Reflectance-mode Confocal Microscopy Research Articles

Konfokális mikroszkópia a bőrgyógyászati gyakorlatban: egyedülálló lehetőség szövettani szintű képalkotásra in vivo nem invazív módon

Among non-invasive in vivo techniques the use of reflectance mode confocal microscopy (RCM) is the most common. In vivo RCM images are horizontal ones similar to dermatoscopy and allow tissue and cellular level analysis almost identical to routine histopathology. In vivo RCM is recommended mainly for the examination of clinically and dermatoscopically equivocal lesions and it can be used to delineate-demark tumor borders prior to surgery, to monitor the results of non-invasive therapies, and to detect recurrent tumors and to diagnose inflammatory skin diseases. In vivo confocal microscopy examination is available at the Department of Dermatology and Allergology of Albert Szent-Györgyi Health Center, University of Szeged and at the Department of Dermatology, Venereology and Oncodermatology Medical School, University of Pécs.

Label‐free laser scanning microscopy targeting sentinel lymph node diagnostics: A feasibility study ex vivo

AbstractThis proof‐of‐concept, observational study investigates the potential for label‐free, laser scanning microscopy to identify melanoma metastatic features in lymph node tissues ex vivo ‐ exploring the translational potential targeting sentinel lymph node diagnostics. Ten lymph node samples, of which seven are positive for melanoma metastases and three negative, were obtained from a tissue biobank. Imaging data acquired using multiphoton microscopy ex vivo were compared with histopathological findings. Morphologic features characteristic for histopathologic diagnosis of melanoma metastasis were confirmed in the positive samples. Four of the samples were complementary studied with reflectance mode confocal microscopy; however, visualization was found restricted to fibrous structures in this case. To conclude, the results imply that multiphoton microscopy based on autofluorescence is a promising label‐free technique for visualization of cellular features in lymph node tissues.

In vivo reflectance-mode confocal microscopy assessments: impact of overweight on human skin microcirculation and histomorphology.

Reflectance-mode confocal microscopy (RCM) enables in vivo assessment of the human skin. Impact of overweight on both human skin microcirculation and histomorphology has not been investigated in vivo. The purpose of this study is to evaluate both microcirculation and histomorphology in vivo in overweight. In 10 normotensive overweight nondiabetic individuals (OW-group, BMI 29.1 ± 2.7 kg/m(2)) and 10 age- and sex-matched healthy lean controls (CO-group, BMI 20.4 ± 1.9 kg/m(2)) the following parameters were evaluated using RCM: dermal blood cell flow (DBCF), density of dermal capillaries (DDC), epidermal thickness (ET), and epidermal cell size (ECS). DBCF was counted at 63.11 ± 4.14 cells/min in OW-group and at 51.06 ± 3.84 cells/min in CO-group (P < 0.05). DDC was reduced in OW-group (4.91 ± 0.39 capillaries/mm(2)) compared to the controls (6.02 ± 0.64 capillaries/mm(2), P < 0.05). Histometric evaluation of ET reveals thickening in OW-group compared to the CO-group (54.79 ± 4.25 μm versus 44.03 ± 3.11 μm, P < 0.05). ECS differed significantly (P < 0.05) in OW-group (821.3 ± 42.02 μm(2)) compared to the controls (772.6 ± 34.79 μm(2)). Inverse correlation of dermal capillary density and overweight point to reduced total tissue perfusion while positive related blood cell flow reveals vasodilatation. Increase of both ET and cell size indicates remodeling of cutaneous histomorphology, maybe as an early stage of adiposity-related skin condition.

Acute effects of splint immobilization of the forearm on in vivo microcirculation and histomorphology of the human skin.

