Visualizing radiofrequency-skin interaction using multiphoton microscopy in vivo

Background

Redundant skin laxity is a major feature of aging. Recently, radiofrequency has been introduced for nonablative tissue tightening by volumetric heating of the deep dermis. Despite the wide range of application based on this therapy, the effect of this technique on tissue and the subsequent tissue remodeling have not been investigated in detail.

Objective

Our objective is to evaluate the potential of non-linear optics, including multiphoton autofluorescence and second harmonic generation (SHG) microscopy, as a non-invasive imaging modality for the real-time study of radiofrequency-tissue interaction.

Methods

Electro-optical synergy device (ELOS) was used as the radiofrequency source in this study. The back skin of nude mouse was irradiated with radiofrequency at different passes. We evaluated the effect on skin immediately and 1 month after treatment with multiphoton microscopy.

Results

Corresponding histology was performed for comparison. We found that SHG is negatively correlated to radiofrequency passes, which means that collagen structural disruption happens immediately after thermal damage. After 1 month of collagen remodeling, SHG signals increased above baseline, indicating that collagen regeneration has occurred. Our findings may explain mechanism of nonablative skin tightening and were supported by histological examinations.

Conclusions

Our work showed that monitoring the dermal heating status of RF and following up the detailed process of tissue reaction can be imaged and quantified with multiphoton microscopy non-invasively in vivo.     

Morphological skin ageing criteria by multiphoton laser scanning tomography: non-invasive in vivo scoring of the dermal fibre network

Background: Morphological changes in the dermal collagen and elastin fibre network are characteristic for skin ageing and for pathological skin conditions of the dermis. Objectives: To characterize pathological and physiological conditions by multiphoton laser scanning tomography (MLT) in vivo, it is necessary to investigate and identify morphological alterations related to ageing. Methods: In vivo MLT was used to image two‐photon excited autofluorescence (AF) and second harmonics generation (SHG) in human dermis of 18 volunteers of different ages. Criteria for the evaluation of age‐dependent morphological changes in MLT images were fibre tension and morphology, network pattern, clot formation and image homogeneity. These criteria were weighted and a score was calculated. Results: The resulting MLT‐based Dermis Morphology Score is correlated with age (R² = ?0.90) and with the previously published SHG to AF Ageing Index of Dermis (R² = 0.66). The two groups of young (age 21–38) and old (age 66–84) volunteers showed a significant difference in MLT score values (P < 0.001). Conclusions: We could demonstrate an in vivo relationship between morphological characteristics of human dermis assessed by MLT and age. The present score allows the semi‐quantitative evaluation of specific morphological changes of the dermal fibre network in ageing skin by in vivo AF and SHG imaging. This method will be useful for diagnostics of pathological conditions and their differentiation from ageing effects.     

Depth‐resolved measurement of the dermal matrix composition by multiphoton laser tomography

Background: In the last years, multiphoton laser tomography (MLT) has emerged as a promising tool for non‐invasive diagnostics in dermatology and other medical specialties. The present work is dedicated to the question to what degree the measurement depth and the thickness of the epidermis influence the evaluation of dermal matrix composition and if recommendations for future measurement procedures can be given. Methods: In a study group of 30 healthy volunteers aged 21–82 years multiphoton depth‐resolved measurements of autofluorescence and second harmonics have been performed in order to evaluate the dermal matrix composition. Results: Characteristic intensity curves depending on the penetration depth were derived and differences between age groups were found. Conclusion: With the present work we provide evidence for the accuracy of the measurement of dermal matrix composition by MLT and give detailed advice for the measurement procedure. Furthermore, we propose the use of depth‐dependent emission intensity curves for monitoring of anti‐aging treatment.     

Evaluation of photoaging in facial skin by multiphoton laser scanning microscopy

Background/purpose: It has been reported that autofluorescence (AF) and second harmonic generation (SHG) generated in the upper dermis are related with skin photoaging. In this study, we assessed the photoaging of facial skin exposed to daily sunlight using in vivo multiphoton laser microscopy to measure AF and SHG. Methods: The intensities of AF and SHG in the upper dermis of cheek skin of 56 healthy volunteers aged 20–69 years were measured using a commercially available multiphoton laser microscope (DermaInspect^®). Correlations between the photo‐signals and volunteer age were calculated. Results: The intensity of SHG and the SHG‐to‐AF aging index of dermis (SAAID) correlated significantly with age (r=−0.48, −0.67, respectively). Conclusion: Our results suggest that SHG and the SAAID index are useful indicators of facial skin aging in vivo.     

