Single-point Edge-excitation Sub-diffraction Research Articles

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. One of the major benefits of SPEED microscopy is that it does not rely on complex optical components and can be implemented on a standard, inverted epifluorescence microscope, simplifying the process of sample preparation and the expertise requirement. SPEED microscopy is specifically designed to obtain 2D spatial locations of individual immobile or moving fluorescent molecules inside submicrometer biological channels or cavities at high spatiotemporal resolution. The collected data are then subjected to postlocalization 2D-to-3D transformation to obtain 3D super-resolution structural and dynamic information. In recent years, SPEED microscopy has provided significant insights into nucleocytoplasmic transport across the nuclear pore complex (NPC) and cytoplasm-cilium trafficking through the ciliary transition zone. This Review focuses on the applications of SPEED microscopy in studying the structure and function of nuclear pores.

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. As a result, high-speed capture of 3D super-resolution information of structures and dynamic processes within live cells remains both desirable and challenging. Recently, a highly effective approach to obtain 3D super-resolution information was developed that can be employed in commonly available laboratory microscopes. This development makes it both scientifically possible and financially feasible to obtain super-resolution 3D information under certain conditions. This is accomplished by converting 2D single-molecule localization data captured at high speed within subcellular structures and rotationally symmetric organelles. Here, a high-speed 2D single-molecule tracking and post-localization technique, known as single-point edge-excitation sub-diffraction (SPEED) microcopy, along with its 2D-to-3D transformation algorithm is detailed with special emphasis on the mathematical principles and Monte Carlo simulation validation of the 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.

Nuclear Import of Adeno-Associated Viruses Imaged by High-Speed Single-Molecule Microscopy.

Understanding the detailed nuclear import kinetics of adeno-associated virus (AAV) through the nuclear pore complex (NPC) is essential for the application of AAV capsids as a nuclear delivery instrument as well as a target for drug development. However, a comprehensive understanding of AAV transport through the sub-micrometer NPCs in live cells calls for new techniques that can conquer the limitations of conventional fluorescence microscopy and electron microscopy. With recent technical advances in single-molecule fluorescence microscopy, we are now able to image the entire nuclear import process of AAV particles and also quantify the transport dynamics of viral particles through the NPCs in live human cells. In this review, we initially evaluate the necessity of single-molecule live-cell microscopy in the study of nuclear import for AAV particles. Then, we detail the application of high-speed single-point edge-excitation sub-diffraction (SPEED) microscopy in tracking the entire process of nuclear import for AAV particles. Finally, we summarize the major findings for AAV nuclear import by using SPEED microscopy.

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.

Nuclear export of mRNA molecules studied by SPEED microscopy.

The nuclear exit of messenger RNA (mRNA) molecules through the nuclear pore complex (NPC) is an essential step in the translation process of all proteins. The current limitations of conventional fluorescence and electron microscopy have prevented elucidation of how mRNA exports through the NPCs of live cells. In the recent years, various single-molecule fluorescence (SMF) microscopy techniques have been developed to improve the temporal and spatial resolutions of live-cell imaging allowing a more comprehensive understanding of the dynamics of mRNA export through native NPCs. In this review, we firstly evaluate the necessity of single-molecule live-cell microscopy in the study of mRNA nuclear export. Then, we highlight the application of single-point edge-excitation sub-diffraction (SPEED) microscopy that combines high-speed SMF microscopy and a 2D-to-3D transformation algorithm in the studies of nuclear transport kinetics and route for mRNAs. Finally, we summarize the new features of mRNA nuclear export found with SPEED microscopy as well as the reliability and accuracy of SPEED microscopy in mapping the 3D spatial locations of transport routes adopted by proteins and mRNAs through the NPCs.

Application of High-speed Super-resolution SPEED Microscopy in Live Primary Cilium

The primary cilium is a microtubule-based protrusion on the surface of many eukaryotic cells and contains a unique complement of proteins that function critically in cell motility and signaling. Since cilia are incapable of synthesizing their own protein, nearly 200 unique ciliary proteins need to be trafficked between the cytosol and primary cilia. However, it is still a technical challenge to map three-dimensional (3D) locations of transport pathways for these proteins in live primary cilia due to the limitations of currently existing techniques. To conquer the challenge, recently we have developed and employed a high-speed virtual 3D super-resolution microscopy, termed single-point edge-excitation sub-diffraction (SPEED) microscopy, to determine the 3D spatial location of transport pathways for both cytosolic and membrane proteins in primary cilia of live cells. In this article, we will demonstrate the detailed setup of SPEED microscopy, the preparation of cells expressing fluorescence-protein-labeled ciliary proteins, the real-time single-molecule tracking of individual proteins in live cilium and the achievement of 3D spatial probability density maps of transport routes for ciliary proteins.

