Lipophilic and hydrophilic moisturizers show different actions on human skin as revealed by cryo scanning electron microscopy

To study the mode of action of moisturizers on human skin, hydrophilic moisturizers in water and neat lipophilic moisturizers were applied on excised skin for 24 h at 32 degrees C. Samples of the treated skin were subsequently visualized in a cryoscanning electron microscope. The stratum corneum (SC) appeared as a region of swollen corneocytes (the swollen region) sandwiched between two layers of relatively dry corneocytes (the upper and lower non-swelling regions respectively). Lipophilic moisturizers increased the water content of the SC, whereas hydrophilic moisturizers can also reduce the water content of the SC. When focusing on the effect of the moisturizers on the three different regions, it was observed that cells in the swelling region are most sensitive to the application of the moisturizers and that the change in SC thickness is most influenced by the change in the thickness of the swelling region. Summarizing, SC cells are not equally sensitive to moisturizer application: centrally located corneocytes are more sensitive than corneocytes in the upper and the lowest regions of the SC.     

Age related changes of human skin investigated with histometric measurements by confocal laser scanning microscopy in vivo

Background/aims: The confocal laser scanning microscope Vivascope (Lucid, Henrietta) allows skin to be studied in real-time with a resolution of 0.5 µm horizontal and 1.3 µm vertical in vivo. In this study, we present the results of a comparison between the skin of an older and a younger group of volunteers by in vivo histometric measurements.
Methods: To investigate changes caused by age, 13 young (18–25 years) and 13 older (> 65 years) volunteers were examined. The following parameters were measured using the Vivascope at the volar forearm: minimal thickness of the epidermis (E_min), size of cells in the granular layer (A_gran), thickness of the horny layer (DSC), thickness of the basal layer (DSB) and number of dermal papillae per area (PapI). The image analysis program image tool was used to measure the size of the cells and the thickness of the basal layer.
Results: The older group of volunteers showed a significant increase in E_min, no significant change in DSC, a significant decrease in dermal papillae and in the thickness of the basal layer, and an increase in A_gran compared to the younger group.
Conclusions: Histometric measurements by in vivo confocal laser scanning microscopy are a sensitive and non-invasive tool for characterizing and quantifying histological changes of the epidermis and papillary dermis due to ageing.     

Elucidated lipid structures of various human stratum corneum investigated by electron paramagnetic resonance spectroscopy

Background: Electron paramagnetic resonance (EPR) in conjunction with a slow‐tumbling simulation is useful for defining stratum corneum (SC) lipid structure. The objectives of this investigation were to evaluate and elucidate the lipid structure of various human SC as a function of the depth. Methods: The SC from mid‐volar forearms of human volunteers and a cadaver was stripped consecutively from one to three times using a glass plate coated with a cyanoacrylate resin. Spin probes were used to monitor SC ordering. EPR spectra were analyzed by an EPR slow‐tumbling simulation. Keratin solution from human epidermis was mixed with 5‐doxylstearic acid (5‐DSA) aqueous solution. Results: EPR analyses of 5‐DSA and CHL probes showed immobilized and mobile spectra, respectively. The simulation for 5‐DSA spectra showed differences in ordering values of the SC as a function of depth. EPR of keratin/5‐DSA showed mobile spectral pattern. In addition, EPR of CHL showed a weak and a broad signal. Conclusions: The keratin/5‐DSA results imply that the most likely location of 5‐DSA probe is in SC lipid. CHL probe may not easily permeate the SC. The EPR results along with the simulation analysis provide elucidated SC lipid structure.     

Three-dimensional imaging of human skin and mucosa by two-photon laser scanning microscopy

BACKGROUND: Various structural components of human skin biopsy specimens are difficult to visualize using conventional histologic approaches. METHODS: We used two-photon microscopy and advanced imaging software to render three-dimensional (3D) images of in situ nerves, blood vessels, and hair follicles labeled with various fluorescent markers. Archived frozen human skin biopsy specimens were cryosectioned up to 150 micro m in thickness and fluorescently stained with rhodamine- or fluorescein-labeled antibodies or lectins. Optical sections were collected by two-photon microscopy and the resulting data sets were analyzed in three dimensions using Voxx software. RESULTS: Reconstructed image volumes demonstrated the complex 3D morphology of nerves, blood vessels and adnexal structures in normal mucocutaneous tissue. CONCLUSION: Two-photon microscopy and Voxx rendering software allow for detailed 3D visualization of structures within human mucocutaneous biopsy specimens, as they appear in situ, and facilitate objective interpretation of variations in their morphology. These techniques may be used to investigate disorders involving cutaneous structures that are difficult to visualize by means of traditional microscopy.     