Evaluation of unique elastic aggregates (elastic globes) in normal facial skin by multiphoton laser scanning tomography

Background

There is no reliable marker to estimate the degree of skin aging in vivo. It now has become possible to quantitatively determine the dermal characteristics of the extracellular matrix (ECM) in vivo using multiphoton laser tomography (MLT).

Methods

Fifty-seven healthy Japanese female volunteers, aged from 20 to 60 years old, were examined using multiphoton depth-resolved measurements of autofluorescence (AF) and second harmonic generation (SHG) at three sites on their right cheek. Paraffin-embedded skin specimens obtained from the faces of 12 normal individuals aged 38-68 years old were stained with Elastica van Gieson (EVG).

Results

We found unique elastic aggregates at a 20µm depth from the dermo-epidermal junction (DEJ) in vivo which increased in size with aging of subjects from 20 to 60 years old. SHG fibers seemed to surround those elastic aggregates. Histological examination of specimens from normal individuals stained with EVG confirmed the occurrence of elastic aggregates with varied sizes just beneath the epidermis or hair follicles.

Conclusions

The elastic aggregates are morphologically similar to previously described ‘elastic globes’ and can serve as a marker of the early stage of photoaging. MLT will contribute to determine age-related dermal changes using a non-invasive technique.

     

Spatial and temporal analysis of skin glycation by the use of multiphoton microscopy and spectroscopy

Background

Tissue glycation, the main cause of many diabetes-related complications, results in the accumulation of advanced glycation endproducts (AGE).

Objectives

These AGEs are endogenous fluorophores that can serve as a viable pathological indicator for disease diagnostics. Here we explore the capabilities of multiphoton microscopy to non-invasively localize and quantify the skin glycation.

Methods

In our study, multiphoton microscopy and spectroscopy were used to investigate glycation events-induced changes in the intensities of autofluorescence and second harmonic generation on ex vivo human skin.

Results

Temporal and spatial dependence of degrees of glycation of the epidermis, collagen and elastin fibers of dermis were evaluated for their relevance to the changes in amplitudes of autofluorescence signals. We found that glycation drastically and linearly increases multiphoton autofluorescence intensity of epidermis and dermal collagen whereas changes in dermal elastin are moderate. We also found decrease in the level of second harmonic generation signal.

Conclusion

Our study suggests that due to intrinsically weak autofluorescence the dermal collagen is the most sensitive skin tissue to be used for detecting changes in tissue glycation.     

Repair of photoaged dermal matrix by topical application of a cosmetic 'antiageing' product

BACKGROUND: Photoaged skin is characterized by coarse and fine wrinkles. The mechanism of wrinkle formation appears to involve changes to components of the dermal extracellular matrix. Topical treatment with all-trans retinoic acid (RA) can repair photoaged dermal matrix; this is regarded as the 'gold standard' against which repair agents are judged. To date, little is known regarding the ability of over-the-counter 'antiageing' products to repair photoaged skin. OBJECTIVES: We used a modified occluded patch test to ascertain whether topical applications of cosmetic 'antiageing' products are able to repair photoaged human skin. METHODS: Commercially available test products [basic moisturizer, 'antiageing' cream containing different active complex levels (6% active: lipopentapeptide, white lupin peptides, antioxidants, retinyl palmitate; 2% active: lipopentapeptide, white lupin peptides, antioxidants)] were applied under occlusion for 12 days prior to biopsy and histological assessment in photoaged volunteers (n=9). RA was used as a positive control. RESULTS: In agreement with previous studies, the patch-test study revealed that RA produced significant fibrillin-1 deposition in the papillary dermis (P<0.01) but had little effect on procollagen I or matrix metalloproteinase-1 expression. The 6% total active complex formulation, however, increased the deposition of fibrillin-1 and procollagen I (P<0.01, P<0.05, respectively). CONCLUSIONS: This study indicates that in an in vivo 12-day patch test an over-the-counter cosmetic product can induce changes in photoaged dermal extracellular matrix, which are indicative of repair.     

