What we have learned and will learn from cell ultrastructure in embedment-free section electron microscopy

Area of Science:

• Cell Biology
• Microscopy Techniques
• Biochemistry

Background:

• Conventional electron microscopy (EM) faces limitations in visualizing biological structures with low electron density.
• Epoxy resin embedding can obscure or alter fine cellular details.
• Embedment-free sectioning offers an alternative approach to overcome these limitations.

Purpose of the Study:

• To introduce and demonstrate the utility of embedment-free electron microscopy (PEG-EM) for visualizing cellular ultrastructure.
• To present key findings on strand- or microtrabecular lattices in the cytoplasmic matrix revealed by PEG-EM.
• To explore the biological significance and interpretations of these observed lattices.

Main Methods:

• Utilized water-soluble polyethylene glycol (PEG) as a transient embedding medium for electron microscopy.
• Performed de-embedding of PEG by water immersion followed by critical point-drying (CPD).
• Applied PEG-EM to analyze cellular structures, including in vitro solutions and pretreated cells.

Main Results:

• PEG-EM revealed distinct strand- or microtrabecular lattices in the cytoplasmic matrix, previously not visible with conventional EM.
• Lattice compactness varied across different cells and intracellular domains.
• In vitro experiments with albumin and gelatin solutions mimicked lattice formation, correlating with concentration and gelation states.
• Cellular pretreatment (hyper/hypotonic) affected lattice compactness.
• Specific intracellular proteins were localized within compact lattices in centrifuged ganglion cells.

Conclusions:

• PEG-EM provides novel insights into the ultrastructure of the cytoplasmic matrix, particularly concerning protein distribution and organization.
• Observed strand-lattices may represent soluble proteins, with compactness indicating concentration and sol-gel states.
• These findings suggest new mechanisms for understanding intracellular organelle localization and movement.
• PEG-EM has potential applications in 3D reconstruction of nonmembranous structures and tomography.