Identification of neuromuscular junctions by correlative confocal and transmission electron microscopy

The physiological processes regulating neuromuscular transmission are highly dependent on the structural features of the motor neuron and motor endplate, and detailing the structure of neuromuscular junctions (NMJs) in muscle biopsies is a powerful method for research and diagnostics. The observation of NMJ ultrastructure, however, is complicated by the difficulty in locating NMJs for analysis by electron microscopy. Consequently, a correlative confocal-transmission electron microscopy method was developed. Fixed muscle samples were cryo-protected in sucrose, sectioned on a cryostat, and stained with fluorescent α-bungarotoxin for confocal microscopy. Sections containing junctions were mapped and then processed for transmission electron microscopy (TEM). Cryostat sections allowed large expanses of muscle tissue to be rapidly screened and enabled specific junctions to be targeted for TEM. The morphology of the junctions was well preserved with all essential features of the pre- and postsynaptic elements readily identifiable without freeze damage. Unlike NMJ correlative methods using histochemical stains and DAB photo-oxidation, no electron dense precipitate was deposited over the NMJ, enabling an unobstructed view of the pre- and postsynaptic structures.