Samdri 795

Hybrid tubes were dehydrated by incubation in a series of ethanol solutions of increasing concentration. Ethanol was subsequently removed by critical point drying (Tousimis Samdri-795). Extra caution was taken to ensure the samples were not heated above the lower critical solution temperature of the polymer material during the exchange. Dehydrated hybrid tubes were mounted on stubs using carbon glue and coated with 21 nm of osmium (Filgen, OPC-60A) to create a conductive sample surface. All SEM images were taken using a Hitachi SU8030 or LEO 1525 instrument operating at an accelerating voltage of 2 kV.

C. elegans were fixed in 2% paraformaldehyde/2.5% glutaraldehyde in 0.15 M sodium phosphate buffer, pH 7.4, for 1 h at room temperature and stored at 4 °C. Following three washes in 0.15 M sodium phosphate buffer, pH 7.4, the samples were post-fixed in 1% osmium tetroxide in 0.15 M sodium phosphate buffer, pH 7.4 for 1 h and gently washed three times in deionized water. Samples were then deposited onto a poly d lysine coverslip or transferred to a microporous chamber and gradually dehydrated with ethanol (30%, 50%, 75%, 100%, 100%). Coverslips and microporous capsules were then transferred to a Samdri-795 critical point dryer and dried using carbon dioxide as the transitional solvent (Tousimis Research Corporation, Rockville, MD). Samples were mounted on 13 mm aluminum stubs with double-sided carbon adhesive and sputter coated with 5 nm of gold-palladium alloy (60 Au:40 Pd, Cressington Sputter Coater 208HR, model 8000-220, Ted Pella, Redding, CA). Specimens were observed and images taken using a Zeiss Supra 25 FESEM operating at 5 kV, using the SE2 detector, 5 mm working distance, and 30 μm aperture (Carl Zeiss SMT Inc., Peabody, MA).

SEM images for the native tissue samples and hydrogel microfibers sheets (with or without cells) were acquired using an SEI Quanta 200 Environmental SEM. All samples were fixed with cold methanol and then thoroughly washed with Dulbecco’s Phosphate Buffer Saline (5× wash, 30 minutes each at room temperature). Then, the samples underwent gradual ethanol dehydration (25%, 50%, 75% 100%), after which they were critical point dried using a Tousimis Samdri-795 and sputtered with 4 nm of palladium layer using an Anatech USA Hummer 6.2 prior to imaging

Three additional plaque biofilms treated with 10 μg/ml (6.5 μM) of peptide 1018 for 24, 48 and 72 hours were collected for scanning electron microscopy (SEM) examination. Samples were prefixed with phosphate buffered 2.5% glutaraldehyde for 10 minutes before further fixation in 1% osmium tetroxide for 1 hour. The specimens were then subjected to increasing concentrations of ethanol (50%, 70%, 80%, and 100%) for dehydration. The dehydrated specimens were dried using a critical point drier (Samdri-795; Tousimis Research Corporation, Rockville, MD, USA), sputter-coated with gold palladium (Hummer VI; Technic Inc, Anaheim, CA, USA), and examined by SEM (Helios Nanolab 650, FEI, Eindhoven, Netherlands) at an accelerating voltage of 5 kV using 5,000 × and 20,000 × magnifications. A control experiment was done by preparing 10 μg/ml peptide in BHI solution and incubating it at 37°C for 72 hours. A droplet of the 72-hour 10 μg/ml of peptide was dropped on a piece of aluminum paper and air-dried for SEM imaging.

The spontaneously formed NE gel was fixed with 2.5% glutaraldehyde in 1× PBS for 2 h at room temperature. The fixed samples were washed three times with PBS and dehydrated with a series of ethanol solutions of increasing concentration, 70%, 80%, 90%, and 100% and twice 100% with each step lasting 10 min. We removed as much ethanol as possible from the sample and added tert-butanol that just covered the sample. The sample was stored in tert-butanol at 4°C until further use. Freeze drying of samples was performed by using critical point drying (Tousimis Samdri-795). The dried samples were covered with a 30 nm layer of Au using a Quorum Q150T ES sputter. The electron microscopy analysis was performed using a Quanta 200 Scanning Electron Microscope, operating with an acceleration voltage of 5 keV and working distance of 5 mm.

To prepare cell samples for scanning electron microscope, the cardiomyocytes cultured on the nanopatterned electrodes are treated with 2.5% glutaraldehyde solution for 4 h and rinsed twice with deionized water. Increasing concentrations of ethanol (30, 50, 70, 80, 90, 95, 100, 100%) are then used and critical point drying (CPD) (Samdri-795, Tousimis Research Corp.)

To evaluate the effect of the temperature and kanamycin concentration on the morphology of E. coli DH5α-pVAX1-NH36 culture, SEM was performed. For all treatments included in experimental design, 1 mL of culture broth was centrifuged for 10 min at 12,000 rpm and 4 °C. Then, the samples were washed twice with PBS buffer (pH = 7.2) and re-suspended in 1 mL of same buffer. Next, 10 µL of suspension was used to coat a glass slide (1 cm × 1 cm). A fixative was placed on the samples (2.5% glutaraldehyde (v/v) in PBS) overnight, then washed three times with PBS followed by fixation in 1% osmium tetroxide at 4 °C for 2 h.
The fixed cells were washed three times with PBS. Dehydration of samples was achieved by washing with increasing concentrations of ethyl alcohol (70%, 80%, 90% and 100%, Sigma Aldrich, 200 proof, molecular biology grade). Lastly, all samples were freeze-dried (Tousimis, Samdri-795, Rockville, Maryland), then fixed on carbon tape and sputter-coated with gold particles for 2 min (Denton Vacuum Desk V, Los Angeles, CA, USA).
Images were taken by Scanning Electron Microscope JSM-6510LV (JEOL, Tokyo, Japan).