Light (Optical) Microscopy

Conventional optical or light microscopy (LM) allows the observation of objects in the range of submicrometer to micrometer size. However, two variants of light

Table 3.1 Comparison of advantages and disadvantages of electron microscopy and light microscopy (Inoue2010; Larue et al.2014a)

Electron microscopy Light microscopy

Sample requirement and preparation

Completely dry sample; Living specimens can be observed Coating with thin metal layer (usually gold)

before detection if sample is non-conductive or poor-conductive;

No coating needed

Staining is unnecessary when preparing nanomaterial suspension

Non-autofluorescence materials have to be stained

Operation environment

Vacuum environment (except environmental electron microscopes)

Ambient environment

Sample damage Greater sample damage by electron beams Lower sample damage by photon beams

Image quality Black/white image Colorful image

microscopy, confocal microscopy (CM) and two-photon excitation microscopy (TPEM), have shown to be effective for the detection of MNMs in plants. For detailed information about the imaging processes using these techniques, the reader is referred to Inoué (2010).

Light microscopy imaging has several advantages, compared with electron microscopy (EM) imaging (Table3.1). The most striking difference between these two techniques is that LM allows the observation of MNMs in living specimens, which is not possible with EM. However, non-autofluorescence MNMs have to be stained before the exposure to plants in order to be detected with LM. The study of the uptake of MNMs by plant cells/roots by LM goes back to 2009. Since then, a few studies have shown the use of this technique to corroborate the uptake of CNTs and metallic NMs by plant roots. Examples and their respective references are shown in Table3.2. A brief description of each procedure is shown in the next sections.

3.2.1.1 Confocal Microscopy (CM)

Confocal microscopy images are obtained by scanning the specimen with a point of light in a raster pattern (Inoué2010). Since it was patented in 1957, CM has been widely used in the biological and biomedicalfields due to its 3D resolution capa-bility, in situ/in vivo imaging, less phototoxicity, and higher optical resolution, compared to EM (Inoué2010). CM has shown great capabilities to study the impact of nanomaterials in human organs (Lee et al. 2014), and some researchers have shown its potential for monitoring the uptake of MNMs by plants.

One of thefirst reports about the use of CM to verify the uptake of NPs was published by Hischemöller et al. (2009). These researchers exposed moth orchid (Phalaenopsis spp.) and Arabidopsis (Arabidopsis thaliana) plants to a colloidal solution of NaYF₄:Yb,Er for a few days. Subsequently, they observed root samples with confocal laser scanning microscopy and detected fluorescent nanocrystals in tissues of the velamen radicum (root epidermis) and in the stele of roots, demon-strating that the NPs were taken up and translocated within the plant. The same year, Liu et al. (2009) used CM to demonstrate that fluorescein isothiocyanate (FITC)-stained SWCNTs penetrated both cell walls and cell membranes of tobacco Bright Yellow (BY-2) cells’ line in a time- and temperature-dependent manner.

The CM has also been used to detect the uptake of widely used metallic NPs by plants. Ma et al. (2010) exposed four weeks Arabidopsis seedlings to silver nanoparticles (nAg) colloid (40 nm). After exposure, they observed, with a confocal/multiphoton microscope, that the majority of the nAg accumulated in the columella (cells of the root cap arranged longitudinally), but some of them were able to reach the vasculature of the seedlings and consequently, potentially translocated to the upper plant parts. Zhao et al. (2012a) exposed the roots of one-month-old corn (Zea mays) plants to FITC-stained ZnO ENPs and observed the samples with a CM after 48 h of exposure. Confocal images showed that the stained NPs were accumulated in the cell walls in root cortex and most of them retained at

Table3.2Examplesoftheuseofconfocalandtwo-photonexcitationmicroscopyforthedetectionofENMSinplants NanomaterialsParticlesize (nm)PlantConcentration (ppm)Modeof exposureGrowth mediaAccumulationDetection methodsReference NaYF4:Yb,ErMothorchid (Phalaenopsis spp.) RootNPs suspensionNaYF4:Yb,Ernanoparticles wastranslocatedfrom velamenradicumtopassage cells,andeventuallyto vasculartissues

CMHischemoller etal.(2009) MWCNT, CeO2, TiO2

MWCNT (diameteris between110 and170, lengthisup to9μm), CeO2(<25), TiO2(100) Wheat (Triticumspp.)100RootNPs suspensionOnlyMWCNTshave capabilitiestopiercethe rootepidermalcell

