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BACKGROUND: Psoralen + ultraviolet-A (PUVA) is associated with photocarcinogenesis. However, carcinogenic risk with other ultraviolet phototherapies remains unclear. OBJECTIVE: Evaluate whether phototherapy without psoralens increases skin cancer risk. METHODS: Retrospective cohort study of patients treated at a teaching-hospital phototherapy center (1977-2018). Skin cancer records were validated against pathology reports. Age-standardized incidence rates (ASIRs) of skin cancer were evaluated for gender, skin phototype, diagnosis, ultraviolet modality, anatomical site; and compared to provincial population incidence rates (2003). RESULTS: In total, 3506 patients treated with broadband-ultraviolet-B, narrowband-UVB and/or combined UVAB were assessed with a mean follow-up of 7.3 years. Majority of patients had psoriasis (60.9%) or eczema (26.4%). Median number of treatments was 43 (1-3598). Overall, 170 skin cancers (17 melanoma, 33 squamous cell carcinoma and 120 basal cell carcinoma) occurred in 79 patients. Patient-based and tumor-based ASIR of skin cancer was 149 (95% CI: 112-187)/100,000 and 264 (219-309)/100,000 person-years, respectively. There was no significant difference between tumor-based ASIRs for melanoma, squamous cell carcinoma, and basal cell carcinoma compared to the general population; or in phototherapy patients with-psoriasis or eczema; or immunosuppressants. No cumulative dose-response correlation between UVB and skin cancer was seen. LIMITATIONS: Treatment and follow-up duration. CONCLUSION: No increased risk of melanoma and keratinocyte cancer was found with phototherapy.
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Carcinoma Basocelular , Carcinoma de Células Escamosas , Eczema , Furocumarinas , Melanoma , Psoríase , Neoplasias Cutâneas , Terapia Ultravioleta , Humanos , Incidência , Melanoma/etiologia , Melanoma/complicações , Estudos Retrospectivos , Terapia Ultravioleta/efeitos adversos , Neoplasias Cutâneas/epidemiologia , Neoplasias Cutâneas/etiologia , Fototerapia/efeitos adversos , Psoríase/complicações , Carcinoma Basocelular/etiologia , Carcinoma Basocelular/complicações , Carcinoma de Células Escamosas/etiologia , Carcinoma de Células Escamosas/complicações , Eczema/complicaçõesRESUMO
BACKGROUND/PURPOSE: A recent direction in skin disease classification is to develop quantitative diagnostic techniques. Skin relief, colloquially known as roughness, is an important clinical feature. The aim of this study is to demonstrate a novel polarization speckle technique to quantitatively measure roughness on skin lesions in vivo. We then calculate the average roughness of different types of skin lesions to determine the extent to which polarization speckle roughness measurements can be used to identify skin cancer. METHODS: The experimental conditions were set to target the fine relief structure on the order of ten microns within a small field of view of 3 mm. The device was tested in a clinical study on patients with malignant and benign skin lesions that resemble cancer. The cancer group includes 37 malignant melanomas (MM), 43 basal cell carcinomas (BCC), and 26 squamous cell carcinomas (SCC), all categories confirmed by gold standard biopsy. The benign group includes 109 seborrheic keratoses (SK), 79 nevi, and 11 actinic keratoses (AK). Normal skin roughness was obtained for the same patients (301 different body sites proximal to the lesion). RESULTS: The average root mean squared (rms) roughness ± standard error of the mean for MM and nevus was equal to 19 ± 5 µm and 21 ± 3 µm, respectively. Normal skin has rms roughness of 31 ± 3 µm, other lesions have roughness of 35 ± 10 µm (AK), 35 ± 7 µm (SCC), 31 ± 4 µm (SK), and 30 ± 5 µm (BCC). CONCLUSION: An independent-samples Kruskal-Wallis test indicates that MM and nevus can be separated from each of the tested types of lesions, except each other. These results quantify clinical knowledge of lesion roughness and could be useful for optical cancer detection.
