Automated trichome counting in soybean using advanced image-processing techniques.

PREMISE: Trichomes are hair-like appendages extending from the plant epidermis. They serve many important biotic roles, including interference with herbivore movement. Characterizing the number, density, and distribution of trichomes can provide valuable insights on plant response to insect infestation and define the extent of plant defense capability. Automated trichome counting would speed up this research but poses several challenges, primarily because of the variability in coloration and the high occlusion of the trichomes. METHODS AND RESULTS: We developed a simplified method for image processing for automated and semi-automated trichome counting. We illustrate this process using 30 leaves from 10 genotypes of soybean (Glycine max) differing in trichome abundance. We explored various heuristic image-processing methods including thresholding and graph-based algorithms to facilitate trichome counting. Of the two automated and two semi-automated methods for trichome counting tested and with the help of regression analysis, the semi-automated manually annotated trichome intersection curve method performed best, with an accuracy of close to 90% compared with the manually counted data. CONCLUSIONS: We address trichome counting challenges including occlusion by combining image processing with human intervention to propose a semi-automated method for trichome quantification. This provides new opportunities for the rapid and automated identification and quantification of trichomes, which has applications in a wide variety of disciplines.

Formation of Size and Density Controlled Nanostructures by Galvanic Displacement.

Gold (Au) and copper (Cu)-based nanostructures are of great interest due to their applicability in various areas including catalysis, sensing and optoelectronics. Nanostructures synthesized by the galvanic displacement method often lead to non-uniform density and poor size distribution. Here, density and size-controlled synthesis of Au and Cu-based nanostructures was made possible by galvanic displacement with limited exposure to hydrofluoric (HF) acid and the use of surfactants like L-cysteine (L-Cys) and cetyltrimethylammonium bromide (CTAB). An approach involving cyclic exposure to HF acid regulated the nanostructure density. Further, the use of surfactants generated monodisperse nanoparticles in the initial stages of the deposition with increased density. The characterization of Au and Cu-based nanostructures was performed by scanning electron microscopy, atomic force microscopy, UV-Visible spectroscopy, X-ray photoelectron spectroscopy, Raman spectroscopy and X-ray diffraction. The surface enhanced Raman spectroscopic measurements demonstrated an increase in the Raman intensity by two to three orders of magnitude for analyte molecules like Rhodamine 6G dye and paraoxon.

Rheological transition in simple shear of moderately dense assemblies of dry cohesive granules.

The rheology of homogeneous cohesive granular assemblies under shear at moderate volume fractions is investigated using the discrete element method for both frictionless and frictional granules. A transition in rheology from inertial to quasistatic scaling is observed at volume fractions below the jamming point of noncohesive systems, which is a function of the granular temperature, energy dissipation, and cohesive potential. The transition is found to be the result of growing clusters, which eventually percolate the domain, and change the mode of momentum transport in the system. Differences in the behavior of the shear stress normalized by the pressure are observed when frictionless and frictional cases are compared. These differences are explained through contact anisotropy after percolation occurs. Both frictionless and frictional systems are found to be vulnerable to instabilities after full system percolation has occurred, where the former becomes thermodynamically unstable and the latter may form shear bands. Finally, implications for constitutive modeling are discussed.

An alternative method to determining optical lever sensitivity in atomic force microscopy without tip-sample contact.

Force studies using atomic force microscopy generally require knowledge of the cantilever spring constants and the optical lever sensitivity. The traditional method of evaluating the optical lever sensitivity by pressing the tip against a hard surface can damage the tip, especially sharp ones. Here a method is shown to calculate the sensitivity without having to bring the tip into contact. Instead a sharpened tungsten wire is used to cause a point contact directly onto the cantilever and cause cantilever bending. Using beam theory, the sensitivity thus found can be converted to the equivalent sensitivity that would be obtained using the tip location. A comparison is presented between sensitivity values obtained from the conventional tip contact method and those derived from the wire-based technique for a range of cantilevers in air. It was found that the difference between the calculated sensitivity from the wire-based technique and the sensitivity obtained conventionally was less than 12%. These measurements indicate the presented method offers a simple alternative approach to obtain optical lever sensitivity without compromising the tip shape.

Atom scale characterization of the near apex region of an atomic force microscope tip.

