Xl30 environmental scanning electron microscope

The ability of SpHyastatin to alter the morphology of microbes was visualized using SEM as described earlier (Wei et al., 2013 (link)). Briefly, microbial suspensions were prepared and incubated with SpHyastatin as described in the PI uptake assay. The cells were then fixed with 2.5% glutaraldehyde for 3 h and washed three times with 100 mM NaPB before being placed on a poly-L-lysine coated glass slide at 4°C for 30 min. The samples were subsequently dehydrated with graded ethanol and tert-butyl alcohol followed by freezing at 4°C and lyophilized using the critical point method. Finally, the cells were gold-coated and observed with an FEI XL-30 Environmental Scanning Electron Microscope (USA).

The combined techniques of electron-beam lithography and photography and ion beam milling are employed to prepare nanoelectrode platforms with gap sizes in the range of 20 nm by using the top-down approaches on 300 nm thick SiO₂ thermally grown on silicon wafer21 29 . Gold wire are patterned using a FEI XL30 Environmental Scanning Electron Microscope (ESEM) operated at an acceleration voltage of 30 kV is used to pattern gold wire with dimensions of 100 nm wide and 2 μm long. Gold deposition is carried out by resistive evaporation, where 2–3 nm thin layer of chromium is used as adhesion layer between gold and SiO₂. To fabricate nanogaps, the highly focused beam (1pA) of Ga⁺ ions at an acceleration voltage of 30 kV is used to cut nanogaps 15–20 nm wide in the gold wire by usage of a FEI Strata DB235 FIB/SEM. 100 μm × 200 μm gold contact pads are prepared by the standard photolithography technique.

Cotton ovules were fixed with 3% (v/v) glutaraldehyde in 0.1 M sodium phosphate, pH 7.0, dehydrated in a graded ethanol series starting from 20% (v/v) up to 100% (v/v) ethanol and mounted on standard Cambridge SEM stubs. SEM images were taken with a XL30 Environmental Scanning Electron Microscope (FEI Company, Hillsboro, OR) at an accelerating voltage from 10–15 kV under high vacuum conditions.