Q150t turbo pumped sputter coater

Samples removed from the dual culture were fixed (3% glutaraldehyde, 0.1 M cacodylate buffer, pH 7.4) for 48–96 h at 4 °C. They were centrifuged (14,300× g, 5 min, room temperature), and the sediment was resuspended in 500 µL of 3% glutaraldehyde and fixed overnight. After fixation, the samples were centrifuged again (12,000× g, 5 min, room temperature) and washed with cacodylate buffer (20 min, three times). The cells were sedimented onto poly-_L-lysine-treated glass coverslips for 24 h at 4 °C. The coverslips were then washed with double-distilled water at room temperature, post-fixed with 1% osmium tetroxide for 1 h, washed again with ddH₂O (20 min, three times), dehydrated in a graded ethanol series and absolute acetone, and critical-point dried (K850, Quorum Technologies Ltd., Ringmer, UK). The dried coverslips were mounted onto aluminum specimen stubs using Wire Glue (Anders Products, Andover, MA, USA) and sputter-coated with 3 nm of platinum in a high-resolution Turbo-Pumped Sputter Coater Q150T (Quorum Technologies Ltd., Ringmer, UK). The samples were examined using an FEI Nova NanoSEM scanning electron microscope (FEI, Brno, Czech Republic) at 3 or 5 kV using Everhart–Thornley Detector (ETD), Circular Backscatter Detector (CBS), and Through the Lens Detector (TLD).

Scanning electron micrographs (SEM, Zeiss Leica, Jena, Germany) of the silicone surfaces facilitated a detailed view of the distribution of pores and their variety in pore size and morphology. For SEM preparation, the samples were placed on metal stubs and sputtered with gold for one minute using a Turbo-Pumped Sputter Coater Q150T (Quorum Technologies, Laughton, United Kingdom). SEM analysis was then performed using a magnification of 500 fold.

AMNP0, AMNP1, and AMNP2 particles were assessed using Scanning Electron Microscopy (SEM). AMNP0 (3 mg) was secured onto a carbon based adhesive substrate and positioned on a specimen stage, while powder samples (3 mg) of AMNP1 and AMNP2 were dispersed in 10 mL of ethanol prior analysis. From each dispersion, 10 µL was placed on separate SEM pin stubs and left to air dry. The samples were sputter-coated with 20 nm of gold for 180 s under argon using a Quorum Q150T Turbo-Pumped Sputter Coater (Essex, UK). AMNP0 was imaged using a JEOL JSM-6301F instrument (Welwyn Garden City, UK), whilst AMNP1 and AMNP2 were imaged using a Quanta 200 FEG ESEM (OR, USA). All images were collected with an accelerating voltage of 5 kV.

After immunofluorescence analysis of the seeded scaffolds after 28 days of myogenic differentiation, microstructural analysis of the scaffolds was performed using an Auriga Fib-scanning electron microscope (SEM) (Zeiss, Oberkochen, Germany) as described previously [4 (link)]. Probes were sputter-coated with gold using a Q150T Turbo-pumped Sputter Coater (Quorum Technologies Inc., Guelph, Canada).

Bacteria were cultivated in small-scale under conditions described in Table 1. A volume corresponding approximately to 2 × 10⁹ CFU was pelleted (6000 × g, 15 min, 4°C) and washed twice in 100 mM sodium cacodylate/5 g/L NaCl, pH 7.2. Bacteria were then fixed by 3% glutaraldehyde in the same buffer at RT for 1 h and then at 4°C overnight with slow rotation. Sterility of the fixed solution was checked by aliquot plating. The washed bacterial cells were then allowed to sediment overnight onto poly-L-lysine treated circular coverslips at 4°C. The coverslips with attached bacteria were post-fixed with 1% OsO₄ for 1 h at room temperature and three times washed with ddH₂O. Washed coverslips with the bacteria were dehydrated through an alcohol series (25, 50, 75, 90, 96, 100, and 100%) followed by absolute acetone and critical point dried from liquid CO₂ in a K850 Critical Point Dryer (Quorum Technologies Ltd.). The dried samples were sputter coated with 3 nm of platinum in a Q150T Turbo-Pumped Sputter Coater (Quorum Technologies Ltd.). The final samples were examined in a FEI Nova NanoSEM 450 scanning electron microscope (Thermo Fisher Scientific) at 5 kV using CBS and TLD detectors.

Cells were fixed with 2.5% glutaraldehyde in 0.1 M phosphate buffer, pH 7.2 for 2 h at RT, followed by rinsing with 0.1 M PBS two times. Cells were dehydrated by passing through a series of ethanol solutions, starting at 50% and sequentially incubating in 75%, 80%, 90%, 96%, and 100% ethanol for 5 min, twice repeating each step. The dehydrated cells were incubated twice for 30 min in hexamethyldisilazane. Cells were air dried overnight, coated with chromium for 20 min using a Q150T Turbo-Pumped Sputter Coater (Quorum Technologies) and then imaged with Supra 50 VP LEO (LEO Carl Zeiss SMT Ltd) scanning electron microscope.

Samples of intact and denuded HAM scaffolds (from two placentas) mounted in a CellCrown™ inserts (Scaffdex Oy, Tampere, Finland) were fixed in PBS buffered 3% glutaraldehyde, washed in PBS, postfixed with 1% OsO4, dehydrated in a graded ethanol series (25, 50, 75, 90, 96, and 100%) and critical point dried in a K850 Critical Point Dryer (Quorum Technologies Ltd, Ringmer, UK). The dried samples were sputter-coated with 3 nm of platinum in a Q150T Turbo-Pumped Sputter Coater (Quorum Technologies Ltd, Ringmer, UK). The final samples were examined in a FEI Nova NanoSEM scanning electron microscope (FEI, Brno, Czech Republic) at 5 kV using ETD, CBS and TLD detectors. Stereo-pair images were taken at tilts of -6°, 0° and +6° of compucentric goniometer stage. Final R-GB anaglyphs were constructed in a “Stereo module” of AnalySis3.2 software suite (EMSIS GmbH, Germany).