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* Erasmus MC, University Medical Center Rotterdam, Department of General Surgery, 3015 GE Rotterdam, The Netherlands; and Personal Care Institute, Philips Research, 5656 AA Eindhoven, The Netherlands
Correspondence: Address reprint requests to Gerwin J. Puppels, E-mail: g.puppels{at}erasmusmc.nl.
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ABSTRACT |
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INTRODUCTION |
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40-µm thick, but it can be much thicker on load-bearing areas such as palms and soles. The epidermis can be divided in various sublayers. The principal cells of the epidermis are keratinocytes. These cells originate in the lower epidermis by division of basal cells in the basal layer. As the keratinocytes mature, they move upward toward the skin surface, thereby changing shape and molecular composition. Finally, the viable keratinocytes undergo a rapid, terminal transition into a cornified layer of dead, flattened cells, called the stratum corneum (SC) (Jakubovic and Ackerman, 1992
; Odland, 1991). This outermost skin layer has a thickness of 10–15 µm on most areas of the body, except for the palms and the soles, where the SC can be more than 10 times thicker. Despite its normally minute thickness, the SC provides a highly efficient barrier for water and protection against microbial and chemical assault. The major constituent of the SC is keratin, which accounts for 80% of its dry weight (Steinert and Freedberg, 1991).
Optical microscopic techniques offer unique possibilities for noninvasive skin research and skin characterization at high spatial resolution. Examples of such techniques are optical coherence tomography, two-photon fluorescence, and in vivo confocal scanning laser microscopy (CSLM). Optical coherence tomography takes advantage of the short temporal coherence of a broadband light source to reject multiple-scattered light. The spatial resolution in the axial direction is 10–20 µm, which is close to the thickness of the stratum corneum (Huang et al., 1991; Schmitt et al., 1995) although more recently, depth resolutions better than 5 µm have been reported (Wang and Elder, 2002; Bordenave et al., 2002; Knuttel et al., 2001). Two-photon fluorescence microscopy uses two-photon excitation of endogenous chromophores in the tissue, which results in imaging with a high spatial resolution (Masters et al., 1998; Hendriks and Lucassen, 2001; So et al., 2000).
In vivo confocal microscopy enables real-time imaging of living tissue at high resolution and high contrast, without physically dissecting the tissue. The first in vivo confocal microscopic images of human skin were obtained with a tandem scanning confocal microscope. A mercury lamp was employed as light source and a Nipkow disk as scanning device (Corcuff and Leveque, 1993). Subsequently, CSLM has been developed, offering the advantage of precise wavelength selectivity and high illumination power (Rajadhyaksha et al., 1995). In vivo CSLM can be used to study the morphology of living skin for basic and clinical research. Confocal images show cellular and nuclear structure in the epidermis, collagen fibers in the dermis, and circulating blood cells in dermal capillaries, as well as structures such as sebaceous glands, hair and hair follicles, and sweat ducts. Different skin layers (stratum corneum, viable epidermis, dermis) can be clearly identified. In vivo confocal microscopy is a potential tool in the clinic for imaging skin lesions and their margins before biopsy, and for diagnosis of lesions (Rajadhyaksha et al., 1999).
These imaging techniques are methods to study skin morphology. In vivo confocal Raman spectroscopy provides detailed information about the molecular composition of the skin. Raman spectroscopy is widely used to study biological samples and, more recently, also to study the skin (Gniadecka et al., 1998; Barry et al., 1992). Application in clinical dermatology has been probed in studies of vitiligo (Schallreuter et al., 1999), atopic and psoriatic skin (Wohlrab et al., 2001), and basal cell carcinoma (Nijssen et al., 2002). We have introduced in vivo confocal Raman spectroscopy as a method to obtain depth-resolved information about the molecular composition of the skin (Caspers et al., 1998). The axial resolution of 5 µm (full width at half maximum) enabled the determination of in vivo concentration profiles of constituents of natural moisturizing factor (free amino acids and amino acid derivatives), of sweat (lactate and urea), and of water in the SC (Caspers et al., 2000, 2001, 2002).
In this paper we demonstrate that Raman spectroscopy for in vivo skin research can be further extended by combining the technique with CSLM. In this way, precise targeting of a (subsurface) skin structure for Raman measurements is enabled. In addition, molecular concentration profiles, as determined by Raman spectroscopy, can be directly related to skin architecture, in particular the skin layers.
