo imaging of human skin. With the ability to provide rapid access to depth resolved images of skin over cm2 area and to generate 3D distribution maps of key sub-cellular skin components such as melanocytic dendrites and melanin, FLAME is ready to be translated into a clinical imaging tool for enhancing diagnosis accuracy, guiding therapy and understanding skin biology.

Non-invasive optical biopsy by multiphoton microscopy identifies the live morphology of common melanocytic nevi.

Multiphoton microscopy (MPM) is a promising non-invasive imaging tool for discriminating benign nevi from melanoma. In this study, we establish a MPM morphologic catalogue of common nevi, information that will be critical in devising strategies to distinguish them from nevi that are evolving to melanoma that may present with more subtle signs of malignancy. Thirty common melanocytic nevi were imaged in vivo using MPM. Quantitative parameters that can distinguish between different types of nevi were developed and confirmed by examining the histology of eleven of the imaged nevi. MPM features of nevi examined included cytologic morphology of melanocytes in the epidermis and dermis, the size and distribution of nevomelanocytes both within and around nests, the size of rete ridges, and the presence of immune cells in the dermis. Distinguishing features include cytological morphology, the size of nevomelanocytes, the size of nevomelanocyte nests, and the distribution of nevomelanocytes. Notably, these distinguishing characteristics were not easily appreciated in fixed tissues, highlighting essential differences in the morphology of live skin. Taken together, this work provides a morphologic compendium of normal nevi, information that will be critical in future studies directed at identifying melanocytic nevi that are evolving to melanoma.

In vivo multiphoton microscopy of melasma.

Melasma is a skin disorder characterized by hyperpigmented patches due to increased melanin production and deposition. In this pilot study, we evaluate the potential of multiphoton microscopy (MPM) to characterize non-invasively the melanin content, location, and distribution in melasma and assess the elastosis severity. We employed a clinical MPM tomograph to image in vivo morphological features in melasma lesions and adjacent normal skin in 12 patients. We imaged dermal melanophages in most dermal melasma lesions and occasionally in epidermal melasma. The melanin volume fraction values measured in epidermal melasma (14% ± 4%) were significantly higher (p < 0.05) than the values measured in perilesional skin (11% ± 3%). The basal keratinocytes of melasma and perilesions showed different melanin distribution. Elastosis was predominantly more severe in lesions than in perilesions and was associated with changes in melanin distribution of the basal keratinocytes. These results demonstrate that MPM may be a non-invasive imaging tool for characterizing melasma.

Imaging mitochondrial dynamics in human skin reveals depth-dependent hypoxia and malignant potential for diagnosis.

Active changes in mitochondrial structure and organization facilitate cellular homeostasis. Because aberrant mitochondrial dynamics are implicated in a variety of human diseases, their assessment is potentially useful for diagnosis, therapy, and disease monitoring. Because current techniques for evaluating mitochondrial morphology are invasive or necessitate mitochondria-specific dyes, their clinical translation is limited. We report that mitochondrial dynamics can be monitored in vivo, within intact human skin by relying entirely on endogenous two-photon-excited fluorescence from the reduced metabolic coenzyme nicotinamide adenine dinucleotide (NADH). We established the sensitivity of this approach with in vivo, fast temporal studies of arterial occlusion-reperfusion, which revealed acute changes in the mitochondrial metabolism and dynamics of the lower human epidermal layers. In vitro hypoxic-reperfusion studies validated that the in vivo outcomes were a result of NADH fluorescence changes. To demonstrate the diagnostic potential of this approach, we evaluated healthy and cancerous human skin epithelia. Healthy tissues displayed consistent, depth-dependent morphological and mitochondrial organization patterns that varied with histological stratification and intraepithelial mitochondrial protein expression. In contrast, these consistent patterns were absent in cancerous skin lesions. We exploited these differences to successfully differentiate healthy from cancerous tissues using a predictive classification approach. Collectively, these results demonstrate that our label-free, automated, near real-time assessments of mitochondrial organization-relying solely on endogenous contrast-could be useful for accurate, noninvasive in vivo diagnosis.

In Vivo Multiphoton Microscopy of Basal Cell Carcinoma.

