Cosmetic Assessment of the Human Hair by Confocal Microscopy

SCANNING VOL. 24, 59 64 (2002) Received: May 5, 2001 FAMS, Inc. Accepted with revision: August 9, 2001 Cosmetic Assessment of the Human Hair by Confocal Microscopy CHRISTOPHE HADJUR,GÉRARD DATY,GENEVIÈVE MADRY,PIERRE CORCUFF L Oréal Recherche, Laboratoires de Recherche Avancée, Aulnay-sous-bois, France Summary: The optical sectioning property of the confocal microscope offers a breakthrough from the classic observation of the hair in a scanning electron microscope (SEM). Confocal microscopy requires minimal sampling preparation, and the hair can be observed in its natural environment with less damage than by other microscopic methods such as SEM. While used in the reflection mode, the true morphology of the cuticle and the various exogenous deposits at the surface can be identified and quantified. This relatively noninvasive, nondestructive technique is routinely used by us to monitor the efficiency of cleansing shampoos, to assess the homogeneity of layering polymers, and to evaluate the changes they induce in the optical properties of the hair surface in terms of opacity, transparency, and brilliancy. A second important field of investigation uses the fluorescence channel which reveals the internal structure of the hair. Fluorescent probes (rhodamine and its derivatives) demonstrate the routes of penetration and outline the geometry of cortical cells and of the medulla according to their lipophilic or hydrophilic properties. A volume rendering of a hair cylinder provides a better understanding of the interrelationships between cuticle cells, cortical cells, and the medullar channel. This recent technology is becoming an invaluable tool for the cosmetic assessment of the hair. Key words: hair, human, confocal microscopy, fluorescence PACS: 06.30.Bp, 06.60.Mr, 07.60.-j, 87.64.Dz Introduction Confocal microscopy provides high-resolution images, for the most part in focus, and can be advantageously adapted to studies of cylindric objects such as human hair. For the past 10 years, this technique has been successfully applied to noninvasive imaging of the surface as well as the internal structures of the hair (Corcuff et al. 1993). It presents an advantageous alternative to classic scanning electron microscopy (SEM) observation. Preserving the integrity of the sample opens up a wide range of applications in cosmetic research, including almost any kind of surface deposits and repeated chemical or physical treatments. The aim of this paper is to present an iconography, based upon confocal imaging, of various fields of investigation involved in hair research and routinely used by us to assess cosmetic treatments. Materials and Methods The confocal laser scanning microscope LSM 310 (Carl Zeiss Inc., Thornwood, N.Y., USA) was equipped with two laser sources: He-Ne 540 nm and Ar-Kr 488/568/ 647 nm. Objective lenses were selected according to their performance for hair imaging: 20 and 50 dry, 40 and 63 oil immersion. The motorized stage (X,Y) was computer controlled for repositioning the sample automatically with a precision of 0.05 µm. A direct current motor with a run of 20 mm and a Z resolution of 50 nm assured fast focusing. Z- series of 80 optical sections separated by 1 µm steps provided either a single in-focus projection image of the surface or a stack of images for a volume rendering of the internal structures of the hair. Using the OTIP3D software (LISA Labs, CPE, Lyon, France), three-dimensional (3-D) reconstruction of segmented features was possible. The segmentation consisted at first of only the wavelets algorithm (Mallat 1989) for noise reduction and then the region-growing algorithm (Zucker 1976) for the binarization of convex features. The confocal scanner had a two-channel configuration that permitted simultaneous imaging of reflected light and fluorescence contrast. Hair samples were simply attached to microscopic slides coated with double-sided adhesive tape. Internal structures of the hair shaft were imaged using oil immersion in order to attenuate the strong reflection generated by the refractive index of the hair surface. Address for reprints: Christophe Hadjur L Oréal - Laboratoires de Recherche Avancée 1 Avenue Eugène Schueller 93600 Aulnay sous Bois, France e-mail: chadjur@recherche.loreal.com Results and Discussion Reflection Images of the Surface More than 30 years ago, Sikorski (1969) used SEM to observe the morphology of wool cuticle. Since then it has

