High-pressure freezing followed by freeze substitution of a complex and variable density miniorgan: the wool follicle.

Cryofixation by high-pressure freezing (HPF) followed by freeze substitution (FS) is a preferred method to prepare biological specimens for ultrastructural studies. It has been shown to achieve uniform vitrification and ultrastructure preservation of complex structures in different cell types. One limitation of HPF is the small sample volume of <200 µm thickness and about 2000 µm across. A wool follicle is a rare intact organ in a single sample about 200 µm thick. Within each follicle, specialized cells derived from multiple cell lineages assemble, mature and cornify to make a wool fibre, which contains 95% keratin and associated proteins. In addition to their complex structure, large density changes occur during wool fibre development. Limited water movement and accessibility of fixatives are some issues that negatively affect the preservation of the follicle ultrastructure via conventional chemical processing. Here, we show that HPF-FS of wool follicles can yield high-quality tissue preservation for ultrastructural studies using transmission electron microscopy.

Structure of photo-damaged white and naturally pigmented human hair.

The effect of photo-degradation processes on the morphology and ultrastructure of pigmented and non-pigmented hair was evaluated in this work by small angle X-ray scattering (SAXS) and transmission electron microscopy (TEM). Samples consisted of three types of grey hair (separated out into subsamples of totally white and totally black strands) and one type of dark-brown hair exposed to solar irradiation for 75 h or to a mercury lamp for 600 h. SAXS patterns showed that irradiation mainly damaged the amorphous regions of hair, and in some types of hair, an ordered structure appeared, with spaces of ca. 4.5 nm. This structure occurred independently of the presence of pigments and its scattering intensity increased after irradiation. Layers of lipids present in the cuticle may be responsible for this scattering around 4.5 nm, as indicated by TEM images. TEM also showed morphological changes in the outermost cuticle layers, such as the degradation of endocuticle and the detachment of the cell membrane complex. Therefore, the results presented herewith showed that irradiation caused different damages to the hair amorphous fractions, especially in the cuticle layers, which are the most exposed areas of the hair strands.

Morphological degradation of human hair cuticle due to simulated sunlight irradiation and washing.

Morphological changes in hair surface are undesirable, since they cause shine loss, roughness increase and split ends. These effects occur more frequently in the cuticle, which is the outermost layer of the hair strand, and thus the most exposed to the environmental damages. Sunlight irradiation contributes significantly to these morphological alterations, which motivates the investigation of this effect on hair degradation. In this work, the influence of irradiation and hand-washing steps on the morphology of pigmented and non-pigmented hair cuticle was investigated using field emission scanning electron microscopy (FESEM) and transmission electron microscopy (TEM). To simulate daily conditions, where hair is hand-washed and light exposed, samples of dark brown and gray hair underwent three different conditions: 1) irradiation with a mercury lamp for up to 600h; 2) irradiation with the mercury lamp combined with washes with a sodium lauryl sulphate solution; and 3) only washing. A new preparation procedure was applied for TEM samples to minimize natural variations among different hair strands: a single hair strand was cut into two neighbouring halves and only one of them underwent irradiation and washing. The non-exposed half was used as a control, so that the real effects caused by the controlled irradiation and washing procedures could be highlighted in samples that had very similar morphologies initially. More than 25images/sample were analysed using FESEM (total of 300 images) and ca. 150images/sample were obtained with TEM (total of 900 images). The results presented herein show that the endocuticle and the cell membrane complex (CMC) are the cuticle structures more degraded by irradiation. Photodegradation alone results in fracturing, cavities (Ø≈20-200nm) and cuticle cell lifting, while the washing steps were able to remove cuticle cells (≈1-2 cells removed after 60 washes). Finally, the combined action of irradiation and washing caused the most severe damages, resulting in a more pronounced cuticle extraction (≈1-4 cuticle cells after a 600h irradiation and a 60 times washing). This irradiation dose corresponds to ca. 2months of sunlight exposure (considering 5h/day) in Campinas-SP, Brazil, during the day period of maximum irradiation intensity. The combined action of irradiation and washing can be explained by the creation of fragile photodegraded spots in the endocuticle and in the CMC, where the mechanical stress associated to the washing steps are more prone to induce rupture.

