The rapid growth and early development period of the dual-scale surface topography was studied on the adaxial leaf surfaces of two aspen tree species with non-wetting leaves: the columnar European aspen (Populus tremula “Erecta”) and quaking aspen (Populus tremuloides). Particular attention was focused on the formation of micro- and nano-scale asperities on their cuticles, which was correlated with the development of superhydrophobic wetting behaviour. Measurements of the wetting properties (contact angle and tilt-angle) provided an indication of the degree of hydrophobicity of their cuticles. Scanning electron microscopy and optical profilometry micrographs were used to follow the growth and major morphological changes of micro-scale papillae and nano-scale epicuticular wax (ECW) crystals, which led to a significant improvement in non-wetting behaviour. Both species exhibited syntopism in the form of small and larger nano-scale ECW platelet morphologies. These findings provide additional support for earlier suggestions that due to fluctuations in leaf hydrophobicity throughout the growing season, canopy storage capacity may also vary considerably throughout this time period.
Recent research efforts have focused on the characterization and understanding of non-wetting (Contact Angle (CA): 110˚ - 150˚) and superhydrophobic (CA ≥ 150˚) leaf surfaces which repel water droplets, allowing them to remain essentially dry (e.g. [
It has been suggested in numerous studies that the chemical composition of the ECW layer dictates the morphological type of these nano-scale asperities on the leaf’s surface [
Leaves from the aspen (Populus) species, were selected as a prime candidate for our study due to their unique biological significance. For example, it has been recently demonstrated [
Recent studies have reported on the significant changes in the surface morphology and wetting characteristics of several superhydrophobic and hydrophobic leaf surfaces throughout the growing season [
Currently, there is a great deal of literature in the field of materials science dedicated to enhancing our understanding of the ways in which water droplets are repelled off various surfaces; these findings provide inspiration for the development of future artificial water-repellent materials and coatings [
All leaf samples were collected three times per week during their initial growth and development period (April 18th-May 21st, 2012) from trees in two wooded areas. Columnar European aspen (CEA) leaves were collected at the University of Toronto (Toronto, Ontario, Canada) in a garden plantation (N43˚39.56', W079˚23.55') while quaking aspen (QA) leaves came from a forest near Peterborough, Ontario, Canada (N44˚12.24', W078˚23.23'). For both species all leaves were collected from one particular branch on the same tree at a height of less than 10 feet. The age of the trees was at least 15 and 10 years for columnar European and quaking aspen, respectively. All leaves were air dried, cut and mounted onto 1'' × 1'' Plexiglass™ coupons using double-sided carbon tape for wetting property measurements on their adaxial surfaces, and subsequently carbon coated for electron microscopy analysis. Additionally, leaf size measurements were recorded to compare the growth of leaf samples with growth of cuticular surface features (micro- and nano-scale asperities). A ruler was used to measure the length parallel and perpendicular to the petiole for a minimum of six leaves on each collection date. The average size and its standard deviation were recorded.
Both static and dynamic wetting properties of adaxial leaf surfaces were measured and correlated with the initial growth of micro-scale papillae and nano-scale epicuticular wax (ECW) morphologies. Static wetting properties were analyzed by measuring the contact angle (CA) created between sessile water droplets and the leaf surface. In addition, the tilt-angle (TA), or relative inclination angle for water droplet roll-off was measured. Note that the standard deviation (+/−) was also included for all wetting property (CA and TA) measurements. For CA measurements, leaf samples were aligned with a high resolution digital camera (Nikon D3000) equipped with a macro-lens (Nikon AF-S Micro Nikkor 40 mm), which was situated on a tripod and used to capture images of no less than four 5 μL droplets in various positions on each aspen leaf surface. Micrographs were analyzed using ImageJ software for contact angle measurements [
The surface morphology development of aspen leaf surfaces was captured by Scanning Electron Microscopy (Hitachi S-4500 Cold-Field Emission SEM). Micrographs were taken at a sample tilt angle of 55˚ in order to clearly distinguish variations in surface morphology. Characterization of nano-scale roughness asperities was completed using high magnification SEM micrographs to estimate the average height (perpendicular to leaf surface) and length (parallel to leaf surface) of both: i) shorter nano-scale crystals, as well as ii) larger elongated nano-morphologies during the initial growth period.
