Cell monolayers were fixed with a solution of 25% glutaraldehyde and 0.2M sodium cacodylate in PBS for 30 min and stored at 4°C. After washing with sodium phosphate buffer and short purging with ethanol (30%), samples were dehydrated in graded ethanol (50%, 75%, 90% and 100%). After that the samples were critical-point dried with CO2 according to the manual of the device CPD 7501 (Quorum Technologies Ltd, Laughton, Lewes, East Sussex, Great Britain). As the investigated samples are insulating, the sample surface was coated with a thin film of gold. The SEM images were acquired with a scanning electron microscope (Quanta FEG 250, FEI Deutschland GmbH) operating in high vacuum and 10 kV, using an Everhart-Thornley secondary electron detector (ETD).
Cpd 7501
Manufactured by Quorum Technologies
Sourced in United Kingdom
The CPD 7501 is a critical point dryer, a piece of lab equipment used to transition samples from a liquid to a dry state while avoiding damage from surface tension. It operates by transitioning the sample solvent from a liquid to a supercritical fluid state, then removing the supercritical fluid to leave a dry sample.
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7 protocols using cpd 7501
1
SEM Imaging of Cell Monolayers
2
Quantifying Olfactory Sensilla on Fly Antennae
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Flies were placed in 70% ethanol and sonicated for 5 minutes to remove surface wax from the cuticle. Heads were carefully excised, dehydrated in 100% ethanol, dried in a critical-point drier (CPD7501, Quorum), mounted on aluminum stubs with argent glue, and oriented so that the external side of the funiculus was exposed. Three specimens per species were coated with platinum (7 nm thickness; EM ACE600, Leica, Germany) and examined with a GeminiSEM 500 microscope (Zeiss, Germany). Olfactory sensilla on the surface of the external side of the funiculus were counted, localized, and assigned to the four main morphological classes (trichoid, basiconic, clavate, and coeloconic sensilla). The number of coeloconic sensilla is probably underestimated since some of these small sensilla may have been hidden. To generate sensilla density maps, antenna length (from the arista to the tip) and width were normalized. Sensilla density maps were computed with convolving spatial positions of sensilla with a 2D-Gaussian Kernel of standard deviation equal to 2% of funiculus length and 5% of funiculus width. The sensilla dominance index was defined as (Db + Dc − Dt)/(Db + Dc + Dt) with Db, Dc, and Dt being the average density of basiconic, clavate, and trichoid sensilla, respectively, along the proximo-distal axis.
3
Scanning Electron Microscopy of Explants
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Scanning electron microscopy explants were cultured on a 14 mm diameter coverslip (as described above), fixed for 1h at 4°C in 2% glutaraldehyde prepared in 0.1 M sodium cacodylate buffer, rinsed in cacodylate buffer, dehydrated in a series of graded ethanol baths, and dried using a critical point dryer (Quorum Technologies CPD7501, Laughton, UK). They were finally mounted on a carbon stub and sputter-coated. Observations were made using a Cambridge Instruments Stereoscan 260 scanning electron microscope equipped with a digital camera.
4
Preparation and Characterization of 1D Al2O3 Nanostructures
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As-deposited and re-phage over coated 1D Al2O3 nanostructures were sputtered with gold-palladium (Polaron, Sputter Coater) and analyzed under SEM (Quanta FEI-SEM, Holland). For the cells, 24 h cultured cells on prepared substrates were fixed following our standard protocol: substrates were washed 2 times with PBS at 37°C and the fixation was done using 2.5% glutaraldehyde in 0.5 M cacodylate buffer with 6% sucrose for 2 h at room temperature under movement. Afterwards, the substrates were incubated in osmium tetroxide (4% in deionized water (dH2O)) for 2 h under movement in a dark chamber. The substrates were stored in dH2O at 4°C overnight. The water was removed by washing substrates twice in ethanol under movement at 4°C for 5 min (30%, 50%, 70%, 80%, and 90%). Dehydrating was finished by washing 3 times in ethanol (100%) for 15 min under movement at 4°C. The substrates were subjected to critical point drying (Polaron CPD 7501, Quorum Technologies, Ringmer, UK). Afterwards, they were sputtered with gold-palladium and visualized under SEM.
