In vivo Drug Screening in Human Skin Using Femtosecond Laser Multiphoton Tomography

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1 Skin Pharmacol Physiol 2006;19:78 88 DOI: / Received: September 2, 2005 Accepted: November 17, 2005 Published online: May 9, 2006 In vivo Drug Screening in Human Skin Using Femtosecond Laser Multiphoton Tomography a, b a a a K. König A. Ehlers F. Stracke I. Riemann a Fraunhofer Institute of Biomedical Technology (IBMT), St. Ingbert, b Faculty of Mechatronics and Physics, Saarland University, Saarbrücken, Germany Free Author Copy - for personal use only ANY DISTRIBUTION OF THIS ARTICLE WITHOUT WRITTEN CONSENT FROM S. KARGER AG, BASEL IS A VIOLATION OF THE COPYRIGHT. Written permission to distribute the PDF will be granted against payment of a permission fee, which is based on the number of accesses required. Please contact permission@karger.ch Key Words Femtosecond laser multiphoton imaging system Two-photon excitation process In vivo tomography of human skin In situ drug screening DermaInspect Abstract The novel femtosecond laser multiphoton imaging system DermaInspect for in vivo tomography of human skin was used to study the diffusion and intradermal accumulation of topically applied cosmetic and pharmaceutical components. Near-infrared 80 MHz picojoule femtosecond laser pulses were employed to excite endogenous fluorophores and fluorescent components of a variety of ointments via a two-photon excitation process. In addition, collagen was imaged by second harmonic generation. A high submicron spatial resolution and 50 ps temporal resolution was achieved using galvoscan mirrors and piezodriven focusing optics together with a timecorrelated single-photon counting module with a fast microchannel plate detector. Individual intratissue cells, intracellular mitochondria, melanosomes, and the morphology of the nuclei as well as extracellular matrix elements were clearly visualized due to NAD(P)H, melanin, elastin, and collagen imaging and the calculation of fluorescence lifetime images. Nanoparticles and intratissue drugs were detected by two-photon-excited fluores- cence. In addition, hydration effects and UV effects were studied by monitoring modifications of cellular morphology and autofluorescence. The system was used to observe the diffusion through the stratum corneum and the accumulation and release of functionalized nanoparticles along hair shafts and epidermal ridges. The novel noninvasive 4-D multiphoton tomography tool provides high-resolution optical biopsies with subcellular resolution, and offers for the first time the possibility to study in situ the diffusion through the skin barrier, long-term pharmacokinetics, and cellular response to cosmetic and pharmaceutical products. Introduction Copyright 2006 S. Karger AG, Basel The human skin is an effective barrier to environmental compounds and protects the body from physical, thermal and chemical hazards. For the same reason the uptake of pharmaceutical and cosmetic substances via this route is strongly limited. Since the transdermal application is noninvasive, simple in use and does not require sterile equipment, a variety of carrier systems for effective topical delivery of drugs and methods for the efficient accumulation in intradermal cellular targets and within the extracellular matrix (ECM) are under investigation. Fax karger@karger.ch S. Karger AG, Basel /06/ $23.50/0 Accessible online at: Karsten König Fraunhofer IBMT, Ensheimer Strasse 48 DE St. Ingbert (Germany) Tel , Fax karsten.koenig@ibmt.fraunhofer.de

