Em vct100 cryo shuttle

The 30 wt.% pPrOzi₁₀₀- b-pEtOx₁₀₀ hydrogel was also visualized with cryo-SEM. Samples were rapidly frozen in slushed nitrogen at −210 °C after placing them between aluminum plates (d = 3 mm) with a 2 mm notch for sample fixation. All the following transfer steps were performed at −140 °C with a EM VCT100 cryo-shuttle (Leica Microsystems). To generate a freshly fractured hydrogel surface, one of the aluminum plates was knocked off and freeze etched for 15 min at −85 °C under high vacuum (<1 × 103 mbar) in a Sputter Coater machine (ACE 400, Leica Microsystems). Afterward, samples were sputtered with 3 nm platinum and transferred to the SEM chamber (Crossbeam 340, Zeiss). Images of the hydrogel surface morphology were taken at −140 °C using an acceleration voltage of 2–8 kV. The images were further processed in ImageJ software (1.46 r, revised edition). The same process was also applied for cell-laden hydrogels.

In
comparison to cryo-TEM, relatively much higher plain polymer concentrations,
i.e., 50 and 100 g/L (as free-flowing liquid solution) were visualized
with cryo-SEM. The samples were rapidly frozen in slushed nitrogen
at −210 °C after being placed between aluminum plates
(d = 3 mm) with a 2 mm notch for sample fixation.
All the following transfer steps were performed at −140 °C
with an EM VCT100 cryo-shuttle (Leica Microsystems). To generate a
freshly fractured polymer surface, one of the aluminum plates was
knocked off and freeze etched for 15 min at −85 °C under
high vacuum (<1 × 10³ mbar) in a Sputter Coater
machine (ACE 400, Leica Microsystems). Afterward, samples were sputtered
with 3 nm of platinum and transferred to the SEM chamber (Crossbeam
340, Zeiss). The images of the polymer surface morphology were taken
at −140 °C using an acceleration voltage of 8 kV. The
images were further processed in ImageJ software (1.46 r, revised
edition).