ИСТИНА

Conventional cell sample preparation methods for transmission electron microscopy (TEM) based on chemical fixation at room temperature are notoriously prone to structural artifacts. On the contrary, numerous reports claim that rapid freezing techniques preserve biological specimens as close as possible to the native state [1]. An important potential application of analytical TEM is monitoring of subcellular depots of phosphorus in microalgae constituted mainly by polyphosphate (PolyP) inclusions. However, the applications of the cryo-methods to study PolyP in microalgal cells by analytical TEM are insufficiently developed so far. We compared (sub)cellular localization, elemental composition and morphology of PolyP inclusions in the cells of Chlorella vulgaris IPPAS C-1 prepared by conventional protocol based on chemical fixation at room temperature or using a protocol involving high-pressure freezing and freeze-substitution with cryofixation. The survey TEM analysis revealed significant differences neither in the degree of preservation of the PolyP inclusions nor in their morphology. The only exception was comprised by the inclusions in the chloroplast stroma which were preserved better after the cryofixation-based protocol application than after the standard chemical-fixation protocol. As revealed by analytical TEM survey (energy-dispersive X-ray analysis), minor element (Ca, Mg, K, Na) composition of the inclusions was similar after application of the both preparation protocols under our experimental conditions. This was not the case in previous works employing the chemical fixation [2] which were unable to detect K in the sample, probably due to the differences between cultivation conditions (culture media composition). To conclude, we showed that cryofixation-based protocol allowed preserving the PolyP inclusions in the chloroplast stroma to a higher degree than the standard chemical-fixation protocol. However, both protocols provided consistent results regarding the elemental composition of the PolyP inclusions. Supported by Russian Foundation for Basic Research (grants 15-54-06004, 15-34-20096). The electron microscopy studies were carried out at the User Facilities Center of M.V.Lomonosov Moscow State University. High-pressure freezing and freeze-substitution of microalgal samples were performed in research resource center «Molecular and cell technologies» of St. Petersburg State University. [1] Mielanczyk, L., Matysiak, N., Michalski, M., Buldak, R. and Wojnicz, R. (2014). “Closer to the native state. Critical evaluation of cryo-techniques for transmission electron microscopy: preparation of biological samples”. Folia Histochem. Cytobiol, 52, 1-17. [2] Adamec, J., Peverly, J.H. and Parthasarathy M.V. (1979). “Potassium in polyphosphate bodies of Chlorella pyrenoidosa (Chlorophyceae) as determined by X-ray microanalysis”. J. Phycol, 15(4), 466-468.