Autofluorescence of human skin is age-related after correction for skin pigmentation and redness

When measuring the skin fluorescence in vivo, the absorption of chromophores such as melanin and hemoglobin often contribute predominantly to the changes in fluorescence and obscure the information from the fluorophores. We measured in vivo the collagen-linked 375 nm fluorescence (excitation: 330 nm) and 455 nm fluorescence (excitation: 370 nm) from nonexposed buttock skin of healthy volunteers. Skin pigmentation and redness of the same sites were quantified by reflectance of the skin at 555 nm and 660 nm. Multiple regression analysis was used to find the correlation between the fluorescence and skin pigmentation and redness. The fluorescence was corrected for the impact of pigmentation and redness according to the equation found in the regression analyses. The age-related trend of the fluorescence was evaluated. The 375 nm fluorescence showed positive relation to age, whereas the 455 nm fluorescence showed no significant relation to age. The increasing rate of the 375 nm fluorescence (logarithm transformed) was 2% per year, which is comparable with previously published data. The results suggest that the correction of the autofluorescence intensity for skin pigmentation and redness is valid, and the 375 nm skin autofluorescence may be used as a biologic marker of skin aging in vivo.     

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.     

New developments in fluorescence diagnostics

In the last decade, significant advances have been achieved in the direct viewing of the skin. Non‐invasive analysis of various skin diseases in vivo has become possible by special skin display devices, allowing the physician to view the structure and properties of the skin in greater detail than can be achieved by simple visual examination. We review the last 100 years of fluorescence imaging development from clinical observation to advanced spectral imaging, addressing the role of fluorescence diagnostics (FD) in modern dermatology as well as the detection of autofluorescence.     

Multiphoton laser scanning microscopy on non-melanoma skin cancer: morphologic features for future non-invasive diagnostics

This study describes the morphologic features of human non-melanoma skin cancer obtained using multiphoton laser scanning microscopy (MPLSM) on freshly excised specimens from 14 patients. Optical sectioning parallel to the tissue surface was performed, resulting in en face autofluorescence images of the epidermis and upper dermis, reaching tissue depths of 135 microm. The microscopy was carried out ex vivo using a femtosecond pulsed laser at 780 nm and a x 40/0.8 objective. The autofluorescence was detected in the range of 450-530 nm. Traditional histopathological criteria such as bowenoid dysplasia, multinucleated cells, or hyperkeratosis in squamous cell carcinoma in situ (SCCIS) (five specimens), and peripheral palisading of tumor cells in superficial basal cell carcinoma (SBCC) (six specimens) were clearly discerned. The morphologic features differed significantly between these lesions and perilesional skin. However, characteristic tumor aggregates were found in only one of the three investigated nodular basal cell carcinomas (NBCCs) due to limited imaging depth. In addition, speckled perinuclear fluorescence was observed in both lesions and normal perilesional skin. In conclusion, MPLSM could potentially be applied for non-invasive diagnostics of SCCIS and SBCC, whereas the ability to characterize NBCC is unclear at this point.     

In vivo fluorescence of human skin. A potential marker of photoaging

Fluorescence is a feature of elastin and collagen, both major compounds of human dermis that are altered by age and photoexposure. We studied the intrinsic fluorescence of skin in vivo in 28 human volunteers to determine whether photoaging and chronologic aging of the skin could be evaluated by this noninvasive technique. We demonstrate that the excitation of skin autofluorescence by laser ultraviolet radiation yields characteristic tissue fluorescence spectra that are unrelated to age, pigmentation, or skin thickness. The differences in skin autofluorescence appear to be related to photoexposure. Thus, laser-induced fluorimetry, a noninvasive technique, may be adaptable as a marker of photoaging.     

