In vivo characterization of minipig skin as a model for dermatological research using multiphoton microscopy

Minipig skin is one of the most widely used non-rodent animal skin models for dermatological research. A thorough characterization of minipig skin is essential for gaining deeper understanding of its structural and functional similarities with human skin. In this study, three-dimensional (3-D) in vivo images of minipig skin was obtained non-invasively using a multimodal optical imaging system capable of acquiring two-photon excited fluorescence (TPEF) and fluorescence lifetime imaging microscopy (FLIM) images simultaneously. The images of the structural features of different layers of the minipig skin were qualitatively and quantitatively compared with those of human skin. Label-free imaging of skin was possible due to the endogenous fluorescence and optical properties of various components in the skin such as keratin, nicotinamide adenine dinucleotide phosphate (NAD(P)H), melanin, elastin and collagen. This study demonstrates the capability of optical biopsy techniques, such as TPEF and FLIM, for in vivo non-invasive characterization of cellular and functional features of minipig skin, and the optical image-based similarities of this commonly utilized model of human skin. These optical imaging techniques have the potential to become promising tools in dermatological research for developing a better understanding of animal skin models, and for aiding in translational pre-clinical to clinical studies.     

Preliminary evaluation of vitiligo using in vivo reflectance confocal microscopy

BACKGROUND: Vitiligo is the most common pigmentary disorder with a global incidence from 0.1% to 2% in different geographical areas. Histopathology and histochemistry have shown the reduction of melanocytes in achromic patches, but microscopic changes of lesional and non-lesional skin are still not completely understood. Reflectance confocal microscopy (RCM), based on the different light reflectance index of cutaneous structures, allowed in vivo, en face microscopic evaluation of superficial skin layers with a resolution similar to skin histology. AIM: The purpose of this study was to evaluate RCM features of lesional and non-lesional skin of vitiligo patients. Moreover, re-pigmented areas were taken into consideration in order to evaluate melanocyte response to ultraviolet B (UVB) radiation. SUBJECTS AND METHODS: Sixteen patients of different phototypes affected by active non-segmental vitiligo and 10 controls were enrolled in the study. In vivo skin imaging was done using a commercially available RCM (Lucid, Vivascope 1500. Re-pigmented areas from 6 to 16 patients (after UVB narrow-band therapy) were also examined. RESULTS: Vitiligo lesions showed the disappearance of the bright rings normally seen at the dermo-epidermal junction. Moreover, non-lesional skin of vitiligo patients showed unexpected changes as the presence of half-rings or scalloped border-like features of the bright papillary rings. In re-pigmented areas after UVB narrow band therapy, the presence of activated, dendritic melanocytes was seen. CONCLUSIONS: Considering our results, and following further studies, RCM clinical applications could be used in the therapeutic monitoring and evaluation of the evolution of vitiligo.     

Clinical study on the effects of a cosmetic product on dermal extracellular matrix components using a high‐resolution multiphoton tomograph

Background/purpose: The aim of this study was to demonstrate the effects of selected plant extracts in a cosmetic cream on the dermal network components after a 3‐month treatment using an in vivo multiphoton tomographic device. Methods: Twenty‐four Caucasian women aged between 45 and 65 applied randomly a cosmetic emulsion B containing active ingredients (soy and jasmine) twice a day on one arm and its vehicle A (without active ingredients) on the other arm during 3 months. Measurements were performed on the internal side of the forearm before starting the treatment (T0), after 4 week (T4) and 12 weeks (T12) treatment. Measurements consisted in a multi‐layers acquisitions using a multiphoton tomograph with subcellular resolution. Optical sections (about 6 μm thick) were recorded from 0 to about 200 μm using two different wavelengths: 760 and 820 nm. To compare the series of images and obtain an objective quantification of the signal of second harmonic generation (SHG) and autofluorescence, the method used consisted in taking the integrated brightness of an image (same rectangular area for all images) as a measure of the signal. Following this step a ratio between brightness of images from the area treated with cream A or B and brightness of untreated area was calculated and used as an assessment of treatment efficacy. The parameter used for statistical analysis (variance analysis) is the difference before and after 12 weeks of treatment by either cream A or B of the signal ratios calculated in the upper dermis (118–130 μm) and those from a deeper region of the upper dermis (165–178 μm). Results: Signals (autofluorescence+SHG) of extracellular matrix do not change significantly with time (weeks 0, 4 and 12) when cream A (vehicle with no active ingredient) is applied. Treatment with cream B results in an enhancement in the signal level of extracellular matrix at week 12. The comparison of signals, in both areas (118–130 μm and 160–178 μm), show an higher increase in the deeper region than in the more superficial one for product B while we do not notice any change with product A. Conclusion: The multiphoton tomograph provided excellent high‐resolution images, which describe clearly the different skin layers, single cells and extracellular matrix components in all the 24 volunteers. Statistic analyses reveal a real effect for product B with selected plant extracts, known to increase collagen synthesis. Changes observed are characteristics of modifications in dermal collagen and elastin content. To our knowledge, it is the first time that it was possible to demonstrate in vivo the effect of a cosmetic product on the superficial dermal layer, in a non‐invasive and non‐destructive process, i.e. without cutting the skin.     