Tattoos: light and transmission electron microscopy studies with X-ray microanalysis

Although the microscopic appearance of tattoos is well described, in many instances the nature of the pigment remains obscure. The presence of a specific pigment is sometimes implied from the colour of the tattoo and when sensitivity is present by a positive patch test (Cronin, 1980). Analysis of the pigment in tattoos has occasionally been described using techniques such as laser microprobe analysis and selected area diffraction studies (Silberberg & Leider, 1970). When made available from the tattooist the dye can also be analysed using X-ray fluorescence spectrometry (Clark & Black, 1979). Electron microscopy with X-ray microanalysis can detect numerous elements with certainty but surprisingly the technique has rarely been applied to tattoos. Taaffe, Knight & Marks (1978) identified mercury using scanning electron microscopy and X-ray microanalysis but due to the small amounts present there was uncertainty as to whether the metal represented contamination. This paper describes the use of X-ray microanalysis in the investigation of tattoos and correlates the findings with light and transmission electron microscopy appearances. The theoretical aspects of the technique have been described previously (Bleehen et al., 1981).     

Minocycline-related cutaneous hyperpigmentation as demonstrated by light microscopy, electron microscopy and X-ray energy spectroscopy

A 70-year-old patient with a chronic cutaneous ulcer treated by minocycline hydrochloride developed hyperpigmentation of the forearms. Biopsy material was studied by light microscopy, electron microscopy and X-ray energy spectroscopy. Granular gold-brown pigment was found in dermal histiocytes and eccrine myoepithelial cells, which gave positive reaction with Prussian blue and Fontana-Masson stains. Electron microscopy revealed intracytoplasmic granules of dark, homogeneous material and small fine particles. X-ray energy spectroscopy showed iron and other elements in smaller amounts. The different types of minocycline-related hyperpigmentation and the possible pathomechanism are discussed with special regard to the importance of the diagnostic methods.     

A replica technique for the evaluation of human skin by scanning electron microscopy

A detailed reproducible procedure has been developed for the replication of human skin for scanning electron-microscopic evaluation. Utilizing a test surface, a number of negative and positive materials were evaluated for consistence and clarity of replication. Optimal replication parameters were determined and the method was successfully applied to the evaluation of human skin in vivo. Furthermore, a method was established which permitted repetitive examination of selected skin sites. Experimental results suggest that this replicating procedure can be used to monitor changes in the stratum corneum overtime, and extend the range of stratum corneum magnifications.     

Scanning electron microscopy of the epidermal lamina densa in normal human skin.

The lamina densa of normal human epidermis was exposed by treatment with 1 M sodium chloride and was examined by high-power scanning electron microscopy before and after trypsinization. Localization of type IV collagen in the lamina densa was also studied by transmission and scanning immunoelectron microscopy. Before trypsinization, the surface of the lamina densa consisted of microridges and microvalleys. The microridges varied in height and were connected with each other. They were arranged in a concentric fashion around the tips of the dermal microprojections. At a higher magnification, the surface of the lamina densa was composed of densely packed cobblestone-like structures approximately 7-15 nm in size, between which were interspaces 4-11 nm wide. These structures expressed type IV collagen. After trypsinization, the lamina densa was found to be composed of microfilaments approximately 10 nm thick showing beaded appearances. These microfilaments exhibited the same cobblestone-like structures as the lamina densa surface. Observation of the torn lamina densa demonstrated anchoring fibrils and oxytalan fibers that were attached to the lamina densa itself. Another kind of filament about 7 nm thick linked the anchoring fibrils and the oxytalan fibers. Beneath the lamina densa was a network of fibers about 40-50 nm thick, which was composed of collagen fibers and possibly also elaunin fibers. In conclusion, this study revealed the detailed surface ultrastructure of the epidermal lamina densa and its underlying filamentous elements.     

