open access

Vol 52, No 1 (2014)
Review paper
Submitted: 2014-02-06
Accepted: 2014-04-07
Published online: 2014-05-07
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Closer to the native state. Critical evaluation of cryo-techniques for Transmission Electron Microscopy: preparation of biological samples

Lukasz Mielanczyk, Natalia Matysiak, Marek Michalski, Rafal Buldak, Romuald Wojnicz
DOI: 10.5603/FHC.2014.0001
·
Folia Histochem Cytobiol 2014;52(1):1-17.

open access

Vol 52, No 1 (2014)
REVIEW
Submitted: 2014-02-06
Accepted: 2014-04-07
Published online: 2014-05-07

Abstract

Over the years Transmission Electron Microscopy (TEM) has evolved into a powerful technique for the structural analysis of cells and tissues at various levels of resolution. However, optimal sample preservation is required to achieve results consistent with reality. During the last few decades, conventional preparation methods have provided most of the knowledge about the ultrastructure of organelles, cells and tissues. Nevertheless, some artefacts can be introduced at all stagesofstandard electron microscopy preparation technique. Instead, rapid freezing techniques preserve biological specimens as close as possible to the native state. Our review focuses on different cryo-preparation approaches, starting from vitrification methods dependent on sample size. Afterwards, we discuss Cryo-Electron Microscopy Of VItreous Sections (CEMOVIS) and the main difficulties associated with this technique. Cryo-Focused Ion Beam (cryo-FIB) is described as a potential alternative for CEMOVIS. Another post-processing route for vitrified samples is freeze substitution and embedding in resin for structural analysis or immunolocalization analysis. Cryo-sectioning according to Tokuyasu is a technique dedicated to high efficiency immunogold labelling. Finally, we introduce hybrid techniques, which combine advantages of primary techniques originally dedicated to different approaches. Hybrid approaches permit to perform the study of difficult-to-fix samples and antigens or help optimize the sample preparation protocol for the integrated Laser and Electron Microscopy (iLEM) technique.(Folia Histochemica et Cytobiologica 2014, Vol. 52, No, 1, 1–17)

Abstract

Over the years Transmission Electron Microscopy (TEM) has evolved into a powerful technique for the structural analysis of cells and tissues at various levels of resolution. However, optimal sample preservation is required to achieve results consistent with reality. During the last few decades, conventional preparation methods have provided most of the knowledge about the ultrastructure of organelles, cells and tissues. Nevertheless, some artefacts can be introduced at all stagesofstandard electron microscopy preparation technique. Instead, rapid freezing techniques preserve biological specimens as close as possible to the native state. Our review focuses on different cryo-preparation approaches, starting from vitrification methods dependent on sample size. Afterwards, we discuss Cryo-Electron Microscopy Of VItreous Sections (CEMOVIS) and the main difficulties associated with this technique. Cryo-Focused Ion Beam (cryo-FIB) is described as a potential alternative for CEMOVIS. Another post-processing route for vitrified samples is freeze substitution and embedding in resin for structural analysis or immunolocalization analysis. Cryo-sectioning according to Tokuyasu is a technique dedicated to high efficiency immunogold labelling. Finally, we introduce hybrid techniques, which combine advantages of primary techniques originally dedicated to different approaches. Hybrid approaches permit to perform the study of difficult-to-fix samples and antigens or help optimize the sample preparation protocol for the integrated Laser and Electron Microscopy (iLEM) technique.(Folia Histochemica et Cytobiologica 2014, Vol. 52, No, 1, 1–17)

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Keywords

cryo-TEM; cryo-fixation; cryo-FIB; CEMOVIS; freeze substitution; iLEM; Tokuyasu method; hybrid technique

About this article
Title

Closer to the native state. Critical evaluation of cryo-techniques for Transmission Electron Microscopy: preparation of biological samples

Journal

Folia Histochemica et Cytobiologica

Issue

Vol 52, No 1 (2014)

Article type

Review paper

Pages

1-17

Published online

2014-05-07

DOI

10.5603/FHC.2014.0001

Bibliographic record

Folia Histochem Cytobiol 2014;52(1):1-17.

Keywords

cryo-TEM
cryo-fixation
cryo-FIB
CEMOVIS
freeze substitution
iLEM
Tokuyasu method
hybrid technique

Authors

Lukasz Mielanczyk
Natalia Matysiak
Marek Michalski
Rafal Buldak
Romuald Wojnicz

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