In vivo skin measurements with a novel probe head for simultaneous skin impedance and near‐infrared spectroscopy

Background/purpose: Near‐infrared (NIR) spectroscopy and skin impedance (IMP) measurements are useful techniques for objective diagnostics of various skin diseases. Here, we present a combined probe head for simultaneous, time‐saving NIR spectroscopy and skin impedance measurements. The probe also ensures that both measurements are performed under equal conditions and at the same skin location. Methods: Finite element method simulations were performed for evaluation of the impedance. In vivo skin measurements were performed and combined NIR and impedance spectra were analysed by means of multivariate methods with respect to body location, age and gender. The classification rate was determined by a planar discriminant analysis. Reproducibility was investigated by calculation of scatter values and statistical significance between overlapping groups was assessed by the calculation of intra‐model distances, q. Results: The novel probe yielded rapid reproducible results and was easy to manage. Significant differences between skin locations and to a lesser extent age groups and gender were demonstrated. Conclusion: With the novel probe, statistically significant differences between overlapping classes in score plots can be confirmed by calculating intra‐model distances. The influence of molecular differences in the skin at different body locations is larger than the influence of gender or age and therefore relevant reference measurements are discussed.     

Mid-infrared in vivo depth-profiling of topical chemicals on skin

Background/purpose: Thermal emission decay–Fourier transform infrared (TED–FTIR) spectroscopy is a non-contact and non-destructive analytical technique and was used in this study to detect the presence of external chemicals on human skin in vivo . The detection was possible due to the ability of the TED–FTIR technique to acquire the mid-infrared spectrum of the outmost layers (less than 10 μm) of Stratum Corneum (SC) and the ability to identify the absorption bands of the chemical.
Methods: As an illustration of such measurements, propylene glycol (PG) was applied on human stratum corneum and depth-resolved TED–FTIR spectra of the SC were measured to quantify the concentration of PG in deeper layers of SC.
Results: The mid-infrared spectrum of the surface 0.7 μm layer of skin had 50% contribution from SC and 50% from PG. At 3 h after application, the contribution of PG at the surface decreased to 7% as PG molecules diffused deeper into the skin and were lost at the surface. At a depth of 6 μm, the maximum concentration was 20% after 25 min after PG application.
Conclusions: This work shows the feasibility of the TED–FTIR technique to detect the presence of chemicals on human SC in vivo and without contact, and for a wide range of other applications, such as detection of toxic chemicals used as warfare (vesicant agents like sulphur mustard and organophosphate nerve agents), pesticides, and other toxins on fruit and vegetable skins, water, or even other contaminated surfaces.     

In vivo study for the discrimination of cancerous and normal skin using fibre probe‐based Raman spectroscopy

Raman spectroscopy has proved its capability as an objective, non‐invasive tool for the detection of various melanoma and non‐melanoma skin cancers (NMSC) in a number of studies. Most publications are based on a Raman microspectroscopic ex vivo approach. In this in vivo clinical evaluation, we apply Raman spectroscopy using a fibre‐coupled probe that allows access to a multitude of affected body sites. The probe design is optimized for epithelial sensitivity, whereby a large part of the detected signal originates from within the epidermal layer's depth down to the basal membrane where early stages of skin cancer develop. Data analysis was performed on measurements of 104 subjects scheduled for excision of lesions suspected of being malignant melanoma (MM) (n = 36), basal cell carcinoma (BCC) (n = 39) and squamous cell carcinoma (SCC) (n = 29). NMSC were discriminated from normal skin with a balanced accuracy of 73% (BCC) and 85% (SCC) using partial least squares discriminant analysis (PLS‐DA). Discriminating MM and pigmented nevi (PN) resulted in a balanced accuracy of 91%. These results lie within the range of comparable in vivo studies and the accuracies achieved by trained dermatologists using dermoscopy. Discrimination proved to be unsuccessful between cancerous lesions and suspicious lesions that had been histopathologically verified as benign by dermoscopy.     

Remittance spectroscopy mapping of human skin in vivo

Remittance spectroscopy of human skin may be influenced by probe application pressure and body site.

