Electron Microscopy (EM)

There are two primary types of electron microscopy instruments: scanning electron microscope (SEM) and transmission electron microscope (TEM). Both instruments have an energy electron source, known as the electron gun, which creates an electron beam (ebeam). The electron gun is typically composed of a tungsten (W)filament, although modern electron microscopes are fitted with a lanthanum hexaboride (LaB₆) orfield emission gun (FEG) that provide a brighter beam for better resolution. Electron microscopes utilize a vacuum column through which the ebeam passes vertically, promoting the straight travel of electrons (Roming1986).

The electrons will then either interact with the sample in the case of SEM or pass through the ultrathin sample (for biological specimens up to 1µm thick) in the case of TEM. While SEM can give rise to apparent three-dimensional (3D) images, the flat portraits’ resolution of TEM can be higher than SEM by one order of magnitude (Luykx et al.2008).

3.2.3.1 Scanning Electron Microscopy (SEM)

With SEM, it is possible to obtain images of the sample’s surface as well as material composition information. While scanning, the data gathered is based on the resulting energy emitted from the sample hit by the electron beam. Most commonly, the electrons that bounce as backscattered or secondary electrons (BSE or SE) are detected for imaging. EMs equipped with energy dispersive X-ray spectrometer (named EDXS, EDAX, EDS, or EDX) can provide compositional information of the sample by the X-rays produced to identify the chemical nature of the elements present. EDX can also provide an elemental mapping of the atomic composition.

Mapping is usually represented by colored dots, and their distribution in the image elucidates the element location within the sample (Fig.3.6a). Abd-Alla et al. (2016) imaged different tissues of faba bean plant previously exposed to Ag NPs by using

contact mode

intermittent stage

diode laser

photodiode detector

cantilever

sample (a)

(b)

(c)

(tapping mode)

Fig. 3.5 Overall illustration of basic AFM setup. a A laser points toward the tip of the probe, and its motion is sensed by a photodiode detector for the creation of an image. b Contact mode: the cantilever passes dragging the tip over the surface of the sample. c Intermittent mode: the cantilever vibrates up and down at the time of scanning

EDX coupled to SEM. The authors reported clear accumulation of Ag in roots, shoots, and nodules. However, even when the quantification was reported, the published data correspond to Ag concentration, with no information about particle size. In a similar way, Yan et al. (2013) exposed the leaves of soybeans to droplets containing Cs NPs and were able to identify the presence of NPs in the outer surface of the leaves as well as in the pods, roots, and stems, confirming the uptake and translocation of the NPs. Moreover, they performed EDX analysis on the particles to evidence the content of Cs. Cañas et al. (2008) studied CNTs in plants exposing different food crops to the nanomaterial for 0, 24 and 48 h. Their SEM analysis revealed CNTs in root surfaces but not in inner tissues of the roots. Perhaps the exposure time was too short for the uptake.

X-rays

Fig. 3.6 Common ebeam–sample interaction effects used for (a) SEM and (b) TEM imaging.

The sketch simulates nanoparticles“X” encountered in the vascular system of the plant. a SEM—

electron beam (primary electrons, red line in sketch) at low acceleration voltage (*5–15 kV) scans over the sample’s surface. The ebeam hitting the sample generates signals that, if detected, create images. Backscattered electrons, secondary electrons, and X-rays are usually detected for the acquisition of a micrograph. Backscattered and secondary electrons produce surface images by differences in atomic number (z) represented in shades of gray. In Figure (a), the X-ray spectrum confirms the presence of an “X” NP, while the mapping shows the distribution of “X” as dots within the vascular system of a leaf. b TEM—electron beam at high acceleration voltage (*80–

200 kV) passes through an ultrathin sample resulting in transmitted or deflected electrons.

Transmitted electrons create an image of the trespassed sample; if the electron energy lost in the path is quantified and related to the elements in the sample, the material composition can be identified. The deflected electrons provide information about the structure of the sample; ring pattern for amorphous materials or dots pattern for crystalline materials

3.2.3.2 Transmission Electron Microscopy (TEM)

In TEM, the ebeam hits and trespasses an ultrathin sample. The electrons trans-mitted through the sample create an image, and these electrons lose energy while passing through the sample. The difference in energy before and after crossing the specimen is correlated with the material’s composition, known as electron energy loss spectroscopy (EELS). Therefore, TEMs with EELS detectors can provide elemental analysis of the sample. Transmitted electrons that are deflected exhibit structural information about the specimen’s components. For example, NPs can be characterized by TEM with selected-area electron diffraction (SADS), which pro-vides information about the nanocrystalline structure. Figure3.6b shows an over-view of TEM.

