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Journal of Dermatological Science xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Journal of Dermatological Science journal homepage: www.jdsjournal.com
Letter to the Editor Topographical and nano-mechanical characterization of native corneocytes using atomic force microscopy
To the editor, The stratum corneum (SC), the outermost layer of the skin, mainly acts as a barrier between the organism and its external environment. It is composed of corneocytes surrounded by a lipid matrix and held together by corneodesmosomes. The SC must be both resistant and flexible to protect the skin while adapting to the body movements without cracking. How this is achieved is not well known. Since it became available in the late eighties, atomic force microscopy (AFM) has been increasingly used for imaging surfaces ranging from micro- to nano-meter scales. Currently, AFM is one of the few techniques providing submicrometer resolution under physiological conditions, available for studying complex biological structures. It consists of a sharp tip at the end of a soft silicon cantilever touching and scanning the surface of a sample. The deflection of the cantilever due to the change in topography is detected by a laser optical device, and transformed into a threedimensional image. AFM has been applied on few occasions to analyze the morphology of isolated corneocytes, and most studies were conducted on denatured corneocytes [1,2]. Gorzelanny et al. have reported performing AFM directly on native tape strips [3]. However, the analysis was limited to corneocytes from only two subjects with a 30-years age difference. We present a process for imaging and analyzing topographical and mechanical properties of corneocytes in their most native conditions, combining a novel method for tape stripping, AFM characterization and statistical analysis. The proposed methodology is based on collecting corneocytes from the skin surface by 10 successive tape-stripping on the same cutaneous site, the tape being fixed on a glass slide, and imaging several individual cells directly on the tape without any additional treatments (Supplementary Materials and methods online). This allows the analysis of the corneocyte surface in direct contact with the underlying cells. To illustrate the power of this methodology, we qualitatively and quantitatively compared corneocytes obtained from two different anatomical regions: the internal side of the forearm and hand palm; from five different healthy females with similar age; at three different stripping depths into the SC.
Abbreviations: AFM, atomic force microscopy; SC, stratum corneum.
At a low magnification (Fig. 1 and Supplementary Fig. S1 online), corneocytes appear with hexagonal or pentagonal shapes as expected. However, the corneocyte surface was clearly different according to the anatomical origin. Most forearm corneocytes presented lines, like crests and valleys, at their surface that may correspond to the limits of the areas of contact with the underlining corneocytes. This was reminiscent of both Nomarski (Supplementary Fig. S2 online) and scanning electron microscopy images [4]. These lines are less frequent on the palmar corneocytes. Corneocyte surface relief was then quantitatively evaluated (Fig. 1c). Confirming the qualitative observations, palmar corneocytes were found to be flatter on average than forearm corneocytes, whatever the depth of stripping (p < 0.001). In addition, the median values of height distribution decreased according to the depth of stripping, whatever the anatomical sites. These data fit well with scanning electron microscopy examinations of SC replicas suggesting a flattening of the lower-corneocyte surface [4]. However, they differ from those of Kashibuchi et al. who have shown that corneocytes become flatter as they reach the skin surface. This discrepancy could be explained by the fact that corneocytes have been denatured with xylene before AFM imaging [2]. At a higher magnification (Fig 1d and e), forearm corneocyte surface appeared smooth, whereas palmar corneocyte surface displayed numerous small protrusions. Similar villus-like structures have previously been observed by scanning electron microscopy of plantar corneocytes [4]. They may well correspond to corneodesmosome areas that are retained on the surface of corneocytes up to the upper SC only in the palmo-plantar skin territories [5]. AFM characterization of palm corneocytes is consistent with the previous description of the so-called rough corneocytes [4] and immature fragile cornified envelopes [6], which both are abundant in the plantar but rare in the forearm SC [4–6]. Smooth corneocytes corresponding to rigid envelopes are the most frequent in the upper SC of arm/leg [4–6]. Force/volume measurements performed on native corneocytes (Supplementary Fig. S3 online) revealed that the average Young modulus of corneocytes (as an indicator of rigidity) was independent of the anatomical region and decreased with the depth of the stripped layer from 12 to 6 MPa. These values are about 100 at 1000 times higher than the usual values obtained for HaCaT keratinocytes and other living cells [7,8]. They are consistent with the current vision of corneocytes as rigid and resistant cells. To test for the effect of corneocyte denaturation, we then analyzed corneocytes from strips 2 and 10 treated with cold methanol for 10 min (Fig. 2). Height distributions of denatured corneocytes (median values = 463 and 261 nm) strongly reduced,
http://dx.doi.org/10.1016/j.jdermsci.2014.04.009 0923-1811/ ã 2014 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.
