Biophysical Measurements of Cells, Microtubules, and DNA with an Atomic Force Microscope

Atomic force microscopes (AFMs) are ubiquitous in research laboratories and have recently been priced for use in teaching laboratories. Here we review several AFM platforms (Dimension 3000 by Digital Instruments, EasyScan2 by Nanosurf, ezAFM by Nanomagnetics, and TKAFM by Thorlabs) and describe various biophysical experiments that could be done in the teaching laboratory using these instruments. In particular, we focus on experiments that image biological materials and quantify biophysical parameters: 1) imaging cells to determine membrane tension, 2) imaging microtubules to determine their persistence length, 3) imaging the random walk of DNA molecules to determine their contour length, and 4) imaging stretched DNA molecules to measure the tensional force.Comment: 29 page preprint, 7 figures, 1 tabl

Algorithmic approaches to high speed atomic force microscopy

Thesis (Ph.D.)--Boston UniversityThe atomic force microscope (AFM) has a unique set of capabilities for investigating biological systems, including sub-nanometer spatial resolution and the ability to image in liquid and to measure mechanical properties. Acquiring a high quality image, however, can take from minutes to hours. Despite this limited frame rate, researchers use the instrument to investigate dynamics via time-lapse imaging, driven by the need to understand biomolecular activities at the molecular level. Studies of processes such as DNA digestion with DNase, DNA-RNA polymerase binding and RNA transcription from DNA by RNA polymerase redefined the potential of AFM in biology. As a result of the need for better temporal resolution, advanced AFMs have been developed. The current state of the art in high-speed AFM (HS-AFM) for biological studies is an instrument developed by Toshio Ando at Kanazawa University in Japan. This instrument can achieve 12 frames/sec and has successfully visualized the motion of protein motors at the molecular level. This impressive instrument as well as other advanced AFMs, however, comes with tradeoffs that include a small scan size, limited imaging modes and very high cost. As a result, most AFM users still rely on standard commercial AFMs. The work in this thesis develops algorithmic approaches that can be implemented on existing instruments, from standard commercial systems to cutting edge HS-AFM units, to enhance their capabilities. There are four primary contributions in this thesis. The first is an analysis of the signals available in an AFM with respect to the information they carry and their suitability for imaging at different scan speeds. The next two are algorithmic approaches to HS-AFM that take advantage of these signals in different ways. The first algorithm involves a new sample profile estimator that yields accurate topology at speeds beyond the bandwidth of the limiting actuator. The second involves more efficient sampling, using the data in real time to steer the tip. Both algorithms yield at least an order of magnitude improvement in imaging rate but with different tradeoffs. The first operates beyond the bandwidth of the controller managing the tip-sample interaction and therefore the applied force is not well-regulated. The second keeps this control intact but is effective only on a limited set of samples, namely biopolymers or other string-like samples. Experiments on calibration samples and λ-DNA show that both of the algorithms improve the imaging rate by an order of magnitude. In the fourth contribution, extended applications of AFMs equipped with the algorithmic approaches are the tracking of a macromolecule moving along a string-like sample and a time optimal path for repetitive non-raster scans along string-like samples

Modeling and Control of MEMS-based Multi-layered Prestressed Piezoelectric Cantilever Beam

