In this work, different processing approaches were used with the aim of providing a range of technical alternatives that different laboratories can adapt to their equipment. All of the alternatives hereby presented have been proven valid for the observation of nanoparticles in plant tissues and cells. Aggregation of metal particles, with a diameter size within the micrometer range, in the culture medium and internalization into human osteoblasts have been reported by Vallés et al. (2007)
by reflection on a confocal microscope. In the present study, the Nomarski and reflection techniques on the confocal microscope have proved to be sufficiently sensitive to detect the aggregates formed by nanoparticles, thus providing a reliable methodological approach for their visualization. Since reflection requires the collection of the emitted signal in a very narrow range of the spectrum including the excitation wavelength, this technology is restricted to state-of-the art spectral confocal microscopes, which permit control over where the signal is collected. These microscopes have been on the market for over 5 years and their availability in most research institutes is increasing. Non-spectral confocals would provide images overlapping reflection and (auto)fluorescence. Then, the use of these microscopes for nanoparticle detection would only be suitable in those specimens with a low autofluorescence signal. The aggregates formed by the nanoparticles were also observed within 3-D stacks spanning cortex cells. Thus, the confocal analysis demonstrates the internalization of nanoparticles inside plant tissues. Whether or not the aggregates of nanoparticles are formed in the cytoplasm, presumably by converging carriers of smaller groups of particles which have not been taken up, is still unknown. It cannot be ruled out that some particles are washed out during the manipulation of the specimens (chopping off the plant material, fixing, washing and further processing). Then, those particles identified in the present work must reflect strong interactions and/or retention in the plant tissues.
Autofluorescence, either natural or induced by glutaraldehyde fixation, can be used to infer the presence of nanoparticles as dark areas embedded within a fluorescent background. This approach had been successfully applied to detect the internalization of micrometric metal particles and aggregates into human macrophages (Vallés et al., 2006
) and osteoblasts (Vallés et al., 2007
). However, most plant cells are highly vacuolated with reduced dense cytoplasmic areas, thus making the identification of deposits of nanoparticles, apart from specific structures and cell types not always evident. Only some particles associated with the cell wall of xylem vessels, which are highly autofluorescent due to their major component – lignin – were observed.
Different resins can be used to embed fixed specimens depending on their final use and the resolution level of the observations. Paraffin embedding is commonly used for light microscopy analysis. Epoxi and acrylic resins, such as Epon, LRWhite or Lowicryl permit the correlation between light and electron microscopy observations, but usually require protocols that last up to 1 week (González-Melendi et al., 2005
). In this work, a correlative approach was followed by observing semi-thin and ultra-thin sections on the light and electron microscopes, respectively, collected from the same blocks of specimens embedded in Epon resin. Dark and refringent small signals were seen on the light microscope, which were further identified as iron-core nanoparticles on the electron microscope.
As far as is known, to date there has not been any report about penetration and transport of nanoparticles inside whole plants. The most recent paper dealing with this topic appeared recently in Nature Nanotechnology
(Torney et al., 2007
), but the work was limited to isolated plant cells and intact leaves. Here, it has been shown how magnetic nanoparticles can penetrate into the plant and the bioferrofluid travels through the vascular system to other parts of the plant, and how this bioferrofluid can be concentrated in the desired areas by using magnets. Since no particles with a diameter size bigger than 50 nm have been detected inside plant tissues, a size-based selection mechanism seems to be operating, probably involving cell walls and waxes acting as a barrier. More studies are underway to unveil the penetration mechanisms of the nanoparticles inside the plant and plant cells, and their transportation and movement to and through the vascular system.
Many things must be addressed prior to utilization of nanoparticles in agriculture. First, those concerning the plant physiology and growth in mid- and long-term, i.e. do the nanoparticles significantly affect the plant and are they phytotoxic? Damage in the plants used during the present experiments was not observed. Furthermore, some of the treated plants were transplanted in pots and grown until maturity like normal plants. It is possible that nanoparticles produce some local damage in cells, as has been reported for in vitro
treatments (Pavel et al., 1999
; Cotae and Creanga, 2005
; Pavel and Creanga, 2005
), but it does not mean that the whole plant would be affected. However, phytotoxicity of more intense treatments should not be discarded, and specific assays must be done in this way. Secondly, the effect of nanoparticles entering the food chain must be considered and studied. There are recent reports about cytotoxicity of nanoparticles developed for biomedical applications (Brunner et al., 2006
; Panessa-Warren et al., 2006
), but no studies about their effect after entering the digestive system are available nor are there any considering whole organisms.
With respect to the use of magnetic nanoparticles in a large-scale situation, it is impractical to place magnets in annual extensive crops (e.g. cereals). But it would be possible for specific treatments in fruit trees (e.g. olive trees) or high-input crops under greenhouse conditions. However, the main advantage of using magnetic nanoparticles is in laboratory and research applications because they allow a very precise localization of the particles to unload their charge, which is of great interest in the study of local treatments in whole living plants. Nevertheless, nanoparticles lacking magnetic properties could be used in large-scale situations with extensive crops, designed with other systems which allow their accumulation and/or guidance into specific areas. In that case, field applications could be done through the leaves and/or root system.
In conclusion, bioferrofluids can be introduced into whole living plants, can travel using the vascular system and can be concentrated in specific areas by application of magnetic gradients. In this work, a number of microscopy tools to identify the tissue and subcellular location of nanoparticles in plants is also described. These cover a range of reliable methodological approaches based on fluorescence, confocal, light and electron microscopy which can be selected by the research teams to achieve their scientific goals within their technical availabilities and skills. In summary, nanoparticles can be visualized by reflection on a confocal microscope; inferred as dark areas in an autofluorescent background (either natural or induced), although with some limitations depending on the cell types and as a dense signal on the light microscope, further identified as clusters of nanoparticles on the electron microscope, due to their iron core. The preliminary results of the present work showed the presence of nanoparticles both in the extracellular space and within some cells. Further work is needed to evaluate how the nanoparticles penetrate and are transported within the plants, and the mechanism(s) of intracellular internalization to explore the potential of nanoparticles as smart treatment-delivery systems in plants.