Ultrastructure of the human stratum corneum.

The ultrastructural study of the intercellular spaces of the human stratum corneum was based on transmission electron microscopy of thin vertical sections and freeze-fracture replicas, field emission scanning electron microscopy and immunofluorescence confocal laser scanning microscopy. The maturation of the corneosomes and their enzymatic degradation could be depicted at strategic interfaces. These sharp and rapid metamorphoses are now relatively well understood from a morphological point of view. But morphology raises a lot of unsolved physiological problems.

Skin optics revisited by in vivo confocal microscopy: melanin and sun exposure.

A new confocal prototype dedicated to the exploration of in vivo human skin has been constructed around a laser confocal module (Oz Noran, Inc.) and a skin contact device, assuring perfect stability of skin images. The power of the Argon/Krypton laser source has been limited to 2mW to secure safety, and the laser provides three visible wavelengths: 488, 568, and 647 nm. Optical sections were digitized at video rate, providing easy and rapid measurements of the thickness of epidermal layers and time-resolved information. Unexpected details of the epidermis were recorded with the blue laser line. Melanin provided strong reflection of the basal keratinocytes instead of the absorption expected. The 3D reconstruction of the melanin cap in basal keratinocytes confirmed the behavior of melanosomes acting as myriads of nanomirrors that reflected light. Confocal images of the posterior aspect of the forearm were recorded before sun exposure and then for one month after exposure. There was a 25% increase in the thickness of the stratum corneum. Bright inclusions into the dark nucleus of numerous spinous cells were interpreted as local condensation of chromatin. Numerous bright intercellular filaments were attributed to melanosomes filling up dendrites of melanocytes. A striking observation concerned the lack of melanosome caps in basal keratinocytes. In vivo confocal microscopy affords new insight to the role of melanin and its gradual migration after sun exposure.

Persistence of both peripheral and non-peripheral corneodesmosomes in the upper stratum corneum of winter xerosis skin versus only peripheral in normal skin.

To understand the biochemical abnormalities that underlie the reduced desquamation observed in dry skin, we analyzed corneodesmosome degradation in normal and winter xerosis skin. Western blotting of total proteins from corneocytes obtained by varnish-strippings from the legs of 56 volunteers with normal (26) or xerotic (30) skin was performed using antibodies specific for (corneo)desmosome proteins. In the whole population, the amounts of desmoglein 1 and plakoglobin were found to be correlated, but were not related to the amounts of corneodesmosin. This suggests simultaneous proteolysis for the former proteins differing from that of corneodesmosin. Neither entire desmoplakins nor any proteolysis-derived fragments were detected. The amounts of corneodesmosin, desmoglein 1, and plakoglobin detected were found to be significantly higher in xerotic compared with normal skin extracts. Conventional and freeze-fracture electron microscopy showed the absence of nonperipheral corneodesmosomes in the upper stratum corneum of normal skin but the presence of a significant number of these structures in the same layer of winter xerosis skin. These results provide a more precise description of the proteolysis of corneodesmosome components in the upper cornified layer of the epidermis. They support previous studies demonstrating the importance of corneodesmosome degradation in desquamation and reveal that the nonperipheral corneodesmosomes, which are totally degraded during maturation of the stratum corneum in normal skin, persist in winter xerosis, probably leading to abnormal desquamation.

A clinical, biometrological and ultrastructural study of xerotic skin.

The purpose of this in vivo study was to investigate, non-invasively on human subjects, xerotic skin and its physiological evolution over time, compared to normal skin. Two groups of 17 female subjects were studied during the winter season, one made up of subjects with normal skin and the other subjects with xerotic skin. A clinical assessment and biometrological measurements of hydration and transepidermal water loss (TEWL) were performed on the same area of the external antero-lateral surface of the leg at the start of the study then after three weeks. At the end of the study, the ultrastructure of stratum corneum samples taken from the same area was examined by transmission electron microscopy. Subjects with xerotic skin were selected according to their impaired cutaneous barrier function, reflected in a TEWL higher than 12 g/m ; 2/h. Compared to normal subjects, they presented a hydration level more than 25% lower. After an interval of 21 days, no significant change in the hydration level or clinical appearance of the xerotic skin was observed. In contrast, the TEWL had decreased significantly (D _ 21- D _ 0=-3.6 g/m ; 2/h; p < 0.001) but still stayed higher than normal values. Changes in the ultrastructure of the stratum corneum were also observed in the subjects with xerotic skin. Unlike normal skin, corneosomes could be detected right up to the surface layers, accompanied by intercellular lipids in an amorphous form. These observations confirm the important roles played by both corneosomes and lipid organization in the cohesion/desquamation processes. In the subjects with normal skin, the hydration level and barrier function remained unchanged during the three week study but an onset of skin dryness was observed, the mean clinical score increasing by +1.3 (p = 0.01). These results confirm that there is no direct relationship between TEWL and the severity of skin dryness. It appears that a clinical evaluation is more sensitive than biometrological measurement for describing early state of cutaneous dryness. This study highlights the importance of a regular cosmetic or dermopharmaceutical treatment during the winter to prevent xerosis apparition on legs.

