Yingke Xu

Time-gated stimulated emission depletion nanoscopy

Abstract. We have designed and built a time-gated continuous wave stimulated emission depletion (CW-STED) nanoscopy to visualize microstructures beyond the diffraction limit. An off-line time-gating detection was performed with the help of time-correlated single-photon counting technique. Experimental results showed that before time-gating, the resolution of our system was about 75 nm with a depletion beam (592 nm) power of 200 mW. By using the off-line time-gating detection, the resolution was further improved to 38 nm. Biological samples were also used to test the performance of our time-gated CW-STED, and a resolution of 70 nm was achieved with a depletion beam (592 nm) power of 85 mW. Detailed principles of time-gated CW-STED were discussed in the text. The time-gated STED provides a better resolution with finite laser power.

Real-time super-resolution imaging by high-speed fluorescence emission difference microscopy

The recently proposed fluorescence emission difference (FED) microscopy has been demonstrated to be capable of breaking the diffraction barrier, which restricts the spatial resolution of far-field fluorescence microscopy. In this paper, we report a novel high-speed FED system that can realize real-time super-resolution imaging. By replacing the conventional nanopositioning stage with a galvo mirror, the temporal resolution of FED is improved to nearly one frame per second, which is 100 times faster than that of best existing FED, while the system maintains a super-high spatial resolution of 150 nm, which is far beyond the diffraction barrier. Therefore, the high-speed FED is suitable for large-area observations while avoiding photobleaching. Detailed theoretical analysis, simulations, and experimental real-time resolution tests on 100 nm nanoparticles and biological cells are reported.

Virtual k -Space Modulation Optical Microscopy

We report a novel superresolution microscopy approach for imaging fluorescence samples. The reported approach, termed virtual k-space modulation optical microscopy (VIKMOM), is able to improve the lateral resolution by a factor of 2, reduce the background level, improve the optical sectioning effect and correct for unknown optical aberrations. In the acquisition process of VIKMOM, we used a scanning confocal microscope setup with a 2D detector array to capture sample information at each scanned x-y position. In the recovery process of VIKMOM, we first modulated the captured data by virtual k-space coding and then employed a ptychography-inspired procedure to recover the sample information and correct for unknown optical aberrations. We demonstrated the performance of the reported approach by imaging fluorescent beads, fixed bovine pulmonary artery endothelial (BPAE) cells, and living human astrocytes (HA). As the VIKMOM approach is fully compatible with conventional confocal microscope setups, it may provide a turn-key solution for imaging biological samples with ∼100  nm lateral resolution, in two or three dimensions, with improved optical sectioning capabilities and aberration correcting.

Systemic lupus erythematosus associated with moyamoya syndrome: a case report and literature review

Moyamoya syndrome (MMS) is a rare, chronic progressive cerebrovascular occlusive disease that is characterized by a stenosis or occlusion of the bilateral internal carotid arteries and the circle of Willis arteries leading to the development of collateral vessels as visualized by cerebral angiography. We report a case of a 24-year-old woman with nephrotic syndrome whose biopsy showed membranous nephropathy. Ten months after the diagnosis she suffered sudden right hemiplegia and seizure. She was diagnosed with MMS by angiogram seven months ago and received decompressive craniotomy. The patient was admitted to our hospital and a diagnosis of systemic lupus erythematosus (SLE) was made. Glucocorticoids and tacrolimus were used to control the symptoms of SLE. Following one month of immunosuppressant treatment, the patient died of brain hemorrhage. This case alongside another six reviewed cases shows that an underlying cerebrovascular lesion of moyamoya in the vessels of patients with SLE is susceptible to cerebrovascular accidents.

A 3D aligning method for stimulated emission depletion microscopy using fluorescence lifetime distribution.

Optimal resolution by stimulated emission depletion (STED) microscopy requires precise alignment of the donut‐shaped depletion focus to the excitation focus. In this article, we demonstrate that fluorescence lifetime distribution can be implemented to align the STED system. Different from the traditional aligning methods in which a scattering imaging module is often equipped, the lifetime‐based method is free from probable mismatches between the scattering mode and the fluorescent mode, drift errors caused by separate imaging and complex fitting methods. Based on this method, a spatial resolution of 38 nm by time‐gated detection has been achieved. Microsc. Res. Tech. 77:935–940, 2014.

