Cellular level robotic surgery: Nanodissection of intermediate filaments in live keratinocytes.

We present the nanosurgery on the cytoskeleton of live cells using AFM based nanorobotics to achieve adhesiolysis and mimic the effect of pathophysiological modulation of intercellular adhesion. Nanosurgery successfully severs the intermediate filament bundles and disrupts cell-cell adhesion similar to the desmosomal protein disassembly in autoimmune disease, or the cationic modulation of desmosome formation. Our nanomechanical analysis revealed that adhesion loss results in a decrease in cellular stiffness in both cases of biochemical modulation of the desmosome junctions and mechanical disruption of intercellular adhesion, supporting the notion that intercellular adhesion through intermediate filaments anchors the cell structure as focal adhesion does and that intermediate filaments are integral components in cell mechanical integrity. The surgical process could potentially help reveal the mechanism of autoimmune pathology-induced cell-cell adhesion loss as well as its related pathways that lead to cell apoptosis.

Characterization of mechanical behavior of an epithelial monolayer in response to epidermal growth factor stimulation.

Cell signaling often causes changes in cellular mechanical properties. Knowledge of such changes can ultimately lead to insight into the complex network of cell signaling. In the current study, we employed a combination of atomic force microscopy (AFM) and quartz crystal microbalance with dissipation monitoring (QCM-D) to characterize the mechanical behavior of A431 cells in response to epidermal growth factor receptor (EGFR) signaling. From AFM, which probes the upper portion of an individual cell in a monolayer of cells, we observed increases in energy dissipation, Young's modulus, and hysteresivity. Increases in hysteresivity imply a shift toward a more fluid-like mechanical ordering state in the bodies of the cells. From QCM-D, which probes the basal area of the monolayer of cells collectively, we observed decreases in energy dissipation factor. This result suggests a shift toward a more solid-like state in the basal areas of the cells. The comparative analysis of these results indicates a regionally specific mechanical behavior of the cell in response to EGFR signaling and suggests a correlation between the time-dependent mechanical responses and the dynamic process of EGFR signaling. This study also demonstrates that a combination of AFM and QCM-D is able to provide a more complete and refined mechanical profile of the cells during cell signaling.

Atomic force microscopy as nanorobot.

Atomic force microscopy (AFM) is a powerful and widely used imaging technique that can visualize single molecules under physiological condition at the nanometer scale. In this chapter, an AFM-based nanorobot for biological studies is introduced. Using the AFM tip as an end effector, the AFM can be modified into a nanorobot that can manipulate biological objects at the single-molecule level. By functionalizing the AFM tip with specific antibodies, the nanorobot is able to identify specific types of receptors on the cell membrane. It is similar to the fluorescent optical microscopy but with higher resolution. By locally updating the AFM image based on interaction force information and objects' model during nanomanipulation, real-time visual feedback is obtained through the augmented reality interface. The development of the AFM-based nanorobotic system enables us to conduct in situ imaging, sensing, and manipulation simultaneously at the nanometer scale (e.g., protein and DNA levels). The AFM-based nanorobotic system offers several advantages and capabilities for studying structure-function relationships of biological specimens. As a result, many biomedical applications can be achieved by the AFM-based nanorobotic system.

Nanomechanical analysis of insulinoma cells after glucose and capsaicin stimulation using atomic force microscopy.

AIM: Glucose stimulates insulin secretion from pancreatic islet ß cells by altering ion channel activity and membrane potential in the ß cells. TRPV1 channel is expressed in the ß cells and capsaicin induces insulin secretion similarly to glucose. This study aims to investigate the biophysical properties of the ß cells upon stimulation of membrane channels using an atomic force microscopic (AFM) nanoindentation system. METHODS: ATCC insulinoma cell line was used. Cell stiffness, a marker of reorganization of cell membrane and cytoskeleton due to ion channel activation, was measured in real time using an integrated AFM nanoindentation system. Cell height that represented structural changes was simultaneously recorded along with cell stiffness. RESULTS: After administration of glucose (16, 20 and 40 mmol/L), the cell stiffness was markedly increased in a dose-dependent manner, whereas cell height was changed in an opposite way. Lower concentrations of capsaicin (1.67 × 10(-9) and 1.67 × 10(-8) mol/L) increased the cell stiffness without altering cell height. In contrast, higher concentrations of capsaicin (1.67 × 10(-6) and 1.67 × 10(-7) mol/L) had no effect on the cell physical properties. CONCLUSION: A unique bio-nanomechanical signature was identified for characterizing biophysical properties of insulinoma cells upon general or specific activation of membrane channels. This study may deepen our understanding of stimulus-secretion coupling of pancreatic islet cells that leads to insulin secretion.

Quantitative analysis of human keratinocyte cell elasticity using atomic force microscopy (AFM).