Three-dimensional microscopic biopsy of in vivo human skin: a new technique based on a flexible confocal microscope.

A new noninvasive microscopic technique of three-dimensional optical biopsy from in vivo human skin based on real-time confocal microscopy and computer reconstruction is demonstrated. A tandem scanning confocal microscope is a prototype of a mobile, flexible design for the in-depth microscopic exploration of the skin on the human body. The various skin layers were observed in real-time, at the subcellular level down to a depth of 200 microns with a vertical resolution of 2 microns. Rapid video recording of the Z-series through the ventral aspect of the forearm avoided shifts caused by subject movement and blood flow pulsations. Two video frames were averaged, and the average was digitized, providing a stack of 64 optical sections in 1-micron vertical steps. Three-dimensional reconstructions of in vivo human skin were obtained with sets of orthogonal slices, and slices at arbitrary planes through a volume containing the stack of slices. This method clearly shows the spatial relationships between the different cell layers. The use of orthogonal cutting planes is preferred because of its analogy with classical vertical sections of histopathology. Linear structures (surface lines) within the stratum corneum are described and their global orientations were determined by the use of Fourier transform analysis. En face optical sections constitute unusual views of this tissue, since typical pathohistological studies are based on sagittal (vertical) slices. The noninvasive optical microscopic technique provides a three-dimensional optical biopsy of in vivo human skin.

In vivo confocal microscopy of human skin: a new design for cosmetology and dermatology.

In-depth exploration of cellular structures in living human skin in situ is possible with the tandem scanning microscope (TSM). However, the rigid design of the microscope limited observations to the arms, hands, and fingers. A mobile version allowing the investigation of any parts of the body has been designed. The head containing the Nipkow disk and the optical path were the only part saved from the original TSM. This prototype can be used to observe, in real time, the different skin structures down to a depth of 200 microns and to measure the thickness of the different layers with micron precision level. The hydration of the stratum corneum (SC) could be assessed. For example, lengthy immersion of the hand in water led to an increase in SC thickness without affecting that of the living epidermis. Occlusive patch tests also showed that water and, even more so. propylene glycol, led to transient swelling of the SC. In dermatology, the example of psoriasis illustrated the value of the TSM for describing, measuring, and assessing pathologic skin changes. The availability of this noninvasive method for observing changes with time in a given skin site should prove useful for monitoring treatment efficacy. This tool opens up new insight for the investigation of cutaneous pathophysiology.

An improved technique for optical interferometric imaging of isolated cells.

We have improved the optical interferometric imaging technique that was recently used to measure local organic material concentrations in quasicylindrical cells. This allowed similar measurements for cells of arbitrary shape. The setup was used to measure the thickness of skin corneocytes.

Mechanism of action of a lipophilic derivative of salicylic acid on normal skin.

BACKGROUND/AIMS: Keratolytics are agents used for a very long period of time to improve various skin disorders such as acne, hyperkeratoses, ichtyose etc. Very little is known about their mechanism of action on healthy skin. On man, the chronic application of a cosmetic cream containing a lipophilic derivative of Salicyclic acid (LSA) markedly improves the aspect and texture of the skin. Different methods were used to investigate the mechanisms of action of this new compound, compared to salicyclic acid. METHODS: Both non-invasive and histologic methods were used on the dorsal forearm of human volunteers treated with the products. Concerning the non-invasive methods, TEWL, silflo replica and confocal microscopy were used. On shave biopsies, various histometric parameters were measured by image analysis after different staining. The use of antibody MIB-1 reacting with the proliferating nuclear antigen Ki 67 allows one to measure the epidermis proliferation index. RESULTS: Compared to the excipient alone, presence of LSA 1% improves smoothness and firmness of the skin. The appearance in terms of clearness and healthy complexion is also improved. The thickening of all the living epidermis layers is obtained by both histometric measurement and confocal microscopy. This acanthosis is only recorded on the LSA-treated zones. The Ki 67 labelling study shows that LSA significantly increases the skin proliferation index. CONCLUSIONS: Salicylic acid, and more markedly its lipophilic derivative (LSA), appear to have a significative effect on the renewal of the living epidermis. This probably explains the cosmetic improvement of the skin obtained after a 1-month treatment with a cream containing this new molecule.

In vivo spatio-temporal visualization of the human skin by real-time confocal microscopy.

A modified tandem scanning confocal microscope is used to obtain in vivo images of the human skin in real time. Three-dimensional and temporal visualizations are demonstrated with volume reconstruction and blood flow images. Two image processing methods based on Fourier transform and logarithmic processing are presented. Their applications in noise removal of the scanning disk lines and of the heterogeneity of light are illustrated.

Three-dimensional measurements of skin surface topography by triangulation with a new laser profilometer.

