Targeted imaging of specialized plant cell walls by improved cryo-CLEM and cryo-electron tomography

Abstract Cryo-focused ion beam scanning electron microscopy (Cryo-FIBSEM) has become essential for preparing cryo-lamellae. Here, we present a series of improvements that speed up and enhance the efficiency of the Serial Lift-Out and SOLIST (Serialized On-grid Lift-In Sectioning for Tomography) procedures. We extend the cryo-FIBSEM session to 5 days and eliminate the need of copper or gold block intermediates and reduce curtaining effects. Finally, we report a routine to target lamellae with a precision of approximately 1 µm in X/Y/Z. We demonstrate the power of our improvements by targeting Casparian strips, suberin lamellae, as well as xylem vessels of Arabidopsis thaliana roots. This requires reaching a target of 5 micrometers in a 3 mm long and 80-120 µm thick root section. Despite ice formation in vacuoles and to some degree in the cytosol, plasma membranes and cell walls are remarkably well preserved, providing stunning insights into the native, hydrated nano-structure of plant cell walls, previously only observable with contrasting agents in a dehydrated state.