Atomic Force Microscopy In Biomedical Research Methods And Protocols 1st Edition Bruno Torre
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- 6. Me t h o d s i n Mo l e c u l a r Bi o l o g y ™ Series Editor John M. Walker School of Life Sciences University of Hertfordshire Hatfield, Hertfordshire, AL10 9AB, UK For further volumes: www.springer.com/series/7651
- 8. Atomic Force Microscopy in Biomedical Research Methods and Protocols Edited by Pier Carlo Braga DepartmentofPharmacology,SchoolofMedicine,UniversityofMilan,Milan,Italy Davide Ricci Robotics,BrainandCognitiveSciencesDepartment,ItalianInstituteofTechnology,Genoa,Italy and DepartmentofBiophysicalElectronicEngineering,UniversityofGenoa,Genoa,Italy
- 9. Editors Pier Carlo Braga Department of Pharmacology School of Medicine University of Milan Milan, Italy piercarlo.braga@unimi.it Davide Ricci Robotics, Brain and Cognitive Sciences Department, Italian Institute of Technology Genoa, Italy and Department of Biophysical Electronic Engineering University of Genoa Genoa, Italy davide.ricci@iit.it ISSN 1064-3745 e-ISSN 1940-6029 ISBN 978-1-61779-104-8 e-ISBN 978-1-61779-105-5 DOI 10.1007/978-1-61779-105-5 Springer New York Dordrecht Heidelberg London Library of Congress Control Number: 2011926794 © Springer Science+Business Media, LLC 2011 All rights reserved. This work may not be translated or copied in whole or in part without the written permission of the publisher (Humana Press, c/o Springer Science+Business Media, LLC, 233 Spring Street, New York, NY 10013, USA), except for brief excerpts in connection with reviews or scholarly analysis. Use in connection with any form of information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed is forbidden. The use in this publication of trade names, trademarks, service marks, and similar terms, even if they are not identified as such, is not to be taken as an expression of opinion as to whether or not they are subject to proprietary rights. Printed on acid-free paper Humana Press is part of Springer Science+Business Media (www.springer.com)
- 10. v Preface The invention and development of the optical microscope in the seventeenth century revealed the presence of a previously unseen and unimaginable world within and around us. Our lives would not be what they are today if optical microscopy had never existed or if it had not helped us to understand better what we are, how we function, and how we can improve our condition – first in the fields of biology and medicine, and then in many other fields. Another great step was made with the introduction of transmission and scanning elec- tron microscopy in the 1930s, which was initially integrated with optical microscopy but subsequently developed its own identity and technology and opened up new horizons in human knowledge. Starting in 1986, further technological advances led to the development of atomic force microscopy (AFM), which is completely different from its predecessors: instead of being based on lenses, photons, and electrons, it directly explores the surface of the sam- ple by means of a local scanning probe while the use of dedicated software allows the results to be visualized on a monitor. AFM has a number of special characteristics: very high magnification with very high resolution; minimal sample preparation (none of the dyes of optical microscopy, or the vacuum, critical point, or gold sputtering required by scanning electron microscopy); real three-dimensional topographical data that allow us to obtain different views of the samples from a single collected dataset; and the ability to work in a liquid in real time, thus making it possible to study the dynamic phenomena of living specimens in their biological environment and under near-physiological conditions. Over the years, an increasing number of researchers have started to use AFM and, in addition to a wide range of scientific articles, there are now also various books on the subject. In 2004, we edited a book published by Humana Press (Atomic Force Microscopy: Biomedical Methods and Applications) that described a series of practical AFM procedures in various applications with the aim of stimulating researchers to use the technique. We were therefore surprised when Humana Press proposed the publication of a second book on the subject so quickly after the first, and hesitated to accept the challenge. However, upon further reflection, we had to agree that the sheer breadth and originality of the new applications that have emerged since the first book was published more than justified