Application of High-speed Super-resolution SPEED Microscopy in Live Primary Cilium.

The primary cilium is a microtubule-based protrusion on the surface of many eukaryotic cells and contains a unique complement of proteins that function critically in cell motility and signaling. Since cilia are incapable of synthesizing their own protein, nearly 200 unique ciliary proteins need to be trafficked between the cytosol and primary cilia. However, it is still a technical challenge to map three-dimensional (3D) locations of transport pathways for these proteins in live primary cilia due to the limitations of currently existing techniques. To conquer the challenge, recently we have developed and employed a high-speed virtual 3D super-resolution microscopy, termed single-point edge-excitation sub-diffraction (SPEED) microscopy, to determine the 3D spatial location of transport pathways for both cytosolic and membrane proteins in primary cilia of live cells. In this article, we will demonstrate the detailed setup of SPEED microscopy, the preparation of cells expressing fluorescence-protein-labeled ciliary proteins, the real-time single-molecule tracking of individual proteins in live cilium and the achievement of 3D spatial probability density maps of transport routes for ciliary proteins.

SPEED Microscopy and Its Application in Nucleocytoplasmic Transport.

In eukaryotic cells, the nuclear pore complexes (NPCs) selectively mediate the bidirectional trafficking of macromolecules between the cytoplasm and the nucleus. The selective barrier is formed by intrinsically disordered phenylalanine-glycine (FG) nucleoporins anchored on the wall of the submicrometer NPC, which allows for passive diffusion and facilitated translocation through the nuclear pore. Dysfunction of nucleocytoplasmic transport has been associated with many human diseases. However, due to the technical challenge of imaging the native tomography of the FG-nucleoporin barrier and its interactions with transiting molecules in the native NPC, the precise nucleocytoplasmic transport mechanism remains unresolved. To refine the transport mechanism, single-molecule fluorescence microscopy methods have been employed to obtain the transport kinetics and the spatial transport route of individual fluorescent molecules through the NPC. In this method paper, we particularly highlight a newly developed high-speed super-resolution three-dimensional microscopy approach, termed as SPEED (single-point edge-excitation subdiffraction) microscopy, and its application in characterizing nucleocytoplasmic transport.

Super-resolution imaging of nuclear import of adeno-associated virus in live cells

Adeno-associated virus (AAV) has been developed as a promising human gene therapy vector. Particularly, recombinant AAV vector (rAAV) achieves its transduction of host cells by crossing at least three physiological barriers including plasma membrane, endosomal membrane, and nuclear envelope (NE). So far, the AAV transduction mechanism has not been explored thoroughly at the single viral particle level. In this study, we employed high-speed super-resolution single-point edge-excitation sub-diffraction (SPEED) microscopy to map the events of single rAAV2 particles infecting live human cells with an unprecedented spatiotemporal resolution of 9–12 nm and 2–20 ms. Data reveal that rAAV2 particles are imported through nuclear pore complexes (NPCs) rather than nuclear membrane budding into the nucleus. Moreover, approximately 17% of the rAAV2 molecules starting from the cytoplasm successfully transverse the NPCs to reach the nucleoplasm, revealing that the NPCs act as a strict selective step for AAV delivery. This study lastly suggests a new pathway to improve AAV vectors for human gene therapy.

Characterization of the Selective Barrier in Nuclear Pore Complex

The nuclear pore complexes (NPCs) selectively mediate the bidirectional trafficking of macromolecules between the cytoplasm and nucleus in eukaryotic cells. The selective barrier formed by natively unfolded phenylalanine-glycine (FG) nucleoporins (Nups) inside the NPC allows for passive and facilitated transport through the NPC. However, the mechanism of formation and spatial distribution of FG-barrier in the NPC remains unresolved. By a newly developed single-molecule microscopy, single-point edge-excitation sub-diffraction (SPEED) microscopy, we have used various fluorescent transport receptors and FG segments, as probes to determine the structure of FG-Nup barrier in the native NPC with a spatiotemporal resolution of 9 nm and 400 μs. The interactions among FG-filaments enhance a donut-like structure formed at the central of NPC, acting as the primary selective gate for nuclear transport. On either side of the NPC, additionally FG Nups may function as the secondary selectivity for incoming molecules. Finally, the conformations of both selective barriers can be further regulated by major transport receptors.