Autofluorescence spectrum of skin: component bands and body site variations

Background/aims: The autofluorescence spectrum of skin consists of a number of overlapping single spectra emitted by fluorescent molecules. The overlapping makes it difficult to distinguish changes in the overall spectrum and the molecular basis for it. We applied curve fitting analysis to decompose the autofluorescence spectrum of normal human skin and studied the variation in different body sites. Methods: Skin autofluorescence spectra were obtained in vivo from the normal skin of 17 subjects, using 5 excitation wavelengths: 340, 350, 360, 370 and 380 nm. The spectra were displayed in the wavelength range of 400 nm to 800 nm. Curve fitting was performed for each spectrum using the Levenberg–Marquardt algorithm. Results: The skin autofluorescence spectra, provoked by all the excitation wavelengths, consisted of three component bands with center wavelengths at 450 nm, 520 nm and 625 nm. The 450 nm band contributed about 75% to the intensity of the overall spectrum, the 520 nm band contributed 25%, and the 625 nm band contributed about 2%. The three bands may correspond mainly to the fluorescence of nicotinamide adenine dinucleotide (NADH), flavin adenine dinucleotide (FAD), and porphyrins. Significant differences were found between the spectra of forehead and buttock skin. In forehead skin, the 625 nm band contributed more to the intensity of the overall spectrum, while the 450 nm and 520 nm bands had much lower peak heights, and the 450 nm and 625 nm bands were broader. Conclusions: The autofluorescence of skin is an overlapped spectra of molecules such as NADH, FAD, and porphyrins. These molecules contribute differently to the overall spectrum in different body sites.     

Spectroscopic assessment of dermal melanin using blue vitiligo as an in vivo model

BACKGROUND: Spectroscopic methods have been used to analyze in vivo melanin in the past but the specific effect of melanin depth on autofluorescence and reflectance spectroscopy has not been determined. In patients with blue vitiligo, three distinctive clinicopathologic patterns are present: (1) normal skin with normal epidermal melanin pigmentation (2), skin of blue vitiligo with dermal melanin pigmentation, and (3) tissue of regular vitiligo with no melanin pigmentation. Blue vitiligo may thus serve as an in vivo model to assess dermal pigment using spectroscopic techniques. OBJECTIVES: To evaluate the reflectance and autofluorescence spectra of a patient with blue vitiligo in order to assess the effect of melanin pigmentation and its localization on the optical properties of the skin. METHODS: The blue-gray, normal and depigmented lesions of a patient with blue vitiligo were analyzed using reflectance and fluorescent spectroscopy. The condition was likely induced by a phototoxic reaction in a patient with pre-existing vitiligo. These data were then correlated to the histologic and electron microscopic findings present in the various types of lesions. RESULTS: Reflectance spectroscopy detected little difference in spectral shape between skin sites affected by blue vitiligo vs. vitiligo. Autofluorescence spectroscopy detected an apparent difference between the two types of lesions, with the blue-gray lesions (blue vitiligo) showing lower fluorescence intensity and spectral maximum position red-shifted compared with regular vitiligo, whereas regular vitiligo showed more intense hemoglobin absorption than the blue vitiligo. CONCLUSIONS: Dermal melanin present in blue vitiligo can be well characterized by autofluorescence spectroscopy, while little difference in reflectance spectral shape exists between vitiligo and blue vitiligo. Thus, autofluorescence spectroscopy may better identify deeper structures in skin tissue, such as melanin, than reflectance spectroscopy.     

Fluorescence lifetime imaging distinguishes basal cell carcinoma from surrounding uninvolved skin

Background  Fluorescence lifetime imaging (FLIM) is a novel imaging technique that generates image contrast between different states of tissue due to differences in fluorescence decay rates.
Objectives  To establish whether FLIM of skin autofluorescence can provide useful contrast between basal cell carcinomas (BCCs) and surrounding uninvolved skin.
Methods  Unstained excision biopsies of 25 BCCs were imaged en face with FLIM following excitation of autofluorescence with a 355 nm pulsed ultraviolet laser.
Results  Using FLIM we were able to distinguish areas of BCC from surrounding skin in an ex vivo study. Significant reductions in mean fluorescence lifetimes between areas of BCC and areas of surrounding uninvolved skin were demonstrated ( P  <   0·0001). These differences were apparent irrespective of the decay model used to calculate the fluorescence lifetimes (single vs. stretched exponential) or the long-pass filter through which the emitted autofluorescence was collected (375 vs. 455 nm). Conversely, there was no significant difference between the BCC and uninvolved areas of each sample when mean autofluorescence intensities were examined. Moreover, wide-field false-colour images of fluorescence lifetimes clearly discriminated areas of BCC from the surrounding uninvolved skin.
Conclusions  We therefore believe that FLIM has a potential future clinical role in imaging BCCs for rapid and noninvasive tumour delineation and as an aid to determine adequate excision margins with best preservation of normal tissue.     

