Freeze Substitution Accelerated via Agitation: New Prospects for Ultrastructural Studies of Lichen Symbionts and Their Extracellular Matrix
Abstract
:1. Introduction
2. Results
2.1. FS under Continuous Agitation—A Means to Cryopreserve Lichens for TEM
2.2. Improvement in Section Quality by Using an Ultrasonic Diamond Knife
2.3. Cryoimmobilized Hyphal/Algal Junction in X. parietina Displays Patterns of Intraparietal Cell Wall Contact
2.4. Cryopreparation Suits the Preservation of Photobionts at Various Degrees of Hydration
2.5. FS Enables Superb Preservation of the ECM in Association with Hyphae
2.6. FS Draws Attention on Structural Diversity of Hyphae
2.7. FS Preserves Crystalline Metabolites inside the Thallus
3. Discussion
4. Materials and Methods
4.1. Sample Collection
4.2. Chemical Fixation and Embedding at Room Temperature
4.3. Freezing
4.4. Freeze Substitution under Agitation
4.5. Light Microscopy
4.6. Sectioning
4.7. Contrasting and TEM
Supplementary Materials
Author Contributions
Funding
Data Availability Statement
Acknowledgments
Conflicts of Interest
Abbreviations
References
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Short FS under agitation | Long FS under agitation |
HPF sample transfer onto liquid N2-frozen substitution medium (1% OsO4 in acetone) in Sarstedt tubes | |
Insertion of the filled tubes in the agitation module within the precooled AFS (ca. −104 °C programmed) | |
FS for 10 h at−85 °C | FS for 60 h at−85 °C |
Warming up slope (5°−6 °C/h) | |
−90 °C for 2 h | |
Warming up slope (15 °C/h) | |
60 °C for 2 h | |
Warming up slope to room temperature (20 °C/h) | |
1 h room temperature (to enhance contrast by OsO4) | |
Removal of the Sarstedt tubes from the AFS for washing and embedding of samples at room temperature |
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Reipert, S.; Gruber, D.; Cyran, N.; Schmidt, B.; de la Torre Noetzel, R.; Sancho, L.G.; Goga, M.; Bačkor, M.; Schmidt, K. Freeze Substitution Accelerated via Agitation: New Prospects for Ultrastructural Studies of Lichen Symbionts and Their Extracellular Matrix. Plants 2023, 12, 4039. https://doi.org/10.3390/plants12234039
Reipert S, Gruber D, Cyran N, Schmidt B, de la Torre Noetzel R, Sancho LG, Goga M, Bačkor M, Schmidt K. Freeze Substitution Accelerated via Agitation: New Prospects for Ultrastructural Studies of Lichen Symbionts and Their Extracellular Matrix. Plants. 2023; 12(23):4039. https://doi.org/10.3390/plants12234039
Chicago/Turabian StyleReipert, Siegfried, Daniela Gruber, Norbert Cyran, Brigitte Schmidt, Rosa de la Torre Noetzel, Leopoldo G. Sancho, Michal Goga, Martin Bačkor, and Katy Schmidt. 2023. "Freeze Substitution Accelerated via Agitation: New Prospects for Ultrastructural Studies of Lichen Symbionts and Their Extracellular Matrix" Plants 12, no. 23: 4039. https://doi.org/10.3390/plants12234039