High-speed super-resolution imaging of rotationally symmetric structures using SPEED microscopy and 2D-to-3D transformation

Various super-resolution imaging techniques have been developed to break the diffraction-limited resolution of light microscopy. However, it still remains challenging to obtain three-dimensional (3D) super-resolution information of structures and dynamic processes in live cells at high speed. We recently developed high-speed single-point edge-excitation sub-diffraction (SPEED) microscopy and its two-dimensional (2D)-to-3D transformation algorithm to provide an effective approach to achieving 3D sub-diffraction-limit information in subcellular structures and organelles that have rotational symmetry. In contrast to most other 3D super-resolution microscopy or 3D particle-tracking microscopy approaches, SPEED microscopy does not depend on complex optical components and can be implemented onto a standard inverted epifluorescence microscope. SPEED microscopy is specifically designed to obtain 2D spatial locations of individual immobile or moving fluorescent molecules inside sub-micrometer biological channels or cavities at high spatiotemporal resolution. After data collection, post-localization 2D-to-3D transformation is applied to obtain 3D super-resolution structural and dynamic information. The complete protocol, including cell culture and sample preparation (6–7 d), SPEED imaging (4–5 h), data analysis and validation through simulation (5–13 h), takes ~9 d to complete. This protocol describes how to implement high-speed single-point edge-excitation sub-diffraction (SPEED) microscopy in combination with 2D-to-3D transformation to obtain 3D sub-diffraction-limited information about rotational symmetric structures.