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Carcinoma Basocelular , Carcinoma de Células Escamosas , Ceratose Actínica , Melanoma , Nevo , Dermatopatias , Neoplasias Cutâneas , Humanos , Neoplasias Cutâneas/patologia , Carcinoma Basocelular/diagnóstico por imagem , Carcinoma Basocelular/patologia , Melanoma/diagnóstico por imagem , Melanoma/patologia , Carcinoma de Células Escamosas/diagnóstico por imagemRESUMO
BACKGROUND: The treatment of vitiligo can be challenging and depends on several factors such as the subtype, disease activity, vitiligo extent, and treatment goals. Vitiligo usually requires a long-term approach. To improve the management of vitiligo worldwide, a clear and up-to-date guide based on international consensus with uniform stepwise recommendations is needed. OBJECTIVES: To reach an international consensus on the nomenclature and to develop a management algorithm for the diagnosis, assessment, and treatment of vitiligo. METHODS: In this consensus statement, a consortium of 42 international vitiligo experts and four patient representatives participated in online and live meetings to develop a consensus management strategy for vitiligo. At least two vitiligo experts summarized the evidence of topics included in the algorithms. A survey was utilized to resolve remaining issues among a core group of eight experts. Subsequently, the unanimous recommendations were finalized and validated based on further input from the entire group during two live meetings. RESULTS: The algorithms highlight the importance of shared decision-making. Dermatologists are encouraged to provide patients with detailed explanations of the prognosis and expected therapeutic outcomes based on clinical examination. The treatment goal should be discussed and clearly emphasized to patients given the different approaches for disease stabilization and repigmentation. The evaluation of disease activity remains a cornerstone in the tailor-made approach to vitiligo patients. CONCLUSIONS: These new treatment algorithms are intended to guide clinical decision-making in clinical practice. Promising novel therapies for vitiligo are on the horizon, further highlighting the need for reliable outcome measurement instruments and greater emphasis on shared decision-making.
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Vitiligo , Humanos , Vitiligo/diagnóstico , Vitiligo/terapia , Consenso , Algoritmos , Tomada de Decisão Clínica , Inquéritos e QuestionáriosRESUMO
BACKGROUND: The treatment of vitiligo can be challenging. Up-to-date agreed consensus recommendations on the use of topical and systemic therapies to facilitate the clinical management of vitiligo are currently lacking. OBJECTIVES: To develop internationally agreed-upon expert-based recommendations for the treatment of vitiligo. METHODS: In this consensus statement, a consortium of 42 international vitiligo experts and four patient representatives participated in different online and live meetings to develop a consensus management strategy for vitiligo. At least two vitiligo experts summarized the evidence for different topics included in the algorithms. A survey was then given to a core group of eight experts to resolve the remaining issues. Subsequently, the recommendations were finalized and validated based on further input from the entire group during two live meetings. RESULTS: The recommendations provided summarize the latest evidence regarding the use of topical therapies (steroids, calcineurin inhibitors and Jak-inhibitors) and systemic therapies, including steroids and other systemic immunomodulating or antioxidant agents. The different modalities of phototherapies (NB-UVB, photochemotherapy, excimer devices and home phototherapy), which are often combined with other therapies, are also summarized. Interventional approaches as well as depigmentation strategies are presented for specific indications. Finally, the status of innovative and targeted therapies under development is discussed. CONCLUSIONS: This international consensus statement culminated in expert-based clinical practice recommendations for the treatment of vitiligo. The development of new therapies is ongoing in vitiligo, and this will likely improve the future management of vitiligo, a disease that still has many unmet needs.
