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Abstract Synaptosomes are subcellular fractions prepared from brain tissues that are enriched in synaptic terminals, widely used for the study of neural transmission and synaptic dysfunction. Immunofluorescence imaging is increasingly applied to synaptosomes to investigate protein localization. However, conventional methods for imaging synaptosomes over glass coverslips suffer from formaldehyde-induced aggregation. Here, we developed a facile strategy to capture and image synaptosomes without aggregation artefacts. First, ethylene glycol bis(succinimidyl succinate) (EGS) is chosen as the chemical fixative to replace formaldehyde. EGS/glycine treatment makes the zeta potential of synaptosomes more negative. Second, we modified glass coverslips with 3-aminopropyltriethoxysilane (APTES) to impart positive charges. EGS-fixed synaptosomes spontaneously attach to modified glasses via electrostatic attraction while maintaining good dispersion. Individual synaptic terminals are imaged by conventional fluorescence microscopy or by super-resolution techniques such as direct stochastic optical reconstruction microscopy (dSTORM). We examined tau protein by two-color and three-color dSTORM to understand its spatial distribution within mouse cortical synapses, observing tau colocalization with synaptic vesicles as well postsynaptic densities.

Abstract Reactive oxygen species (ROS) have crucial roles in cell signaling and homeostasis. Overproduction of ROS can induce oxidative damage to various biomolecules and cellular structures. Therefore, developing an approach capable of monitoring and quantifying ROS in living cells is significant for physiology and clinical diagnoses. Some cell-permeable fluorogenic probes developed are useful for the detection of ROS while in conjunction with horseradish peroxidase (HRP). Their intracellular scenario is however hindered by the membrane-impermeable property of enzymes. Herein, a new approach for intracellular sensing of ROS by using horseradish peroxidase-encapsulated hollow silica nanospheres (designated HRP@HSNs), with satisfactory catalytic activity, cell membrane permeability, and biocompatibility, was prepared via a microemulsion method. These HRP@HSNs, combined with selective probes or targeting ligands, could be foreseen as ROS-detecting tools in specific organelles or cell types. As such, dihydrorhodamine 123-coupled HRP@HSNs were used for the qualitative and semi-quantitative analysis of physiological H2O2 levels in activated RAW 264.7 macrophages. We envision that this HSNs encapsulating active enzymes can be conjugated with selective probes and targeting ligands to detect ROS in specific organelles or cell types of interest.

Replication forks are vulnerable to wayward nuclease activities. We report here our discovery of a new member in guarding genome stability at replication forks. We previously isolated a Drosophila mutation, wuho (wh, no progeny), characterized by a severe fertility defect and affecting expression of a protein (WH) in a family of conserved proteins with multiple WD40 repeats. Knockdown of WH by siRNA in Drosophila, mouse, and human cultured cells results in DNA damage with strand breaks and apoptosis through ATM/Chk2/p53 signaling pathway. Mice with mWh knockout are early embryonic lethal and display DNA damage. We identify that the flap endonuclease 1 (FEN1) is one of the interacting proteins. Fluorescence microscopy showed the localization of WH at the site of nascent DNA synthesis along with other replication proteins, including FEN1 and PCNA. We show that WH is able to modulate FEN1's endonucleolytic activities depending on the substrate DNA structure. The stimulatory or inhibitory effects of WH on FEN1's flap versus gap endonuclease activities are consistent with the proposed WH's functions in protecting the integrity of replication fork. These results suggest that wh is a new member of the guardians of genome stability because it regulates FEN1's potential DNA cleavage threat near the site of replication.

Wavelength tunable temporal focusing multiphoton excitation microscopy (TFMPEM) is conducted to visualize optical sectioning images of multiple fluorophore-labeled specimens through the optimal two-photon excitation (TPE) of each type of fluorophore. The tunable range of excitation wavelength was determined by the groove density of the grating, the diffraction angle, the focal length of lenses, and the shifting distance of the first lens in the beam expander. Based on a consideration of the trade-off between the tunable-wavelength range and axial resolution of temporal focusing multiphoton excitation imaging, the presented system demonstrated a tunable-wavelength range from 770 to 920 nm using a diffraction grating with groove density of 830 lines/mm. TPE fluorescence imaging examination of a fluorescent thin film indicated that the width of the axial confined excitation was 3.0 ± 0.7 µm and the shifting distance of the temporal focal plane was less than 0.95 µm within the presented wavelength tunable range. Fast different wavelength excitation and three-dimensionally rendered imaging of Hela cell mitochondria and cytoskeletons and mouse muscle fibers were demonstrated. Significantly, the proposed system can improve the quality of two-color TFMPEM images through different excitation wavelengths to obtain higher-quality fluorescent signals in multiple-fluorophore measurements. [ABSTRACT FROM AUTHOR]