Please cite this article in press as: Fredonnet J, et al. Topographical and nano-mechanical characterization of native corneocytes using atomic force microscopy. J Dermatol Sci (2014), http://dx.doi.org/10.1016/j.jdermsci.2014.04.009
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J. Fredonnet et al. / Journal of Dermatological Science xxx (2014) xxx–xxx
Fig. 1. Atomic force microscopy analysis revealed differences in the topography of corneocytes from forearm and hand palm skin. Individual native corneocytes on tape strips 2, 6 and 10 obtained from five different volunteers and both the forearm and hand palm were analyzed. (a, b) Representative images of heights given in colour brightness (in mm) at a low magnification. Graphs represent the height distributions corresponding to the two dimensional sections along the dotted lines indicated on the upper images. Bars, 10 mm. (c) Quantification, using Nanoscope Analysis software, of the height distributions according to the anatomical region and to the stripping depth. Ten corneocytes per strip and per individual were analyzed. Boxplots in the upper parts indicate the means (diamond), medians, and 25th and 75th percentiles. Rm, median relief. (d and e) Individual corneocytes were analyzed at a low (upper images) and high magnification (lower images). Height (in mm) and deflection error (in nm) images are shown on the left and right parts, respectively. Bars, 10 mm (upper images), and 1.75 mm (lower images).
as compared to the non-treated ones. In addition, the decrease was higher for the deeper corneocytes (3.6 vs. 2.7 times). Remarkably, the thickness of dry corneocytes from dehydrated abdominal SC has been estimated after cryo-scanning electronmicroscopy as 360 nm [9]. Hydration of the SC (e.g., during bathing) induces its swelling. This may correspond to the expansion of either the extracellular compartment [10] or the corneocyte matrix [1] or both [9]. To investigate the possible swelling of corneocytes, a drop of distilled water was added on the tape during AFM imaging, and data were collected every 30 min for 2 h for each subject and stripping depth. No major modifications in the heights and sizes
of the corneocytes were observed (Supplementary Fig. S4 online), suggesting that the cellular compartment in the outer/mid SC does not change during hydration in agreement with [10]. The effects observed by others [1,9] may be explained by the treatments they used to prepare the SC and corneocytes, or the longer time of hydration. In conclusion, direct AFM measurement on tape-stripping is a powerful non-invasive methodology to study corneocytes in their most native state. In addition to imaging their surface topology, AFM is helpful to get quantitative data about their mechanical properties. This can be considered an advantage over other imaging technics, e.g., Nomarski analysis.
Please cite this article in press as: Fredonnet J, et al. Topographical and nano-mechanical characterization of native corneocytes using atomic force microscopy. J Dermatol Sci (2014), http://dx.doi.org/10.1016/j.jdermsci.2014.04.009
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Fig. 2. Methanol treatment of native corneocytes from the forearm region induced a strong decrease in their relief. Individual corneocytes (n = 10) on tape strips 2 and 10 from one volunteer were analyzed by atomic force microscopy before and after treatment with cold methanol for 10 min. The height distributions were quantified. Boxplots indicate the means (diamond), medians, 25th and 75th percentiles, and outlier values. Rm, median relief.