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Fotoquimioterapia , Terapia Ultravioleta , Vitiligo , Humanos , Vitiligo/terapia , Vitiligo/tratamento farmacológico , Fototerapia , Esteroides/uso terapêutico , Resultado do Tratamento , Terapia CombinadaRESUMO
BACKGROUND: Biases inherent in self-assessment of sun exposure and sun-safe behavior may lead to inaccurate conclusions about the effectiveness of sun-safety educational programs. OBJECTIVES: We aimed to compare self-reports to objective measures of sun exposure, when examining the effectiveness of passive versus active educational interventions. METHODS: From May to June 2018, 73 participants recruited at a dermatology clinic were sequentially assigned to receive sun-safety education through one of 3 modes: interactive online module, video, or no education. A baseline Sun Exposure and Behavior Inventory (SEBI) questionnaire was administered, and spectrophotometric measurements of sun-exposed and sun-protected areas were taken and reported in the CIE L*a*b* color space. Participants were followed 4-8 and 16 weeks after the initial visit where the SEBI was re-administered, and serial measurements of skin color were taken. The change in SEBI scores and L*a*b values, as calculated by the individual typology angle (ITA°), was analyzed. RESULTS: There was a significant increase in skin darkening in all the groups at 4-8 and 16 weeks follow-up. There was no statistically significant difference between the groups in the magnitude of color change. However, subjectively at 4-8 weeks post-intervention, participants in the interactive module and video groups had significantly improved self-reported SEBI scores compared to control (p < .05, Kruskal-Wallis). By 16 weeks, only the interactive module group showed significant improvement in SEBI scores compared to control (p < .05, ANOVA). CONCLUSION: In determining the effectiveness of sun-safety programs, spectrophotometric evaluation of sun-induced skin pigmentation can allow for a more complete evaluation of self-reported sun exposure and sun-protective behavior.
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Comportamentos Relacionados com a Saúde , Neoplasias Cutâneas , Escolaridade , Conhecimentos, Atitudes e Prática em Saúde , Humanos , Neoplasias Cutâneas/tratamento farmacológico , Pigmentação da Pele , Protetores Solares/uso terapêutico , Inquéritos e QuestionáriosRESUMO
BACKGROUND: Although many hair disorders can be readily diagnosed based on their clinical appearance, their progression and response to treatment are often difficult to monitor, particularly in quantitative terms. We introduce an innovative technique utilizing a smartphone and computerized image analysis to expeditiously and automatically measure and compute hair density and diameter in patients in real time. METHODS: A smartphone equipped with a dermatoscope lens wirelessly transmits trichoscopy images to a computer for image processing. A black-and-white binary mask image representing hair and skin is produced, and the hairs are thinned into single-pixel-thick fiber skeletons. Further analysis based on these fibers allows morphometric characteristics such as hair shaft number and diameters to be computed rapidly. The hair-bearing scalps of fifty participants were imaged to assess the precision of our automated smartphone-based device in comparison with a specialized trichometry device for hair shaft density and diameter measurement. The precision and operation time of our technique relative to manual trichometry, which is commonly used by hair disorder specialists, is determined. RESULTS: An equivalence test, based on two 1-sided t tests, demonstrates statistical equivalence in hair density and diameter values between this automated technique and manual trichometry within a 20% margin. On average, this technique actively required 24 seconds of the clinician's time whereas manual trichometry necessitated 9.2 minutes. CONCLUSION: Automated smartphone-based trichometry is a rapid, precise, and clinically feasible technique which can significantly facilitate the assessment and monitoring of hair loss. Its use could be easily integrated into clinical practice to improve standard trichoscopy.
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Doenças do Cabelo , Smartphone , Alopecia , Cabelo , Humanos , Couro CabeludoRESUMO
The coronavirus disease 2019 (COVID-19) pandemic and subsequent physical distancing recommendations created major gaps in traditional dermatologic undergraduate and postgraduate medical education delivery. Nevertheless, the educational consequences of various public health restrictions have indirectly set aside the inertia, resistance, and risk averse approach to pedagogical change in medicine. In Canada, rapid collaboration and innovation in dermatologic education has led to novel programs including the implementation of a range of internet-facilitated group learning activities and a dramatic expansion of digital telehealth and virtual care. Going forward, three key issues arising from these developments will need to be addressed: the ongoing assessment of these innovations for efficacy; sustaining the momentum and creativity that has been achieved; and, determining which of these activities are worth maintaining when traditional "tried and true" learning activities can be resumed.