Effect of photodamage on the outermost cuticle layer of human hair.

The surface of the hair is the region most exposed to solar radiation and to the environment in general. Many of the well-known damaging effects of sun exposure on hair must start or even be restricted to the most external cuticle layers. As such, this work investigates morphological, ultrastructural and chemical changes in the outermost cuticle layer of dark brown hair, using atomic force microscopy (AFM), field emission scanning electron microscopy (FESEM) and attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR). The results showed that after 230 h of irradiation with a mercury lamp, small bumps with globular shape (heights lying in the 1-5 nm range) appeared on the cuticle surface and their size increased with increasing irradiation times. In addition, the enlargement of pre-existing holes was also observed (holes increase around 350% in depth) and the height of the steps formed between the edges of two cuticle scales increased around 65%, as a result of 500 h of irradiation. The damages in hair strands were accurately identified by analyzing exactly the same surface region before and after irradiation by AFM images. Finally, the results were discussed in terms of the chemical differences between the non-irradiated and the irradiated hair, for instance, the increased level of cystine oxidation as a consequence of photodegradation.

Yellowing and bleaching of grey hair caused by photo and thermal degradation.

Yellowing is an undesirable phenomenon that is common in people with white and grey hair. Because white hair has no melanin, the pigment responsible for hair colour, the effects of photodegradation are more visible in this type of hair. The origin of yellowing and its relation to photodegradation processes are not properly established, and many questions remain open in this field. In this work, the photodegradation of grey hair was investigated as a function of the wavelength of incident radiation, and its ultrastructure was determined, always comparing the results obtained for the white and black fibres present in grey hair with the results of white wool. The results presented herein indicate that the photobehaviour of grey hair irradiated with a mercury lamp or with solar radiation is dependent on the wavelength range of the incident radiation and on the initial shade of yellow in the sample. Two types of grey hair were used: (1) blended grey hair (more yellow) and (2) grey hair from a single-donor (less yellow). After exposure to a full-spectrum mercury lamp for 200 h, the blended white hair turned less yellow (the yellow-blue difference, Db(*) becomes negative, Db(*)=-6), whereas the white hair from the single-donor turned slightly yellower (Db(*)=2). In contrast, VIS+IR irradiation resulted in bleaching in both types of hair, whereas a thermal treatment (at 81 °C) caused yellowing of both types of hair, resulting in a Db(*)=3 for blended white hair and Db(*)=9 for single-donor hair. The identity of the yellow chromophores was investigated by UV-Vis spectroscopy. The results obtained with this technique were contradictory, however, and it was not possible to obtain a simple correlation between the sample shade of yellow and the absorption spectra. In addition, the results are discussed in terms of the morphology differences between the pigmented and non-pigmented parts of grey hair, the yellowing and bleaching effects of grey hair, and the occurrence of dark-follow reactions.

Photo yellowing of human hair.

In general, human hair is claimed to turn yellower after sun exposure. This is particularly affirmed for white hair. However, quantitative data relating yellowness to hair type and to the radiation wavelength are missing. This work shows results of the effect of full or UVB-filtered radiation of a mercury vapor or a xenon-arc lamp on the yellowness of virgin white, dark-brown, blond and red hair. All hair types showed a substantial change in yellowness after irradiation, which is dependent on the hair type and radiation wavelength. Surprisingly, white hair turns less yellow after both full and UVB-filtered radiation exposure. This effect is more pronounced when UVB is filtered from the radiation system. The only radiation that shows a photo-yellowing effect on white hair is infrared. As the yellowness of white hair is commonly related to tryptophan degradation, fluorescence experiments with hair solutions were performed to identify the natural degradation of tryptophan which occurs in hair after light irradiation. Pigmented hairs were also studied, as well as hair treated with a bleaching solution. Although we observe a decrease in tryptophan content of hair after lamp radiation, a direct correlation with hair yellowness was not achieved. Results are discussed in terms of hair type, composition and melanin content.