SignificanceAccurate identification of epidermal cells on reflectance confocal microscopy (RCM) images is important in the study of epidermal architecture and topology of both healthy and diseased skin. However, analysis of these images is currently done manually and therefore time-consuming and subject to human error and inter-expert interpretation. It is also hindered by low image quality due to noise and heterogeneity.AimWe aimed to design an automated pipeline for the analysis of the epidermal structure from RCM images.ApproachTwo attempts have been made at automatically localizing epidermal cells, called keratinocytes, on RCM images: the first is based on a rotationally symmetric error function mask, and the second on cell morphological features. Here, we propose a dual-task network to automatically identify keratinocytes on RCM images. Each task consists of a cycle generative adversarial network. The first task aims to translate real RCM images into binary images, thus learning the noise and texture model of RCM images, whereas the second task maps Gabor-filtered RCM images into binary images, learning the epidermal structure visible on RCM images. The combination of the two tasks allows one task to constrict the solution space of the other, thus improving overall results. We refine our cell identification by applying the pre-trained StarDist algorithm to detect star-convex shapes, thus closing any incomplete membranes and separating neighboring cells.ResultsThe results are evaluated both on simulated data and manually annotated real RCM data. Accuracy is measured using recall and precision metrics, which is summarized as the F1-score.ConclusionsWe demonstrate that the proposed fully unsupervised method successfully identifies keratinocytes on RCM images of the epidermis, with an accuracy on par with experts’ cell identification, is not constrained by limited available annotated data, and can be extended to images acquired using various imaging techniques without retraining.
SignificanceReflectance 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.AimWe 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.ApproachWe 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).ResultsAll 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 (precision = 0.720 ± 0.068, recall = 0.850 ± 0.11) and synthetic images (precision = 0.835 ± 0.067, recall = 0.925 ± 0.012). The tested machine learning methods failed to identify and segment keratinocytes on RCM images with a satisfactory accuracy.ConclusionsWe 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.
Significance: Reflectance confocal microscopy (RCM) is a noninvasive, in vivo technology that offers near histopathological resolution at the cellular level. It is useful in the study of phenomena for which obtaining a biopsy is impractical or would cause unnecessary tissue damage and trauma to the patient.Aim: This review covers the use of RCM in the study of skin and the use of machine learning to automate information extraction. It has two goals: (1) an overview of information provided by RCM on skin structure and how it changes over time in response to stimuli and in disease and (2) an overview of machine learning approaches developed to automate the extraction of key morphological features from RCM images.Approach: A PubMed search was conducted with additional literature obtained from references lists.Results: The application of RCM as an in vivo tool in dermatological research and the biologically relevant information derived from it are presented. Algorithms for image classification to epidermal layers, delineation of the dermal–epidermal junction, classification of skin lesions, and demarcation of individual cells within an image, all important factors in the makeup of the skin barrier, were reviewed. Application of image analysis methods in RCM is hindered by low image quality due to noise and/or poor contrast. Use of supervised machine learning is limited by time-consuming manual labeling of RCM images.Conclusions: RCM has great potential in the study of skin structures. The use of artificial intelligence could enable an easier, more reproducible, precise, and rigorous study of RCM images for the understanding of skin structures, skin barrier, and skin inflammation and lesions. Although several attempts have been made, further work is still needed to provide a definite gold standard and overcome issues related to image quality, limited labeled datasets, and lack of phenotype variability in available databases.
Reflectance confocal microscopy (RCM) allows real-time in vivo visualization of the skin at cellular level. The study of RCM images provides information on the topological and geometrical 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 propose an automated pipeline to analyze the structure of the skin in RCM images. The first step is to identify the region of interest (ROI) containing the epidermal cells. The second step is to identify individual cells in the segmented tissue area using an image filter. We then use prior biological knowledge to process the resulting detected cells, removing cells that are too small and reapplying the used filter locally on detected regions that are too big to be considered as a single cell. The results are evaluated both on simulated data and on manually annotated real RCM data. This study shows that automatic cell identification can be achieved, with an accuracy (precision and recall) that matches the inter-expert variability.
Two-photon fluorescence (TPF) and second harmonic generation (SHG) microscopy provide direct visualization of the skin dermal fibers in vivo. A typical method for analyzing TPF/SHG images involves averaging the image intensity and therefore disregarding the spatial distribution information. The goal of this study is to develop an algorithm to document age-related effects of the dermal matrix. TPF and SHG images were acquired from the upper inner arm, volar forearm, and cheek of female volunteers of two age groups: 20 to 30 and 60 to 80 years of age. The acquired images were analyzed for parameters relating to collagen and elastin fiber features, such as orientation and density. Both collagen and elastin fibers showed higher anisotropy in fiber orientation for the older group. The greatest difference in elastin fiber anisotropy between the two groups was found for the upper inner arm site. Elastin fiber density increased with age, whereas collagen fiber density decreased with age. The proposed analysis considers the spatial information inherent to the TPF and SHG images and provides additional insights into how the dermal fiber structure is affected by the aging process.