Splint immobilization of the forearm is often performed in clinical practice. Previous studies investigated the effect of immobilization on bone, cartilage, muscle, and tendon, however, the acute effects on human skin microcirculation and histomorphology remains elusive. In 12 healthy, nonsmoking individuals (aged 29.7 ± 9.1 years) a randomly selected forearm was immobilized by splinting for 72 h, whereas the other forearm served as control. In vivo Reflectance-Mode Confocal-Microscopy (RMCM) was performed prior (baseline value) and postimmobilization to evaluate: quantitative blood cell flow; density of functional dermal capillaries; epidermal thickness; and granular cell size. At 72h forearm immobilization, quantitative blood cell flow was significantly reduced (42.86 ± 3.68 cells/min) compared to the control blood flow (53.11 ± 3.68 cells/min, P < 0.05) and dermal capillaries indicates less functional density (5.73 ± 0.63 capillaries/mm2) compared to the controls (7.04 ± 0.81 capillaries/mm2, P < 0.05). Histometric assessment reveals significantly thinner epidermis following immobilization compared to the control site (40.02 ± 2.91 vs. 46.64±3.09 µm, P < 0.05). Granular cell size was significantly altered at 72 h splinting (730.1 ± 42.53 µm2) compared to the control cell size at 770.2 ± 38.21 µm2. Comparison of baseline values of both forearms indicate statistically insignificance (P > 0.05) for each parameter. At 72 h splint immobilization, for the first time, significant adaptive mechanisms were evaluated on human skin microcirculation and histomorphology using in vivo RMCM. These adaptations may be considered as an incipient atrophy of the human skin. Long-term effects of immobilization including the regenerative potential should be evaluated in further RMCM studies.

Anal melanosis diagnosed by reflectance confocal microscopy

Until now, in vivo reflectance-mode confocal microscopy (IVCM) has been applied only to pigmented lesions of the vulvar and oral mucosa, but not to anal mucosa lesions. We present the first case in which IVCM has been used to diagnose anal melanosis. Clinical and dermoscopic features were of concern while IVCM found the draped pattern already described for genital melanosis. IVCM adds information to the clinical and dermatoscopic examination and allows skin biopsies to be avoided. Further studies are needed to define the IVCM features of anal melanosis and to compare the performance of IVCM with the findings of histological examinations.

Reflectance Confocal Microscopy for the Diagnosis of Vulvar Melanoma and Melanosis: Preliminary Results

In the early stages, vulvar melanoma can mimic vulvar melanosis and therefore the diagnosis is often late and carries a poor prognosis. In vivo reflectance-mode confocal microscopy (RCM) is an emerging technique that allows noninvasive high-resolution imaging of the skin and mucosa, but it has not been employed in the study of genital pigmentation. To analyze the characteristics of vulvar melanosis and vulvar melanoma using RCM to define the confocal aspects that allow a correct differential diagnosis. Features of eight melanoses and two melanomas of the vulva were analyzed using RCM. RCM diagnosis was then compared with clinical and histologic diagnosis. Two major characteristics are associated with vulvar melanosis: papillae rimmed by bright monomorphous cells and possible presence of a few dendritic bright cells in the basal layer of the epithelium. Two major features of vulvar melanoma have been identified: atypical cells in the epithelium and loss of normal architecture of chorion papillae. Reflectance Confocal Microscopy can play a role in noninvasive differentiation between vulvar melanoma and vulvar melanosis, but further broader studies are needed to validate our observations.

Reflectance mode confocal microscopy and digital image analysis in naevus of Hori and pathogenetic evaluation

Reflectance mode confocal microscopy and digital image analysis in naevus of Hori and pathogenetic evaluation

Reflectance confocal microscopy for quantification of Sarcoptes scabiei in Norwegian scabies

In vivo reflectance-mode confocal microscopy (RCM) can be used for the diagnosis of scabies. This study quantifies S. scabiei and its eggs and droppings in a patient affected by Norwegian Scabies (NS), and describes their distribution within the epidermis and in different body areas. Different skin sites were randomly chosen in four sections (head, upper limbs, trunk and inferior limbs) of the body surface area (BSA) to acquire a total of 60 RCM z-stacks. The number of mites and eggs, the presence of droppings, as well as the minimum epidermal depth at which mites, eggs and faeces were detectable, was established for each z-stack. The total number of mites and eggs on the entire BSA was calculated considering the weighted mean for the four sections of the BSA. A total of 15.8 millions of S. scabiei and 7.2 millions of eggs were calculated. Mites, eggs and faeces were homogeneously distributed all over the body surface. Droppings, easily recognized by the RCM, were present in more than an half of the analyzed cutaneous sites and were associated with the presence of parasites (chi-squared test, P = 0.002). Our study illustrates the ability of RCM to identify, locate, and quantify the various forms of S. scabiei in human skin. NS is an extremely contagious disease, considering that the number of mites can be around 15.8 millions. Moreover, all areas of the body are parasitized in NS, including the face.

