Axial superresolution via multiangle TIRF microscopy with sequential imaging and photobleaching

• Published in: Proceedings of the National Academy of Sciences - PNAS 2016-04, Vol.113 (16), p.4368-4373
• Main Authors: Fu, Yan, Winter, Peter W., Rojas, Raul, Wang, Victor, McAuliffe, Matthew, Patterson, George H.
• Format: Article
• Language: English
• Subjects: Bayesian analysis; Biological samples; Biological Sciences; Cell Line; Clathrin - metabolism; Endocytosis - physiology; Epidermal Growth Factor - metabolism; Fluorescence; Fluorescent Dyes - chemistry; Humans; Imaging, Three-Dimensional; Ligands; Microscopy; Microscopy, Fluorescence - methods; Molecules; Photobleaching
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.1516715113

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 (