Fluorescence excitation spectroscopy provides information about human skin in vivo

Fluorescence spectroscopy of human skin has the potential to provide useful morphologic and biochemical information. The endogenous fluorescence of human skin has been investigated in vivo on normal human volunteers as well as on patients with psoriasis and it was found that characteristic bands can be identified in the fluorescence spectra that are associated with specific skin fluorophores. One epidermal band (295 nm excitation, attributed to tryptophan) and two dermal bands (335 and 370 nm excitation, attributed to collagen cross-links) were consistently present in all fluorescence spectra. In addition, the fluorescence spectra obtained from lesions and nonlesional sites of psoriatic patients differed from those obtained from healthy volunteers and the hyperproliferative state of the lesions was characterized by a significantly larger signal at 295 nm excitation. These results indicate that fluorescence spectroscopy is a promising technique for the investigation of human skin in vivo.     

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.     

An improved human skin explant culture method for testing and assessing personal care products

Background

Cultured human skin models have been widely used in the evaluation of dermato-cosmetic products as alternatives to animal testing and expensive clinical testing. The most common in vitro skin culture approach is to maintain skin biopsies in an airlifted condition at the interface of the supporting culture medium and the air phase. This type of ex vivo skin explant culture is not, however, adequate for the testing of cleansing products, such as shampoos and body washes. One major deficiency is that cleansing products would not remain confined on top of the epidermis and have a high chance of running off toward the dermal side, thus compromising the experimental procedure and data interpretation.

Materials and Methods

Here, we describe an improved ex vivo method for culturing full-thickness human skin for the effective testing and evaluation of skin care products by topical application.

Results

This newly developed ex vivo human skin culture method has the ability to maintain healthy skin tissues for up to 14 days in culture. Importantly, the model provides a quick and safe way to evaluate skin care products at different time points after single or repetitive topical applications using a combined regimen of leave-on and wash-off. We found that the results obtained using the new skin culture method are reproducible and consistent with the data collected from clinical testing.

Conclusion

Our new ex vivo skin explant method offers a highly efficient and cost-effective system for the evaluation and testing of a variety of personal care products and new formulations.     

Skin autofluorescence as a biological UVR dosimeter

BACKGROUND/PURPOSE: Collagen is one of the major endogenous skin fluorophores. Alteration in the structure of collagen due to chronic ultraviolet radiation (UVR) exposure may influence the intensity of the autofluorescence. The aim of this study was to investigate the relation between collagen-linked autofluorescence and sun exposure to clarify whether the skin can be used as a biological UVR dosimeter. METHODS: We conducted an in vivo study with 131 healthy volunteers. Fluorescence was measured from sun-exposed (dorsal forearm, forehead and shoulder) and sun-protected (buttock) skin and corrected for the impact of pigmentation and redness. The excitation wavelengths (Ex) and emission wavelengths (Em) were: Ex330:Em370, Ex330:Em455 and Ex370:Em455 nm. Individual UVR exposure data were collected both retrospectively and prospectively using questionnaires and electronic personal UVR dosimeters for a summer period. RESULTS: Age, but not sex, skin type or smoking habits correlated significantly positively with skin autofluorescence at Ex370:Em455 at all body sites (P<0.001, r(2)=0.08-0.26), and at Ex330:Em455 only at the buttock (P=0.001, r(2)=0.08), whereas age was not correlated with Ex330:Em370. Sun-protected buttock skin had significantly higher autofluorescence than sun-exposed skin (P-values<0.0001). Because of great between-subject differences in autofluorescence at different body sites, and because the autofluorescence at the unexposed buttock represents the baseline value, individual correction of skin autofluorescence measurement with that of the buttock was performed. Different measures of individual chronic cumulative UVR doses correlated significantly negatively with the skin autofluorescence ratio (F(ratio)), but the correlations were poor (r(2)=0.03-0.10). CONCLUSION: The results indicate that the collagen-linked skin F(ratio) might be best to use as a measure of individual photodamage, a UVR dose effect, and that it is also a better marker of individual cumulative UVR dose than the used UVR exposure measurements. The methods used to obtain UVR exposure data might not be sensitive and specific enough.     