The effect of UV-A and UV-B irradiation on the skin barrier. Skin physiologic, electron microscopy and lipid biochemistry studies]

In order to gain insight into the effects of UV-irradiation on the skin barrier, functional (skin reactivity), electron microscopic and lipid-biochemical studies were performed. In three different irritation models, both UV-A-irradiated and UV-B-irradiated areas proved to be more resistant to damage than normal skin, providing evidence for improvement of barrier function after UV irradiation. Electron microscopic evaluation showed that UV-B induced a significant increase in horny cell layers, whereas after UV-A no change was detected. However, both UV-B and UV-A exposure resulted in an increase in the amount of all stratum corneum lipids. This was also observed in all major ceramide subfractions, which are believed to be the essential lipid constituents for the epidermal barrier function. These findings may explain the known beneficial effects of phototherapy in dermatoses with impaired barrier function, i.e., atopic dermatitis.     

Plasma membrane ultrastructure of the human skin keratinocyte as observed by freeze-fracture electron microscopy.

The ultrastructure of the plasma membrane of human keratinocytes was investigated by freeze-fracture electron microscopy. Desmosomes were identified as an intramembranous particle aggregation on both of the fracture faces of the plasma membrane. The particle density of desmosomes ranged from 1,100 to 1,500/μ², while those of the P and E faces of the non-desmosomal membrane areas were 350 and 100/μ², respectively. The size of desmosomes varied from 0.3 to 0.7 μ in diameter. The frequency of the desmosomes was one to two per 2 μ². Gap junctions were a hexagonal array of the intramembranous particles in freeze-fracture replicas, and found much less frequently than desmosomes. On the basal cell membranes, round pits due to pinocytosis and particle aggregates of hemidesmosomes were observed.     

A statistical analysis of the characteristics of pigmented skin lesions using epiluminescence microscopy

Due to the fact that not all pigmented skin lesions (PSL) can be diagnosed solely by their clinical appearance, additional criteria are required to optimize the clinical diagnosis of atypical nevus and melanoma. Epiluminescence microscopy is a non-invasive in vivo examination that often helps to improve the accuracy of clinical diagnosis of such lesions. Years of experience have indicated some differential epiluminescent patterns for benign and malignant PSL but there is some controversy about certain borderline lesions for which histological examination is always necessary. In our study we performed a statistical analysis of data concerning 183 PSL to determine characteristics significantly associated with these lesions allowing identification of epiluminescent criteria suggestive of atypical nevus and malignant melanoma. Using the chi-quadro test and stepwise regression logistic model, we identified the following epiluminescent pattern as a risk factor for atypical nevus and malignant melanoma: irregular pigment network, presence of capillaries, irregular and abrupt ending of overall pigmentation, irregular brown globules and irregular shape and size of black dots.     

Effect of lipid solvents on protein, DNA, and collagen synthesis in human skin: an electron microscopic autoradiographic study.

The effect of acetone and kerosene on the synthesis of protein, DNA, and collagen was studied by electron microscopic autoradiography using [3H]leucine, [3H]thymidine, and [3H]proline as tracers in human skin. Quantitative analyses following concomitant administration of tritiated leucine and acetone or kerosene demonstrated, at 90 min, a marked decrease in silver grains as compared to control or nonexposed areas. Incorporation of tritiated thymidine is moderately stimulated only by acetone, whereas radioactive proline distribution is not significantly affected. Electron microscopic autoradiograms revealed that tritiated leucine is distributed over all epidermal cells, mostly in the stratum spinosum of control epidermis; a marked decrease of silver grains from [3H]leucine followed both lipid solvent exposures. The autoradiographic reaction is specifically located over cytoplasmic organelles, such as polysomes, endoplasmic reticulum, and especially tonofilaments. Tritiated thymidine resulted in silver grains mostly over nuclear chromatin and these were moderatly increased after acetone application, whereas the incorporation of radioactive proline in the fibroblasts and collagen fibrils were not significantly influenced. These investigations indicate a dissociated effect of lipid solvents on protein, DNA, and collagen synthesis in human skin.     

