Cryo-SEM specimen preparation and analysis:
For cryogenic scanning electron microscopy (cryo-SEM) imaging: A Zeiss
Ultra Plus high-resolution SEM, equipped with a Schottky field-emission
gun and with a BalTec VCT100 cold-stage maintained below -145 °C.
Specimens were imaged at low acceleration voltage of 1.4kV, and working
distances of 3-5 mm. We used the Everhart Thornley (“SE2”) secondary
electron detector.
Specimens were prepared by the drop plunging method: A 3 μL drop of
solution is set on top of a special planchette maintaining its droplet
shape and is manually plunged into liquid ethane, after which it is set
on top of a specialized sample table set in a liquid nitrogen pool. The
frozen specimens are then mounted on a specialized sample table under
liquid nitrogen, and transferred by a high vacuum cryo-transfer shuttle
(VCT100; Bal-Tec) to a freeze-fracture system (BAF060; Leica) where it
is kept at -170 °C. In the BAF060, the frozen droplets are fractured by
a rapid stroke from a cooled knife, exposing their inner part. They are
then transferred into the pre-cooled HR-SEM as described above. Ideally,
imaging is performed as close as possible to the drop surface, where
cooling rate should be maximal.