Prior to the measurements of samples at 77 K, we first validated our microscopy setup by detecting fluorescence from fluorescently labelled beads of two different sizes (Fig. ). Fluorescent beads were prepared on the TEM grid using plunge-freezing (section 2.3). We confirmed that both 780 nm and 200 nm-sized FL beads were detectable in our cryo-FM image when illuminated with 40 W/cm 2 at 520 nm laser excitation wavelength.