Super-resolution microscopy for analyzing neuromuscular junctions and synapses

Abstract

Super-resolution microscopy techniques offer subdiffraction limited resolution that is two- to ten-fold improved compared to that offered by conventional confocal microscopy. This breakthrough in resolution for light microscopy has contributed to new findings in neuroscience and synapse biology. This review will focus on the Structured Illumination Microscopy (SIM), Stimulated emission depletion (STED) microscopy, and Stochastic optical reconstruction microscopy (STORM) / Single molecule localization microscopy (SMLM) techniques and compare them for the better understanding of their differences and their suitability for the analysis of synapse biology. In addition, we will discuss a few practical aspects of these microscopic techniques, including resolution, image acquisition speed, multicolor capability, and other advantages and disadvantages. Tips for the improvement of microscopy will be introduced; for example, information resources for recommended dyes, the limitations of multicolor analysis, and capabilities for live imaging. In addition, we will summarize how super-resolution microscopy has been used for analyses of neuromuscular junctions and synapses.

Keywords: Neuromuscular junction; SIM; STED; STORM; Super-resolution microscopy; Synapse.

Figure 1.

Abbe’s diffraction limit, the sizes of synaptic structures and tools used in super-resolution microscopy. The X-axis indicates the size in micrometers to nanometers. The sizes of the synaptic structures and objects shown in the figure are as follows: NMJs in the en face view are 10 – 60 μm (human, mouse) [22, 56], the central nervous system (CNS) synapse is 1 – 2 μm (mouse) [59], the synaptic cleft of the NMJ is 50 – 100 nm [72], the junctional fold crest width is 207 nm and the opening width is 55 nm [139], the active zone is 50 – 100 nm [73, 82, 86], the synaptic vesicle (SV) is 40 – 50 nm [72, 102], the antibody is 12 × 14 nm [68, 80], the nanobody (variable domain, VD) is 2.5 × 4 nm [60], and green fluorescent protein (GFP) is 2.4 × 4.2 nm [51].