Optical Profilometry (WYKO NT-2000 with Wyko Vision 32 Software) was utilized to image 2-D and 3-D topographical surface plots of each leaf surface and detect changes to the size and shape of micro-scale features during the early stage growth phase. Using X-Y surface plots, the average peak to valley (p-v) height (μm) and micro-scale feature base diameters (μm) were measured. In order to obtain meaningful averages, a minimum of five micro-features were measured for each leaf specimen in both the X and Y directions, and at least two leaf specimens were analyzed per collection date. The standard deviation for all measurements was also included.
Columnar European aspen leaves first emerged April 16th, 2012 and were collected and analyzed from April 18th-May 21st, 2012. Correspondingly, quaking aspen leaves were collected beginning two days after bud break (May 3rd, 2012) until May 20th, 2012.
The wetting properties (contact angle and tilt-angle) of columnar European and quaking aspen leaf surfaces measured during their initial growth and development period (spanning from April 18th to May 21st) are shown in
were characterized by a ramp up in CA from a slightly hydrophobic surface (94˚) to a superhydrophobic state (160˚), which occurred over a 9 day period (May 5th to 14th). This trend correlated with a simultaneous decrease in TA (as high as 18˚ to as low as 6˚), over the same timeframe. In addition, leaves were found to grow in both parallel (19 à 45 mm) and perpendicular (from 17 to 47 mm) dimensions relative to the petiole (
Quaking and columnar European aspen adaxial leaf surfaces experienced a similar trend in wetting properties, as was highlighted in
Optical profilometry results related to micro-scale papillae roughness for columnar European aspen and quaking aspen are summarized in
Micro-Scale Roughness | Nano-Scale Roughness | Wetting Properties | ||||||
---|---|---|---|---|---|---|---|---|
Date | Base Diameter (μm) | Peak-Valley Height (μm) | Nano-crystals | Elongated Nano-morphologies | Contact Angle (˚) | Tilt-Angle (˚) | ||
Height (nm) | Length (nm) | Height (μm) | Length (μm) | |||||
May 2 | 9.3 ± 4.4 | 1.4 ± 1.1 | 232 ± 54 | 369 ± 89 | - | - | 92 ± 6 | 30 ± 6 |
May 4 | 11.5 ± 3.0 | 2.1 ± 0.7 | 409 ± 68 | 568 ± 169 | - | - | 109 ± 9 | 41 ± 4 |
May 7 | 15.8 ± 4.4 | 2.2 ± 1.1 | 452 ± 77 | 659 ± 149 | - | - | 125 ± 5 | 22 ± 4 |
May 14 | 20.1 ± 4.9 | 6.1 ± 1.2 | 591 ± 109 | 636 ± 87 | 1.1 ± 0.3 | 2.9 ± 1.1 | 144 ± 2 | 4 ± 2 |
May 21 | 17.4 ± 4.0 | 5.4 ± 1.1 | 626 ± 102 | 567 ± 122 | 0.9 ± 0.3 | 2.1 ± 0.4 | 151 ± 2 | 7 ± 1 |
Micro-Scale Roughness | Nano-Scale Roughness | Wetting Properties | ||||||
---|---|---|---|---|---|---|---|---|
Date | Base Diameter (μm) | Peak-Valley Height (μm) | Nano-Crystals | Elongated Nano-Morphologies | Contact Angle (˚) | Tilt-Angle (˚) | ||
Height (nm) | Length (nm) | Height (μm) | Length (μm) | |||||
May 8 | 11.4 ± 3.2 | 2.9 ± 0.6 | 330 ± 100 | 420 ± 70 | - | - | 119 ± 13 | 17 ± 3 |
May 11 | 14.1 ± 2.8 | 4.7 ± 1.1 | 452 ± 65 | 549 ± 180 | - | - | 134 ± 3 | 6 ± 3 |
May 14 | 17.8 ± 2.9 | 6.1 ± 1.0 | 527 ± 93 | 521 ± 117 | 1.0 ± 0.3 | 3.4 ± 1.0 | 160 ± 4 | 12 ± 3 |
