High vacuum carbon tabs

SEM was used to investigate the morphology of the samples. Bar specimens of PLA-c, PLA-g-TPCS, PLA-GRH, and PLA-g-TPCS-GRH blends were produced by compression molding using pellets from the twin-screw extruder. Bars of PLA-g-TPCS and PLA-g-TPCS-GRH were immersed in liquid nitrogen for ~3 min, then fractured by hand, treated with hydrochloric acid (6 N) for 6 h to remove the TPCS phase, and air dried for 12 h in a fume hood [17 (link)]. Finally, the samples were mounted on aluminum stubs using carbon suspension cement (SPI Supplies, West Chester, PA, USA). Samples of films evaluated before and after tensile testing were also mounted on aluminum stubs with high vacuum carbon tabs (SPI Supplies) and coated with iridium at a thickness of ~2.7 nm. Samples were examined in a JEOL 6610LV (tungsten hairpin emitter) and a JEOL 7500F (field emission emitter) scanning electron microscope (JEOL Ltd., Tokyo, Japan) at various magnifications at 10 and 3.0 kV, respectively.
AFM was conducted using a Cypher™ atomic force microscope (Oxford Instruments Asylum Research, Inc., Santa Barbara, CA, USA) in the contact mode. Roughness parameters, calculated as the root mean square (Rq) and average roughness (Ra), were determined for each type of film and were calculated from the Htr mode image. Images were obtained in the Dfr mode. The film area for the determination of roughness was 900 μm².

Fresh nine-week stems were cut horizontally with a razor blade and fixed at 4°C for 1–2 h in 4% glutaraldehyde buffered with 0.1 M sodium phosphate at pH 7.4. Following a brief rinse in the buffer, the samples were dehydrated in an ethanol series (25, 50, 75, and 95%) for 15 min at each gradation and with three 15 min changes in 100% ethanol. Samples were critical point dried in a Leica Microsystems model EM CPD300 critical point dryer (Leica Microsystems, Vienna, Austria) using carbon dioxide as the transitional fluid. Samples were mounted on aluminum stubs using high vacuum carbon tabs (SPI Supplies, West Chester, PA, USA) and System Three Quick Cure 5 epoxy glue (System Three Resins, Inc. Auburn, WA, USA). Samples were coated with osmium (~10 nm thickness) in an NEOC-AT osmium chemical vapor deposition (CVD) coater (Meiwafosis Co., Ltd., Osaka, Japan), and examined in a JEOL 7500F (field emission emitter) SEM (JEOL Ltd., Tokyo, Japan) at an angle of ~70° between the longitudinal direction of the stem and the plane of image acquisition. Samples were prepared using three biological stems for each sample, and the images were acquired in triplicate (n = 3) at 200×, 14,000×, and 22,000× magnification.

Scanning electron microscopy was performed to investigate the physical interaction between N. oceanica and fungi at the Center for Advanced Microscopy of Michigan State University (CAM, MSU). Algae–fungi aggregates were collected after 6-day co-culture of the alga N. oceanica with M. elongata (AG77 and NVP64) or M. americana 3668S and were fixed in 4% (v/v) glutaraldehyde solution, followed by drying in a critical point dryer (Model 010, Balzers Union). The samples were then mounted on aluminum stubs with high vacuum carbon tabs (SPI Supplies), and were coated with osmium using a NEOC-AT osmium coater (Meiwafosis). The samples were observed with a JSM-7500F scanning electron microscope (Japan Electron Optics Laboratories).