Automated measurement and analysis of sidewall roughness using three-dimensional atomic force microscopy.

As semiconductor device architecture develops, from planar field-effect transistors (FET) to FinFET and gate-all-around (GAA), there is an increased need to measure 3D structure sidewalls precisely. Here, we present a 3-Dimensional Atomic Force Microscope (3D-AFM), a powerful 3D metrology tool to measure the sidewall roughness (SWR) of vertical and undercut structures. First, we measured three different dies repeatedly to calculate reproducibility in die level. Reproducible results were derived with a relative standard deviation under 2%. Second, we measured 13 different dies, including the center and edge of the wafer, to analyze SWR distribution in wafer level and reliable results were measured. All analysis was performed using a novel algorithm, including auto flattening, sidewall detection, and SWR calculation. In addition, SWR automatic analysis software was implemented to reduce analysis time and to provide standard analysis. The results suggest that our 3D-AFM, based on the tilted Z scanner, will enable an advanced methodology for automated 3D measurement and analysis.

Quantitative Visualization of the Nanomechanical Young's Modulus of Soft Materials by Atomic Force Microscopy.

The accurate measurement of nanoscale mechanical characteristics is crucial in the emerging field of soft condensed matter for industrial applications. An atomic force microscope (AFM) can be used to conduct nanoscale evaluation of the Young's modulus on the target surface based on site-specific force spectroscopy. However, there is still a lack of well-organized study about the nanomechanical interpretation model dependence along with cantilever stiffness and radius of the tip apex for the Young's modulus measurement on the soft materials. Here, we present the fast and accurate measurement of the Young's modulus of a sample's entire scan surface using the AFM in a newly developed PinPointTM nanomechanical mode. This approach enables simultaneous measurements of topographical data and force-distance data at each pixel within the scan area, from which quantitative visualization of the pixel-by-pixel topographical height and Young's modulus of the entire scan surface was realized. We examined several models of contact mechanics and showed that cantilevers with proper mechanical characteristics such as stiffness and tip radius can be used with the PinPointTM mode to accurately evaluate the Young's modulus depending on the sample type.

Characterization of Ligand Adsorption at Individual Gold Nanocubes.

Cetyltrimethylammonium bromide (CTAB) is a widely used surfactant that aids the aqueous synthesis of colloidal nanoparticles. However, the presence of residual CTAB on nanoparticle surfaces can significantly impact nanoparticle applications, such as catalysis and sensing, under hydrated conditions. As such, consideration of the presence and quantity of CTAB on nanoparticle surfaces under hydrated conditions is of significance. Herein, as part of an integrated material characterization framework, we demonstrate the feasibility of in situ atomic force microscopy (AFM) to detect CTAB on the surface of Au nanocubes (Au NCs) under hydrated conditions, which enabled superior characterization compared to conventional spectroscopic methods. In situ force-distance (FD) spectroscopy and Kelvin probe force microscopy (KPFM) measurements support additional characterization of adsorbed CTAB, while correlative in situ AFM and scanning electron microscopy (SEM) measurements were used to evaluate sequential steps of CTAB removal from Au NCs across hydrated and dehydrated environments, respectively. Notably, a substantial quantity of CTAB remained on the Au NC surface after methanol washing, which was detected in AFM measurements but was not detected in infrared spectroscopy measurements. Subsequent electrochemical cleaning was found to be critically important to remove CTAB from the Au NC surface. Correlative measurements were also performed on individual nanoparticles, which further validate the method described here as a powerful tool to determine the extent and degree of CTAB removal from nanoparticle surfaces. This AFM-based method is broadly applicable to characterize the presence and removal of ligands from nanomaterial surfaces under hydrated conditions.

Anti-inflammatory effect of Lycium barbarum on polarized human intestinal epithelial cells.

