The optical method
The optical method following Brodribb et al. (2016a, b) was
applied to quantify the amount of leaf xylem embolism. A healthy, mature
and undamaged leaf from each branch was fixed under a stereo microscope
(Axio Zoom.V16, Zeiss, Jena, Germany) or in optical clamps (for more
details, see http://www.opensourceov.org/). The surface area of the
leaf, which was scanned for both the stereo microscope and clamps, was
about 1 cm2. In general, images were taken every 5
min, and the water potential was simultaneously monitored with a stem
psychrometer at 10 min intervals (see below). Then, images were
processed using the Fiji version of ImageJ (Schindelin et al. ,
2012) and the “OpenSourceOV ImageJ Toolbox” was used to analyse the
images. Image stacks were made to determine changes in the brightness of
leaf veins, which was due to embolism formation. The Percentage of
Embolised Pixels (PEP) was quantified over time at decreasing xylem
water potentials, with PEP50 representing the xylem
water potential corresponding to 50% of total embolised pixels
(Brodribb et al. , 2016b).