Novel Method for the Direct Visualization of in Vivo Nanomaterials and Chemical

1. Novel Method for the Direct Visualization of in Vivo Nanomaterials and Chemical Interactions in Plants by Daniel Parra, Jake Owczarek, Patrick Hering, and Rahul Sirasao

2. Introduction -General focus was to track and image nanomaterials and their interactions with chemicals in organic materials -Multiwalled Carbon Nanotubes were the model nanomaterial -Phenanthrene was the model chemical compound -Living wheat root cells as host -Two-photon excitation microscopy to observe

3. Experimental Procedure -Nanomaterials involved in experimentation: -Multiwalled Carbon Nanotubes (MWCNTs) -diameters of 110-170nm, lengths < 9µm -Titanium (IV) Dioxide Nanopowder (TiO₂) -99.9% purity; particle sizes < 100nm -Cerium (IV) Dioxide (CeO₂) - particle sizes <25nm -Fluorescent grade Phenanthrene -98.9% purity

4. Experimental Procedure Cont. The nanomaterial solutions were prepared at 100 µg/mL. • Sonicated for 12 hours at 47 kHz ± 6% • Analyzed for optimum autofluorescence excitation and emission using TPEM Chart 1 • Ranges of excitation and emission frequencies for each solution, wheat, and phenanthrene are displayed in Chart 1 to the right

5. Experimental Procedure Cont. -Interaction between MWCNTs and plant growth was also studied -Roots grown in individually sealed, hydroponic systems containing -100 µg/mL MWCNTs and Phenanthrene -Only 100 µg/mL Phenanthrene -Hydroponic solution control system -Plants grown under 14 hr photoperiod, illuminated by 400 W lighting -Samples were analyzed at 96 hour intervals -Living roots sealed in glass slides with 5 mL DI water -All plants analyzed for each time period and treatment -Images and 3D data series were produced at different root zones

6. Detecting and Visualizing Nanomaterials -MWCNTs, TiO₂, and CeO₂ were visualized at the same time with wheat roots and TiO₂ and CeO₂ were observed to form aggregates at the root surface -While using MWCNTs, root growth and uptake studies were performed, and these results were visualized using TPEM (two-photon excitation microscopy) -The roots, shoots, and leaves of each plant were measured at each sampling interval for each growth system and no statistically significant difference in root, shoot, or leaf growth was observed between the different systems throughout the experiment.

7. Nanomaterial-Host Interaction -It was observed on the root surfaces that the MWCNTs pierced the epidermal and root hair cell walls and also the nanotubes traversed the cell walls, entering the cellular cytoplasm, by up to 4 μm -Over the course of the 28 day experiment, the number of MWCNTs piercing the cell walls increased, however the depth of penetration did not change -The MWCNTs did not enter or become encapsulated within the cells, due to the relatively large size of the MWCNTs. -The MWCNTs did not significantly affect root or shoot growth throughout the duration of the experiment

8. Nanomaterial-Phenanthrene Interaction -Phenanthrene was visualized to both partition to the MWCNTs and to be associated with an increased gathering of nanotubes over the root surface -Phenanthrene also was taken up into the cellular cytoplasm of cells pierced by nanotubes -Uptake was 50% faster for roots growing through MWCNTs with uptake visualized after 1-3 days. -Uptake without MWCNTs was consistent with previous studies -MWCNTs which come into contact with growing roots can pierce the cell walls -They also provide a hydrophobic substrate which is highly attractive to certain chemicals within soil, aqueous, or atmospheric systems

9. Conclusion -MWCNTs are very effective at piercing cell walls and creating a highway for chemicals into cells -Major benefit was the success of TPEM -Capable of tracking multiple subjects and various materials -Does not require tagging -The relationship between TPEM and MWCNTs autofluorescence could be utilized in many novel ways