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Background and significanceThere is a need to develop non-invasive diagnostic tools to achieve early and accurate detection of skin cancer in a non-surgical manner. In this study, we evaluate the capability of stimulated Raman scattering (SRS) microscopy, a potentially non-invasive optical imaging technique, for identifying the pathological features of s squamous cell carcinoma (SCC) tissue.Study designWe studied ex vivo SCC and healthy skin tissues using SRS microscopy, and compared the SRS contrast with the contrast obtained in reflectance confocal microscopy (RCM) and standard histology.Results and conclusionSRS images obtained at the carbon-hydrogen stretching vibration at 2945 cm(-1) exhibit contrast related protein density that clearly delineates the cell nucleus from the cell cytoplasm. The morphological features of SCC tumor seen in the SRS images show excellent correlation with the diagnostic features identified by histological examination. Additionally, SRS exhibits enhanced cellular contrast in comparison to that seen in confocal microscopy. In conclusion, SRS represents an attractive approach for generating protein density maps with contrast that closely resembles histopathological contrast of SCC in human skin.

Collagen type I is a material widely used for 3D cell culture and tissue engineering. Different architectures, such as gels, sponges, membranes, and nanofibers, can be fabricated with it. In collagen hydrogels, the formation of fibrils and fibers depends on various parameters, such as the source of collagen, pH, temperature, concentration, age, etc. In this work, we study the fibrillogenesis process in collagen type I hydrogels with different types of microbeads embedded, using optical techniques such as turbidity assay and confocal reflectance microscopy. We observe that microbeads embedded in the collagen matrix hydrogels modify the fibrillogenesis. Our results show that carboxylated fluorescent microbeads accelerate 3.6 times the gelation, while silica microbeads slow down the formation of collagen fibrils by a factor of 1.9, both compared to pure collagen hydrogels. Our observations suggest that carboxylate microbeads act as nucleation sites and the early collagen fibrils bind to the microbeads., (Creative Commons Attribution license.)

Cutaneous melanoma (CM) is potentially the most dangerous form of skin tumor and causes 90% of skin cancer mortality. A unique collaboration of multidisciplinary experts from the European Dermatology Forum (EDF), the European Association of Dermato-Oncology (EADO), and the European Organization of Research and Treatment of Cancer (EORTC) was formed to make recommendations on CM diagnosis and treatment, based on systematic literature reviews and the experts' experience. The diagnosis of melanoma can be made clinically and shall always be confirmed through dermatoscopy. If a melanoma is suspected, a histopathological examination is required. Sequential digital dermatoscopy and full-body photography can be used in risk persons to detect the development of melanomas at an earlier stage. Where available, confocal reflectance microscopy can improve clinical diagnosis in special cases. Melanoma shall be classified according to the 8th version of the AJCC classification. Thin melanomas up to 0.8 mm tumor thickness does not require further imaging diagnostics. From stage IB onwards, examinations with lymph node sonography are recommended, but no further imaging examinations. From stage IIC whole-body examinations with CT or PET-CT in combination with brain MRI are recommended. From stage III and higher, mutation testing is recommended, particularly for BRAF V600 mutation. It is important to provide a structured follow-up to detect relapses and secondary primary melanomas as early as possible. There is no evidence to support the frequency and extent of examinations. A stage-based follow-up scheme is proposed, which, according to the experience of the guideline group, covers the minimum requirements; further studies may be considered. This guideline is valid until the end of 2021. [ABSTRACT FROM AUTHOR]

We propose a compact laser feedback interferometry imaging system for concurrent reflectance confocal microscopy and laser Doppler flowmetry. This system acquires both confocal reflectance and Doppler signals in a confocal architecture to image dynamic turbid media with higher contrast than a system operating in either modality and is coherent in nature. In a confocal optical configuration, reflectance confocal microscopy provides information about scattering from within a small volume centered around the focal point of the confocal system, and laser Doppler flowmetry provides information about the velocity of moving scatterers within the same volume. Raster scanning the sample enables the concurrent creation of two images, containing independent information, from a well-specified depth within the sample. Concurrent spatial mapping of these independent sensing modalities affords improvement in the capability of the imaging system by obtaining additional information from both morphological and functional features of the dynamic turbid medium at depths penetrable by near-infrared lasers. We realize the idea using a laser feedback interferometry imaging system scanning a microfluidic channel that contains a dynamic turbid medium. We show the effectiveness of this integrated imager quantitatively through the improvement of the signal-to-background ratio of a combined (multiplication) image.

