UCL Queen Square Institute of Neurology, University College London, London, United Kingdom; FutureNeuro Research Centre, Royal College of Surgeons in Ireland, Dublin, Ireland.
UCL Queen Square Institute of Neurology, University College London, London, United Kingdom; Department of Physiology, Faculty of Basic Medical Sciences, Alex Ekwueme Federal University Ndufu-Alike Ikwo, PMB 1010 Abakaliki, Nigeria.
Methods. 2020 Mar 1;174:81-90. doi: 10.1016/j.ymeth.2019.05.024. Epub 2019 May 31.
Astroglia are vital facilitators of brain development, homeostasis, and metabolic support. In addition, they are also essential to the formation and regulation of synaptic circuits. Due to the extraordinary complex, nanoscopic morphology of astrocytes, the underlying cellular mechanisms have been poorly understood. In particular, fine astrocytic processes that can be found in the vicinity of synapses have been difficult to study using traditional imaging techniques. Here, we describe a 3D three-colour super-resolution microscopy approach to unravel the nanostructure of tripartite synapses. The method is based on the SMLM technique direct stochastic optical reconstruction microscopy (dSTORM) which uses conventional fluorophore-labelled antibodies. This approach enables reconstructing the nanoscale localisation of individual astrocytic glutamate transporter (GLT-1) molecules surrounding presynaptic (bassoon) and postsynaptic (Homer1) protein localisations in fixed mouse brain sections. However, the technique is readily adaptable to other types of targets and tissues.
星形胶质细胞是大脑发育、内稳态和代谢支持的重要促进剂。此外，它们对于突触回路的形成和调节也是必不可少的。由于星形胶质细胞具有非常复杂的纳米级形态，其潜在的细胞机制还了解甚少。特别是，在突触附近发现的精细星形胶质细胞过程，使用传统的成像技术很难进行研究。在这里，我们描述了一种 3D 三色超分辨率显微镜方法，用于揭示三突触的纳米结构。该方法基于 SMLM 技术直接随机光学重建显微镜 (dSTORM)，该技术使用常规荧光标记抗体。这种方法可以重建围绕突触前 (bassoon) 和突触后 (Homer1) 蛋白定位的单个星形胶质细胞谷氨酸转运体 (GLT-1) 分子的纳米级局部定位，在固定的小鼠脑切片中。然而，该技术很容易适用于其他类型的靶标和组织。