Detection of living Sarcoptes scabiei larvae by reflectance mode confocal microscopy in the skin of a patient with crusted scabies

Scabies is an intensely pruritic disorder induced by a delayed type hypersensitivity reaction to infestation of the skin by the mite Sarcoptes scabiei. The diagnosis of scabies is established clinically and confirmed by identifying mites or eggs by microscopic examination of scrapings from the skin or by surface microscopy using a dermatoscope. Reflectance-mode confocal microscopy is a novel technique used for noninvasive imaging of skin structures and lesions at a resolution compatible to that of conventional histology. Recently, the technique was employed for the confirmation of the clinical diagnosis of scabies.We demonstrate the first ever documentation of a larva moving freely inside the skin of a patient infected with scabies.

Intense pulsed light therapy or Q-switched laser for solar lentigo evaluated by skin physiologic examination and reflectance-mode confocal microscopy

Intense pulsed light therapy or Q-switched laser for solar lentigo evaluated by skin physiologic examination and reflectance-mode confocal microscopy

ASSESSMENT OF OPTICAL CLEARING AGENTS USING REFLECTANCE-MODE CONFOCAL SCANNING LASER MICROSCOPY

The mechanism of action of clearing agents to improve optical imaging of mouse skin during reflectance-mode confocal microscopy was tested. The dermal side of excised dorsal mouse skin was exposed for one hour to saline, glycerin, or 80% DMSO, then the clearing agent was removed and the dermis placed against a glass cover slip through which a confocal microscope measured reflectance at 488 nm wavelength. An untreated control was also measured. The axial attenuation of reflectance signal, R(zf) versus increasing depth of focus zf behaved as R = ρ exp (-μzf2G), where ρ is tissue reflectivity and μ is attenuation [cm-1]. The factor 2G accounts for the in/out path of photons, and the numerical aperture of the lens. The ρ, μ data were mapped to values of scattering coefficient (μs [cm-1]) and anisotropy of scattering (g). Images showed that glycerin significantly increased the g of dermis from about 0.7 to about 0.99, with little change in the μs of dermis at about 300 cm-1. DMSO and saline had only slight and inconsistent effects on g and μs.

In Vivo Microscopic Approaches for Facial Melanocytic Lesions after Quality-Switched Ruby Laser Therapy: Time-Sequential Imaging of Melanin and Melanocytes of Solar Lentigo in Asian Skin

The quality-switched ruby laser (QSRL) has been widely used for the treatment of pigmented lesions, but clinical evaluations in most studies have been conducted on macroscopic skin color observation comparing the laser-treated skin with its nontreated surrounding area. A few investigations examined skin changes after laser therapy at a cellular level, but almost none did so noninvasively. To elucidate the dynamic changes after QSRL irradiation of facial solar lentigo using noninvasive optical techniques. Time-sequential imaging of Japanese female patients with a clinical diagnosis of solar lentigo was performed using ultraviolet photography, high-magnification videomicroscopy, and reflectance-mode confocal microscopy to examine pigmentary change after QSRL irradiation. The present study showed that remaining melanocytes were visible in the solar lentigo of all subjects when crusts peeled off, despite hardly observable skin pigmentation to the naked eye. Moreover, noninvasive confocal imaging revealed that pigmented melanocytes varied in each solar lentigo after QSRL treatment, as indicated by melanin reflection level. Optical techniques facilitate the evaluation of the in vivo dynamics of epidermal-melanocytic changes in solar lentigo after QSRL therapy and may be useful for monitoring outcomes after laser irradiation.