Cancer cell metastasis is responsible for approximately 90% of deaths related to cancer. The migration of cancer cells away from the primary tumor and into healthy tissue is driven in part by contact guidance, or directed migration in response to aligned extracellular matrix. While contact guidance has been a focus of many studies, much of this research has explored environments that present 2D contact guidance structures. Contact guidance environments in 3D more closely resemble in vivo conditions and model cell-ECM interactions better than 2D environments. While most cells engage in directed migration on potent 2D contact guidance cues, there is diversity in response to contact guidance cues based on whether the cell migrates with a mesenchymal or amoeboid migration mode. In this paper, rotational alignment of collagen gels was used to study the differences in contact guidance between MDA-MB-231 (mesenchymal) and MTLn3 (amoeboid) cells. MDA-MB-231 cells migrate with high directional fidelity in aligned collagen gels, while MTLn3 cells show no directional migration. The collagen stiffness was increased through glycation, resulting in decreased MDA-MB-231 directionality in aligned collagen gels. Interestingly, partial inhibition of cell contractility dramatically decreased directionality in MDA-MB-231 cells. The directionality of MDA-MB-231 cells was most sensitive to ROCK inhibition, but unlike in 2D contact guidance environments, cell directionality and speed are more tightly coupled. Modulation of the contractile apparatus appears to more potently affect contact guidance than modulation of extracellular mechanical properties of the contact guidance cue. Statement of Significance Collagen fiber alignment in the tumor microenvironment directs migration, a process called contact guidance, enhancing the efficiency of cancer invasion and metastasis. 3D systems that assess contact guidance by locally orienting collagen fiber alignment are lacking. Furthermore, cell type differences and the role of extracellular matrix stiffness in tuning contact guidance fidelity are not well characterized. In this paper rotational alignment of collagen fibers is used as a 3D contact guidance cue to illuminate cell type differences and the role of extracellular matrix stiffness in guiding cell migration along aligned fibers of collagen. This local alignment offers a simple approach by which to couple collagen alignment with gradients in other directional cues in devices such as microfluidic chambers. [ABSTRACT FROM AUTHOR]

Oral cancer is among the most common malignancies worldwide, therefore early detection and treatment is imperative. The 5-year survival rate has remained at a dismal 50% for the past several decades. The main reason for the poor survival rate is the fact that most of the oral cancers, despite the general accessibility of the oral cavity, are not diagnosed until the advanced stage. Early detection of the oral tumors and its precursor lesions may be the most effective means to improve clinical outcome and cure most patients. One of the emerging technologies is the use of non-invasive in vivo tissue imaging to capture the molecular changes at high-resolution to improve the detection capability of early stage disease. This review will discuss the use of optical probes and highlight the role of optical imaging such as autofluorescence, fluorescence diagnosis (FD), laser confocal endomicroscopy (LCE), surface enhanced Raman spectroscopy (SERS), optical coherence tomography (OCT) and confocal reflectance microscopy (CRM) in early oral cancer detection. FD is a promising method to differentiate cancerous lesions from benign, thus helping in the determination of adequate resolution of surgical resection margin. LCE offers in vivo cellular imaging of tissue structures from surface to subsurface layers and has demonstrated the potential to be used as a minimally invasive optical biopsy technique for early diagnosis of oral cancer lesions. SERS was able to differentiate between normal and oral cancer patients based on the spectra acquired from saliva of patients. OCT has been used to visualize the detailed histological features of the oral lesions with an imaging depth down to 2–3 mm. CRM is an optical tool to noninvasively image tissue with near histological resolution. These comprehensive diagnostic modalities can also be used to define surgical margin and to provide a direct assessment of the therapeutic effectiveness.

