Table Of Content

ATOMIC FORCE MICROSCOPY INVESTIGATIONS INTO BIOLOGY – FROM CELL TO PROTEIN Edited by Christopher L. Frewin ATOMIC FORCE MICROSCOPY INVESTIGATIONS INTO BIOLOGY – FROM CELL TO PROTEIN Edited by Christopher L. Frewin Atomic Force Microscopy Investigations into Biology – From Cell to Protein Edited by Christopher L. Frewin Published by InTech Janeza Trdine 9, 51000 Rijeka, Croatia Copyright © 2012 InTech All chapters are Open Access distributed under the Creative Commons Attribution 3.0 license, which allows users to download, copy and build upon published articles even for commercial purposes, as long as the author and publisher are properly credited, which ensures maximum dissemination and a wider impact of our publications. After this work has been published by InTech, authors have the right to republish it, in whole or part, in any publication of which they are the author, and to make other personal use of the work. Any republication, referencing or personal use of the work must explicitly identify the original source. As for readers, this license allows users to download, copy and build upon published chapters even for commercial purposes, as long as the author and publisher are properly credited, which ensures maximum dissemination and a wider impact of our publications. Notice Statements and opinions expressed in the chapters are these of the individual contributors and not necessarily those of the editors or publisher. No responsibility is accepted for the accuracy of information contained in the published chapters. The publisher assumes no responsibility for any damage or injury to persons or property arising out of the use of any materials, instructions, methods or ideas contained in the book. Publishing Process Manager Oliver Kurelic Technical Editor Teodora Smiljanic Cover Designer InTech Design Team First published February, 2012 Printed in Croatia A free online edition of this book is available at www.intechopen.com Additional hard copies can be obtained from [email protected] Atomic Force Microscopy Investigations into Biology – From Cell to Protein, Edited by Christopher L. Frewin p. cm. ISBN 978-953-51-0114-7 Contents Preface IX Part 1 General Techniques 1 Chapter 1 Atomic Force Spectroscopies: A Toolbox for Probing the Biological Matter 3 Michele Giocondo, Said Houmadi, Emanuela Bruno, Maria P. De Santo, Luca De Stefano, Emmanuelle Lacaze, Sara Longobardi and Paola Giardina Chapter 2 Artifacts in Atomic Force Microscopy of Biological Samples 29 E. Ukraintsev, A. Kromka, H. Kozak, Z. Remeš and B. Rezek Chapter 3 Tapping Mode AFM Imaging for Functionalized Surfaces 55 Nadine Mourougou-Candoni Part 2 Biological Molecules, Proteins and Polymers 85 Chapter 4 AFM Measurements to Investigate Particulates and Their Interactions with Biological Macromolecules 87 L. Latterini and L. Tarpani Chapter 5 AFM Imaging of Biological Supramolecules by a Molecular Imprinting-Based Immobilization Process on a Photopolymer 99 Taiji Ikawa Chapter 6 Protein Interactions on Phospholipid Bilayer, Studied by AFM Under Physiological Conditions 123 Špela Irman, Miha Škarabot, Igor Muševič and Borut Božič VI Contents Chapter 7 Nanomechanics of Amyloid Materials Studied by Atomic Force Microscopy 153 Guanghong Zeng, Yusheng Duan, Flemming Besenbacher and Mingdong Dong Part 3 DNA, Chromatin and Membranes 175 Chapter 8 Analyzing DNA Structure Quantitatively at a Single-Molecule Level by Atomic Force Microscopy 177 Yong Jiang and Yuan Yin Chapter 9 Atomic Force Microscopy of Chromatin 195 Delphine Quénet, Emilios K. Dimitriadis and Yamini Dalal Chapter 10 Artificial and Natural Membranes 219 György Váró and Zsolt Szegletes Part 4 Viral Physiology 233 Chapter 11 Atomic Force Microscopy in Detection of Viruses 235 Norma Hernández-Pedro, Edgar Rangel-López, Benjamín Pineda and Julio Sotelo Chapter 12 Force Microscopy – A Tool to Elucidate the Relationship Between Nanomechanics and Function in Viruses 253 J.L. Cuéllar and E. Donath Part 5 Cellular Physiology 279 Chapter 13 Single-Molecule Force Microscopy: A Potential Tool for the Mapping of Polysaccharides in Plant Cell Walls 281 Julian C. Thimm, Laurence D. Melton and David J. Burritt Chapter 14 AFM and Cell Staining to Assess the In Vitro Biocompatibility of Opaque Surfaces 297 Christopher L. Frewin, Alexandra Oliveros, Edwin Weeber and Stephen E. Saddow Chapter 15 The Transversal Stiffness of Skeletal Muscle Fibers and Cardiomyocytes in Control and After Simulated Microgravity 325 Irina V. Ogneva and Igor B. Ushakov Preface Atomic force microscopy, AFM, is a modern technique for generating high resolution surface topography images and can image many orders of magnitude below the optical diffraction