Bioengineering and subjective approaches to the clinical evaluation of dry skin

Dry skin (also known as xerosis) is a cutaneous reaction pattern indicative of abnormal desquamation, which has not only cosmetic considerations, but can also lead to the penetration of irritants and allergens through the stratum corneum (SC). Over the last few decades, our understanding of the structure, composition, formation and function of the SC has advanced tremendously; however, despite these advancements, the occurrence of dry skin remains prevalent in the adult population. The clinical evaluation of dry skin is therefore of significant importance to the cosmetic industry not only for understanding the condition but also for measuring the effects of treatment. Traditionally, dry skin has been evaluated by visual inspection, however, recently a variety of bioengineering techniques have emerged enabling the investigator to objectively assess the extent of xerotic conditions. The most frequently employed methods for the evaluation of dry skin are discussed in this review, including regression testing, squametry, measurement of transepidermal water loss, epidermal hydration, profilometry, confocal Raman spectroscopy, optical coherence tomography, in vivo confocal microscopy and magnetic resonance imaging.

To Heal or Not to Heal: Predictive Value of In Vivo Reflectance-Mode Confocal Microscopy in Assessing Healing Course of Human Burn Wounds

The purpose of this study was to assess if the healing course of burn wounds of indeterminate depth can be predicted based on serial in vivo reflectance-mode confocal microscopy (RMCM) analysis. Twenty-four patients (mean age, 33.1+/-11.4 years; mean burn size: 6% TBSA) were investigated at 12, 36, and 72 hours after burn of indeterminate depth and retrospectively grouped into healing group (HG: 16 patients) and nonhealing group (NHG: eight patients). Noninjured skin served as controls. The following parameters were assessed: quantitative blood cell flow (BCF), basal layer thickness (BLT), and inflammatory cells. At 12 hours postburn, BCF increased to 101.67+/-7.64 cells/min in HG vs 85+/-50 cells/min in NHG compared with controls (56.5+/-2.3 cells/min). At 36 and 72 hours, BCF increased to 115+/-10 cells/min and 125+/-50 cells/min in HG vs decreased to 80+/-5 cell/min and 75+/-5 cells/min in NHG (P<.05). At 12 hours postburn, BLT increased to 19.43+/-0.93 microm in HG vs 29+/-1 microm in NHG compared with controls (15.40+/-0.60 microm, P<.05). In HG, further gradual increase of BLT to 20+/-1 microm (36 hours) and 21+/-1 microm (72 hours) was observed, whereas BLT was destroyed after 36 hours in NHG. Qualitative assessment found insignificant amount of IC in controls and low amount in HG until 72 hours postburn, whereas progressive increase in IC from low amount (12 hours) to numerous (36 hours) and massive (72 hours) was observed in NHG. RMCM enables simultaneous evaluation of microcirculation, histomorphology, and inflammatory cell trafficking in burn wounds. RMCM may help to predict whether burns of indeterminate depth have the potential to heal and can be a valuable tool to clinicians to guide early therapeutic decision-making process in burn patients.

Assessment of microcirculatory influence on cellular morphology in human burn wound healing using reflectance‐mode‐confocal microscopy

Previous studies have assessed the effects of changes in microcirculation on wound healing; however, the influence of microcirculation on tissue histomorphology remains widely unknown. Reflectance-mode-confocal microscopy (RMCM) enables in vivo tissue observation on a cellular level. We present RMCM data evaluating the local microcirculation and assess the influence on histomorphology during burn healing. RMCM was performed in 12 patients (aged; 36.2+/-14.2 years, maximum-burn-extent: 4% total body surface area) at times 12, 36, and 72 hours after a superficial burn. The following parameters were assessed: quantitative blood-cell-flow (cbf), epidermal thickness (Emin), basal-layer thickness (tbl), and granular cell-size (Agran). Cbf was found to be 54+/-3.6 cells/minutes (control), increased to 91+/-3.6 cells/minutes (p<0.05) 12 hours postburn; decreased to 71+/-6.1 cells/minutes (p<0.05) (36 hours), and to 63+/-2.3 cells/minutes (p>0.05) 72 hours postburn. Emin was 43.74+/-3.87 mum (control), increased to 51.67+/-4.04 mum (p<0.05) 12 hours, decreased to 48.67+/-3.51 mum (p<0.05) 36 hours, and to 45.33+/-3.21 mum (p>0.05) at 72 hours postburn. Tbl was 14.17+/-0.6 mum (control), increased to 16.93+/-1.15 mum (p<0.05) 12 hours, decreased to 15.93+/-1.20 mum (p<0.05) 32 hours, and to 15.00+/-0.85 mum (p>0.05) 72 hours postburn. Agran was 718+/-56.20 mum(2) (control), increased to 901+/-66.02 mum(2) (p<0.05) 12 hours, decreased to 826+/-56.86 mum(2) 36 hours, and 766+/-65.06 mum(2) at 72 hours postburn. RMCM enables in vivo observation of wound microcirculation and allows direct assessment of vascular effects on cutaneous histomorphology during the healing course of superficial burns.

