The ex vivo porcine lung tissue exposure to nicotine-flavour free e-liquid was examined in-depth using confocal Raman micro-spectroscopy. It was found that the lung-related Raman bands and autofluorescence intensities were enhanced after exposure to e-liquid for all depths and treatment time (first and second treatments) due to the optical clearing effect of glycerol and propylene glycol as an OC agent. The nicotine-flavour free e-liquids that contain glycerol and propylene glycol could potentially be used in clinical protocols for lung disease discrimination in-depth using Raman-based in vivo bronchoscopy due to light scattering reduction as an optical clearing agent.
Non-invasive in vivo monitoring of the antioxidant level of human skin, e.g. by measuring carotenoid marker substances, can deliver significant data on human health status and can provide diagnostic data during medical treatments. Among others, resonance Raman spectroscopy is a promising contactless tool to detect carotenoids in human skin within the necessary nanomolar concentration range. Unfortunately, laser induced fluorescence and ambient light can obscure the Raman signals even when using the resonance Raman effect and exciting the carotenoids at wavelengths around 500 nm. Here, shifted excitation resonance Raman difference spectroscopy (SERRDS) is a powerful and easyto- use tool separating the wanted from the unwanted signals. For this purpose, a portable clinical diagnostic system including a compact Raman handheld probe and a miniaturized wavelength-tunable frequency-doubled diode laserbased 488 nm light source was developed. The diode laser can be tuned over 2 nm providing the two excitation lines with a flexible spectral distance for SERRDS, resulting fluorescence-free skin Raman spectra. For reliable and representative measurements of human skin, an excitation spot diameter of 3 mm was selected. An excitation power of 9 mW at the sample provides a power density of 1.3 mW/mm2 meeting the laser safety regulations with a maximum permissible exposure of 20 mW/mm2. A calibration procedure was performed using skin phantoms containing ßcarotene at selected concentrations and a limit of detection of 0.05 nmol g-1 of β-carotene is achieved using the 3-sigma criterion.
Optical clearing (OC) of biological tissues is a promising technology for a wide spread use in medical practice to increase the screening depth, spatial resolution and contrast of the resulting images/spectra. Nevertheless, despite the significant OC effect, some biocompatible optical clearing agents (OCAs) can adversely affect biological tissues, causing local hemostasis, morphological changes, d ehydration, and in some cases even tissue necrosis. The aim of this study was to study the effect of Omnipaque 300 and fructose solutions of various concentrations and exposure times on the intact skin using confocal Raman microspectroscopy. It was shown that the application of each of these OCAs on intact skin for 5 min also leads to an appreciable OC effect. The increase in OC was achieved using a mixture of Omnipaque 300 with DMSO; it was shown that the optical properties of the skin can be controlled at a depth of about 80 μm.
Imaging Raman spectroscopy can be used to identify cancerous tissue. Traditionally, a step-by-step scanning of the sample is applied to generate a Raman image, which, however, is too slow for routine examination of patients. By transferring the technique of integral field spectroscopy (IFS) from astronomy to Raman imaging, it becomes possible to record entire Raman images quickly within a single exposure, without the need for a tedious scanning procedure. An IFS-based Raman imaging setup is presented, which is capable of measuring skin ex vivo or in vivo. It is demonstrated how Raman images of healthy and cancerous skin biopsies were recorded and analyzed.
Although chemotherapeutics for cancer treatment are becoming increasingly efficient these days, they often cause severe dermal side effects. Systemically applied doxorubicin is known for inducing free radicals, which leads to the development of the hand-foot syndrome. This syndrome manifests itself through skin irritations, extending from blistering to open wounds. As doxorubicin exhibits a fluorescence signal in the 520-600 nm region if excited at 488 nm, the doxorubicin’s leakage onto the skin surface could be analyzed. It was found that part of the doxorubicin is ejected with the sweat onto the skin surface, where it spreads and penetrates into the skin like topically applied. By topical application of antioxidants, the doxorubicin could be prevented from inducing free radicals in the skin and consequently the hand-foot syndrome. Raman spectroscopy was used to show that the action mechanism of chemotherapeutics not showing fluorescence signals is similar to the action mechanism of doxorubicin.
