Multiplexed and high-throughput neuronal fluorescence imaging with diffusible probes
Multiplexed and high-throughput neuronal fluorescence imaging with diffusible probes
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DOI：
10.1038/s41467-019-12372-6
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发表时间：
2019-09-26
影响因子：
16.6
通讯作者：
Bathe, Mark
中科院分区：
文献类型：
--
作者：
Guo, Syuan-Ming;Veneziano, Remi;Bathe, Mark
突触包含数百种不同的蛋白质，其异质表达水平是突触可塑性和与一系列疾病相关的信号传递的决定因素。在这里，我们使用可扩散的核酸成像探针，使用多路共聚焦和超分辨率显微镜的神经元突触配置文件。使用高亲和力锁定核酸成像探针进行共聚焦成像，所述探针稳定但可逆地结合与抗体和肽缀合的寡核苷酸。使用低亲和力DNA成像探针对相同靶点进行超分辨率PAINT成像，以解析九种不同蛋白质靶点的纳米级突触蛋白组织。我们的方法能够定量分析神经元培养物中的数千个突触，以从十几种蛋白质中识别推定的突触亚型和共定位模式。应用程序来表征神经元活动阻断后的突触重组揭示了突触后蛋白PSD-95，SHANK 3和Homer-1b/c的协调上调，以及活性和突触囊泡区中突触标记物之间的相关性增加。
Synapses contain hundreds of distinct proteins whose heterogeneous expression levels are determinants of synaptic plasticity and signal transmission relevant to a range of diseases. Here, we use diffusible nucleic acid imaging probes to profile neuronal synapses using multiplexed confocal and super-resolution microscopy. Confocal imaging is performed using high-affinity locked nucleic acid imaging probes that stably yet reversibly bind to oligonucleotides conjugated to antibodies and peptides. Super-resolution PAINT imaging of the same targets is performed using low-affinity DNA imaging probes to resolve nanometer-scale synaptic protein organization across nine distinct protein targets. Our approach enables the quantitative analysis of thousands of synapses in neuronal culture to identify putative synaptic sub-types and co-localization patterns from one dozen proteins. Application to characterize synaptic reorganization following neuronal activity blockade reveals coordinated upregulation of the post-synaptic proteins PSD-95, SHANK3 and Homer-1b/c, as well as increased correlation between synaptic markers in the active and synaptic vesicle zones.