Ashless filter paper

Olive leaves were harvested in April from trees of the cv Coratina present in the CREA OFA experimental olive orchard located in Rende (Italy). The cv Coratina was chosen for its high phenolic, and, in particular, oleuropein content in leaves [30 (link),31 (link),32 (link)]. The leaves, immediately after harvesting, were cleaned and subjected to mechanical crushing in water by means of a blender (1.5 kg of leaves in 3 L of distilled water). To facilitate the phenolic extraction, the mixture was kept under shaking in an ultrasonic bath (Fisher Scientific, Milan, Italy) for 15 min. To obtain the mechanical separation of the liquid component from the solid one, the paste obtained was subjected to centrifugation at 10,000 rpm for 5 min. The supernatant (aqueous phase) was collected and subjected to a second centrifugation at 10,000 rpm for 5 min and then to filtration on ashless filter paper (Whatman International Ltd., Maidston, England) to better clean the solution. Aliquots of extract subsequently filtered through PTFE filters (0.45 µm, Millipore Merk, Darmstadt, Germany) were analyzed for single phenols by LC-MS/MS and for total polar phenols by HPLC.

Holey carbon film 200-mesh R2/1 C-Flat grids (Protochips) were first glow discharged on the carbon film side before depositing 3 µl of 0.5 mg ml⁻¹ suspended graphene oxide sheets (Sigma-Aldrich) for 60 s before blotting and air drying over several hours. The grid was UV treated before depositing 3 µl of protein solution at ~0.5 mg ml⁻¹ for 60 s at the opposite of the carbon film side. The grid was blotted with ash-less filter paper (Whatman) and plunge frozen in liquid ethane using EMGP (LEICA) under controlled temperature, hygrometry respectively at 4 °C and 80% humidity. The grids were transferred and stored in liquid nitrogen before imaging. Electron microscopy images were recorded on a FEI Titan Krios electron microscope at 300 keV equipped with a post-column GIF Quantum energy filter (20 eV) (Gatan). Dose fractionated images were recorded on a K2 Summit direct detector (GATAN) in a counting mode with a pixel size of 1.36 Å. A data set of 3358 micrographs was collected over a 72 h session. Each micrograph was collected as 40 movie frames for 7 s with a dose rate of 6 e Å⁻² s⁻¹. The total dose was about 42 e Å⁻². Images were recorded using the automated acquisition program EPU (FEI) with defocus values ranging from −1 to −2.4 µm, a spot size of 6, a condenser aperture of 50 µm, and an objective aperture of 100 µm.