Super-Resolution Live Cell Imaging of Subcellular Structures.
Super-Resolution Live Cell Imaging of Subcellular Structures.
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DOI：
10.3791/61563
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发表时间：
2021-01-13
期刊：
影响因子：
--
通讯作者：
Chen X
中科院分区：
文献类型：
--
作者：
Ranjan R;Chen X
长期以来，成像中的空间分辨率和时间分辨率之间存在着至关重要的权衡。传统上，超过光衍射极限的成像仅限于用于固定的样品或标记有强荧光信号的组织外的活细胞。当前的超分辨率活细胞成像技术需要使用特殊的荧光探针、高照明、具有采集后处理的多个图像采集，或者通常是这些过程的组合。这些先决条件极大地限制了该技术可以应用的生物样品和背景。在这里，我们描述了一种方法来执行超分辨率（140 nm XY分辨率）延时荧光活细胞原位成像。该技术还与低荧光强度相容，例如，在低表达基因处内源性标记EGFP或mCherry。作为一个证明的原则，我们已经使用这种方法来可视化多个亚细胞结构的果蝇睾丸。在组织制备过程中，细胞结构和组织形态都保持在解剖的睾丸内。在这里，我们使用这种技术来成像微管动力学，微管和核膜之间的相互作用，以及微管对着丝粒的附着。该技术需要在样品制备、样品安装和样品固定方面的特殊程序。此外，标本必须在解剖后保持几个小时，而不损害细胞功能和活性。虽然我们已经优化了特别是在果蝇雄性生殖系干细胞（GSC）和睾丸组织中的生殖祖细胞中进行实时超分辨率成像的条件，但该技术广泛适用于各种不同的细胞类型。在生理条件下观察细胞而不牺牲空间或时间分辨率的能力将成为研究人员寻求解决细胞生物学关键问题的宝贵工具。这里提出的是一个协议的超分辨率活细胞成像在完整的组织。我们已经标准化了在其天然组织环境中对高度敏感的成体干细胞群体进行成像的条件。该技术涉及平衡时间和空间分辨率，以允许直接观察活组织中的生物现象。
There has long been a crucial tradeoff between spatial and temporal resolution in imaging. Imaging beyond the diffraction limit of light has traditionally been restricted to be used only on fixed samples or live cells outside of tissue labeled with strong fluorescent signal. Current super-resolution live cell imaging techniques require the use of special fluorescence probes, high illumination, multiple image acquisitions with post-acquisition processing, or often a combination of these processes. These prerequisites significantly limit the biological samples and contexts that this technique can be applied to. Here we describe a method to perform super-resolution (∼140 nm XY-resolution) time-lapse fluorescence live cell imaging in situ. This technique is also compatible with low fluorescent intensity, for example, EGFP or mCherry endogenously tagged at lowly expressed genes. As a proof-of-principle, we have used this method to visualize multiple subcellular structures in the Drosophila testis. During tissue preparation, both the cellular structure and tissue morphology are maintained within the dissected testis. Here, we use this technique to image microtubule dynamics, the interactions between microtubules and the nuclear membrane, as well as the attachment of microtubules to centromeres. This technique requires special procedures in sample preparation, sample mounting and immobilizing of specimens. Additionally, the specimens must be maintained for several hours after dissection without compromising cellular function and activity. While we have optimized the conditions for live super-resolution imaging specifically in Drosophila male germline stem cells (GSCs) and progenitor germ cells in dissected testis tissue, this technique is broadly applicable to a variety of different cell types. The ability to observe cells under their physiological conditions without sacrificing either spatial or temporal resolution will serve as an invaluable tool to researchers seeking to address crucial questions in cell biology. Presented here is a protocol for super-resolution live-cell imaging in intact tissue. We have standardized the conditions for imaging a highly sensitive adult stem cell population in its native tissue environment. This technique involves balancing temporal and spatial resolution to allow for the direct observation of biological phenomena in live tissue.
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影响因子：
48
作者：
Shroff, Hari;Galbraith, Catherine G.;Betzig, Eric
通讯作者：
Betzig, Eric
DOI：
10.1083/jcb.201002018
发表时间：
2010-07-26
期刊：
The Journal of cell biology
影响因子：
--
作者：
Schermelleh L;Heintzmann R;Leonhardt H
通讯作者：
Leonhardt H
影响因子：
2.4
作者：
Korobchevskaya, Kseniya;Lagerholm, B. Christoffer;Fritzsche, Marco
通讯作者：
Fritzsche, Marco
影响因子：
48
作者：
Vicidomini, Giuseppe;Bianchini, Paolo;Diaspro, Alberto
通讯作者：
Diaspro, Alberto
影响因子：
16.8
作者：
Wooten, Matthew;Snedeker, Jonathan;Chen, Xin
通讯作者：
Chen, Xin