Quanta 200

Trichome biogenesis and development were visualised under a stereomicroscope (SMZ25; Nikon, Japan). The morphology and microstructure of the trichomes were inspected using a LEO-1430VP scanning electron microscope (SEM; Carl Zeiss, Germany) and a Quanta 200 environmental scanning electron microscope (ESEM; Eindhoven, Netherlands). The length and density of the trichomes were measured using ImageJ software (NIH ImageJ system, Bethesda, MD, USA). Three biological replicates of each treatment and three to five observations of each sample were applied.

Field emission scanning electron microscopy (FESEM, Quanta 200, Zeiss, Jena, Germany) was utilized to analyze the shape and size of all fabricated GN-loaded scaffolds (G1, G2, and G3). Before analysis, the scaffolds were gold sputter coated (up to 20 nm) under argon to reduce their electrical conductivity. Gold-coated samples were positioned in the microscopic cavity to which a high vacuum was applied, and their images were then captured at an excitation voltage of 15 kV. The dimension of approximately 30 fibers was analyzed and their mean average diameter was computed with image J software (https://imagej.net/ij/ accessed on 11 June 2023). The hydrophilic behavior of GN-loaded scaffolds was determined by measuring their water contact angle (WCA) through the sessile drop method (Rame Hart Inc., Succasunna, NJ, USA). The average WCA value/or extent of hydrophilicity was obtained by measuring the angle of the water droplets on the area of 5 cm² scaffolds (each G1, G2, and G3) at five randomly distributed positions. Subsequently, swelling capacity was determined in triplicate manner by retaining each scaffold in 10 mL phosphate buffer media (pH 7.4) by gravimetric method for 24 h at 37 °C. Percentage swelling capacity was estimated by calculating the ratio of the weight of water adhered to the scaffolds to the weight of dried scaffolds.

To analyze the structure and composition of the sample, electron microscopy was used. Scanning electron microscopes FEI Quanta 200, equipped with an FIB, and Zeiss Supra 55 VP were used for top-view and cross-sectional imaging of the specimen. Since FIB ablation rates of Ag and Al were different, a height step at a twin boundary was formed, which led to a shift in the image as observed in Fig. 1. In addition, detailed TEM was carried out. To prepare plan-view TEM specimens, the Al film was peeled from the substrate and thinned to electron transparency using ion milling. In addition, a cross-sectional TEM lamella was extracted from the specimen by FIB methods. TEM images and diffraction patterns were acquired to analyze the grain and twin structure. In addition, aberration-corrected HRTEM, JEOL 2100F, was used to obtain atomic resolution images. Last, composition maps of the specimen were recorded in a scanning TEM, FEI Titan, using the superX four detector EDS spectrometer enabling to obtain quantitative maps with nanometer resolution and high-count rates. Quantification was carried out using the Bruker Esprit software.