{"title":"DNA-PAINT 定量成像在突触神经科学中的应用。","authors":"Eduard M Unterauer, Ralf Jungmann","doi":"10.3389/fnsyn.2021.798267","DOIUrl":null,"url":null,"abstract":"<p><p>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.</p>","PeriodicalId":12650,"journal":{"name":"Frontiers in Synaptic Neuroscience","volume":null,"pages":null},"PeriodicalIF":2.8000,"publicationDate":"2022-02-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8860300/pdf/","citationCount":"0","resultStr":"{\"title\":\"Quantitative Imaging With DNA-PAINT for Applications in Synaptic Neuroscience.\",\"authors\":\"Eduard M Unterauer, Ralf Jungmann\",\"doi\":\"10.3389/fnsyn.2021.798267\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><p>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.</p>\",\"PeriodicalId\":12650,\"journal\":{\"name\":\"Frontiers in Synaptic Neuroscience\",\"volume\":null,\"pages\":null},\"PeriodicalIF\":2.8000,\"publicationDate\":\"2022-02-07\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8860300/pdf/\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Frontiers in Synaptic Neuroscience\",\"FirstCategoryId\":\"3\",\"ListUrlMain\":\"https://doi.org/10.3389/fnsyn.2021.798267\",\"RegionNum\":4,\"RegionCategory\":\"医学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"2021/1/1 0:00:00\",\"PubModel\":\"eCollection\",\"JCR\":\"Q2\",\"JCRName\":\"NEUROSCIENCES\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Frontiers in Synaptic Neuroscience","FirstCategoryId":"3","ListUrlMain":"https://doi.org/10.3389/fnsyn.2021.798267","RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"2021/1/1 0:00:00","PubModel":"eCollection","JCR":"Q2","JCRName":"NEUROSCIENCES","Score":null,"Total":0}
引用次数: 0
摘要
超分辨率(SR)显微镜技术推动了对神经元蛋白质网络和相互作用的了解。以分子分辨率揭示蛋白质的排列,为了解神经元细胞骨架结构和突触棘中肌动蛋白聚合动态提供了新的视角。最近,定量 SR 成像技术的改进被应用于突触蛋白簇,通过改进的多路复用技术,突触活性区中多个蛋白伙伴的相互作用得以阐明。虽然所有 SR 技术都有利有弊,但真正的分子定量是一项重大挑战,对标记试剂和精心的实验设计有着最复杂的要求。在本文中,我们将概述定量 SR 多路复用技术,并更详细地讨论 SR 技术 DNA-PAINT 的定量和多路复用能力。DNA-PAINT 利用短寡核苷酸可预测的结合动力学,提供了两种独特的方法来解决多重分子定量问题:qPAINT 和 Exchange-PAINT。DNA-PAINT 具有精确定量和光谱无限复用的特点,为揭示神经元中复杂的蛋白质相互作用网络提供了极具吸引力的途径。最后,虽然 SR 界一直在从成像技术的角度推动技术进步,但开发普遍可用、小巧、高效和定量的标签仍是该领域的一大挑战。
Quantitative Imaging With DNA-PAINT for Applications in Synaptic Neuroscience.
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.