BACKGROUND/OBJECTIVES: Inflammatory Bowel Disease (IBD) has rapidly escalated in Asia (including Korea) due to increasing westernized diet patterns subsequent to industrialization. Factors associated with endoplasmic reticulum (ER) stress are demonstrated to be one of the major causes of IBD. This study was conducted to investigate the effect of Lycium barbarum (L. barbarum) on ER stress. MATERIALS/METHODS: Mouse embryonic fibroblast (MEF) cell line and polarized Caco-2 human intestinal epithelial cells were treated with crude extract of the L. chinense fruit (LF). Paracellular permeability was measured to examine the effect of tight junction (TJ) integrity. The regulatory pathways of ER stress were evaluated in MEF knockout (KO) cell lines by qPCR for interleukin (IL) 6, IL8 and XBP1 spliced form (XBP1s). Immunoglobulin binding protein (BiP), XBP1s and CCAAT/enhancer-binding homologous protein (CHOP) expressions were measured by RT-PCR. Scanning Ion Conductance Microscopy (SICM) at high resolution was applied to observe morphological changes after treatments. RESULTS: Exposure to LF extract strengthened the TJ, both in the presence and absence of inflammation. In polarized Caco-2 pretreated with LF, induction in the expression of proinflammatory marker IL8 was not significant, whereas ER stress marker XBP1s expression was significantly increased. In wild type (wt) MEF cells, IL6, CHOP and XBP1 spliced form were dose-dependently induced when exposed to 12.5-50 µg/mL extract. However, absence of XBP1 or IRE1α in MEF cells abolished this effect. CONCLUSION: Results of this study show that LF treatment enhances the barrier function and reduces inflammation and ER stress in an IRE1α-XBP1-dependent manner. These results suggest the preventive effect of LF on healthy intestine, and the possibility of reducing the degree of inflammatory symptoms in IBD patients.

Quantitative Evaluation of Viral Protein Binding to Phosphoinositide Receptors and Pharmacological Inhibition.

There is significant interest in developing analytical methods to characterize molecular recognition events between proteins and phosphoinositides, which are a medically important class of carbohydrate-functionalized lipids. Within this scope, one area of high priority involves quantitatively evaluating drug candidates that pharmacologically inhibit protein-phosphoinositide interactions. As full-length proteins are often difficult to produce, establishing methods to study these interactions with shorter, bioactive peptides would be advantageous. Herein, we report an atomic force microscopy (AFM)-based force spectroscopic approach to detect the specific interaction between an amphipathic, α-helical (AH) peptide derived from the hepatitis C virus NS5A protein and its biological target, the phosphatidylinositol (4,5)-bisphosphate [PI(4,5)P2] phosphoinositide receptor. After optimization of the peptide tethering strategy and measurement parameters, the binding specificity of AH peptide for PI(4,5)P2 receptors was comparatively evaluated across a panel of phosphoinositides and the influence of ionic strength on AH-PI(4,5)P2 binding strength was tested. Importantly, these capabilities were translated into the development of a novel experimental methodology to determine the inhibitory activity of a small-molecule drug candidate acting against the AH-PI(4,5)P2 interaction, and extracted kinetic parameters agree well with literature values obtained by conventional biochemical methods. Taken together, our findings provide a nanomechanical basis for explaining the high binding specificity of the NS5A AH to PI(4,5)P2 receptors, in turn establishing an analytical framework to study phosphoinositide-binding viral peptides and proteins as well as a broadly applicable approach to evaluate candidate inhibitors of protein-phosphoinositide interactions.

Controlling the Formation of Phospholipid Monolayer, Bilayer, and Intact Vesicle Layer on Graphene.

Exciting progress has been made in the use of graphene for bio- and chemical sensing applications. In this regard, interfacing lipid membranes with graphene provides a high-sealing interface that is resistant to nonspecific protein adsorption and suitable for measuring biomembrane-associated interactions. However, a controllable method to form well-defined lipid bilayer coatings remains elusive, and there are varying results in the literature. Herein, we demonstrate how design strategies based on molecular self-assembly and surface chemistry can be employed to coat graphene surface with different classes of lipid membrane architectures. We characterize the self-assembly of lipid membranes on CVD-graphene using quartz crystal microbalance with dissipation, field-effect transistor, and Raman spectroscopy. By employing the solvent-assisted lipid bilayer (SALB) method, a lipid monolayer and bilayer were formed on pristine and oxygen-plasma-treated CVD-graphene, respectively. On these surfaces, vesicle fusion method resulted in formation of a lipid monolayer and intact vesicle layer, respectively. Collectively, these findings provide the basis for improved surface functionalization strategies on graphene toward bioelectronic applications.

"Multipoint Force Feedback" Leveling of Massively Parallel Tip Arrays in Scanning Probe Lithography.

Nanoscale patterning with massively parallel 2D array tips is of significant interest in scanning probe lithography. A challenging task for tip-based large area nanolithography is maintaining parallel tip arrays at the same contact point with a sample substrate in order to pattern a uniform array. Here, polymer pen lithography is demonstrated with a novel leveling method to account for the magnitude and direction of the total applied force of tip arrays by a multipoint force sensing structure integrated into the tip holder. This high-precision approach results in a 0.001° slope of feature edge length variation over 1 cm wide tip arrays. The position sensitive leveling operates in a fully automated manner and is applicable to recently developed scanning probe lithography techniques of various kinds which can enable "desktop nanofabrication."

