Chapter 16
High-Pressure Freezing and Freeze Substitution for
Transmission Electron Microscopy Imaging and
Immunogold-Labeling
Marisa S. Otegui
Abstract
Electron microscopy enables the unbiased imaging of organelles and cellular structures at nano-meter scale
resolution. The combination of cryofixation/freeze-substitution methods with other imaging techniques
such as correlative light and electron microscopy (CLEM), electron tomography (ET), and immunogoldlabeling provides unique opportunities to understand structural changes associated with cellular processes.
This chapter presents the main steps in the preparation of Arabidopsis thaliana roots, cotyledons, anthers,
and developing seeds by high-pressure freezing and freeze-substitution for structural analysis and
immunogold-labeling using transmission electron microscopy.
Key words High-pressure freezing, Freeze-substitution, Immunolabeling
1 Introduction
Modern cell biology heavily relies on electron microscopy imaging
to analyze localization of cellular components, dynamic changes of
membranes and proteins, the architecture and function of organelles, the structural features of cells and their cell walls. Traditional
transmission electron microscopy (TEM) of chemically fixed
biological materials provides limited applicability, as it is known to
introduce artifactual changes to the cellular structure. This is
because chemical fixation depends on the slow (seconds to minutes) penetration into cells and tissues and crosslinking action of
fixatives, resulting in membrane swelling, shrinking, and fusion
[1]. The problems associated with chemical fixation can be overcome by processing samples by high-pressure freezing, which is
carried out at liquid nitrogen temperature (À196
C) under high
pressure (~2100 bar) and within milliseconds, achieving nearnative cellular preservation. As high-pressure freezing instantaneously and simultaneously immobilizes all cellular components,
Jose J. Sanchez-Serrano and Julio Salinas (eds.), Arabidopsis Protocols, Methods in Molecular Biology, vol. 2200,
https://doi.org/10.1007/978-1-0716-0880-7_16, © Springer Science+Business Media, LLC, part of Springer Nature 2021
337
High-Pressure Freezing and Freeze Substitution for
Transmission Electron Microscopy Imaging and
Immunogold-Labeling
Marisa S. Otegui
Abstract
Electron microscopy enables the unbiased imaging of organelles and cellular structures at nano-meter scale
resolution. The combination of cryofixation/freeze-substitution methods with other imaging techniques
such as correlative light and electron microscopy (CLEM), electron tomography (ET), and immunogoldlabeling provides unique opportunities to understand structural changes associated with cellular processes.
This chapter presents the main steps in the preparation of Arabidopsis thaliana roots, cotyledons, anthers,
and developing seeds by high-pressure freezing and freeze-substitution for structural analysis and
immunogold-labeling using transmission electron microscopy.
Key words High-pressure freezing, Freeze-substitution, Immunolabeling
1 Introduction
Modern cell biology heavily relies on electron microscopy imaging
to analyze localization of cellular components, dynamic changes of
membranes and proteins, the architecture and function of organelles, the structural features of cells and their cell walls. Traditional
transmission electron microscopy (TEM) of chemically fixed
biological materials provides limited applicability, as it is known to
introduce artifactual changes to the cellular structure. This is
because chemical fixation depends on the slow (seconds to minutes) penetration into cells and tissues and crosslinking action of
fixatives, resulting in membrane swelling, shrinking, and fusion
[1]. The problems associated with chemical fixation can be overcome by processing samples by high-pressure freezing, which is
carried out at liquid nitrogen temperature (À196
C) under high
pressure (~2100 bar) and within milliseconds, achieving nearnative cellular preservation. As high-pressure freezing instantaneously and simultaneously immobilizes all cellular components,
Jose J. Sanchez-Serrano and Julio Salinas (eds.), Arabidopsis Protocols, Methods in Molecular Biology, vol. 2200,
https://doi.org/10.1007/978-1-0716-0880-7_16, © Springer Science+Business Media, LLC, part of Springer Nature 2021
337