Melanin in human hair plays a role in protecting hair proteins and the scalp from sunlight (Hoting et al., 1995a). Melanin granules (melanosomes) are mainly present in the hair cortex, which is surrounded by five to ten cuticle layers. (Swift, 1997, Popescu and Höcker, 2007, Robbins, 2012a). Melanin pigments, found within melanin granules, determine the color of natural hair, and result in the numerous natural hair colors observed in the world. Analysis of the melanin content in hairs confirmed that various natural hair colors were caused by differences in the quantity of melanin pigment and the ratio of black-dark brown eumelanin and reddish-brown pheomelanin. (Borges et al., 2001, Ito and Wakamatsu, 2011).

Oxidative (permanent) hair coloring and bleaching products are used world- wide to change natural hair color to a desired color (Morel and Chrestie, 2011, Robbins, 2012b, Robbins, 2012c). Melanin granules are one of the primary target of oxidative hair coloring and bleaching products, which cause the melanin pigments to undergo decolorization during the treatment of those products. A previous study in 24 Japanese individuals showed that even upon using same bleaching product for black hair, the degree of brightness of the final hair color differed between individuals (Mita et al., 2020). Furthermore, it was confirmed that differences in the amounts of melanin pigment contributed to the observed difference in the degree of lightening following the bleaching treatment. In this way, difference in melanin within hair affects the final hair color when treated with coloring or bleaching. Therefore, a detailed understanding of the melanin in hair is important for the development of more effective and optimized hair coloring and bleaching products for each individual.

Melanin granules are generally examined by electron microscopy, such as scanning electron microscopy (SEM) and transmission electron microscopy (TEM), as they are in the nanometer scale and contain only small weight percent in hair (Arnaud and Bore, 1981, Kita et al., 1990, Imai, 2011, Ito, 2018, Koch et al., 2021). However, these methods only provide two-dimensional information on melanin granules in the hair shaft. In a previous hair study using ultra-high voltage electron microscopy, the three-dimensional distribution of melanin granules in hair was investigated, but the morphological information of melanin granules was not examined. (Koike, et al., 2004). The shape of melanin granules is ellipsoidal, approximately 400–1200 nm in length and 100–500 nm in diameter. Studies on the morphology of melanin granules have typically been performed after isolation using acid/base or enzyme extractions, which may cause partial degradation and some elution of the melanin granules (Hoting, et al., 1995b).

Therefore, there is requirement for techniques that can simultaneously observe three-dimensional distribution and morphology of melanin granules without isolating them from the hair sample. Focused ion beam scanning electron microscopy (FIB-SEM) can be used to repeatedly perform specimen sectioning using FIB and image acquisition using SEM in instrument. By reconstructing a three-dimensional image using serial two-dimensional SEM images obtained by FIB-SEM, tissues can be evaluated three-dimensionally. This method was recently used to examine the three-dimensional structure of skin and hair samples (Marsh et al., 2018, Mizutani et al., 2021). However, detailed characterization of hair melanin granules using FIB-SEM is yet to be conducted.

Differences in hair fiber characteristics of various races with respect to axial shape, hair diameter, cross-sectional shape, and color has previously been studied (Robbins, 2012d, Koch et al., 2021). In addition, the morphology and properties of Asian and Caucasian hair cuticles were examined, and differences in the configuration and hardness of the cuticular components were evaluated (Takahashi et al., 2006). However, there are limited studies on variations in the distribution and morphology of melanin granules between races (Kita et al., 1990).

In this study, the three-dimensional distribution of melanin granules in human black hair was investigated using FIB-SEM. Moreover, morphometric parameters of the melanin granules were calculated using the three-dimensionally reconstructed images to evaluate the difference of the morphology of melanin granules in hair obtained from Asians and Caucasians.