Css450

Cryo-scanning electron microscopy (cryo-SEM) was applied to unravel the 3D morphology of the fat crystal flocs. The samples were crystallized following the protocols described above. Crystallization toward 20 °C or 25 °C was performed on the PE120 Peltier system of the PLM, without cover glass. Crystallization toward 0 °C was performed in a shear cell CSS450 equipped with liquid nitrogen cooling system (Linkam, Redhill, UK). After one hour of isothermal time, the sample (about the volume of a droplet) was transferred to a pre-tempered aluminum cryo-SEM stub covered with carbon tape. The sample was de-oiled by dripping 1 mL of isobutanol on top and letting it evaporate at the crystallization temperature. This was repeated four times. Lastly, 1 mL of acetone was dripped on top and also allowed to evaporate. After vitrification by means of a nitrogen slush, the sample was transferred into the cryo-preparation chamber (PP3010T Cryo-SEM Preparation System, Quorum Technologies, Lewes, UK) under vacuum and conditioned at −140 °C. All samples were sublimated for 45 min at −70 °C, sputter-coated with platinum for 90 s and visualized with a cryo-SEM JEOL JSM 7100F (JEOL Ltd., Tokyo, Japan). The SEM stage had a temperature of −140 °C, and the electron beam had an accelerated voltage of 3 keV.

1 × 10⁶ reticulocytes were resuspended in 200 μL of polyvinylpyrrolidone (PVP) solution (viscosity, 28.1; Mechatronics Instruments, the Netherlands). Samples were assayed in an ARCA (automated rheoscope and cell analyser) assay,³⁹ (link) which consists of a plate-to-plate optical shearing stage (model CSS450) mounted on a Linkam imaging station assembly and temperature controlled using Linksys32 software (Linkam Scientific Instruments, Surrey, UK) at 37°C with 3 Pa. The microscope was equipped with an LMPlanFl 50× objective lens with a 10.6-mm working distance objective (Olympus, Essex, UK) illuminated by an X-1500 stroboscope (PerkinElmer, the Netherlands) through a band-pass interference filter (center wavelength [CWL], 420 nm; full width at half maximum [FWHM], 10 nm; Edmund Optics, Poppleton, UK). Images were acquired using a uEye camera (UI-2140SE-M-GL; IDS, Obersulm, Germany). At least 1,000 cell images per sample were acquired and analyzed using bespoke ARCA software. In this analysis the distribution of the surface area of the red blood cells within the sample as observed in the ARCA assay was estimated as well as the distribution of the degree of deformation of the red blood cells due to the imposed shear stress.