Fu Yan, Winter Peter W, Rojas Raul, Wang Victor, McAuliffe Matthew, Patterson George H
Section on Biophotonics, National Institute of Biomedical Imaging and Bioengineering, National Institutes of Health, Bethesda, MD 20892;
Section on High-Resolution Optical Imaging, National Institute of Biomedical Imaging and Bioengineering, National Institutes of Health, Bethesda, MD 20892;
Proc Natl Acad Sci U S A. 2016 Apr 19;113(16):4368-73. doi: 10.1073/pnas.1516715113. Epub 2016 Apr 1.
We report superresolution optical sectioning using a multiangle total internal reflection fluorescence (TIRF) microscope. TIRF images were constructed from several layers within a normal TIRF excitation zone by sequentially imaging and photobleaching the fluorescent molecules. The depth of the evanescent wave at different layers was altered by tuning the excitation light incident angle. The angle was tuned from the highest (the smallest TIRF depth) toward the critical angle (the largest TIRF depth) to preferentially photobleach fluorescence from the lower layers and allow straightforward observation of deeper structures without masking by the brighter signals closer to the coverglass. Reconstruction of the TIRF images enabled 3D imaging of biological samples with 20-nm axial resolution. Two-color imaging of epidermal growth factor (EGF) ligand and clathrin revealed the dynamics of EGF-activated clathrin-mediated endocytosis during internalization. Furthermore, Bayesian analysis of images collected during the photobleaching step of each plane enabled lateral superresolution (<100 nm) within each of the sections.
我们报道了使用多角度全内反射荧光（TIRF）显微镜进行超分辨率光学切片。通过对荧光分子进行顺序成像和光漂白，从正常TIRF激发区内的几层构建TIRF图像。通过调整激发光入射角来改变不同层处倏逝波的深度。将角度从最高值（最小的TIRF深度）调至临界角（最大的TIRF深度），以优先光漂白来自下层的荧光，并能直接观察更深层的结构，而不会被靠近盖玻片的更亮信号所掩盖。TIRF图像的重建实现了对生物样品的三维成像，轴向分辨率为20纳米。表皮生长因子（EGF）配体和网格蛋白的双色成像揭示了内化过程中EGF激活的网格蛋白介导的内吞作用的动力学。此外，对每个平面光漂白步骤中收集的图像进行贝叶斯分析，可在每个切片内实现横向超分辨率（<100纳米）。
