Reipert, SiegfriedGruber, DanielaCyran, NorbertSchmidt, BrigitteDe la Torre Noetzel, RosaGarcía Sancho, LeopoldoGoga, MichalBačkor, MartinSchmidt, Katy2024-05-282024-05-282023-11-30Reipert 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):4039.10.3390/plants12234039https://hdl.handle.net/20.500.14352/1045102023 Descuento MDPI Slovak Research and Development Agency, contract No. APVV-21-0289 Slovak Grant Agency KEGA, contracts No. 008SPU-4/2023 and No. 009UPJŠ-4/2023 Field campaigns for the collection of C. gyrosa were financed by grants from the Spanish Ministry of Science and InnovationBackground: 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.engAttribution 4.0 Internationalhttp://creativecommons.org/licenses/by/4.0/journal article2223-7747https://doi.org/10.3390/plants12234039open access582.29581.6LichensAlgal photobiontsExtracellular matrixFreeze substitutionSample preparationTransmission electron microscopyBotánica (Farmacia)Biología celular (Farmacia)Biotecnología2417 Biología Vegetal (Botánica)2407 Biología Celular2203.04 Microscopia Electrónica