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COVID-19/prevenção & controle , Estágio Clínico/métodos , Dermatologia/educação , Internato e Residência/métodos , Canadá , Humanos , Internet , SARS-CoV-2 , Estudantes de Medicina , Ensino , TelemedicinaRESUMO
BACKGROUND: Clinical photography is an important component of the initial assessment and follow-up of patients with vitiligo in clinical practice and research settings. Standardization of this photographic process is essential to achieve useful, high-quality, and comparable photographs over time. OBJECTIVE: The aim is to develop an international consensus for a core set of recommendations for standardized vitiligo clinical photography. METHODS: Based an international meeting of vitiligo experts, a standard operating procedure was developed for vitiligo photography in daily practice and research settings. This protocol was subsequently reviewed by 20 vitiligo experts until agreement was reached. RESULTS: The resulting protocol includes a set of 10 and 15 photographs for clinical practice and research purposes, respectively. The photographic series are based on anatomic units included in the Vitiligo Extent Score. Furthermore, graphic representations of standardized positioning and suggestions for guidelines to standardize the process (background color, lighting, position marking, scales, materials, instruments) for both color and ultraviolet photographs are described. CONCLUSIONS: This consensus-based protocol for vitiligo photography will harmonize imaging for both clinical practice, translational research, and clinical trials. It can improve outcome assessment, foster multicenter collaboration, and promote better communication with patients regarding outcomes of treatment.
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Dermatologia/normas , Fotografação/normas , Guias de Prática Clínica como Assunto , Pele/diagnóstico por imagem , Vitiligo/diagnóstico , Ensaios Clínicos como Assunto/normas , Consenso , Dermatologia/métodos , Humanos , Cooperação Internacional , Iluminação/normas , Avaliação de Resultados em Cuidados de Saúde/métodos , Avaliação de Resultados em Cuidados de Saúde/normas , Padrões de Referência , Reprodutibilidade dos Testes , Índice de Gravidade de Doença , Pesquisa Translacional Biomédica/métodos , Pesquisa Translacional Biomédica/normas , Raios Ultravioleta , Vitiligo/terapiaRESUMO
BACKGROUND: Studies have shown disparities in the perception of skin disease burden between patients and physicians, with patients often feeling that the severity, emotional impact, and social repercussions of their skin condition are underestimated. Although physician's professional behavior is guided by documents such as the Hippocratic Oath, there are no patient-driven principles to guide healthcare interactions involving skin concerns. OBJECTIVE: To develop a concise and practical charter for patients based on their perceptions of unmet needs with the goals of helping patients express their needs and exercise their rights to accessing and utilizing the healthcare system for conditions, diseases, or traumas involving the skin. METHODS: An initial literature review examined healthcare delivery concerns of patients with skin conditions. Results were used to draft a charter that was reviewed by a Canadian patient focus group representing various skin condition advocacy groups. A revised charter was reviewed by Canadian dermatologists before being formally approved by the Canadian Skin Patient Alliance Board and endorsed by the Canadian Dermatology Association. RESULTS: The Patient Charter comprises 8 principles for providing and receiving professional services for the skin in the healthcare setting. CONCLUSIONS: This Patient Charter provides direct insights into patient priorities and will be used as an educational and advocacy tool in healthcare, occupational, and social settings. The intended goal is for the Patient Charter to empower patients and to educate health professions, government, industry, and society at large. Accordingly, the charter will be disseminated through print materials, informational videos, and social media campaigns.
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Atenção à Saúde/normas , Dermatologia/normas , Defesa do Paciente/normas , Medidas de Resultados Relatados pelo Paciente , Dermatopatias , Pele/lesões , Canadá , Efeitos Psicossociais da Doença , Necessidades e Demandas de Serviços de Saúde/normas , Humanos , Participação do Paciente/métodos , Direitos do Paciente/normasRESUMO
Raman spectroscopy provides molecular finger-printing of biological tissues. To achieve high spatial resolution, confocal Raman spectroscopy (micro-Raman) has been developed. To guide where micro-Raman is acquired, imaging guidance is necessary, especially for high scattering biological tissue. Reflectance confocal microscopy (RCM) has been integrated with micro-Raman. However, existing systems do not allow point-of-interest micro-Raman measurement with simultaneous RCM guidance. Here we demonstrate how a single laser can be used to localize and acquire micro-Raman signals within tissue with simultaneous real-time RCM imaging. Applications of this RCM-guided micro-Raman system for ex vivo samples and in vivo skin measurements are presented.
