A super-resolution protocol to correlate structural underpinnings of fast second-messenger signalling in primary cell types
A super-resolution protocol to correlate structural underpinnings of fast second-messenger signalling in primary cell types
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DOI：
10.1101/2020.09.29.319400
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发表时间：
2020-09
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影响因子：
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通讯作者：
M. Hurley;T. M. Sheard;R. Norman;Hannah M. Kirton;Shihab S. Shah;E. Pervolaraki;Zhaokang Yang;N. Gamper;Ed White;D. Steele;I. Jayasinghe
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文献类型：
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作者：
M. Hurley;T. M. Sheard;R. Norman;Hannah M. Kirton;Shihab S. Shah;E. Pervolaraki;Zhaokang Yang;N. Gamper;Ed White;D. Steele;I. Jayasinghe
通过超分辨率显微镜获得的纳米级细胞信息通常不伴有功能信息，特别是在纳米微米空间尺度上协调生命的瞬态和扩散信号。我们描述了一种相关的成像方案，该方案允许无处不在的细胞内第二信使钙（Ca2+）直接可视化，以抵抗在野生型原代细胞中产生这些Ca2+信号的ryanodine受体（RyR） Ca2+通道的纳米级模式。这是通过将基本Ca2+信号的全内反射荧光（TIRF）成像与随后的rrs DNA-PAINT成像相结合来实现的。我们报告了相关数据集之间的特征提取和图像对齐的直接图像分析方案，并演示了如何使用这些数据来直观地识别细胞质Ca2+信号发生过程中局部激活的Ca2+通道的集合。
Nanometre-scale cellular information obtained through super-resolution microscopies are often unaccompanied by functional information, particularly transient and diffusible signals through which life is orchestrated in the nano-micrometre spatial scale. We describe a correlative imaging protocol which allows the ubiquitous intracellular second messenger, calcium (Ca2+), to be directly visualised against nanoscale patterns of the ryanodine receptor (RyR) Ca2+ channels which give rise to these Ca2+ signals in wildtype primary cells. This was achieved by combining total internal reflection fluorescence (TIRF) imaging of the elementary Ca2+ signals, with the subsequent DNA-PAINT imaging of the RyRs. We report a straightforward image analysis protocol of feature extraction and image alignment between correlative datasets and demonstrate how such data can be used to visually identify the ensembles of Ca2+ channels that are locally activated during the genesis of cytoplasmic Ca2+ signals.