Since the 1990’s superhydrophobic and self-cleaning leaf surfaces have been studied extensively; the most popular and first to be thoroughly examined being the lotus leaf [1-5]. It has been shown that the extreme non-wetting properties of these leaves arise from a unique dual-scale surface structure consisting of micro-scale papillae covered with nano-scale wax crystals [3,6-8]. Superhydrophobic leaf structures, possessing these unique surface features, have been used as a biological blueprint in the structuring of a variety of materials, rendering their surfaces highly water-repellent [9-13]. One of the most recent examples is the mimicking of the quaking aspen (Populus tremuloides) leaf structure to make superhydrophobic polymer and metal surfaces [14]. Although there has been a considerable effort to characterize and mimic these leaf surfaces, less is known about the exact mechanism(s) for the formation of these surface features, and how they react to different external environmental stresses.

Over the past five years, we have been monitoring and analyzing the surface structures and wetting properties of quaking aspen leaves. Normally, shortly after leaf emergence in the spring these leaves exhibit superhydrophobicity (water contact angle > 150˚) that remains for the rest of their growth season, is maintained even after leaf abscission and is preserved in the dried state for many years thereafter [8]. However, this trend was not observed during the 2011 growth season. At some point during this particular season (second half of August) the non-wetting properties of the aspen leaves were suddenly lost. It has been previously shown that the wetting characteristics of hydrophobic and superhydrophobic leaves are strongly influenced by the competition between 1) the degradation of nano-scale wax crystals due to environmental factors and 2) the self-repairing ability of the leaves through wax crystal regeneration [15-19]. In an effort to understand this unusual loss of the superhydrophobic properties of aspen leaves observed in 2011, the focus of the current study was to establish a link between wax crystal loss/regeneration and environmental factors such as temperature, precipitation, wind and relative humidity.

All quaking aspen leaf samples were collected throughout the 2011 growing season (May 14-October 9) from the same tree located in a forest near Peterborough, Ontario (44˚11.43'N/78˚24.26'W and 214 m altitude) and analyzed within 48 hours. To fully characterize a surface’s wetting behaviour both dynamic and static wetting measurements are necessary. For the assessment of static wetting characteristics, the contact angles between water droplets and levelled leaf surfaces were measured, while for dynamic measurements the sample’s tilt angle (relative to horizontal) to initiate water droplet roll-off was recorded. Wetting property measurements and scanning electron microscopy (SEM) were performed on 1 inch × 1 inch leaf sections that were cut and mounted using double sided tape on flat Plexiglas^TM coupons with their adaxial sides facing up. For contact angle measurements, leaf samples were placed on a pre-levelled stage and aligned with a horizontally positioned digital camera (Nikon D3000) equipped with a macro lens (Nikon—AF-S Micro Nikkor 40 mm) which was used to image no less than four 5 µl water droplets on each aspen leaf surface. Water droplet images were subsequently analyzed using ImageJ’s contact angle function [20]. Tilt angle measurements were carried out using a tilting stage and 25 µl water droplets.

Electron microscopy was performed using a Hitachi SU-6600 environmental scanning electron microscope (ESEM) which requires no coatings for surface electrical conductivity. This allowed freshly harvested, uncoated leaf samples to be imaged directly. All samples were analyzed using an environmental secondary electron detector (ESED) at a pressure of 60 Pa. Images were taken at a 45˚ sample tilt to enhance surface topography and highlight structural features.

Figures 1-5 display multiple SEM images at different magnifications of quaking aspen leaves collected on five different days during the 2011 summer growth season. SEM images of a quaking aspen leaf harvested on May 14, 2011 (just a few days after leaf emergence) are shown in Figure 1. This young leaf surface does not exhibit the typical superhydrophobic leaf surface structure consisting of micro-scale papillae and nano-scale wax crystals that are responsible for non-wetting properties [7,8]. Instead, the leaf shows multiple, randomly oriented folds in the cuticle and no nano-scale wax crystals are present on its surface (Figure 1). Additionally, this surface contains many small contamination particles, indicating that it does not possess superhydrophobic/selfcleaning properties. In fact, the average water contact and tilt angles for these early leaves were 102˚ ± 6˚ and 26˚, respectively.

Figure 2 displays SEM images of a quaking aspen leaf harvested on May 22, 2011. It is clearly visible that multiple morphological changes have occurred during this relatively short growth period of only 8 days. The cuticular folds of the samples harvested on May 14, 2011 have acted as precursors for the random array of microscale papillae displayed on surfaces collected on May 22, 2011. Moreover, the entire leaf surface is now covered by a dense layer of nano-scale wax crystals, both on and in between surface papillae. Due to the development of these surface features, the non-wetting properties of these leaves have augmented to an average water contact angle of 148˚ ± 5˚ and a tilt angle less than 5˚. Effectively, the growth of this dual scale structure has rendered the quaking aspen leaves superhydrophobic and self-cleaning: a