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Phloem pp 83-94 | Cite as

Super-Resolution Microscopy of Phloem Proteins

  • Ryan C. Stanfield
  • Alexander SchulzEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 2014)

Abstract

Super-resolution microscopy bridges the gap between light and electron microscopy and gives new opportunities for the study of proteins that contribute to phloem function. The established super-resolution techniques are derived from fluorescence microscopy and depend on fluorescent dyes, proteins tagged with GFP variants or fluorochrome-decorated antibodies. Compared with confocal microscopy they improve the resolution between 2.5 and 10 times and, thus, allow a much more precise (co-) localization of membranes, plasmodesmata, and structural proteins. However, they are limited to thin tissue slices rather than intact plant organs and can only show immobilized or only slowly moving targets. Accordingly, the first super-resolution micrographs of the phloem were recorded from fixed tissue which was sectioned using a vibratome or microtome. As with transmission electron microscopy, preparation of phloem tissue for super-resolution microscopy is challenged by the sudden pressures release when dissecting the functional tissue (see Chapter  2).

This chapter describes a protocol for investigation of proteins in the plasma membranes of sieve elements and companion cells. It illustrates how high-resolution fluorescence imaging can provide information that could not be obtained with confocal or electron microscopy. Further, a brief introduction outlines the theoretical background of super-resolution techniques suitable for phloem imaging and summarizes the findings of the first available super-resolution studies on the phloem. The protocol focusses on the crucial steps for super-resolution microscopy of immunolocalized phloem proteins, adjusted to the use of three-dimensional structured illumination microscopy (3D-SIM).

Key words

Aquaporins Early Nodulin Like 9 Plasma membrane domains Pore-plasmodesma units Sieve-element reticulum 

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Department of Renewable ResourcesUniversity of AlbertaEdmontonCanada
  2. 2.Department of Plant and Environmental SciencesUniversity of CopenhagenFrederiksbergDenmark

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