Visualizing SARS-CoV-2 Delta Variant Ultrastructure

Heat-inactivated Delta variant of SARS-CoV-2 was diluted in 10 mM PBS (pH 7.6) to three concentrations (6.45 × 10³, 6.45 × 10⁴, and 6.45 × 10⁵ TCID₅₀/mL). The method for sample preparation was adopted from Haddad et al. (2020) (link) with slight modifications. The following steps were carried out in the fume hood. All samples were fixed by adding 2.5% glutaraldehyde (G5882, Sigma-Aldrich, USA) to the sample (1:1) for 1 h. 40 µL of the fixed sample was deposited on the pure carbon films (01840-F, Ted Pella Inc, USA) for 30 min, and a filter paper was used to carefully remove the excess liquid. The grids were then immediately stained with 1% osmium tetroxide (75632, Sigma-Aldrich, USA) and allowed to dry at room temperature. The three negatively stained carbon grids were then placed on the microscope stub using a double-sided adhesive tape and sputtered with a 10 nm-thick gold layer using a turbomolecular pumped coater Q150T ES (Quorum Technologies, UK) for 10 min, to reduce charging of the non-conductive samples. The micrographs were acquired using the scanning electron microscope crossbeam 540 (Carl Zeiss, Germany). The SEM allowed to observe samples under high vacuum pressure, and the images were acquired at magnifications ranging from 5 kX to 200 kX using an electron high tension (EHT) of 5 kV and a high-resolution energy selective backscattered (ESB) electron detector.