Evaluation of allergic vesicular reaction to patch test using in vivo confocal microscopy

Background: Confocal microscopy has been successfully applied both in oncologic and inflammatory diseases. In particular, it has been proved as a useful tool for the in vivo detection of microscopical changes occurring in allergic reactions. Aims of the study: To evaluate microscopic changes occurring in positive patch test reactions. Methods: Eight patients with history of allergic dermatitis and positive patch test reaction were analysed by means of confocal microscopy. Results: Confocal microscopy showed the presence of spongiotic vesicle preferentially localized around the adnexal ducts that appeared to be in the middle of the spongiotic phenomena. Conclusion: Confocal microscopy offered for the first time new insight into vesicle formation and development, showing that adnexal ducts can play a role in allergic reaction.     

Evaluation of photoaging in facial skin by multiphoton laser scanning microscopy

Background/purpose: It has been reported that autofluorescence (AF) and second harmonic generation (SHG) generated in the upper dermis are related with skin photoaging. In this study, we assessed the photoaging of facial skin exposed to daily sunlight using in vivo multiphoton laser microscopy to measure AF and SHG. Methods: The intensities of AF and SHG in the upper dermis of cheek skin of 56 healthy volunteers aged 20–69 years were measured using a commercially available multiphoton laser microscope (DermaInspect^®). Correlations between the photo‐signals and volunteer age were calculated. Results: The intensity of SHG and the SHG‐to‐AF aging index of dermis (SAAID) correlated significantly with age (r=−0.48, −0.67, respectively). Conclusion: Our results suggest that SHG and the SAAID index are useful indicators of facial skin aging in vivo.     

Visualizing nuclei in skin cryosections: viable options to 4'6-diamidino-2-phenylindol for confocal laser microscopy

Background: Visualization of nuclei in skin (cryo‐) sections is essential for both, rapid overview and reliable orientation within skin samples. Therefore, nuclear staining is a very common counterstain for immunohistochemical studies of human skin as this nuclear staining precisely depicts the cellular distribution within the epidermis. Moreover, it clearly shows the epidermal–dermal border as well as the transition zone between the living and the cornified layers of the epidermis. For standard epifluorescence microscopy, 4′6‐diamidino‐2‐phenylindol (DAPI) is commonly used. For confocal laser scanning microscopy (CLSM), however, DAPI is often not suitable because its excitation maximum is in the ultraviolet (UV) range (Ex_max 359 nm) when bound to DNA, and UV lasers and the corresponding optics are not part of CLSM standard configuration. Methods: In order to find an adequate DAPI substitute that is excitable with standard visible light lasers, the following nuclear stains were tested: LOLO^?‐1 iodide (Ex_max 565 nm), TOTO^®‐3 iodide (Ex_max 642 nm), LO‐PRO^?‐1 iodide (Ex_max 567 nm), SYTO^® 84 (Ex_max 567 nm), SYTO^® 85 (Ex_max 567 nm), SYTOX^® Green (Ex_max 488 nm) and SYTOX^® Orange (Ex_max 547 nm), Propidium iodide (Ex_max 535 nm). Besides optimal concentration and incubation time, following criteria were also evaluated: photobleaching, background, e.g. cytoplasmic staining of RNA, and sensitivity to different fixation conditions (unfixed, IEM fixation, PLP fixation and PFA fixation). Results: According to these criteria Sytox^® Green showed the best overall staining score and can be used for variously fixed skin samples and shows a distinct and stable green nuclear fluorescence.     

