Euglena gracilis is a unicellular fresh water photosynthetic flagellate at which a bleaching phenomenon was described. Using antibacterial compounds like quinolones and cumarins, the photosynthetic activity may be destroyed and results in irreversible elimination of chloroplasts. Beside the loss of chloroplasts in E. gracilis, damage and ultrastructural transformation of mitochondria leading to a formation of giant mitochondria has been also shown [1,2,3]. In this study, we used serial block face scanning electron microscopy (SBF-SEM) for 3D reconstruction of E.gracilis to see whether the giant mitochondria are inside the bleached non-photosynthetic mutant cells or they were just an artifact/myth caused during specimen preparation.  

For SBF-SEM, biological samples are standardly prepared according the protocol described by Deerinck et al. (2010) In this case, we needed the ultrastructure preservation of our sample as close as possible to the native state, therefore we decided to use high pressure freezing followed by the freeze substitution method. To increase the image contrast in backscatter electron imaging, we added soluble salts containing heavy metals in freeze substitution solutions. We tested four modified protocols in which we used various combinations of the following compounds: osmium tetroxide, thiocarbohydrazide, potassium ferricyanide, lead nitrate, uranyl acetate, phosphotungstic acid.   Resin embedding was done using low viscosity Spurr (EMS) at room temperature.

At first, the ultrathin sections were cut from polymerized blocks using the ultramicrotome (Leica EM UC6).  Sections were examined in transmission electron microscope (TEM, JEOL 1010) and the contrast of mitochondria was compared in recorded images of mutant cells prepared by modified and standard Deerinck protocols (Fig.1). The sample with the highest contrast was roughly trimmed by a razor blade and mounted on the stub using a superglue and conductive colloidal silver paint (EMS). Small pyramids with square block face in size appr. 100x100x100 µm were prepared using a diamond trimming tool (Diatome) or glass knife in the microtome.

The high resolution SEM (Tescan Maia3 XMU FEG) equipped with ultramicrotome (Gatan 3View2XP) in the microscope chamber was used for the collection of images recorded at the accelerating voltages 1.5 and 3.0 kV. More than 1000 slices with the thickness of either 30 or 50 nm were cut from sample pyramids with the oscillating diamond knife working at cutting speed 0.5 mm/s. To prevent the sample charging, the image acquisition was performed at the chamber pressure 50 Pa. Gaussian 3D filter in Image J software was applied to recorded images to reduce a noise. 3D model was created using automatic segmentation tool in the Amira software package.

The resulting 3D reconstructions did not prove the presence of giant mitochondria in the mutant cells. However SBF-SEM in the combination with the current methods of specimen preparation has proven to be the method of choice allowing the study of the distribution of cell organelles and their mutual position in the whole cell volume.

References:

[1] L. Ebringer, J. Polónyi, J. Krajcovic, Arzneimittel-Forschung, 43(7), (1993), 777–81.

[2] J.Polonyi  et al., Folia Microbiol. 43(6), (1998),661-66.

[3] J.Polónyi et al., Zeitschrift für mikroskopisch-anatomische Forschung, 104(1),(1990), 61-78.

[4] The study was supported by the Technology Agency of the Czech Republic (TE01020118) and from the program for large research infrastructures of the Ministry of Education, Youth and Sports within the project „National Infrastructure for Biological and Medicinal Imaging  (Czech-BioImaging LM2015062)

Figures:

The comparison of the mitochondria contrast in mutant cells of E.gracilis stained by various ways during freeze substitution: A. OTO in acetone. B. UA and ferricyanide in acetone. C. OTO, UA and lead nitrate in methanol. D. OTO, UA and PTA in methanol.

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EMC Abstracts - https://emc-proceedings.com/abstract/3d-reconstruction-of-euglena-gracilis-using-serial-block-face-scanning-electron-microscopy/