Two-photon excitation improves multifocal structured illumination microscopy in thick scattering tissue

Multifocal structured illumination microscopy (MSIM) provides a twofold resolution enhancement beyond the diffraction limit at sample depths up to 50 µm, but scattered and out-of-focus light in thick samples degrades MSIM performance. Here we implement MSIM with a microlens array to enable efficient two-photon excitation. Two-photon MSIM gives resolution-doubled images with better sectioning and contrast in thick scattering samples such as Caenorhabditis elegans embryos, Drosophila melanogaster larval salivary glands, and mouse liver tissue.Fluorescence microscopy is an invaluable tool for biologists. Protein distributions in cells have an interesting structure down to the nanometer scale, but features smaller than 200–300 nm are blurred by diffraction in widefield and confocal fluorescence microscopes. Superresolution techniques like photoactivated localization microscopy (1), stochastic optical reconstruction microscopy (2), or stimulated emission depletion (STED) (3) microscopy allow the imaging of details beyond the limit imposed by diffraction, but usually trade acquisition speed or straightforward sample preparation. And although STED can provide resolution down to 40 nm, STED-specific fluorophores are recommended and it often requires light intensities that are orders of magnitude above widefield and confocal microscopy. On the other hand, structured illumination microscopy (SIM) (4) gives twice the resolution of a conventional fluorescence microscope with light intensities on the order of widefield microscopes and can be used with most common fluorophores. SIM uses contributions from both the excitation and emission point spread functions (PSFs) to substantially improve the transverse resolution and is generally performed by illuminating the sample with a set of sharp light patterns and collecting fluorescence on a multipixel detector, followed by image processing to recover superresolution detail from the interaction of the light pattern with the sample. A related technique, image scanning microscopy (ISM), uses a scanned diffraction-limited spot as the light pattern (5, 6). Multifocal SIM (MSIM) parallelizes ISM by using many excitation spots (7), and has been shown to produce optically sectioned images with ∼145-nm lateral and ∼400-nm axial resolution at depths up to ∼50 µm and at ∼1 Hz imaging frequency. In MSIM, images are excited with a multifocal excitation pattern, and the resulting fluorescence in the multiple foci are pinholed, locally scaled, and summed to generate an image [multifocal-excited, pinholed, scaled, and summed (MPSS)] with root 2-improved resolution relative to widefield microscopy, and improved sectioning compared with SIM due to confocal-like pinholing. Deconvolution is applied to recover the final MSIM image which has a full factor of 2 resolution improvement over the diffraction limit.MSIM works well in highly transparent samples (such as zebrafish embryos), but performance degrades in light scattering samples (such as the Caenorhabditis elegans embryo). Imaging in scattering samples can be improved by two-photon microscopy (8) and although the longer excitation wavelength reduces the resolution in nondescanned detection configurations, this can be partially offset by descanned detection and the addition of a confocal pinhole into the emission path. Whereas the nondescanned mode collects the most signal, the addition of a pinhole in the emission path of a point-scanning system can improve resolution when the pinhole is closed (9). In practice this is seldom done for biological specimens because signal-to-noise decays as the pinhole diameter decreases (9–11).SIM is an obvious choice in improving resolution without a dramatic loss in signal-to-noise, but the high photon density needed for efficient two-photon excitation is likely difficult to achieve in the typical widefield SIM configuration. This has led to other methods, such as line scanning (12) to achieve better depth penetration than confocal microscopy and up to twofold improvements in axial resolution (but with only ∼20% gain in lateral resolution). Multiphoton Bessel plane illumination (13) achieved an anisotropic lateral resolution of 180 nm (only in one direction) but requires an instrument design with two objectives in an orthogonal configuration. Cells and embryos can be readily imaged, but the multiaxis design may hinder the intravital imaging of larger specimens. Here, a combination of multiphoton excitation with MSIM is shown to improve both lateral and axial resolutions twofold compared with conventional multiphoton imaging while improving the sectioning and contrast of MSIM in thick, scattering samples.     