Transmission electron microscopy allows the detection and quantification of carbonaceous materials in plant cells. Lin et al. (2009) confirm the uptake of carbon-based MNMs evidenced by TEM micrographs showing C₇₀in vacuoles and leaves’ cell walls. Lahiani et al. (2015) proposed the use of single-walled carbon nanohorns (SWCNHs) as plant growth regulators after studying the effects of SWCNHs in different edible plant species, confirming the presence of SWCNH in the roots and seeds of tomato and tobacco cells by TEM. Larue et al. (2012b) used MW¹⁴CNT and TEM to quantify the uptake and translocation of gum Arabic (GA), and humic acid (HA) stabilized MWCNTs in wheat (Triticum aestivum) and rapeseed (Brassica napus) plants. They reported the presence of CNTs in the leaves of both plant species, though at low concentration. In rapeseed, total accumulation was 140± 32 and 108 ± 47 µg CNTs/kg, and in wheat, the uptake was 200 ± 83 and 43± 15 µg CNTs/kg dry biomass for GA and HA stabilized CNTs, respec-tively. In a very recent study, Le Van et al. (2016) utilized TEM to localize CuO NPs in cotton leaves. These researchers found that CuO NPs aggregated on the epidermis of leaves in non-modified plants, while they penetrated the cells after endocytosis on transgenic cotton plants. After correlating CuO uptake and toxicity to gene expression, the authors were able to conclude that CuO NPs enhanced the expression of a gene related to cotton insect resistance.

Several studies have shown the versatility of TEM for the detection of the uptake and translocation of NMs in plants. High-resolution TEM (HRTEM) is a variant of TEM that is suitable for visualizing materials where contrast is not an issue to overcome. The HRTEM can provide substantial information on the crystalline structure of materials utilizing a higher acceleration voltage than conventional TEM. By using this technique Gardea-Torresdey et al. (2002,2003) showed, for the first time, the formation of Au and Ag NPs in alfalfa (Medicago sativa) plants grown in agar medium enriched with either potassium tetrachloroaurate or silver nitrate. With HRTEM, Gardea-Torresdey et al. (2002) measured Au particles of 4, 20, and 40 nm along the alfalfa stem, which endorsed them to hypothesize the continuous growth of the Au NPs within alfalfa plant. Low-magnification TEM and HRTEM also allowed Gardea-Torresdey et al. (2003) to show images of alfalfa shoot with icosahedral silver nanoparticles ranging from 2 to 3 nm in size. These studies opened the door for the detection of metal NPs in plants using TEM. Other

studies have also shown the capability of TEM for localizing NPs within the ultrastructure of plant cells. Taylor et al. (2014) studied the effects of K(AuCl4) and AuCl3in A. thaliana. They grew alfalfa in agar medium enriched with ionic gold and Au NPs of 5 and 100 nm. The researchers found Au NPs in the roots of plants treated with ionic Au, leading them to conclude that the plants did not take up the NPs.

The SEM and TEM have been used together for complementing the analysis of NPs in plants. For example, Du et al. (2011) detected Ti in periderm cells of wheat root by SEM-X act analysis and with TEM they observed TiO₂NPs (20± 5 nm) in cortex cells of the root. Li et al. (2013), using SEM found TiO₂NPs accumulated on the Lemna minor leaves, and TEM micrographs showed no cellular uptake of TiO₂NPs.

3.2.3.3 Scanning Transmission Electron Microscopy (STEM)

The STEM combines capabilities of SEM and TEM. Modified SEM and TEM equipped with additional detectors are enabled to run in STEM mode. The SEM in transmission mode (SEM/STEM) provides images with better spatial resolution (http://www.fei.com/introduction-to-electron-microscopy/stem/) still using the rel-atively low accelerating voltages. Bandyopadhyay et al. (2015) used dark-field STEM (DF-STEM) to study tissues of alfalfa plants exposed to ZnO NPs.

DF-STEM, which is sensitive to atomic number due to Z-contrast, showed that striations accumulated along the cell walls of stem cells were formed by small particles of 9–12 nm. Punctual EDX spectroscopy confirmed that these high-contrast structures corresponded to Zn and O combined, showing the aggre-gation of ZnO NPs.

3.2.3.4 Sample Preparation for EM Analysis

The detection and analysis of NPs inside organic matrices are difficult. One of the biggest challenges of EM analysis is sample preparation of biological specimens because it is time-consuming, and the process itself can damage or contaminate the sample, introducing artifacts. Moreover, studying NMs’ morphology, size, and other properties may vary depending on the instrument being used, the image acquisition, image analysis, and the selected sample for examination (Dudkiewicz et al.2015). Analytes of living organisms have to withstand the vacuum environ-ment needed for the analysis, which is not possible in their native state for con-ventional instruments. However, variants in electron microscopes such as cryo-EMs and environmental-EMs allow for lesser sample preparation and a more suitable ambient for living specimens. Moreover, the relatively recent development of SEM capsules enables the observation of biological samples with ongoing metabolic activity by protecting the sample from the harsh conditions of the SEM (Kokina et al. 2013). When analyzing NPs in plants, minimum specimen disturbance is

desired in order to analyze the exact location of the nanomaterial [that may change according to the matrix environment (Kumari et al. 2011)] and to elucidate the effects produced in the plant.

Protocols vary, but the main objective is to preserve the integrity of the sample without disturbing the morphology of cells and structure of their components while being analyzed (Pathan et al.2008; Wu et al.2012). Ensikat et al. (2010) presented and evaluated sample preparation methods that are not routinely used but are fea-sible for the visualization of even fresh plant surfaces in conventional SEM.

Dudkiewicz et al. (2011) reviewed the EM technologies that have been applied to characterize NPs in food matrices. Their work is a useful reference for the analysis of NPs in the agriculturalfield, as it involves the study of MNMs inside soft and moistened organic matrix.

The above literature shows that EM is a state of the art technique for the detection of ENMs in plants. However, this technique does not allow quantification of the MNMs within plant tissues.