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. 2021 Mar 27;11(4):859.
doi: 10.3390/nano11040859.

A Guide for Using Transmission Electron Microscopy for Studying the Radiosensitizing Effects of Gold Nanoparticles In Vitro

Ioanna Tremi  1   2 , Sophia Havaki  2 , Sofia Georgitsopoulou  3 , Nefeli Lagopati  2 , Vasilios Georgakilas  3 , Vassilis G Gorgoulis  2   4   5   6 , Alexandros G Georgakilas  1
Affiliations

A Guide for Using Transmission Electron Microscopy for Studying the Radiosensitizing Effects of Gold Nanoparticles In Vitro

Ioanna Tremi et al. Nanomaterials (Basel). .

Abstract

The combined effects of ionizing radiation (IR) with high-z metallic nanoparticles (NPs) such as gold has developed a growing interest over the recent years. It is currently accepted that radiosensitization is not only attributed to physical effects but also to underlying chemical and biological mechanisms' contributions. Low- and high-linear energy transfer (LET) IRs produce DNA damage of different structural types. The combination of IR with gold nanoparticles may increase the clustering of energy deposition events in the vicinity of the NPs due to the production mainly of photoelectrons and Auger electrons. Biological lesions of such origin for example on DNA are more difficult to be repaired compared to isolated lesions and can augment IR's detrimental effects as shown by numerous studies. Transmission electron microscopy (TEM) offers a unique opportunity to study the complexity of these effects on a very detailed cellular level, in terms of structure, including nanoparticle uptake and damage. Cellular uptake and nanoparticle distribution inside the cell are crucial in order to contribute to an optimal dose enhancement effect. TEM is mostly used to observe the cellular localization of nanoparticles. However, it can also provide valuable insights on the NPs' radiosensitization pathways, by studying the biochemical mechanisms through immunogold-labelling of antigenic sites at ultrastructural level under high resolution and magnification. Here, our goal is to describe the possibilities, methodologies and proper use of TEM in the interest of studying NPs-based radiosensitization mechanisms.

Keywords: gold nanoparticles; immunocytochemistry; immunogold-labelling; radiosensitization; silver-enhancement; transmission electron microscopy (TEM).

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Representative TEM images of gold nanoparticle uptake in PC3 cells. (a) Image depicts cellular uptake of 15 nm citrate-capped GNPs; (b) Image depicts cellular uptake of 15 nm PEG-capped GNPs. Nanoparticles are located in vesicles (double arrows) and autophagosomes (thick arrows) but PEG-capped GNPs are also found dispersed (single or grouped particles) in the cytoplasm (thin arrows) compared to citrate-capped GNPs. N: nucleus, G: Golgi apparatus, RER: rough endoplasmic reticulum, m: mitochondrion. Fixation: 2.5% glutaraldehyde in 0.01 M PBS, embedding in epoxy resins, staining with alcoholic uranyl acetate/lead citrate. Scale bar: 500 nm.
Figure 2
Figure 2
Representative data of gold nanoparticles (GNPs) uptake in PC3 cells. Data here are presented, in the form of histograms, as mean values of GNPs/area in μm2 of different cellular compartments: (a) autophagosomes (AUTO), cytoplasmic vesicles (VES); (b) cytoplasmic area (CYTO) and nucleus (NCL). Figure shows indicatively the cellular uptake of both citrate-capped GNPs and PEG-capped GNPs presented in Figure 1.
Figure 3
Figure 3
Silver-enhancement technique. Figure shows how silver nucleates gold nanoparticles, resulting in larger visible particles. Final particle size is time-dependent (increases after time).
Figure 4
Figure 4
Representative TEM images of PC3 cells incubated with 5 nm PEG-capped gold nanoparticles following silver-enhancement technique: (a) Image shows the signal amplification after silver-enhancement of GNPs revealing their distribution both in the cytoplasm and inside the nucleus of the cell (arrows) (b) Part of the cytoplasm of the cell where gold nanoparticles, after silver-enhancement, are found to be dispersed or located inside vesicles and autophagosomes (arrows). N: nucleus; m: mitochondrion. Fixation: 3% paraformaldehyde and 0.5% glutaraldehyde in 0.1 M PB, embedding method: PLT, staining with alcoholic uranyl acetate/lead citrate. Scale bar: 1 μm.
Figure 5
Figure 5
Representative TEM images of PC3 cells after immunogold labelling for DNA damage detection. 15 nm citrate-capped nanoparticles are found mostly inside cytoplasmic vesicles (double arrows), whereas 10 nm immunogold particles and 25 nm immunogold particles, labelling γH2AX and OGG1 respectively, are located inside the nucleus: (a) Image represents single immunolocalization for γH2Ax detection (thin arrows) inside the nucleus. (b) Image represents double immunolocalization for γH2Ax (10 nm gold) (thin arrows) and OGG1 (25 nm gold) (thick arrows) detection inside the nucleus. N: nucleus; n: nucleolus. Fixation: 3% paraformaldehyde and 0.5% glutaraldehyde in 0.1 M PB, embedding method: PLT, staining with alcoholic uranyl acetate/lead citrate. Scale bar: 1 μm.
Figure 6
Figure 6
Representative data of DNA damage induced by 1 Gy IR after incubation with citrate-capped GNPs in PC3 cells. Specific markers, in this case, indicating DNA lesions, can be semi-quantified and data can be presented in the form of histograms. Figure shows indicatively the DNA damage detection by single immunolocalization of γH2AX per nuclear area (μm2) (a), or by double immunolocalization of γH2AX and OGG1 per nuclear area (μm2) (b).
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