Topical glucocorticoids (GC) are known to induce changes in human skin with the potential to develop skin atrophy. Here, atrophogenic effects and subsequent structural changes in the skin after topical application of GC were investigated in vivo. Sixteen healthy volunteers were topically treated daily on the forearms with clobetasol propionate, betamethasone dipropionate, and the petrolatum vehicle for 4 weeks. All treated skin areas and a nontreated control area were examined by ultrasound, optical coherence tomography, confocal laser scanning microscopy, multiphoton tomography (MPT), and resonance Raman spectroscopy at baseline 1 day after last application and 1 week after last application. Investigated parameters included stratum corneum thickness, epidermal, and full skin thickness, keratinocyte size and density, keratinocyte nucleus-to-cytoplasm ratio, skin surface classification, relative collagen and elastin signal intensity, second-harmonic generation-to-autofluorescence aging index of dermis (SAAID), and the antioxidant status of the skin. A reduction in epidermal and dermal skin thickness was observed in GC treated as well as in vehicle-treated and untreated skin areas on the volar forearm. MPT analysis showed an increased epidermal cell density and reduced cell size and nucleus-to-cytoplasm ratio and a significant increase of SAAID after GC treatment indicating a restructuring or compression of collagen fibers clinically being observed as atrophic changes.
Autofluorescence photobleaching describes the decrease of fluorescence intensity of endogenous fluorophores in biological tissue upon light irradiation. The origin of autofluorescence photobleaching is not fully understood. In the skin, the spatial distribution of various endogenous fluorophores varies within the skin layers. Most endogenous fluorophores are excited in the ultraviolet and short visible wavelength range, and only a few, such as porphyrins (red) and melanin (near-infrared), are excited at longer wavelengths. The excitation wavelength- and depth-dependent irradiation of skin will therefore excite different fluorophores, which will likely influence the photobleaching characteristics. The autofluorescence photobleaching of porcine ear skin has been measured ex vivo using 325, 473, 633, and 785 nm excitation at different skin depths from the surface to the dermis at 150 μm. Confocal Raman microscopes were used to achieve sufficient spatial resolution of the measurements. The autofluorescence area under the curve was measured for 21 consecutive acquisitions of 15 s. In all cases, the photobleaching follows a two-exponential decay function approximated by nonlinear regression. The results show that photobleaching can be applied to improve the signal-to-noise ratio in Raman spectroscopy for all of the applied excitation wavelengths and skin depths.
In the last decade, cutaneous carotenoid measurements have become increasingly popular, as carotenoids were found to be a biomarker of nutrition rich in fruits and vegetables, permitting monitoring of the influence of various stress factors. For such measurements, in addition to the specific and selective resonance Raman spectroscopy (RRS), newly developed low expensive small and mobile sensors that are based on spatially resolved reflectance spectroscopy (SRRS) are used for cutaneous carotenoid measurements. Human volunteers of different age exhibiting skin types I to III were investigated using RRS and two SRRS-based sensors to determine the influence of these parameters on the measuring results. In two studies on volunteers of either the same age or skin type, however, the respective other parameter being varied and no significant influences of age or skin type could be detected. Furthermore, the kinetic changes resulting from the intake and discontinued intake of a vegetable extract rich in carotenoids showed a good correlation among the three sensors and with the detected blood carotenoids. This illustrates that the SRRS-based sensors and RRS device provide reliable cutaneous carotenoid values independent of age and skin types I to III of the volunteers.