Methods:

We investigated remittance spectroscopy qualities of human skin in vivo in different areas: a) forearm, b) frontal, c) back, d) back of the hand, e) palms and f) cheek. Twenty volunteers of skin type 2–3 free of inflammatory skin diseases, were enrolled into the study. Spectroscopy readings were performed with a fiber optic spectrometer (Ocean Optics, USA). The readings were taken with standardized force (0 and 100 pont) by applying the probe vertically to the skin surface. The remittance in relation to wavelength was registered. White light with wavelengths from 420 to 750 nm were used. Individual remittance values and their standard deviations were obtained from 20 readings each.

Results/Conclusions:

Spectroscopic patterns of skin are influenced by external force and regional factors. Standardization remains critical for the use of this approach in bioengineering of skin.     

Remittance spectroscopy of human skin in vivo

Background/aims: The skin is an easily accessible organ on which non-invasive examination methods can be applied. Recently spectroscopic methods have been introduced in characterization of skin under physiological and pathological conditions. To examine remittance spectroscopic qualities of human skin in vivo and to clarify the influence of selected test conditions, a single-beam spectrometer MCS 410 (Carl Zeiss, Jena, Germany) has been used.
Methods: Remittance spectra readings were performed in 35 volunteers. Wavelength ranged from 362 nm to 780 nm. Individual remittance values and their standard deviations were obtained from 20 readings under standardized test conditions. The effect of pressure, rubbing, cooling, washing, greasing and degreasing on average remittance values was investigated. Statistical analysis was done with paired Student's f-test and Fisher's test.
Results: Pressure increased remittance values over a wide range of wavelength, peaking at 518 nm. Greasing and degreasing modified spectral remittance of shorter wavelength peaking around 362 nm. Rubbing and cooling did not induce significant variations of spectral remittance of skin.
Conclusion: Spectral remittance provides an individual profile in human skin, which may be influenced by pressure and greasing/ degreasing. To establish standardized test conditions with a narrow range of intra-individual variation these items have to be kept constant.     

Freeze‐drying as a preserving preparation technique for in vitro testing of human skin

In vitro testing of drugs with excised human skin is a valuable prerequisite for clinical studies. However, the analysis of excised human skin presents several obstacles. Ongoing drug diffusion, microbial growth and changes in hydration state influence the results of drug penetration studies. In this work, we evaluate freeze‐drying as a preserving preparation method for skin samples to overcome these obstacles. We analyse excised human skin before and after freeze‐drying and compare these results with human skin in vivo. Based on comprehensive thermal and spectroscopic analysis, we demonstrate comparability to in vivo conditions and exclude significant changes within the skin samples due to freeze‐drying. Furthermore, we show that freeze‐drying after skin incubation with drugs prevents growth of drug crystals on the skin surface due to drying effects. In conclusion, we introduce freeze‐drying as a preserving preparation technique for in vitro testing of human skin.     

Exploratory multivariate spectroscopic study on human skin

Background/aims: Spectroscopy on human skin is a field that is being adopted increasingly because of its rapidity and high reproducibility. Infrared reflectance (IR), near-infrared reflectance (NIR), and fluorescence spectroscopy have previously been applied to human skin in vivo to compare healthy and sick skin, including skin cancer, atopy, and leprosy. Exploratory data analysis/chemometrics is a tool for evaluating multivariate data such as spectroscopic measurements. The objective of this study was to explore the spectral variance spanned by people with normal integument, and to demonstrate the advantages of multivariate analysis to skin research.
Methods: IR, NIR and fluorescence spectroscopy have been carried out in vivo on 216 volunteers' forearms before and after four tape strippings. The subjects were asked to fill in a questionnaire regarding factors suspected to influence the measurement results. Principal Component Analysis (PCA) was used to investigate whether the population can be divided into groups on the basis of their skin chemistry. Unless otherwise stated, the results are from the measurements prior to stripping.
Results: In contrast to IR and fluorescence spectra, NIR spectra proved able to detect gender differences. By use of PCA, classifications on male and female subjects were observed from the IR and NIR measurements, and as an indication from the fluorescence measurements. The NIR and fluorescence measurements varied between elderly and young subjects. The largest variance in the fluorescence landscapes was seen between pigmented and non-pigmented skin. No connection was found between the spectroscopic measurements and smoking or drinking habits.
Conclusions: Future spectroscopic skin investigations should be balanced as regards to gender and age, as these can possibly affect the measurement results. Chemometrics proved to be superior to traditional attempts of interpreting the spectra.     