In vivo reflectance-mode confocal microscopy provides insights in human skin microcirculation and histomorphology

Purpose Various approaches are used to study microcirculation, however, no modality evaluates microcirculation and histomorphology on cellular levels. We hypothesized that reflectance-mode confocal microscopy (RCM) enables simultaneous evaluation in vivo of both microcirculation and histomorphology. Principals The forearm of 20 volunteers was exposed to either local heat stress (HS-group), or to local cold stress (CS-group). RCM was performed prior and after temperature stress to evaluate quantitative blood-cell flow, capillary loop diameter, granular cell size, and basal layer thickness. Results In the HS-group, we observed significant increase in capillary loop diameter and increased blood-cell flow after heat stress. In the CS-group, significant decreases of capillary loop diameter and in blood-cell flow were determined following cold stress. Granular cell size and basal layer thickness differed insignificantly prior and after local temperature stress. Conclusions RCM provides real-time and in vivo high resolution imaging of temperature-dependent changes in the human skin microcirculation and histomorphology on cellular levels.

In Vivo Microscopic Features of Nodular Melanomas

To characterize nodular melanoma (NM) using dermoscopy, in vivo reflectance-mode confocal microscopy, and histopathologic analysis. Consecutive pure NMs and superficial spreading melanomas (SSMs) with nodular or blue areas were studied using dermoscopy and confocal microscopy, and a correlation with histopathologic findings was performed. Ten NMs, 10 SSMs with a nodular area, and 10 SSMs with a blue palpable but not yet nodular area. Confocal differences within the nodular component between pure NMs and SSMs with a nodular area, hypothesizing different biological behaviors. Whereas NMs had predominantly nonspecific global dermoscopic patterns, SSMs exhibited a multicomponent pattern and higher dermoscopic scores. Globules, blue-white veil, atypical vessels, and structureless areas were frequent in NMs and in nodular areas from SSMs. At confocal microscopy, NMs exhibited few pagetoid cells within a typical epidermal architecture in the superficial layers in most cases, differing from SSMs frequently characterized by epidermal disarrangement and pagetoid infiltration. At the dermoepidermal junction, dermal papillae were rarely seen in nodular areas both from NMs and from SSMs, frequently substituted by nonaggregated atypical cells distributed in sheetlike structures. In the upper dermis, all groups exhibited plump bright cells, dense dishomogeneous cell clusters, and atypical nucleated cells, whereas cerebriform clusters were characteristic of NMs. Conclusion Distinctive dermoscopic and confocal features seen in NMs compared with SSMs are helpful in making the diagnosis and suggest different biological behavior.

In vivo assessment of pigmentary and vascular compartments changes in UVA exposed skin by reflectance‐mode confocal microscopy

