Freezing xylem conduits with liquid nitrogen creates artifactual embolisms in water-stressed broadleaf trees

Key message: Freezing with liquid nitrogen promotes the expansion of air in water columns of water-stressed, intact trees that are not transpiring. Abstract: In analyses of the distribution of water-filled and embolized conduits, xylem sap is frozen with liquid nitrogen and visualized using cryo-scanning electron microscopy (cryo-SEM). However, artifacts may be introduced during preparation of samples for these analyses. If the xylem is frozen intact, conduits may embolize during freezing when xylem water potential (Ψ_xylem) is substantially negative, whereas rehydration to release negative pressure may induce artifactual refilling. To evaluate these sampling phenomena during dehydration and rehydration, we monitored dynamic changes in xylem functional status in the stem of Dendropanax trifidus by compact magnetic resonance imaging (CMRI). We also visualized the water distribution in xylem (under low Ψ_xylem and with no transpiration) after freezing D. trifidus and Carpinus tschonoskii stems with liquid nitrogen. Artifactual refilling of embolized conduits was sometimes observed in D. trifidus samples cut underwater prior to freezing; however, the extent of refilling may have no influence on hydraulic measurements. Living cells in all samples were dramatically restored after rehydration by cutting underwater. In the current-year xylem, intact saplings of C. tschonoskii frozen with liquid nitrogen after dehydration had a greater number of embolized vessels than samples cut underwater. When a low Ψ_xylem (≤−2.5 MPa) was maintained, artifactual embolisms were rarely observed in samples directly frozen after rehydration. Thus, relaxation of negative Ψ_xylem and rehydration are important factors for consideration when assessing the distribution of embolized conduits by cryo-SEM.