Rab8A regulates insulin-stimulated GLUT4 translocation in C2C12 myoblasts

Rab proteins are important regulators of GLUT4 trafficking in muscle and adipose cells. It is still unclear which Rabs are involved in insulin‐stimulated GLUT4 translocation in C2C12 myoblasts. In this study, we detect the colocalization of Rab8A with GLUT4 and the presence of Rab8A at vesicle exocytic sites by TIRFM imaging. Overexpression of dominant‐negative Rab8A (T22N) diminishes insulin‐stimulated GLUT4 translocation, while constitutively active Rab8A (Q67L) augments it. In addition, knockdown of Rab8A inhibits insulin‐stimulated GLUT4 translocation, which is rescued by replenishment of RNAi‐resistant Rab8A. Together, these results indicate an indispensable role for Rab8A in insulin‐regulated GLUT4 trafficking in C2C12 cells.

Phase microscopy using light-field reconstruction method for cell observation

The refractive index (RI) distribution can serve as a natural label for undyed cell imaging. However, the majority of images obtained through quantitative phase microscopy is integrated along the illumination angle and cannot reflect additional information about the refractive map on a certain plane. Herein, a light-field reconstruction method to image the RI map within a depth of 0.2 μm is proposed. It records quantitative phase-delay images using a four-step phase shifting method in different directions and then reconstructs a similar scattered light field for the refractive sample on the focus plane. It can image the RI of samples, transparent cell samples in particular, in a manner similar to the observation of scattering characteristics. The light-field reconstruction method is therefore a powerful tool for use in cytobiology studies.

Differential requirement for N‐ethylmaleimide‐sensitive factor in endosomal trafficking of transferrin receptor from anterograde trafficking of vesicular stomatitis virus glycoprotein G

N‐ethylmaleimide‐sensitive fusion factor (NSF) is an ATPase that plays a crucial role in vesicular transport. Here, we examined the effects of NSF knockdown on Golgi structure and different vesicle trafficking pathways in mammalian cells. NSF knockdown caused Golgi fragmentation and abolished transferrin receptor exocytosis, defects that were rescued by RNAi‐resistant NSF. Strikingly, NSF deficiency in HeLa cells barely affected cell viability, anterograde trafficking of vesicular stomatitis virus glycoprotein G and transferrin endocytosis. These results confirm the central role of NSF in Golgi structure and reveal differential requirement of NSF for exocytic recycling and constitutive trafficking pathways.

Spatiotemporal Regulators for Insulin-Stimulated GLUT4 Vesicle Exocytosis

Insulin increases glucose uptake and storage in muscle and adipose cells, which is accomplished through the mobilization of intracellular GLUT4 storage vesicles (GSVs) to the cell surface upon stimulation. Importantly, the dysfunction of insulin-regulated GLUT4 trafficking is strongly linked with peripheral insulin resistance and type 2 diabetes in human. The insulin signaling pathway, key signaling molecules involved, and precise trafficking itinerary of GSVs are largely identified. Understanding the interaction between insulin signaling molecules and key regulatory proteins that are involved in spatiotemporal regulation of GLUT4 vesicle exocytosis is of great importance to explain the pathogenesis of diabetes and may provide new potential therapeutic targets.

Sub-diffraction imaging with total internal reflection fluoresence (TIRF) microscopy by stochastic photobleaching

Total internal reflection fluorescence (TIRF) microscopy is widely used in fluorescent imaging. Evanescent wave fields generated by the internal reflection are used to illuminate the sample, and only fluorophores within a thickness of 100 nm thick from the surface are activated, improving the signal-to-noise ratio (SNR) of the image. Sub-diffraction imaging with TIRF microscopy by stochastic photobleaching is studied. Each fluorophore can be localized from the recorded streaming followed by image subtraction. This method can yield contrast-enhanced images with a higher SNR and improve the lateral resolution to approximately 120 nm.