Transmission Electron Microscopy of the Phloem with Minimal Artifacts
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It is a universal feature of seed plants that their phloem consists of a continuous sieve-tube system throughout the plant that is highly pressurized by its sugar contents. Cellular continuity and the pressure flow, osmotically generated in the source leaves, allow the assimilates to reach all sinks organs. However, both phloem features, the cellular continuity and the high pressure, are challenges when fixing the phloem for transmission electron microscopy. With very few exceptions, the tissue preparation necessary for the fixation evokes rapid wound responses that eventually result in artifacts.
This chapter describes the steps necessary to minimize development of artifacts in the phloem and includes preparation of fixatives, a dissection procedure that optimizes penetration of the fixatives and application to axial and lateral plant organs. Moreover, as alternative to the established fixation of fresh hand sections, we suggest a xylem-assisted perfusion fixation method for herbaceous plants. After the initial fixation, the subsequent dehydration, embedding, and ultrathin sectioning of the material follow routine procedures, which are briefly discussed, as is the orientation of samples for obtaining transverse and longitudinal phloem sections.
Key wordsAldehyde fixatives Companion cell Phloem fixation Perfusion fixation Pressure release Sieve element Transmission electron microscopy Wound response
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