Axial superresolution via multiangle TIRF microscopy with sequential imaging and photobleaching.

ABSTRACT: 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. Tw

SUBMITTER: Fu Y 

PROVIDER: S-EPMC4843470 | biostudies-literature | 2016 Apr

REPOSITORIES: biostudies-literature