Label-Free Imaging of Single Proteins Secreted from Living Cells via iSCAT Microscopy
Label-Free Imaging of Single Proteins Secreted from Living Cells via iSCAT Microscopy
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DOI：
10.3791/58486
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发表时间：
2018-11-01
影响因子：
1.2
通讯作者：
Sandoghdar, Vahid
中科院分区：
文献类型：
--
作者：
Gemeinhardt, Andre;McDonald, Matthew P.;Sandoghdar, Vahid
我们展示了干涉散射(ISCAT)显微镜，这是一种能够实时检测单个活细胞分泌的单个未标记蛋白质的方法。在这个方案中，我们涵盖了实现iSCAT显微镜的基本步骤，并用额外的成像通道来补充它，以监测正在研究的细胞的活力。然后，我们使用这种方法实时检测单个蛋白质，因为它们是从一个活细胞中分泌出来的，我们用一个永生化的B细胞系(Laz388)演示了这一点。讨论了显微镜和样品制备以及记录数据分析的必要步骤。视频协议表明，iSCAT显微镜提供了一种在单分子水平上研究分泌的直接方法。
We demonstrate interferometric scattering (iSCAT) microscopy, a method capable of detecting single unlabeled proteins secreted from individual living cells in real time. In this protocol, we cover the fundamental steps to realize an iSCAT microscope and complement it with additional imaging channels to monitor the viability of a cell under study. Following this, we use the method for real-time detection of single proteins as they are secreted from a living cell which we demonstrate with an immortalized B-cell line (Laz388). Necessary steps concerning the preparation of microscope and sample as well as the analysis of the recorded data are discussed. The video protocol demonstrates that iSCAT microscopy offers a straightforward method to study secretion at the single-molecule level.