IMPORTANCE: Basal cell carcinomas (BCCs) are diagnosed by clinical evaluation, which can include dermoscopic evaluation, biopsy, and histopathologic examination. Recent translation of multiphoton microscopy (MPM) to clinical practice raises the possibility of noninvasive, label-free in vivo imaging of BCCs that could reduce the time from consultation to treatment. OBJECTIVES: To demonstrate the capability of MPM to image in vivo BCC lesions in human skin, and to evaluate if histopathologic criteria can be identified in MPM images. DESIGN, SETTING, AND PARTICIPANTS: Imaging in patients with BCC was performed at the University of California-Irvine Health Beckman Laser Institute & Medical Clinic, Irvine, between September 2012 and April 2014, with a clinical MPM-based tomograph. Ten BCC lesions were imaged in vivo in 9 patients prior to biopsy. The MPM images were compared with histopathologic findings. MAIN OUTCOMES AND MEASURES: MPM imaging identified in vivo and noninvasively the main histopathologic feature of BCC lesions: nests of basaloid cells showing palisading in the peripheral cell layer at the dermoepidermal junction and/or in the dermis. RESULTS: The main MPM feature associated with the BCC lesions involved nests of basaloid cells present in the papillary and reticular dermis. This feature correlated well with histopathologic examination. Other MPM features included elongated tumor cells in the epidermis aligned in 1 direction and parallel collagen and elastin bundles surrounding the tumors. CONCLUSIONS AND RELEVANCE: This study demonstrates, in a limited patient population, that noninvasive in vivo MPM imaging can provide label-free contrast that reveals several characteristic features of BCC lesions. Future studies are needed to validate the technique and correlate MPM performance with histopathologic examination.

Distinguishing between benign and malignant melanocytic nevi by in vivo multiphoton microscopy.

Monitoring of atypical nevi is an important step in early detection of melanoma, a clinical imperative in preventing the disease progression. Current standard diagnosis is based on biopsy and histopathologic examination, a method that is invasive and highly dependent upon physician experience. In this work, we used a clinical multiphoton microscope to image in vivo and noninvasively melanocytic nevi at three different stages: common nevi without dysplastic changes, dysplastic nevi with structural and architectural atypia, and melanoma. We analyzed multiphoton microscopy (MPM) images corresponding to 15 lesions (five in each group) both qualitatively and quantitatively. For the qualitative analysis, we identified the morphologic features characteristic of each group. MPM images corresponding to dysplastic nevi and melanoma were compared with standard histopathology to determine correlations between tissue constituents and morphology and to evaluate whether standard histopathology criteria can be identified in the MPM images. Prominent qualitative correlations included the morphology of epidermal keratinocytes, the appearance of nests of nevus cells surrounded by collagen fibers, and the structure of the epidermal-dermal junction. For the quantitative analysis, we defined a numerical multiphoton melanoma index (MMI) based on three-dimensional in vivo image analysis that scores signals derived from two-photon excited fluorescence, second harmonic generation, and melanocyte morphology features on a continuous 9-point scale. Indices corresponding to common nevi (0-1), dysplastic nevi (1-4), and melanoma (5-8) were significantly different (P < 0.05), suggesting the potential of the method to distinguish between melanocytic nevi in vivo.

Immunohistochemistry of angiogenesis mediators before and after pulsed dye laser treatment of angiomas.

BACKGROUND AND OBJECTIVE: Tissue effects of vascular lesion laser treatment are incompletely understood. Injury caused by pulsed dye laser (PDL) treatment may result in altered expression of mediators associated with angiogenesis. MATERIALS AND METHODS: Eight human subjects had one angioma treated with PDL (7 mm, 1.5 millisecond pulse duration, 9 J/cm(2), cryogen spray cooling of 30 millisecond with a 30 millisecond delay). One week later, three biopsies were taken: normal skin, untreated angioma, angioma post-PDL. Tissue was frozen and sections processed for immunohistochemistry staining of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), matrix metalloproteinase 9 (MMP-9), and angiopoietin 2 (ANG-2). Images were graded in a blinded fashion by a board certified dermatopathologist. RESULTS: There were no clear trends in VEGF expression in the epidermis, dermis, or endothelial cells. As compared to normal skin, angiomas demonstrated the following: bFGF was decreased in the epidermis; MMP-9 was decreased or unchanged in the epidermis and increased in the endothelial cells; ANG-2 was increased in the endothelial cells. When comparing normal skin to angiomas + PDL, bFGF was decreased in the epidermis and increased in the dermis; MMP-9 was decreased or unchanged in the epidermis; ANG-2 was again increased in the endothelial cells. Comparison of staining in angioma to angioma + PDL samples revealed increased dermal bFGF expression. CONCLUSION: Alterations in angiogenesis mediators were noted after PDL. Angiogenesis mediator changes associated with PDL treatment differed from those previously reported for incisional biopsies. This pilot study can guide future work on laser-induced alterations in vascular lesions and such information may ultimately be used to optimize treatment outcomes.

