Axial superresolution via multiangle TIRF microscopy with sequential imaging and photobleaching

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.

AXIAL SUPERRESOLUTION BY PHASE FILTER IN OPTICAL COHERENCE TOMOGRAPHY

Axial superresolution in optical coherence tomography (OCT) by a three-zone annular phase filter is demonstrated. In the proposed probe of a spectral domain OCT system, the width of the central lobe of the axial intensity point spread function is apodized by the filter to be within the coherence gate determined by the light source, while its sidelobes are lying outside the coherence gate without contributing to the coherence imaging. By measurement of the depth response of the OCT system before and after inserting the filter, an improvement of about 20% in axial resolution is confirmed. OCT imaging on biological sample of orange fresh is also conducted, demonstrating increased depth discrimination without the negative contribution from sidelobes realized by the phase filter in combination with the coherence gate intrinsic to OCT. It comes to a conclusion that we can obtain axial superresolution by filter in OCT system without the degrading influence of large sidelobes.