Duane P. Harland

Biomaterials can be complex to study, due to a range of considerations such as shape, non-conductivity and internal changes in axial alignment, including, for fibres, natural curvature. To better understand these materials, we can use serial block-face scanning electron microscopy (SBF-SEM) for the visualization of the internal and external structures over micrometers, through a cyclic process. However, the application of this technique remains technically challenging, due to sample charging and natural morphology where the sample’s centre varies in location with respect to the resin block axis. In this study we used sheep wool fibres as a model protein-based non-infiltrated solid material to determine which stages of sample preparation could be altered to improve image quality from fibres by trialing different adherence methods, dehydration methods, and alterations to embedding resin. We found that the use of double-sided tape to adhere fibres to acetate frames improved ability to fine-tune fibre alignment and keep them straight, which improved maintenance of sample centering during long-runs of automated sectioning. Dehydration using silica gel and a vacuum resulted in cleaner images than solvent dehydration. The addition of a carbon powder to the embedding resin, even levels as low as 5% substantially reduced imaging artifacts associated with static charge accumulation. These optimizations for bio-based fibres improved the reliability of automated imaging runs to generate data for wool fibres at a suitable quality for ongoing analysis using automated segmentation tools.

The rise of serial block-face scanning electron microscopy (SBF-SEM) allows for the visualisation of internal and external 3-dimensional structures of samples over micrometers through a cyclic process. The application of this technique to electrically insulative natural samples, such as fibres, remains technically challenging largely due to sample charging. In this study we determined which stages could be altered to improve image quality of fibres by trialling different adherence methods, dehydration methods, and alterations to embedding resin. We found that the use of double-sided tape to adhere fibres to acetate frames improved ability to fine-tune fibre alignment and keep them straight, which improved maintenance of sample centring during long-runs of automated sectioning. Dehydration using silica gel and a vacuum resulted in cleaner images than solvent dehydration. The addition of Ketjen carbon black powder to the embedding resin, even levels as low as 5% substantially reduced imaging artifacts associated with static charge accumulation. These optimisations for bio-based fibres improved reliability of automated imaging runs to generate data for wool fibres at a suitable quality for ongoing analysis using automated segmentation tools.

The hair follicle undergoes repeated cycles, with anagen representing the active growth phase. During anagen, transit amplifying cells within the germinative matrix at the follicle bulb drive rapid proliferation for hair growth. This region exhibits some of the highest mitotic rates observed in any tissue, offering a rare opportunity to study mitosis in its native epithelial context, previously studied only in cultured cell lines. We applied volume electron microscopy to intact chemically fixed hair follicles enabling exceptional ultrastructural preservation of the entire mini-organ. Morphometric analysis revealed stage-specific changes in chromosomal and organelle volume and spatial distribution, highlighting coordinated roles for the mitochondria, vesicles and endoplasmic reticulum, and enabled, to our knowledge, the first ultrastructure-based karyotype of ovine chromosomes. This work advances understanding of mitosis by resolving ultrastructure in a highly proliferative, spatially constrained epithelial microenvironment, demonstrating the power of serial block face scanning electron microscopy to bridge in vitro models and native tissue architecture.

Investigation of the three-dimensional structure of hair fibres and follicles started in the 1960’s using sheep wool as a model. Pre-digital approaches to this included manually tracing of cell outlines from projected serial sections onto rolls of paper to line up cells. New electron microscopy techniques, automation and big-data handling allow a more efficient workflow, enabling spatial reconstructions that revolutionise how we visualize cell changes within the three-dimensional context of hair structures and during hair growth within follicles. Serial block-face scanning electron microscope (SBF-SEM) is one of the new microscopic techniques employed to determine how sheep wool and the cells within a fibre can chance along the length of the fibre. Sheep wool fibres (fine hairs) have a complex architecture with para- and ortho-cortical cells being responsible for the curliness of wool fibres ( 1, 2). However, there is no definite explanation regarding the mechanism by which they dictate the curliness of the fibre and what drives one cortical cell type over the other to cause curl. We are using SBF-SEM to be able to model where the cortical cells sit in relation to the curl. Alongside modelling of the whole fibre, we aim to segment individual cortical cells to explore how internal variation in sub-cellular morphology (macrofibrils composed of keratin fibrils) relates to cell type (ortho- or para-) and distribution across fibres with different curvatures. Within the growing fibre we aim to understand what leads to the formation of one cortical cell over the other and what this could mean for the cells and fibre formation.

Transmission electron microscopy (TEM) has greatly advanced our knowledge of hair growth and follicle morphogenesis, but complex preparations such as fixation, dehydration and embedding compromise ultrastructure. While recent developments with cryofixation have been shown to preserve the ultrastructure of biological materials close to native state, they do have limitations. This review will focus on each stage of the TEM sample preparation process and their effects on the structural integrity of follicles.

Mammalian hair fibers are internally sophisticated. We introduce a modeling approach aimed at use in research that derives value from understanding how microstructural organization generates effects at the macroscopic level in the context of natural biological variation. Critical buckling load is solved using a numerical approach applied to a modular fiber microstructure model where fibers of arbitrary length are made up of snippets composed of serial cross-sections at 25 micrometer intervals. As an example, the model is applied to investigate how much effect changes to single microstructural properties (fiber ellipticity, cortical cell type distribution and cell type proportion) have on critical buckling load in the context of prickle. Potential uses and key weak areas in our knowledge of wool fiber morphology and biophysics are discussed.

Summary: 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.