Porokeratotic eccrine ostial and dermal duct nevus treated with a combination erbium/CO2 laser: a case and brief review.

Porokeratotic eccrine ostial and dermal duct nevus (PEODDN) is an uncommon disease that presents early in childhood and is characterized by keratotic papules, often in a linear configuration. We describe a 12-year-old girl with characteristic lesions of PEODDN and describe her response to treatment with a combination CO2/Erbium laser. We also briefly review the literature on PEODDN.

Sampling of melanocytic nevi for research purposes: a prospective, pilot study to determine effect on diagnosis.

BACKGROUND: Research on melanocytic nevi predominantly utilizes formalin-fixed, paraffin-embedded tissue, largely limiting research to morphologic and immunohistochemical observations. Withholding portions of fresh nevus tissue for molecular studies could result in the loss of important diagnostic material. OBJECTIVE: This study prospectively evaluated melanocytic nevi for histologic homogeneity to determine if using a portion for research would have affected diagnosis. METHODS: Thirty-three subjects were enrolled in a prospective study in which pigmented lesions were chosen for biopsy on a clinical basis. Lesions were sectioned and each piece submitted in a separate block (mean, 2.7; range 2-5 blocks per lesion). Slides from nevi were examined in two phases. In phase I, sections of nevi were randomized and a diagnosis was rendered for each section of nevus. In phase II, the dermatopathologist reviewed all slides for each nevus as a case, similar to the original interpretation of the lesion provided to the clinician. Diagnoses from phases I and II were compared with the original diagnosis. RESULTS: Case material included 51 melanocytic lesions from 31 subjects. The phase I diagnosis matched the original diagnosis for 99 of 121 slides that showed a melanocytic lesion (82%). The phase II diagnosis matched the original diagnosis for 45 of 51 specimens (88%). LIMITATIONS: The study was limited by: a small number of specimens; the clinician could have chosen clinically homogeneous nevi for biopsy; effect of interobserver and intraobserver variability on diagnosis. CONCLUSIONS: For the majority of melanocytic nevi in this study, the diagnostic information present in one section of a melanocytic nevus could be extrapolated to the remainder of the specimen without adverse consequences from a diagnostic or therapeutic perspective.

Multiple primary melanomas in a CDKN2A mutation carrier exposed to ionizing radiation.

BACKGROUND: Recent research has shown a possible causal relationship between ionizing radiation exposure and melanoma. Individuals with mutations in CDKN2A (cyclin-dependent kinase inhibitor 2A), the major melanoma predisposition gene, have an increased susceptibility to melanoma-promoting exposures, such as UV light. We describe a patient from a familial melanoma pedigree with 7 primary melanomas on the right side of her body, the first occurring 5 years after exposure to atmospheric nuclear bomb testing in the 1950s. OBSERVATIONS: Physical examination revealed phototype I skin, red hair, and 26 nevi (14 on the right and 12 on the left side of her body). One nevus was larger than 5 mm, and 2 were clinically atypical. Sequence analysis demonstrated a known deleterious mutation in CDKN2A (G-34T) and homozygosity for a red hair color variant in MC1R (melanocortin 1 receptor) (R151C). Fluorescence in situ hybridization analysis of blood, fibroblasts, and melanocytes from both upper extremities ruled out mosaicism. CONCLUSIONS: Individuals such as this patient, who has CDKN2A and MC1R mutations, are likely to be more susceptible to environmental insults. A careful review of environmental exposures in these vulnerable cases may reveal cancer-promoting agents, such as ionizing radiation, that go unnoticed in less susceptible populations.

Usefulness (or lack thereof) of immunophenotyping in atypical cutaneous T-cell infiltrates.

