Unterauer Eduard M, Jungmann Ralf
Max Planck Institute of Biochemistry, Martinsried, Germany.
Faculty of Physics and Center for Nanoscience, Ludwig Maximilian University, Munich, Germany.
Front Synaptic Neurosci. 2022 Feb 7;13:798267. doi: 10.3389/fnsyn.2021.798267. eCollection 2021.
Super-resolution (SR) microscopy techniques have been advancing the understanding of neuronal protein networks and interactions. Unraveling the arrangement of proteins with molecular resolution provided novel insights into neuron cytoskeleton structure and actin polymerization dynamics in synaptic spines. Recent improvements in quantitative SR imaging have been applied to synaptic protein clusters and with improved multiplexing technology, the interplay of multiple protein partners in synaptic active zones has been elucidated. While all SR techniques come with benefits and drawbacks, true molecular quantification is a major challenge with the most complex requirements for labeling reagents and careful experimental design. In this perspective, we provide an overview of quantitative SR multiplexing and discuss in greater detail the quantification and multiplexing capabilities of the SR technique DNA-PAINT. Using predictable binding kinetics of short oligonucleotides, DNA-PAINT provides two unique approaches to address multiplexed molecular quantification: qPAINT and Exchange-PAINT. With precise and accurate quantification and spectrally unlimited multiplexing, DNA-PAINT offers an attractive route to unravel complex protein interaction networks in neurons. Finally, while the SR community has been pushing technological advances from an imaging technique perspective, the development of universally available, small, efficient, and quantitative labels remains a major challenge in the field.
超分辨率（SR）显微镜技术一直在推动人们对神经元蛋白质网络和相互作用的理解。以分子分辨率解析蛋白质的排列，为神经元细胞骨架结构和突触棘中的肌动蛋白聚合动力学提供了新的见解。定量SR成像的最新进展已应用于突触蛋白簇，并且随着多路复用技术的改进，突触活性区中多个蛋白质伙伴之间的相互作用已得到阐明。虽然所有SR技术都有其优缺点，但真正的分子定量是一项重大挑战，对标记试剂有最复杂的要求，并且需要精心的实验设计。从这个角度来看，我们概述了定量SR多路复用，并更详细地讨论了SR技术DNA-PAINT的定量和多路复用能力。利用短寡核苷酸可预测的结合动力学，DNA-PAINT提供了两种独特的方法来解决多路复用分子定量问题：qPAINT和交换PAINT。凭借精确准确的定量和光谱无限的多路复用，DNA-PAINT为解析神经元中复杂的蛋白质相互作用网络提供了一条有吸引力的途径。最后，虽然SR领域一直从成像技术的角度推动技术进步，但开发普遍可用、小型、高效且定量的标记物仍然是该领域的一项重大挑战。