The effect of probe depth on histamine levels in humans. In vivo cutaneous microdialysis

Background/aims: Microdialysis technique, for the in vivo estimation of endogenous and exogenous substances in tissues, is gaining increasing use in human experimentation. The cutaneous microdialysis technique has interesting applications in several fields, including the study of skin inflammation and percutaneous absorption. One of the aspects of cutaneous microdialysis which needs clarification is the influence on results of the exact position of the microdialysis probe in the dermis.
Methods: In a total material of 67 histamine experiments, probe depth has been measured by the ultrasound technique. In 24 uniform experiments, histamine values at 10-min intervals after probe insertion and prior to other provocation have been analyzed in relation to probe depth.
Results: The mean probe depth was 1.1 mm (range 0.8-1.9 mm). In regression analysis of histamine values in 10-min intervals up to 90 min after probe insertion in relation to probe depth, the histamine values were found to be independent of probe depth. This may be due to rapid local diffusion of histamine, together with axon reflex mediated histamine release. Further analysis revealed no correlation between histamine results and the subject's age, sex or presence of atopy.
Conclusion: The present results indicate that the exact placement of the microdialysis probe into the dermis is not too critical in experiments where histamine is to be studied. It must be pointed out that the same may not necessarily be true of other inflammatory mediators: for each new target substance and application, the influence of probe placement needs to be considered.     

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.     

In vivo measurement of the photostability of sunscreen products using diffuse reflectance spectroscopy

BACKGROUND/AIM: The issue of the photostability of sunscreens has been frequently raised because of the possible loss of photoprotection, mainly in the UVA range, during sun exposure. Up to now, in vitro techniques have been mainly proposed to evaluate photostability. These techniques have generated controversial debates concerning the predictive value of these in vitro observations in relation to the in vivo reality during sun exposure. METHODS: Diffuse reflectance spectroscopy (DRS) is a recently developed technique that allows measurement of the UVA efficacy of sunscreen products in vivo on human volunteers. The absorption spectrum of the product is obtained by measuring the change in reflection of the skin with and without product. From this absorption spectrum, the UVA protective efficacy of the test product can be calculated for an appropriate source and a given biological action spectrum. We have used the DRS technique in vivo to determine the photostability of sunscreen products by measuring reflection spectra in the UVA range (320-400 nm) before and after product application and before and after UV exposure of the test products. Comparison between these spectra or between the corresponding calculated UVA protection factors has made it possible to determine the remaining level of protection in the UVA range after exposure. This study was designed to compare in vivo the protective efficiency and the photostability of three marketed sunscreen products (SPF 23-30) after solar-simulated exposure for SPF testing or after actual sun exposure. These in vivo data were then compared to in vitro photoinstability results. RESULTS: According to the in vitro measurements, one sunscreen product was found photostable and two products photo-unstable. After UVe exposure for in vivo SPF determination, a decrease in UVA absorption and UVA-PF was observed for the two photo-unstable products, while the photostable product did not present any decrease in UVA absorption. These results were confirmed through exposure to actual sun. CONCLUSION: Our study confirms the prediction of the in vitro methods previously used to assess the photostability of sunscreen products. In addition, DRS provides a powerful new tool for the in vivo simultaneous evaluation of photostability and estimation of the UVA protection factor of sunscreen products performed during the test for SPF determination.     

Migration and penetration of a fluorescent textile dye into the skin –in vivo versus in vitro methods

Abstract:  The amount of textile dye migration from the textile and penetration into the skin is relevant when assessing the risk of textile dyes. In this paper, in vivo methods were developed using a harmless textile dye with a strong fluorescence and were then compared with in vitro methods. For the in vivo method, the textile was applied to the lower back of six volunteers wearing the textile 12 h and to the lower back of 12 volunteers during 30 min active sport. The maximum skin absorption of 55 ± 17 ng/cm² was obtained in the group engaged in sports. The in vitro methods, which involved the application of the textile to the pig ear skin, was shown to yield similar results to the 12 h in vivo group (31.2 ± 9.6 ng/cm² vs 27 ± 14 ng/cm²). The migration of the textiles into artificial sweat resulted in approximately 20  μ g/cm². The disadvantage of such textile extract applications on pig ear skin is discussed. It could be demonstrated that the absorption of the dye is strongly correlated to the amount of sweat, whereas the contact time was less important.     