Funding sources This work was supported by the French National Center for Scientific Research (CNRS), the Toulouse University, the French National Institute of Health and Medical Research (INSERM). The authors acknowledge Professor Jean Marie François (CNRS UMR5504, Toulouse, France) and l’Agence Nationale de la Recherche (ANR) for the financing of Julie Fredonnet. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j. jdermsci.2014.04.009. References [1] Richter T, Müller JH, Schwarz UD, Wepf R, Wiesendanger R. Investigation of the swelling of human skin cells in liquid media by tapping mode scanning force microscopy. Appl Phys A 2001;72(Suppl):S125–8. [2] Kashibuchi N, Hirai Y, O' Goshi K, Tagami H. Three-dimensional analyses of individual corneocytes with atomic force microscope: morphological changes related to age, location and to the pathologic skin conditions. Skin Res Technol 2002;8:203–11. [3] Gorzelanny C, Goerge T, Schnaeker E-M, Thomas K, Luger TA, Schneider SW. Atomic force microscopy as an innovative tool for nanoanalysis of native stratum corneum. Exp Dermatol 2006;15:387–91. [4] King CS, Barton SP, Nicholls S, Marks R. The change in properties of the stratum corneum as a function of depth. Br J Dermatol 1979;100:165–72. [5] Serre G, Mils Valérie, Haftek M, Vincent C, Croute F, Réano A, et al. Identification of late differentiation antigens of human cornified epithelia, expressed in re-organized desmosomes and bound to cross-linked envelope. J Invest Dermatol 1991;97:1061–72. [6] Michel S, Schmidt R, Shroot B, Reichert U. Morphological and biochemical characterization of the cornified envelopes from human epidermal keratinocytes of different origin. J Invest Dermatol 1988;91:11–5. [7] Fung CKM, Xi N, Yang R, Seiffert-Sinha K, Lai KWC, Sinha AA. Quantitative analysis of human keratinocyte cell elasticity using atomic force microscopy. IEEE Trans Nanobioscience 2011;10:9–15.
[8] Kuznetsova TG, Starodubtseva MN, Yegorenkov NI, Chizhik SA, Zhdanov RI. Atomic force microscopy probing of cell elasticity. Micron 2007;38:824–33. [9] Bouwstra JA, de Graaff A, Gooris GS, Nijsse J, Wiechers JW, van Aelst AC. Water distribution and related morphology in human stratum corneum at different hydration levels. J Invest Dermatol 2003;120:750–8. [10] Lin TK, Crumrine D, Ackerman LD, Santiago JL, Roelandt T, Uchida Y, et al. Cellular changes that accompany shedding of human corneocytes. J Invest Dermatol 2012;132:2430–9.
Julie Fredonnet USR3505 CNRS, ITAV, Toulouse 31106, France Géraldine Gascb,c,d Guy Serreb,c,d Childérick Séveraca,**,1 Michel Simonb,c,d,* ,1 a USR3505 CNRS, ITAV, Toulouse 31106, France b
UMR5165 CNRS, Toulouse 31059, France
c
U1056 INSERM, Toulouse 31059, France
d
University of Toulouse, Toulouse 31059, France These authors contributed equally to this work.