Confocal microscopy is a technique able to realize 'optic sections' of a tissue with increasing applications. We wondered if we could apply an ex vivo confocal microscope designed for dermatological purpose in a routine use for the most frequent brain tumors. The aim of this work was to identify tumor tissue and its histopathological hallmarks, and to assess grading criteria used in neuropathological practice without tissue loss on freshly removed brain tissue. Seven infiltrating gliomas, nine meningiomas and three metastases of carcinomas were included. We compared imaging results obtained with the confocal microscope to frozen sections, smears and tissue sections of formalin-fixed tissue. Our results show that ex vivo confocal microscopy imaging can be applied to brain tumors in order to quickly identify tumor tissue without tissue loss. It can differentiate tumors and can assess most of grading criteria. Confocal microscopy could represent a new tool to identify tumor tissue on freshly removed sample and could help in selecting areas for biobanking of tumor tissue. [ABSTRACT FROM AUTHOR]

Background: Xeroderma pigmentosum (XP) is a genodermatosis caused by abnormal DNA repair. XP complementation group C (XPC) is the most frequent type in Mediterranean countries. We describe a case with a novel mutation in the XPC gene.Case: A healthy Caucasian male patient was diagnosed with multiple primary melanomas. Digital follow-up and molecular studies were carried out.Results: During digital follow-up 8 more additional melanomas were diagnosed. Molecular studies did not identify mutations in CDKN2A, CDK4 or MITF genes. Two heterozygous mutations in the XPC gene were detected: c.2287delC (p.Leu763Cysfs*4) frameshift and c.2212A>G (p.Thr738Ala) missense mutations.Conclusion: The p.Thr738Ala missense mutation has not been previously described. Missense mutations in the XPC gene may allow partial functionality that could explain this unusual late onset XP. Atypical clinical presentation of XPC could be misdiagnosed when genetic aberrations allow partial DNA repair capacity. [ABSTRACT FROM AUTHOR]

Autonomous microrobots are at the forefront of biomedical research as they are expected to be applied for specific tasks at the intracellular level such as cargo delivery, sensing, molecular manipulation, among others. Here, we report on a preparation of microrobots based on quinacridone and indigo, which are members of the organic hydrogen-bonded pigment family. The microrobots were fabricated by asymmetric platinum deposition on corresponding quinacridone and indigo microparticles that possessed a homogeneous size and shape distribution. The microrobots exhibited autonomous locomotion in the presence of hydrogen peroxide, which was further supported by UV irradiation. The organic pigment-based microrobots were studied in the presence of mouse colorectal carcinoma cells, and it was observed that they were internalized into the cells. Internalization was visualized using confocal laser scanning microscopy. This study reveals the possibility of fabricating hydrogen-bonded organic pigment-based microrobots for biomedical applications by employing the principles of nanoarchitectonics.

Oral cancer is one of the most common malignancies that affect human beings across the world, mainly because of the wide-spread consumption of tobacco and its products. Tobacco and its products have been implicated as predisposing factors in the etiology of oral cancer. The commonest type of oral cancer is oral squamous cell carcinoma. Early detection is very important in the management of oral cancer. However, when the lesion is detected in the oral cavity, it is very much advanced for effective management with minimum morbidity and mortality. It is clearly known that cancer and cancer treatment are always associated with considerable morbidity and mortality, especially because of the late detection. In order to improve the clinical outcome, early detection is very important. Though the oral cavity is easily accessible for direct visual examination, medical practitioners mostly neglect this area in the general examination of the patient. The general dental practitioners also do not give much importance to the oral mucosal changes while carrying out dental treatment. One of the emerging technologies in the early detection of oral cancer is the use of non-invasive in vivo tissue imaging that capture the molecular changes at high-resolution to improve the detection capability of early stage oral cancer. Various newer cancer detection methods currently available are the use of optical imaging such as autofluorescence, fluorescence diagnosis, laser confocal endomicroscopy, surface enhanced Raman spectroscopy, optical coherence tomography and confocal reflectance microscopy in the early oral cancer. Fluorescence diagnosis is emerging as a promising method in the differentiation of cancerous lesions from benign lesions, thus helping in the determination of resolution for the surgical resection of affected area of malignancy very accurately. Laser confocal endomicroscopy offers in vivo cellular imaging of tissue structures from surface to subsurface layers and can be used as a minimally invasive biopsy technique for the early detection of the lesion. Based on the pattern of the spectrum obtained from the saliva, the detection of the lesion is possible in surface enhanced Raman spectroscopy. Optical coherence tomography can be used in the imaging of histopathological features of oral lesions. The above mentioned techniques can help in defining the surgical margin and in the assessment of successful management of the lesion. The review process was carried out by literature search using appropriate key words for articles published between 1995 1nd 2015. Out of 4,46,000 articles obtained, 1480 articles were selected. In the final review process, 179 articles were selected. After subjecting the articles to systematic review, finally 19 articles were selected. The study designs in the articles were interventional experimental studies. [ABSTRACT FROM AUTHOR]

