Sputter coater 108

Mouse temporal bones were fixed with 2.5% glutaraldehyde in 0.1 M phosphate buffer overnight at 4 °C. Cochleae were dissected out of the temporal bone and post‐fixed with 1% osmium tetroxide in 0.1 M phosphate buffer at 4 °C for 2 h. Samples were then dehydrated in ethanol and critically point‐dried using a Leica EM CPD300 (Leica, Germany). After that, samples were mounted and sputter coated with platinum (15 nm) using a Cressington 108 sputter coater (Cressington, United Kingdom). The images were taken with a Quanta250 field‐emission scanning electron microscope (FEI, The Netherlands).

A voltammetric behavior experiment was performed using a 621E electrochemical analyzer (CH Instruments, Austin, TX, USA). The identification and quantification of cannabinoids were conducted using a high performance liquid chromatography and electrochemical detection (HPLC-EC) system consisting of a 5160 pump and a 5260 auto sampler (Hitachi, Tokyo, Japan) coupled with a LC-4C amperometric detector (BAS, West Lafayette, IN, USA). MAE experiments were performed using a MARS 5 microwave system (CEM, Matthews, NC, USA). UAE experiments were performed using a 3210 ultrasonic system (Branson, Danbury, CT, USA). SFE experiments were performed using a Spe-ed SFE supercritical fluid system consisting of an SFE oven module, SFE pump module, and SFE control and collection module (Applied Separations, Allentown, PA, USA) coupled with air compressors (JW, Taichung, Taiwan). A D-606 cooling bath (Deng Yng, New Taipei, Taiwan) was applied for HRE, SE, and SFE experiments. Microstructure analysis was performed using a Quanta 200 environmental scanning electron microscope (SEM, FEI, Hillsboro, OR, USA). A 108 Sputter Coater (Cressington, Watford, Hertfordshire, UK) was used for gold coating.

Mouse inner ears were dissected and fixed with 2.5% glutaraldehyde in 0.1 M phosphate buffer overnight at 4°C. Cochleae were dissected out of the temporal bone, post-fixed with 1% osmium tetroxide in 0.1 M phosphate buffer at 4°C for 2 h, and dehydrated in ethanol and critically point dried using a Leica EM CPD300 (Leica, Germany). Samples were then mounted and sputter coated with platinum (15 nm) using a Cressington 108 sputter coater (Cressington, United Kingdom) and imaged using a Quanta250 field-emission scanning electron microscope (FEI, Netherlands).

Small clusters of carbon fibers from the BMFC anode that had been stored in fixative were observed under bright field microscopy at 100x, a few days after recovery, as an exploratory procedure. In these initial images, filamentous structures were visible and often appeared to span between and across fibers (Figure S1) prompting further investigation. Later, other fiber clusters from the 10 sampling locations were dehydrated in a graded series of ethanol from 10 to 100%. Specimens were mounted on aluminum SEM stubs with double-sided carbon tape, critical-point dried using an EMS 850 Critical Point Dryer, and sputter-coated with gold and palladium using a Cressington 108 sputter coater. The resultant specimens were observed under a FEI Quanta 600FEG ESEM at 5–10 kV. This instrument also provided elemental spectra by X-Ray Energy Dispersive Spectrometry (EDS).

Mouse inner ears were fixed with 2.5% glutaraldehyde in 0.1 M phosphate buffer overnight at 4°C. Cochleae were dissected out of the temporal bone, then post-fixed with 1% osmium tetroxide in 0.1 M phosphate buffer at 4°C for 2 h. After dehydration in ethanol, samples were critically point dried using a Leica EM CPD300 (Leica, Germany), then mounted and sputter coated with platinum (15 nm) using a Cressington 108 sputter coater (Cressington, United Kingdom). Images were taken using a Quanta250 field-emission scanning electron microscope (FEI, Netherlands).

The WT (H99), the mar1Δ mutant (MAK1), and the cap59Δ mutant (cap59) strains were incubated in YPD medium at 30°C and CO₂-independent medium (Gibco) at 37°C until saturation. Samples were fixed with 2.5% glutaraldehyde for 1 h at room temperature and were subsequently washed 3 times with PBS. Each sample was mounted onto 12-mm poly-l-lysine-coated coverslips (Neuvitro Corporation) and subsequently dehydrated by immersing the coverslips in ethanol (30% for 5 min, 50% for 5 min, 70% for 5 min, 95% for 10 min, 100% for 10 min, and 100% for 10 min). Samples were then critical point dried with a Tousimis 931 critical point dryer (Rockville, Maryland) and coated with gold-palladium using a Cressington 108 sputter-coater (Watford, United Kingdom). Coverslips containing the prepared samples were mounted and imaged on a Hitachi S-4700 scanning electron microscope (Tokyo, Japan).

FTIR spectra were recorded on dried samples in KBr pellets (1% dried material) using a Bruker Vertex 70 spectrophotometer (Berlin, Germany) and scanned within the range of 400–4000 cm⁻¹ in transmittance mode. The cross-section morphology of the lyophilized hydrogels was analyzed using a HITACHI SU 1510 scanning electron microscope (Hitachi SU-1510, Hitachi Company, Tokyo, Japan) in a Secondary Electron (SE) system. For sample preparation, a transversal sectioning of the dry material was conducted using a sharp scalpel; then, the exposed sections were mounted on an aluminum stub and fixed. All the analyzed samples were coated with a 7 nm gold layer using a Cressington 108 Sputter Coater. The SEM images were analyzed using ImageJ software.