Cyanobacterial Cells Imaged Using Uranyl-less Low Voltage Electron Microscopy

abstract

English

The studies of intracellular structures of various microorganisms rely heavily on electron microscopy, especially transmission electron microscopy (TEM). Generally, the techniques of electron miscopy require extensive sample preparation starting with fixation of ultrastructure followed by dehydration and in the case of TEM also resin embedding, sectioning and contrasting with salts of heavy metals. To date, uranyl acetate (UA) is one of the most used contrasting agents. Since the 1960s it was used for negative staining, on-section staining as well as en bloc staining. Unfortunately, because of its highly toxic properties, UA is becoming a subject of rising legal restrictions for its storage, use as well as disposal. Several studies have already focused on various lanthanoid compounds (e.g., ytterbium chloride) as possible UA replacements, but also more exotic substances such as oolong tea extracts were tested as well. Another approach, how to avoid the use of UA, is to change the imaging technique itself. If the accelerating voltage of the electron beam in TEM is lowered than the conventional 60-300kV, the contrast of the studied carbon-based specimen increases and it is possible to observe the sample without any post-staining using heavy metals. It was also proven, that low-voltage TEM was able to distinguish even areas of different compositions of the polymer-blend samples. In our study, we have focused on the study of cyanobacterial cells. Strain Synechocystis sp. PCC 6803 is a unicellular cyanobacterium significant for both industry and science because of its capability to produce various biotechnologically valuable substances such as polyhydroxyalkanoates. The samples were observed by conventional high-voltage TEM as well as by low-voltage (scanning) transmission electron microscopy to prove that electron beam voltages of 15-25kV are capable to image biological specimens without heavy metal staining and obtain results comparable to conventional staining and imaging methods.

low voltage electron microscopy; uranyl acetate; synechocystis; polyhydroxyalkanoates;

MRÁZOVÁ, K.; BAČOVSKÝ, J.; ŠEDRLOVÁ, Z.; SLANINOVÁ, E.; OBRUČA, S.; FRITZ, I.; KRYŽÁNEK, V.

13. 9. 2023

ESBP

Brno

1