Dual integrated actuators for extended range high speed atomic force microscopy

Cataloged from PDF version of article.A flexible system for increasing the throughput of the atomic force microscope without sacrificing imaging range is presented. The system is based on a nested feedback loop which controls a micromachined cantilever that contains both an integrated piezoelectric actuator and an integrated thermal actuator. This combination enables high speed imaging (2 mm/s) over an extended range by utilizing the piezoelectric actuator’s high bandwidth (15 kHz) and thermal actuator’s large response (300 nm/V). A constant force image, where the sample topography exceeds the range of the piezoelectric actuator alone, is presented. It has also been demonstrated that the deflection response of the thermal actuator can be linearized and controlled with an integrated diode. © 1999 American Institute of Physic

Decoupling Internalization, Acidification and Phagosomal-Endosomal/lysosomal Fusion during Phagocytosis of InlA Coated Beads in Epithelial Cells

BACKGROUND: Phagocytosis has been extensively examined in 'professional' phagocytic cells using pH sensitive dyes. However, in many of the previous studies, a separation between the end of internalization, beginning of acidification and completion of phagosomal-endosomal/lysosomal fusion was not clearly established. In addition, very little work has been done to systematically examine phagosomal maturation in 'non-professional' phagocytic cells. Therefore, in this study, we developed a simple method to measure and decouple particle internalization, phagosomal acidification and phagosomal-endosomal/lysosomal fusion in Madin-Darby Canine Kidney (MDCK) and Caco-2 epithelial cells. METHODOLOGY/PRINCIPAL FINDINGS: Our method was developed using a pathogen mimetic system consisting of polystyrene beads coated with Internalin A (InlA), a membrane surface protein from Listeria monocytogenes known to trigger receptor-mediated phagocytosis. We were able to independently measure the rates of internalization, phagosomal acidification and phagosomal-endosomal/lysosomal fusion in epithelial cells by combining the InlA-coated beads (InlA-beads) with antibody quenching, a pH sensitive dye and an endosomal/lysosomal dye. By performing these independent measurements under identical experimental conditions, we were able to decouple the three processes and establish time scales for each. In a separate set of experiments, we exploited the phagosomal acidification process to demonstrate an additional, real-time method for tracking bead binding, internalization and phagosomal acidification. CONCLUSIONS/SIGNIFICANCE: Using this method, we found that the time scales for internalization, phagosomal acidification and phagosomal-endosomal/lysosomal fusion ranged from 23-32 min, 3-4 min and 74-120 min, respectively, for MDCK and Caco-2 epithelial cells. Both the static and real-time methods developed here are expected to be readily and broadly applicable, as they simply require fluorophore conjugation to a particle of interest, such as a pathogen or mimetic, in combination with common cell labeling dyes. As such, these methods hold promise for future measurements of receptor-mediated internalization in other cell systems, e.g. pathogen-host systems

Hidden multiple bond effects in dynamic force spectroscopy

In dynamic force spectroscopy, a (bio-)molecular complex is subjected to a steadily increasing force until the chemical bond breaks. Repeating the same experiment many times results in a broad distribution of rupture forces, whose quantitative interpretation represents a formidable theoretical challenge. In this study we address the situation that more than a single molecular bond is involved in one experimental run, giving rise to multiple rupture events which are even more difficult to analyze and thus are usually eliminated as far as possible from the further evaluation of the experimental data. We develop and numerically solve a detailed model of a complete dynamic force spectroscopy experiment including a possible clustering of molecules on the substrate surface, the formation of bonds, their dissociation under load, and the post processing of the force extension curves. We show that the data, remaining after elimination of obvious multiple rupture events, may still contain a considerable number of "hidden" multiple bonds, which are experimentally indistinguishable from "true" single bonds, but which have considerable effects on the resulting rupture force statistics and its consistent theoretical interpretation.Comment: 31 pages, 7 figure

Parallel atomic force microscopy with optical interferometric detection

Cataloged from PDF version of article.We have developed an atomic force microscope that uses interferometry for parallel readout of a cantilever array. Each cantilever contains a phase sensitive diffraction grating consisting of a reference and movable set of interdigitated fingers. As a force is applied to the tip, the movable set is displaced and the intensity of the diffracted orders is altered. The order intensity from each cantilever is measured with a custom array of siliconphotodiodes with integrated complementary metal–oxide–semiconductor amplifiers. We present images from five cantilevers acquired in the constant height mode that reveal surface features 2 nm in height. The interdigital method for cantilever array readout is scalable, provides angstrom resolution, and is potentially simpler to implement than other methods. © 2001 American Institute of Physic