Intrinsic graphene possesses many outstanding physical and chemical properties, but their full explorations are often hindered by the effects of substrate and/or contamination. The authors employ the ultrahigh vacuum transmission electron microscopy equipped with a residual gas analyzer to in-situ characterize an effective decontamination process on a suspended graphene. Raman spectroscopic spectra further verify the cleanness of the resultant graphene membrane. The authors also present two contrasting growth morphologies of copper nanoparticles obtained on both clean and unclean graphene surfaces and show that the intrinsic growth dynamics can only manifest on the surface without contaminations. [ABSTRACT FROM AUTHOR]

The blinking phenomena of the quantum dots have been utilized in the super-resolution localization microscopy to map out the locations of individual quantum dots on a total internal reflection microscope. Our result indicated that the reconstructed image of quantum dots agreed with the topographic image measured by atomic force microscopy. Because of the superior optical properties of the quantum dots, the high localization resolution can be achieved in the shorter acquisition time with larger detected photon numbers. When the cells were labeled with quantum dots, the sub-cellular structures could be clearly seen in the reconstructed images taken by a commercial microscope without using complicated optical systems, special photo-switchable dye pairs or photo-activated fluorescence proteins. [ABSTRACT FROM AUTHOR]

In this study, the exciton lifetime images within the photoactive layers of poly(3-hexylthiophene) (P3HT) and [6,6)-phenyl-C61-butyric acid methyl ester (PCBM) are revealed by confocal optical microscopy combined with the fluorescence module. The images reveal that the active layers during slow solvent evaporation provide 3D pathways for charge transport and the concentration gradient through the film which reflects the better cell performance. This technique offers a great help to investigate the 3D optical-physical property without destroying the blends. [ABSTRACT FROM AUTHOR]

An energy upconversion system based on triplet-triplet annihilation exploiting an organic triplet sensitizer is devised and has achieved a white-light emission with a low power laser excitation. [ABSTRACT FROM AUTHOR]

Flexible polymer nanopillar substrates were used to systematically demonstrate cell alignment and migration guided by the directional formation of focal adhesions. The polymer nanopillar substrates were constructed to various height specifications to provide an extensive variation of flexibility; a rectangular arrangement created spatial confinement between adjacent nanopillars, providing less spacing in the horizontal and vertical directions. Three polymer nanopillar substrates with the diameter of 400 nm and the heights of 400, 800, and 1200 nm were fabricated. Super-resolution localization imaging and protein pair–distance analysis of vinculin proteins revealed that Chinese hamster ovary (CHO) cells formed mature focal adhesions on 1200 nm high nanopillar substrates by bending adjacent nanopillars to link dot-like adhesions. The spacing confinement of the adjacent nanopillars enhanced the orthogonal directionality of the formation tendency of the mature focal adhesions. The directional formation of the mature focal adhesions also facilitated the organization of actin filaments in the horizontal and vertical directions. Moreover, 78% of the CHO cells were aligned in these two directions, in conformity with the flexibility and nanotopographical cues of the nanopillars. Biased cell migration was observed on the 1200 nm high nanopillar substrates. [ABSTRACT FROM AUTHOR]

We report on red phosphorescent trilayer organic light-emitting field-effect transistors (LEFETs), which exhibit ambipolar transport characteristics but strong emission in the unipolar hole regime, with an exceptionally wide recombination zone of 60–70 µm and a maximum external quantum efficiency of 0.21%. From the results of detailed electroluminescence characterization, we clarify how the energy-level matching condition and transport geometry of the heterostructure govern the charge distribution and recombination, and affect the overall device performance. [ABSTRACT FROM AUTHOR]