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MATERIALS AND METHODS |
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, 2001), and a confocal scanning laser microscope (type VivaScope 1000, Lucid Inc., Rochester, NY). The confocal Raman system employs a tunable titanium-sapphire laser (model 3900S, Spectra-Physics, Mountain View, CA) pumped by an Argon-ion laser (model 2020, Spectra-Physics). The spectra were recorded using a custom-designed F/2.1 spectrograph with 6 cm-1 spectral resolution and a liquid nitrogen cooled, deep depletion charge-coupled device (CCD) camera with a chip size of 1024 x 256 pixels (Princeton Instruments, Trenton, NJ). A 30x numerical aperture (NA) 0.90 water immersion microscope objective with coverglass correction was used (Lomo, Vermont Optechs, Charlotte, VT) to focus the laser light from both the Raman laser and the CSLM laser. The objective was mounted in a fast and accurate piezoelectric focusing drive, accurate to less than 0.1 µm operating in closed-loop (position-controlled) mode (Physik Instrumente, Waldbronn, Germany). Focusing of the microscope objective and adjustment of the power of the laser diode of the CSLM were controlled by a personal computer. This PC was also used to digitize and store the confocal images produced by the VivaScope. The operating software was custom developed (Philips Research, Eindhoven, The Netherlands). A second PC was used to record the Raman spectra.
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The standard operating procedure of the CSLM was applied, in which a coverglass was mounted in a metal ring that was stuck to the skin with double-sided tape. The metal ring was then attached to the microscope and could be moved laterally with a xy-translation stage. In this way a target of interest in the skin could be positioned in the focused laser beam for Raman spectroscopic evaluation. Because the skin was in contact with the coverglass, the distance between the microscope objective and the skin surface could be accurately controlled. Unintentional vertical motion of the skin could be limited to 2 µm (Caspers et al., 2001). Coverglasses were made of CaF2, because of its weak Raman signal in the spectral region of interest. Demineralized water between the coverglass and the skin surface was used to improve refractive index matching. This is of particular importance for the quality of the confocal images of the skin. Without water, a thin layer of air exists between coverglass and skin, resulting in large changes in the refractive index at the coverglass-air-skin interface. This would strongly degrade the image quality (Rajadhyaksha et al., 1999). The refresh rate of the CSLM images was 14 frames/s.
It was experimentally verified that the origin of the Raman signal was located within 2 µm of the focal plane of the CSLM. For this purpose polystyrene beads of 1-µm diameter were stuck to the coverglass of the CSLM and the surface of the coverglass was brought into the focal plane of the CSLM. A bead was positioned in the focused Raman laser by moving the coverglass laterally. Once the bead was in focus, the intensity of its Raman signal was monitored while the bead was moved through the focal plane in the axial direction. In this way it was determined that the maximum Raman signal was obtained when the bead was within 2 µm of the focal plane of the CSLM. This is well within the current axial resolution of the confocal Raman spectrometer in the combined system (9 µm). The axial resolution, defined as the full width at half maximum (FWHM) of the response curve of an infinitesimal thin plane, was determined from the derivative of a measured step response curve across an oil-CaF2 interface. Details of this method have been described elsewhere (Caspers et al., 2000). In the current setup we used the standard microscope objective that was provided with the VivaScope (30x NA 0.9 water immersion). The combination of magnification and NA of the microscope objective, the focal length of the lens in front of the signal collection fiber, and the core diameter of this fiber (see Fig. 1 A) was not fully optimized. Therefore, the axial resolution for the Raman measurements was worse than the 5-µm resolution achieved in previous studies (Caspers et al., 2000).
In all in vivo experiments the depth, or distance to the skin surface, was determined from the displacement of the piezoelectric drive relative to the zero position, at which the focal plane was at the skin surface. No attempt was made to correct for a focus shift in the axial direction due to refractive index differences between water, the coverglass, and skin (Rajadhyaksha et al., 1999).
Experiments
In vivo experiments were carried out on the volar aspect of the arm and on the palm of the hand of healthy volunteers. The skin was not pretreated in any way. Approval for these experiments was obtained from the Medical Ethics Review Board of the Erasmus MC.
In vivo concentration profiles of water and constituents of sweat and natural moisturizing factor (NMF) were determined as follows. Raman spectra were recorded from a range of depths across the SC of the palm, from the skin surface down to the viable epidermis. Depth increments were typically 10 µm. A corresponding set of CLSM images was used to visualize the boundary between the SC and the viable epidermis. Images were recorded at depth intervals of 1 µm. The images were stored on disk and processed afterwards to obtain an in vivo cross-sectional image of the SC and part of the viable epidermis.