Closed-loop ARS mode for scanning ion conductance microscopy with improved speed and stability for live cell imaging applications.

Scanning ion conductance microscopy (SICM) is an increasingly useful nanotechnology tool for non-contact, high resolution imaging of live biological specimens such as cellular membranes. In particular, approach-retract-scanning (ARS) mode enables fast probing of delicate biological structures by rapid and repeated approach/retraction of a nano-pipette tip. For optimal performance, accurate control of the tip position is a critical issue. Herein, we present a novel closed-loop control strategy for the ARS mode that achieves higher operating speeds with increased stability. The algorithm differs from that of most conventional (i.e., constant velocity) approach schemes as it includes a deceleration phase near the sample surface, which is intended to minimize the possibility of contact with the surface. Analysis of the ion current and tip position demonstrates that the new mode is able to operate at approach speeds of up to 250 µm s(-1). As a result of the improved stability, SICM imaging with the new approach scheme enables significantly improved, high resolution imaging of subtle features of fixed and live cells (e.g., filamentous structures & membrane edges). Taken together, the results suggest that optimization of the tip approach speed can substantially improve SICM imaging performance, further enabling SICM to become widely adopted as a general and versatile research tool for biological studies at the nanoscale level.

Study of Mast Cells and Granules from Primo Nodes Using Scanning Ionic Conductance Microscopy.

Acupuncture points have a notable characteristic in that they have a higher density of mast cells (MCs) compared with nonacupoints in the skin, which is consistent with the augmentation of the immune function by acupuncture treatment. The primo vascular system, which was proposed as the anatomical structure of the acupuncture points and meridians, also has a high density of MCs. We isolated the primo nodes from the surfaces of internal abdominal organs, and the harvested primo nodes were stained with toluidine blue. The MCs were easily recognized by their stained color and their characteristic granules. The MCs were classified into four stages according to the degranulation of histamine granules in the MCs. Using conventional optical microscopes details of the degranulation state of MCs in each stage were not observable. However, we were able to investigate the distribution of the granules on the surfaces of the MCs in each stage, and to demonstrate the height profiles and three-dimensional structures of the MCs without disturbance of the cell membrane by using the scanning ion conductance microscopy.

Cellular interactions of doxorubicin-loaded DNA-modified halloysite nanotubes.

Halloysite nanotube (HNT)-based supramolecular complexes are synthesized and evaluated with respect to their cytotoxicity and effects on cellular structures. As HNTs are water-insoluble, DNA is applied for wrapping the surface of HNTs to enhance their water-dispersibility. To investigate the potential of DNA-wrapped HNTs (HD) as a promising drug delivery carrier, doxorubicin (DOX) is introduced as a model anticancer agent and loaded onto HD. The DOX-loaded, DNA-wrapped HNTs (HDD) show sustained DOX release over two weeks without initial burst of DOX indicating delayed DOX release inside cells. In addition, effects of DNA-wrapped HNTs (HD) or HDD on the cytoskeleton organization of A549 cells are studied by visualizing the distribution of F-actin filaments using confocal laser scanning microscopy, and cellular morphological changes are observed by scanning electron microscopy and scanning ion conductance microscopy.

A trachea-inspired bifurcated microfilter capturing viable circulating tumor cells via altered biophysical properties as measured by atomic force microscopy.

Circulating tumor cells (CTCs), though exceedingly rare in the blood, are nonetheless becoming increasingly important in cancer diagnostics. Despite this keen interest and the growing number of potential clinical applications, there has been limited success in developing a CTC isolation platform that simultaneously optimizes recovery rates, purity, and cell compatibility. Herein, a novel tracheal carina-inspired bifurcated (TRAB) microfilter system is reported, which uses an optimal filter gap size satisfying both 100% theoretical recovery rate and purity, as determined by biomechanical analysis and fluid-structure interaction (FSI) simulations. Biomechanical properties are also used to clearly discriminate between cancer cells and leukocytes, whereby cancer cells are selectively bound to melamine microbeads, which increase the size and stiffness of these cells. Nanoindentation experiments are conducted to measure the stiffness of leukocytes as compared to the microbead-conjugated cancer cells, with these parameters then being used in FSI analyses to optimize the filter gap size. The simulation results show that given a flow rate of 100 µL min(-1), an 8 µm filter gap optimizes the recovery rate and purity. MCF-7 breast cancer cells with solid microbeads are spiked into 3 mL of whole blood and, by using this flow rate along with the optimized microfilter dimensions, the cell mixture passes through the TRAB filter, which achieves a recovery rate of 93% and purity of 59%. Regarding cell compatibility, it is verified that the isolation procedure does not adversely affect cell viability, thus also confirming that the re-collected cancer cells can be cultured for up to 8 days. This work demonstrates a CTC isolation technology platform that optimizes high recovery rates and cell purity while also providing a framework for functional cell studies, potentially enabling even more sensitive and specific cancer diagnostics.