2 Unfortunately, current high-resolution studies on the diffusion behavior and intratissue accumulation of topically applied cosmetic and pharmaceutical products are based on in vitro studies, such as light and electron microscopy and HPLC measurements of mechanically-removed biopsies or tape stripping after treatment, the use of diffusion models such as Franz chambers, the employment of artificial skin and cell culture models, etc. There is a demand for in vivo high-resolution 3-D imaging methods to trace chemical compounds within the skin. Such methods should enable (a) the painless investigation of the stratum corneum (SC) and intradermal compartments without tissue removal, (b) the access to fast (immediate) information, (c) the examination under natural physiological (in vivo) conditions and (d) the possibility of long-term studies on the same tissue area to study pharmacokinetics. Optical methods provide better resolution than ultrasound or magnetic resonance imaging. Fluorescence, second harmonic generation (SHG), Raman [1] and reflection can be used to perform optical imaging of tissue components and exogenous components. In order to obtain high-resolution depth-resolved (3-D) images, methods with the capability of optical sectioning have to be applied, such as the confocal detection method [2]. Confocal laser scanning microscopes have been used to perform optical sectioning of the skin in the reflection mode [3 5] and in the fluorescence mode [6]. However, one-photon fluorescence excitation has the disadvantages of low light penetration depth, out-of-focus photodamage, out-of-focus photobleaching, and low photon collection efficiency due to the spatial filtering by the pinhole. In addition, there is a loss of information due to scattered out-of-focus photons which are able to pass through the pinhole towards the detector. These disadvantages can be overcome by multiphoton excitation [7] of intradermal fluorophores and SHG-sensitive molecules, such as collagen, using near-infrared (NIR) femtosecond laser pulses in the spectral range of 700 1,000 nm. This spectral range is considered as the optical window with high light penetration depth where one-photon absorption coefficients and scattering coefficients of skin are low compared with the UV/visible range. Therefore, the required transient laser intensities of GW/cm 2 for multiphoton deep tissue imaging can be applied without damage to tissues and fluorophores. When using focusing optics with high numerical aperture (NA), multiphoton excitation occurs only in a tiny intratissue focal volume of the order of less than 1 fl. Using fast galvoscanner (x, y) and piezodriven focusing optics (z), the intratissue position of the multiphoton excitation volume can be changed in three directions to carry out deep tissue optical sectioning. By means of pinhole-free detectors in the de-scanned mode (luminescence is not transmitted through the scanners), fluorescence and SHG imaging at high photon collection efficiency can be carried out. Blue-/green-emitting fluorophores such as NAD(P)H and elastin, which normally require UV excitation, can be imaged even in the dermis [8]. Using a two-photon femtosecond laser scanning microscope, Masters et al. [9] detected the autofluorescence of human skin down to 200 m depth. Optical sectioning of animal and human skin by NIR femtosecond laser autofluorescence microscopy has been reported, e.g. by So and Kim [10], Masters et al. [11], Hendriks and Lucassen [12], König [13], König et al. [14], and Peuckert et al. [15]. Teuchner et al. [16] reported on femtosecond pulse excitation of melanin fluorescence. However, multiphoton microscopes are not suitable for human skin imaging in vivo. Here we report on the use of the novel CE-marked femtosecond laser scanning system DermaInspect (Jenlab GmbH, Jena, Germany, [17] ) for clinical high-resolution multiphoton tomography of human skin and in vivo and in situ drug screening. We demonstrate its potential for skin diagnostics, and present first hydration studies using water at different ionic strengths, measurements on the impact of UVA radiation, and on the distribution, migration and release of topically applied functionalized nanoparticles and cosmetic substances. Materials and Methods The Multiphoton Imaging System DermaInspect The multiphoton imaging system DermaInspect is a novel highresolution diagnostic tool and can be used for in vitro and in vivo studies on skin. So far, the CE-marked device is world-wide the only femtosecond laser system for clinical diagnostics ( fig. 1 ). It is considered as a class 1M device according to the European laser safety regulations and consists of three major modules: laser, scanning unit, and control module [17]. A compact turn-key mode-locked 80/90-MHz titanium:sapphire laser (MaiTai, Spectra Physics, Mountain View, Calif., USA) with a tuning range of nearly 700 1,000 nm, a maximum laser output of about 1.2 W and a pulse width below 100 fs serves as the laser source. For clinical studies, the maximum output power was limited to 40 mw at the sample which corresponds to a low laser pulse energy of 500 pj only. Typically, 2 mw mean power (25 pj) was used to scan the skin surface. The mean power was increased to values of around 40 mw, when focused into deep tissue structures. In vivo Drug Screening in Human Skin Skin Pharmacol Physiol 2006;19:

3 ments occurred within 1 h after excision. Excised human skin was supplied by K.-H. Kostka, Department of Plastic and Hand Surgery, Caritaskrankenhaus, Lebach, Germany. Pharmaceutical and cosmetic agents of interest were applied topically under occlusion. Fluorescein isothiocyanate-labeled poly( D, L -lactide-co-glycolide) (PLGA) 50: nm nanoparticles (Sigma) were synthesized and suspended in Natrosol hydrogel (Aqualon, Hercules Inc.) by J. Luengo and B. Weiss from the research group of Prof. Lehr, Department of Biopharmaceutics and Pharmaceutical Technology, University of Saarbrücken. Results Fig. 1. Photograph of the DermaInspect system. The scanning module consists of a trigger diode, fast x, y galvo scanners with beam expander and scan optics, a piezodriven 40! focusing optics with NA 1.3 (oil, 200 m working distance and z steps of minimum 40 nm), two photon detectors including one photomultiplier with short picosecond rise time and a microchannel plate multialkali detector ( nm) with 50 ps temporal resolution, a variety of filter sets, as well as a two-part metallic sample adapter with a special m glass window. One part is attached to the skin of interest by an adhesive film, and the magnetic counterpart is connected to the focusing optics. The control module provides power supply, a single photon counting board for fluorescence lifetime imaging (FLIM) [18] and image-processing hardware/software including the special features of online power control and power adjustment according to signal depth, scanning of regions of interest, and single-point illumination. The detector signal was synchronized with the x, y, z beam position calculated from signals of the galvoscanner (frame sync, line sync, pixel clock) and the input voltage from the piezodriven objective. The spatial resolution was determined by the measurement of the point spread function of nanobeads embedded in the upper layers of skin biopsies to be about 0.5- m lateral resolution and 1 to 2 m axial resolution. A pulse width of 240 fs was determined at the focal plane of the system using a special autocorrelator for the measurements of high NA optics. Samples and Chemical Substances Optical in vivo sectioning of Caucasian human skin (type II and III) was performed in about 100 subjects aged years. Typically, the measurement was performed on the lower forearm or the leg of the volunteer. Biopsies obtained from patients with a variety of diseases were introduced into sterile biopsy chambers for microscopic imaging (MiniCeM Biopsy, JenLab GmbH, Jena, Germany) consisting of silicon with 4-mm holes and two m-thick glass windows. Isotonic NaCl solution was used to avoid drying. The measure- Multiphoton Tomography on Nontreated Skin NIR femtosecond laser pulses at a typical short NIR wavelength of 740 or 760 nm have been used to excite endogenous skin fluorophores, such as the coenzymes NAD(P)H and the ECM protein elastin, via a two-photon absorption process. In addition, the luminescence of the pigment melanin was induced. The ECM protein collagen was imaged by the generation of the second harmonic at a laser excitation wavelength of typically 820 or 840 nm and by the use of a broadband detection filter at 410 or 420 nm. Figure 2 demonstrates horizontal high-resolution in vivo autofluorescence images out of a stack of optical sections from the epidermis and the epidermal-dermal junction. As can be seen, even deep-tissue single autofluorescent mitochondria was imaged. The round and ellipsoidal-shaped nuclei can be determined by the absence of visible luminescence. The SC with its fluorescent component keratin