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BACKGROUND AND PURPOSE: Although cutaneous autofluorescence has been utilized for evaluation of skin conditions, there is a paucity of data on normal human skin autofluorescence and its dependence on anatomical site. The objective of this study is to use excitation-emission matrix spectroscopy to quantify and characterize skin autofluorescence at different body sites. METHODS: Ten anatomical sites from 30 healthy volunteers were measured with a double-grating excitation-emission matrix spectrofluorometer. RESULTS: For the 10 body sites evaluated, there were four overall patterns of autofluorescence: skin from the head and neck exhibits high superficial and low bilayer fluorescence; the dorsal forearm and dorsal hand have both low superficial and bilayer fluorescence; the upper inner arm and back have high superficial and intermediate bilayer fluorescence; while the palm and thumbnail have both high superficial and bilayer fluorescence. The corresponding fluorescence excitation-emission peaks for these patterns were as follows: head and neck, 3 peaks at 290-300/330-350, 360-380/460-485, and 380-420/610-630 nm; dorsal forearm and dorsal hand, 2 peaks around 295-300/345-360 and 385-395/460-485 nm; upper inner arm and back, 3 peaks around 295-300/335-355, 335-340/390-410, and 375-390/455-480 nm; palm and thumbnail, 3 peaks around 285-300/345-355, 335-345/390-410, and 365-390/450-480 nm. CONCLUSION: Cutaneous fluorescence varies in distinct patterns according to anatomical site, due to the component fluorophores present, skin thickness, and the degree of melanization and long term sun exposure. These EEM patterns for normal skin should be accounted for when interpreting fluorescence signals from disease states and can also be used to guide the selection of optimal wavebands when applying this optical modality.
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Fluorescência , Fenômenos Fisiológicos da Pele , Adulto , Idoso , Braço , Dorso , Feminino , Mãos , Cabeça , Humanos , Masculino , Pessoa de Meia-Idade , Unhas , Pescoço , Espectrometria de Fluorescência , Adulto JovemRESUMO
BACKGROUND: There is no accepted method to objectively assess the visual appearance of sunscreens on the skin. METHODS: We present a method for sunscreen application, digital photography, and computer analysis to quantify the appearance of the skin after sunscreen application. Four sunscreen lotions were applied randomly at densities of 0.5, 1.0, 1.5, and 2.0 mg/cm2 to areas of the back of 29 subjects. Each application site had a matched contralateral control area. High-resolution standardized photographs including a color card were taken after sunscreen application. After color balance correction, CIE L*a*b* color values were extracted from paired sites. Differences in skin appearance attributed to sunscreen were represented by ΔE, which in turn was calculated from the linear Euclidean distance within the L*a*b* color space between the paired sites. RESULTS: Sunscreen visibility as measured by median ΔE varied across different products and application densities and ranged between 1.2 and 12.1. The visibility of sunscreens varied according to product SPF, composition (organic vs inorganic), presence of tint, and baseline b* of skin (P < .05 for all). CONCLUSION: Standardized sunscreen application followed by digital photography and indirect computer-based colorimetry represents a potential method to objectively quantify visibility of sunscreen on the skin.