The increased ability of TMX-202 (derivative of imiquimod) to penetrate the intact stratum corneum (SC) and the follicular orifices of porcine ear skin was shown ex vivo using confocal Raman microscopy and laser scanning microscopy. Moreover, to assess whether TMX-202 is able to reach the immune cells, Langerhans cells extracted from pretreated human skin were investigated ex vivo using confocal Raman microscopy combined with multivariate statistical methods. Tracking the Raman peak of dimethyl sulfoxide centered at 690 cm−1, the absorption of TMX-202 containing formulation by Langerhans cells was shown. To answer the question whether the TMX-202 active ingredient is able to reach Langerhans cells, the attraction of immune cells to TMX-202 containing formulation treated skin was measured in the in vivo rodent model Mastomys coucha. The results show that TMX-202 active ingredient is able to reach Langerhans cells after penetrating through the intact skin and subsequently attract immune cells. Both the intercellular/transcellular as well as the follicular pathways allow the penetration through the intact barrier of the SC.
In order to investigate the penetration depth of silver nanoparticles (Ag NPs) inside the skin, porcine ears treated with Ag NPs are measured by two-photon tomography with a fluorescence lifetime imaging microscopy (TPT-FLIM) technique, confocal Raman microscopy (CRM), and surface-enhanced Raman scattering (SERS) microscopy. Ag NPs are coated with poly-N-vinylpyrrolidone and dispersed in pure water solutions. After the application of Ag NPs, porcine ears are stored in the incubator for 24 h at a temperature of 37°C. The TPT-FLIM measurement results show a dramatic decrease of the Ag NPs’ signal intensity from the skin surface to a depth of 4 μm. Below 4 μm, the Ag NPs’ signal continues to decline, having completely disappeared at 12 to 14 μm depth. CRM shows that the penetration depth of Ag NPs is 11.1±2.1 μm. The penetration depth measured with a highly sensitive SERS microscopy reaches 15.6±8.3 μm. Several results obtained with SERS show that the penetration depth of Ag NPs can exceed the stratum corneum (SC) thickness, which can be explained by both penetration of trace amounts of Ag NPs through the SC barrier and by the measurements inside the hair follicle, which cannot be excluded in the experiment.
The standard diagnostic procedure for basal cell carcinoma (BCC) is invasive tissue biopsy with time-consuming histological examination. To reduce the number of biopsies, noninvasive optical methods have been developed providing high-resolution skin examination. We present direct comparison of a reflectance confocal microscope (RLSM) and a multiphoton tomograph (MPT) for BCC diagnosis. Both systems are applied to nine patients prior to surgery, and the results are analyzed, including histological results. Both systems prove suitable for detecting typical characteristics of BCC in various stages. The RLSM allows large horizontal overview images to be obtained, enabling the investigator to find the regions of interest quickly, e.g., BCC nests. Elongated cells and palisading structures are easily recognized using both methods. Due to the higher resolution, changes in nucleus diameter or cytoplasm could be visualized with the MPT. Therefore, the nucleus diameter, nucleus/cytoplasm ratio, and cell density are estimated for normal and BCC cells using the MPT. The nucleus of elongated BCC cells is significantly longer than other measured normal skin cells, whereas the cell density and nucleus/cytoplasm ratio of BCC cannot be significantly distinguished from granular cells.
We combined two-photon fluorescence and coherent anti-Stokes Raman scattering (CARS) imaging in a clinical hybrid
multiphoton tomograph for in vivo imaging of human skin. The clinically approved TPEF/CARS system provides
simultaneous imaging of endogenous fluorophores and non-fluorescent lipids. The Stokes laser for the two-beam
configuration of CARS is based on spectral broadening of femtosecond laser pulses in a photonic crystal fiber (PCF). We
report on the highly flexible medical TPEF/CARS tomograph MPTflex®-CARS with an articulated arm and first in vivo
measurements on human skin.
Microscopic imaging based on multiphoton fluorescence, second harmonic generation (SHG) and coherent anti-Stokes
Raman scattering (CARS) imaging has been realized in one common platform which is appropriate for use in hospitals.