Comparison of cutaneous bioavailability of cosmetic preparations containing caffeine or alpha-tocopherol applied on human skin models or human skin ex vivo at finite doses

The use of human skin models for performing cutaneous bioavailability studies has been little investigated. For instance, only few studies have been reported on human skin models dealing with vehicle effects on percutaneous penetration. The present study aimed at evaluating the influence on caffeine's and alpha-tocopherol's cutaneous bioavailability of cosmetic vehicles such as a water-in-oil emulsion, an oil-in-water emulsion, a liposome dispersion and a hydrogel applied at finite dose using the reconstructed human skin models EpiDerm and Episkin. The results were compared with those obtained in human skin ex vivo using similar experimental conditions. It was demonstrated that the rank order of solute permeability could be correctly predicted when the preparation was applied at a finite dose in human skin models, at least when solutes with far different physicochemical properties such as caffeine and alpha-tocopherol were used. If only slight effects of cosmetic vehicle on skin bioavailability were observed in human skin ex vivo, they were less predictable using skin models. Especially, alcohol-containing vehicles seemed to behave differently in EpiDerm as well as in Episkin than on human skin ex vivo. Stratum corneum intercellular lipid composition and organization of human skin models differ to some extent from that of human stratum corneum ex vivo, which contributes to less pronounced barrier properties, together with the increased hydration of the outermost stratum corneum layers of the models. These features, as well as still unknown factors, may explain the differences observed in vehicle effects in human skin ex vivo versus human skin models.     

Ocular lens blue autofluorescence cannot be used as a measure of individual cumulative UVR exposure

BACKGROUND/PURPOSE: The accumulation of fluorophores in the ocular lens with age might be caused by ultraviolet solar radiation (UVR) exposure, but evidence of a relation between individual cumulative UVR exposure and lens autofluorescence is lacking. Individually determined UVR exposure has never before been related to lens autofluorescence, and the aim of this study was to investigate if ocular lens blue autofluorescence can be used as a biological UVR dosimeter. METHODS: Ocular lens autofluorescence was quantified in vivo by fluorescence spectroscopy in 145 volunteers (108 healthy subjects, 18 with basal cell carcinoma (BCC) and 19 with cutaneous malignant melanoma (MM)). The excitation wavelength was 350 nm and the fluorescence emission was 450 nm. Individual UVR exposure data were collected both retrospectively and prospectively using questionnaires and electronic personal UVR dosimeters. RESULTS: Lens blue autofluorescence increased significantly with age (P=0.01), and females had significantly higher autofluorescence than males (P=0.024); the two factors explained 10% of the total variation in lens autofluorescence. Neither smoking habits nor use of glasses/contact lenses or sunglasses influenced autofluorescence. No correlations between autofluorescence and UVR exposure measurements were found, and neither was there a difference in autofluorescence between groups with high and low UVR exposure (P-values>0.1), respectively. MM patients had significantly (P=0.019) higher autofluorescence than healthy subjects when age and sex differences were taken into account; no such difference (P=0.097) was detected between BCC patients and healthy subjects. CONCLUSION: The results indicate that age and gender only play a minor role in the level of lens blue autofluorescence. Exposure to UVR has been suggested to be responsible for a part of the age-related increase in autofluorescence, but this could not be confirmed in this study. The higher level of lens autofluorescence found in MM patients might be due to genetics rather than higher cumulative UVR exposure. In conclusion, ocular lens blue autofluorescence cannot be used as a biological UVR dosimeter.     