1
* Corresponding author. Tel.: +33 5 6181 2993; ?fax: +33 5 6149 9036. ** Corresponding author. Tel.: +33 5 8299 1016. E-mail addresses: [email protected] (C. Séverac) [email protected] (M. Simon). Received 17 January 2014 Received in revised form 16 April 2014 Accepted 19 April 2014
Please cite this article in press as: Fredonnet J, et al. Topographical and nano-mechanical characterization of native corneocytes using atomic force microscopy. J Dermatol Sci (2014), http://dx.doi.org/10.1016/j.jdermsci.2014.04.009
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J. Fredonnet et al. / Journal of Dermatological Science xxx (2014) xxx–xxx
Please cite this article in press as: Fredonnet J, et al. Topographical and nano-mechanical characterization of native corneocytes using atomic force microscopy. J Dermatol Sci (2014), http://dx.doi.org/10.1016/j.jdermsci.2014.04.009
Journal of Dermatological Science xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Journal of Dermatological Science journal homepage: www.jdsjournal.com
Letter to the Editor Topographical and nano-mechanical characterization of native corneocytes using atomic force microscopy
To the editor, The stratum corneum (SC), the outermost layer of the skin, mainly acts as a barrier between the organism and its external environment. It is composed of corneocytes surrounded by a lipid matrix and held together by corneodesmosomes. The SC must be both resistant and flexible to protect the skin while adapting to the body movements without cracking. How this is achieved is not well known. Since it became available in the late eighties, atomic force microscopy (AFM) has been increasingly used for imaging surfaces ranging from micro- to nano-meter scales. Currently, AFM is one of the few techniques providing submicrometer resolution under physiological conditions, available for studying complex biological structures. It consists of a sharp tip at the end of a soft silicon cantilever touching and scanning the surface of a sample. The deflection of the cantilever due to the change in topography is detected by a laser optical device, and transformed into a threedimensional image. AFM has been applied on few occasions to analyze the morphology of isolated corneocytes, and most studies were conducted on denatured corneocytes [1,2]. Gorzelanny et al. have reported performing AFM directly on native tape strips [3]. However, the analysis was limited to corneocytes from only two subjects with a 30-years age difference. We present a process for imaging and analyzing topographical and mechanical properties of corneocytes in their most native conditions, combining a novel method for tape stripping, AFM characterization and statistical analysis. The proposed methodology is based on collecting corneocytes from the skin surface by 10 successive tape-stripping on the same cutaneous site, the tape being fixed on a glass slide, and imaging several individual cells directly on the tape without any additional treatments (Supplementary Materials and methods online). This allows the analysis of the corneocyte surface in direct contact with the underlying cells. To illustrate the power of this methodology, we qualitatively and quantitatively compared corneocytes obtained from two different anatomical regions: the internal side of the forearm and hand palm; from five different healthy females with similar age; at three different stripping depths into the SC.
Abbreviations: AFM, atomic force microscopy; SC, stratum corneum.
At a low magnification (Fig. 1 and Supplementary Fig. S1 online), corneocytes appear with hexagonal or pentagonal shapes as expected. However, the corneocyte surface was clearly different according to the anatomical origin. Most forearm corneocytes presented lines, like crests and valleys, at their surface that may correspond to the limits of the areas of contact with the underlining corneocytes. This was reminiscent of both Nomarski (Supplementary Fig. S2 online) and scanning electron microscopy images [4]. These lines are less frequent on the palmar corneocytes. Corneocyte surface relief was then quantitatively evaluated (Fig. 1c). Confirming the qualitative observations, palmar corneocytes were found to be flatter on average than forearm corneocytes, whatever the depth of stripping (p < 0.001). In addition, the median values of height distribution decreased according to the depth of stripping, whatever the anatomical sites. These data fit well with scanning electron microscopy examinations of SC replicas suggesting a flattening of the lower-corneocyte surface [4]. However, they differ from those of Kashibuchi et al. who have shown that corneocytes become flatter as they reach the skin surface. This discrepancy could be explained by the fact that corneocytes have been denatured with xylene before AFM imaging [2]. At a higher magnification (Fig 1d and e), forearm corneocyte surface appeared smooth, whereas palmar corneocyte surface displayed numerous small protrusions. Similar villus-like structures have previously been observed by scanning electron microscopy of plantar corneocytes [4]. They may well correspond to corneodesmosome areas that are retained on the surface of corneocytes up to the upper SC only in the palmo-plantar skin territories [5]. AFM characterization of palm corneocytes is consistent with the previous description of the so-called rough corneocytes [4] and immature fragile cornified envelopes [6], which both are abundant in the plantar but rare in the forearm SC [4–6]. Smooth corneocytes corresponding to rigid envelopes are the most frequent in the upper SC of arm/leg [4–6]. Force/volume measurements performed on native corneocytes (Supplementary Fig. S3 online) revealed that the average Young modulus of corneocytes (as an indicator of rigidity) was independent of the anatomical region and decreased with the depth of the stripped layer from 12 to 6 MPa. These values are about 100 at 1000 times higher than the usual values obtained for HaCaT keratinocytes and other living cells [7,8]. They are consistent with the current vision of corneocytes as rigid and resistant cells. To test for the effect of corneocyte denaturation, we then analyzed corneocytes from strips 2 and 10 treated with cold methanol for 10 min (Fig. 2). Height distributions of denatured corneocytes (median values = 463 and 261 nm) strongly reduced,
http://dx.doi.org/10.1016/j.jdermsci.2014.04.009 0923-1811/ ã 2014 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.
