Transmission Electron Microscopy of the Phloem.

A universal feature of seed plants is that their phloem consists of a continuous sieve-tube system extending throughout the plant, which is highly pressurized due to its sugar content. This cellular continuity, combined with the pressure-driven, osmotically generated flow in the source leaves, enables assimilates to reach all sink organs. However, these two key features of the phloem, the cellular continuity and high pressure, pose significant challenges when attempting to fix the phloem for transmission electron microscopy. In most cases, the tissue preparation required for fixation triggers rapid wound responses that eventually result in artefacts. This chapter describes the steps necessary to minimize artefact formation during phloem fixation. It includes the preparation of fixatives, a dissection procedure designed to optimize fixative penetration, and their application to both axial and lateral plant organs. Additionally, as an alternative to the conventional fixation of fresh hand sections, we suggest a xylem-assisted perfusion fixation method for herbaceous plants. Following the initial fixation, the subsequent steps of dehydration, embedding, and ultrathin sectioning are carried out using standard procedures, which are briefly discussed. The chapter also addresses sample orientation to obtain transverse and longitudinal sections of the phloem.