this further review. The reason is quite simple: AFM is no longer simply just another form of microscopy, but has given rise to a completely new way of using microscopy that fulfils the dreams of all microscopists: being able to touch, move, and interact with the sample while it is being examined, thus making it possible to discover not only morphological, but also chemical and physical structural information. Optical microscopy made it possible to talk at the “micron” level (cells), and transmission and scanning electron microscopy introduced the idea of the “nano” level (sub-cellular), but still only in two dimensions; however, when speaking of AFM, it is not only usual to talk in three-dimensional “nano” terms, but it is also already possible to talk
- 11. vi Preface at the “pico” level (molecular). Together with continuous technical improvements, the reaching of this new dimensional range means that AFM can provide an opportunity to interact with individual molecules, observing them while we touch them and move them around in order to be able to discover their physical characteristics. All of this has also led to the development of a parallel “nano-technology” insofar as an AFM workstation has become a “nano-robot” that can dynamically interact with and manipulate samples on a “nano-scale”, and acquire information of sub-pico Newton “force spectroscopy” data on which to base the study of “nano-biology”. Functionalizing the AFM tip has made it pos- sible to obtain “nano-biosensors” that can be used in the field of dynamic biomolecular processes in ways that could not even be imagined just a few years ago. Finally, combining AFM with other microscopic techniques, such as confocal or fluorescence microscopy is now being actively explored, and a number of interesting synergies have been discovered. This book brings together different types of applications in order to provide examples from different fields in the hope that this will stimulate researchers to apply their ingenuity in their own specialization and allow them to add significant originality to their studies. We gratefully acknowledge all of the contributions of our colleagues, each of whom donated their experience in order to cross-fertilize this new and fascinating technology. “GOD BLESS MICROSCOPY (ALL TYPES) …AND MICROSCOPISTS TOO” because they show us what and how wonderful life is. Milan, Italy Pier Carlo Braga Genoa, Italy Davide Ricci
- 12. vii Contents Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v Contributors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xi Part I The Basics of Atomic Force Microscopy 1 How the Atomic Force Microscope Works? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 Bruno Torre, Davide Ricci, and Pier Carlo Braga 2 Measurement Methods in Atomic Force Microscopy . . . . . . . . . . . . . . . . . . . . . . 19 Bruno Torre, Claudio Canale, Davide Ricci, and Pier Carlo Braga 3 Recognizing and Avoiding Artifacts in Atomic Force Microscopy Imaging . . . . . . 31 Claudio Canale, Bruno Torre, Davide Ricci, and Pier Carlo Braga Part ii Molecule Imaging 4 Imaging the Spatial Orientation of Subunits Within Membrane Receptors by Atomic Force Microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47 Stewart M. Carnally, J. Michael Edwardson, and Nelson P. Barrera 5 High Resolution Imaging of Immunoglobulin G Antibodies and Other Biomolecules Using Amplitude Modulation Atomic Force Microscopy in Air . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61 Sergio Santos and Neil H. Thomson 6 Atomic Force Microscopy of Ex Vivo Amyloid Fibrils . . . . . . . . . . . . . . . . . . . . . 81 Claudio Canale, Annalisa Relini, and Alessandra Gliozzi 7 Studying Collagen Self-Assembly by Time-Lapse High-Resolution Atomic Force Microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97 Clemens M. Franz and Daniel J. Muller 8 Atomic Force Microscopy Imaging of Human Metaphase Chromosomes in Liquid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109 Osamu Hoshi and Tatsuo Ushiki 9 Atomic Force Microscopy of Proteasome Assemblies . . . . . . . . . . . . . . . . . . . . . . 117 Maria Gaczynska and Pawel A. Osmulski 10 Atomic Force Microscopy of Isolated Mitochondria . . . . . . . . . . . . . . . . . . . . . . . 133 Bradley E. Layton and M. Brent Boyd 11 Imaging and Interrogating Native Membrane Proteins Using the Atomic Force Microscope . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 153 Andreas Engel
