Protocol for live-cell super-resolution imaging of transport of pre-ribosomal subunits through the nuclear pore complex.

Here, we present a protocol for single-molecule super-resolution imaging of the nuclear export of pre-ribosomal subunits pre-40S and pre-60S through nuclear pore complexes. We describe steps for plating cells and co-transfecting cells. We then detail steps for using single-point edge-excitation sub-diffraction microscopy, allowing visualization of real-time dynamics of the pre-ribosomal subunits.

Unveiling the complexity: assessing models describing the structure and function of the nuclear pore complex.

The nuclear pore complex (NPC) serves as a pivotal subcellular structure, acting as a gateway that orchestrates nucleocytoplasmic transport through a selectively permeable barrier. Nucleoporins (Nups), particularly those containing phenylalanine-glycine (FG) motifs, play indispensable roles within this barrier. Recent advancements in technology have significantly deepened our understanding of the NPC's architecture and operational intricacies, owing to comprehensive investigations.

Application of Super-resolution SPEED Microscopy in the Study of Cellular Dynamics.

Super-resolution imaging techniques have broken the diffraction-limited resolution of light microscopy. However, acquiring three-dimensional (3D) super-resolution information about structures and dynamic processes in live cells at high speed remains challenging. Recently, the development of high-speed single-point edge-excitation subdiffraction (SPEED) microscopy, along with its 2D-to-3D transformation algorithm, provides a practical and effective approach to achieving 3D subdiffraction-limit information in subcellular structures and organelles with rotational symmetry.

Obtaining 3D super-resolution images by utilizing rotationally symmetric structures and 2D-to-3D transformation.

Super-resolution imaging techniques have provided unprecedentedly detailed information by surpassing the diffraction-limited resolution of light microscopy. However, in order to derive high quality spatial resolution, many of these techniques require high laser power, extended imaging time, dedicated sample preparation, or some combination of the three. These constraints are particularly evident when considering three-dimensional (3D) super-resolution imaging.