PATTERNS AND INTENSITY OE AUTOFLUORESCENCE AND ITS RELATION TO MELANIN IN HUMAN EPIDERMIS AND HAIR

The skin of 41 patients including 16 blacks, 15 Caucasians, and 10 Hispanics, was observed using a fluorescent microscope. Three patterns of autofluorescence were observed: intercellular, cytoplasmic, and a combination of intercellular and cytoplasmic. The hair of 75 subjects, including 18 Negroes and 55 Caucasians, was observed. Two patterns were found: medullar and at the cortex. Skin form black patients was associated with the cytoplasmic pattern of autofluorescence. Compared to lighter skin, black skin was also significantly associated with increased intensity of autofluorescence, indicating that autofluorescence of the epidermis parallels the clinical degree of pigmentation. In the hair of 75 subjects, similar results were obtained: Negro hair exhibited more fluorescence than Caucasian hair, and darker hair (brown to black) exhibited more fluorescence than lighter hair (blond). This may be related to melanin and it breakdown products.     

In vitro testing of tensides employing monolayer cultures: a comparison with results of patch tests on human volunteers

Evaluation of the irritant potential of new products or ingredients prior to human testing is generally performed in vivo on animals. However, according to the 6th amendment and following updates of the European Community directive on cosmetic products (93/35/EEC), animal testing will be banned when suitable substitutes will be available. To know whether in vitro tests for assessment of skin irritancy provide results approaching human conditions, comparisons have to be made between data deriving from in vitro tests and skin response in humans. The aim of our study was to assess the validity of the monolayer culture system of normal human keratinocytes as a model for the evaluation of the irritant effects of detergents, by comparing in vitro cell culture data to in vivo acute skin irritancy effects of cocamidopropyl betaine (CAPB), an amphoteric compound, Tween 20 (TW20) (polysorbate 20) and Tween 80 (TW80) (polysorbate 80), representing nonionic compounds, applied to the skin of 24 healthy volunteers at a concentration similar to that employed in commercial products. As parameters for cytotoxicity, cell proliferation, cell membrane integrity and cell metabolism were assessed by cell counts, thymidine incorporation, MTT conversion, and Neutral Red uptake. In order to increase the sensitivity of the in vivo evaluation, bioengineering methods for assessment of the effects of test products on the skin were employed. Whereas all 4 in vitro methods ranked the tensides according to their toxicity in the following order: CAPB>SLS>TW20>TW80, both in vivo methods agreed in identifying SLS as the most irritating substance. Moreover, as compared with the irritation potential on human skin, all 4 in vitro tests overestimated the toxicity of CAPB. This suggests that the keratinocyte monolayer cell culture technique cannot directly replace in vivo methods, and that data obtained by this method should be interpreted cautiously.     

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 role of the skin and adjacent mucous membranes in host defense

This review shows that useful information about immunological mechanisms of pathogenesis can be obtained from observations of human skin and appropriate animal models. Although the immunological mechanisms in skin and mucous membranes are becoming better known, much research remains to be done.     

Spectral fluorescence lifetime detection and selective melanin imaging by multiphoton laser tomography for melanoma diagnosis

Abstract:  Multiphoton excited tissue fluorescence summarises the emission of all naturally occurring endogenous fluorescent bio-molecules with their often overlapping fluorescence spectra. Common fluorescence intensity measurements could not be utilised to distinguish between different fluorophores or metabolic states. To overcome this limitation, we investigated new procedures of selective melanin imaging and spectral fluorescence lifetime imaging in combination with high resolution multiphoton laser tomography. Overall 46 melanocytic lesions of human skin were analysed. We suggested that fluorescence light, detected in such a way, may yield additional information for melanoma diagnostics. Remarkable differences in lifetime behaviour of keratinocytes in contrast to melanocytes were observed. Fluorescence lifetime distribution was found in correlation with the intracellular amount of melanin. Spectral analysis of melanoma revealed a main fluorescence peak around 470 nm in combination with an additional peak close to 550 nm throughout all epidermal layers. Excitation at 800 nm shows a selectively observable fluorescence of melanin containing cells and offers the possibility of cell classification. Procedures of selective imaging as well as spectral fluorescence lifetime imaging by means of multiphoton laser tomography support diagnostic decisions and may improve the process of non-invasive early detection of melanoma.     

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.