The biophysical effects of localized electrochemical therapy on porcine skin

BackgroundMinimally-invasive methods to treat scars address a common pathway of altering collagen structure, leading to collagen remodeling.ObjectiveIn this study, we employed in situ redox chemistry to create focal pH gradients in skin, altering dermal collagen, in a process we refer to as electrochemical therapy (ECT). The effects of ECT to induce biochemical and structural changes in ex vivo porcine skin were examined.MethodsDuring ECT, two platinum electrodes were inserted into fresh porcine skin, and following saline injection, an electrical potential was applied. pH mapping, high frequency ultrasonography, and two photon excitation microscopy and second harmonic generation (SHG) microscopy were used to evaluate treatment effects. Findings were correlated with histology.ResultsFollowing ECT, pH mapping depicted acid and base production at anode and cathode sites respectively, with increasing voltage and application time. Gas formation during ECT was observed with ultrasonography. Anode sites showed significant loss of SHG signal, while cathode sites showed disorganized collagen structure with fewer fibrils emitting an attainable signal. Histologically, collagen denaturation at both sites was confirmed.ConclusionWe demonstrated the production of in situ acid and base in skin occurring via ECT. The effects chemically and precisely alter collagen structure through denaturation, giving insight on the potential of ECT as a simple, low-cost, and minimally-invasive means to remodel skin and treat scars.     

Retinyl retinoate,a novel hybrid vitamin derivative,improves photoaged skin: a double‐blind,randomized‐controlled trial

Background: All‐trans‐retinoic acid (RA) and all‐trans‐retinol (ROL) are not widely used as anti‐wrinkle agents due to their irritancy and photo‐stability, respectively. Therefore, the safety and photo‐stability in the development of RA or ROL derivatives have been an important issue. Aim: To identify the efficacy of retinyl retinoate as an anti‐aging agent of cosmetics in treating females over 30 years old with periorbital wrinkles. Methods: The clinical study was a prospective, double‐blind, randomized, and controlled study with a total of 11 Korean women. At every 4 weeks, the effectiveness was assessed with a global photodamage score, photographs, and image analysis using replicas and visiometers. The dermal distance and intensity was also evaluated using Dermascan C. Results: A statistically significant improvement in facial wrinkles (P<0.05) in eleven volunteers was observed in a clinical trial. The successive application of 0.06% retinyl retinoate cream for 3 months showed decreased depth and area of wrinkles in comparison with 0.075% retinol cream. The visual wrinkle improvement and the maximum roughness improvement rate (R2) for retinyl retinoate cream were 22% higher than that of retinol cream after 12 weeks. A statistically significant increase was observed after 8 and 4 weeks for dermal distance and dermal intensity, respectively (P<0.05). Conclusions: Retinyl retinoate had characteristic features of new anti‐aging agents, and effectively improved facial wrinkle conditions.     

Texture analysis of second‐harmonic‐generation images for quantitative analysis of reticular dermal collagen fibre in vivo in human facial cheek skin

Second‐harmonic‐generation (SHG) microscopy is a powerful tool for in vivo visualisation of collagen fibres in human skin because of its specific collagen selectivity without the need for staining, non‐invasiveness and high‐resolution three‐dimensional imaging. Although texture analysis of SHG images is a promising method for the quantitative analysis of well‐orientated collagen fibre structure in the tendon and cornea, there are few attempts to assess cutaneous ageing. In this study, we applied two texture analysis techniques, namely autocorrelation (2D‐AC) analysis and two‐dimensional Fourier transform (2D‐FT), to evaluate the age‐dependent changes in reticular dermal collagen fibres in in vivo human cheek skin. Age‐dependent changes in the reticular dermal collagen fibres of female subjects in their 20s, 40s and 60s clearly appeared in these texture analyses. Furthermore, the parameter from 2D‐AC analysis showed a significantly higher correlation with skin elasticity measured by a Cutometer^®. These results clearly indicate that 2D‐AC analysis of SHG images is highly promising for the quantitative evaluation of age‐dependent change in facial collagen fibres as well as skin elasticity. An appropriate texture analysis will help to provide quantitative insight into collagen fibre structure and will be useful for the diagnosis of pathological conditions as well as cutaneous ageing in skin.     

Fluorescence induction of protoporphyrin IX by a new 5‐aminolevulinic acid nanoemulsion used for photodynamic therapy in a full‐thickness ex vivo skin model

