Low voltage electron microscopy: uranyl-less alternative for imaging cyanobacterial cells

Low voltage electron microscopy: uranyl-less alternative for imaging cyanobacterial cells

Abstract

Uranyl acetate (UAc) has been used as a contrasting agent for biological samples in transmission electron microscopy since the 1960s and is still one of the crucial chemical compounds used in electron microscopy. Unfortunately, because of its high toxicity and mild radioactive properties, UAc is becoming subject to legal restrictions for its usage and disposal. Several studies have therefore focused on finding a suitable substitute and found several promising lanthanoid substances capable of similar staining as UAc. Moreover, some more exotic substances, such as oolong tea extracts, were suggested as possible contrasting solutions. However, if the imaging technique itself is altered, specifically if the voltage of the primary electron beam is lowered, it is possible to observe carbon-based specimens without using additional staining agents. Low-voltage electron microscopy (LV-EM) was employed in the study of polymer substances and was proven to be capable of distinguishing even areas of different chemical compositions using a voltage of 5kV. Unfortunately, for such low voltages of the electron beam, sample preparation is considerably more challenging, because it is necessary for the sample to be even thinner than common ultrathin sections. However, if the voltage is increased to 25kV, the electrons have sufficient energy to penetrate conventional samples of 70nm thickness. In our study, we employed low-voltage transmission microscopy in the study of cyanobacteria. Samples were observed using both TEM (25kV) as well as STEM (15kV) to investigate the possibility of reducing the chromatic aberration and the results were compared with the standard imaging of stained samples followed by high-voltage TEM (200kV).

Uranyl acetate (UAc) has been used as a contrasting agent for biological samples in transmission electron microscopy since the 1960s and is still one of the crucial chemical compounds used in electron microscopy. Unfortunately, because of its high toxicity and mild radioactive properties, UAc is becoming subject to legal restrictions for its usage and disposal. Several studies have therefore focused on finding a suitable substitute and found several promising lanthanoid substances capable of similar staining as UAc. Moreover, some more exotic substances, such as oolong tea extracts, were suggested as possible contrasting solutions. However, if the imaging technique itself is altered, specifically if the voltage of the primary electron beam is lowered, it is possible to observe carbon-based specimens without using additional staining agents. Low-voltage electron microscopy (LV-EM) was employed in the study of polymer substances and was proven to be capable of distinguishing even areas of different chemical compositions using a voltage of 5kV. Unfortunately, for such low voltages of the electron beam, sample preparation is considerably more challenging, because it is necessary for the sample to be even thinner than common ultrathin sections. However, if the voltage is increased to 25kV, the electrons have sufficient energy to penetrate conventional samples of 70nm thickness. In our study, we employed low-voltage transmission microscopy in the study of cyanobacteria. Samples were observed using both TEM (25kV) as well as STEM (15kV) to investigate the possibility of reducing the chromatic aberration and the results were compared with the standard imaging of stained samples followed by high-voltage TEM (200kV).

low voltage electron microscopy; (S)TEM; synechocystis; polyhydroxyalkanoates;

low voltage electron microscopy; (S)TEM; synechocystis; polyhydroxyalkanoates;

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

03.10.2023

Czech-BioImaging

Hradec Králové

Imaging Principles of Life: Book of Abstracts

72

73

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