Technical Description

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Cryo SEM

Cryo-samples were imaged using an FEI NOVA nanoSEM field emission scanning electron microscope (FEI Company, Hillsboro Oregon) using ET (Everhart-Thornley) dectector or the high-resolution thorough-the lens (TLD) detector operating at 5kV accelerating voltage, spot 3, ~5.0 mm working distance and 30mm aperture.

Quanta Imaging

The samples were imaged with an FEI Quanta 3D FEG scanning electron microscope (FEI Company, Hillsboro, OR) using the Everhart-Thornley (ET) detector at high vacuum. Parameters for imaging, 5kV, spot 5, 50µm aperture and working distance of ~10 mm.

Quanta EDX

x-ray analysis (EDX) was done with an Oxford INCA Xstream-2 with Xmax80 detector (Oxford Instruments, Peabody, MA) using parameters with analytical mode at 10kV, Spot 1, 1000µm objective aperture.

SEM sample preparation HMDS

Specimens were fixed in 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer, post-fixed in 1% osmium tetroxide, dehydrated in a graded ethanol series, transferred into hexamethyldisilazane and air dried. Dried specimens were coated with platinum in a Cressington 208HR sputter coater.

SEM sample preparation CPD

Specimens were fixed in 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer, post-fixed in 1% osmium tetroxide, dehydrated in a graded ethanol series, transferred into a Tousimis 931 and critical point dried. Dried specimens were coated with platinum in a Cressington 208HR sputter coater.

TEM sample preparation

Samples were fixed in 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer, post-fixed in buffered 1% osmium tetroxide containing 0.8% potassium ferricyanide, and en bloc stained in 1% aqueous uranyl acetate. They were then dehydrated with a graded series of acetonitrile and embedded in EMbed-812 resin. Thin sections (80nm) were cut on a Leica EM UC6 ultramicrotome and stained with 2% uranyl acetate and lead citrate.

TEM imaging

Images were acquired using a Gatan US1000 2K CCD camera on a FEI Tecnai G2 20 electron microscope equipped with a LaB6 source and operating at 100 kV or 200kV.