A new technical approach for preparing frozen biological samples for electron microscopy
Abstract Background Many methodological approaches have focused so far on physiological and molecular responses of plant tissues to freezing but only little knowledge is available on the consequences of extracellular ice-formation on cellular ultrastructure that underlies physiological reactions. In...
Published in: | Plant Methods |
---|---|
Main Authors: | , , , , , , |
Other Authors: | |
Format: | Article in Journal/Newspaper |
Language: | English |
Published: |
Springer Science and Business Media LLC
2020
|
Subjects: | |
Online Access: | http://dx.doi.org/10.1186/s13007-020-00586-5 http://link.springer.com/content/pdf/10.1186/s13007-020-00586-5.pdf http://link.springer.com/article/10.1186/s13007-020-00586-5/fulltext.html |
id |
crspringernat:10.1186/s13007-020-00586-5 |
---|---|
record_format |
openpolar |
spelling |
crspringernat:10.1186/s13007-020-00586-5 2023-05-15T18:04:32+02:00 A new technical approach for preparing frozen biological samples for electron microscopy Buchner, Othmar Steiner, Philip Andosch, Ancuela Holzinger, Andreas Stegner, Matthias Neuner, Gilbert Lütz-Meindl, Ursula Austrian Science Fund 2020 http://dx.doi.org/10.1186/s13007-020-00586-5 http://link.springer.com/content/pdf/10.1186/s13007-020-00586-5.pdf http://link.springer.com/article/10.1186/s13007-020-00586-5/fulltext.html en eng Springer Science and Business Media LLC http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ CC-BY Plant Methods volume 16, issue 1 ISSN 1746-4811 Plant Science Genetics Biotechnology journal-article 2020 crspringernat https://doi.org/10.1186/s13007-020-00586-5 2022-01-04T15:46:20Z Abstract Background Many methodological approaches have focused so far on physiological and molecular responses of plant tissues to freezing but only little knowledge is available on the consequences of extracellular ice-formation on cellular ultrastructure that underlies physiological reactions. In this context, the preservation of a defined frozen state during the entire fixation procedure is an essential prerequisite. However, current techniques are not able to fix frozen plant tissues for transmission electron microscopy (TEM) without interrupting the cold chain. Chemical fixation by glutaraldehyde and osmium tetroxide is not possible at sub-zero temperatures. Cryo-fixation methods, such as high pressure freeze fixation (HPF) representing the state-of-the-art technique for best structural preservation, are not equipped for freezing frozen samples. In order to overcome this obstacle, a novel technical approach for maintaining the cold chain of already frozen plant samples prior and during HPF is presented. Results Different algae ( Micrasterias denticulata , Klebsormidium crenulatum ) and higher plant tissues ( Lemna sp., Ranunculus glacialis , Pinus mugo ) were successfully frozen and prepared for HPF at freezing temperatures (− 2 °C, − 5 °C, − 6 °C) within a newly developed automatic freezing unit (AFU), that we manufactured from a standard laboratory freezer. Preceding tests on photosynthetic electron transport and ability to plasmolyse show that the temperatures applied did not impair electron transport in PSII nor cell vitality. The transfer of the frozen specimen from the AFU into the HPF-device and subsequently cryo-fixation were performed without intermediate thawing. After cryo-substitution and further processing, the resulting TEM-micrographs showed excellent ultrastructure preservation of the different organisms when compared to specimens fixed at ambient temperature. Conclusions The method presented allows preserving the ultrastructure of plant cells in the frozen state during cryo-fixation. The resulting high quality TEM-images represent an important step towards a better understanding of the consequences of extracellular ice formation on cellular ultrastructure. It has the potential to provide new insights into changes of organelle structure, identification of intracellular injuries during ice formation and may help to understand freezing and thawing processes in plant tissues. It may be combined with analytical TEM such as electron energy loss spectroscopy (EELS), X-ray analyses (EDX) and various other electron microscopic techniques. Article in Journal/Newspaper Ranunculus glacialis Springer Nature (via Crossref) Plant Methods 16 1 |
institution |
Open Polar |
collection |
Springer Nature (via Crossref) |
op_collection_id |
crspringernat |
language |
English |
topic |
Plant Science Genetics Biotechnology |
spellingShingle |