Background Stretch marks or striae distensae (SD) are characterized by epidermal atrophy following repeated over-stretching of the skin tissue. The objective of this study was to investigate the skin texture and microstructure of SD lesions compared to those of normal adjacent skin in vivo using non-invasive methods. Methods A population of 26 women and 3 men with SD were examined after giving written informed consent. Following clinical grading, skin replicas were collected, confocal microscopy was performed on SD lesions and healthy neighboring skin. Skin surface texture parameters were calculated using 3D image analysis of the skin replicas and epidermal and dermal microstructure were evaluated by analysis of the confocal images. In a parallel study, histological analysis was performed on 6 skin biopsies taken from abdominal reduction surgeries in areas where skin exhibited SD. Results Analysis of the skin surface texture showed that the SD area was more anisotropic and with higher skin roughness than the adjacent skin in terms of directions of skin microglyphics. Confocal microscopy demonstrated that SD were characterized by shallower dermal papillae ( P < 0.05) and that dermal papillae height inversely correlated to the intensity of collagen alignment on the SD sites ( P < 0.05). The histology findings confirmed the in vivo confocal microscopy findings. Conclusions The skin structure of SD is qualitatively and quantitatively different compared to healthy skin. Altered skin relief reflects structural modifications in the dermis. Flattening of the dermal-epidermal junction maybe functionally related to the observed collagen fiber alignment. Observations by non-invasive methods were in line with the histological findings and therefore relevant in studies assessing the efficacy of SD treatment options. [ABSTRACT FROM AUTHOR]

Rhythmical contraction of the heart is controlled by the cardiac conduction system (CCS) that consists of the three main parts: the sino-atrial node, the atrioventricular node and the His-Purkinje system. A heartbeat signal, originated from CCS, spreads through its branches to the different parts of the heart, initiating depolarization of the ventricles. However, this highly important system could not be distinguished visually from the surrounding heart tissues: myocardium (MC) and connective tissue (CT). Thus, during surgical procedures, CCS could be easily damaged; namely, the reliable method for identification of CCS either in vivo or ex vivo does not exist. Accordingly, there is a definite need for developing a CCS imaging method. Reflection confocal microscopy (RCM) offers non-destructive imaging of the tissue at depths of up to 0.35 mm with the capability of identification of a single cell. During the visualization procedure, a given tissue is illuminated with infrared laser light and the image is obtained because of different reflections from the tissue structures. However, the reflective structures in the heart tissues are still not identified. In the present study, for the first time we investigated cardiac tissues by RCM. The resolution of the method allowed us to distinguish MC cells and CCS cells. The method also allowed us to distinguish the networklike structures that are main components of CT. The ability to visualize different tissue components indicates a great potential for RCM to be used in non-destructive cardiac investigations and for imaging CCS. [ABSTRACT FROM AUTHOR]

Purpose: To measure penetration and metabolic effects of ion-stabilized, polar, 15 nm gold nanoparticles in aqueous solution (AuNP-Aq) and sterically stabilized, non-polar, 6 nm gold nanoparticles in toluene (AuNP-TOL) on excised human skin. Methods: Gold nanoparticles were characterized with dynamic light scattering and transmission electron microscopy (TEM). Skin penetration studies were done on frozen or fresh excised skin using static Franz diffusion cells. Viable treated skin was assessed by dermoscopy, reflectance confocal microscopy (RCM), multiphoton tomography (MPT) with fluorescence lifetime imaging microscopy (FLIM), and TEM. Results: Dermoscopy and RCM showed large aggregates in the furrows of AuNP-Aq-treated skin. Treatment of thawed and viable skin only showed enhanced permeability to nanoparticles in the AuNP-TOL group with MPT and FLIM imaging to stratum spinosum of epidermis. TEM analysis revealed gold nanoparticles within AuNP-treated stratum corneum. FLIM analysis of NAD(P)H showed a significant decrease in total NAD(P)H in all toluene-treated groups. Conclusions: Gold nanoparticles, 15 nm, in aqueous solution aggregated on the skin surface. Toluene treatment eliminated skin metabolism; skin treated with toluene/gold nanoparticles (6 nm) for 24 h, but not at 4 h, showed increased nanoparticle permeability. These results are of value to nanotoxicology. [ABSTRACT FROM AUTHOR]

