Jsm 5600

A thin gold layer was deposited on the CaPP surface (BAL-TEC SCD 050; Capovani Brothers Inc., NY, USA) to assess CaPP morphology. Images at ×5000 magnification were obtained on an SEM (JSM-5600, JEOL; Tokyo, Japan) with a 15 kV accelerating voltage, 13 mm Z, and 15 mm WD.
The powder was coated with carbon by vapor deposition (Delton vacuum Desk II, Moorestown, NJ, USA) to characterize the chemical elements of the CaPP particles. Elemental analysis was performed using an EDX detector (Vantage, Acquisition Engine Company, Tokyo, Japan) connected to an SEM (JSM-5600, JEOL, Tokyo, Japan). The EDX system was operated at 15 kV with a collection time of 100 s, 30° incidence, Z = 20 mm, and WD = 20 mm. Three areas of approximately 10 µm² were evaluated.

The cell morphology was observed using a scanning electron microscope (SEM, JEOL JSM-5600, Japan) at an accelerating voltage of 15 kV. After 1, 3, 7 and 14 days of culturing, the medium was removed from the wells and the samples were washed with PBS solution three times. Then, the samples were fixed with 4% paraformaldehyde (PFA) for 30 min at 4 °C. The samples were dehydrated with gradient ethanol (30, 50, 70, 80, 90, 95 and 100% v/v) for 10 min. Finally, the samples were freeze-dried overnight and coated with gold in an automatic sputter coater, and were then observed using SEM.
The morphology and cytoskeleton of the adherent cells were observed using confocal laser microscopy imaging. After 3 days of culturing, the samples were rinsed with PBS twice and then fixed with 4% paraformaldehyde for 30 min at 4 °C. Then, the samples were incubated in 1% Triton X-100 (Sigma, USA) for 10 min to permeate the cell membrane. After being washed with PBS 3 times, the cytoskeletons and nuclei of the cells were stained with rhodamine-conjugated phalloidole (25 μg mL⁻¹) and 4′,6′-diamidino-2-phenylindo hydrochlorides (10 μg mL⁻¹) (Invitrogen, USA) for 30 min. The samples were observed using laser scanning confocal microscopy (LSCM) (Carl Zeiss, Germany).

Leaves were collected from 2 to 3 week-old plants for scanning electron microscopy observation. To preserve the appearance of the cuticles, samples were processed without fixation and dehydration by flash-freezing in supercooled nitrogen slush at −210°C for 5 s according to Hayat (2000 ). The frozen samples were placed in 1 kg brass receptacles supercooled in liquid nitrogen (−196°C) to keep the leaves frozen during the initial stages of lyophilization at −50°C. The lyophilized leaf samples were then placed on aluminum stubs with their adaxial surfaces exposed and coated with a layer of gold-palladium (15 mA under 75 mTorr pressure) using a Hummer 6.2 sputter coater (Anatech USA, Union City, CA 94587). The adaxial surfaces of several samples of each species were observed with a scanning electron microscope (JEOL JSM-5600, Peabody, MA 01960). The images were digitally colorized using Adobe Photoshop CS4 (Adobe, San Jose, CA 95110).