Unlocking New Potential in Bio-TEM: The Impact of Low Voltage Electron Microscopy and Alternative Staining Methods

Electron microscopy (EM) is a powerful imaging technique to understand and investigate the structure and organization of biological samples [1,2]. Among the various EM techniques, Transmission Electron Microscopy (TEM) has been most widely applied technique with relation to biological specimens [3]. TEM provides for visualization of the internal structure and a deeper understanding of the inner organization of biological samples [4]. Traditionally, biological TEM (Bio-TEM) images are acquired using high-voltage TEM (HV-TEM), operated at accelerating voltages of 80 kV or above. HV-TEM can only be carried out on biological samples after extensive sample processing procedures, including sample fixation, dehydration and staining. Biological specimens require contrast enhancement through staining [5,6]. In addition, staining distributes the high energy of the electron beam over a larger area of the sample, reducing beam damage to the sample – which is a challenge for biological specimen [7]. The original TEM staining protocol was introduced over 50 years ago and remains as the most common processing procedure for Bio-TEM [2], where uranyl acetate (UA) is the gold standard staining solution for imaging biological samples [5]. The classic combination of HV-TEMs with UA staining protocols is widely established for providing high resolution and detailed visualization into biological ultrastructure. However, the related disadvantages must be discussed to better understand if Bio-TEM is obligated to continue using guidelines established many decades ago. The HV-TEM is a significant investment, including capital cost, the requirement for complex infrastructure and the need for highly experienced staff. In addition, even though there currently exist an array of different staining procedures [4], these are all with only marginal adaptions to the protocol established in 1974 [6], and by and large, these protocols continue the use of toxic heavy metals, are time-consuming, and are typically performed manually, affecting reproducibility and quality [8,9]. A proposed alternative to the compulsory methodology is to replace expensive HV-TEM systems with Low Voltage Electron Microscopy systems (LVEM), and to eliminate or replace the use of heavy metal stains with lower density and safer alternatives. LVEM employes lower energies than HV-TEM and as such, provides higher contrast and less damage to sensitive soft materials [10]. In this work, LVEM was applied to understand the interaction of macrophages and viruses. To better understand the benefits of combining the use of LVEM and various alternate staining procedures, staining with either the presence or absence of UA was investigated. Briefly, cells were incubated and fixed using a paraformaldehyde and glutaraldehyde solution. After fixation, the cells underwent a series of post-fixation and staining steps, including osmium tetroxide alone or osmium tetroxide with potassium ferrocyanide, followed by “en bloc” UA staining. As a control, the UA staining step was omitted for some specimens. Embedded specimens were sectioning into ultrathin sections and post-stained with UA and lead citrate. The post-stained with UA and lead citrate was omitted for some specimens. Low voltage TEM and STEM images were acquired using the all-in-one LVEM 25E (Delong Instrument, Czech Republic). TEM images were acquired at accelerating voltage of 25 kV. STEM images were acquired at accelerating voltage of 15 kV. High voltage TEM images were acquired using Hitachi HT7800 operating at 80 kV. For all sample preparation methodologies applied, the low voltage approach was sufficient to reveal the internal structure of the cells and to localize the virus particles surround the macrophages. Contrast is particularly good for all images; however, samples submitted to post-stain procedure using UA/Lead showed a slight better delineated contour, exhibiting sharper images when compared to no post-stained samples. For the ultrathin sections, ultrastructural details of the viruses could be revealed in all images. Results were all in accordance with previous literature [7,11]. In particular, for unstained samples, both microscopes provided sufficient resolution to identify the viruses and it was possible to observe nanoscale details of the macrophage’s structure; although the cell ultrastructure was not very well defined in HV-TEM images. Overall, image contrast was satisfactory, indicating that the possibility of modifying the traditional staining procedures, by eliminating or reducing UA, was feasible; however, contrast was weaker on HV-TEM when compared to post-stained samples or unstained samples imaged on the LV-TEM. In conclusion, all the different staining procedures applied in this work allowed the visualization of the macrophages with varying detail, in the HV and LV-TEMs. The absence of UA provided lower contrast for both TEMs, but the reduction was far less significant on LVEM as compared to HV-TEM. Although HV-TEM provided increased resolution and sharpness at higher magnifications; for a virus diagnostic point of view, the low-voltage TEM was demonstrated to be a suitable alternative to high-voltage TEMs. As a diagnostic tool, LVEM showed fast speed of imaging, allowing rapid screening of diagnostic samples.

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