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Excessive exposure to ultraviolet (UV) light leads to acute and chronic UV damage and is the main risk factor for the development of skin cancer. In most countries with western lifestyle, the topical application of sunscreens on UV‐exposed skin areas is by far the most frequently used preventive measure against sunburn. Further than preventing sunburns, increasing numbers of consumers are appreciating sunscreens with a medium‐ to high‐level sun protective factor (SPF) as basis for sustainable‐skin ageing or skin cancer prevention programs. However, recent investigations indicate that clinically significant DNA damages as well as a lasting impairment of cutaneous immunosurveillance already occur far below the standard of one minimal erythema dose (MED) sunburn level, which contributes to the current discussion of the clinical value of high‐protective SPF values. Ex vivo investigations on human skin showed that the application of SPF30 reduces DNA damage for a day long sun exposure (24 MED) drastically by about 53% but is significantly surpassed by SPF100 reducing DNA damage by approx. 73%. Further analysis on different SPF protection levels in UV‐exposed cell culture assays focusing on IL‐18, cell vitality and cis/trans‐urocanic acid support these findings. Whereas SPF30 and SPF50+ sunscreens already offer a solid UVB cover for most indications, our results indicate that SPF100 provides significant additional protection against mutagenic (non‐apoptotic‐) DNA damage and functional impairment of the cutaneous immunosurveillance and therefore qualifies as an optimized sunscreen for specifically vulnerable patient groups such as immunosuppressed patients, or skin cancer patients. [ABSTRACT FROM AUTHOR]

Fibroblasts are among the most common cell types in the stroma responsible for creating and maintaining the structural organization of the extracellular matrix in the dermis, skin regeneration, and a range of immune responses. Until now, the processes of fibroblast adaptation and functioning in a varying environment have not been fully understood. Modern laser microscopes are capable of studying fibroblasts in vitro and ex vivo. One‐photon‐ and two‐photon‐excited fluorescence microscopy, Raman spectroscopy/microspectroscopy are well‐suited noninvasive optical methods for fibroblast imaging in vitro and ex vivo. In vivo staining‐free fibroblast imaging is not still implemented. The exception is fibroblast imaging in tattooed skin. Although in vivo noninvasive staining‐free imaging of fibroblasts in the skin has not yet been implemented, it is expected in the future. This review summarizes the state‐of‐the‐art in fibroblast visualization using optical methods and discusses the advantages, limitations, and prospects for future noninvasive imaging. [ABSTRACT FROM AUTHOR]

Far‐UVC radiation sources of wavelengths 222 nm and 233 nm represent an interesting potential alternative for the antiseptic treatment of the skin due to their high skin compatibility. Nevertheless, no studies on far‐UVC‐induced DNA damage in different skin types have been published to date, which this study aims for. After irradiating the skin with far‐UVC of the wavelengths 222 and 233 nm as well as broadband UVB, the tissue was screened for cyclobutane pyrimidine dimer‐positive (CPD+) cells using immunohistochemistry. The epidermal DNA damage was lower in dark skin types than in fair skin types after irradiation at 233 nm. Contrary to this, irradiation at 222 nm caused no skin type‐dependent differences, which can be attributed to the decreased penetration depth of radiation. UVB showed the relatively strongest differences between light and dark skin types when using a suberythemal dose of 3 mJ/cm2. As melanin is known for its photoprotective effect, we evaluated the ratio of melanin content in the stratum basale and stratum granulosum in samples of different skin types using two‐photon excited fluorescence lifetime imaging (TPE‐FLIM) finding a higher ratio up to skin type IV–V. As far‐UVC is known to penetrate only into the upper layers of the viable skin, the aforementioned melanin ratio could explain the less pronounced differences between skin types after irradiation with far‐UVC compared to UVB. [ABSTRACT FROM AUTHOR]