Inkjet assisted fabrication of planar biocompatible memristors

In this study we address a novel design of a planar memristor and investigate its biocompatibility. An experimental prototype of the proposed memristor assembly has been manufactured using a hybrid nanofabrication method, combining sputtering of electrodes, patterning the insulating trenches, and filling them with a memristive substance. To pattern the insulating trenches, we have examined two nanofabrication techniques employing either a focused ion beam or a cantilever tip of an atomic force microscope. Inkjet printing has been used to fill the trenches with the functional titania ink. The experimental prototypes have qualitatively demonstrated memristive current–voltage behavior, as well as high biocompatibility.

A planar memristor was fabricated by a hybrid method combining AFM patterning and inkjet printing.     

Synthesis and study of C-substituted methylthio derivatives of cobalt bis(dicarbollide)

The C-methylthio derivatives of cobalt bis(dicarbollide) were synthesized by reaction of anhydrous CoCl₂ with nido-carborane [7-MeS-7,8-C₂B₉H₁₁]⁻ and isolated as a mixture of rac-[1,1′-(MeS)₂-3,3′-Co(1,2-C₂B₉H₁₀)₂]⁻ and meso-[1,2′-(MeS)₂-3,3′-Co(1,2-C₂B₉H₁₀)₂]⁻ isomers. The structures of both isomers were studied using DFT quantum chemical calculations. The most preferable geometry of rotamers and the stabilization energy of C-methylthio derivatives of cobalt bis(dicarbolide) were calculated. The (BEDT-TTF)[1,1′-(MeS)₂-3,3′-Co(1,2-C₂B₉H₁₀)₂] salt was prepared and its structure was determined by single crystal X-ray diffraction. The cisoid conformation of the rac-[1,1′-(MeS)₂-3,3′-Co(1,2-C₂B₉H₁₀)₂]⁻ anion is stabilized by short intramolecular CH⋯S hydrogen and BH⋯S chalcogen bonds between the dicarbollide ligands, that is in good agreement with the data of quantum chemical calculations.

The C-methylthio derivatives of cobalt bis(dicarbollide) rac-[1,1′-(MeS)₂-3,3′-Co(1,2-C₂B₉H₁₀)₂]⁻ and meso-[1,2′-(MeS)₂-3,3′-Co(1,2-C₂B₉H₁₀)₂]⁻ were synthesized and studied by DFT calculations and X-ray diffraction.     

Qualitative and quantitative analysis of glycine- and GABA-immunoreactive nerve terminals on motoneuron cell bodies in the cat spinal cord: A postembedding electron microscopic study

The distribution of glycine- and gamma-aminobutyric acid (GABA)-like immunoreactivity (LI) in nerve terminals on the cell soma of motoneurons in the aldehyde-fixed cat L7 spinal cord was examined using postembedding immunogold histochemistry in serial ultrathin sections. Quantitative examination of 405 terminals on eight neurons of α-motoneuron size in the L7 motor nuclei from one animal was performed. A majority of the terminals (69%) were immunoreactive to glycine and/or GABA. These terminals contained flat or oval synaptic vesicles, thus classifying them as F type or as C type in one case. In no case was a type-F terminal unlabeled for both glycine and GABA. Most of the immunolabeled terminals were immunoreactive to glycine only (62.5%), whereas 35.4% contained both glycine- and GABA-LI. A very small number of immunolabeled terminals (2%) were immunoreactive to GABA only. In those terminals, where glycine- and GABA-LI coexisted, the gold particle density for each amino acid was only half of that seen in boutons containing only one of the two amino acids. The involvement of glycine and GABA in postsynaptic inhibition of spinal α-motoneurons is discussed, with particular reference to the possibility that these two inhibitory amino acids may be coreleased from a significant proportion of the nerve terminals impinging on the cell bodies. © 1996 Wiley-Liss, Inc.     