May 17 | 19.8 ± 2.8 | 5.9 ± 1.1 | 560 ± 70 | 513 ± 110 | 1.1 ± 0.3 | 3.7 ± 1.2 | 156 ± 5 | 10 ± 2 |
May 20 | 18.1 ± 4.0 | 5.0 ± 1.1 | 620 ± 93 | 478 ± 89 | 1.0 ± 0.3 | 2.8 ± 0.8 | 160 ± 4 | 10 ± 3 |
2nd to 21st for columnar European aspen and May 8th to 20th for quaking aspen. Values were not provided for the initial steady state period for columnar European aspen leaf species due to difficulties in resolving the height of relatively small micro-scale papillae.
The surface morphology development on the nano-scale of columnar European aspen leaf surfaces was further
characterized using SEM during its initial growth phase (April 18th-May 21st). Plate 1 illustrates the four major morphological changes to the leaf’s surface during this period of leaf expansion ( Plates 1(a)-(h) ), each at low (left) and high (right) magnifications.
The first stage of development was captured on April 25, where leaves exhibited a relatively flat surface topography ( Plate 1(a) and Plate 1(b) ). SEM micrographs show little surface roughness over large areas. The low magnification micrograph ( Plate 1(a) ) clearly shows the presence of a rare stoma on the upper leaf cuticle. Leaf surfaces were determined to be slightly hydrophilic in nature (CA = 86˚) with high tilt-angle (TA = 65˚) at this stage.
The second stage of growth, captured on May 4th ( Plate 1(c) and Plate 1(d) ), demonstrates the development of micro-scale papillae and nucleation of sparsely distributed nano-scale ECW crystals on top of micro-scale papillae, typically 100 - 300 nm in thickness at the top and up to 500 nm at the bottom. Optical profilometry results (
On the third stage of leaf surface structure development (captured May7th), an increased density of nano-scale ECW platelets atop micro-scale papillae is observed ( Plate 1(e) and Plate 1(f) ). While their thickness remained relatively constant, ECW nano-crystals grew in height (452 nm) and length (659 nm), during this period (
Finally the fourth stage of development (captured on May 14) displays evidence of syntopism [
Plate 1. Low magnification (left) and high magnification (right) SEM micrographs showing development of CEA leaf surface captured: April 25th ((a) & (b)), May 4th ((c) & (d)), May 7th (e) & (f) and May 14th ((g) & (h)).
rized by an average height and length of 591 and 636 nm, respectively (
The surface morphology development of quaking aspen leaf surfaces was also characterized using SEM during early season leaf development (May 5th to 14th). The original surface and the fully developed structure are shown in Plate 2 . The initial stage of development was captured on May 5 ( Plate 2(a) and Plate 2(b) ), two days
Plate 2. Low magnification (left) and high magnification (right) SEM micrographs showing development of QA leaf surface captured: May 5th ((a) & (b)), and May 14th ((c) & (d)).
following bud break. SEM Micrographs show a relatively flat leaf surface and the absence of well defined micro-scale papillae and nano-scale ECW morphologies. As a result, the wetting properties were found to be wettable in nature (CA = 94˚).
