SEM/FIB Imaging for Studying Neural Interfaces.

Area of Science:

• Tissue and neural engineering
• Regenerative medicine
• Biomedical applications

Background:

• Studying cell-material interfaces is vital for regenerative therapies.
• Scanning electron microscopy with focused ion beam offers high resolution but requires optimized sample preparation.
• Preserving the natural cell structure during fixation, dehydration, and staining is challenging.

Purpose of the Study:

• To compare and optimize sample drying and staining protocols for scanning electron microscopy.
• To preserve cells in a life-like state for studying cell interfaces with 3D structures and electrodes.
• To determine the best methods for visualizing intracellular organelles and membranes.

Main Methods:

• Chemical fixation using glutaraldehyde and formaldehyde.
• Comparison of four drying techniques: critical point drying, hexamethyldisiloxane, osmium tetroxide-thiocarbohydrazide (OTOTO), and resin.
• Evaluation of four staining techniques: osmium tetroxide, osmium tetroxide and salts, osmium and uranyl acetate, and OTOTO.
• Experiments conducted on photoreceptor precursors, neural cells, and retinal pigment epithelial cells.

Main Results:

• Resin drying combined with OTOTO staining emerged as the optimal processing combination.
• This combination demonstrated excellent preservation of cell morphology and cell interface.
• It resulted in the lowest percentage of cellular protrusion breakage and high-quality imaging.

Conclusions:

• The optimized protocol enhances the understanding of cell-structure interfaces.
• This advancement is expected to improve various biomedical applications in regenerative medicine.
• The findings provide a foundation for future research in tissue and neural engineering.