J. Cosmet. Sci., 71, 117–131 (May/June 2020) 117 Investigation of the Internal Structure of Human Hair with Atomic Force Microscopy ROGER L. MCMULLEN and GUOJIN ZHANG, Ashland Specialty Ingredients G.P., Bridgewater, New Jersey 08807 (R.L.M., G.Z.) Accepted for publication February 6, 2020. S ynopsis The internal ultrafi ne structure of human hair was explored with atomic force microscopy (AFM). Cross sections of hair were prepared by a proprietary technique that provided a smooth surface for effective imaging in contact-mode AFM. Investigations of virgin hair revealed structural details of cortical and cuticle cells consistent with previous transmission electron microscopy (TEM) studies, in addition to the identifi cation of a boundary region surrounding macrofi brils of the cortex. The effects of bleaching and solvent extraction on the internal structure of hair were also investigated. In the cuticle cell, bleaching causes the most damage to the endocuticle and cell membrane complex, evident by erosion of these components. Similarly, bleaching results in crevices, cracks, and asperities in the cortex of hair. In addition, the cortical cell membrane complex appears compromised along with either lipid or protein structures at the outer boundaries of macrofi brils. In delipidated hair, most structural components of the fi ber appear intact with the exception of an overall swollen nature of the various morphological components. INTRODUCTION The ultrafi ne structure of human hair has captured the attention of scientists for decades. Most of what we currently know about hair morphology comes from a plethora of studies of thin sections of hair using transmission electron microscopy (TEM) (1–5). Despite advances made in our understanding of the hair structure, there still remain a number of unanswered questions about the physicochemical properties of hair. The advent of atomic force microscopy (AFM) techniques gave researchers an alternative route to explore the nanomechanical, nanotribological, and nanostructural properties of the hair fi ber. AFM studies of human hair started to appear in the literature in the late 1990s to early 2000s and mostly focused on the quantitative analysis of three-dimensional topographi- cal images of the hair surface (6–8). A key advantage to using AFM as an investigative technique of the morphological properties of the hair fi ber surface is that there is no need to coat the specimen with metal (e.g., gold and platinum) and studies can be conducted in ambient conditions (and even in solution) rather than vacuum (9). A study of cuticle Address all correspondence to rmcmullen@ashland.com. Current affi liation: Guojin Zhang, L’Oréal, Clark, New Jersey 07066.

JOURNAL OF COSMETIC SCIENCE 118 step height and its swelling behavior in water as a function of pH was key to understand- ing possible access points to the fi ber for cosmetic ingredients (7). In addition to topographic imaging, one of the earliest AFM modes available to research- ers was lateral force microscopy (LFM), also known as frictional force microscopy. In this technique, the lateral forces or twisting experienced by an AFM probe, as it moves from one side of a sample to the other, can be monitored. This mode of operation provided researchers with many insights into the nanotribology of the outer surface of hair, spe- cifi cally the exposed surface of the cuticle. Early LFM studies sought to elucidate the role that free and covalently attached lipids play on the hair surface (10,11). In addition, AFM friction and adhesion mapping has also provided insight into the structural features of the lamellar layers of the cuticle. In any given area, the different cuticle layers (A layer, exocu- ticle, endocuticle, and inner layer) may be exposed to the surface and observed by AFM (12). Furthermore, the relationship between macro- and microscale friction of hair was investigated with LFM (13). In another LFM study, it was suggested that stretching hair could possibly affect the nanotribological properties (coeffi cient of friction, adhesion, and surface roughness) of the hair surface (14). A rather modern approach for its time, Sadaie et al. (15) modifi ed an AFM probe with a self-assembled monolayer and monitored the frictional interaction between the modifi ed probe and the surface of hair, specifi cally probing the outermost 18-methyleicosanoic acid (18-MEA) layer of the cuticle. In fact, AFM has been an essen- tial tool to demonstrate that 18-MEA plays a paramount role in the structural integrity of the cuticle (16). AFM has also been at the forefront of allowing researchers to monitor intrinsic and extrin- sic aging of hair. Intrinsic aging refers to the natural aging process of the fi ber, and it has been shown by AFM that the surface roughness of hair increases with age, which is ac- companied by a decline in the overall health state of the ultrafi ne morphological struc- tures of hair (17). Extrinsic aging of hair refers to external elements that accelerate the aging process of hair, such as photodegradation or chemical treatments. Photodegrada- tion leads to a substantial increase in the cuticle step height of hair and an increase in the size and depth of micropores apparent in the cuticle cell structure (18). The presence of micropores in the cuticle has been demonstrated by several techniques and their presence increases with photodegradation and bleaching (11,19). Using chemical force micros- copy—achieved by chemically modifying AFM probes with –CH3 and –NH2 groups— allowed for the determination that bleaching of hair leads to the partial removal of the hydrophobic moiety—presumably 18-MEA—on the outermost surface of the cuticle (20). Not surprisingly, great efforts have been made for understanding how AFM techniques could better be used to understand the deposition of cosmetic ingredients onto hair. Mostly, this includes monitoring the deposition of cationic ingredients onto the hair surface and performing frictional measurements with LFM (21–26). Although some ingredients decrease the dry friction of hair, it should be noted that application of cationic polymers to the hair normally increases the dry friction. In the wet state, however, cationic polymers reduce the capillary forces between the fi bers and, as a result, lower the energy required to comb through the hair. Therefore, when conducting AFM measurements, a good under- standing of the targeted state, dry or wet, and its relation to the application is needed. Nanomechanical measurements of hair have also been carried out using AFM. Initial studies in this area included the determination of the mechanical properties (e.g.,