Five days following initial bud break (May 8), micro-scale papillae had developed together with nucleation of superimposed nano-scale ECW morphologies (330 nm in height and 420 nm in length,
On May 11, there is a further improvement in non-wetting behaviour (CA = 134˚) which can be attributed to an increase in average micro-papillae size (p-v height = 4.7 μm, base diameter = 14.1 μm,
Finally, the fourth stage of growth, captured on May 14 ( Plate 2(c) and Plate 2(d) ), is characterized by significant changes in nano-scale ECW morphologies, leading to the development of a cuticle surface with superhydrophobic wetting properties (CA = 160˚). High magnification SEM micrograph ( Plate 2(d) ) reveal the co-existence of small nano-scale ECW platelets with average height and length of 527 and 521 nm, respectively, (see
In summary, SEM micrographs were used to correlate the development of micro- and nano-scale roughness asperities with ramp up in non-wetting behaviour. Not surprisingly, quaking and columnar European aspen also showed similarities in their initial stages of growth and development. Within a short period of time, micro features began to form, grew in size and nucleated nano-scale ECW nanocrystal and platelet morphologies superimposed on micro-scale papillose epidermal cells. The nucleated crystals grew in size and populated densely, which was followed by the formation of secondary nano-scale ECW platelets, providing evidence of syntopism. The increase in nano-crystal height and length observed on the surfaces of both columnar European and quaking aspen leaf cuticles, in conjunction with micro-papillae growth, positively influenced the non-wetting behaviour. In addition, co-existing elongated nano-scale ECW platelets were shown to improve the non-wetting behaviour of inherently hydrophobic leaf cuticles.
The exact mechanisms for nucleation and growth of the ECW morphologies are not known in the context of the studied leaf specimens. Various mechanisms have been suggested in the literature [
The aim of the current study was to address this issue from a morphological point of view. It was found that the early season formation of dual-scale surface topography on both quaking and columnar European aspen leaf surfaces resulted in a ramp up from wettable to superhydrophobic leaf wetting behaviour. These findings in conjunction with previous results [
Varying wetting characteristics due to leaf surface morphology changes during the leaf lifetime is also important to consider in studies that attempt to relate stomata density of leaves and their corresponding wetting angle, both on adaxial and abaxial surfaces. An earlier hypothesis that stomata density is strongly correlated with wetting angle [
In conclusion, columnar European and quaking aspen leaf surfaces experienced a similar growth of their micro- and nano-scale asperities during their early season growth period, which led to the development of their unique superhydrophobic property. The increase in both relative size and density of nano-scale wax crystals was positively correlated with an increase in contact angle and decrease in tilt-angle. Quaking aspen leaf cuticles were found to achieve superhydrophobicity in a shorter time period (9 days) than columnar European aspen leaf samples (32 days). It was also found that syntopism, or the coexistence of ECW morphologies of different size lengths (specifically short nano-crystals as well as elongated nano-scale morphologies), appeared to enhance this unique extreme non-wetting behaviour. This particular study has served to support the importance of hierarchical roughness (at both the micro- and nano-scale levels) for this class of hydrophobic cuticles as an important criterion for the development of superhydrophobic wetting behaviour. Future work within this field of research should focus on characterization of changes in ECW chemistry during leaf development, which may help explain how these wax morphologies are developed. Specifically, establishing a link between wax micro/nano- morphology, and the type(s) of chemical constituents present in the ECW layer of each leaf species should be studied. In addition, further studies may also aim to establish the mechanism(s) for the nucleation and growth of ECW morphologies.
We would like to thank Mr. Sal Boccia for his help with scanning electron microscopy micrography. The financial support from the Natural Sciences and Engineering Research Council of Canada (NSERC) is greatly acknowledged.
George ChristopherTranquada,Jared JenningsVictor,UweErb, (2015) Early Season Development of Micro/Nano-Morphology and Superhydrophobic Wetting Properties on Aspen Leaf Surfaces. American Journal of Plant Sciences,06,2197-2208. doi: 10.4236/ajps.2015.613222