Nanoscale Imaging of Neurons Under Near-Physiological Conditions Using Field-Emission Scanning Electron Microscopy

Area of Science:

• Neuroscience
• Cell Biology
• Microscopy

Background:

• Understanding neural function requires visualizing neuronal ultrastructure at the nanometer scale.
• Traditional scanning electron microscopy (SEM) sample preparation causes morphological distortions.
• Environmental SEM requires specialized and costly equipment.

Purpose of the Study:

• To present and validate a modified NanoSuit protocol for SEM examination of cultured neurons.
• To compare the modified NanoSuit method with standard dehydration-based preparation.
• To assess the NanoSuit method's effectiveness for visualizing neuronal morphology and enabling correlative microscopy.

Main Methods:

• A modified NanoSuit protocol was developed for SEM sample preparation of cultured neurons.
• The modified NanoSuit method was compared against traditional dehydration-based methods.
• Correlative light and electron microscopy (CLEM) was implemented using the modified NanoSuit protocol.

Main Results:

• The modified NanoSuit approach preserved neuronal morphology, avoiding transection and loss of fine processes seen with traditional methods.
• High-resolution SEM imaging clearly visualized thin neurites and their interactions.
• CLEM successfully colocalized cytoskeletal proteins (actin, tubulin) with SEM-observed surface features.

Conclusions:

• The modified NanoSuit protocol offers superior morphological preservation for SEM of cultured neurons compared to traditional methods.
• This method enables detailed visualization of neuronal structures and molecular localization via CLEM.
• The technique benefits studies on neural development, synaptic connectivity, and biomedical applications.