Speaker
Description
Synchrotron-based X-ray fluorescence spectroscopy (SXRF) imaging is a unique tool for understanding the localisation and dynamics of chemical elements within plant tissues, e.g., leaves, roots, stems, flowers (including pollen), fruits, and seeds, down to subcellular-level resolution. Unlike cell cultures, 2D XRF imaging of plant specimens often requires thin sections and, hence, an imbibing medium to support and maintain the sample’s structural integrity during freezing, cryo-sectioning, and transport to the beamline’s instruments. Although optimal cutting temperature (OCT) compounds are widely regarded as the ‘gold standard’ imbibing medium for immunohistochemical analyses, usually performed between -20 and -40 °C, their use for fast-plunging cryofixation at much lower temperatures (ca. -150 to -180 °C) often results in fractured blocks, compromising or even preventing proper sectioning of the samples. Moreover, widely adopted cryofixation protocols include OCT-poured into plastic containers, such as microcentrifuge tubes, thereby creating additional insulation layers that can reduce the freezing speed and lead to elemental redistribution. Here, we present a simple, insulation-free setup and a sucrose-doped imbibition medium for the cryofixation of plant materials. The XRF imaging results obtained using leaves of crop and ornamental species at the ID-21 beamline of the European Synchrotron Radiation Laboratory (ESRF, Grenoble, France) revealed optimal preservation of structural integrity and elemental distribution in the leaf cross-sections, as well as a significant reduction in the block’s cracking incidence. Furthermore, no striking differences were observed between samples cryofixed directly in liquid nitrogen and those plunged into supercooled isopentane, suggesting that the use of isopentane, a toxic and difficult-to-handle compound, could be avoided without compromising data quality. Therefore, as a simpler and more efficient methodology, this approach is expected to be reproducible at other experimental stations dedicated to 2D X-ray fluorescence spectroscopy imaging of plant materials.