Cryogenic-temperature electron microscopy direct imaging of carbon nanotubes and graphene solutions in superacids.

Cryogenic electron microscopy (cryo-EM) is a powerful tool for imaging liquid and semiliquid systems. While cryogenic transmission electron microscopy (cryo-TEM) is a standard technique in many fields, cryogenic scanning electron microscopy (cryo-SEM) is still not that widely used and is far less developed. The vast majority of systems under investigation by cryo-EM involve either water or organic components. In this paper, we introduce the use of novel cryo-TEM and cryo-SEM specimen preparation and imaging methodologies, suitable for highly acidic and very reactive systems. Both preserve the native nanostructure in the system, while not harming the expensive equipment or the user. We present examples of direct imaging of single-walled, multiwalled carbon nanotubes and graphene, dissolved in chlorosulfonic acid and oleum. Moreover, we demonstrate the ability of these new cryo-TEM and cryo-SEM methodologies to follow phase transitions in carbon nanotube (CNT)/superacid systems, starting from dilute solutions up to the concentrated nematic liquid-crystalline CNT phases, used as the 'dope' for all-carbon-fibre spinning. Originally developed for direct imaging of CNTs and graphene dissolution and self-assembly in superacids, these methodologies can be implemented for a variety of highly acidic systems, paving a way for a new field of nonaqueous cryogenic electron microscopy.

Complexes between anionic liposomes and spherical polycationic brushes. An assembly of assemblies.

This paper has at its objective the assembling of liposomal assemblies onto nanoparticles. In this manner, one generates nanoparticles with a high loading capacity. Thus, spherical spherical polycationic "brushes" (SPBs) were synthesized by graft polymerizing a cationic monomer, (trimethylammonium)ethylmethacrylate chloride, onto the surface of monodisperse polystyrene particles, ca. 100 nm in diameter. These particles were complexed with small unilamellar anionic liposomes, 40-60 nm in diameter, composed of egg lecithin (EL) and anionic phosphatidylserine (PS(1-)) in PS(1-)/EL ratios from 0.10 to 0.54, a key parameter designated as ν. These complexes were then characterized according to electrophoretic mobility, dynamic light scattering, conductivity, fluorescence, and cryogenic transmission electron microscopy, with the following main conclusions: (a) All added liposomes are totally associated with SPBs up to a certain saturation concentration (specific for each ν value). (b) The number of liposomes per SPB particle varies from 40 (ν = 0.1) to 14 (ν = 0.5). (c) At sufficiently high liposome concentrations, the SPBs experience an overall change from positive to negative charge. (d) SPB complexes tend to aggregate when their initial positive charge has been precisely neutralized by the anionic liposomes. Aggregation is impeded by either positive charge at lower lipid concentrations, or negative charge at higher lipid concentrations. (e) The liposomes remain intact (i.e., do not leak) when associated with SPBs, at ν ≤ 0.5. (f) Complete SPB/liposome dissociation occurs at external [NaCl] = 0.3 M for ν = 0.1 and at 0.6 M for ν = 0.5. Liposomes with ν = 0.54 do not dissociate from the SPBs even in NaCl solutions up to 1.0 M. (g) Complexation of the PS(1-)/EL liposomes to the SPBs induces flip-flop of PS(1-) from the inner leaflet to the outer leaflet. (h) The differences in the ability of PS(1-) (a cylindrical lipid) and CL(2-) (a conical lipid) to create membranes defects are attributed to geometric factors.

Therapeutic ultrasound-mediated DNA to cell and nucleus: bioeffects revealed by confocal and atomic force microscopy.

Therapeutic ultrasound (TUS) has the potential of becoming a powerful nonviral method for the delivery of genes into cells and tissues. Understanding the mechanism by which TUS delivers genes, its bioeffects on cells and the kinetic of gene entrances to the nucleus can improve transfection efficiency and allow better control of this modality when bringing it to clinical settings. In the present study, direct evidence for the role and possible mechanism of TUS (with or without Optison) in the in vitro gene-delivery process are presented. Appling a 1 MHz TUS, at 2 W/cm(2), 30%DC for 30 min was found to achieve the highest transfection level and efficiency while maintaining high cell viability (>80%). Adding Optison further increase transfection level and efficiency by 1.5 to three-fold. Confocal microscopy studies indicate that long-term TUS application localizes the DNA in cell and nucleus regardless of Optison addition. Thus, TUS significantly affects transfection efficiency and protein kinetic expression. Using innovative direct microscopy approaches: atomic force microscopy, we demonstrate that TUS exerts bioeffects, which differ from the ones obtained when Optison is used together with TUS. Our data suggest that TUS alone affect the cell membrane in a different mechanism than when Optison is used.