Vegetation plays a key role in the environmental cycling and fate of many organic chemicals. A compound's location on or within leaves will affect its persistence and significance; retention in surface compartments (i.e., the epicuticular wax and cuticle) renders the compound more susceptible to photodegradation and volatilization, while penetration into the epidermal cell walls or cytoplasm will enhance susceptibility to metabolism. Here, for the first time, methodologies which combine plant and PAH autofluorescence with two-photon excitation microscopy (TPEM) are used to visualize and quantify compound photodegradation on and within living plant leaves. Anthracene,fluoranthene, and phenanthrene were introduced to living leaves of Zea mays and monitored in real time, in control treatments, and when subject to UV-A radiation. Compound photodegradation was observed directly; different degradation rates occurred for different compounds (anthracene > fluoranthene > phenanthrene) and in different locations (at the leaf surface > within the epidermal cells). Results suggest that photodegradation on vegetation may be a more important loss mechanism for PAHs than previously thought. Compound fate in vegetation is potentially highly complex, influenced by diffusion into and location within leaf structures, the rates of supply/loss with the atmosphere, exposure to sunlight, and other environmental conditions. The techniques described here provide a real-time tool to advance insight into these issues.

The increasing use of nanomaterials in almost all sectors of society (e.g., health or energy to agriculture and transport) has generated a need for innovative detection methods for nanomaterials, to enable their continued development, environmental and toxicological monitoring, and risk assessment. In vivo nanoparticle visualization is needed to support applications in drug delivery to plant biology where real-time monitoring is essential. Techniques are sought that do not require the addition of molecular tags or nanotags to enhance detection, because these may modify the surface properties or behavior of the nanomaterials. Here two-photon excitation microscopy coupled with plant nanomaterial, or chemical autofluorescence is used to detect and visualize multiwalled carbon nanotubes (MWCNTs), titanium dioxide, and cerium dioxide in living wheat tissues. The potential of the technique to track chemical-nanomaterial interactions in living tissues is then demonstrated, using phenanthrene as a model compound. MWCNTs were observed to pierce wheat root cell walls and enhance the transport of phenanthrene into the living cells. The ability of this technique to monitor real-time in vivo nanomaterial behavior and its potential applications and limitations for use in various disciplines is highlighted.

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Two-photon excitation microscopy (TPEM) was used to monitor the air-to-leaf transfer and within-leaf movement and distribution of phenanthrene in two plant species (maize and spinach) grown within a contaminated atmosphere. Phenanthrene was visualized within the leaf cuticle, epidermis, mesophyll, and vascular system of living maize and spinach plants. No detectable levels of phenanthrene were observed in the roots or stems of either species, suggesting phenanthrene entered the leaves only from the air. Phenanthrene was observed in both the abaxial and adaxial cuticles of both species. Particulate material (aerosols/dust) contaminated with phenanthrene was located at the surface of the cuticle and became encapsulated within the cuticularwaxes. Overtime, diffuse areas of phenanthrene formed within the adjacent cuticle. However, most of the visualized phenanthrene reaching the leaves arrived via gas-phase transfer. Phenanthrene was found within the wax plugs of stomata of both species and on the external surface of the stomatal pore, but not on the internal surface, or within the sub-stomatal cavity. Phenanthrene diffused through the cuticles of both species in 24-48 h, entering the epidermis to reside predominantly within the cell walls of maize (indicative of apoplastic transport) and the cellular cytoplasm of spinach (indicative of symplastic transport). Phenanthrene accumulated within the spinach cytoplasm where it concentrated into the vacuoles of the epidermal cells. Phenanthrene was not observed to accumulate in the cytoplasm of maize cells. Phenanthrene entered the internal mesophyll of both species, and was found within the mesophyll cell walls, at the surface of the chloroplasts, and within the cellular cytoplasm. Phenanthrene was observed within the xylem of maize following 12 days exposure. The cuticle and epidermis at the edges of spinach leaves had a systematically higher concentration of phenanthrene than the cuticle and epidermal cells at the center of the leaf. These results provide important new information about how such compounds enter, move, and distribute within leaves, and suggest that contemporary views of such processes based on data obtained from traditional analytical methods may need to be revised.

Vegetation plays a key role in the environmental fate of many organic chemicals, from pesticides applied to plants, to the air-vegetation exchange and global cycling of atmospheric organic contaminants. Our ability to locate such compounds in plants has traditionally relied on inferences being made from destructive chemical extraction techniques or methods with potential artifacts. Here, for the first time, two-photon excitation microscopy (TPEM) is coupled with plant autofluorescence to visualize and track trace levels of an organic contaminant in living plant tissue, without any form of sample modification or manipulation. Anthracene-a polynuclear aromatic hydrocarbon (PAH)-was selected for study in living maize (Zea mays) leaves. Anthracene was tracked over 96 h, where amounts as low as approximately 0.1-10 pg were visible, as it moved through the epicuticular wax and plant cuticle, and was observed reaching the cytoplasm of the epidermal cells. By this stage, anthracene was identifiable in five separate locations within the leaf: (1) as a thin (approximately 5 microm) diffuse layer, in the upper surface of the epicuticular wax; (2) as thick (approximately 28 microm) diffuse bands extending from the epicuticular wax through the cuticle, to the cell walls of the epidermal cells; (3) on the external surface of epidermal cell walls; (4) on the internal surface of epidermal cell walls; and (5) within the cytoplasm of the epidermal cells. This technique provides a powerful nonintrusive tool for visualizing and tracking the movement, storage locations, and degradation of organic chemicals within vegetation using only plant and compound autofluorescence. Many other applications are envisaged for TPEM, in visualizing organic chemicals within different matrixes.