5
Scanning Electron Microscopy of Endothelial Cells
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Cell morphology of human endothelial EA.hy926 cells grown for 48 h on either polymeric nonwovens or tissue culture polystyrene control surface (NC) was observed by scanning electron microscopy. After the incubation on the polymeric surfaces, cells were fixed with 2.5% glutaraldehyde and 0.2 M sodium cacodylate, in PBS for 30 min. Samples were then washed with sodium phosphate buffer, dehydrated in a graded series of ethanol (50%, 75%, 90% and 100%) and dried with CO2 in a critical point dryer (CPD 7501, Quorum Technologies Ltd., Laughton, Lewes, East Sussex, UK). Samples were sputter-coated with gold by Agar Sputter Coater (Canemco Inc., QC, Canada), and image acquisition was performed with the scanning electron microscope QuantaTM FEG 250 (FEI Company, Hillsboro, OR, USA) at 10 kV under high vacuum conditions by using the Everhart–Thornley secondary electron detector (ETD).
6
Ultrastructural Analysis of C. watsonii
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C. watsonii cells were harvested in exponential phase, 2 hours after the beginning of the dark period, onto 0.4 µm polycarbonate membranes, and incubated overnight into a fixative with adjusted osmolarity (3% glutaraldehyde in 0.1M cacodylate pH 7.4, NaCl 1.75%). Membranes were then washed, post-fixed for 1 h with 1% osmium tetroxide in 0.1M cacodylate buffer with 1.75% NaCl, and then dehydrated with graded increasing concentrations of ethanol (50, 70, 96, 100%) and critical point dried (CPD 7501, Quorum Technologies). Finally, membranes were mounted on stubs, gold-sputtered (Scancoat Six, Edwards) and observed with a conventional SEM (Scanning Electron Microscope, Cambridge Stereoscan S260). Pictures were analysed with ImageJ software [41] (link) in order to determine cell diameters and biovolumes. Due to experimental constraints, cell diameters and biovolumes were determined on four cultures (dFe = 3.3, 13.3, 43.3 and 403.3 nM).
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C. watsonii cells were harvested in exponential phase, 2 hours after the beginning of the dark period, onto 0.4 µm polycarbonate membranes, and incubated overnight into a fixative with adjusted osmolarity (3% glutaraldehyde in 0.1M cacodylate pH 7.4, NaCl 1.75%). Membranes were then washed, post-fixed for 1 h with 1% osmium tetroxide in 0.1M cacodylate buffer with 1.75% NaCl, and then dehydrated with graded increasing concentrations of ethanol (50, 70, 96, 100%) and critical point dried (CPD 7501, Quorum Technologies). Finally, membranes were mounted on stubs, gold-sputtered (Scancoat Six, Edwards) and observed with a conventional SEM (Scanning Electron Microscope, Cambridge Stereoscan S260). Pictures were analysed with ImageJ software [41] (link) in order to determine cell diameters and biovolumes. Due to experimental constraints, cell diameters and biovolumes were determined on four cultures (dFe = 3.3, 13.3, 43.3 and 403.3 nM).
7
Imaging Magnetic Core-Shell Beads by SEM
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The magnetic core-shell beads were observed by SEM just after preparation (wet) or after overnight drying. The wet samples were dehydrated through concentration gradient of ethanol (30-50-70-96-100%) before critical point drying (CPD 7501, Quorum Technologies).
Samples were directly mounted on stubs, or manually freeze-fractured after a fast plunging in liquid nitrogen and then gold-sputtered (Scancoat Six, Edwards). Observation was carried out with a conventional SEM operating at 15 kV (Cambridge Stereoscan S260).
Samples were directly mounted on stubs, or manually freeze-fractured after a fast plunging in liquid nitrogen and then gold-sputtered (Scancoat Six, Edwards). Observation was carried out with a conventional SEM operating at 15 kV (Cambridge Stereoscan S260).
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