Freeze Substitution Accelerated via Agitation: New Prospects for Ultrastructural Studies of Lichen Symbionts and Their Extracellular Matrix

Abstract

settingsOrder Article Reprints Open AccessArticle Freeze Substitution Accelerated via Agitation: New Prospects for Ultrastructural Studies of Lichen Symbionts and Their Extracellular Matrix by Siegfried Reipert 1,*ORCID,Daniela Gruber 1,Norbert Cyran 1,Brigitte Schmidt 1,Rosa de la Torre Noetzel 2,Leopoldo G. Sancho 3ORCID,Michal Goga 4,Martin Bačkor 4,5ORCID andKaty Schmidt 1 1 Cell Imaging and Ultrastructural Research, University of Vienna, A-1030 Vienna, Austria 2 Department of Earth Observation, National Institute for Aerospace Technology, 28850 Madrid, Spain 3 Section of Botany, Faculty of Pharmacy, University Complutense Madrid, 28040 Madrid, Spain 4 Institute of Biology and Ecology, Pavol Jozef Šafárik University, 040 01 Košice, Slovakia 5 Institute of Biotechnology, Faculty of Biotechnology and Food Sciences, Slovak University of Agriculture, 949 76 Nitra, Slovakia * Author to whom correspondence should be addressed. Plants 2023, 12(23), 4039; https://doi.org/10.3390/plants12234039 Submission received: 21 October 2023 / Revised: 22 November 2023 / Accepted: 27 November 2023 / Published: 30 November 2023 (This article belongs to the Special Issue Microscopy Techniques in Plant Studies) Downloadkeyboard_arrow_down Browse Figures Versions Notes

Abstract (1) Background: Lichens, as an important part of the terrestrial ecosystem, attract the attention of various research disciplines. To elucidate their ultrastructure, transmission electron microscopy of resin-embedded samples is indispensable. Since most observations of lichen samples are generated via chemical fixation and processing at room temperature, they lack the rapid immobilization of live processes and are prone to preparation artefacts. To improve their preservation, cryoprocessing was tested in the past, but never widely implemented, not least because of an extremely lengthy protocol. (2) Methods: Here, we introduce an accelerated automated freeze substitution protocol with continuous agitation. Using the example of three lichen species, we demonstrate the preservation of the native state of algal photobionts and mycobionts in association with their extracellular matrix. (3) Results: We bring to attention the extent and the structural variability of the hyphae, the extracellular matrix and numerous crystallized metabolites. Our findings will encourage studies on transformation processes related to the compartmentation of lichen thalli. They include cryopreserved aspects of algal photobionts and observations of putative physiological relevance, such as the arrangement of numerous mitochondria within chloroplast pockets. (4) Conclusions: In summary, we present accelerated freeze substitution as a very useful tool for systematic studies of lichen ultrastructures.