Three-dimensional atom probe tomography (APT) is successfully used to analyze the near-apex regions of an atomic force microscope (AFM) tip. Atom scale material structure and chemistry from APT analysis for standard silicon AFM tips and silicon AFM tips coated with a thin film of Cu is presented. Comparison of the thin film data with that observed using transmission electron microscopy indicates that APT can be reliably used to investigate the material structure and chemistry of the apex of an AFM tip at near atomic scales.

Friction and wear behavior of ultrahigh molecular weight polyethylene as a function of crystallinity in the presence of the phospholipid dipalmitoyl phosphatidylcholine.

In this study, the friction and wear behavior of ultrahigh molecular weight polyethylene (UHMWPE) were evaluated as a function of polymer crystallinity in the presence of the phospholipid dipalmitoyl phosphatidylcholine (DPPC) dissolved in ethanol. Samples of UHMWPE were separately heat treated to get high and low crystallinity samples. Degree of crystallinity was evaluated using differential scanning calorimetry. Quantitative friction and wear experiments were conducted using a custom-made microtribometer with commercially available spherical Si(3)N(4) probes in controlled and phospholipid-dissolved lubricants. The higher crystallinity sample exhibited slightly lower friction than the lower crystallinity in the control and decreased significantly when phospholipids were present. The higher crystallinity sample showed a higher wear resistance than the lower crystallinity sample during all reciprocating wear tests. DPPC acting as a lubricant had a marginal effect on the wear resistance of high crystallinity UHMWPE, whereas the low crystallinity sample became more prone to wear. Atomic force microscopy topography images and contact angle measurements of both samples before and after phospholipid exposure indicate that the higher crystallinity sample absorbed a greater density of DPPC. Increasing crystallinity is a way of escalating adsorption of surface active phospholipids onto UHMWPE to make it a more wear-resistant load-bearing material for total joint replacements.

Nanoscale friction switches: friction modulation of monomolecular assemblies using external electric fields.

This paper presents experimental investigations to actively modulate the nanoscale friction properties of a self-assembled monolayer (SAM) assembly using an external electric field that drives conformational changes in the SAM. Such "friction switches" have widespread implications in interfacial energy control in micro/nanoscale devices. Friction response of a low-density mercaptocarboxylic acid SAM is evaluated using an atomic force microscope (AFM) in the presence of a DC bias applied between the sample and the AFM probe under a nitrogen (dry) environment. The low density allows reorientation of individual SAM molecules to accommodate the attractive force between the -COOH terminal group and a positively biased surface. This enables the surface to present a hydrophilic group or a hydrophobic backbone to the contacting AFM probe depending upon the direction of the field (bias). Synthesis and deposition of the low-density SAM (LD-SAM) is reported. Results from AFM experiments show an increased friction response (up to 300%) of the LD-SAM system in the presence of a positive bias compared to the friction response in the presence of a negative bias. The difference in the friction response is attributed to the change in the structural and crystalline order of the film in addition to the interfacial surface chemistry and composition presented upon application of the bias.

Visualization by atomic force microscopy and FISH of the 45S rDNA gaps in mitotic chromosomes of Lolium perenne.

The mitotic chromosome structure of 45S rDNA site gaps in Lolium perenne was studied by atomic force microscope (AFM) combining with fluorescence in situ hybridization (FISH) analysis in the present study. FISH on the mitotic chromosomes showed that 45S rDNA gaps were completely broken or local despiralizations of the chromatid which had the appearance of one or a few thin DNA fiber threads. Topography imaging using AFM confirmed these observations. In addition, AFM imaging showed that the broken end of the chromosome fragment lacking the 45S rDNA was sharper, suggesting high condensation. In contrast, the broken ends containing the 45S rDNA or thin 45S rDNA fibers exhibited lower density and were uncompacted. Higher magnification visualization by AFM of the terminals of decondensed 45S rDNA chromatin indicated that both ends containing the 45S rDNA also exhibited lower density zones. The measured height of a decondensed 45S rDNA chromatin as obtained from the AFM image was about 55-65 nm, composed of just two 30-nm single fibers of chromatin. FISH in flow-sorted G2 interphase nuclei showed that 45S rDNA was highly decondensed in more than 90% of the G2/M nuclei. Our results suggested that a failure of the complex folding of the chromatin fibers occurred at 45S rDNA sites, resulting in gap formation or break.

Iterative control approach to high-speed force-distance curve measurement using AFM: time-dependent response of PDMS example.