60 Scanning Vol. 24, 2 (2002) been widely used to improve our knowledge about the architecture of the hair surface (Swift 1991, Wolfram 1972). Except recent advances in electron microscopy (environmental and field-emission), SEM often induces unacceptable modification of the sample; for example, coating with an electrically conducting film, drying by exposure to high vacuum, and chemical change wrought by exposure to high-energy electrons. Confocal microscopy provides the capability for nondestructive optical sectioning. A stacking of a series of such optical sections allows precise depictions of the hair cuticle. A brown untreated hair (Fig. 1a) exhibits smooth borders to its cuticle cells, which are regularly spaced along the hair shaft. This pattern is readily observed near the root on freshly plucked hair. Hair exhibiting fatigue after repeated shampooing (Fig. 1b) or cyclical thermal stresses (Gamez- Garcia 1998) showed crenelated cuticle borders and lacked the regular spacing between cells. This type of degradation usually occurs with increasing severity with increasing distance along the hair shaft from its root end. Excessive combing or brushing is often the cause of this type of hair surface abrasion. These mechanical injuries often provoke the loss of several layers of cuticle cells. A severe situation resulted in local absences of the cuticle that revealed the cortex (Fig. 1c). To protect the hair surface from mechanical damage, a thin polymeric film on a previously untreated hair (Fig. 1d) provided firmness and brilliancy. The interference patterning revealed in (c) (d) FIG. 1 Reflection images of the surface: A brown untreated hair exhibits smooth borders of the cuticle cells; an example of hair exhibiting fatigue with crenelated cuticle borders; (c) advanced hair degradation: local absence of the cuticle reveals the cortex; (d) a thin polymeric film on a previously untreated hair provides firmness and brilliancy (interference aspect). Horizontal field width = 125 µm.

C. Hadjur et al.: Confocal microscopy of human hair 61 this case by the confocal microscope is caused by optical interference between the outer smooth surface of the deposited semitransparent polymer film and the underlying rougher surface of the hair proper. The outer smooth surface did not allow one to distinguish individual cuticle cells. A more critical use of SEM concerns observations of deposits on the hair surface. Dehydration and coating precludes the true observation of hydrophilic and lipophilic compounds (Pille et al. 1998). The noninvasive confocal mode for imaging the hair surface in its natural environment, coupled with no change being induced in the sample by microscope examination, permits repeated observations of the same field of view. Thus, reflection images with repositioning allows a comparison of the hair surface before and after treatment. For example, polymer deposits can be evaluated before and after shampooing. Polymers resistant to removal (remanence) thereby can be conveniently classified. Figure 2 presents two experimental types of response. Polymer A, covering a large part of the surface (Fig. 2a), had been eliminated by the shampoo (Fig. 2b); polymer B irregularly covered the surface (Fig. 2c). Two patterns of deposits could be identified: smooth droplets corresponding to the polymer and rough features caused by dust particles. After shampooing (Fig. 2d), almost all of the dust particles disappeared and the polymer droplets remained at the same place with an identical pattern and distribution. (c) (d) FIG. 2 Reflection images with repositioning. Polymer A deposits on the hair before and after shampooing: the hair surface has been cleaned. Polymer B deposits on the hair (c) before and (d) after shampooing remanence of the polymer, only dust particles have been eliminated. Horizontal field width = 125 µm.

