Carbon coating

TBEV samples were applied on standard copper grids for transmission electron microscopy with a carbon coating (Ted Pella, Redding, CA, USA), and treated in a glow discharge atmosphere for 45 s in a Pelco EasyGlow apparatus (Ted Pella, Redding, CA, USA). For NC-TEM, the samples were negatively stained with either a 1% aqueous solution of uranyl acetate or a 2% aqueous solution of ammonium molybdate. Ammonium molybdate was used as a substitute for uranyl acetate in STEM-EELS experiments because the uranium M_4,5 line precedes the phosphorus line in the characteristic electron energy loss spectra, and, thus, makes correct approximations of the background component difficult.
TEM images were obtained using a Jeol JEM-2100 electron microscope (JEOL, Japan) equipped with a LaB₆ electron source and an accelerating voltage of 200 kV. To obtain characteristic electron energy loss spectra (EELS) and to build maps of the spatial distribution of elements, the dark-field scanning transmission electron microscopy (STEM) mode, in combination with a Gatan GIF Quantum ER spectrometer (Gatan, Pleasanton, CA, USA), was used. To reduce the effects of contamination and to stabilize the sample under the electron beam in STEM, a cooling holder JEOL 21090 (JEOL, Tokyo, Japan), supporting the sample at −180 °C, was used.