Sputter coater

The methicillin resistant S. aureus, clinical isolates of P. aeruginosa, L. monocytogenes, and B. Cereus strains were grown for 7 h in tryptone soya yeast extract broth (TSYEB) at 37 °C. Methanolic ASE of T. ferdinandiana fruits and leaves were reconstituted in 75 µL 20% v/v ethanol, added to 1 mL bacteria and broth samples, and incubated for 24 h at 37 °C. The negative control was comprised of 75 µL 20% v/v ethanol. The samples and controls were washed three times in sterile phosphate buffered saline and fixed in 3% v/v glutaraldehyde [27 (link)]. Glutaraldehyde-fixed samples were fixed again in 1% v/v osmium tetroxide and dehydrated with ethanol. Samples were adhered to coverslips coated with poly-L-lysine (1 mg/mL) and dehydrated in the same manner, before being dried in a critical point dryer (Tousimis Research Corporation, Rockville, MD, USA) according to manufacturer’s instructions. Coverslips were attached to stubs with double-sided carbon tabs and coated with gold using a sputter coater (Agar Scientific Ltd, Essex, UK), following the manufacturer’s instructions. Samples were imaged in a Jeol Neoscope JCM 5000 (Jeol Ltd., Tokyo, Japan) at an accelerating voltage of 10 kV and for high resolution images in a Jeol JSM 7100F (Jeol Ltd., Tokyo, Japan) field emission scanning electron microscopy (SEM) at an accelerating voltage of 1 kV.

For scanning electron microscopy (SEM) observation, cells cultured on the different substrates were fixed in 0.5% glutaraldehyde for 1 h at 4 °C, washed in cacodylate buffer, and then post-fixed with 1% OsO4 for an additional hour. Fixed specimens were dehydrated through a series of passages in increasing ethanol baths and dried in pure hexamethyldisilazane (HMDS, Fluka Chemie AG, Buchs, Switzerland). Finally, samples were mounted on stubs, coated with gold in a sputter coater (Agar Scientific Ltd., Stansted, England) and then examined on a Cross-Beam 1540EsB electron microscope (Carl Zeiss Microscopy, Oberkochen, Germany).

The polymer particles were characterised using images captured by high resolution field emission gun-scanning electron microscopy (FEG-SEM, LEO Electron Microscopy Ltd., Cambridge, UK). Each type of fractionated polymer particle was suspended in RPMI 1640 medium with 20 mM HEPES (N-(2-hydroxyethyl) piperazine-N′-(2-ethanulfonic acid) (Bio-Whittaker, Lonza, Belgium) and 100 U ml⁻¹ penicillin/streptomycin (Bio-Whittaker, Lonza, Belgium). A volume of 24 μl of FluoSpheres, 100 μl of fractionated nanometre-sized Ceridust and 100 μl of each of the UHMWPE wear particle suspensions was resuspended in 70% (v/v) ethanol (VWR International Poole, UK) and filtered through a 0.015 μm filter. Sections of each filter were mounted on aluminium stubs using double sided carbon pads and coated with 3.0 nm platinum/palladium using a sputter coater (Agar Scientific Ltd., UK). A minimum of 100 particles were analysed for each sample using a wide range of magnifications (×1000–200,000). The particle distributions were determined using Image Pro Plus software (Media Cybernetics, Maryland, USA) by measuring the size (length) and area of particles on the filters.

For SEM observations, cells cultured on ibidi as well as on Nichoid were fixed in 0.5% glutaraldehyde for 1 h at 4 °C, washed in cacodylate buffer, and post-fixed with 1% OsO4 for an additional hour. The fixed specimens were dehydrated through a series of passages in increasing ethanol baths and dried in pure hexamethyldisilazane (HMDS, Fluka Chemie AG, Buchs, Switzerland). The samples were mounted on stubs and coated with gold in a sputter coater (Agar Scientific Ltd., Stansted, England). The coated specimens were observed by SEM using secondary electron detection (Supra55, Carl Zeiss GmbH, Jena, Germany).

In
these experiments, LESA microextraction was followed by sample
collection in a well plate. The sample was then loaded into a gold-coated
borosilicate nanoelectrospray emitter, i.e., the LESA sampling and
ionization processes were decoupled. These experiments are referred
to as “nanoESI” throughout this Article. Details of
the microextraction are given in the Supporting Information.
Borosilicate glass capillaries were prepared
in house using a P-1000 pipette puller (Sutter Instrument) before
coating with gold using a sputter coater (Agar Scientific Ltd.).
Sample-loaded tips were inserted into a nanospray ion source equipped
with the static spray option (Thermo) attached to either of the mass
spectrometers described below. The electrospray voltage for the tips
was typically in the range of 1.0–1.2 kV and performed with
no additional backing pressure. The use of borosilicate emitters improved
nanoelectrospray stability, duration, and signal intensity when compared
with that of chip-based nanoESI. This observation can be attributed
to the narrower spray orifice (1–2 μm) and the tapered
geometry of the borosilicate emitter versus the square-cut geometry
of the chip-based nanoESI emitters.^{33 (link)}