limit. It uses a principal similar to the one used by the phonograph developed by Thomas Alva Edison. Essentially, the phonograph has a sharp object which is dragged across a moving surface, and the tip is deformed by the features it encounters. The AFM uses this principal as well, with the AFM tip generating a physical deflection in the cantilever according to Hooke’s law, but unlike the phonograph, the AFM can capitalize on a wider variety of forces generated between the sharp tip and the scanned surface. The AFM cantilever deflection is quantified through the use of a laser reflected off of the back of the cantilever onto an array of photodiodes. Of course, as was seen with Edison’s original invention, scanning an object at a constant height presents the danger of a collision with the surface. With this problem in mind, the AFM cantilever is mounted onto a piezoelectric column commonly called a head stage, which moves the cantilever to maintain constant force according to feedback from the measured deflection. The combination of all of these components can be recorded and produces a topographical image of the surface with nanoscale resolution. This measurement technique has also been used to develop a method of force spectroscopy which measures the force between the tip and the sample as a function of distance. However, this technique is not the only measurement these devices can produce. The AFM is not limited to only one operating mode, but has a second distinctive mode, which itself has been developed into another mode of operation. The previous mode we discussed is known as contact or static mode, and the next mode we will briefly introduce is called non-contact, or dynamic, mode. In this mode, the tip is oscillated near its natural resonance, and brought close to the surface. As the tip approaches the surface, the interaction between the surface and tip forces generates a change in the natural resonance of the cantilever. The feedback circuit is used to reestablish the original oscillation set point by changing the distance between the sample and the tip. The difference that the cantilever moves can be recorded and compiled to produce topographical images. Oscillation differences can be detected as a function of amplitude or frequency. Detections of changes in the frequency of oscillation produce very high-resolution measurements. Through the measurement of changes in amplitude, non-contact mode becomes the third main mode, known as X Preface tapping or intermittent mode. In tapping mode, the physical distance of the amplitude of oscillation is large enough to produce brief contact with the surface. This contact leads to changes the amplitude which is measured by the feedback circuit. The head stage moves the cantilever to maintain constant amplitude, and once again can be used to generate topographical images. Changes in the phase of oscillations can also be measured in this mode and is useful in detecting differences in surface friction or between different materials. The AFM is a very dynamic measurement tool and provides many methods to quantitatively measure a wide range of physical, electromagnetic, and atomic forces. However, the one aspect of this device is that puts it in a class by itself among nanoscale microscopy is the fact that AFM can be used in almost any environment. Vacuum, air, and liquid are not a barrier for this measurement style, and because of this, the AFM lends itself very well for biological investigations where environmental factors can influence biological reactions. This book was developed to showcase the growing use of AFM techniques and methodologies in the investigation of many different aspects of biological and medical sciences. Another, less known advantage for the use of biological AFM is it can be used to investigate structures as large as a whole cell down to the very proteins which constitute the cells themselves. Another advantage is this measurement technique does not require complicated, invasive, and often permanent sample preparation like that required for other microscopy techniques. Personally, I was unknowledgeable about this fantastic device until I was almost a senior in college. Even further from my expectations is that I never thought I would have used AFM as a tool to investigate biology, specifically neurological cells and their properties. After taking undergraduate physics, and influenced greatly by my childhood idol Nikola Tesla, I wanted to explore the mysteries of electricity and magnetism, so I chose electrical engineering. As with most students, I entered college with preconceptions as to what I would be exposed to within this field, but I found that my expectations were not in line