We present the use of atomic force microscopy (AFM) to visualize and quantify the dynamics of epithelial cell junction interactions under physiological and pathophysiological conditions at the nanoscale. Desmosomal junctions are critical cellular adhesion components within epithelial tissues and blistering skin diseases such as Pemphigus are the result in the disruption of these components. However, these structures are complex and mechanically inhomogeneous, making them difficult to study. The mechanisms of autoantibody mediated keratinocyte disassembly remain largely unknown. Here, we have used AFM technology to image and measure the mechanical properties of living skin epithelial cells in culture. We demonstrate that force measurement data can distinguish cells cultured with and without autoantibody treatment. Our demonstration of the use of AFM for in situ imaging and elasticity measurements at the local, or tissue level opens potential new avenues for the investigation of disease mechanisms and monitoring of therapeutic strategies in blistering skin diseases.

The Emergence of AFM Applications to Cell Biology: How new technologies are facilitating investigation of human cells in health and disease at the nanoscale.

Atomic Force Microscopy (AFM) based nanorobotics has been used for building nano devices in semiconductors for almost a decade. Leveraging the unparallel precision localization capabilities of this technology, high resolution imaging and mechanical property characterization is now increasingly being performed in biological settings. AFM also offers the prospect for handling and manipulating biological materials at nanometer scale. It has unique advantages over other methods, permitting experiments in the liquid phase where physiological conditions can be maintained. Taking advantage of these properties, our group has visualized membrane and cytoskeletal structures of live cells by controlling the interaction force of the AFM tip with cellular components at the nN or sub-nN range. Cell stiffness changes were observed by statistically analyzing the Young's modulus values of human keratinocytes before and after specific antibody treatment. Furthermore, we used the AFM cantilever as a robotic arm for mechanical pushing, pulling and cutting to perform nanoscale manipulations of cell-associated structures. AFM guided nano-dissection, or nanosurgery was enacted on the cell in order to sever intermediate filaments connecting neighboring keratinocytes via sub 100 nm resolution cuts. Finally, we have used a functionalized AFM tip to probe cell surface receptors to obtain binding force measurements. This technique formed the basis for Single Molecule Force Spectroscopy (SMFS). In addition to enhancing our basic understanding of dynamic signaling events in cell biology, these advancements in AFM based biomedical investigations can be expected to facilitate the search for biomarkers related to disease diagnosis progress and treatment.

Investigation of human keratinocyte cell adhesion using atomic force microscopy.

Desmosomal junctions are specialized structures critical to cellular adhesion within epithelial tissues. Disassembly of these junctions is seen consequent to the development of autoantibodies directed at specific desmosomal proteins in blistering skin diseases such as pemphigus. However, many details regarding cell junction activity under normal physiological and disease conditions remain to be elucidated. Because of their complex structure, desmosomal junctions are not well suited to existing techniques for high-resolution three-dimensional structure-function analyses. Here, atomic force microscopy (AFM) is used for detailed characterization and visualization of the cell junctions of human epithelial cells. We demonstrate the ability to image the detailed three-dimensional structure of the cell junction at high magnification. In addition, the effect of specific antibody binding to desmosomal components of the cell junction is studied in longitudinal analyses before and after antibody treatment. We show that antibodies directed against desmoglein 3 (a major component of the desmosomal structural unit, and the major target of autoantibodies in patients with pemphigus vulgaris) are associated with changes at the cell surface of the human keratinocytes and alterations within keratinocyte intercellular adhesion structures, supporting the assertion that cell structures and junctions are modified by antibody binding. The present study indicates that the molecular structure of gap junctions can be more completely analyzed and characterized by AFM, offering a new technological approach to facilitate a better understanding of disease mechanisms and potentially monitor therapeutic strategies in blistering skin diseases. FROM THE CLINICAL EDITOR: Disassembly of desmosomal junctions is seen in blistering skin diseases such as Pemphigus. This present study demonstrates that the molecular structure of gap junctions can be more completely analyzed and characterized by atomic force microscopy.

Nanoresonant signal boosters for carbon nanotube based infrared detectors.

We report the development of a sensitive carbon nanotube (CNT) infrared detector whose signals are boosted by nanoantenna-like features. This assembly is fabricated using nanoassembly of CNTs and a standard photolithographic process, together with nanoantenna-like features that are designed to create a resonance structure necessary to boost the electric field intensity at the CNT sensor. A novel approach is employed to find the near-field effect of the antenna. As a result, these effects are verified and demonstrated experimentally in this paper. The first experimental demonstration of a practical infrared device with nanoantenna-like structures is reported; it shows that the photocurrent is increased by an order of magnitude. The proposed fabrication and design process enables a ready integration of resonance structures into the manufacture of infrared devices, and opens the possibility of developing high fidelity infrared sensors with wide sensing range.