exhibited the strongest autofluorescence. The cell borders of the hexagonal-shaped corneocytes with a mean cell diameter of m were clearly seen. In contrast, the membrane of living cells did not fluoresce. The fluorescence pattern on the skin surface was interrupted by nonfluorescent areas with a thickness of up to 100 m due to epidermal ridges. Hairs appeared as highly fluorescent structures. The thickness of the SC in the area of the lower forearm was found to be m. Below this tissue layer of cornified cells, a more diffuse luminescent zone became obvious. No clear morphological features were detectable within this zone with a typical thickness of! 15 m. It is likely that this area contains fluorescent subwavelength features which cannot be resolved by the system. The transition to the stratum granulosum was detected by the occurrence of living cells with a well-defined cell shape with their fluorescent cytoplasms and nonfluores- 80 Skin Pharmacol Physiol 2006;19:78 88 König /Ehlers /Stracke /Riemann

4 Free Author Copy - for personal use only ANY DISTRIBUTION OF THIS ARTICLE WITHOUT WRITTEN CONSENT FROM S. KARGER AG, BASEL IS A VIOLATION OF THE COPYRIGHT. Written permission to distribute the PDF will be granted against payment of a permission fee, which is based on the number of accesses required. Please contact permission@karger.ch Free Author Copy - for personal use only ANY DISTRIBUTION OF THIS ARTICLE WITHOUT WRITTEN CONSENT FROM S. KARGER AG, BASEL IS A VIOLATION OF THE COPYRIGHT. Written permission to distribute the PDF will be granted against payment of a permission fee, which is based on the number of accesses required. Please contact permission@karger.ch Fig. 2. Multiphoton sections of human skin in tissue depths of 5 m, 20 m, 55 m, 65 m and 85 m. The insert was taken with an optical zoom and depicts single living cells. Note that single fluorescent mitochondria can be seen. The dark non-fluorescent circular shaped areas reflect nuclei. cent nuclei. The horizontal cell diameter decreases with increasing tissue depth and was found to be m in the upper part of this layer and m in the deepest cell layer. Also the ratio between the volume of the cytoplasm to the volume of the nuclei decreased. The single innermost cell layer of the epidermis, the stratum basale, was determined by the presence of the cuboidal cells in the perinucleic region, probably the basal cells, with higher light intensity than the living cells in the upper tissue layers. During optical sectioning, the basal cells with melanin occurred as clusters around the tips of papillae. The transition between epidermis and corium was typically found at a depth of m, depending on sex In vivo Drug Screening in Human Skin Skin Pharmacol Physiol 2006;19:

5 Stratum corneum τ 2 = 1.6 ns Stratum spinosum τ 2 = 2.3 ns Stratum basale τ 2 = 1.9 ns Dermis τ 1 <150 ps Fig. 3. Fluorescence decay kinetics and FLIM images from different tissue areas. and age. Connective tissue, in particular single fluorescent long elastin fibers with a thickness of! 1 m were clearly visualized. Collagen structures have been imaged by SHG. NIR femtosecond laser pulses can also be used to excite intense luminescence of the pigment melanin. In the case of a malignant superficial spreading melanoma, clear transitions between the different tissue layers and dendritic cells were recognized, in contrast to nevi. Fluorescence Lifetime Imaging We counted single fluorescence photons with the fast photon detectors using the method of time-correlated single-photon counting. The system allows count rates of more than 10 6 photons per second. Typically, the twophoton-excited autofluorescence of skin at moderate laser powers provided count rates of about 100,000 photons per second. This information was used to provide spatially resolved autofluorescence decay curves per pixel, to fit these curves using a biexponential approach, and to calculate mean fluorescence lifetimes per pixel or per region of interest. The calculated mean fluorescence lifetimes were depicted as color-coded -images (FLIM images) ( fig. 3 ). From this large amount of data, two major fluorescence lifetime components were determined. The