Use of the unroofing technique for atomic force microscopic imaging of the intra-cellular cytoskeleton under aqueous conditions.

Atomic force microscopy (AFM) combined with unroofing techniques enabled clear imaging of the intracellular cytoskeleton and the cytoplasmic surface of the cell membrane under aqueous condition. Many actin filaments were found to form a complex meshwork on the cytoplasmic surface of the membrane, as observed in freeze-etching electron microscopy. Characteristic periodic striations of about 5 nm formed by the assembly of G-actin were detected along actin filaments at higher magnification. Actin filaments aggregated and dispersed at several points, thereby dividing the cytoplasmic surface of the membrane into several large domains. Microtubules were also easily detected and were often tethered to the membrane surface by fine filaments. Furthermore, clathrin coats on the membrane were clearly visualized for the first time in water by AFM. Although the resolution of these images is lower than electron micrographs of freeze-etched samples processed similarly, the measurement capabilities of the AFM in a more biologically relevant conditions demonstrate that it is an important tool for imaging intracellular structures and cell surfaces in the native, aqueous state.

Scanning ion conductance microscopy studies of amyloid fibrils at nanoscale.

Atomic force microscopy (AFM) has developed to become a very versatile nano-scale technique to reveal the three-dimensional (3D) morphology of amyloid aggregates under physiological conditions. However, the imaging principle of AFM is based on measuring the 'force' between a sharp tip and a given nanostructure, which may cause mechanical deformation of relatively soft objects. To avoid the deformation, scanning ion conductance microscopy (SICM) is an alternative scanning probe microscopy technique, operating with alternating current mode. Here we can indeed reveal the 3D morphology of amyloid fibrils and it is capable of exploring proteins with nanoscale resolution. Compared with conventional AFM, we show that SICM can provide precise height measurements of amyloid protein aggregates, a feature that enables us to obtain unique insight into the detailed nucleation and growth mechanisms behind amyloid self-assembly.

Scanning ion conductance microscopy for imaging biological samples in liquid: a comparative study with atomic force microscopy and scanning electron microscopy.

The present study was designed to show the applicability of scanning ion conductance microscopy (SICM) for imaging different types of biological samples. For this purpose, we first applied SICM to image collagen fibrils and showed the usefulness of the approach-retract scanning (ARS)/hopping mode for such samples with steep slopes. Comparison of SICM images with those obtained by AFM revealed that the ARS/hopping SICM mode can probe the surface topography of collagen fibrils and chromosomes at nanoscale resolution under liquid conditions. In addition, we successfully imaged cultured HeLa cells, with 15 µm in height by ARS/hopping SICM mode. Because SICM can obtain non-contact (or force-free) images, delicate cellular projections were visualized on the surface of the fixed cell. SICM imaging of live HeLa cells further demonstrated its applicability to study the morphological dynamics associated with biological processes on the time scale of minutes under liquid conditions. We further applied SICM for imaging the luminal surface of the trachea and succeeded in visualizing the surface of both ciliated and non-ciliated cells. These SICM images were comparable with those obtained by scanning electron microscopy. Although the dynamic mode of AFM provides better resolution than the ARS/hopping mode of SICM in some samples, only the latter can obtain contact-free images of samples with steep slopes, rendering it an important tool for observing live cells as well as unfixed or fixed soft samples with complicated shapes. Taken together, we demonstrate that SICM imaging, especially using an ARS/hopping mode, is a useful technique with unique capabilities for imaging the three-dimensional topography of a range of biological samples under physiologically relevant aqueous conditions.

Three-dimensional imaging of undercut and sidewall structures by atomic force microscopy.

Sidewall surface roughness is an important parameter in electronic device manufacture. At present, no high resolution technique exists to quantitatively characterize this property for undercut structures created by semiconductor processing techniques. We developed a three-dimensional atomic force microscope (3D-AFM) to measure the surface roughness of undercut sidewalls with nanometer precision. Decoupled from the positional scanner, the 3D-AFM probe had a variable tilt up to 40° off the normal. Nonorthogonal scans resolved the sidewall surface roughness, base width, and acute critical angle for undercut structures, including a metal overhang and the transmission line of a photonic device. Compatible with standard cantilevers, the 3D-AFM demonstrates great potential for characterizing the sidewalls of soft materials such as photoresist.

