Low Temperature Scanning Electron Microscopy (LTSEM) Findings on the Ultrastructure of Trebouxia lynnae (Trebouxiophyceae, Lichenized Microalgae)

Abstract

The lichenized green microalga Trebouxia lynnae Barreno has been recently described and is considered a model organism for studying lichen chlorobionts. Its cellular ultrastructure has already been studied in detail by light, electron, and confocal microscopy, and its nuclear, chloroplast and mitochondrial genomes have been sequenced and annotated. Here, we investigated in detail the ultrastructure of in vitro grown cultures of T. lynnae observed by Low Temperature Scanning Electron Microscopy (LTSEM) applying a protocol with minimum intervention over the biological samples. This methodology allowed for the discovery of ultrastructural features previously unseen in Trebouxiophyceae microalgae. In addition, original Transmission Electron Microscopy (TEM) images of T. lynnae were reinterpreted based on the new information provided by LTSEM. The nucleolar vacuole, dictyosomes, and endoplasmic reticulum were investigated and reported for the first time in T. lynnae and most likely in other Trebouxia lineages.

Visualisation of microalgal lipid bodies through electron microscopy

“…32 Nevertheless, if a flat sample surface is obtained by freeze-fracturing, then this cutting method seems also useful for the detection of organelles such as mitochondria in microalgae. 37 These were however not observed in the species tested in our study, indicating that obtaining a clear internal structure with cryo-planing remains sample dependent. Using a combination of both freeze-fracturing and cryo-planing can have an added value to observe different aspects of the same sample.…”

“…For freeze-substitution, frozen carriers were transferred to a Leica EM AFS2 (Leica Microsystems GmbH, Austria), where the samples were incubated in 2 mL Eppendorf tubes in a mixture of 1% osmium tetroxide, 0.5% glutaraldehyde (EMS, Hatfield, Pennsylvania, USA) and 1% water in water free acetone (VWR, Leuven, Belgium) at −90 • C for 24 h. The temperature was gradually raised by 2 • C/h to −60 • C, where the samples were incubated for 8 h, followed by a further increase by 1.3 • C/h to -30 • C. The samples were washed three times with dried acetone and incubated for an additional 24 h. Subsequently, the temperature was raised by 2 • C/h to 0 • C, where the samples were incubated for 15 h, followed by a final increase by 2 • C/h to room temperature. 35,37 Next, the samples were embedded by incubating them in increasing concentrations of Spurr resin (1/3, 2/3, 3×100%) (EMS, Hatfield, Pennsylvania, USA) diluted in dried acetone for 8 h each. After resin infiltration, the specimen-discs were released from the carriers using a forceps and embedded in flowthrough capsules (5×15 mm, Leica Microsystems, Vienna, Austria), which were placed in size-1 gelatine capsules (Agar scientific, Stansted, UK) before being polymerised at 65 • C for 24 h. Resin blocks were prepared for sectioning and trimmed with a Trim 45 diamond knife (Diatome AG, Nidau, Switzerland) into a trapezoidal pyramid shape.…”

“…Only in one study lipid bodies were identified in microalgae using cryo-SEM preceded by freezefracturing. 37 Lipid bodies were recognised by the lack of a clear membrane. This was feasible as the freeze-fracturing resulted in a rather flat cell and lipid body surface, 37 in contrast to the rough cell surface observed in the P. tricornutum cells.…”

“…37 Lipid bodies were recognised by the lack of a clear membrane. This was feasible as the freeze-fracturing resulted in a rather flat cell and lipid body surface, 37 in contrast to the rough cell surface observed in the P. tricornutum cells. The cryo-planed samples, however, showed more flat cell surfaces.…”

“…Hereby, sample layers are either removed by cryo‐ultramilling (a rotating chisel mills away sample) 34 or by cryo‐(ultra)microtomy (removing sample slices by use of a cryo‐(ultra)‐microtome) 36 until the region of interest is exposed. Particularly for lipid body research in microalgae, freeze‐fracturing followed by cryo‐SEM has only been scarcely used, 37 while cryo‐planing has not been explored so far. The latter technique has however been used on several other samples such as plants and yeasts 32,38,39 …”