Oral cancer is among the most common malignancies worldwide, therefore early detection and treatment is imperative. The 5-year survival rate has remained at a dismal 50% for the past several decades. The main reason for the poor survival rate is the fact that most of the oral cancers, despite the general accessibility of the oral cavity, are not diagnosed until the advanced stage. Early detection of the oral tumors and its precursor lesions may be the most effective means to improve clinical outcome and cure most patients. One of the emerging technologies is the use of non-invasive in vivo tissue imaging to capture the molecular changes at high-resolution to improve the detection capability of early stage disease. This review will discuss the use of optical probes and highlight the role of optical imaging such as autofluorescence, fluorescence diagnosis (FD), laser confocal endomicroscopy (LCE), surface enhanced Raman spectroscopy (SERS), optical coherence tomography (OCT) and confocal reflectance micr scopy (CRM) in early oral cancer detection. FD is a promising method to differentiate cancerous lesions from benign, thus helping in the determination of adequate resolution of surgical resection margin. LCE offers in vivo cellular imaging of tissue structures from surface to subsurface layers and has demonstrated the potential to be used as a minimally invasive optical biopsy technique for early diagnosis of oral cancer lesions. SERS was able to differentiate between normal and oral cancer patients based on the spectra acquired from saliva of patients. OCT has been used to visualize the detailed histological features of the oral lesions with an imaging depth down to 2-3 mm. CRM is an optical tool to noninvasively image tissue with near histological resolution. These comprehensive diagnostic modalities can also be used to define surgical margin and to provide a direct assessment of the therapeutic effectiveness. [ABSTRACT FROM AUTHOR]

Two-photon fluorescence microscopy and confocal reflectance microscopy were compared to detect intracellular gold nanorods in rat basophilic leukaemia cells. The two-photon photoluminescence images of gold nanorods were acquired by an 800 nm fs laser with the power of milliwatts. The advantages of the obtained two-photon photoluminescence images are high spatial resolution and reduced background. However, a remarkable photothermal effect on cells was seen after 30 times continuous scanning of the femto-second laser, potentially affecting the subcellular localization pattern of the nanorods. In the case of confocal reflectance microscopy the images of gold nanorods can be obtained with the power of light source as low as microwatts, thus avoiding the photothermal effect, but the resolution of such images is reduced. We have noted that confocal reflectance images of cellular gold nanorods achieved with 50 μW 800 nm fs have a relatively poor resolution, whereas the 50 μW 488 nm CW laser can acquire reasonably satisfactory 3D reflectance images with improved resolution because of its shorter wavelength. Therefore, confocal reflectance microscopy may also be a suitable means to image intracellular gold nanorods with the advantage of reduced photothermal effect. [ABSTRACT FROM AUTHOR]

The article presents a case study of a 36-year-old man with psoriasis who developed two patches of alopecia areata on his scalp after receiving ustekinumab as treatment for his disease. The patient has been treated for seven months and has achieved significant improvement before the occurrence of lesions. It has been concluded that the development of lesions was caused by emotional stress.

OBJECTIVE The objective of this study was to evaluate the feasibility of using confocal reflectance microscopy (CRM) ex vivo to differentiate adenoma from normal pituitary gland in surgical biopsy specimens. CRM allows for rapid, label-free evaluation of biopsy specimens with cellular resolution while avoiding some limitations of frozen section analysis. METHODS Biopsy specimens from 11 patients with suspected pituitary adenomas were transported directly to the pathology department. Samples were immediately positioned and visualized with CRM using a confocal microscope located in the same area of the pathology department where frozen sections are prepared. An H & E-stained slide was subsequently prepared from imaged tissue. A neuropathologist compared the histopathological characteristics of the H & E-stained slide and the matched CRM images. A second neuropathologist reviewed images in a blinded fashion and assigned diagnoses of adenoma or normal gland. RESULTS For all specimens, CRM contrasted cellularity, tissue architecture, nuclear pleomorphism, vascularity, and stroma. Pituitary adenomas demonstrated sheets and large lobules of cells, similar to the matched H & E-stained slides. CRM images of normal tissue showed scattered small lobules of pituitary epithelial cells, consistent with matched H & E-stained images of normal gland. Blinded review by a neuropathologist confirmed the diagnosis in 15 (94%) of 16 images of adenoma versus normal gland. CONCLUSIONS CRM is a simple, reliable approach for rapidly evaluating pituitary adenoma specimens ex vivo. This technique can be used to accurately differentiate between pituitary adenoma and normal gland while preserving biopsy tissue for future permanent analysis, immunohistochemical studies, and molecular studies.