Wavelength effects on contrast observed with reflectance in vivo confocal laser scanning microscopy

Background/purpose: The ability to optically section live biological tissue in vivo with laser light is made possible by confocal laser scanning microscopy (CLSM). In this work, the effects of changing the wavelength of incident light used for CLSM imaging of human skin are reported and analyzed.
Methods: Optical phantoms and the skin of eight human volunteers were imaged using CLSM systems having three different incident light wavelengths (405, 785, and 830 nm).
Results: Qualitative and quantitative differences were observed between images obtained at each wavelength, despite the proximity of the two near infrared 785 and 830 nm wavelengths. Furthermore, the penetration depth achieved with the 405 nm CLSM permitted imaging into the papillary dermis.
Conclusion: The laser wavelength used in CLSM reflectance imaging is important to properly understand and resolve different biological structures within human skin.     

Morphometry of human epidermis in vivo by real-time confocal microscopy

Real-time confocal microscopy has brought substantial improvements to the imaging of the human skin in vivo. On early images, the stratum corneum could be distinguished from the living epidermis and the circulatory network of the superficial dermis. We have adapted the Tandem Scanning Microscope to obtain images of the living skin, showing thinner structures such as the stratum lucidum and the dermo-epidermal junction, both of which are essential markers for micron-order measurements of the thickness of the stratum corneum and living epidermis. The measurements were corrected for the differences in the refractive index of the various cutaneous layers, and the undulation of the dermo-epidermal junction. Furthermore, nucleus size and number could be assessed from horizontal optical sections. To illustrate the sensitivity of the thickness measurements, changes in the thickness of the epidermis were recorded during and after stripping of the horny layers. This non-invasive methodology is a very promising tool for morphometric studies of the living human skin at the cellular level.     

In vivo confocal laser scanning microscopy of melanocytic skin tumours: diagnostic applicability using unselected tumour images

BACKGROUND: In vivo confocal laser scanning microscopy (CLSM) represents a novel imaging tool that allows the noninvasive examination of skin cancer morphology in real time at a 'quasi-histopathological' resolution viewing microanatomical structures and individual cells. OBJECTIVES: To validate diagnostic confocal examination of melanocytic skin tumours using unselected tumour images. METHODS: In the present study, we used a total of 3709 unselected CLSM tumour images obtained from 20 malignant melanomas and 50 benign naevi. The entire set of images derived from each tumour was evaluated by independent observers. Classification tree analysis based on a subsample of 857 tumour images was performed to develop a diagnostic algorithm. RESULTS: Overall, sensitivity and specificity of 97.5% and 99% could be achieved by the independent observers (positive predictive value 97.5%, negative predictive value 99%). Classification tree analysis yielded a three-step algorithm based on only three morphological CLSM features, facilitating a correct classification in 92.4% of the benign naevus images and 97.6% of melanoma images. CONCLUSIONS: In vivo CLSM augurs a sea change in the way we will view skin tumour processes clinically at the bedside and merits application for use as a screening tool in skin oncology.     

The biophysical effects of localized electrochemical therapy on porcine skin

BackgroundMinimally-invasive methods to treat scars address a common pathway of altering collagen structure, leading to collagen remodeling.ObjectiveIn this study, we employed in situ redox chemistry to create focal pH gradients in skin, altering dermal collagen, in a process we refer to as electrochemical therapy (ECT). The effects of ECT to induce biochemical and structural changes in ex vivo porcine skin were examined.MethodsDuring ECT, two platinum electrodes were inserted into fresh porcine skin, and following saline injection, an electrical potential was applied. pH mapping, high frequency ultrasonography, and two photon excitation microscopy and second harmonic generation (SHG) microscopy were used to evaluate treatment effects. Findings were correlated with histology.ResultsFollowing ECT, pH mapping depicted acid and base production at anode and cathode sites respectively, with increasing voltage and application time. Gas formation during ECT was observed with ultrasonography. Anode sites showed significant loss of SHG signal, while cathode sites showed disorganized collagen structure with fewer fibrils emitting an attainable signal. Histologically, collagen denaturation at both sites was confirmed.ConclusionWe demonstrated the production of in situ acid and base in skin occurring via ECT. The effects chemically and precisely alter collagen structure through denaturation, giving insight on the potential of ECT as a simple, low-cost, and minimally-invasive means to remodel skin and treat scars.     