Glabrous skin is hair‐free skin with a high density of sweat glands, which is found on the palms, and soles of mammalians, covered with a thick stratum corneum. Dry hands are often an occupational problem which deserves attention from dermatologists. Urea is found in the skin as a component of the natural moisturizing factor and of sweat. We report the discovery of dendrimer structures of crystalized urea in the stratum corneum of palmar glabrous skin using laser scanning microscopy. The chemical and structural nature of the urea crystallites was investigated in vivo by non‐invasive techniques. The relation of crystallization to skin hydration was explored. We analysed the index finger, small finger and tenar palmar area of 18 study participants using non‐invasive optical methods, such as laser scanning microscopy, Raman microspectroscopy and two‐photon tomography. Skin hydration was measured using corneometry. Crystalline urea structures were found in the stratum corneum of about two‐thirds of the participants. Participants with a higher density of crystallized urea structures exhibited a lower skin hydration. The chemical nature and the crystalline structure of the urea were confirmed by Raman microspectroscopy and by second harmonic generated signals in two‐photon tomography. The presence of urea dendrimer crystals in the glabrous skin seems to reduce the water binding capacity leading to dry hands. These findings highlight a new direction in understanding the mechanisms leading to dry hands and open opportunities for the development of better moisturizers and hand disinfection products and for diagnostic of dry skin. [ABSTRACT FROM AUTHOR]

Background: The knowledge about the location and kinetics of tattoo pigments in human skin after application and during the recovery is restricted due to the limitation of in vivo methods for visualizing pigments. Here, the localization and distribution of tattoo ink pigments in freshly and old tattooed human skin during the regeneration of the epidermis and dermis were investigated in vivo. Methods: Two-photon excited fluorescence lifetime imaging (TPE-FLIM) was used to identify tattoo ink pigments in human skin in vivo down to the reticular dermis. One subject with a freshly applied tattoo and 10 subjects with tattoos applied over 3 years ago were investigated in the epidermal and dermal layers in vivo. One histological slide of tattooed skin was used to localize skin-resident tattoo pigment using light microscopy. Results: The carbon black particles deposited around the incision have still been visible 84 days after tattoo application, showing delayed recovery of the epidermis. The TPE-FLIM parameters of carbon black tattoo ink pigments were found to be different to all skin components except for melanin. Distinction from melanin in the skin was based on higher fluorescence intensity and agglomerate size. Using TPE-FLIM in vivo tattoo pigment was found in 75% of tattoos applied up to 9 years ago in the epidermis within keratinocytes, dendritic cells, and basal cells and in the dermis within the macrophages, mast cells, and fibroblasts. Loading of highly fluorescent carbon black particles enables in vivo imaging of dendritic cells in the epidermis and fibroblasts in the dermis, which cannot be visualized in native conditions. The collagen I structures showed a higher directionality similar to scar tissue resulting in a greater firmness and decreased elasticity of the tattooed skin. Conclusions: Here, we show the kinetics and location of carbon black tattoo ink pigment immediately after application for the first time in vivo in human skin. Carbon black particles are located exclusively intracellularly in the skin of fresh and old tattoos. They are found within macrophages, mast cells, and fibroblasts in the dermis and within keratinocytes, dendritic cells, and basal cells in the continuously renewed epidermis even in 9-year-old tattoos in skin showing no inflammation. [ABSTRACT FROM AUTHOR]

Macrophages (Φs) are important immune effector cells that promote (M1 Φs) or inhibit (M2 Φs) inflammation and are involved in numerous physiological and pathogenic immune responses. Their precise role and relevance, however, are not fully understood for lack of noninvasive quantification methods. Here, we show that two-photon excited fluorescence lifetime imaging (TPE-FLIM), a label-free noninvasive method, can visualize Φs in the human dermis in vivo. We demonstrate in vitro that human dermal Φs exhibit specific TPE-FLIM properties that distinguish them from the main components of the extracellular matrix and other dermal cells. We visualized Φs, their phenotypes and phagocytosis in the skin of healthy individuals in vivo using TPE-FLIM. Additionally, machine learning identified M1 and M2 Φs with a sensitivity of 0.88±0.04 and 0.82±0.03 and a specificity of 0.89±0.03 and 0.90±0.03, respectively. In clinical research, TPE-FLIM can advance the understanding of the role of Φs in health and disease. [ABSTRACT FROM AUTHOR]

The stratum corneum (SC) forms a strong barrier against topical drug delivery. Therefore, understanding the penetration depth and pathways into the SC is important for the efficiency of drug delivery and cosmetic safety. In this study, TPT-FLIM (two-photon tomography combined with fluorescence lifetime imaging) was applied as a non-invasive optical method for the visualization of skin structure and components to study penetration depths of exemplary substances, like hydrophilic propylene glycol (PG), sodium fluorescein (NaFl) and lipophilic Nile red (NR) into porcine ear skin ex vivo. Non-fluorescent PG was detected indirectly based on the pH-dependent increase in the fluorescence lifetime of SC components. The pH similarity between PG and viable epidermis limited the detection of PG. NaFl reached the viable epidermis, which was also proved by laser scanning microscopy. Tape stripping and confocal Raman micro-spectroscopy were performed additionally to study NaFl, which revealed penetration depths of ≈5 and ≈8 μm, respectively. Lastly, NR did not permeate the SC. We concluded that the amplitude-weighted mean fluorescence lifetime is the most appropriate FLIM parameter to build up penetration profiles. This work is anticipated to provide a non-invasive TPT-FLIM method for studying the penetration of topically applied drugs and cosmetics into the skin. [ABSTRACT FROM AUTHOR]