Confocal microscopy utilizing fluorescent dyes is widely gaining use in the clinical setting as a diagnostic tool. Reflectance confocal microscopy is a method of visualizing tissue specimens without fluorescent dyes while relying on the natural refractile properties of cellular and subcellular structures. We prospectively evaluated 76 CNS lesions with confocal reflectance microscopy (CRM) to determine cellularity, architecture, and morphological characteristics. A neuropathologist found that all cases showed similar histopathological features when compared to matched hematoxylin and eosin-stained sections. RNA isolated from 7 tissues following CRM imaging retained high RNA integrity, suggesting that CRM does not alter tissue properties for molecular studies. A neuropathologist and surgical pathologist masked to the imaging results independently evaluated a subset of CRM images. In these evaluations, 100% of images reviewed by the neuropathologist and 95.7% of images reviewed by the surgical pathologist were correctly diagnosed as lesional or nonlesional. Furthermore, 97.9% and 91.5% of cases were correctly diagnosed as tumor or not tumor by the neuropathologist and surgical pathologist, respectively, while 95.8% and 85.1% were identified with the correct diagnosis. Our data indicate that CRM is a useful tool for rapidly screening patient biopsies for diagnostic adequacy, molecular studies, and biobanking., (© 2017 American Association of Neuropathologists, Inc. All rights reserved.)

During aging, alterations of extracellular matrix proteins contribute to various pathological phenotypes. Among these alterations, type I collagen cross-linking and associated glycation products accumulation over time detrimentally affects its physico-chemical properties, leading to alterations of tissue biomechanical stability. Here, different-age collagen 3D matrices using non-destructive and label-free biophotonic techniques were analysed to highlight the impact of collagen I aging on 3D constructs, at macroscopic and microscopic levels. Matrices were prepared with collagens extracted from tail tendons of rats (newborns, young and old adults) to be within the physiological aging process. The data of diffuse reflectance spectroscopy reveal that aging leads to an inhibition of fibril assembly and a resulting decrease of gel density. Investigations by confocal reflectance microscopy highlight poor-fibrillar structures in oldest collagen networks most likely related to the glycation products accumulation. Complementarily, an infrared analysis brings out marked spectral variations in the Amide I profile, specific of the peptidic bond conformation and for carbohydrates vibrations as function of collagen-age. Interestingly, we also highlight an unexpected behavior for newborn collagen, exhibiting poorly-organized networks and microscopic features close to the oldest collagen. These results demonstrate that changes in collagen optical properties are relevant for investigating the incidence of aging in 3D matrix models.

Reflectance confocal microscopy (RCM) has seen increasing clinical application for noninvasive diagnosis of skin cancer. Identifying the location of the dermal-epidermal junction (DEJ) in the image stacks is key for effective clinical imaging. For example, one clinical imaging procedure acquires a dense stack of 0.5×0.5mm FOV images and then, after manual determination of DEJ depth, collects a 5×5mm mosaic at that depth for diagnosis. However, especially in lightly pigmented skin, RCM images have low contrast at the DEJ which makes repeatable, objective visual identification challenging. We have previously published proof of concept for an automated algorithm for DEJ detection in both highly- and lightly-pigmented skin types based on sequential feature segmentation and classification. In lightly-pigmented skin the change of skin texture with depth was detected by the algorithm and used to locate the DEJ. Here we report on further validation of our algorithm on a more extensive collection of 24 image stacks (15 fair skin, 9 dark skin). We compare algorithm performance against classification by three clinical experts. We also evaluate inter-expert consistency among the experts. The average correlation across experts was 0.81 for lightly pigmented skin, indicating the difficulty of the problem. The algorithm achieved epidermis/dermis misclassification rates smaller than 10% (based on 25×25 mm tiles) and average distance from the expert labeled boundaries of ~6.4 μm for fair skin and ~5.3 μm for dark skin, well within average cell size and less than 2x the instrument resolution in the optical axis.