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Processamento de Imagem Assistida por Computador , Pigmentação da Pele/efeitos dos fármacos , Pele , Protetores Solares/administração & dosagem , Adulto , Idoso , Colorimetria , Feminino , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
Vascular structures of skin are important biomarkers in diagnosis and assessment of cutaneous conditions. Presence and distribution of lesional vessels are associated with specific abnormalities. Therefore, detection and localization of cutaneous vessels provide critical information towards diagnosis and stage status of diseases. However, cutaneous vessels are highly variable in shape, size, color and architecture, which complicate the detection task. Considering the large variability of these structures, conventional vessel detection techniques lack the generalizability to detect different vessel types and require separate algorithms to be designed for each type. Furthermore, such techniques are highly dependent on precise hand-crafted features which are time-consuming and computationally inefficient. As a solution, we propose a data-driven feature learning framework based on stacked sparse auto-encoders (SSAE) for comprehensive detection of cutaneous vessels. Each training image is divided into small patches of either containing or non-containing vasculature. A multilayer SSAE is designed to learn hidden features of the data in hierarchical layers in an unsupervised manner. The high-level learned features are subsequently fed into a classifier which categorizes each patch into absence or presence of vasculature and localizes vessels within the lesion. Over a test set of 3095 patches derived from 200 images, the proposed framework demonstrated superior performance of 95.4% detection accuracy over a variety of vessel patterns; outperforming other techniques by achieving the highest positive predictive value of 94.7%. The proposed Computer-Aided Diagnosis (CAD) framework can serve as a decision support system assisting dermatologists for more accurate diagnosis, especially in teledermatology applications in remote areas.
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Dermoscopia/métodos , Diagnóstico por Computador/métodos , Reconhecimento Automatizado de Padrão/métodos , Pele/irrigação sanguínea , Pele/diagnóstico por imagem , Biomarcadores , Humanos , Interpretação de Imagem Assistida por Computador/métodos , Aprendizado de MáquinaRESUMO
Real-time Raman spectroscopy can be used to assist in assessing skin lesions suspicious for cancer. Most of the diagnostic algorithms are based on full band of the Raman spectra, either in the fingerprint region or the high wavenumber region. In this paper we explored wavenumber selection based analysis in Raman spectroscopy for skin cancer diagnosis. Wavenumber selection was implemented using windows of wavenumber and leave-one-out cross-validated stepwise regression or least and shrinkage selection operator (LASSO). The diagnostic algorithms were then generated from the selected windows of wavenumber using multivariate statistical analyses, including principal component and general discriminate analysis (PC-GDA) and partial least squares (PLS). In total a combined cohort of 645 confirmed lesions from 573 patients encompassing skin cancers, precancers and benign skin lesions were included, which were divided into training cohort (n = 518) and testing cohort (n = 127) according to the measurement time. It was found that the area under the receiver operating characteristic curve (ROC) was improved from 0.861-0.891 to 0.891-0.911 and the diagnostic specificity for fixed sensitivity 0.99-0.90 was improved from 0.17-0.65 to 0.20-0.75 with wavenumber selection based analysis.
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Algoritmos , Neoplasias Cutâneas/diagnóstico , Análise Espectral Raman/métodos , Estatística como Assunto/métodos , Análise Discriminante , Humanos , Análise de Regressão , Sensibilidade e Especificidade , Fatores de TempoRESUMO
In a recent study, we have demonstrated that real-time Raman spectroscopy could be used for skin cancer diagnosis. As a translational study, the objective of this study is to validate previous findings through a completely independent clinical test. In total, 645 confirmed cases were included in the analysis, including a cohort of 518 cases from a previous study, and an independent cohort of 127 new cases. Multi-variant statistical data analyses including principal component with general discriminant analysis (PC-GDA) and partial least squares (PLS) were used separately for lesion classification, which generated similar results. When the previous cohort (n = 518) was used as training and the new cohort (n = 127) was used as testing, the area under the receiver operating characteristic curve (ROC AUC) was found to be 0.889 (95 % CI 0.834-0.944; PLS); when the two cohorts were combined, the ROC AUC was 0.894 (95 % CI 0.870-0.918; PLS) with the narrowest confidence intervals. Both analyses were comparable to the previous findings, where the ROC AUC was 0.896 (95 % CI 0.846-0.946; PLS). The independent study validates that real-time Raman spectroscopy could be used for automatic in vivo skin cancer diagnosis with good accuracy.