Correlation of image analysis features and visual morphology in melanocytic skin tumours using in vivo confocal laser scanning microscopy

Background/purpose: In vivo confocal laser scanning microscopy (CLSM) represents a novel imaging tool that allows the non-invasive examination of skin cancer morphology at a quasi histological resolution without biopsy. Previous studies dealt with the search for diagnostic, but subjective visual criteria. In this study we examined the correlation between objectively reproducible image-analysis features und visual morphology in melanocytic skin tumours using CLSM.
Methods: Eight hundred and fifty-seven CLSM tumour images including 408 benign nevi and 449 melanoma images were evaluated. Image analysis was based on features of the wavelet transform and classification tree analysis (CART) was used for classification purposes. In a second step, morphologic details of CLSM images, which have turned out to be of diagnostic significance by the classification algorithm were evaluated.
Results: CART analysis of the whole set of CLSM images correctly classified 97.55% of all melanoma images and 96.32% of all nevi images. Seven classification tree nodes seemed to indicate benign nevi, whereas six nodes were suggestive for melanoma morphology. The visual examination of selected nodes demonstrated that monomorphic melanocytic cells and melanocytic cell nests are characteristic for benign nevi whereas polymorphic melanocytic cells, disarray of melanocytic architecture and poorly defined or absent keratinocyte cell borders are characteristic for melanoma.
Conclusion: Well-known, but subjective CLSM criteria could be objectively reproduced by image analysis features and classification tree analysis. Moreover, features not accessible to the human eye seem to contribute to classification success.     

In vivo reflectance confocal microscopy findings in a case of atypical fibroxanthoma

Clinical and dermoscopic features of atypical fibroxanthoma (AFX) are mostly non-diagnostic, so other in vivo diagnostic tools may give additional clues for accurate clinical diagnosis, such as in vivo reflectance confocal microscopy (RCM). However, there has been scarce data on RCM features of AFX in the literature, in only clear cell type. Herein we present a case of epithelioid cell predominant type AFX with RCM findings.     

Digital image enhancement for in vivo laser scanning microscopy

BACKGROUND/PURPOSE: In vivo confocal laser scanning microscopy (CLSM) is a new method that provides skin images in horizontal plane at a level of resolution that allows to view microanatomic structures. This study examines whether certain digital image-processing steps can increase the visibility of various structures in CLSM. STUDY DESIGN: Fifty images were taken from normal skin of 25 probands, and 39 image enhancement procedures were created. Eight procedures that seemed to provide some quality enhancement were deliberately selected for further evaluation. Subsequently, a collection of random pairs of the original image and an image submitted to any of the eight selected procedures was rated by five independent observers. RESULTS: In three of the eight procedures tested, the modified image was significantly preferred to the original image (chi2-test,: P< or =0.001). In particular, smoothing, shading correction, delineate and grey-level normalization in various combinations were helpful in showing the characteristic honeycomb pattern, pigmented basal cell layer, cell borders and the nuclei more clearly. CONCLUSION: Digital image processing may help to increase visibility of in vivo CLSM images.     

Establishing the dynamics of neutrophil accumulation in vivo by reflectance confocal microscopy

Reflectance confocal microscopy (RCM) is an imaging tool, which visualizes the epidermal skin layers in vivo with a cellular resolution. Neutrophil accumulation is a characteristic feature in psoriasis and is thought to play a role in the pathophysiology of psoriasis. Until now, imaging of neutrophil accumulation in vivo is not performed. We evaluated the dynamics of neutrophil migration in active psoriatic lesions by non‐invasive RCM imaging. Additionally, we evaluated the time phasing and duration of neutrophil trafficking. We performed RCM imaging prior to the start of topical treatment and for seven consecutive days with a 24‐h time interval at the Radboud University Medical Center, Nijmegen, the Netherlands. Twelve psoriatic lesions in three patients with a severe exacerbation of psoriasis were included. The four most active lesions were selected in each patient based on the highest degree of redness, induration and expansion in the previous 2 weeks. In all lesions, a cyclic pattern of neutrophil migration was observed, consisting of squirting papillae, transepidermal migration, accumulation in the stratum spinosum, accumulation in the stratum corneum and degeneration of the abscesses. The time interval of a neutrophil‐trafficking cycle was 5–7 days and showed a synchronic time phasing. This study is the first to establish the dynamics and time phasing of neutrophil migration in vivo in psoriatic lesions. Previously reported theories were confirmed by these novel in vivo data. RCM might distinguish between active or chronic psoriatic areas, which might contribute to new insights into the pathogenesis of psoriasis.