For determination of water concentrations in the SC, a laser wavelength of 720 nm was used for Raman excitation. Raman spectra were recorded at 1 s per spectrum in the wavenumber region from 2000 to 4000 cm-1. From these Raman spectra, water concentration profiles were determined. The water concentration in mass percentage was calculated using the intensity ratio between the Raman bands of water (3350–3550 cm-1) and protein (2910–2965 cm-1). The details of this procedure are described elsewhere (Caspers et al., 2000, 2001).
For the determination of concentration profiles of NMF and sweat constituents, Raman spectra were recorded in the spectral region from 400–2000 cm-1 ("fingerprint region"). The excitation wavelength in these experiments was 850 nm. The data collection time was 30 s per spectrum. Concentration profiles of NMF were determined semiquantitatively, using the relative signal contributions of NMF and keratin to the total in vivo Raman spectrum of stratum corneum (80% of the dry weight of the stratum corneum consists of keratin). This was done using multiple-least-squares fitting, in which the in vivo skin spectrum was fitted with the in vitro Raman spectra of NMF, keratin, ceramide, lactate, and urea. A combination of these in vitro Raman spectra provides an adequate model for in vivo spectra of human SC (Caspers et al., 2001). The resulting fit coefficients represent the relative proportions in which these species contribute to the total SC Raman signal. The NMF/keratin ratio was the ratio between the fit coefficients of NMF and keratin. This ratio was normalized to 1 at the maximum value.
Raman spectra of structures in the skin (sweat ducts, sebaceous glands, and blood capillaries) were measured in the fingerprint region. The excitation wavelength was 850 nm and the exposure time was 20 s per spectrum. Together with each recorded Raman spectrum, the corresponding CSLM image was captured and stored. For comparison with in vivo Raman spectra of dermal capillaries, in vitro spectra were recorded from fresh, human blood in glass capillaries. In all in vivo experiments the Raman laser power at the skin was 100 mW.
Data processing
The software used to control the CSLM enabled automatic recording and storage of confocal images as a stack of uncompressed bitmap files. For easy handling, a descriptor file was generated containing the filenames and depth coordinates for each image in a stack. A complete stack represents a volume section of the tissue, with each pixel having an (x,y,z)-coordinate and a brightness level. Plotting the image stack in an xy-plane (that is, all pixels having the same z-coordinate) results in an image of a section parallel to the skin surface. Similarly, cross-sectional images were obtained by plotting the image stack in an xz-plane or in a yz-plane.
Processing of images and of spectral data was done with software that was developed in-house using Matlab (MathWorks, Natick, MA). The Raman spectra were calibrated to relative wavenumbers and corrected for the wavelength dependent detection efficiency of the Raman system (Wolthuis et al., 1999).
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RESULTS |
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). Blood flow within dermal capillaries can be observed in the center of dermal papillae. These capillaries ascend from the papillary plexus, which is a network of microcirculatory elements oriented parallel to the skin surface. The plexus is located in the upper part of the dermis. From this plexus, capillary loops are directed toward the epidermis and extend into the dermal papillae (Jakubovic and Ackerman, 1992). Fig. 6 shows a confocal image of a dermal capillary. Although capillaries may be difficult to recognize in a single image, they clearly show up in the live video image because of the circulating blood cells, which appear as bright dots moving along the same path (Rajadhyaksha et al., 1995). By positioning capillaries in the center of the cross-hair, Raman spectra were obtained of blood inside the capillary. Fig. 6 shows an in vivo spectrum obtained from a capillary and for comparison, a spectrum of blood that was obtained in vitro.
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DISCUSSION |
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). However, the SC is an optically homogeneous layer; the cells are flattened, and nuclei and other cell organelles are absent. As a result CSLM images of the SC are relatively dark and show little contrast. In comparison, the living cells of the viable epidermis contain cytoplasm, organelles, and nuclei, from which light is scattered due to refractive index changes. Overall, images of the viable epidermis are therefore brighter than images of the SC. As a result, the cross-sectional image shows a well-defined junction between the darker SC and the brighter viable epidermis (Figs. 2 A and 3 A).
The water concentration profile in the SC provides insight into the water-holding capacity and barrier properties of the skin. This has implications for pharmacology, cosmetics, and fundamental skin biology. Transepidermal water loss measurements and skin impedance measurements have been used in combination with mathematical modeling of water diffusion through a membrane to study the barrier properties of the SC (Kalia et al., 1996; Schwindt et al., 1998). These studies have provided indirect information about the location of the water barrier and the water concentration profile. However, they have not resulted in a clear understanding of the in vivo water concentration in the SC as a function of depth.
