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

Visualization of neuronal ultrastructure facilitates molecular and biochemical analyses that may help to better elucidate neural function and information processing. While the neuron exists at the micron scale, critical events such as synaptic vesicle release and dendritic spine remodeling occur at the nanometer scale, necessitating submicron resolution. Scanning electron microscopy (SEM) provides high-resolution imaging at these scales. However, the commonly used dehydration-based sample preparation method induces morphological distortions, while environmental SEM requires specialized equipment that is costly and difficult to operate. The NanoSuit method has recently emerged as a promising alternative, enabling SEM observations under high-vacuum conditions without standard (dehydration-based) pretreatment. Although known to be successful when applied to specimens with protective surface layers such as insects, flowers, and wet tissues, its effectiveness when examining "bare" cultured cells has not been thoroughly explored. Here, we present a modified NanoSuit protocol for SEM examination of cultured neurons and compare it with standard pretreatment. We demonstrate that traditional methods frequently cause neuronal transection and loss of fine dendritic processes, particularly during early development of neurons. However, the modified NanoSuit approach preserves neuronal morphology, enabling clear visualization of thin neurites and their interactions. Further, we successfully implemented correlative light and electron microscopy (CLEM) using this method, enabling the colocalization of cytoskeletal proteins such as actin and tubulin with the surface features observed by SEM. This combination of morphological preservation and molecular localization provides a more accurate and holistic understanding of neuronal structures, benefiting studies on neural development, synaptic connectivity, and related biomedical applications.