Noninvasive Determination of Epidermal and Stratum Corneum Thickness in vivo Using Two-Photon Microscopy and Optical Coherence Tomography: Impact of Body Area, Age, and Gender.

BACKGROUND: High-resolution images of the epidermis are important to understand the transdermal penetration and changes in epidermal components. Both ex vivo and in vivo technologies are available to picture the epidermal thickness (ET). So far, the illustration of the stratum corneum (SC) has not been possible without artifacts. OBJECTIVE: Precision in vivo measurement of the ET and SC, duly considering the impact of location on the body, age, and gender. METHODS: In this pilot study on 20 skin-healthy subjects aged 18-66 years, the ET was imaged by two-photon microscopy (2PM) and optical coherence tomography (OCT), and the SC by using 2PM at five different body sites. RESULTS: On solar-exposed body areas, both the epidermis and SC are thicker compared to solar-protected areas (p < 0.05), the epidermis at the gluteal region being the thickest (p < 0.05). The ET decreases with age (p < 0.05). Males show a thicker epidermis than females (p < 0.05). CONCLUSION: 2PM provides a noninvasive method for imaging the epidermis and especially the SC in vivo and is optimally suited for the application of histological criteria.

Body regions have an impact on the collagen/elastin index of the skin measured by non-invasive in vivo vertical two-photon microscopy.

It is known that the collagen and elastin fibre structures are changing with age. There is little knowledge about the influence of body area as these investigations have been limited for ethical reasons, so far. Thus, modified non-invasive two-photon microscopy was used providing vertical optical sections of second harmonic generation and autofluorescence to calculate the collagen-to-elastin ratio and its alterations depending on the investigated body site in vivo. The results of this study indicate that the impact of different body areas could be higher than the influence of age and should be considered in future studies.

Impact of Body Site, Age, and Gender on the Collagen/Elastin Index by Noninvasive in vivo Vertical Two-Photon Microscopy.

BACKGROUND/AIMS: Extrinsic and intrinsic skin aging is subject to constant remodeling and degradation processes, primarily in components of the extracellular matrix. While collagen fibers thin out during the aging process, the amorphous elastin fibers accumulate. These are essential formative components of the dermis. So far, these processes have been detected in vertical histological sections of invasive biopsies and recently in noninvasive horizontal scans. METHODS: In this pilot study, a modified noninvasive 2-photon microscope was applied to measure the collagen/elastin index of skin in vivo. The obtained images permit an immediate vertical survey and allow a conclusion on the dermal composition at once. The collagen/elastin index was quantified by the second harmonic to autofluorescence aging index of dermis (SAAID) depending on volunteers' age (18-66 years), gender, and body area. RESULTS: The highest SAAID was measured at the volar forearm as compared to the abdominal SAAID, which was significantly lower (p < 0.05). The gluteal region showed the significantly lowest SAAID (p < 0.05). The SAAID in female skin was higher compared to male skin and decreased with increasing age. CONCLUSION: These effects are to be considered in subsequent studies to be able to specifically detect and evaluate influences.

Sentinel-Lymphknoten-Biopsie des Melanoms mittels Indocyaningrün und "FOVIS"-System.

