Fei tecnai g20

For TEM, cells were fixed and processed as described for SBF-SEM up to the embedding step. The cells were embedded in Epon without fillers, sectioned to 50 nm on a Leica Ultracut Microtome (Leica), and transferred to copper mesh grids. Electron microscopy was performed on an FEI Tecnai G20 (FEI/ Thermo Fisher Scientific), and images were acquired on an Olympus Veleta side-mounted camera (Olympus). A simplified staining protocol was used for the TEM-CLEM experiments with the HCMV-Merlin-pAL1502 variants: Before spinning-disk microscopy, the infected cells were fixed with 4% PFA in PBS at 37°C and 5% CO₂ for 10 min. Afterwards, cells were postfixed at 4°C with 2.5% GA in PBS O/N and stained on ice using 1% OsO₄/PBS for 30 min and 2% UA/H₂O for 30 min. Subsequently the sample was dehydrated through a series of increasing Ethanol concentrations in water (30%, 50%, 70% and 3x 100%; each 10 minutes on ice). Subsequently, the sample was infiltrated by increasing concentrations of Epon in Ethanol (50% for 30 min, 70% 1:30h, 100% O/N) before polymerization at 60°C for 2 days. Sectioning was performed as described above. Before TEM, the sections were post-stained for 7 minutes with a saturated solution of UA in 70% Ethanol/H₂O.

Au NPs were prepared according to a previously published sodium citrate reduction method with minor adjustment [24 (link)]. Briefly, 30 mL of distilled water and 300 µL of 1% (w/v) HAuCl₄·4H₂O were added into a 100 mL clean conical flask. The mixture was stirred by a magnetic stirrer at a speed of 250 rpm and rapidly heated to boil. As soon as the solution was boiling, 180 µL of 1% (w/v) sodium citrate tribasic dihydrate solution was added into the solution. After boiling for about 5 min, when the color of the colloid turned to cloudy claret, the conical flask was removed from heat and the synthesized Au NPs colloid was cooled to room temperature. After cooling, the synthesized Au NPs colloid was stored at 4 °C for further use. The diameter of the prepared Au NPs was analyzed using a Litesizer 500 Zeta potentiometer (Anton Paar, Graz, Austria) using 3 mL at 1:50 (Stock:H₂O) dilution. TEM images were collected using a FEI Tecnai G20 (FEI Company, Hillsboro, OR, USA) with 1:5 (Stock:H₂O) dilution dried on carbon-coated copper grid.