Conductive silver paint

All samples were mounted on 15 mm steel pucks (Ted Pella) using conductive silver paint (Agar Scientific Ltd.) used to ground the sample with respect to the tip. The charge contrast in the HOPG sample seen in Figs 5 and 6 was only formed after contact mode scanning a rough (e.g. having an unusually large number of step edges) area of a freshly cleaved sample while applying an AC voltage. Prior to scanning the sample in contact no contrast was shown in classical KPFM measurements on the same region (not shown). After contact mode scanning, CPD contrast was observed in both classical and G-Mode KPFM and remained for over a day. We believe the contact mode scanning, delaminated, or partially delaminated, flakes of graphite which were weakly bound after the initial cleaving process. In Fig. 7., the diode sample50 was prepared by cross sectioning a commercial Schottky barrier diode. The top of the diode was removed by polishing with diamond paste down to 1 μm gritsize. Further polishing was precluded by selective polishing of the interconnect material, resulting in large topographical variations from the metal to silicon.

For transmission electron microscopy, enucleated zebrafish eyes were fixed in 2% paraformaldehyde-2% glutaraldehyde prior to incubation with 1% osmium tetroxide-1% potassium ferrocyanide. Following dehydration in an ethanol series and propylene oxide, the zebrafish were embedded in Epon 812 resin. Using a Leica EM UC7 ultramicrotome, 100 nm sections were cut, collected on formvar-coated copper grids (Electron Microscopy Sciences, Hatfield, PA, USA) and stained with lead citrate (Agar Scientific, Stansted, UK). Sections were examined on a Jeol 1010 TEM and imaged using a Gatan Orius SC1000B charge-coupled device camera.
For scanning electron microscopy, whole 6 dpf larvae were fixed and dehydrated as above. Samples were incubated twice in anhydrous methanol before hexamethyldisilazane (Sigma-Aldrich) incubation and overnight drying on watch glasses. Samples were mounted on stubs (Agar Scientific) and painted with conductive silver paint (Agar Scientific) and left to dry overnight before sputter-coating with gold/palladium using a Cressington Sputter Coater. Larval neuromasts were imaged on a Zeiss Sigma VP.

For SEM, the dissected mouse cochleae were initially fixed by a very gentle intralabyrinthine perfusion using a 100-μL pipette tip through the round window. The fixative contained 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer plus 2 mM CaCl₂ (pH 7.4). Following perfusion, the cochleae were immersed in the above fixative for 2 h at room temperature. After the fixation, the organ of Corti was exposed by removing the bone from the apical coil, and the cochleae, then incubated in solutions of saturated aqueous thiocarbohydrazide (20 min) alternating with 1% osmium tetroxide in cacodylate buffer (2 h) twice (the OTOTO technique).⁸¹ (link) The cochleae were then dehydrated through an ethanol series and critical point dried using CO₂ as the transitional fluid (Leica EM CPD300) and mounted on specimen stubs using conductive silver paint (Agar Scientific, Stansted, UK). The apical coil of the organ of Corti was examined at 10 kV using a Tescan Vega3 LMU scanning electron microscope.

The isolated inner ear was very gently perfused with fixative for 1–2 min through the round window. A small hole in the apical portion of the cochlear bone was made prior to perfusion to allow the fixative to flow out from the cochlea. The fixative contained 2.5% vol/vol glutaraldehyde in 0.1 M sodium cacodylate buffer plus 2 mM CaCl₂ (pH 7.4). The inner ears were then immersed in the above fixative and placed on a rotating shaker for 2 h at room temperature. After fixation, the organ of Corti was exposed by removing the bone from the apical coil of the cochlea and then immersed in 1% osmium tetroxide in 0.1 M cacodylate buffer for 1 h. For osmium impregnation, which avoids gold coating, cochleae were incubated in solutions of saturated aqueous thiocarbohydrazide (20 min) alternating with 1% osmium tetroxide in buffer (2 h) twice (the OTOTO technique: Furness & Hackney, 1986 (link)). The cochleae were then dehydrated through an ethanol series and critical point dried using CO₂ as the transitional fluid (Leica EM CPD300) and mounted on specimen stubs using conductive silver paint (Agar Scientific, Stansted, UK). The apical coil of the organ of Corti was examined at 10 kV using a Tescan Vega3 LMU scanning electron microscope in the electron microscopy unit at the University of Sheffield.

The morphology of RBV raw material, spray-dried excipient microparticles and agglomerates was examined by Field Emission – Scanning Electron Microscopy (FE-SEM), using a Zeiss SUPRA 40 microscope (Zeiss, Oberkochen, Germany). The powder samples were fixed onto high-purity aluminum pin stubs using double-sided tape, with the exception of the agglomerates which were dipped in silver glue (Conductive Silver Paint, Agar Scientific, Stansted, UK), as it was not possible to stick them onto the bioadhesive tape without breaking them. This paint formed a thin highly conductive film on the agglomerate’s surface. In all cases, the magnifications selected ranged between 250× and 5000× to appreciate both the whole particle and its surface detail.