Photooxidation of Genetically Encoded MiniSOG-Fused VGLUT2 for Identification of Glutamatergic Synapses by Transmission and 3D Electron Microscopy

Immuno-electron microscopy (EM) is fundamentally limited by problems related to hindrance of antigenicity in fixed and resin-embedded specimens and destruction of ultrastructure by detergents. Furthermore, immunolabeling is problematic to use with three-dimensional (3D) EM techniques, such as serial block-face scanning EM (SBEM). Here we present an approach for the identification of glutamatergic synaptic vesicles (SVs) in nerve terminals that does not rely on immunolabeling and is compatible with both transmission and 3D EM applications. This technique utilizes heterologous expression of recombinant vesicular glutamate transporter-2 (VGLUT2) fused with mini singlet oxygen generator (miniSOG) and photooxidation of diaminobenzidine (DAB) to label glutamatergic SVs in nerve terminals in both fixed cultured cells and brain tissue.