Determination of the antioxidative capacity of the skin in vivo using resonance Raman and electron paramagnetic resonance spectroscopy

Abstract: Background: Non‐invasive measurements are of major interest for investigating the effects of stress, nutrition, diseases or pharmaceuticals on the antioxidative capacity of the human skin. However, only a few non‐invasive methods are available. Material and Methods: The resonance Raman spectroscopy is well established to monitor carotenoids in the skin, but correlations with other antioxidants have not yet been described. Electron paramagnetic resonance spectroscopy used for measurements of free radicals has already been used elsewhere to investigate the reduction of applied long‐living nitroxide radicals, caused by skin antioxidants and UV irradiation, but only a single or up to four volunteers were included in these studies. Therefore, in this study, the two methods were applied in parallel on 17 volunteers, and the rate constant of the nitroxide decrease was correlated with the cutaneous carotenoid concentration. Results and Discussion: A correlation with R = 0.65 was found, supporting the thesis that different antioxidants protect each other and build an antioxidative network in the skin. The results also give first indications that the carotenoids serve as marker substances for the antioxidative capacity, if the nutrition is well balanced.     

In vivo confocal laser scanning microscopy in the diagnosis of melanocytic skin tumours

Summary Melanoma of the skin represents one of the greatest challenges in early or preventive detection. Whereas surgical excision in early stages of melanoma development is almost always curative, delayed recognition puts the patient at risk for destructive growth and death from disease once the tumour has progressed to competence for metastasis. The worldwide introduction of dermoscopy has led to improved diagnostic accuracy for melanocytic skin tumours. Whereas dermoscopy has probably reached the method's inherent potential diagnostic accuracy because of the lack of cellular level evaluation, further improvements could be expected by in vivo confocal laser scanning microscopy. In vivo confocal microscopy represents a novel imaging tool that allows the noninvasive examination of skin cancer morphology in real time at a 'quasihistopathological' resolution viewing microanatomical structures and individual cells. Numerous morphological confocal features of melanocytic skin tumours have been described and histopathological correlates of confocal structures have been previously elucidated. Recently, several studies have evaluated the diagnostic accuracy of in vivo confocal microscopy for melanocytic skin tumours, investigating approximately 50 000 tumour images. Remarkably, sensitivity superior to the diagnostic accuracy achieved with dermoscopy could be reached by this imaging modality. These studies represent a significant contribution to the body of research necessary for the evaluation and implementation of in vivo confocal microscopy in clinical practice to avoid many currently unnecessary biopsies. In vivo confocal microscopy probably augurs a sea change in the way we evaluate melanocytic skin tumours in the future and will ultimately move the art of histological diagnosis closer to the bedside.     

Characterization of the mechanical properties of a dermal equivalent compared with human skin in vivo by indentation and static friction tests

Background/aims: The study of changes in skin structure with age is becoming all the more important with the increase in life. The atrophy that occurs during aging is accompanied by more profound changes, with a loss of organization within the elastic collagen network and alterations in the basal elements. The aim of this study is to present a method to determine the mechanical properties of total human skin in vivo compared with dermal equivalents (DEs) using indentation and static friction tests.
Methods: A new bio-tribometer working at a low contact pressure for the characterization the mechanical properties of the skin has been developed. This device, based on indentation and static friction tests, also allows to characterize the skin in vivo and reconstructed DEs in a wide range of light contact forces, stress and strain.
Results: This original bio-tribometer shows the ability to assess the skin elasticity and friction force in a wide range of light normal load (0.5–2 g) and low contact pressure (0.5–2 kPa). The results obtained by this approach show identical values of the Young's modulus E ^* and the shear modulus G ^* of six DEs obtained from a 62-year-old subject ( E ^*=8.5±1.74 kPa and G ^*=3.3±0.46 kPa) and in vivo total skin of 20 subjects aged 55 to 70 years ( E ^*=8.3±2.1 kPa, G ^*=2.8±0.8 kpa).