TPEMWildand Jones(2009) ZnO380Maize (Zeamays)100,200,400, 800RootSandyloam soilNPaggregatespiercedcorn rootsepidermisandcortex throughapoplasticand symplasticpathways CMZhaoetal. (2012a) CeO28±1Maize(Zeamays)100,200,400, 800RootSandyloam soil/organic soil

UncoatednCeO2have preferentialtranslocationby cornrootsthancoatedones andhigherconcentrationin organicsoilthanin unenrichedsoil Converseresultshave shownincornshoots

CMZhaoetal. (2012b) (continued)

Table3.2(continued) NanomaterialsParticlesize (nm)PlantConcentration (ppm)Modeof exposureGrowth mediaAccumulationDetection methodsReference Mn100Mungbean (Vignaradiata var.Sonali) 0.05,0.1,0.5, 1RootNPs suspensionMnNPswereobservedin theroot(corticalandstellar) andleaves(stomataand mesophyll)

CMPradhanetal. (2013) Mesoporoussilica nanoparticles(MSNs)20Maize(Zeamays), wheat (Triticumspp.), lupin(Lupinus spp.)Arabidopsis (Arabidopsis thaliana)

200,500, 1000,2000, 10000,20000

RootNPs suspensionMSNswereobservedinthe roots,stemsandleavesof lupin,wheat,maize,and Arabidopsisthaliana Theaccumulation percentageofMSNwas between25and37.5%in therootofmaize CM,μ- PIXESunetal. (2014)

the endodermis, but some of them reached the transport system. Subsequently, Zhao et al. (2012b) exposed the corn roots to FITC-stained CeO2 NPs and cor-roborated the previous results found with ZnO NPs (Figs.3.2 and 3.3). Authors hypothesized that the ENPs entered through the Casparian band at the emission points of the lateral roots, where it was not fully formed. Pradhan et al. (2013) observed, after 15 days of exposure, that FITC-labeled Mn NPs were taken up by cortical and stellar root tissues and translocated to leaves (stomata and mesophyll) of mung beans (Vigna radiata var. Sonali). Sun et al. (2014) investigated the uptake of mesoporous silica nanoparticles (MSNs) in four plant species, maize (Zea mays), wheat (Triticum spp.), lupin (Lupinus spp.), and Arabidopsis. After five days of treatment, MSNs were observed in the roots of maize and in roots, stems, and leaves of lupin and wheat. Afterward, they incubated Arabidopsis seedlings in FITC-stained MSNs for 12 h and obtained confocal images of the NPs in chloro-plasts, corroborating the capability of CM for the observation of ENM uptake and translocation in plants.

3.2.1.2 Two-Photon Excitation Microscopy (TPEM)

TPEM is another variation of LM that offers less sample phototoxicity, greater depth penetration (down to millimeter scale), and 3D resolution, which allows in vivo and situ observation of living plant cells. Different from traditional CM, which is one-photon excitation, TPEM only requires half energy and does not need a pinhole to block the background signal in the detection pathway (Zipfel et al.

Fig. 3.2 Confocal images of cross sections of root treated for 24 h with 200 mg/L FITC (a) and FITC-stained nZnO (b) suspensions. Stained NP aggregates were observed in root epidermis, cortex, endodermis, and xylem. The transport was restrained by the Casparian strip. (Adopted from Zhao et al. (2012a). Copyright @ 2012 American Chemical Society)

2003; Rubart 2004; Stutzmann and Parker 2005; Fahrni 2009; Wild and Jones 2009). To the best of authors’ knowledge, only one report has shown the capability of TPEM to image the uptake of MNMs by plants. Wild and Jones (2009) employed TPEM to determine the uptake of MWCNTs, TiO₂, and CeO₂ENPs by wheat roots. These researchers exposed the plants for 28 days and observed single and aggregate MWCNTs in epidermal cells (Fig.3.4). They also found that some MWCNTs penetrated cell walls and entered up to 4µm into the cytoplasm, but they were not found to enter fully into the cells, perhaps due to the size of MWCNTs (diameter between 110 and 170 nm). TiO₂NP and CeO₂NP aggregates were just found adhered onto the root surface.

Compared with EM, light imaging microscopy has outstanding advantages, such as unsophisticated sample preparation and less sample damage. It provides images with high resolution and has a high potential for tracking the fate of non-fluorescence emission MNMs in plants. However, few reports include the use of this technique to investigate the uptake and translocation of nanomaterials by the whole plant. A possible reason is that non-fluorescence MNMs have to be stained before exposure to plants, which might modify their surface properties; addition-ally, unpredictable artificial contaminants might be introduced. As a result, microscopes and observation platforms without any staining are urgently needed.

We believe this will be achievable shortly (Min et al.2011).