shorter has a time within the response time of the detector and can be attributed mainly to the SHG signal as well as to a small amount of scattered light able to pass through the filters. The long component is due to autofluorescence. Its lifetime has been calculated and depicted as false-color fluorescence lifetime image. Typically the fluorescence lifetime of an intratissue cell was found to be on the order of ns. Figure 3 demonstrates typical fluorescence decay curves from tiny intratissue regions (pixel) in different tissue layers and FLIM images. The mean value of the major component was found to be 1.6 ns in the SC probably determined by keratin fluorescence, 2.3 ns in 82 Skin Pharmacol Physiol 2006;19:78 88 König /Ehlers /Stracke /Riemann

6 the stratum spinosum due to NAD(P)H fluorescence, 1.9 ns in the stratum basale which may reflect the influence of melanin and NAD(P)H, and! 50 ps in the dermis due to SHG signals. These in vivo studies on normal human skin demonstrate the capability of 4-D fluorescence imaging in deep tissue with subcellular resolution in the range of 1 m and 50 ps temporal resolution. Time-correlated single-photon counting enables measurements of intratissue fluorescence decay kinetics and the calculation of mean fluorescence lifetimes. In addition, it supports the detection of SHG in the tissue depth by the detection of a short luminescence component limited by the temporal resolution of the detector. The process of SHG occurs immediately in contrast to fluorescence with its typical lifetime in the nanosecond range. The Grinlens Endoscope for Multiphoton Imaging of the Dermis Gradient index lenses [19] offer possibilities to expand the multiphoton imaging depth to intradermal deep-tissue areas. We built a small Grinlens endoscope system of 1.8 mm diameter and up to 6 cm length which can be integrated into the DermaInspect system ( fig. 4 ). The miniature endoscope is capable of transferring the NIR femtosecond laser excitation pulses and transmitting SHG and fluorescence radiation. In particular, 20! NA 0.5 focusing optics in the DermaInspect system were used to match the NA of the Grinlens endoscope (NA 0.5). The endoscope was positioned in such a way that the laser beam scanned the distal end of the endoscope. The laser focus was translated to the intratissue focal plane. The transmittance at 800 nm through the endoscope was determined to be 88%. Using an autocorrelator and 86-fs laser output pulses, the pulse width at the target was measured to be 307 fs. In order to gain information on spatial resolution, the two-photon-excited fluorescence of 0.2- m beads was imaged. The point spread function reveals a lateral resolution of 0.9 m and an axial resolution of 2.8 m. Figure 4 also demonstrates a first image taken from 6- m beads using a pitch 1 Grinlens. The system is currently under investigation regarding background emission, biosafety of the Grinlens materials and damage thresholds before it can be used clinically. Multiphoton Studies on Hydration of Skin We performed in vivo multiphoton studies on functional and structural modifications of the SC and the viable epidermis due to water uptake of different ionic Fig. 4. Diagram of the DermaInspect system in combination with the Grinlens mini-endoscope ( a ) and first images taken from fluorescent microbeads ( b ). strength (0 20% NaCl). In particular, we wanted to detect the process of swelling and shrinking of the corneocytes upon water uptake and water loss. The corneocytes are supposed to be responsible for the water holding capacity of the SC (10 35% water content in the SC) [1, 20]. Typically, the SC consists of about 15 cell layers with a typical layer thickness of m [21]. The skin of the upper forearm of two male and two female volunteers (aged years) was treated topically with solutions of distilled water and 5, 10, and 20% NaCl and sealed with adhesive tape for 2 and 4 h prior to multiphoton tomography. In vivo Drug Screening in Human Skin Skin Pharmacol Physiol 2006;19:

7 Fig. 5. Effect of swelling of the SC due to the application of distilled water and NaCl solutions. Control measurements before the application of the solutions revealed a typical thickness of m for the male and m for the female volunteers. We observed strong vertical swelling effects of distilled water and a shrinking effect of 20% NaCl solutions on the SC layer. In fact, we observed an increase in SC thickness of a factor of two within 2 h in the case of distilled water application, a 50% increase in the case of 5% NaCl, and a 30% decrease when applying 20% NaCl solution. No significant changes occurred at 10% NaCl application. The contrast of the autofluorescence images decreased with applied solutions of! 10% NaCl and increased with 20% NaCl. The high salt concentration resulted in the formation of a fluorescent granular pattern within the corneocytes. The difference in SC thickness between 2-hour and 4-hour occlusions was not significant. Interestingly, the swelling and shrinking of the cells were mostly vertical, not horizontal where the cell diameter or the size of cells did not change. Figure 5 shows an autofluorescence image of the SC before and 2 h after incubation with water. After 2 h, the cell borders changed their pattern significantly. When comparing the in vivo multiphoton tomography data with cryoscanning electron microscopy studies on high-pressure frozen biopsies, similar effects were observed. Using the high resolution of the electron microscope, single corneocytes were identified. Interestingly, it was found that the outermost SC layers as well as the inner SC layers close to the stratum granulosum exhibited the largest cell size. Vertical swelling was found in these layers after application of the same salt solutions. However, the middle part of the SC with the highest cell density did not fluctuate significantly with the change in the ionic strength. Based on these multiphoton and cryomicroscopy studies we conclude that the SC regulates its structure upon hydration depending on the depth of the tissue layer. We also hypothesize a three-hydration-zone model of the SC [21]. In vivo Studies on the Diffusion of Ointments In a variety of studies, ointments with different carriers and drugs have been tested. Most of the ointments revealed two-photon fluorescence. Typically, the emission band and the fluorescence lifetime was different from the skin autofluorescence. Here, we present just one example of a fluorescent cream which was topically applied for 4 min. The skin was scanned at laser wavelengths of 750 and 840 nm down to a depth of 115 m. The mean powers after transmission through the objective were adjusted regarding the signal depth, and ranged from 4 mw for SC imaging to 20 mw at the stratum basale and 23 mw at the stratum papillare (115 m). Images were taken before and 10 min after application. In figure 6, autofluorescence images of Fig. 6. In vivo human skin without ( a ) and with cream application ( b ) at different tissue depths. c 750-nm-excited fluorescence of the cream. 84 Skin Pharmacol Physiol 2006;19:78 88 König /Ehlers /Stracke /Riemann

8 In vivo Drug Screening in Human Skin Skin Pharmacol Physiol 2006;19:

9 Fig. 7. Autofluorescence modifications before (A), directly after irradiation (B), and 30 min (C) after light exposure to the 365- nm radiation of the high pressure mercury lamp HBO50. the nontreated control skin and skin with the probe, and the fluorescence of the cream are shown. The most significant differences were observed within the SC and the diffuse weak fluorescent layer underneath. In the control, the thicknesses of the SC and the diffuse fluorescent layer were found to be about 10 and 5 m, respectively. In contrast, the vertical expansion of the SC was found to be increased to 15 m and of the diffuse layer to 20 m after application of the cream. Fluorescent granules within the upper SC layers were probably due to the accumulation of fluorescent components of the cream. First stratum granulosum cells appeared at a m tissue depth. Elastic fibers at 115 m tissue depth were observed in both cases. The Effect of UVA Radiation For UVA exposure, we used a high pressure mercury lamp HBO50 equipped with a BP 365 filter. A radiation dose of 12 J/cm 2 (8 min exposure time) was applied which was below the minimal erythema dose for the