Piezoelectric materials are the preferred smart materials for sensing and actuation in the form of micro and nano-engineering structures like beams and plates. Cantilever beams play a significant role as key components in atomic force microscopy and bio and chemical sensors. Adding an active layer such as lead zirconate titanate (PZT) thin-film to form smart cantilever beams with sensing and actuation capabilities is highly desirable to facilitate miniaturization, enhance performance and functionali- ties such as enabling on-chip high-speed parallel AFM. During the micro-fabrication process, residual stresses develop in the different layers of the cantilever beam, causes initial deflection. The residual stress in the different layers of the cantilever beam and the application of voltage to the PZT thin-film affects their dynamics. This the- sis investigates the dynamic behaviour and develops a control technique and a novel charge readout circuit to improve the performance of a micro-fabricated multi-layer prestressed piezoelectric cantilever beam as an actuator and a deflection sensor. Firstly, the fabrication process of a unimorph PZT cantilever beam is explained. A low thermal budget Ultra-high vacuum e-beam evaporated polysilicon thin-film (UHVEEpoly) process is used for the fabrication of multi-layered PZT cantilever beam in d31 mode. The sharp peaks at resonant frequencies in the frequency response of the PZT cantilever beam show very little damping and a large settling time of the cantilever beam. Secondly, the dynamic behaviour of the prestressed PZT cantilever beam is in- vestigated subjected to change in driving voltage. Experimental investigations show a shift in resonant frequencies of a PZT cantilever beam. However, there is no reported mathematical model that predicts the shift in resonance frequencies of a multi-layered prestressed piezoelectric cantilever beam subjected to a change in driving voltage. This work developed a mathematical model with experimental val- idation to estimate the shift in resonance frequencies of such cantilever beams with the change in the driving voltage. A very good agreement between the model predic- tions and experimental measurements for the frequency response of the cantilever beam at different driving voltages has been obtained. A novel linear formulation has been developed to predict the shift in resonance frequencies of the PZT can- i tilever beam for a wide range of driving voltages. The formulation shows that the shift in resonance frequencies of a multi-layered prestressed piezoelectric cantilever beam per unit of applied voltage is dependent on geometric parameters and material properties. Thirdly, a robust resonant controller has been designed and implemented to re- duce the settling time of a highly vibrating PZT cantilever beam. The controller design is based on a mixed negative-imaginary, passivity, and a small-gain approach. The motivation to design a resonant controller using the above-mentioned analyti- cal framework is its bandpass nature and the use of velocity feedback, as the charge collected from a vibrating PZT cantilever beam gives the velocity information of the beam. The proposed controller design results in finite gain stability for a pos- itive feedback interconnection between two stable linear systems with a large gain and phase margin. Experimental results demonstrate that the designed resonant controller is able to effectively damp the first resonant mode of a cantilever, signifi- cantly reducing settling time from 528 ms to 32 ms. The robustness of the designed resonant controller is tested against changes in the cantilever beam dynamics due to residual stress variation and or stress variation due to driving voltage. Finally, to facilitate the miniaturization of on-chip sensors and parallel high- speed AFM, a single layer of a PZT thin-film in a cantilever beam is used as a deflection sensor and an actuator instead of bulky optical deflection sensors. A novel charge readout circuit is designed for deflection sensing by capturing the electrical charge generated due to the vibration of the PZT beam. The signal-to-noise ratio and sensitivity analysis of the readout circuit shows similar results compared to the commercially available optical deflection sensors. Our work highlights very important aspects in the dynamic behaviour and perfor- mance of a multi-layered prestressed piezoelectric cantilever beam. The agreement between the proposed theoretical formulation and experimental investigations in modeling, control design, and a novel readout circuit will provide the platform for further the development and miniaturization of microcantilever-based technologies, including on-chip parallel HS-AFM

Array atomic force microscopy for real-time multiparametric analysis.

Nanoscale multipoint structure-function analysis is essential for deciphering the complexity of multiscale biological and physical systems. Atomic force microscopy (AFM) allows nanoscale structure-function imaging in various operating environments and can be integrated seamlessly with disparate probe-based sensing and manipulation technologies. Conventional AFMs only permit sequential single-point analysis; widespread adoption of array AFMs for simultaneous multipoint study is challenging owing to the intrinsic limitations of existing technological approaches. Here, we describe a prototype dispersive optics-based array AFM capable of simultaneously monitoring multiple probe-sample interactions. A single supercontinuum laser beam is utilized to spatially and spectrally map multiple cantilevers, to isolate and record beam deflection from individual cantilevers using distinct wavelength selection. This design provides a remarkably simplified yet effective solution to overcome the optical cross-talk while maintaining subnanometer sensitivity and compatibility with probe-based sensors. We demonstrate the versatility and robustness of our system on parallel multiparametric imaging at multiscale levels ranging from surface morphology to hydrophobicity and electric potential mapping in both air and liquid, mechanical wave propagation in polymeric films, and the dynamics of living cells. This multiparametric, multiscale approach provides opportunities for studying the emergent properties of atomic-scale mechanical and physicochemical interactions in a wide range of physical and biological networks

Atomic Force Microscopy Based DNA Sensing and Manipulation

Sequencing DNA provides a positive impact for the biomedical community by understanding a wide variety of applications such as human genetics, disease, and pathogens. The reason the Arkansas Micro & Nano Systems lab is involved with research in DNA sequencing is due to the current, leading industry method. Nanopore sequencing was developed by Oxford Nanopore Technology in which its sequencing method separates double stranded DNA to electrically characterize individual nucleotides traveling through a charged nanopore. Unfortunately, nanopore sequencing uses biological materials that require a shelf life and drives high cost. Therefore, the Arkansas Micro & Nano Systems lab has developed a DNA sequencing method using atomic force microscopy (AFM) to eliminate any shelf life of materials. One of the main functions of sequencing DNA using atomic force microscopy is using force spectroscopy to control the movement of DNA by creating an electrostatic force between a cantilever tip and strand of DNA. Two different force spectroscopy methods were developed by graduate students, Dr. Bo Ma and Lucas Bartmann, to control DNA. The focus of this report is to test and evaluate both spectroscopy methods developed by previous graduate students from the Arkansas Micro & Nano Systems lab and determine which procedure is more effective. Because atomic force microscopy will be used to control and move DNA, this undergraduate thesis will also entail a top-down approach of how to prepare DNA on a mica surface and how to use a CoreAFM in liquid mode to scan images of DNA. Explaining the procedure to locate DNA using an AFM and determining which spectroscopy method is more effective in moving DNA will hopefully provide a more efficient process of sequencing DNA using atomic force microscopy

High-speed atomic force microscopy combined with inverted optical microscopy for studying cellular events.

