Imaging green fluorescent protein-labeled neurons using light and electron microscopy

Area of Science:

• Neuroscience
• Cell Biology
• Microscopy

Background:

• Studying neuronal connectivity requires high-resolution imaging of neuronal structures.
• Correlating live imaging with ultrastructural analysis is challenging.

Purpose of the Study:

• To provide a detailed protocol for reimaging live neurons with transmission electron microscopy (EM).
• To enable three-dimensional (3D) ultrastructural analysis of neuronal processes after live imaging.

Main Methods:

• Neurons expressing green fluorescent protein (GFP) were imaged live using laser-scanning microscopy.
• Brain tissue was chemically fixed, and GFP was rendered electron-dense via immunocytochemistry.
• Neurons were located using light microscopy and serial thin-sectioned for transmission EM.
• Blood vessel patterns aided in relocating previously imaged neurons.

Main Results:

• The protocol allows for sequential live imaging and EM analysis of the same neurons.
• Detailed three-dimensional (3D) ultrastructure of neurites can be analyzed.
• Complete 3D reconstructions of imaged neuronal structures are achievable from serial electron micrographs.

Conclusions:

• This method bridges live neuronal imaging and high-resolution EM.
• It facilitates in-depth analysis of synaptic connectivity and neuronal architecture.
• The protocol is valuable for studying neuronal structure-function relationships.