volunteer. The skin was laser scanned at an excitation wavelength of 760 nm before and twice after UV treatment (immediately and 30 min after exposure). The forearm was not moved during the whole procedure so that exactly the same area was scanned. The parameters of the photomultiplier were not altered. In figure 7, the relative mean fluorescence intensity (yaxis) is shown for the different tissue depths (x-axis) before (A), directly after (B) and 30 min after UVA radiation (B). Very high signals were obtained in the SC (0 15 m) for all three measurements. The autofluorescence of the keratinocytes of the stratum granulosum and the stratum basale (20 65 m) was at average levels. In the stratum papillare ( 1 65 m) higher luminescence signals were found due to dermal ECM structures strong SHG signals of collagenous structures. Directly after UVA radiation, the autofluorescence of the cells of the stratum granulosum was poorly detectable. Higher values were found in the stratum spinosum (40 50 m) and stratum basale. Interestingly, 30 min after radiation the autofluorescence of the upper epidermal layers recovered and nearly doubled compared with the value before UVA exposure. Visualizing Nanoparticles in Human Skin Studies indicate that nanoparticles of diverse composition could lead to enhanced intradermal delivery of compounds [22]. The accumulation in hair follicles and wrinkles can be used as long-term storage depots and the residence time of a particle-borne drug on the skin surface can be increased compared with particle-free ointments [23]. 86 Skin Pharmacol Physiol 2006;19:78 88 König /Ehlers /Stracke /Riemann

10 Fig. 8. Multiphoton imaging of FITC-labeled PLGA nanoparticles in the epidermal ridges of excised human skin in vitro at z = 4 m ( a ), z = 8 m ( b ), and z = 18 m ( c ). Fig. 9. Multiphoton images of PLGA nanoparticles carrying the autofluorescent drug flufenamic acid in the epidermal ridges of human skin in vivo ( a ) compared to drug-free nanoparticles as control ( b ). We performed measurements on a variety of nanoparticles with the aim to find out the location on and in the SC and within deeper tissue layers, and to discriminate between particle-bound drugs and released drugs. Figure 8 shows optical sections of excised human skin after application of fluorescent 200-nm PLGA nanoparticles. Imaging was performed up to 370 min of application. We observed significant fluorescence changes of the outermost layer of the SC within the first 10 min of application. As seen from the image, the 800-nm excited SC fluorescence was found to be influenced by the uptake of released fluorescein from the hydrogel. Single particles and particle clusters were only observable within the outermost optical section (z = 4 m) and within the wrinkles. No penetration of particles through the SC took place. A similar investigation was carried out in human skin in vivo. In this study, PLGA particles (mean diameter of 200 nm) loaded with the antirheumatic drug flufenamic acid in hydrogel were applied to the skin of the forearm of a male volunteer. Because flufenamic acid shows a fluorescence band at 420 nm in nonpolar rigid hosts, we tuned the laser wavelength to 720 nm. At this laser wavelength, the drug was detected, whereas the control nanoparticles without the drug were not observable. As seen in figure 9, single nanoparticles were visualized by the flufenamic acid fluorescence within the wrinkles but not in the intratissue compartments. Note the autofluorescence from particle-free living cells in the same focal plane near the wrinkle. The results of in vivo studies proved that PLGA nanoparticles do not penetrate the SC. A detailed discussion of these in vivo studies can be found in Luengo et al. [24]. In vivo Drug Screening in Human Skin Skin Pharmacol Physiol 2006;19:

