Fig. 1.

Experimental design and manual analysis of infection in Vero E6 cells using SARS-CoV-2 variants D614G and B.1.1.7. A Fluorescence enhancement of the BEEF-CP dye upon Ca²⁺ binding is attributed to the alleviation of the photoinduced electron transfer (PET) quenching. Its low background fluorescence and high two-photon cross-section (TPCS) along with its advantageous cell internalization make BEEF-CP an appropriate candidate for cytosolic Ca²⁺ imaging with 2P microscopy. B Experimental design with 2P microscopy. A pulsed Ti:Sa laser source was used for excitation at 700 nm wavelength, and laser light was diffracted with a pair of galvo mirrors for scanning the region of interest. A high-numerical-aperture objective (Olympus 20×) was used to obtain subcellular resolution. Samples were prepared in 96-well bioassay plates and transferred directly to the microscope. Samples were moved semi-automatically to locate the different wells in the field of view of the microscope. C Exemplary images from samples where most cells correspond to a particular stage of the infection (see “Methods” for more details on the characterization criteria). D The relative number of cells in different stages of infection 48 h post infection for the two variants at two different dye concentrations at various virus titers. Data from wells with the same virus titer, variant, and dye concentration are averaged. Statistical data for the individual cell categories are shown in Supplementary Fig. 5. E 50th percentile values, i.e., the percentage of cells under the median severity of infection, for the data displayed in D. Statistical analysis of the manual cell sorting shows that (i) the ratio of healthy cells is significantly higher with lower virus titer, regardless of the virus variant or dye concentration; and (ii) for high infection levels (TCID₅₀ > 10^–3 mL⁻¹), the proportion of absent cells is higher for the D614G variant