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Neoplasias Cutâneas/diagnóstico , Pele/patologia , Análise Espectral Raman/métodos , Análise Discriminante , Humanos , Análise dos Mínimos Quadrados , Análise de Componente Principal , Neoplasias Cutâneas/classificaçãoRESUMO
BACKGROUND: Recent advances in biomedical optics have enabled dermal and epidermal components to be visualized at subcellular resolution and assessed noninvasively. Multiphoton microscopy (MPM) and reflectance confocal microscopy (RCM) are noninvasive imaging modalities that have demonstrated promising results in imaging skin micromorphology, and which provide complementary information regarding skin components. This study assesses whether combined MPM/RCM can visualize intracellular and extracellular melanin granules in the epidermis and dermis of normal human skin. METHODS: We perform MPM and RCM imaging of in vivo and ex vivo skin in the infrared domain. The inherent three-dimensional optical sectioning capability of MPM/RCM is used to image high-contrast granular features across skin depths ranging from 50 to 90 µm. The optical images thus obtained were correlated with conventional histologic examination including melanin-specific staining of ex vivo specimens. RESULTS: MPM revealed highly fluorescent granular structures below the dermal-epidermal junction (DEJ) region. Histochemical staining also demonstrated melanin-containing granules that correlate well in size and location with the granular fluorescent structures observed in MPM. Furthermore, the MPM fluorescence excitation wavelength and RCM reflectance of cell culture-derived melanin were equivalent to those of the granules. CONCLUSION: This study suggests that MPM can noninvasively visualize and quantify subepidermal melanin in situ.
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Grânulos Citoplasmáticos/metabolismo , Melaninas/metabolismo , Pele/citologia , Pele/metabolismo , Feminino , Humanos , Masculino , Microscopia ConfocalRESUMO
Fiber delivery of ultrashort pulses is important for multiphoton endoscopy. A chirped photonic crystal fiber (CPCF) is first characterized for its transmission bandwidth, propagation loss, and dispersion properties. Its extremely low dispersion (~150 fs(2)/m) enables the delivery of sub-30 fs pulses through a ~1 m-long CPCF. The CPCF is then incorporated into a multiphoton imaging system and its performance is demonstrated by imaging various biological samples including yew leaf, mouse tendon, and human skin. The imaging quality is further compared with images acquired by a multiphoton imaging system with free-space or hollow-core photonic band-gap fiber (PBF) delivery of pulses. Compared with free-space system, the CPCF delivered system maintains the same ultrashort pulsewidth and the image qualities are comparable. Compared with the PBF delivery, CPCF provides a 35 times shorter pulsewidth at the sample location, which results in a ~12 and 50 times improvement in two-photon excitation fluorescence (TPEF) and second harmonic generation (SHG) signals respectively. Our results show that CPCF has great potential for fiber delivery of ultrashort pulses for multiphoton endoscopy.
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Stem cells offer tremendous opportunities for regenerative medicine. Over the past decade considerable research has taken place to identify and characterize the differentiation states of stem cells in culture. Raman micro-spectroscopy has emerged as an ideal technology since it is fast, nondestructive, and does not require potentially toxic dyes. Raman spectroscopy systems can also be incorporated into confocal microscope imaging systems allowing spectra to be obtained from below the tissue surface. Thus there is significant potential for monitoring stem cells in living tissue. Stem cells that reside in hair follicles are suitable for testing this possibility since they are close to the skin surface, and typically clustered around the bulge area. One of the first steps needed would be to obtain Raman micro-spectra from stem cells located in thin sections of tissue, and then see whether these spectra are clearly different from those of the surrounding differentiated cells. To facilitate this test, standard 5 µm thick sections of murine skin tissue were stained to identify the location of hair follicle stem cells and their progeny. Raman spectra were then obtained from adjacent cells in a subsequent unstained 10 µm thick section. The spectra revealed significant differences in peak intensities associated with nucleic acids, proteins, lipids and amino acids. Statistical analyses of the Raman micro-spectra identified stem cells with 98% sensitivity and 94% specificity, as compared with a CD34 immunostaining gold standard. Furthermore analyses of the spectral variance indicated differences in cellular dynamics between the two cell groups. This study shows that Raman micro-spectroscopy has a potential role in identifying adult follicle stem cells, laying the groundwork for future applications of hair follicle stem cells and other somatic stem cells in situ.