VACNFs penetrate Populus epidermis without damaging cells.

(a) and (b) Micrographs of transverse sections through a Populus leaf showing individual nanofibers penetrating the cuticle and epidermis. The arrow in (a) shows a nanofiber reaching the base of an epidermal cell without penetrating the underlying palisade cell. Arrows in (b) indicate a nanofiber traversing the cytoplasm of the cell. (c) DIC micrograph of a leaf epidermis showing that carbon nanofiber impalement occurs in a grid pattern, similar to the original chip. (d) DIC micrograph of the same leaf depicted in (c) with the focal plane moved into the underlying palisade layer showing the absence of over-penetrant fibers. Results are representative of leaves from 2 separate plants. (e) and (f) Bright-field micrographs of Populus leaves after staining with DAB to detect H2O2 production. Leaves penetrated by carbon nanofibers (e) show no DAB staining and are similar to untreated areas of the leaf (f). The boxed inset in (e) shows a magnified image of the nanofiber-treated area, with black arrows indicating the location of carbon nanofibers in this image. (g) and (h) Leaves wounded with a cork borer (g) or abraded with carborundum (h) show areas stained dark brown by DAB deposition in reaction to H2O2 produced in the wound response. Black arrows in (h) indicate carborundum powder remaining on the leaf. Dashed arrows in (g) point to the cut edge of the leaf and solid arrows indicate staining with DAB. Similar results were obtained with leaves from 3 separate plants. (i) and (j) Transverse sections of a Populus leaf after carborundum abrasion, showing areas of (i) severe and (j) mild epidermal damage. The black arrow in (j) points to grit particles within the palisade layer. (k) DIC micrograph of a leaf epidermis after carborundum treatment showing abraded epidermal cells (denoted by white arrows). (l) DIC micrograph of the same leaf depicted in (k) with the focal plane moved into the underlying palisade layer, showing the presence of embedded grit particles (denoted by white arrows). Images (a), (b), (i) and (j) were obtained from thin sections of fixed (formalin), embedded (paraffin) and stained (toluidine blue) tissue; images (c), (d), (k) and (l) were obtained from fixed (ethanol-acetic acid) and cleared (chloral hydrate) tissue; images in (e)–(h) were obtained from stained (DAB) and decolorized (boiling ethanol) tissue. Details are provided in Materials and Methods.