The assessment of dermal alterations is necessary to monitor skin aging, cancer, and other skin diseases and alterations. The gold standard of morphologic diagnostics is still histopathology. Here, we proposed parameters to distinguish morphologically different collagen I structures in the extracellular matrix and to characterize varying collagen I structures in the skin with similar SAAID (SHG-to-AF Aging Index of Dermis, SHG—second-harmonic generation; AF—autofluorescence) values. Test datasets for the papillary and reticular extracellular matrix from images in 24 female subjects, 36 to 50 years of age, were generated. Parameters for SAAID, edge detection, and fast Fourier transformation directionality were determined. Additionally, textural analyses based on the grey level co-occurrence matrix (GLCM) were conducted. At first, changes in the GLCM parameters were determined in the native greyscale images and, furthermore, in the Hilbert-transformed images. Our results demonstrate a robust set of parameters for noninvasive in vivo classification for morphologically different collagen I structures in the skin, with similar and different SAAID values. We anticipate our method to enable an automated prevention and monitoring system with an age- and gender-specific algorithm. [ABSTRACT FROM AUTHOR]

Topical retinoid treatments stimulate biological activities in the skin. The main physical barrier, which limits the efficacy of transdermal drug delivery, is the stratum corneum. Proretinal nanoparticles (PRN) have already been proven to efficiently deliver retinal into the epidermis. In the present study, two transdermal drug delivery systems, microneedles (MN) and PRN, were combined to directly target the dermis. The microchannels induced by the MN, the PRN localization in the microchannels and the skin closure kinetics were investigated by non-invasive imaging techniques, such as dermoscopy, optical coherence tomography and multiphoton tomography. Additionally, the amount of retinal in the epidermis and dermis after application in three different forms (PRN-Loaded microneedles, PRN suspension or conventional retinal solution) was compared. All imaging techniques confirmed the formation of microchannels in the skin, which were partly still detectable after 24 h. Multiphoton tomography showed the release of PRN from the MN within the microchannels. The recovered retinal concentration in the dermis was significantly higher when applied via PRN-loaded microneedles. We hypothesized that this platform of PRN-loaded microneedles can provide a rapid and efficient administration of retinal in the dermis and could be of benefit in some skin conditions such as atrophic scar or photo-aged skin. [ABSTRACT FROM AUTHOR]

• Ex vivo barrier-disrupted skin could be established by ≤ 30 tape strips. • Ex vivo barrier-disrupted skin could be prepared by ≤ 2 cyanoacrylate stripping. • 50 tape strips or 4 cyanoacrylate strippings entirely remove human stratum corneum. • Each cyanoacrylate stripping removes a similar amount (~2 µm) of stratum corneum. • Cyanoacrylate stripping can remove viable skin layers. Numerous studies have employed tape stripping (TS) or cyanoacrylate stripping (CS) to induce skin barrier disruption of the stratum corneum (SC) in human and porcine skin. However, the thickness of the remaining SC and the respective changes of the skin permeability have been rarely quantified. By using high-resolution multiphoton tomography, about 5 µm thick SC was found remaining on human skin after the performance of 30 times TS or 2 times CS. 50 tape strips or 4 times CS removed the entire human SC, but on porcine skin 2–3 µm thick SC was still left. TS can only reach the transition zone between the SC and the stratum granulosum because of the limited adhesion, whereas CS was able to remove viable skin layers. Permeation investigations on porcine skin revealed that the apparent permeability coefficient of the hydrophilic nitroxide spin 2,5,5-Tetramethyl-1-pyrrolidinyloxy-3-carboxylic acid increased 15-, 18-, and 21-fold when the SC amount remaining in the skin was 30%, 16%, and 8%, respectively. It is recommended to use at most 30 times TS or 3 times CS to obtain ex vivo barrier-disrupted skin that mimics diseased skin. The study provides quantitative information for the utility of TS and CS in skin penetration research. [ABSTRACT FROM AUTHOR]