Simultaneous Two-Angle Axial Ratiometry for Fast Live and Long-Term Three-Dimensional Super-Resolution Fluorescence Imaging

Abstract

The application of optical microscopy in four-dimensional (spatial and temporal) super-resolution imaging poses challenges because of the requirement of a long acquisition time or high illumination intensity. In this paper, we introduce simultaneous two-angle axial ratiometry (STARII) for <20 nm axial super-resolution imaging and for fast and long-term imaging of live cells up to hundreds of frames per second. This method involves recording two raw images in two incident angle channels in the context of evanescent wave illumination and obtaining the corresponding intensity ratio. Furthermore, we demonstrate the combination of STARII with the lateral superresolution method to resolve three-dimensional nanoscale structures of microtubules and to visualize the long-term dynamical plasma membrane curvature and fast remodeling of endoplasmic reticulum tubule meshwork and three-way junctions. These demonstrations indicate an important potential application of STARII in investigating nanoscale cellular complex processes in the native state.

Virtual Issue on Super-Resolution Far-Field Optical Microscopy

“…Work in this area includes the development of new algorithms, 23 as well as novel experimental techniques such as combining Total Internal Reflection ("TIRF") microscopy with structured illumination. 24 Another research focus for super-resolution microscopy is the study of emitters interacting with plasmonic nanostructures. Super-resolution fluorescence imaging of surface attached dye molecules has been used to determine the size and shape of gold nanostructures.…”

“…Measurements at cryogenic temperatures have also been reported, demonstrating that high spatial resolution can be achieved at low temperatures. , A significant challenge in super-resolution experiments is to generate 3D structures from the images. Work in this area includes the development of new algorithms, as well as novel experimental techniques such as combining Total Internal Reflection (“TIRF”) microscopy with structured illumination …”