Reflectance confocal microscopy is successfully used in infant skin research. Infant skin structure, function, and composition are undergoing a maturation process. We aimed to uncover how the epidermal architecture and cellular topology change with time. Images were collected from three age groups of healthy infants between one and four years of age and adults. Cell centers were manually identified on the images at the stratum granulosum (SG) and stratum spinosum (SS) levels. Voronoi diagrams were used to calculate geometrical and topological parameters. Infant cell density is higher than that of adults and decreases with age. Projected cell area, cell perimeter, and average distance to the nearest neighbors increase with age but do so distinctly between the two layers. Structural entropy is different between the two strata, but remains constant with time. For all ages and layers, the distribution of the number of nearest neighbors is typical of a cooperator network architecture. The topological analysis provides evidence of the maturation process in infant skin. The differences between infant and adult are more pronounced in the SG than SS, while cell cooperation is evident in all cases of healthy skin examined.
Pharmaceutical and cosmetic industries are concerned with treating skin disease, as well as maintaining and promoting
skin health. They are dealing with a unique tissue that defines our body in space. As such, skin provides not only the
natural boundary with the environment inhibiting body dehydration as well as penetration of exogenous aggressors to the
body, it is also ideally situated for optical measurements. A plurality of spectroscopic and imaging methods is being
used to understand skin physiology and pathology and document the effects of topically applied products on the skin.
The obvious advantage of such methods over traditional biopsy techniques is the ability to measure the cutaneous tissue
in vivo and non-invasively. In this work, we will review such applications of various spectroscopy and imaging methods
in skin research that is of interest the cosmetic and pharmaceutical industry. Examples will be given on the importance
of optical techniques in acquiring new insights about acne pathogenesis and infant skin development.
Typical manifestations of cutaneous inflammation include erythema and edema. While erythema is the result of capillary dilation and local increase of oxygenated hemoglobin concentration, edema is characterized by an increase in extracellular fluid in the dermis, leading to local tissue swelling. Both of these inflammatory reactions are typically graded visually. We demonstrate the potential of spectral imaging as an objective noninvasive method for quantitative documentation of both erythema and edema. As examples of dermatological conditions that exhibit skin inflammation we applied this method on patients suffering from (1) allergic dermatitis (poison ivy rashes), (2) inflammatory acne, and (3) viral infection (herpes zoster). Spectral images are acquired in the visible and near-IR part of the spectrum. Based on a spectral decomposition algorithm, apparent concentrations maps are constructed for oxyhemoglobin, deoxyhemoglobin, melanin, optical scattering, and water. In each dermatological condition examined, the concentration maps of oxyhemoglobin and water represent quantitative visualizations of the intensity and extent of erythema and cutaneous edema, correspondingly. We demonstrate that spectral imaging can be used to quantitatively document parameters relevant to skin inflammation. Applications may include monitoring of disease progression as well as screening for efficacy of treatments.
A rotationally invariant algorithm was developed to evaluate the orientation direction and orientation coherence of
features in a two-dimensional image. The algorithm was validated on test images and was applied on in vivo confocal
microscopy images to extract information on collagen matrix orientation and on skin microrelief images for the
calculation of the primary direction of microglyphics.
Skin inflammation is often accompanied by edema and erythema. While erythema is the result of capillary dilation and subsequent local increase of oxygenated hemoglobin (oxy-Hb) concentration, edema is characterized by an increase in extracellular fluid in the dermis leading to local tissue swelling. Edema and erythema are typically graded visually. In this work we tested the potential of spectral imaging as a non-invasive method for quantitative documentation of both the erythema and the edema reactions. As examples of dermatological conditions that exhibit skin inflammation we imaged patients suffering from acne, herpes zoster, and poison ivy rashes using a hyperspectral-imaging camera. Spectral images were acquired in the visible and near infrared part of the spectrum, where oxy-Hb and water demonstrate absorption bands. The values of apparent concentrations of oxy-Hb and water were calculated based on an algorithm that takes into account spectral contributions of deoxy-hemoglobin, melanin, and scattering. In each case examined concentration maps of oxy-Hb and water can be constructed that represent quantitative visualizations of the intensity and extent of erythema and edema correspondingly. In summary, we demonstrate that spectral imaging can be used in dermatology to quantitatively document parameters relating to skin inflammation. Applications may include monitoring of disease progression as well as efficacy of treatments.
Hyperspectral imaging of skin combines the spectral information of diffuse reflectance spectroscopy with the spatial information of 2D imaging. Skin chromophore maps can be reconstructed in which features such as pigmented lesions, diffuse and localized erythema, areas of increased blood stasis, etc. could be identified and the relative
parameters quantified. Hyperspectral imaging is the only reliable method to produce a quantitative distribution map of chromophores contributing to the color appearance of the skin.
Skin reactions to stimuli such as UV irradiation include vascular changes and stimulation of melanin production. Both reactions alter the color appearance of the skin. Skin color reactions were evaluated visually by a trained dermatologist and using a diffuse reflectance spectrometer in the visible range. Our results provide strong evidence that mixed vascular and pigment reactions cannot be visually separated. The involvement of each chromophore can only be identified spectroscopically.
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