Gemini sem 300 fesem

Stomata cryo‐SEM observations were performed in three plants per cultivar that were kept in a dark chamber overnight. Leaf samples were collected in dark conditions using a red headlight lamp in a very low intensity (0–23 μmol m⁻² s⁻¹) and for no more than 3–5 s (the time needed to punch and drop the leaf sample in N2), avoiding any response of the stomata (Raven 2014). Three leaf discs (9 mm diameter) from different leaves were sampled with a punch holder (“Biopsy Punch”) from the middle zone of the lamina between major veins. Leaf samples were immediately mounted in copper specific supports with cryo‐glue, freshly made up as a 50/50 mixture of colloidal graphite and Tissue‐Tek mounting medium, and frozen in slush nitrogen (mix of solid and liquid nitrogen). Frozen samples were transferred under nitrogen to the preparation chamber (maintained at −140°C) in which sublimation at −95°C for 5 min and metallization with Platinum at 10 μA for 15–20 s was performed. Finally, samples were transferred under vacuum to the cold stage of the microscope chamber in which stomata were observed and photographed in high vacuum mode at 3kV acceleration voltage. Cryo‐SEM observations were carried out in a Gemini SEM 300 FESEM (ZEISS, Germany) microscope coupled with a CRYO‐SEM PP3010T module (Quorum Technologies, England).

The samples were prepared by filling a rivet with the NF0 samples (3 h, 6 h and 24 h), itself mounted on a stub, at room temperature. Freezing was carried out by plunging the stub in liquid nitrogen then vacuum pumping until nitrogen slush formed. Afterwards, the frozen sample was transferred to the preparation chamber (−140 °C), fractured and sublimed (−95 °C). The sublimation was conducted for 7 min (NF0_3 h or 24 h) or 8 min (NF0_6 h), following by sputtering Platinum for the sample metallization for 35 s (NF0_3 h) or 40 s (NF0_6 h or 24 h) to increase conductivity. Specimens were then put into the SEM cryo stage (−140 °C) to be observed, using CRYO-SEM PP3010T (Quorum Technologies, Lewes, England). Observations were done at 3 kV, in high vacuum mode using a GeminiSEM 300 FESEM (ZEISS, Dresden, Germany).