The different optical modalities non-invasively provide in vivo images from human skin with subcellular resolution, at
different depths based on endogenous fluorescent, SHG-active molecules as well as non-fluorescent molecules with
vibrational resonances at 2845 cm-1, in particular lipids. An overview of the system employing a Ti:sapphire laser and
photonic crystal fiber to generate the excitation light as well as several imaging examples are presented.
Carotenoids are important substances for human skin due to their powerful antioxidant properties in reaction of neutralization of free radicals and especially reactive oxygen species, including singlet oxygen. Concentration of carotenoids in the skin could mirror the current redox status of the skin and should be investigated in vivo. Optical methods are ideally suited for determination of carotenoids in mammalian skin in vivo as they are both noninvasive and quick. Four different optical methods could be used for in vivo measurement of carotenoids in the human or animal skin: (1) resonance Raman spectroscopy; (2) Raman microscopy; (3) reflection spectroscopy; (4) skin color measurements. The advantages, shortcomings, and limitations of the above-mentioned optical methods are discussed.
Herd health programs for the maintenance of welfare and productivity in cattle need efficient tools for monitoring the health of individual animals. Recent reports demonstrate that the oxidative status is related to various stress conditions in dairy cows. Biomarkers, among other carotenoids, could serve as indicators of stress originating from the environment (e.g., heat stress or sun radiation) or from the animal itself (e.g., disease). To date, only invasive in vitro tests are available to assess the oxidative status in cattle. The present study compares the results of optical noninvasive in vivo measurements of dermal carotenoids in cattle udder skin using an LED-based miniaturized spectroscopic system (MSS) with those obtained by photometric analysis of beta carotene in whole blood samples using a portable device. Correlations between the concentrations of dermal and blood carotenoids were calculated under consideration of the nutritional status of the animals. Significant correlation (R=0.86) was found for cattle with a moderate to obese body condition. Thus, the blood and skin concentrations of the marker substance beta carotene are comparable under stable stress conditions of the cattle. This demonstrates that the MSS is suitable for noninvasive assessment of dermal carotenoid concentrations in cattle.
For a long time, the antioxidative status in cattle has been discussed as an indicator for stress conditions resulting from disease or exertion. Until now, invasive approaches have been necessary to obtain blood samples or biopsy materials and gain insights into the antioxidative status of cattle. Due to these efforts and the costs of the analyses, serial sampling is feasible in an experimental setting, but not for measurements on a routine basis. The present study focuses on the feasibility of an innovative, noninvasive spectroscopic technique that allows in vivo measurements of carotenoids in the skin by reflection spectroscopy. To this end, in a first trial, repeated measurements of the carotenoid concentration of the udder skin were performed on 25 healthy cattle from different breeds. Carotenoid concentrations showed highly significant differences between individual animals (P<0.001), although they were kept under the same environmental conditions and received the same diet. The carotenoid concentrations in "sensitive" and "robust" cows (evaluated by a temperament test) differed significantly (P<0.005), with higher concentrations observed in robust cows.
Penetration of nanoparticles into tooth tissues is of significant interest in solving problems related to reduction of tooth
sensitivity, enamel strengthening and restoration and cosmetic bleaching. In this work we demonstrate two-photonexcited
autofluorescence and second-harmonic generation microscopy for visualization of penetration of TiO2 and ZnO
nanoparticles into tooth tissues.
The human organism has developed a protection system against the destructive effect of free radicals. The aim of the present study was to investigate the extent of exogenous stress factors such as disinfectant and IR-A radiation on the skin, and their influence on the kinetics of carotenoids distribution during the recovery process. Ten healthy volunteers were assessed with resonance spectroscopy using an Argon-laser at 488 nm to excite the carotenoids in vivo. Additionally, Raman-confocal-micro-spectroscopy measurements were performed using a model 3510 Skin Composition Analyzer with spatially resolved measurements down to 30 μm. The measurements were performed at a baseline of 20, 40, 60, and 120 min after an external stressor consisting either of water-filtered infrared A (wIRA) with 150 mW/cm2 or 1 ml/cm2 of an alcoholic disinfectant. Both Raman methods were capable to detect the infrared-induced depletion of carotenoids. Only Raman-microspectroscopy could reveal the carotenoids decrease after topical disinfectant application. The carotenoid-depletion started at the surface. After 60 min, recovery starts at the surface while deeper parts were still depleted. The disinfectant- and wIRA-induced carotenoid depletion in the epidermis recovers from outside to inside and probably delivered by sweat and sebaceous glands. We could show that the Raman microscopic spectroscopy is suited to analyze the carotenoid kinetic of stress effects and recovery.
