2.9 Secondary ion mass spectrometry (SIMS)
Roots of two-day-old plants treated with 5 μM Al for 2, 6, 24, and 72 h were excised and fixed in 2.5% glutaraldehyde and 1.6% paraformldehyde at 4 °C overnight, followed by 1% osmium tetroxide for 2 h. Then, the fixed root samples were dehydrated acetone for 3 times. The root samples were embedded in Spurr resin overnight, and polymerized at 70 °C for 24 h. Five-micrometer-thick section was cut with Leica RM2165 microtome (Leica, Germany), and placed on the silicon wafer (University Wafer, USA). The element distribution in the cross-section of different root zones was analyzed using SIMS (CAMECA, France) according to Smart et al. (2010). The sample was pre-sputtered with 30 nA O2+ beam for 15 min to remove the potential contaminants on the sample surface, and ion maps of27Al of root cross-section were acquired using 300 pA O2+ beam for 20 min, and ion map of31P was acquired using 200 pA Cs+beam for 30 min, with a resolution of 256 × 256 pixels. SIMS data was analyzed using WinImage 2.0 (CAMECA, France).