Please cite this article in press as: Fredonnet J, et al. Topographical and nano-mechanical characterization of native corneocytes using atomic force microscopy. J Dermatol Sci (2014), http://dx.doi.org/10.1016/j.jdermsci.2014.04.009
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J. Fredonnet et al. / Journal of Dermatological Science xxx (2014) xxx–xxx
Fig. 1. Atomic force microscopy analysis revealed differences in the topography of corneocytes from forearm and hand palm skin. Individual native corneocytes on tape strips 2, 6 and 10 obtained from five different volunteers and both the forearm and hand palm were analyzed. (a, b) Representative images of heights given in colour brightness (in mm) at a low magnification. Graphs represent the height distributions corresponding to the two dimensional sections along the dotted lines indicated on the upper images. Bars, 10 mm. (c) Quantification, using Nanoscope Analysis software, of the height distributions according to the anatomical region and to the stripping depth. Ten corneocytes per strip and per individual were analyzed. Boxplots in the upper parts indicate the means (diamond), medians, and 25th and 75th percentiles. Rm, median relief. (d and e) Individual corneocytes were analyzed at a low (upper images) and high magnification (lower images). Height (in mm) and deflection error (in nm) images are shown on the left and right parts, respectively. Bars, 10 mm (upper images), and 1.75 mm (lower images).
as compared to the non-treated ones. In addition, the decrease was higher for the deeper corneocytes (3.6 vs. 2.7 times). Remarkably, the thickness of dry corneocytes from dehydrated abdominal SC has been estimated after cryo-scanning electronmicroscopy as 360 nm [9]. Hydration of the SC (e.g., during bathing) induces its swelling. This may correspond to the expansion of either the extracellular compartment [10] or the corneocyte matrix [1] or both [9]. To investigate the possible swelling of corneocytes, a drop of distilled water was added on the tape during AFM imaging, and data were collected every 30 min for 2 h for each subject and stripping depth. No major modifications in the heights and sizes
of the corneocytes were observed (Supplementary Fig. S4 online), suggesting that the cellular compartment in the outer/mid SC does not change during hydration in agreement with [10]. The effects observed by others [1,9] may be explained by the treatments they used to prepare the SC and corneocytes, or the longer time of hydration. In conclusion, direct AFM measurement on tape-stripping is a powerful non-invasive methodology to study corneocytes in their most native state. In addition to imaging their surface topology, AFM is helpful to get quantitative data about their mechanical properties. This can be considered an advantage over other imaging technics, e.g., Nomarski analysis.
Please cite this article in press as: Fredonnet J, et al. Topographical and nano-mechanical characterization of native corneocytes using atomic force microscopy. J Dermatol Sci (2014), http://dx.doi.org/10.1016/j.jdermsci.2014.04.009
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Fig. 2. Methanol treatment of native corneocytes from the forearm region induced a strong decrease in their relief. Individual corneocytes (n = 10) on tape strips 2 and 10 from one volunteer were analyzed by atomic force microscopy before and after treatment with cold methanol for 10 min. The height distributions were quantified. Boxplots indicate the means (diamond), medians, 25th and 75th percentiles, and outlier values. Rm, median relief.
