High-pressure freezing and low-temperature processing of plant tissue samples for electron microscopy

Ichirou Karahara, Byung Ho Kang

Research output: Chapter in Book/Report/Conference proceedingChapterpeer-review

15 Scopus citations

Abstract

Use of electron tomography methods improves image resolution of transmission electron microscopy especially in the z-direction, enabling determination of complicated 3D structures of organelles and cytoskeleton arrays. The increase in resolution necessitates preservation of cellular structures close to the native states with minimum artifacts. High-pressure freezing (HPF) that immobilizes molecules in the cell instantaneously has been used to avoid damages caused by convention chemical fixation. Despite the advantages of HPF, cells could still be damaged during dissection prior to HPF. Therefore, it is critical to isolate cells/tissues of interest quickly and carefully. The samples frozen by HPF are often processed by freeze substitution (FS), and FS should be carried out under appropriate conditions. Here we describe dissection, HPF, and FS methods that we have utilized to prepare plant samples for electron tomography/immuno-electron microscopy.

Original languageEnglish
Title of host publicationPlant Cell Morphogenesis
Subtitle of host publicationMethods and Protocols
PublisherHumana Press Inc.
Pages147-157
Number of pages11
ISBN (Print)9781627036429
DOIs
StatePublished - 2014

Publication series

NameMethods in Molecular Biology
Volume1080
ISSN (Print)1064-3745

Keywords

  • Coated pit and vesicle
  • Freeze substitution
  • High-pressure freezing
  • Maize endosperm
  • Microfilament
  • Microtubule
  • Onion

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics

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