Back-scattered electron imaging of skeletal tissues

Area of Science:

• * Materials Science
• * Biomineralization
• * Microscopy

Background:

• * Scanning electron microscopy (SEM) is crucial for analyzing skeletal tissue microstructures.
• * Conventional secondary electron imaging in SEM can present challenges with sample topography and charging, particularly for unembedded or organically prepared samples.
• * Back-scattered electron (BSE) imaging offers an alternative contrast mechanism based on atomic number, potentially overcoming some limitations of secondary electron imaging.

Purpose of the Study:

• * To investigate the utility and optimization of solid-state back-scattered electron (BSE) detectors for scanning electron microscopic analysis of skeletal tissues.
• * To evaluate methods for minimizing topographic artifacts in BSE images of bone and cartilage.
• * To assess the effectiveness of BSE imaging for discriminating different density phases and surface types in skeletal samples.

Main Methods:

• * Utilized solid-state BSE detectors in a scanning electron microscope.
• * Prepared samples by diamond micromilling and diamond polishing for plastic-embedded tissues to achieve high flatness.
• * Employed a ring detector configuration with the sample at normal incidence to the electron beam to minimize topographic contrast.

Main Results:

• * Achieved excellent density discrimination, enabling imaging of different mineralized phases within bone.
• * Successfully differentiated cut surfaces from natural surfaces in unembedded spongy bone using topographic contrast.
• * Demonstrated BSE imaging's advantage for unembedded samples with rough topography, such as anorganic cartilage preparations, avoiding charging problems seen in secondary electron imaging.

Conclusions:

• * Solid-state BSE detectors are highly effective for SEM studies of skeletal tissues, offering superior density discrimination.
• * Specific sample preparation techniques (flat surfaces) and detector configurations optimize BSE imaging by minimizing topographic effects.
• * BSE imaging provides a valuable alternative to secondary electron imaging, particularly for challenging unembedded or organically prepared skeletal samples prone to charging.