The fast measurement of furrows or wrinkles requires the use of a non-contact device. We have chosen a laser detector based on a triangulation principle, using position-sensitive detectors. This apparatus has a sensitivity of about 2-3 microns for a vertical range > 1 mm; this vertical range can reach 8 mm. There is no contact between the detector and the surface and this reduces the measurement time because, while data are being transmitted, there is no decrease in the scanning speed as with earlier methods. We describe the device and its technical characteristics. The limits of use are shown in the measurement of low-amplitude defects (> 3-4 microns) and of wide defects of < or = 8 mm. Examples of software possibilities and practical applications related to skin microrelief, wrinkles and pathological cases, are also described.

[Microscopic diagnosis of onychomycoses]. / Diagnostic microscopique des onychomycoses.

INTRODUCTION: The microbiological identification of onychomycosis may be uncertain as some fungi growing in culture from this material are not necessarily pathogen and invasive. Conversely, the negativity of a culture is not rare even when fungi are seen by microscopy. MATERIALS AND METHODS: We compared the information brought by standard histological examination, immunohistochemistry and in vivo confocal microscopy. RESULTS: The histological examination of nails is of importance in this pathology. We present a laboratory technique that proves to be easy and rapid. We report diagnostic criteria allowing the distinction between dermatophytes, yeasts and non-dermatophyte molds. The standard microscopic examination may further be improved by immunohistochemistry using some antibodies to fungi. In vivo confocal microscopy is a technique for the future. The dermatologist will be able to see fungi in the nail at the clinical examination, without any sampling or peculiar preparation. CONCLUSION: The histological examination is a routine technique useful for defining the nature and localization of fungi in the nail plate. Immunohistochemistry applied to onychomycosis is an experimental approach bringing prominent informations about the identification of fungi. In vivo confocal microscopy is a door opened to the future.

High-spatial-resolution maps of sulphur from human hair sections: an EELS study.

High-resolution sulphur maps have been acquired from human hair using a Zeiss CEM 902A transmission electron microscope equipped with an energy filter. Analysis by electron energy-loss spectroscopy (EELS) was performed on ultrathin sections of hair shafts embedded in three different types of resin: Nanoplast (water-soluble), Spurr (epoxy) and Lowicryl (low-temperature resin). Good-quality energy-loss images have been obtained with the three resins, although it was found that Nanoplast gave the best image contrast. For the first time, the results obtained for the detection of sulphur by silver staining of hair sections, which until now has been the only way to map sulphur at the electron microscopic level, have been confirmed. The results are compared with local sulphur concentrations from bulk analysis.

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 vision of the human skin with the tandem scanning microscope.

The tandem scanning reflected light microscope invented by Petran and Hadravsky provides real-time vision in the confocal mode. It performs nondestructive optical sectioning of biological material to a depth that depends on the transparency of the tissue. This real-time facility adds a fourth dimension (time) to the study of living specimens. In the present work, human skin was explored in vivo, at the cellular level to a depth of 150 microns. Two different anatomical sites were investigated: the back of the hand and the volar aspect of the forearm. The stratification of the horny layers, the nuclei of the living keratinocytes through the whole epidermis and the capillary loops within the superficial dermis constituted clear reference points. These permitted thickness measurements of the stratum corneum and epidermis to an accuracy of 1 microns. This method of exploration provides a new challenge for dermatologists and cosmetologists.

Extension-induced changes in the microrelief of the human volar forearm: variations with age.

The skin microrelief can be assessed in vivo using a noninvasive method based on the image analysis of negative replicas, shadowed by oblique illumination. This technique was used to study changes in the surface furrows of the human volar forearm, related to the degree of extension, in volunteers of three age groups. Results confirmed that the skin in elderly subjects can be characterized by the lack of one of the two perpendicular furrow orientations observed in the young. During extension of the arm, the response of the microrelief in young subjects was clearly different from that in the elderly. Young skin buffered strain through a progressive shift in line density and furrow depth from one orientation to the other, whereas in the elderly, a progressive rotation of the single furrow orientation occurred. These differences would appear to be related to changes in the architecture and physical properties of the dermis. Microrelief, in the elderly subjects, showed a mean line density of 24/cm, regardless of the degree of extension. This constancy may reflect the ultimate stage in the chronological aging of the skin and may be characteristic of a loosened and relaxed dermis.

Racial differences in corneocytes. A comparison between black, white and oriental skin.

It is well known that spontaneous desquamation and corneocyte size can reflect respectively stratum corneum cohesiveness and epidermal cell proliferation. The influence of skin pigmentation on these parameters has been investigated on the upper-outer arm of black, white and oriental volunteers, using the detergent scrub method. We found no difference between race in corneocyte surface area, a mean size of 900 microns 2 agreeing closely with that generally encountered in Whites on the upper-outer arm. By contrast, spontaneous desquamation is increased in black vis-à-vis white and oriental skin (factor 2.5, p less than 0.001). Taking into account the importance of the intercellular cement for the cohesion between corneocytes, racial differences in epidermal lipid composition should be investigated.