Exposure of the skin to ultraviolet A (UVA) results in various biological responses, skin-colour changes being among the major ones. Although intense research has been performed on UVA-induced pigmentation and vascular changes, the process of skin-colour changes after UVA irradiation remains unclear. For a better understanding of the UVA tanning mechanism, we here performed a human study in 27 healthy volunteers with skin phototype (SPT) II to VI. After a single UVA exposure to inner forearm, the skin sites were imaged using reflectance-mode confocal microscopy (RCM), for analysis of melanin and vascular changes. Punch biopsies were also taken from the UVA-exposed or non-exposed sites for histological examination. Skin sections were stained with Fontana-Masson and evaluated by a sensitive tyrosinase assay for comparison with RCM images. Furthermore, the effect of blood flow on skin-colour changes was evaluated visually after administration of an intradermal anesthesia of lidocaine with or without epinephrine. Our RCM analysis showed dendritic melanocytes and a different melanin distribution in the epidermal layer, clearly visible 1 week after the UVA exposure in subjects of SPT V which were supported by histological examination. However, no melanin distribution pattern changes were apparent immediately after the exposure, while RCM images showed accelerated capillary flow patterns. The restriction of this UVA induced-accelerated blood flow by epinephrine inhibited partially or completely the immediate pigment darkening and delayed tanning. These in vivo studies confirmed that vascular change is an important factor for the development of the immediate pigment darkening and delayed tanning.

Dendritic Cells in Pigmented Basal Cell Carcinoma

Reflectance-mode confocal microscopy (RCM) is a new approach for the in vivo diagnosis of skin tumors. A few studies of RCM on basal cell carcinoma (BCC) have provided specific diagnostic criteria, but large studies on pigmented basal cell carcinoma are lacking. Proliferation of large dendritic-shaped cells within a melanocytic tumor has been associated with the diagnosis of melanoma by RCM. Benign melanocytes and Langerhans cells may populate BCC according to previous histological studies. We studied 3 consecutive pigmented BCC by means of RCM and performed a histological and immunohistochemical correlation focusing on the presence of dendritic structures. Reflectance-mode confocal microscopy revealed highly refractive dendritic structures within tumor nests that correlated with the presence of melanocytes within the tumor by immunochemical analysis. In 1 case, dendritic structures on the overlying epidermis corresponding to Langerhans cells were also noted. Leaf-like areas observed on dermoscopy correlated with low-refractive cordlike structures and nodules by RCM and corresponded to nests of basaloid cells, whereas blue-gray globules presented as bright oval structures with ill-defined borders corresponding to melanophages. Reflectance-mode confocal microscopy allows the study of pigmented BCC and the identification of specific criteria described previously. In these tumors, dendritic melanocytes can be easily identified with this technique.

Noninvasive Evaluation of Allergic and Irritant Contact Dermatitis by In Vivo Reflectance Confocal Microscopy

The clinical differentiation of allergic contact dermatitis (ACD) and irritant contact dermatitis (ICD) is often difficult to accomplish. Reflectance-mode confocal microscopy (RCM) is an imaging technique that has previously been used to examine ACD and ICD noninvasively in vivo. To determine characteristic features of ACD and ICD and their kinetic evolution over time. Ethnic susceptibility to contact irritants such as sodium lauryl sulfate and Ivory dishwashing liquid was evaluated noninvasively, and the sensitivity and specificity of RCM parameters were analyzed in a clinical context and in reference to patch testing. Subjects were patch-tested with allergens, irritants, and controls. Clinical scoring and RCM evaluation were performed at various time points, assessing stratum corneum (SC) disruption, spongiosis, exocytosis, vesicle formation, and epidermal thickness. RCM features of both ACD and ICD include spongiosis, exocytosis, vesicle formation, and blood vessel dilatation. SC disruption, epidermal necrosis, and hyperproliferation are hallmarks of ICD whereas ACD more typically presents with vesicle formation. Patients with ICD showed a more rapid recovery than those with ACD. When tested with Ivory soap at selected concentrations, Caucasians, when compared to African Americans, showed significantly lower clinical thresholds for ICD and features that were more severe. RCM may be a promising new technology for longitudinal noninvasive studies of contact dermatitis (CD). Using a diagnostic algorithm and those parameters with high sensitivity for CD, RCM may facilitate the differentiation of acute ACD and ICD. RCM can reliably visualize cutaneous changes at subclinical degrees of CD, which suggests a possible role for RCM as an adjunctive tool in CD diagnosis. The results of this pilot study also indicate ethnic differences in the response to contact irritants. However, further studies are needed to substantiate the relevance and clinical applicability of our findings.