We applied reference-free, CTF corrected, subtomogram averaging to solve the structure of the repeating inner coat unit with a resolution of approximately 23 Å.
For energy dispersive X-ray analysis (EDS, Quantax 200, Bruker, Germany), samples were coated with carbon in carbon coating unit and measurements were carried out at 20 kV at high vacuum.
Subsequently, they were mounted on stubs using colloidal coal as an adhesive, and sputter coated with gold (Polaron SEM Coating Unit E5000).
For the biofilm study, samples of the oropharyngeal section of the NGT were fixed overnight with 2.5% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.4, washed with the same buffer, dehydrated in increasing concentrations of ethanol, dried with a critical point drier, and coated with gold (Polaron-sem coating unit E5100, Thermo VG Scientific, Beverly, MA).
The material was sputter-coated with gold in a SEM coating unit E5100 (Polaron equipment Ltd ,England) and stored over silica beads (Scharlau, Spain) in an air-tight container until imaged.
The cells were affixed to a metal SEM stub and sputter coated in gold by using an SEM coating unit (E5100 Polaron, UK).
Dried samples were mounted on stubs and sputter-coated with chromium in an EMS150T-ES coating unit (Electron Microscopy Sciences, Hatfield, PA, USA).
Samples were critical point dried in liquid CO2 using the Balzers CPD 010 (Balzers Instruments, Liechtenstein), mounted on aluminum stubs, and sputter coated with gold/palladium using the Polaron SEM Coating Unit E5100 (Thermo VG Scientific, Beverly, MA).
The bead samples were then sputter-coated with a thin gold layer using a coating unit (Balzers Union model FL 9496) and analyzed in a JEOL JSM 5310 operated at 15 or 20 kV.
The masticated almond tissues were mounted on stubs, coated with gold in a Polaron E5100 sputter coating unit, and viewed in a JEOL 25SM and a Philips 501 scanning electron microscope (FEI Company).