Acid-Sensing Ion Channels: Focus on Physiological and Some Pathological Roles in the Brain

Acid-sensing ion channels (ASICs) are Na⁺-permeable ion channels activated by protons and predominantly expressed in the nervous system. ASICs act as pH sensors leading to neuronal excitation. At least eight different ASIC subunits (including ASIC1a, ASIC1b, ASIC2a, ASIC2b, ASIC3, ASIC4, ASIC5) are encoded by five genes (ASIC1-ASIC5). Functional ASICs assembled in the plasma membrane are homo- or heteromeric trimers. ASIC1a-containing trimers are of particular interest as, in addition to sodium ions, they also conduct calcium ions and thus can trigger or regulate multiple cellular processes. ASICs are widely but differentially expressed in the central and peripheral nervous systems. In the mammalian brain, a majority of neurons express at least one ASIC subunit. Several recent reviews have summarized findings of the role of ASICs in the peripheral nervous system, particularly in nociception and proprioception, and the structure-function relationship of ASICs. However, there is little coverage on recent findings regarding the role of ASICs in the brain. Here we review and discuss evidence regarding the roles of ASICs: (i) as postsynaptic receptors activated by protons co-released with glutamate at glutamatergic synapses; (ii) as modulators of synaptic transmission at glutamatergic synapses and GABAergic synapses; (iii) in synaptic plasticity, memory and learning; (iv) in some pathologies such as epilepsy, mood disorders and Alzheimer''s disease.     

Degradation kinetic study of ZIF-8 microcrystals with and without the presence of lactic acid

The zeolitic imidazolate framework ZIF-8 (Zn(mim)₂, mim = 2-methylimidazolate) has recently been proposed as a drug delivery platform for anticancer therapy based on its capability of decomposing in acidic media. The concept presumes a targeted release of encapsulated drug molecules in the vicinity of tumor tissues that typically produce secretions with elevated acidity. Due to challenges of in vivo and in vitro examination, many studies have addressed the kinetics of ZIF-8 decomposition and subsequent drug release in phosphate buffered saline (PBS) with adjusted acidity. However, the presence of hydrogen phosphate anions [HPO₄]²⁻ in PBS may also affect the stability of ZIF-8. As yet, no separate analysis has been performed comparing the dissolving capabilities of PBS and various acidification agents used for regulating pH. Here, we provide a systematic study addressing the effects of phosphate anions with and without lactic acid on the degradation rate of ZIF-8 microcrystals. Lactic acid has been chosen as an experimental acidification agent, since it is particularly secreted by tumor cells. Interestingly, the effect of a lactic acid solution with pH 5.0 on ZIF-8 degradation is shown to be weaker compared to a PBS solution with pH 7.4. However, as an additive, lactic acid is able to enhance the decomposition efficacy of other solutions by 10 to 40 percent at the initial stage, depending on the presence of other ions. Additionally, we report mild toxicity of ZIF-8 and its decomposition products, as examined on HDF and A549 cell lines.

ZIF-8 microcrystals demonstrate different degradation kinetics in water, PBS (pH 7.4), and PBS with lactic acid (pH 5.0).     

Evaluation of Mechanical and Electrical Performance of Aging Resistance ZTA Composites Reinforced with Graphene Oxide Consolidated by SPS

This paper presents a study of Al₂O₃–ZrO₂ (ZTA) nanocomposites with different contents of reduced graphene oxide (rGO). The influence of the rGO content on the physico-mechanical properties of the oxide composite was revealed. Graphene oxide was obtained using a modified Hummers method. Well-dispersed ZTA-GO nanopowders were produced using the colloidal processing method. Using spark plasma sintering technology (SPS), theoretically dense composites were obtained, which also reduced GO during SPS. The microstructure, phase composition, and physico-mechanical properties of the sintered composites were studied. The sintered ZTA composite with an in situ reduced graphene content of 0.28 wt.% after the characterization showed improved mechanical properties: bending strength was 876 ± 43 MPa, fracture toughness—6.8 ± 0.3 MPa·m^1/2 and hardness—17.6 ± 0.3 GPa. Microstructure studies showed a uniform zirconia distribution in the ZTA ceramics. The study of the electrical conductivity of reduced graphene oxide-containing composites showed electrical conductivity above the percolation threshold with a small content of graphene oxide (0.28 wt.%). This electrical conductivity makes it possible to produce sintered ceramics by electrical discharge machining (EDM), which significantly reduces the cost of manufacturing complex-shaped products. Besides improved mechanical properties and EDM machinability, 0.28 wt.% rGO composites demonstrated high resistance to hydrothermal degradation.