High-speed tapping mode imaging with active Q control for atomic force microscopy

Cataloged from PDF version of article.The speed of tapping mode imaging with the atomic force microscope(AFM) has been increased by over an order of magnitude. The enhanced operation is achieved by (1) increasing the instrument’s mechanical bandwidth and (2) actively controlling the cantilever’s dynamics. The instrument’s mechanical bandwidth is increased by an order of magnitude by replacing the piezotube z-axis actuator with an integrated zinc oxide (ZnO)piezoelectric cantilever. The cantilever’s dynamics are optimized for high-speed operation by actively damping the quality factor (Q) of the cantilever. Active damping allows the amplitude of the oscillating cantilever to respond to topography changes more quickly. With these two advancements, 80μm×80 μm high-speed tapping mode images have been obtained with a scan frequency of 15 Hz. This corresponds to a tip velocity of 2.4 mm/s. © 2000 American Institute of Physic

Scan speed control for tapping mode SPM

In order to increase the imaging speed of a scanning probe microscope in tapping mode, we propose to use a dynamic controller on 'parachuting' regions. Furthermore, we propose to use variable scan speed on 'upward step' regions, with the speed determined by the error signal of the closed-loop control. We offer line traces obtained on a calibration grating with 25-nm step height, using both standard scanning and our scanning method, as experimental evidence

Amphotericin B induced interdigitation of apolipoprotein stabilized nanodisk bilayers

Amphotericin B nanodisks (AMB-ND) are ternary complexes of AMB, phospholipid (PL) and apolipoprotein organized as discrete nanometer scale disk-shaped bilayers. In gel filtration chromatography experiments, empty ND lacking AMB elute as a single population of particles with a molecular weight in the range of 200 kDa. AMB-ND formulated at a 4:1 PL:AMB weight ratio, separated into two peaks. Peak 1 eluted at the position of control ND lacking AMB while the second peak, containing all of the AMB present in the original sample, eluted in the void volume. When ND prepared with increased AMB (1:1 phospholipid:AMB molar ratio) were subjected to gel filtration chromatography, an increased proportion of phospholipid and apolipoprotein were recovered in the void volume with the AMB. Prior to gel filtration the AMB-ND sample could be passed through a 0.22 {micro}m filter without loss of AMB while the voided material was lost. Native gel electrophoresis studies corroborated the gel permeation chromatography data. Far UV circular dichroism analyses revealed that apoA-I associated with AMB-ND denatures at a lower guanidine HCl concentration than apoA-I associated with ND lacking AMB. Atomic force microscopy revealed that AMB induces compression of the ND bilayer thickness consistent with bilayer interdigitation, a phenomenon that is likely related to the ability of AMB to induce pore formation in susceptible membranes

Atomic force microscopy differentiates discrete size distributions between membrane protein containing and empty nanolipoprotein particles

AbstractTo better understand the incorporation of membrane proteins into discoidal nanolipoprotein particles (NLPs) we have used atomic force microscopy (AFM) to image and analyze NLPs assembled in the presence of bacteriorhodopsin (bR), lipoprotein E4 n-terminal 22k fragment scaffold and DMPC lipid. The self-assembly process produced two distinct NLP populations: those containing inserted bR (bR-NLPs) and those that did not (empty-NLPs). The bR-NLPs were distinguishable from empty-NLPs by an average increase in height of 1.0 nm as measured by AFM. Streptavidin binding to biotinylated bR confirmed that the original 1.0 nm height increase corresponds to br-NLP incorporation. AFM and ion mobility spectrometry (IMS) measurements suggest that NLP size did not vary around a single mean but instead there were several subpopulations, which were separated by discrete diameters. Interestingly, when bR was present during assembly the diameter distribution was shifted to larger particles and the larger particles had a greater likelihood of containing bR than smaller particles, suggesting that membrane proteins alter the mechanism of NLP assembly

A Compact, Low-Power Cantilever-Based Sensor Array for Chemical Detection

A compact and low-power cantilever-based sensor array has been developed and used to detect various vapor analytes. This device employs sorptive polymers that are deposited onto piezoresistive cantilevers. We have successfully detected several organic vapors, representing a breadth of chemical properties and over a range of concentrations. Comparisons of the polymer/vapor partition coefficient to the cantilever deflection responses show that a simple linear relationship does not exist, emphasizing the need to develop an appropriate functional model to describe the chemical-to-mechanical transduction that is unique to this sensing modality