Force-distance curve measurements using atomic force microscope (AFM) has been widely used in a broad range of areas. However, currently force-curve measurements are hampered the its low speed of AFM. In this article, a novel inversion-based iterative control technique is proposed to dramatically increase the speed of force-curve measurements. Experimental results are presented to show that by using the proposed control technique, the speed of force-curve measurements can be increased by over 80 times--with no loss of spatial resolution--on a commercial AFM platform and with a standard cantilever. High-speed force curve measurements using this control technique are utilized to quantitatively study the time-dependent elastic modulus of poly(dimethylsiloxane) (PDMS). The force-curves employ a broad spectrum of push-in (load) rates, spanning two-order differences. The elastic modulus measured at low-speed compares well with the value obtained from dynamic mechanical analysis (DMA) test, and the value of the elastic modulus increases as the push-in rate increases, signifying that a faster external deformation rate transitions the viscoelastic response of PDMS from that of a rubbery material toward a glassy one.

Friction and wear behavior of ultra-high molecular weight polyethylene as a function of polymer crystallinity.

In this study the friction, wear and surface mechanical behavior of medical-grade ultra-high molecular weight polyethylene (UHMWPE) (GUR 1050 resin) were evaluated as a function of polymer crystallinity. Crystallinity was controlled by heating UHMWPE to a temperature above its melting point and varying the hold time and cooling rates. The degree of crystallinity of the samples was evaluated using differential scanning calorimetry (DSC). A higher degree of crystallinity in the UHMWPE resulted in lower friction force and an increase in scratch resistance at the micro- and nanoscales. On the nanoscale, the lamellar structure appeared to affect the observed wear resistance. Reciprocating-wear tests performed using a microtribometer showed that an increase in crystallinity also resulted in lower wear depth and width. Nanoindentation experiments also showed an increase in hardness values with an increase in sample crystallinity.

Method to generate surfaces with desired roughness parameters.

A surface engineering method based on the electrostatic deposition of microparticles and dry etching is described and shown to be able to independently tune both amplitude and spatial roughness parameters of the final surface. Statistical models were developed to connect process variables to the amplitude parameters (center line average and root-mean-square) and a spatial parameter (autocorrelation length) of the final surfaces. Process variables include particle coverage, which affects both amplitude and spatial roughness parameters, particle size, which affects only spatial parameters, and etch depth, which affects only amplitude parameters. Correlations between experimental data and model predictions are discussed.

Comparison of the effect of surface roughness on the micro/nanotribological behavior of ultra-high-molecular-weight polyethylene (UHMWPE) in air and bovine serum solution.

Tribological properties of materials used in biomedical implants critically affect the performance of the implant. A UHMWPE cup paired with a ceramic ball is a popular combination for implants due to its relatively low wear rate. In this study we investigate the effect of surface roughness of UHMWPE on the friction behavior and onset of wear in a UHMWPE/silicon nitride interface in both dry air and bovine serum environments. Microscale multi-asperity contact is examined using a ball-on-flat reciprocating microtribometer. Nanoscale single-asperity contact and surface topography are examined using atomic force microscopy. Friction was found to increase with a decrease in surface roughness of the UHMWPE sample in air, which is due to an increase in real area of contact. This trend was seen to disappear or even reverse in serum. This is due to an increase in the interfacial shear stress of the UHMWPE surface when exposed to the serum. This increase is believed to be caused by an adhered layer of protein on the UHMWPE surface.

Mechanical property measurements of nanoscale structures using an atomic force microscope.

This paper describes nanometer-scale bending tests of fixed single-crystal silicon (Si) and silicon dioxide (SiO2) nanobeams using an atomic force microscope (AFM). The technique is used to evaluate elastic modulus of the beam materials and bending strength of the beams. Nanometer-scale Si beams with widths ranging from 200 to 800 nm were fabricated on a Si diaphragm using field-enhanced anodization using an AFM followed by anisotropic wet etching. Subsequent thermal oxidation of Si beams was carried out to create SiO2 beams. Results from the bending tests indicate that elastic modulus values are comparable to bulk values. However, the bending strength appears to be higher for these nanoscale structures than for large-scale specimens. Observations of the fracture surface and calculations of the crack length from Griffith's theory appear to indicate that the maximum peak-to-valley distance on the beam top surfaces influence the values of the observed bending strengths.