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 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.

Cell Wall Reinforcements Accompany Chilling and Freezing Stress in the Streptophyte Green Alga Klebsormidium crenulatum

“…The development of the AFU method (Buchner et al, 2020) allowed the preparation of frozen Klebsormidium filaments for further TEM investigations. To our knowledge, these are the first ultrastructural images and tomograms of frozen algal samples prepared prior to high pressure freeze fixation (HPF), together with recently published data of algal or plant tissue (Buchner et al, 2020).…”

“…The development of the AFU method (Buchner et al, 2020) allowed the preparation of frozen Klebsormidium filaments for further TEM investigations. To our knowledge, these are the first ultrastructural images and tomograms of frozen algal samples prepared prior to high pressure freeze fixation (HPF), together with recently published data of algal or plant tissue (Buchner et al, 2020). In our opinion, it is important to analyze 3-D ultrastructure of cells with different 2-D and 3-D electron microscopic methods as performed in previous studies before (Wanner et al, 2013;Steiner et al, 2018).…”

“…Low temperature preparation of Klebsormidium was performed in an AFU (for more details see Buchner et al, 2020) prior to high pressure freezing (HPF, see section "Preparation for TEM and FIB-SEM"). In the AFU, Klebsormidium was adapted to 4 • C from 20 • C with −8 • C•h −1 and subsequently cooled down with −2 • C•h −1 (starting at 4 • C).…”

“…In the AFU, Klebsormidium was adapted to 4 • C from 20 • C with −8 • C•h −1 and subsequently cooled down with −2 • C•h −1 (starting at 4 • C). Freezing was induced via transfer of ice crystals to the sample in the specimen holder (for details see Buchner et al, 2020). For each final temperature (−2 and −4 • C) three independent biological replicates (n = 3) were used.…”

Fusion of Mitochondria to 3-D Networks, Autophagy and Increased Organelle Contacts are Important Subcellular Hallmarks during Cold Stress in Plants

“…For providing sufficient resolution, e.g., for discriminating between real membrane fusions or simple surface contacts of adjacent organelles [ 32 ] and for the depiction of structural changes within organelles or degradation processes, it is indispensable to investigate subcellular structural reactions during low temperature stress by means of high-resolution, nano-scale electron microscopic methods after cryo-preservation. The freezing of plant tissue prior to high pressure freezing (HPF) was enabled by the development of an automatic freezing unit (AFU) for subsequent electron microscopic investigations [ 33 ].…”