Funding sources This work was supported by the French National Center for Scientific Research (CNRS), the Toulouse University, the French National Institute of Health and Medical Research (INSERM). The authors acknowledge Professor Jean Marie François (CNRS UMR5504, Toulouse, France) and l’Agence Nationale de la Recherche (ANR) for the financing of Julie Fredonnet. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j. jdermsci.2014.04.009. References [1] Richter T, Müller JH, Schwarz UD, Wepf R, Wiesendanger R. Investigation of the swelling of human skin cells in liquid media by tapping mode scanning force microscopy. Appl Phys A 2001;72(Suppl):S125–8. [2] Kashibuchi N, Hirai Y, O' Goshi K, Tagami H. Three-dimensional analyses of individual corneocytes with atomic force microscope: morphological changes related to age, location and to the pathologic skin conditions. Skin Res Technol 2002;8:203–11. [3] Gorzelanny C, Goerge T, Schnaeker E-M, Thomas K, Luger TA, Schneider SW. Atomic force microscopy as an innovative tool for nanoanalysis of native stratum corneum. Exp Dermatol 2006;15:387–91. [4] King CS, Barton SP, Nicholls S, Marks R. The change in properties of the stratum corneum as a function of depth. Br J Dermatol 1979;100:165–72. [5] Serre G, Mils Valérie, Haftek M, Vincent C, Croute F, Réano A, et al. Identification of late differentiation antigens of human cornified epithelia, expressed in re-organized desmosomes and bound to cross-linked envelope. J Invest Dermatol 1991;97:1061–72. [6] Michel S, Schmidt R, Shroot B, Reichert U. Morphological and biochemical characterization of the cornified envelopes from human epidermal keratinocytes of different origin. J Invest Dermatol 1988;91:11–5. [7] Fung CKM, Xi N, Yang R, Seiffert-Sinha K, Lai KWC, Sinha AA. Quantitative analysis of human keratinocyte cell elasticity using atomic force microscopy. IEEE Trans Nanobioscience 2011;10:9–15.
[8] Kuznetsova TG, Starodubtseva MN, Yegorenkov NI, Chizhik SA, Zhdanov RI. Atomic force microscopy probing of cell elasticity. Micron 2007;38:824–33. [9] Bouwstra JA, de Graaff A, Gooris GS, Nijsse J, Wiechers JW, van Aelst AC. Water distribution and related morphology in human stratum corneum at different hydration levels. J Invest Dermatol 2003;120:750–8. [10] Lin TK, Crumrine D, Ackerman LD, Santiago JL, Roelandt T, Uchida Y, et al. Cellular changes that accompany shedding of human corneocytes. J Invest Dermatol 2012;132:2430–9.
Julie Fredonnet USR3505 CNRS, ITAV, Toulouse 31106, France Géraldine Gascb,c,d Guy Serreb,c,d Childérick Séveraca,**,1 Michel Simonb,c,d,* ,1 a USR3505 CNRS, ITAV, Toulouse 31106, France b
UMR5165 CNRS, Toulouse 31059, France
c
U1056 INSERM, Toulouse 31059, France
d
University of Toulouse, Toulouse 31059, France These authors contributed equally to this work.
1
* Corresponding author. Tel.: +33 5 6181 2993; ?fax: +33 5 6149 9036. ** Corresponding author. Tel.: +33 5 8299 1016. E-mail addresses: [email protected] (C. Séverac) [email protected] (M. Simon). Received 17 January 2014 Received in revised form 16 April 2014 Accepted 19 April 2014
Please cite this article in press as: Fredonnet J, et al. Topographical and nano-mechanical characterization of native corneocytes using atomic force microscopy. J Dermatol Sci (2014), http://dx.doi.org/10.1016/j.jdermsci.2014.04.009
G Model DESC 2678 No. of Pages 4
4
J. Fredonnet et al. / Journal of Dermatological Science xxx (2014) xxx–xxx
Please cite this article in press as: Fredonnet J, et al. Topographical and nano-mechanical characterization of native corneocytes using atomic force microscopy. J Dermatol Sci (2014), http://dx.doi.org/10.1016/j.jdermsci.2014.04.009
