Hein Birka, Willig Katrin I, Hell Stefan W
Department of Nanobiophotonics, Max Planck Institute for Biophysical Chemistry, 37070 Göttingen, Germany.
Proc Natl Acad Sci U S A. 2008 Sep 23;105(38):14271-6. doi: 10.1073/pnas.0807705105. Epub 2008 Sep 16.
We demonstrate far-field optical imaging with subdiffraction resolution of the endoplasmic reticulum (ER) in the interior of a living mammalian cell. The diffraction barrier is overcome by applying stimulated emission depletion (STED) on a yellow fluorescent protein tag. Imaging individual structural elements of the ER revealed a focal plane (x, y) resolution of <50 nm inside the living cell, corresponding to a 4-fold improvement over that of a confocal microscope and a 16-fold reduction in the focal-spot cross-sectional area. A similar gain in resolution is realized with both pulsed- and continuous-wave laser illumination. Images of highly convoluted parts of the ER reveal a similar resolution improvement in 3D optical sectioning by a factor of 3 along the optic axis (z). Time-lapse STED recordings document morphological changes of the ER over time. Thus, nanoscale 3D imaging of organelles in the interior of living cells greatly expands the scope of light microscopy in cell biology.
我们展示了对活的哺乳动物细胞内部内质网（ER）进行亚衍射分辨率的远场光学成像。通过对黄色荧光蛋白标签应用受激发射损耗（STED）来克服衍射障碍。对ER的单个结构元件成像显示，活细胞内部的焦平面（x，y）分辨率小于50 nm，与共聚焦显微镜相比提高了4倍，焦斑横截面积减小了16倍。脉冲激光和连续波激光照明都实现了类似的分辨率提升。ER高度卷曲部分的图像显示，沿光轴（z）方向的三维光学切片分辨率提高了3倍。时间推移STED记录记录了ER随时间的形态变化。因此，对活细胞内部细胞器进行纳米级三维成像极大地扩展了细胞生物学中光学显微镜的应用范围。
