Imaging of In Vitro and In Vivo Neurons in Drosophila Using Stochastic Optical Reconstruction Microscopy.
Imaging of In Vitro and In Vivo Neurons in Drosophila Using Stochastic Optical Reconstruction Microscopy.
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DOI：
10.1002/cpz1.203
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发表时间：
2021-07
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影响因子：
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通讯作者：
Kamiyama D
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文献类型：
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作者：
Inal MA;Bui KC;Marar A;Li S;Kner P;Kamiyama D
果蝇的大脑由不同类型的神经元细胞组成，它们通过精确的突触连接模式相互连接。这些模式对大脑的正常功能至关重要。为了理解连接模式，我们必须在单细胞分辨率下描述它们，荧光显微镜成为不可或缺的工具。此外，由于神经元是在纳米尺度上连接的，因此研究通常需要超分辨率显微镜。在这里，我们采用了一种超分辨率显微镜技术，称为STORM（随机光学重建显微镜），将横向和轴向分辨率提高到~20 nm。本单元广泛描述了我们在体外和体内神经元样品制备，染料与抗体结合，免疫荧光标记以及STORM数据采集和处理的方法和注意事项。有了这些工具和技术，我们打开了在果蝇神经系统中使用STORM研究细胞-细胞相互作用的潜力。
The Drosophila brain comprises different neuronal cell types that interconnect with precise patterns of synaptic connections. These patterns are essential for the normal function of the brain. To understand the connectivity patterns, we must characterize them at single-cell resolution, for which a fluorescence microscope becomes an indispensable tool. Additionally, because the neurons connect at the nanoscale, the investigation often demands super-resolution microscopy. Here, we adopt one of the super-resolution microscopy techniques, called STORM (Stochastic Optical Reconstruction Microscopy), improving the lateral and axial resolution to ~20 nm. This unit extensively describes our methods and considerations for sample preparation of neurons in vitro and in vivo, conjugation of dyes to antibodies, immunofluorescence labeling, and STORM data acquisition and processing. With these tools and techniques, we open up the potential to investigate cell-cell interactions using STORM in the Drosophila nervous system.