Please cite this paper as: Fluorescence induction of protoporphyrin IX by a new 5‐aminolevulinic acid nanoemulsion used for photodynamic therapy in a full‐thickness ex vivo skin model. Experimental Dermatology 2010; 19 : e302–e305. Abstract: An ex vivo porcine skin model was utilized to analyse the penetration of 5‐aminolevulinic acid (5‐ALA) contained in a nanoemulsion‐based formulation BF‐200 ALA (10% 5‐ALA‐hydrochloride) versus 16% aminolevulinate methyl ester‐hydrochloride in a commercially cream (MAL cream) by fluorescence microscopy of their common metabolite protoporphyrin IX (PpIX) after 3, 5, 8 and 12 h. Fluorescence signals of PpIX in pig skin treated with BF‐200 ALA were stronger than those for MAL cream. At 8 and 12 h, the PpIX fluorescence signals were 4.8‐ and 5.0‐fold higher than those measured after MAL cream application. Fluorescence signals of PpIX after application of BF‐200 ALA were detected in deeper tissue layers of the epidermis than after application of MAL cream (97.2 ± 5.7 μm for BF‐200 ALA vs 42.0 ± 4.2 μm for MAL cream). These data implicate that BF‐200 ALA in photodynamic therapy might lead to a superior therapeutically effect of intraepidermal (in situ) squamous cell carcinomas.     

报告比值比法对化妆品不良反应信号的挖掘研究

目的:评价报告比值比法对化妆品不良反应的安全警戒信号的作用。方法:收集2019年烟台市化妆品不良反应/事件报告系统中不良反应报告数据,选定疼痛、干燥、瘙痒及灼热感等4种不良反应症状作为不良反应信号,报告比值比法(ROR)计算值95%可信区间(CI)下限>1提示出现警戒信号。结果:分析纳入的1429条不良反应上报报告,具有不良反应前五位的化妆品类型分别是霜、乳液、面膜、水、露。经ROR计算,霜(ROR=2.1757)、乳液(ROR=2.0365)具有瘙痒警戒信号;面膜(ROR=3.3815)和乳液(ROR=2.3994),染发类产品(ROR=2.9289)具有灼热感警戒信号。结论:采用报告比值比法对化妆品不良反应警戒信号进行分析,可为化妆品不良反应提供参考。     

Qualification of a new and precise automatic tool for the assessment of hair diameters in phototrichograms

Background/purpose: To automatically assess hair growth during cosmetic trials, incorporating parameters such as anagen‐to‐telogen rate, growth rate, and especially hair diameter. Methods: We designed and qualified a new and automatic phototrichogram system based on a high‐resolution DSLR camera system (theoretical resolution of 2.557 μm/pixel) and modular macrolens system with fixed focus, combined with a trainable pattern recognition software for automated analysis. Results: We improved the standard routine for dermatological phototrichogram technique to overcome inaccuracy in thickness measurements due to hair swelling by using an alternative immersion fluid, and increased the effective resolution for hair size and thickness measurement to <4 μm. After having qualified manual measurements as gold standard for the determination of hair diameters, we established a new trainable automatic picture analysis software able to locate and measure individual hairs in length and thickness even in picture series taken from the same skin area at different time points. Comparisons between manual and automatic measurements of the same hairs showed a >90% correlation, and by comparing the automatic results with manual measurements of the same images without individual hair annotation, we could find a correlation of at least 80%. Conclusion: According to the results and findings generated in this qualification study, we have a reliable tool now that enables us to test cosmetic products for hair treatment in a highly automated way with a sufficient degree of precision and accuracy to detect even small changes in hair diameter during cosmetic trials.     

Superiority of a vitamin B12‐barrier cream compared with standard glycerol‐petrolatum‐based emollient cream in the treatment of atopic dermatitis: A randomized,left‐to‐right comparative trial

Atopic dermatitis (AD) is a result of complex genetic, epigenetic, environmental, and immunological interactions with an overlapping epidermal barrier defect. The study evaluates the efficacy and tolerability of topical Vitamin B12‐barrier cream (MB12) compared with standard glycerol‐petrolatum‐based emollient cream (GPC) used three times a day for mild AD. The study was conducted as a on one hemi‐body randomized, controlled, single‐blind, intra‐patient left‐to‐right comparative trial by patients with clinical diagnosis of mild AD measured with total SCORAD index over 4 months. MB12 was compared on one hemi‐body treated (GPC). The comparisons of score values were performed primarily by using non‐parametric procedures: Mann–Whitney‐U test (for independent samples) and Wilcoxon test (for dependent samples). All 22 patients were randomized (left or right side treated with MB12 or GPC). At week 12 a reduction from baseline in SCORAD index was assessed in both body sites with 77.6% SCORAD index reduction in the MB12 treated body sites versus 33.5% in the GPC treated body sites. These results suggest that MB12 could represent a new option in the treatment of mild AD.     

