Orius sc1000 camera

Images were taken under low dose conditions (20 e⁻/ Ų) with a FEI T12 electron microscope (FEI, Hillsboro, OR, USA) at 120 kV using an ORIUS SC1000 camera (Gatan, Inc., Pleasanton, CA, USA). Samples were prepared according to two protocols: TEM1, the carbon flotation technique [56 (link),57 ] or, TEM2, directly deposited on carbon-coated grid (S160-4 grids, Agar Scientific Ltd, Stansted, UK). For the flotation technique, 4 μL of samples were injected to the clean side of carbon protected by a mica surface; the carbon was separated from mica by floating on a water drop; and a grid was placed on top of the carbon film, which was subsequently air-dried. The substrate carbon-coated mica was produced by evaporation of a carbon in an Emitech K950X carbon coater (QUORUM Technologie Ltd., Asford, UK). For the carbon-coated technique, samples were directly deposited on the carbon-coated grid, the excess of sample was first removed by blotting with a filter paper and then air-dried. Instrument calibration was performed during regular maintenance from the manufacturer. Raw dm3 images (4008 × 2672 px) were analyzed with Gwyddion using cross-section profiles.

Embryonic hearts from of heterozygous (n = 3) and homozygous mutant (n = 4) at E11.5 were fixed in 2% glutaraldehyde in PBS (pH 7.0) then stored at 4°C until further processing, embedding, sectioning and imaging as previously described [27 (link)]. Sections were examined using Tecnai 12 Biotwin transmission electron microscope (FEI UK Limited, Cambridge), and images were acquired using an Orius SC1000 camera (11 Megapixels, 4008 x 2672, Gatan, Abingdon).

Copper grids for negative stain were prepared by the carbon flotation technique. Samples were diluted to ~ 0.05 mg/mL in 20 mM Tris (pH 8.0) and small aliquots were adsorbed on carbon-coated mica. The mica was then transferred to a staining solution containing 2% (w/v) sodium silico tungstate, causing detachment of the carbon film. Subsequently, a copper grid was placed on top of the detached carbon which was recovered and dried under air flow. Images were taken under low dose conditions at a nominal magnification of 23,000 × or 30,000 × with a T12 electron microscope (FEI, Hillsboro, OR) at an operating voltage of 120 kV using an ORIUS SC1000 camera (Gatan, Inc., Pleasanton, CA).

Negative-stain grids were prepared using the mica-carbon flotation technique [92 (link)]. 4 μL of spike samples from purifications diluted at about 0.05–0.1 mg/mL were adsorbed on the clean side of a carbon film previously evaporated on mica and then stained using 2% w/v Uranyl Acetate for 30 s. The sample/carbon ensemble was then fished using an EM grid and air-dried. Images were acquired under low dose conditions (<30 e−/Å2) on a Tecnai 12 FEI electron microscope operated at 120 kV using a Gatan ORIUS SC1000 camera (Gatan, Inc., Pleasanton, CA) at 30,000x nominal magnification. To facilitate the visualization of the molecules, a Gaussian filter was applied to the images using Photoshop, then the gray levels were saturated and the background eliminated. For the 2D classification, images were processed with RELION 2.1 [93 (link)]. CTF was estimated with CTFFind-4.1 [94 (link)]. An initial set of 409 particles (box size of 512 pixels, sampling of 2.2 Å/pixel) was obtained by manual picking. After 2D classification the best looking 2D class averages were used as references for Autopicking. A set of around 35 000 particles (box size of 256 pixels, sampling of 4.4 Å/pixel, mask diameter 300Å) was obtained by Autopicking with a gaussian blob. The 8 best obtained classes were calculated from 2854 particles.

AuNP samples for TEM were prepared by suspending the AuNPs in ultra-pure distilled water, and diluting down to a concentration of ∼5 pM. Then 1 μL of sample solution was added to a TEM copper grid (CF-400-Cu, Electron Microscopy Sciences) which was then dried in air for 30 min. Nanoparticle TEM samples were imaged on a JEOL 2100Plus Transmission Electron Microscope at 200 kV with a beam current of 101 μA. TEM micrographs were captured with the Gatan Orius SC1000 camera at magnifications of 25 000× and 60 000×.