62 Scanning Vol. 24, 2 (2002) Today, the importance of the polymers in hair care is a huge cosmetic challenge (McMullen and Jachowicz 1998). The atomic force microscope (AFM) is a more recent approach. It provides images of very high resolution and, in a manner similar to confocal microscopy, preserves the integrity of the sample (Smith 1997). Nevertheless, the minimum size of the working image for the AFM is typically 20 20 µm, and consequently many such fields of view need to be examined to gain satisfactory descriptions over wider areas of the hair surface (Swift and Smith 2000). With the capability for changing the objective lens for ones at alternative magnifications, such restrictions of minimum sampling area do not pose a restriction for confocal microscope examinations of human hair. in water (Fig. 6a) remained at the cuticle and did not penetrate into the cortex. Octadecyl-rhodamine in EtOH/H 2 O (9:1) penetrated into the medulla (Fig. 6b) and marked the membranes and the nuclear remnants of the cortical cells. This final image demonstrated the optical sectioning limitations of the confocal microscope in that a rapid degradation of the lower half of the image is observed. Three-Dimensional Reconstruction (Fig. 7) The OTIP3D software allowed volume rendering of segmented features: in gray, the cuticle envelope and in yellow the medulla channel (white hair labeled with octadecyl-rhodamine). This opens up a new field of investigation Internal Exploration of the Hair Structures The internal constituents of the hair shaft are commonly observed in transverse mechanical sections examined by light microscopy (Sideris et al. 1990), by confocal laserscanning fluorescence microscopy (Swift et al. 2000), and by transmission electron microscopy (Hallegot and Corcuff 1993). Very recently, Kelch et al. (2000) demonstrated the capability of the scanning near-field optical microscope for imaging cross sections of hair labeled with octadecanoylaminofluorescein. The optical sectioning property of the confocal microscope can be conveniently applied to internal hair structures by using immersion oil that decreases the refractive index mismatch and consequently limits the strong reflection at the surface. Natural white hair assured a better penetration of light by limiting scattering generated by melanosomes. Octadecyl-rhodamine increased the contrast of the medulla both in the reflection image (Fig. 3a) and revealed nuclear remnants within the cortical cells. The corresponding fluorescence image (Fig. 3b) showed the location of the probe into the medulla (yellow), in the cortex on the left part of the image (red), and is delineating the border of cuticle cells to the right in the image. The fluorescence channel can also be used to study the penetration pathways of fluorescent probes. Diffusion of molecules into hairs can influence the chemical and physical properties of the hair fibers (Kimura et al. 1999, Swift et al. 2000). Rhodamine B incorporated into polymers allowed a sharp depiction of the location of the polymer at the hair surface (Fig. 4). Clearly, the fluorescent polymer (in red) had infiltrated the border of the cuticle cells, and the corresponding reflection signal (in green) demonstrated the absence of polymer on the surface of the cuticle. An optical section through a natural white hair labeled with octadecyl-rhodamine exhibited fusiform cortical cells and keratin fibers (Fig. 5). A transversal fracture through the cortex, that was not visible from the examination of the surface, alone was clearly revealed from the in-depth optical sections. Transversal optical sections allow a follow-up cracking of the penetration of the fluorescent probes. Rhodamine B FIG. 3 Internal exploration of the hair structures. Natural white hair labeled with octadecyl-rhodamine: The reflection image reveals the medulla and nuclear remnants of the cortical cells; the fluorescence image shows the location of the probe into the medulla (yellow), in the cortex (red), and delineating the border of cuticle cells. Horizontal field width = 125 µm.

C. Hadjur et al.: Confocal microscopy of human hair 63 FIG. 4 Hair treated with a polymer labeled with rhodamine B. In red, the polymer infiltrates the borders of the cuticle cells. In green, the reflection signal demonstrates the absence of polymer on the surface of the cuticle. Horizontal field width = 125 µm. FIG. 7 Three-dimensional reconstruction. The OTIP3D software allowed a volume rendering of segmented features: in grey, the cuticle envelope and in yellow the medulla channel (white hair labeled with octadecyl-rhodamine). Horizontal field width = 160 µm. concerning the interrelationships between the internal structures (cortex and medulla) and the outer envelope (cuticle). Conclusion Confocal microscopy provides rapid, easy, elegant, and nondestructive observations of the hair in its natural environment. Both the surface and the internal structures can be imaged noninvasively, thus providing longitudinal and transversal optical sections for volume rendering. The condition of the hair surface can be evaluated according to the chemical or physical injuries sustained. Surface deposits can be observed in terms of thickness, homogeneity and brilliancy as well as their resistance to cosmetic treatments. The routes of penetration of fluorochromes into the hair structures can be investigated dynamically. FIG. 5 A natural white hair labeled with octadecyl-rhodamine exhibits fusiform cortical cells and keratin fibers. A transversal fracture through the cortex is clearly revealed. Horizontal optical section 35 µm below the surface. Horizontal field width = 90 µm. Acknowledgment They wish to thank Daniel Good for his help in reviewing the manuscript and Yolanda Duvault for hair treatments. They also wish to thank Dr. J. Alan Swift for his fruitful criticism and expert assistance. References FIG. 6 Transversal optical sections allow a follow-up tracking of the penetration of fluorescent probes: Rhodamine B in water was absorbed by the cuticle and did not penetrate into the cortex; octadecyl-rhodamine in EtOH/H 2 O (9:1) penetrated into the medulla. Notice the membrane marking of the cortical cells and nuclear remnants. Horizontal field width = 160 µm. Corcuff P, Gremillet P, Jourlin M, Duvault Y, Leroy F, Lévèque JL: 3D reconstruction of human hair by confocal microscopy. J Soc Cosmet Chem 44, 1 12 (1993) Gamez-Garcia M: The cracking of human hair cuticles by cyclical thermal stresses. J Cosmet Sci 49, 141 153 (1998) Hallegot P, Corcuff P: High spatial resolution maps of sulfur from human hair sections: An EELS study. J Microsc 172(2), 131 136 (1993)

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