Charge Effects on Nucleocytoplamsic Transport Studied by Single-Molecule Microscopy

In eukaryotic cells, the transport of genetic material and proteins between nucleus and cytoplasm is mediated by the nuclear pore complexes (NPCs) embedded in the double-membrane nuclear envelope. A highly selective barrier formed by phenylalanine-glycine (FG)-nucleoporin (Nup) with net positive charge in the NPC allows for passive diffusion of signal-independent small molecules (< 40 kDa) and transportin-facilitated translocation of signal-dependent large molecules (up to 50 MDa). Previously it was suggested that the positively charged FG-barrier would inhibit the transport of positively charged transiting molecules. However, the fundamental inhibition mechanism and the detailed pathways for both transport modes remain poorly understood. Here, we employ an innovative single-molecule technique, single-point edge-excitation subdiffraction (SPEED) microscopy, to track fluorescent single molecules with different charges transiting through single native NPCs. We have obtained the transport kinetics and the 3D transport pathways for both passive diffusion of variously charged GFPs (27 kDa) and facilitated translocation of transportin-cargo complexes with different charges. Our results indicate that (i): the positively charged selective barrier in the NPC possesses an axial central conduit for passive diffusion of small molecules with different charges, and allows for transportin-facilitated translocation of differently charged cargos via the peripheral regions around the central conduit; and (ii) the positively charged environment in the NPC significantly affects the transport pathway, transport time and efficiency for both passive and facilitated transport.

Three-Dimensional Characterization of the Selective Barrier formed by Unfolded Proteins in the Nuclear Pore Complex

The nuclear pore complexes (NPCs) mediate the bidirectional trafficking of macromolecules between the cytoplasm and the nucleus in eukaryotic cells. The selective barrier formed by natively unfolded phenylalanine-glycine (FG) nucleoporins (Nups) inside the NPC allows for passive and facilitated transport through the NPC. However, the mechanism of formation and spatial distribution of FG-barrier in the NPC remains unresolved. By a newly developed single-molecule microscopy, single-point edge-excitation sub-diffraction (SPEED) microscopy, we have used various fluorescent transport receptors as probes to determine formation and spatial distribution of individual FG-Nups in the native NPC with a spatiotemporal resolution of 9-10 nm and 400 μs. We found that Nup62 complex form a donut-like structure at the central scaffold of NPC and act as the primary selective gate for large single-independent cargoes. On either side of the central barrier, Nup98 and other FG-Nups could function as the secondary selective barrier for incoming molecules. Finally, the conformation of the central selective barriers can be significantly regulated by a major transport receptor Importin β1, but not by the other transport receptors including Importin β2, Crm1, NTF2 and CAS.

Three-dimensional distribution of transient interactions in the nuclear pore complex obtained from single-molecule snapshots

The translocation of large macromolecules through the nuclear pore complex (NPC) of eukaryotic cells is hindered by the phenylalanine-glycine (FG) nucleoporin (Nup) barrier unless molecules are chaperoned by transport receptors. The precise mechanism of facilitated translocation remains unclear due to the challenges of measuring the series of transient interactions between a transport receptor and the FG-Nups. This study developed single-point edge-excitation subdiffraction microscopy to obtain a three-dimensional density map of the transient interactions with a spatiotemporal resolution of 9 nm and 400 mus. Three unique features were observed under real-time trafficking conditions that have escaped detection by conventional electron microscopy: (i) the spatial density of interaction sites between Importin beta1 (Imp beta1, a major transport receptor) and the FG-Nups gradually increases from both sides of the NPC and is highest in the central pore region; (ii) cargo-free or cargo-bound Imp beta1 rarely occupies an axial channel with a diameter of approximately 10-20 nm at its narrowest point through the NPC; and (iii) the pathway of facilitated translocation through the NPC depends more on the interaction sites of the FG-Nups than on the NPC architecture.

Single-Molecule Snapshots of Three-Dimensional Distribution of Transient Interactions in the Nuclear Pore Complex

Translocation of macromolecules through the nuclear pore complex (NPC) is hindered by the phenylalanine-glycine (FG) repeats barrier unless they are chaperoned by transport receptors in eukaryotic cells. However, challenged by measuring a series of transient interactions between the transport receptor and the FG repeats, the precise mechanism of the facilitated translocation remains unclear. We applied single-point edge-excitation sub-diffraction (SPEED) microscopy to obtain a three-dimensional density map of the transient interactions with a spatiotemporal resolution of 9 nm and 400 μs. We observed novel features under real-time trafficking conditions that escape detection by conventional electron microscopy: the actual pathway of facilitated translocation through the NPC is not completely restricted by the NPC architecture; the primary interaction sites between Importin β1 (Imp β1, a major transport receptor) and the FG repeats locate symmetrically on the cytoplasmic and nucleoplasmic sides of the nuclear pore; Imp β1 only rarely occupies a central channel of approximate 10-20 nm diameter along the NPC axis, but the Imp β1-assisted cargo molecules expand their pathways into the central channel.