In contrast, confocal Raman spectroscopy combined with CSLM provides a direct method to study the water concentration profile in the SC. With the results presented here, we show for the first time a detailed in vivo water concentration profile for the SC in relation to the skin architecture. The water concentration within the SC of the palm is constant throughout most of the SC (Fig. 2). Only at the junction between the SC and viable epidermis does the water content increase with a steep gradient, reaching 70% (mass %, or grams of water per 100 g of wet tissue) in the viable epidermis. The slight increase of the water content at the skin surface has not been observed in earlier experiments using in vivo confocal Raman spectroscopy without CSLM (Caspers et al., 2000, 2001) and must be dismissed as an artifact. It is presumably caused by the thin film of water between the window and the skin surface that was applied to improve optical refractive index matching between skin surface and coverglass (see Materials and Methods section). The use of a different matching medium, such as oil, would avoid this problem. The presence of a steep gradient at the lower part of the SC implies that the water barrier is not homogeneously distributed over the entire SC of the palm, but located mostly at the junction between the SC and the SG. These observations do not confirm results obtained by transepidermal water loss measurements and mathematical modeling from which it was concluded that the SC of the arm is a homogeneous water barrier (Kalia et al., 1996; Schwindt et al., 1998). Whether the in vivo water concentration profile for the SC of the arm has the same pattern of a slowly varying water concentration in most of the SC followed by a steep gradient at the lower SC boundary remains to be elucidated. Currently the axial resolution of the combined Raman/CSLM system is not sufficient to answer this question, but an improvement with a factor of 3 to 3 µm is feasible.
The relative changes in the concentration of NMF show a pattern quite different from the water concentration profile. Scott and co-workers have shown that a layer of stable filaggrin exists in the lowermost cell layers of the SC (Scott and Harding, 1986; Rawlings et al., 1994). This precursor protein is broken down by hydrolytic enzymes, a process that produces the free amino acids and amino acid derivatives that, together with specific salts, form the NMF. Proteolytic activity of these enzymes is modulated by the water content in the tissue. Radio labeling and immunohistochemical studies in rats have shown that a high-water content in the SC prevents the production of NMF by filaggrin proteolysis. In normal conditions, the relatively dry external environment dries out the outer layers of the SC resulting in a water gradient decreasing from the deeper SC toward the surface. The decreased water content triggers proteolysis of filaggrin in the deeper part of the SC (Scott and Harding, 1986).
Fig. 3 visualizes this localized production of NMF in the lower part of the SC in vivo. The NMF-to-keratin ratio is zero in the viable epidermis. The steep gradient in the NMF profile indicates a very rapid increase of NMF at 15 µm above the SC/SG junction. According to Fig. 2, the water concentration at the distance of 15 µm from the SC/SG junction has dropped considerably with respect to the water concentration in the viable cells. These in vivo observations are in agreement with the findings by Scott and co-workers (Scott and Harding, 1986) that a decrease in water concentration is required before the proteolysis of filaggrin can start and the presence of NMF can be detected.
Absence or decreased production of NMF has been related to skin problems such as xerosis, ichtyosis, and psoriasis (Horii et al., 1989; Rawlings et al., 1994). Decrease in NMF has also been observed in aged skin (Rawlings et al., 1994). In vivo confocal Raman spectroscopy combined with CSLM provides a method to study the distribution of NMF and water in the SC in vivo. It can therefore contribute to gaining insight into the cause and treatment of skin conditions such as those mentioned above.