Skin aging is mainly caused by the destructive action of free radicals, produced by the UV light of the sun. The human skin has developed a protection system against these highly reactive molecules in the form of the antioxidative potential. Carotenoids are one of the main components of the antioxidants of the human skin. From former studies, it is known that skin aging is reduced in individuals with high levels of carotenoids. Because most of the antioxidants cannot be produced by the human organism, they must be up taken by nutrition. Using noninvasive Raman spectroscopic measurements it is demonstrated that not only fruits and vegetables but also eggs contain high concentrations of antioxidants including carotenoids, which are even doubled in the case of ecological eggs. After a 1-week diet with ecological eggs performed by six volunteers, it is found that the concentration of the carotenoids in the skin of the volunteers increased by approx. 20%. Our study does not intend to recommend exorbitant egg consumption, as eggs also contain harmful cholesterol. But in the case of egg consumption, ecological eggs from hens kept on pasture should be preferred to also receive a benefit for the skin.
Variation in the level of the carotenoid antioxidant substances beta-carotene and lycopene in the human skin of ten healthy volunteers was measured with resonance Raman spectroscopy in an in vivo experiment over the course of 12 months. Information on the lifestyle of the volunteers concerning dietary supplementation and stress factors was obtained daily by the completion of questionnaires. The results showed individual variations in the levels of carotenoid antioxidant substances in the skin of the volunteers, which strongly correlated to specific lifestyles, such as the intake of dietary supplementations rich in carotenoids, and the influence of stress factors. A carotenoid-rich nutrition, based on large amounts of fruit and vegetables, increased the measured carotenoid levels of skin, while stress factors such as fatigue, illness, smoking, and alcohol consumption gave rise to a decrease in carotenoid levels of the skin. These decreases occured relatively quickly over the course of one day, while the subsequent increases lasted for up to 3 days. During the summer and autumn months, an increase in the level of carotenoids in the skin was measured for all volunteers. The average “seasonal increase” of the carotenoid content in the skin was determined to be 1.26-fold.
A non-invasive optical method based on resonance Raman spectroscopy was used for the in vivo detection of the
concentration of the carotenoid antioxidant substance lycopene in the human skin.
The physiological variation of the level of lycopene in the skin during a 6 month period was measured daily in 7 volunteers. It
was shown that all volunteers had a different individual level of lycopene in the skin, depending on the lifestyle of volunteers.
It was shown that the supplementation of the foodstuffs containing lycopene, such as tomato products and some fruits,
increases the level of lycopene in the skin. The increase in the lycopene level can be usually observed on the next day after
the supplementation.
The present results demonstrate that a diet rich in products containing a high amount of carotenoids, such as lycopene, can be
an efficient strategy to increase the carotenoid level of the skin.
The antioxidant β-carotene and lycopene substances were detected non-invasively, in vivo in human skin using resonance Raman spectroscopy. Both substances were detected simultaneously. To distinguish between the substances, the Raman signals were excited at 488 nm and 514,5 nm simultaneously using a multilane Ar+ laser. The application of a fiber based optical imaging system allowed the detection of β-carotene and lycopene on any skin area. The disturbance of the measurements because of non-homogeneous skin pigmentation was avoided by using a measuring area of 28 mm2. The minimum power density for registration of the Raman signals and their optimum relation was determined. The Raman spectroscopic method is well suited for the evaluation of the efficacy of topically or systematically applied amounts of β-carotene and lycopene.
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