HINTERGRUND: Der Nachweis metastatischer Infiltrate im Sentinel-Lymphkoten (SLN) gilt als wesentlicher prognostischer Faktor des Melanoms. Alternativ zur Farbstoffmethode mit Patentblau zum Goldstandard der SLN-Biopsie (SLNB) mittels Radiokolloid wird die fluoreszenzoptische Darstellung mit Hilfe von Indocyaningrün (ICG) und Nahinfrarot (NIR)-Kamerasystem kommuniziert. Im Vergleich zur konventionellen Methode wurde die Wertigkeit des ICG-/NIR-Verfahrens in Abhängigkeit vom Body-Mass-Index (BMI) des Patienten und der Konzentration von ICG bezüglich der Visualisierung des Lymphabstroms und des SLNs untersucht. PATIENTEN UND METHODIK: An zehn Patienten wurde die SLNB mittels Technetium-99m, Patentblau und ICG durchgeführt. Die Fluoreszenz-Darstellung von Lymphbahnen und SLN erfolgte in Echtzeit mittels der NIR-Kameratechnik "FOVIS". Je nach erzielter Bildqualität wurde ICG in einer Dosis von 0,25 mg bis 2,5 mg intrakutan appliziert. ERGEBNISSE: Neun der zehn SLN wurden fluoreszenzoptisch identifiziert (90 %), alle zehn radioaktiv (100 %), nur acht (80 %) mittels ICG-Grünfärbung bzw. Patenblau-Markierung. Transdermal wurde ein SLN dargestellt (10 %). In Korrelation zum BMI waren höhere ICG-Mengen, bis zu 2,5 mg intrakutan absolut, in der Darstellung der Lymphbahnen von Vorteil. SCHLUSSFOLGERUNGEN: Die SLN-Fluoreszenzmarkierung mit dem ICG/NIR-Kamera-System "FOVIS" stellt eine sichere Alternative zur Farbstoffmethode mit Patentblau ergänzend zur Radiokolloidmethode mit Technetium-99m dar. Weitere Studien zur optimalen Dosierung von ICG und transdermalen Bildgebung in Relation zum BMI sind notwendig.

Indocyanine green-assisted sentinel lymph node biopsy in melanoma using the "FOVIS" system.

BACKGROUND: Metastatic involvement of the sentinel lymph node (SLN) represents a key prognostic factor in melanoma. The combined use of a radiocolloid (technetium-99m) and blue dye is the gold standard in sentinel lymph node biopsy (SLNB). In this context, near-infrared (NIR) fluorescence imaging with indocyanine green (ICG) has been suggested as an alternative. The objective of the present study was to examine the potential advantages of fluorescence-guided SLNB - compared to the conventional method - with respect to the visualization of lymphatic drainage pathways and the SLN. Particular focus was on the impact of the ICG dose used and the body mass index (BMI). PATIENTS AND METHODS: The study included ten patients who underwent the SLNB procedure using technetium-99m, blue dye, and ICG. Real-time fluorescence imaging of lymphatic drainage pathways and the SLN was done using the "FOVIS"-NIR system. Depending on the quality of the images achieved, ICG was intradermally administered at a dose ranging from 0.25 to 2.5 mg. RESULTS: Nine SLNs were identified by fluorescence (90 %); (100 %) ten, by gamma probe; eight (80 %), by ICG or blue dye. Transdermal SLN detection was possible in one case (10 %). In correlation to the BMI, higher intradermal ICG doses - up to 2.5 mg overall - proved to be advantageous in the visualization of lymphatic vessels. CONCLUSIONS: Supplementing the technetium-99m method, fluorescence SLNB using ICG and the "FOVIS"-NIR system is a safe alternative to the blue-dye technique. Further studies on the optimal ICG dose and transdermal imaging in correlation to the BMI are required.

Identification of polystyrene nanoparticle penetration across intact skin barrier as rare event at sites of focal particle aggregations.

The question whether nanoparticles can cross the skin barrier is highly debated. Even in intact skin rare events of deeper penetration have been reported, but technical limitations and possible artifacts require careful interpretation. In this study, horizontal scanning by 2-photon microscopy (2 PM) of full-thickness human skin samples placed in a lateral position yielded highly informative images for skin penetration studies of fluorescently tagged nanoparticles. Scanning of large fields of view allowed for detailed information on interfollicular and follicular penetration in tissue blocks without damaging the sample. Images in histomorphological correlation showed that 2P-excited fluorescence signals of fluorescently tagged 20 and 200 nm polystyrene nanoparticles preferentially accumulated in the stratum corneum (SC) and in the upper part of vellus hair follicles (HFs). Rare events of deeper penetration in the SC and in the infundibulum of vellus HFs were observed at sites of high focal particle aggregations. Wide-field 2 PM allows for imaging of nanoparticle penetration in large tissue blocks, whereas total internal reflection microscopy (TIRFM) enables selective detection of individual nanoparticles as well as clusters of nanoparticles in the SC and within the epidermal layer directly beneath the SC, thus confirming barrier crossing with high sensitivity.