with reality. I thought that I would learn about electrical circuits, power transfer, and communications and then go out into the world and work in some job designing these things, but in the beginning of my junior year, I was fortunate to be able to join, Professor Stephen E. Saddow, in his silicon carbide (SiC) laboratory. Little did I know it at the time, but Dr. Saddow would become my Ph.D. mentor. At first, like many undergraduates, I was just amazed to be in a real research laboratory, but became slightly disillusioned with research due to my preconceptions. My new lab was focused on materials research, which seemed more like chemistry and only seemed connected to electrical engineering due to the fact that crystalline SiC is a semiconductor. As time moved on, I slowly began to understand the importance of this research as educational maturity set in. Finally I was able to look past the chemistry and see a material which could withstand almost every base or acid compound, conduct large amounts of electricity, dissipate heat as well as copper, and even emit visible light from yellow to blue. I began to become intrigued with the possibilities, and wanted to build electrical devices with this wonderful material. Preface XI After redesigning the control system for Professor Saddow’s SiC reactor, I was teamed with one of his graduate students, Dr. Camilla Coletti, who was studying the surface effects of hydrogen etching on SiC in collaboration with Dr Ulrich Starke of the Max- Planck-Institut für Festkörperforschung in Stuttgart, Germany. Thanks to the graciousness of both Professor Saddow and Dr. Starke, I was able to study at the Institut for a month so that I could gather some data for Dr. Coletti. Here it was that I had my first “hands on” exposure to AFM. It was an experience I will never forget as we immediately got off the plane, drove to the institute, and after introductions to Dr. Starke’s group, Prof. Saddow trained me on how to operate the AFM. In a laboratory where there were many complicated, large, ultra-high vacuum instruments, like time- of-flight secondary ion mass spectrometry (ToF-SIMS) and scanning tunneling microscopy (STM) systems, the small, blue can-like system sitting on a floating table seemed almost out of place and ineffectual for surface science. However, I was amazed to find that such a simple machine could be used to examine so many different properties. I imaged the surfaces of the SiC materials we had hydrogen etched in the CVD reactor back in Florida, and Dr. Colletti was able to answer some issues she had concerning the etching process. I found that AFM was an invaluable tool in not only examining etched SiC surfaces, but also in the development of an improved heteroepitaxial growth process for cubic silicon carbide (3C-SiC) on silicon substrates, as many of the defects at the heteroepitaxial interface are transferred to the 3C-SiC film surface. AFM proved to be a fast and efficient method, especially when compared to instruments like transmission electron microscopy (TEM) and X-ray Diffraction (XRD), to perform quick analysis of the crystal film so one could determine what growth parameters needed to be adjusted for the reduction of defects. AFM reduced the amount of empirical experimentation involved in SiC heteroepitaxial growth, and reduced the analysis workload by allowing us to only analyze our best materials using more time and preparation intensive methods, like TEM and XRD. Of course, this is not a book about the use of AFM in materials science, as it is a mainstay in that field, but about the use of AFM in applications within biological sciences. Personally, the experience once again began with Professor Saddow, who saw the potential of SiC for use in medical technology. His group began in earnest to examine SiC biocompatibility using the AFM, starting with Dr. Coletti, continuing with myself, and now with Alexandra Oliveros, a current PhD candidate in his group, to this day. Dr. Saddow purchased a Park Systems XE-100 AFM system because it not only could examine dry materials, but with a removable liquid cell would allow us to examine the interaction of living cells on our materials. During my early graduate experience, I was exposed to many biological science techniques through Dr. Coletti. Her methods included chemical assays to examine cellular proliferation, fluorescent microscopy to examine cellular morphology, and AFM to examine fixed cells on our novel materials. During my Ph.D., I came across a difficulty that stemmed from many optical microscopes used for biology. Although 3C-SiC is translucent, our films are on silicon, which is opaque. The fluorescent optical microscopes I had available to me at