Figs. 4–6 illustrate the capability of combined confocal Raman spectroscopy and CSLM for study of the molecular composition of structures in the skin in situ. The real-time imaging capability of the CSLM was used to locate a structure of interest, such as a sweat duct (Fig. 4). Confocal Raman spectroscopy was then used to obtain spectroscopic information from this structure. Clearly the Raman band at 856 cm-1 is more intense in the spectrum of the sweat duct (a, right) than in the spectrum measured outside the duct (b, right). Multiple regression fitting of spectrum a with spectrum b and the spectra of lactate, urea, and the predominant constituents of NMF was applied to interpret these spectral differences in terms of differences in molecular composition. Details of the fitting procedure have been described elsewhere (Caspers et al., 2001). The choice of these constituents for fitting purposes is based on the assumption that NMF, which is highly water soluble and present in high concentrations in the SC, can easily dissolve in sweat (Sato et al., 1991). Lactate and urea are also present in sweat, as both are secreted by the sweat glands. The concentrations usually depend on the sweat rate. At low sweat rates, the concentration of lactate can be as high as 40 mM and that of urea 4 mM (Sato et al., 1991). At the current spatial resolution of the instrument, signal from the tissue surrounding the sweat duct also unavoidably is collected. Therefore, spectrum b (Fig. 4) was also included in the fit to the spectrum obtained in the sweat duct. The difference spectrum and the fit with the aforementioned compounds (Fig. 4) showed that the concentration of lactate in the sweat duct is much higher than in the surrounding tissue. The significance of this result is that it demonstrates that information about the molecular composition of sweat can be obtained from a sweat duct in situ. For cosmetic research, in designing and improving antiperspirants, which involves modulation of sweat production, this may offer a new and valuable tool to study the effects of these formulations in situ.
A similar experiment was carried out for a sebaceous gland, located at the lower forearm. Human sebaceous glands secrete a lipid mixture of mainly triglycerides, wax esters, free fatty acids, and squalene (Stewart and Downing, 1991). The process of sebogenesis is complex and not entirely understood. It involves elongation and desaturation of free fatty acids in the sebaceous cells, and incorporation of free fatty acids into triglycerides and wax esters. At a later stage bacterial hydrolysis of triglycerides causes the release of free fatty acids.
The Raman spectrum (Fig. 5) from the sebaceous gland (a, right) shows a strong band at 1295 cm-1, which is not present in the spectrum recorded outside the gland (b, right). The difference spectrum (c, right) shows many Raman bands, indicating a difference in molecular composition between the sebaceous gland and the skin around the gland. Although the Raman bands of spectrum a (a, right) remain to be assigned, the comparison with the spectrum of palmitic acid (d, right) suggests the presence of this 16-carbon fatty acid in the sebaceous gland. The 16-carbon acids are predominant in sebaceous cells (Pappas et al., 2002). This first result demonstrates the potential to study the lipid composition in a sebaceous gland in vivo. It also suggests the possibility to measure Raman spectra in various parts of the sebaceous gland, which would enable one to follow the complex process of sebogenesis in vivo.
Confocal Raman spectroscopy combined with an imaging modality such as CSLM can be used to obtain direct chemical information about structures in the skin in situ. As such, the technique presented also provides a potential method to investigate in vivo the penetration of topically applied substances via shunt routes. This offers the possibility to apply substances to the skin and study their effects in situ, directly in the sebaceous glands, sweat glands, or hair follicles. This capability should be of particular interest in cosmetics research and pharmaceutical research on transdermal drug delivery.
In vivo recording of Raman spectra from dermal capillaries (Fig. 6) offers possibilities that reach well beyond skin research. Spectrum a was recorded in vivo, focusing on a capillary, whereas spectrum b is an in vitro spectrum of blood. The in vivo spectrum a is likely to contain signal contributions from the surrounding tissue, but clearly the majority of the spectrum originates from blood flowing through the dermal capillary. The collection time for spectrum a was only 30 s, demonstrating that high-quality Raman spectra of blood can be rapidly measured in a completely noninvasive manner.
The Raman spectrum of blood largely depends on the oxygenation state of hemoglobin (Venkatesh et al., 1999; Wood and McNaughton, 2002). Due to the high spatial resolution of both CSLM and in vivo confocal Raman spectroscopy, it seems feasible to study the local hemoglobin-oxygen saturation at a microscopic scale. This is currently being investigated. Several in vitro studies have shown the feasibility of using Raman spectroscopy to determine analyte concentrations in whole blood, in plasma, or in serum (Pilotto et al., 2001; Berger et al., 1997; Berger et al., 1999). We therefore believe that the new technique presented here, enabling the in vivo recording of Raman spectra from dermal capillaries, has great potential for noninvasive monitoring of blood analytes, including glucose.
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CONCLUSIONS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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Submitted on October 11, 2002; accepted for publication March 19, 2003.
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  L. Li, H. Wang, and J.-X. Cheng Quantitative Coherent Anti-Stokes Raman Scattering Imaging of Lipid Distribution in Coexisting Domains Biophys. J., November 1, 2005; 89(5): 3480 - 3490. [Abstract] [Full Text] [PDF]
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 A. M. Cotton A Review of the Principles and Use of Lasers in Lower Limb Problems International Journal of Lower Extremity Wounds, September 1, 2004; 3(3): 133 - 142. [Abstract] [PDF]
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