j. Soc. Cosmet. Chem., 48, 199-208 (July/August 1997) Use of atomic force microscopy for high-resolution non-invasive structural studies of human hair JAMES R. SMITH, Scanning Probe Microscopy Laboratory, School of Pharmacy, Biomedical and Physical Sciences, University of Portsmouth, St. Michael's Building, White Swan Road, Portsmouth PO1 2DT, United Kingdom. Accepted for publication September 3 O, 1997. Synopsis The morphology of the fine cuticular structure of human hair has traditionally been investigated using scanning electron microscopy and transmission electron microscopy. Although these techniques are very useful, they require specimens to be coated with metallic films or to be suitably stained. In addition, high vacuum conditions are required that may damage or alter the appearance of delicate cuticular structures. Atomic force microscopy is a relatively new scanning probe technique, capable of imaging surfaces at high resolution under ambient conditions. In this communication, the potential applications of atomic force microscopy for structural investigations of human hair surfaces are discussed. Fine surface structures, such as the exocuticle, the endocuticle, and the marginal band (A- or oMayer), could be easily identified. The technique has also been demonstrated to image hair surfaces in liquid environments, opening the way to in situ studies of the effects of hair-care products and treatments. INTRODUCTION The human hair is protected by an almost formidable barrier comprised of many thou- sands of scale-like plates, called cuticles. These are arranged in approximately six to ten layers (1), each of a thickness of 0.3-0.5 pm (2,3). The cuticular cell consists of essen- tially two layers, the endocuticle and exocuticle (1), the former being the inner one third of the cell and containing cellular debris that can easily undergo hydrolysis by proteases (4,5). A significant degree of keratinization occurs near the surface of the cuticle, and trichohyalin granules (6) are deposited near the lateral cell wall it is this layer, termed the exocuticle, that is responsible for the hair's resistance to detergents, organic solvents, and many other harsh environments (7). Structural information concerning hair cuticles has largely been obtained from scanning electron microscopy (SEM) and transmission electron microscopy (TEM) studies of hu- man hair fibres (3,4,7-9). While these are valuable techniques, their application in structural hair analysis is limited by the requirement for samples to be coated with a thin metallic film, or, in the case of TEM, the need to suitably stain specimens prior to investigation. In addition, both these microscopic techniques require high vacuum 199

200 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS conditions for successful imaging, which could damage or alter the appearance of fine cuticular structures. Atomic force microscopy (AFM) is a relatively new technique (10) and is a member of a generic class of related scanning probe microscopy (SPM) techniques (11,12). The principle of AFM relies on the use of a sharp, pyramidal tip mounted on a cantilever that is brought into close proximity to the sample of interest where intermolecular forces acting between the tip and the surface cause the cantilever to bend. Cantilever deflec- tions are detected by a laser beam focused on the top of the cantilever and are converted to a digital voltage signal that is then applied across a piezoelectric ceramic element to which the sample is mounted. This has the effect of maintaining a constant force, in the order of a few nano-Newtons, between the scanning tip and the sample. A three- dimensional map of the specimen's topography can therefore be obtained by plotting this feedback voltage signal against X and Y coordinates. Confocal microscopy has recently been demonstrated to be a good technique for imaging hair surfaces in their natural environment (13). However, the resolution limit for this method is of the order of 0.25 pm, an improvement on conventional light microscopy (0.6 pm), but much less than that of electron microscopy. Since AFM does not require the focusing of light or other radiation for its operation, a very high resolution is achievable, of the order of 1-2 nm for biological materials. This high resolving power, together with the ability to image surfaces in aqueous environments, makes AFM a very attractive technique for imaging hair surfaces. Only a few reports on the use of AFM in this application area have appeared in the literature (14-17). Here, the potential applications of AFM for structural investigations of human hair surfaces are discussed. MATERIALS AND METHODS Human scalp hairs were obtained from biopsies from European brown-haired subjects who had washed their hair approximately 5 h prior to the investigations. A sodium lauryl sulphate-based shampoo and conditioner containing dimethicones or 1% zinc pyrithione was used, and hair was allowed to dry naturally at room temperature. 'Un- washed' samples refer to hair that had been washed the previous day and had not been groomed. Hair sections of 1 cm in length, no further than 5 cm from the scalp, unless otherwise stated, were cut and fixed on a sample mounting assembly using carbon- loaded double-sided adhesive tape. AFM studies were performed using a Discoverer TMX2000 scanning probe microscope (SPM) (TopoMetrix Corporation, Saffron Wal- den, Essex, UK) under atmospheric conditions, although in some experiments, a liquid cell was used to obtain images in an aqueous environment. A relative humidity of 60-65% was noted during all the experiments, although this was not accurately con- trolled room temperature was recorded to be 22øC. Standard profile pyramidal silicon nitride tips mounted on cantilevers of force constants in the range 0.036 to 0.072 N m -• were used. The sample was held in position on a piezoelectric tripod scanner capable of a maximum XYZ translation of 75 x 75 x 12 pm. Images were left-shaded to enhance topographical features and displayed on a monitor with a resolution of 500 lines x 500 pixels. Quantitative data, such as height measurements and surface roughness, were obtained using TopoMetrix image analysis software (18). The arithmetic surface rough-