Our purpose was to evaluate the interobserver concordance for the diagnoses of mycosis fungoides (MF), atypical dermatoses (AD), and benign dermatoses (BD) and the impact of T-cell immunophenotyping on the diagnoses MF, AD, and BD. Specimens of MF (n = 57), AD (n = 27), BD and normal skin (n = 54) were reviewed by 2 hematopathologists and 1 dermatopathologist to establish diagnostic interobserver concordance by routine morphologic examination. Immunophenotyping was performed to evaluate expression of CD2, CD3, CD4, CD5, CD7, CD8, CD20, CD30, and MIB-1. The interobserver concordance was fair to moderate compared with the original diagnosis. Partial deletion of CD2 alone was associated significantly with MF. Epidermal deletions of 2 or 3 T-cell antigens or 2 T-cell antigens not including CD7 were associated significantly with MF. An elevated CD4/CD8 ratio correlated with MF. Morphologic features were most diagnostic of MF. Immunophenotyping generally resulted in downgrading of the reaction pattern but was helpful in distinguishing MF from benign dermatoses.

Lesions resembling malignant atrophic papulosis in a patient with progressive systemic sclerosis.

Malignant atrophic papulosis (MAP), or Degos syndrome, is a rare disorder of unknown etiology. It is characterized by a deep subcutaneous vasculopathy resulting in atrophic, porcelain-white papules. We report the case of a 42-year-old woman with a history of progressive systemic sclerosis who presented with painful subcutaneous nodules on her abdomen along with chronic atrophic papules on her upper and lower limbs. Biopsy results of both types of lesions revealed vascular thrombi without surrounding inflammation. We briefly review the literature on MAP and its association with various connective tissue diseases. To our knowledge, there have been no previous reports of a patient with the clinical and histologic presentations described here. Although the histologic appearance of the subcutaneous nodules was very similar to that of the atrophic papules, the clinical characteristics of the 2 types of lesions were strikingly different. It is fair to theorize that Degos lesions do not start as atrophic porcelain-white papules but rather evolve from a primary lesion. We hypothesize that these lesions start as painful red nodules and may represent part of the disease spectrum in the evolution of MAP.

Longitudinal assessment of the nevus phenotype in a melanoma kindred.

Phenotypic characteristics of members of a melanoma prone kindred with a V126D CDKN2A gene mutation were monitored over approximately 15 y. Thirty-eight previously studied subjects were recruited. Participants underwent a complete skin examination by the same dermatologist who examined them initially. The size and location of all nevi were recorded on a body map diagram. Total nevus number (TNN) and total nevus density (TND) were determined. CDKN2A sequencing verified 13 mutation carriers and 16 non-carriers. Nine participants were spouse controls without a history of melanoma and did not carry a CDKN2A mutation. Mutation carriers demonstrated a greater mean TNN and TND at initial and follow-up examinations compared with non-carriers and continued to develop nevi rather than show nevus regression seen in non-carriers and spouse controls. Non-carriers showed an intermediate nevus phenotype between mutation carriers and spouse controls. Four of the 13 mutation carriers and one non-carrier have developed invasive melanoma. Over a 15-y interval, TNN and TND were increased in mutation carriers compared with non-carriers and spouse controls. Continued accumulation of nevi in mutation carriers supports a nevogenic role for this CDKN2A mutation. An intermediate nevus phenotype in non-carrier family members suggests the presence of additional modifier genes.

Post-transplant lymphoproliferative disorder limited to the skin.

BACKGROUND: Post-transplant lymphoproliferative disorder (PTLD) is a condition affecting immunosuppressed transplant patients and has a variety of clinical presentations. It is rarely found in the skin, and cases of PTLD in the skin are usually linked with lymph node or other organ involvement. METHODS: We report a case of plasmacytoid PTLD that is limited to the skin. A 63-year-old man with a history of cardiac transplant presented with exophytic tumors involving the lower extremity. The diagnosis and classification of the various forms of PTLD are discussed. RESULTS: Histology, immunohistochemical stains, and in situ hybridization revealed an aggressive plasmacytoid tumor that was Epstein-Barr virus positive. The patient's tumors resolved with decreased immunosuppression and localized radiation. CONCLUSION: This case is unusual for several reasons including involvement limited to the skin, presentation 15 years following transplant, and plasmacytoid phenotype of the tumor. This disorder will likely be seen by dermatologists and dermatopathologists with the increasing use of immunosuppressive medications in the dermatologist's patient population.