A hybrid atomic force microscopy (AFM)-optical fluorescence microscopy is a powerful tool for investigating cellular morphologies and events. However, the slow data acquisition rates of the conventional AFM unit of the hybrid system limit the visualization of structural changes during cellular events. Therefore, high-speed AFM units equipped with an optical/fluorescence detection device have been a long-standing wish. Here we describe the implementation of high-speed AFM coupled with an optical fluorescence microscope. This was accomplished by developing a tip-scanning system, instead of a sample-scanning system, which operates on an inverted optical microscope. This novel device enabled the acquisition of high-speed AFM images of morphological changes in individual cells. Using this instrument, we conducted structural studies of living HeLa and 3T3 fibroblast cell surfaces. The improved time resolution allowed us to image dynamic cellular events

THE DEVELOPMENT OF A NOVEL ELECTRO-MAGNETIC FORCE MICROSCOPE

This thesis describes the development of a new type of Magnetic Force Microscope (MFM) probe based on a unique electromagnetic design. In addition the design, construction and testing of a new MFM system, complete in both hardware and software, is also described. The MFM allowed initial tests on prototypes of the new probe, and is to provide a base for future new probe integration. The microscope uses standard MFM micro-cantilever probes in static modes of imaging. A new computer hosted DSP control system, software, and its various interfaces with the MFM have been integrated into the system. The system has been tested using standard probes with various specimens and satisfactory results have been produced. A novel probe has been designed to replace the standard MFM magnetic coated tip with a field generated about a sub-micron aperture in a conducting film. The field from the new probe is modelled and its imaging capability investigated, with iterative designs analysed in this way. The practical construction and potential problems therein, of the probe are also considered. Test apertures have been manufactured, and an image of the field produced when operating is provided as support to the theoretical designs. Future methods of using the new probe are also discussed, including the examination of the probe as a magnetic write mechanism. This probe, integrated into the MFM, can provide a new method of microscopic magnetic imaging, and in addition opens a new potential method of magnetic storage that will require further research

DEVELOPMENT AND APPLICATIONS OF MULTIFREQUENCY IMAGING AND SPECTROSCOPY METHODS IN DYNAMIC ATOMIC FORCE MICROSCOPY

Force spectroscopy and surface dissipation mapping are two of the most important applications of dynamic atomic force microscopy (AFM), in addition to topographical imaging. These measurements are commonly performed using the conventional amplitude-modulation and frequency-modulation dynamic imaging modes. However, the acquisition of the tip-sample interaction force curves using these methods can generally be performed only at selected horizontal positions on the sample, which means that a 3-dimensional representation of the tip-sample forces requires fine-grid scanning of a volume above the surface, making the process lengthy and prone to instrument drift. This dissertation contains the development of two novel atomic force spectroscopy methods that could enable acquisition of 3-dimensional tip-sample force representations through a single 2-dimensional scan of the surface. The force curve reconstruction approach in the first method is based on 3-pass scanning of the surface using the recently proposed single-frequency imaging mode called frequency and force modulation AFM. A second, more versatile method based on bimodal AFM operation is introduced, wherein the fundamental eigenmode of the cantilever is excited to perform the topographical scan and a simultaneously excited higher eigenmode is used to perform force spectroscopy. The dissertation further presents the development of a trimodal AFM characterization method for ambient air operation, wherein three eigenmodes of the cantilever are simultaneously excited with the objective of rapidly and quantitatively mapping the variations in conservative and dissipative surface properties. The new methods have been evaluated within numerical simulations using a multiscale simulation methodology, and experimental implementation has been accomplished for two multifrequency variants that can provide 2-dimensional surface property contrast

A dual-loop tracking control approach to precise nanopositioning

The author(s) received no financial support for the research, authorship, and/or publication of this article.Peer reviewedPostprin

Generalizing Negative Imaginary Systems Theory to Include Free Body Dynamics: Control of Highly Resonant Structures with Free Body Motion

Negative imaginary (NI) systems play an important role in the robust control of highly resonant flexible structures. In this paper, a generalized NI system framework is presented. A new NI system definition is given, which allows for flexible structure systems with colocated force actuators and position sensors, and with free body motion. This definition extends the existing definitions of NI systems. Also, necessary and sufficient conditions are provided for the stability of positive feedback control systems where the plant is NI according to the new definition and the controller is strictly negative imaginary. The stability conditions in this paper are given purely in terms of properties of the plant and controller transfer function matrices, although the proofs rely on state space techniques. Furthermore, the stability conditions given are independent of the plant and controller system order. As an application of these results, a case study involving the control of a flexible robotic arm with a piezo-electric actuator and sensor is presented