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Super-Resolution Microscopy of Phloem Proteins

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Phloem

Part of the book series: Methods in Molecular Biology ((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).

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Author information

Authors and Affiliations

  1. Department of Renewable Resources, University of Alberta, Edmonton, AB, Canada

    Ryan C. Stanfield

  2. Department of Plant and Environmental Sciences, University of Copenhagen, Frederiksberg, Denmark

    Alexander Schulz

Authors
  1. Ryan C. Stanfield
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  2. Alexander Schulz
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Correspondence to Alexander Schulz .

Editor information

Editors and Affiliations

  1. College of Life Sciences, Northwest A&F University, Yangling, China

    Johannes Liesche

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Stanfield, R.C., Schulz, A. (2019). Super-Resolution Microscopy of Phloem Proteins. In: Liesche, J. (eds) Phloem. Methods in Molecular Biology, vol 2014. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9562-2_7

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  • DOI: https://doi.org/10.1007/978-1-4939-9562-2_7

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-4939-9561-5

  • Online ISBN: 978-1-4939-9562-2

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