Non-invasive evaluation of dermal elastosis by in vivo multiphoton tomography with autofluorescence lifetime measurements

The non-invasive differentiation of dermal elastic fibres from solar elastosis in vivo is of great interest in dermatologic research, especially for efficacy testing of anti-ageing products. To date, no studies on multiphoton excited fluorescence lifetime characteristics of human elastic fibres and solar elastosis are reported. The goal of the present work was the identification of differential criteria for elastic fibres and solar elastosis by the analysis of fluorescence decay curves acquired by time-correlated single photon counting in vivo multiphoton tomography. For this purpose, fluorescence lifetime measurements (FLIM) were performed with 47 volunteers of different age groups at sun-protected and sun-exposed localizations. Bi-exponential curve fitting was applied to the FLIM data, and characteristic differences between age groups and localizations were found in both relevant fit parameters describing the decay slope. The FLIM analyses have shown that dermal autofluorescence has different lifetimes depending on age and in part on localization.     

Favorable effect of creams with skimmed donkey milk encapsulated in nanoliposomes on skin physiology

The use of donkey milk has a long history in external dermal application for cosmetic and medical purposes. The encapsulation of different drugs into nanoliposomes and nutrients may have many benefits for the delivery of the targeted substance. The aim of the present study is to test creams with nanoliposomes encapsulated with skimmed donkey milk, reported in our registered patent, compared with effect of the placebo cream and the untreated skin. To investigate the effect of formulated cream, we have measured the electrical capacitance, transepidermal water loss and the skin pH value. The study included 15 healthy volunteers (11 women and 4 men). The patented creams exhibited satisfactory moisturizing properties very soon after application, while hydration was reached earlier in the spots treated with night cream. Transepidermal water loss slightly decreased only after 4 weeks of treatment in each of the spots assessed. The pH value was similar after each of the treatments. Tested creams may enhance the moisture of skin very soon after application, in this way potentially enable deeper permeation of phospholipids and essential proteins, without changing the pH. It may also contribute to additional anti‐aging effects.     

Nonlinear spectral imaging of human hypertrophic scar based on two-photon excited fluorescence and second-harmonic generation

Background A noninvasive method using microscopy and spectroscopy for analysing the morphology of collagen and elastin and their biochemical variations in skin tissue will enable better understanding of the pathophysiology of hypertrophic scars and facilitate improved clinical management and treatment of this disease. Objective To obtain simultaneously microscopic images and spectra of collagen and elastin fibres in ex vivo skin tissues (normal skin and hypertrophic scar) using a nonlinear spectral imaging method, and to compare the morphological structure and spectral characteristics of collagen and elastin fibres in hypertrophic scar tissues with those of normal skin, to determine whether this approach has potential for in vivo assessment of the pathophysiology of human hypertrophic scars and for monitoring treatment responses as well as for tracking the process of development of hypertrophic scars in clinic. Methods Ex vivo human skin specimens obtained from six patients aged from 10 to 50 years old who were undergoing skin plastic surgery were examined. Five patients had hypertrophic scar lesions and one patient had no scar lesion before we obtained his skin specimen. A total of 30 tissue section samples of 30 μm thickness were analysed by the use of a nonlinear spectral imaging system consisting of a femtosecond excitation light source, a high‐throughput scanning inverted microscope, and a spectral imaging detection system. The high‐contrast and high‐resolution second harmonic generation (SHG) images of collagen and two‐photon excited fluorescence (TPEF) images of elastin fibres in hypertrophic scar tissues and normal skin were acquired using the extracting channel tool of the system. The emission spectra were analysed using the image‐guided spectral analysis method. The depth‐dependent decay constant of the SHG signal and the image texture characteristics of hypertrophic scar tissue and normal skin were used to quantitatively assess the amount, distribution and orientation of their collagen and elastin components. Results Our experiments and data analyses demonstrated apparent differences between hypertrophic scar tissue and normal skin in terms of their morphological structure and the spectral characteristics of collagen and elastin fibres. These differences can potentially be used to distinguish hypertrophic scar tissues from normal skin and to evaluate treatment responses. Conclusions All the measurements were performed in backscattering geometry and demonstrated that nonlinear spectral imaging has the ability to differentiate hypertrophic scar tissue from normal skin based on noninvasive SHG imaging, and TPEF imaging revealed the microstructure and spectral features of collagenand elastin fibres. With the advances in spectral imaging apparatus miniaturization, we have good reason to believe that this approach can become a valuable tool for the in vivo pathophysiology study of human skin hypertrophic scars and for assessing the treatment responses of this disfiguring disease in clinic. It can also be used to track the development of hypertrophic scars and to study wound healing processes in a noninvasive fashion without biopsy, fixation, sectioning and the use of exogenous dyes or stains.