Identification of a class of HCV inhibitors directed against the nonstructural protein NS4B.

New classes of drugs are needed to combat hepatitis C virus (HCV), an important worldwide cause of liver disease. We describe an activity of a key domain, an amphipathic helix we termed 4BAH2, within a specific HCV nonstructural protein, NS4B. In addition to its proposed role in viral replication, we validate 4BAH2 as essential for HCV genome replication and identify first-generation small-molecule inhibitors of 4BAH2 that specifically prevent HCV replication within cells. Mechanistic studies reveal that the inhibitors target 4BAH2 function by preventing either 4BAH2 oligomerization or 4BAH2 membrane association. 4BAH2 inhibitors represent an additional class of compounds with potential to effectively treat HCV.

Mechanism of an amphipathic alpha-helical peptide's antiviral activity involves size-dependent virus particle lysis.

The N-terminal region of the hepatitis C virus (HCV) nonstructural protein NS5A contains an amphipathic alpha-helix that is necessary and sufficient for NS5A membrane association. A synthetic peptide (AH) comprising this amphipathic helix is able to lyse lipid vesicles that serve as a model system for virus particles. Based on quartz crystal microbalance-dissipation (QCM-D) experiments, the degree of vesicle rupturing was found to be inversely related to vesicle size, with maximal activity in the size range of several medically important viruses. In order to confirm and further study vesicle rupture, dynamic light scattering (DLS) and atomic force microscopy (AFM) experiments were also performed. The size dependence of vesicle rupturing helps explain the peptide's observed effect on the infectivity of a wide range of viruses. Further, in vitro studies demonstrated that AH peptide treatment significantly decreased the infectivity of HCV particles. Thus, the AH peptide might be used to rupture HCV particles extra-corporally (for HCV prevention) and within infected individuals (for HCV therapy).

Binding behavior of CRP and anti-CRP antibody analyzed with SPR and AFM measurement.

Atomic force microscope (AFM) was exploited to take picture of the molecular topology of C-reactive protein (CRP) in phosphate-buffered saline (PBS) solution. An explicit molecular image of CRP demonstrated a pentagonal structure composed of five subunits. Dimensions of the doughnut-shaped CRP molecule measured by AFM were about 25 nm in outside diameter and 10 nm in central pore diameter, and the height of CRP molecule was about 4 nm which was comparable to the value determined by X-ray crystallography. Bis(N-succinimido)-11,11'-dithiobis (undecyl succinate) (DSNHS) was synthesized for use as a linker for immobilizing anti-CRP antibody (anti-CRP) onto the gold surface of a surface plasmon resonance (SPR) sensor chip. DSNHS formed self-assembled monolayer (SAM) on the gold surface. By use of an AFM tip, a pattern of ditch was engraved within the SAM of DSNHS, and anti-CRP was immobilized on the engraved SAM through replacement of N-hydroxysuccinimide group on the outside surface of DSNHS by the amine group of anti-CRP. Formation of CRP/anti-CRP complex on the gold surface of SPR sensor chip was clearly demonstrated by measuring SPR angle shift. A consecutive series of SAM, SAM/anti-CRP, and SAM/anti-CRP/CRP complexes was generated on a SPR sensor chip, and the changes in depth of the ditch were monitored by taking AFM images of the complexes. Comparative analysis of the depth differences indicates that binding of CRP to anti-CRP occurs in a planar mode.

Creation of lipid partitions by deposition of amphipathic viral peptides.

Phospholipid vesicles exhibit a natural characteristic to fuse and reform into a continuous single bilayer membrane on hydrophilic solid substrates such as glass, mica, and silica. The resulting solid-supported bilayer mimics physiological tendencies such as lipid flip-flop and lateral mobility. The lateral mobility of fluorescently labeled lipids fused into solid-supported bilayers is found to change upon deposition on the membrane surface of an amphipathic alpha-helical peptide (AH) derived from the hepatitis C virus (HCV) NS5A protein. The binding of the AH peptide to a phospholipid bilayer, with the helical axis parallel to the bilayer, leads to immobilization of the bilayer. We used AFM to better understand the mechanistic details of this specific interaction, and determined that the diminished fluidity of the bilayer is due to membrane thinning. Utilizing this specific interaction between AH peptides and lipid molecules, we demonstrate a novel process for the creation of lipid partition by employing AH peptides as agents to immobilize lipid molecules, thus creating a patterned solid support with partition-defined areas of freely mobile lipid bilayers. This architecture could have a wide range of applications in novel sensing, biotechnology, high-throughput screening, and biomimetic strategies.