11 Conclusions The novel femtosecond laser system DermaInspect provides for the first time in vivo high-resolution images of the human skin. In particular, two-photon-excited fluorescence and SHG can be used to trace pharmaceutical and cosmetic components in the natural dermal environment. The high resolution allows even the detection of single deep-tissue mitochondria. Staining of the skin is not required. Multiphoton tomography offers the unique chance to obtain optical biopsies for the detection of dermatological disorders, to detect in situ the diffusion, accumulation and release of chemical components and to monitor therapeutic effects. Acknowledgements The authors wish to thank Dr. Joachim Fluhr, Dr. Martin Kaatz and Prof. Elsner from the Department of Dermatology of the Friedrich Schiller University Jena. This work was supported in part by BMBF grant 01ZZ0105. References 1 Caspers PJ, Lucassen GW, Carter EA, Bruining HA, Puppels GJ: In vivo confocal Raman microspectroscopy of the skin: noninvasive determination of molecular concentration profiles. J Invest Dermatol 2001; 116: Pawley JB (ed): Handbook of Biological Confocal Microscopy. New York, Plenum Press, Rajadhyaksha M, Gonzalez S, Zavislan JM, Anderson RR, Webb RH: In vivo confocal laser scanning laser microscopy of human skin. II. Advances in instrumentation and comparison with histology. J Invest Dermatol 1999; 113: Rajadhyaksha M, Anderson RR, Webb RH: Video-rate confocal scanning laser microscope for imaging human tissues in vivo. Appl Opt 1999; 38: Peuckert C, Riemann I, Wollina U, König K: Remission microscopy with NIR femtosecond laser pulses for 3D in vivo imaging of human skin (abstract). Cell Mol Biol 2000; 46: Masters BR: Three-dimensional confocal microscopy of human skin in vivo: autofluorescence of human skin. Bioimages 1996; 4: Denk W, Strickler JH, Webb WW: Two-photon laser scanning microscopy. Science 1990; 248: König K: Multiphoton microscopy in life sciences. J Microscopy 2000; 200: Masters BR, So PTC, Gratton E: Multiphoton excitation fluorescence microscopy and spectroscopy of in vivo human skin. Biophys J 1997; 72: So PTC, Kim H: Two-photon deep tissue ex vivo imaging of mouse dermal and subcutaneous structures. Opt Express 1998; 3: Masters BR, So PTC, Gratton E: Multiphoton excitation microscopy of in vivo human skin. Ann NY Acad Sci 1998; 838: Hendriks RFM, Lucassen GW: Two photon fluorescence microscopy of in vivo human skin. SPIE 1999; 4164: König K: Laser tweezers and multiphoton microscopes in life sciences. Histochem Cell Biol 2000; 114: König K, Peuckert C, Riemann I, Wollina U: Non-invasive 3D optical biopsy of human skin with NIR-femtosecond laser pulses for diagnosis of dermatological disorders (abstract). Cell Mol Biol 2000; 46: Peuckert C, Riemann I, König K: Two photon induced autofluorescence of in vivo human skin with femtosecond laser pulses a novel imaging tool of high spatial, spectral and temporal resolution (abstract). Cell Mol Biol 2000; 46: Teuchner K, Freyer W, Leupold D, Volkmer A, Birch DJ, Altmeyer P, Strucker M, Hoffmann K: Femtosecond two-photon excited fluorescence of melanin. Photochem Photobiol 1999; 70: König K, Riemann I: High-resolution multiphoton tomography of human skin with subcellular spatial resolution and picosecond time resolution. J Biomed Optics 2003; 8: Becker W, Bergmann A, König K, Tirlapur U: Picosecond fluorescence lifetime microscopy by TCSPC imaging. SPIE 2001; 4262: Göpel W, Kerr JND, Nimmerjahn A, Helmchen F: Miniaturized two-photon microscope based on a flexible coherent fiber bundle and a gradient index lens objective. Opt Lett 2004; 29: Warner RR, Myers MC, Taylor DA: Electron probe analysis of human skin: Determination of the water concentration profile. J Invest Dermatol 1988; 90: Richter T, Peuckert C, Sattler M, König K, Riemann I, Hintze U, Wittern KP, Wiesendanger R, Wepf R: Dead but highly dynamic the stratum corneum is divided into three hydration zones. Skin Pharmacol Physiol 2004; 17: Alvarez-Roman R, Naik A, Kalia YN, Guy RH, Fessi H: Enhancement of topical delivery from biodegradable nanoparticles. Pharm Res 2004; 21: Lademann J, Schäfer H, Otberg N, Blume-Peytavi U, Sterry W: Penetration von Mikropartikeln in die menschliche Haut. Hautarzt 2004; 55: Luengo J, Weiss B, Schneider M, Ehlers A, Stracke F, König K, Kostka KH, Lehr CM, Schäfer UF: Influence of the encapsulation of flufenamic acid into PLGA nanoparticles on human skin absorption. Skin Pharmacol Physiol, in press. 88 Skin Pharmacol Physiol 2006;19:78 88 König /Ehlers /Stracke /Riemann