Technologies Enabling Single-Molecule Super-Resolution Imaging of mRNA.

The transient nature of RNA has rendered it one of the more difficult biological targets for imaging. This difficulty stems both from the physical properties of RNA as well as the temporal constraints associated therewith. These concerns are further complicated by the difficulty in imaging endogenous RNA within a cell that has been transfected with a target sequence. These concerns, combined with traditional concerns associated with super-resolution light microscopy has made the imaging of this critical target difficult. Recent advances have provided researchers the tools to image endogenous RNA in live cells at both the cellular and single-molecule level. Here, we review techniques used for labeling and imaging RNA with special emphases on various labeling methods and a virtual 3D super-resolution imaging technique.

Speed Microscopy: High-Speed Single Molecule Tracking and Mapping of Nucleocytoplasmic Transport.

The nuclear pore complex (NPC) functions as a gateway through which molecules translocate into and out of the nucleus. Understanding the transport dynamics of these transiting molecules and how they interact with the NPC has great potentials in the discovery of clinical targets. Single-molecule microscopy techniques are powerful tools to provide sub-diffraction limit information about the dynamic and structural details of nucleocytoplasmic transport. Here we detail single-point edge-excitation subdiffraction (SPEED) microscopy, a high-speed superresolution microscopy technique designed to track and map proteins and RNAs as they cross native NPCs.

Three-Dimensional Mapping of mRNA Export through the Nuclear Pore Complex.

The locations of transcription and translation of mRNA in eukaryotic cells are spatially separated by the nuclear envelope (NE). Plenty of nuclear pore complexes (NPCs) embedded in the NE function as the major gateway for the export of transcribed mRNAs from the nucleus to the cytoplasm. Whereas the NPC, perhaps one of the largest protein complexes, provides a relatively large channel for macromolecules to selectively pass through it in inherently three-dimensional (3D) movements, this channel is nonetheless below the diffraction limit of conventional light microscopy. A full understanding of the mRNA export mechanism urgently requires real-time mapping of the 3D dynamics of mRNA in the NPC of live cells with innovative imaging techniques breaking the diffraction limit of conventional light microscopy. Recently, super-resolution fluorescence microscopy and single-particle tracking (SPT) techniques have been applied to the study of nuclear export of mRNA in live cells. In this review, we emphasize the necessity of 3D mapping techniques in the study of mRNA export, briefly summarize the feasibility of current 3D imaging approaches, and highlight the new features of mRNA nuclear export elucidated with a newly developed 3D imaging approach combining SPT-based super-resolution imaging and 2D-to-3D deconvolution algorithms.