Analysis of human skin surface lipid during treatment with anti-androgens

Skin surface lipid was collected from the foreheads of patients during a study of anti-androgen therapy for acne, and analysed for free fatty acid composition and triglyceride fatty acids. Both sebum excretion rate and bacterial flora decreased during therapy but only the ratio of free fatty acid to total glyceride plus free fatty acid changed, falling in all groups during therapy. No differences in lipid composition were detected in relation to individual therapy but patients admitted at the start of the trial, had, overall, a different lipid composition to those admitted later and had lower surface microbial counts.     

不同时期黄褐斑皮损三种皮肤影像的形态学分析

【摘要】 目的 运用三种皮肤影像技术探究女性黄褐斑不同时期皮损的形态。方法 2017年6月至2018年1月,在杭州市第三人民医院门诊收集女性黄褐斑患者253例。结合临床分期标准,应用反射式共聚焦显微镜（RCM）、VISIA皮肤图像检测仪、皮肤镜观察不同时期黄褐斑皮损,分析临床分期与树突状黑素细胞、亚临床黄褐斑、血管形态改变的相关性。采用SPSS19.0统计学软件,计数资料比较采用卡方检验及独立样本Mann?Whitney U检验。结果 253例患者中,进展期100例,稳定期153例。进展期患者中78例（78%）RCM下有树突状黑素细胞,稳定期中22例（14.4%）可见树突状黑素细胞,两组间差异有统计学意义（χ2 = 102.40,P＜0.01）。VISIA皮肤图像检测仪观察显示,进展期患者中78例（78%）有亚临床黄褐斑,稳定期患者25例有亚临床黄褐斑（16.3%）,进展期亚临床黄褐斑出现率高于稳定期（χ2 = 95.26,P＜0.01）。皮肤镜显示,进展期患者血管改变发生率为74%（74/100）,稳定期为68.6%（105/153）,两组差异无统计学意义（χ2 = 0.84,P = 0.39）。结论 RCM、VISIA皮肤图像检测仪下分别观察的树突状黑素细胞及亚临床表现可作为黄褐斑临床分期的参考指标。     

Scanning electron microscopy of skin surface and the internal structure of corneocyte in normal human skin. An application of the osmium-dimethyl sulfoxide-osmium method

Summary A horny layer of normal human skin prepared according to the newly developed osmium-dimethyl sulfoxide-osmium method was examined using scanning electron microscopy. On the skin surface, cytomembranes of the uppermost corneocytes frequently had unilateral, very slightly flat-elevated, zonal areas along the junctions between the corneocytes. The uppermost corneocytes peeled off along the junctions, leaving the remnants of their cell bodies in the junctional areas. In the cracked surface, cytomembranes of the corneocytes protruded from the plane of their cytoplasmic surface. In the lateral junction between the corneocytes, the cytomembranes of the corneocytes were in tight contact with each other, while occasionally the marginal bands had become detached from the cytomembranes. In the vertical connection, cleavages formed between the cytomembranes of the corneocytes. There were thick woollen thread-like structures about 10–30 nm thick in the cytoplasm of the corneocytes. They formed fine irregular meshworks, with their tips projecting digitally: Transmission electron microscopy revealed these structures as most likely being keratin bundles transformed during processing.     

Comparative evaluation of different hair removal lasers in skin types IV,V, and VI

BACKGROUND: Lasers permit treatment of unwanted excess hair with less discomfort than other methods of epilation. Many lasers with different parameters are now available from which the dermatologist can choose. Improved clinical results are made possible by the high specificity and selectivity of the laser systems to pigmented hair because of the use of an appropriate wavelength with the proper pulse and duration. OBJECTIVES: We aimed to compare the results of treatment of skin types IV, V, and VI using three different laser systems. METHODS: One hundred female patients were compared using different laser systems: 35 patients underwent epilation using a Nd-Yag laser, 33 patients using an Alexandrite laser, and 32 patients using a Diode laser. RESULTS: Follow up 12 months after the multiple treatments (three to six sessions) showed an insignificant difference between these three groups (35-40%). CONCLUSION: Our findings indicate that all three laser systems tested can be used for dark skin; however, one should select a system that minimizes side-effects, primarily hypo- and hyper-pigmentation, especially when used for skin types IV, V, and VI.