Super-resolution Microscopy at Cryogenic Temperatures Using Solid Immersion Lenses.
Super-resolution Microscopy at Cryogenic Temperatures Using Solid Immersion Lenses.
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DOI：
10.21769/bioprotoc.3426
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发表时间：
2019-11
期刊：
影响因子：
0.8
通讯作者：
Benji C. Bateman;Laura C. Zanetti-Domingues;A. N. Moores;S. R. Needham;D. Rolfe;Lin Wang;D. Clarke;M. Martin-Fernandez
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文献类型：
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作者：
Benji C. Bateman;Laura C. Zanetti-Domingues;A. N. Moores;S. R. Needham;D. Rolfe;Lin Wang;D. Clarke;M. Martin-Fernandez
我们对细胞功能的机械理解依赖于用分子分辨率成像细胞中的生物过程。超分辨率荧光显微镜通过以20-30 nm的分辨率报告细胞超微结构起着至关重要的作用。然而，这种分辨率不足以对工作中的大分子机器进行成像。提高分辨率的一个途径是在低温条件下成像，这大大增加了大多数荧光团的亮度，并比化学固定剂更好地保留了天然的超微结构。然而，由于缺乏兼容的高数值孔径（NA）物镜，低温条件未得到充分利用。在这里，我们描述了一个协议，使用超半球形固体浸没透镜（superSILs），以实现超分辨率成像在低温下的有效NA为2.17和分辨率为10 nm。
Our mechanistic understanding of cell function depends on imaging biological processes in cells with molecular resolution. Super-resolution fluorescence microscopy plays a crucial role by reporting cellular ultrastructure with 20-30 nm resolution. However, this resolution is insufficient to image macro-molecular machinery at work. A path to improve resolution is to image under cryogenic conditions, which substantially increases the brightness of most fluorophores and preserves native ultrastructure much better than chemical fixatives. Cryogenic conditions are, however, underutilized because of the lack of compatible high numerical aperture (NA) objectives. Here we describe a protocol for the use of super-hemispherical solid immersion lenses (superSILs) to achieve super-resolution imaging at cryogenic temperatures with an effective NA of 2.17 and resolution of ~10 nm.