Practical Guidelines for Two-Color SMLM of Synaptic Proteins in Cultured Neurons

  • Xiaojuan Yang
  • Christian G. SpechtEmail author
Part of the Neuromethods book series (NM, volume 154)


The application of single-molecule localization microscopy (SMLM) to the study of synaptic proteins has shown that the postsynaptic density (PSD) is organized heterogeneously in subsynaptic domains (SSDs) that are thought to play important roles in neurotransmission and synaptic plasticity. However, the dense packing of neurotransmitter receptors and scaffold proteins at synapses, together with the small total number of target molecules, makes SMLM of synaptic components particularly challenging. Here, we discuss the technical difficulties of SMLM imaging that are specific to synapses. We present a method for dual-color direct stochastic optical reconstruction microscopy (dSTORM) of two inhibitory synaptic proteins, the glycine receptor (GlyR) and the scaffold protein gephyrin (GPHN), highlighting strategic choices and practical solutions for imaging quality control. Our aim is to provide biologists with guidelines for the implementation of two-color dSTORM imaging of synaptic proteins from sample preparation to data analysis.

Key words

Super-resolution imaging Single-molecule localization microscopy (SMLM) Two-color direct stochastic optical reconstruction microscopy (dSTORM) Synaptic proteins Subsynaptic domain (SSD) Trans-synaptic nanocolumn Correlation analysis 



We thank Manuel Maidorn and Felipe Opazo for the illustrations in Fig. 2 [55], and Ignacio Izeddin for Fig. 4. Our research is funded by grants (to Antoine Triller, IBENS, Paris) from the Agence Nationale de la Recherche (ANR-12-BSV4-0019-01, ANR-11-IDEX-0001-02, ANR-10-LABX-54) and the European Research Council (ERC, PlastInhib). X.Y. is supported by the China Scholarship Council (CSC).


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Authors and Affiliations

  1. 1.Institute of Biology of the École Normale Supérieure (IBENS), CNRS, InsermPSL Research UniversityParisFrance
  2. 2.East China Normal UniversityShanghaiChina

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