Plant Science Genetics Biotechnology Buchner, Othmar Steiner, Philip Andosch, Ancuela Holzinger, Andreas Stegner, Matthias Neuner, Gilbert Lütz-Meindl, Ursula A new technical approach for preparing frozen biological samples for electron microscopy |
topic_facet |
Plant Science Genetics Biotechnology |
description |
Abstract Background Many methodological approaches have focused so far on physiological and molecular responses of plant tissues to freezing but only little knowledge is available on the consequences of extracellular ice-formation on cellular ultrastructure that underlies physiological reactions. In this context, the preservation of a defined frozen state during the entire fixation procedure is an essential prerequisite. However, current techniques are not able to fix frozen plant tissues for transmission electron microscopy (TEM) without interrupting the cold chain. Chemical fixation by glutaraldehyde and osmium tetroxide is not possible at sub-zero temperatures. Cryo-fixation methods, such as high pressure freeze fixation (HPF) representing the state-of-the-art technique for best structural preservation, are not equipped for freezing frozen samples. In order to overcome this obstacle, a novel technical approach for maintaining the cold chain of already frozen plant samples prior and during HPF is presented. Results Different algae ( Micrasterias denticulata , Klebsormidium crenulatum ) and higher plant tissues ( Lemna sp., Ranunculus glacialis , Pinus mugo ) were successfully frozen and prepared for HPF at freezing temperatures (− 2 °C, − 5 °C, − 6 °C) within a newly developed automatic freezing unit (AFU), that we manufactured from a standard laboratory freezer. Preceding tests on photosynthetic electron transport and ability to plasmolyse show that the temperatures applied did not impair electron transport in PSII nor cell vitality. The transfer of the frozen specimen from the AFU into the HPF-device and subsequently cryo-fixation were performed without intermediate thawing. After cryo-substitution and further processing, the resulting TEM-micrographs showed excellent ultrastructure preservation of the different organisms when compared to specimens fixed at ambient temperature. Conclusions The method presented allows preserving the ultrastructure of plant cells in the frozen state during cryo-fixation. The resulting high quality TEM-images represent an important step towards a better understanding of the consequences of extracellular ice formation on cellular ultrastructure. It has the potential to provide new insights into changes of organelle structure, identification of intracellular injuries during ice formation and may help to understand freezing and thawing processes in plant tissues. It may be combined with analytical TEM such as electron energy loss spectroscopy (EELS), X-ray analyses (EDX) and various other electron microscopic techniques. |
author2 |
Austrian Science Fund |
format |
Article in Journal/Newspaper |
author |
Buchner, Othmar Steiner, Philip Andosch, Ancuela Holzinger, Andreas Stegner, Matthias Neuner, Gilbert Lütz-Meindl, Ursula |
author_facet |
Buchner, Othmar Steiner, Philip Andosch, Ancuela Holzinger, Andreas Stegner, Matthias Neuner, Gilbert Lütz-Meindl, Ursula |
author_sort |
Buchner, Othmar |
title |
A new technical approach for preparing frozen biological samples for electron microscopy |
title_short |
A new technical approach for preparing frozen biological samples for electron microscopy |
title_full |
A new technical approach for preparing frozen biological samples for electron microscopy |
title_fullStr |
A new technical approach for preparing frozen biological samples for electron microscopy |
title_full_unstemmed |
A new technical approach for preparing frozen biological samples for electron microscopy |
title_sort |
new technical approach for preparing frozen biological samples for electron microscopy |
publisher |
Springer Science and Business Media LLC |
publishDate |
2020 |
url |
http://dx.doi.org/10.1186/s13007-020-00586-5 http://link.springer.com/content/pdf/10.1186/s13007-020-00586-5.pdf http://link.springer.com/article/10.1186/s13007-020-00586-5/fulltext.html |
genre |
Ranunculus glacialis |
genre_facet |
Ranunculus glacialis |
op_source |
Plant Methods volume 16, issue 1 ISSN 1746-4811 |
op_rights |
http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ |
op_rightsnorm |
CC-BY |
op_doi |
https://doi.org/10.1186/s13007-020-00586-5 |
container_title |
Plant Methods |
container_volume |
16 |
container_issue |
1 |
_version_ |
1766175922081234944 |