- 13. viii Contents Part III Nanoscale Surface Analysis and Cell Imaging 12 Atomic Force Microscopy Investigation of Viruses . . . . . . . . . . . . . . . . . . . . . . . . 171 Alexander McPherson and Yurii G. Kuznetsov 13 Determination of the Kinetic On- and Off-Rate of Single Virus–Cell Interactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 197 Christian Rankl, Linda Wildling, Isabel Neundlinger, Ferry Kienberger, Hermann Gruber, Dieter Blaas, and Peter Hinterdorfer 14 Atomic Force Microscopy as a Tool for the Study of the Ultrastructure of Trypanosomatid Parasites . . . . . . . . . . . . . . . . . . . . . . . . 211 Wanderley de Souza, Gustavo M. Rocha, Kildare Miranda, Paulo M. Bisch, and Gilberto Weissmuller 15 Normal and Pathological Erythrocytes Studied by Atomic Force Microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 223 Andreas Ebner, Hermann Schillers, and Peter Hinterdorfer 16 The Growth Cones of Living Neurons Probed by the Atomic Force Microscope . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 243 Davide Ricci, Massimo Grattarola, and Mariateresa Tedesco 17 Highlights on Ultrastructural Pathology of Human Sperm . . . . . . . . . . . . . . . . . . 259 Narahari V. Joshi, Ibis Cruz, and Jesus A. Osuna 18 High-Speed Atomic Force Microscopy and Biomolecular Processes . . . . . . . . . . . 285 Takayuki Uchihashi and Toshio Ando Part IV Non-topographical Applications (Force-Spectroscopy) 19 Atomic Force Microscopy in Mechanobiology: Measuring Microelastic Heterogeneity of Living Cells . . . . . . . . . . . . . . . . . . . . . 303 Evren U. Azeloglu and Kevin D. Costa 20 Force-Clamp Measurements of Receptor–Ligand Interactions . . . . . . . . . . . . . . . 331 Félix Rico, Calvin Chu, and Vincent T. Moy 21 Measuring Cell Adhesion Forces: Theory and Principles . . . . . . . . . . . . . . . . . . . . 355 Martin Benoit and Christine Selhuber-Unkel 22 Nanoscale Investigation on E. coli Adhesion to Modified Silicone Surfaces . . . . . . 379 Ting Cao, Haiying Tang, Xuemei Liang, Anfeng Wang, Gregory W. Auner, Steven O. Salley, and K.Y. Simon Ng Part V Investigating Drug Action 23 Imaging Bacterial Shape, Surface, and Appendages Before and After Treatment with Antibiotics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 391 Pier Carlo Braga and Davide Ricci 24 Thymol-Induced Alterations in Candida albicans Imaged by Atomic Force Microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 401 Pier Carlo Braga and Davide Ricci
- 14. ix Contents 25 Atomic Force Microscope-Enabled Studies of Integrin–Extracellular Matrix Interactions in Vascular Smooth Muscle and Endothelial Cells . . . . . . . . . 411 Zhe Sun and Gerald A. Meininger 26 Atomic Force Microscopy Studies on Circular DNA Structural Changes by Vincristine and Aspirin . . . . . . . . . . . . . . . . . . . . . . . . . . . 425 Zhongdang Xiao, Lili Cao, Dan Zhu, and Zuhong Lu Part VI Atomic Force Microscopy as a Nanotool 27 Combined Atomic Force Microscopy and Fluorescence Microscopy . . . . . . . . . . . 439 Miklós S.Z. Kellermayer 28 Chemical Modifications of Atomic Force Microscopy Tips . . . . . . . . . . . . . . . . . . 457 Régis Barattin and Normand Voyer 29 Atomic Force Microscopy as Nanorobot . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 485 Ning Xi, Carmen Kar Man Fung, Ruiguo Yang, King Wai Chiu Lai, Donna H. Wang, Kristina Seiffert-Sinha, Animesh A. Sinha, Guangyong Li, and Lianqing Liu Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 505
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- 16. xi Contributors Toshio Ando • Department of Physics, Kanazawa University, Kanazawa, Japan; CREST, JST, Tokyo, Japan Gregory W. Auner • College of Engineering, Wayne State University, Detroit, MI, USA Evren U. Azeloglu • Department of Pharmacology and Systems Therapeutics, Mount Sinai School of Medicine, New York, NY, USA Régis Barattin • CEA-Grenoble, Grenoble, France Nelson P. Barrera • Department of Physiology, Pontificia Universidad Católica de Chile, Santiago, Chile Martin Benoit • Lehrstuhl für Angewandte Physik, LMU, Sektion Physik, München, Germany Paulo M. Bisch • Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Ilha do Fundão, Rio de Janeiro, Brazil Dieter Blaas • Department of Medical Biochemistry, Max F. Perutz Laboratories, Vienna Biocenter, Medical University of Vienna, Vienna, Austria M. Brent Boyd • Department of Mechanical Engineering and Mechanics, Drexel University, Philadelphia, PA, USA Pier Carlo Braga • Department of Pharmacology, School of Medicine, University of Milan, Milan, Italy Claudio Canale • Nanophysics Unit, Italian Institute of Technology, Genoa, Italy Lili Cao • State Key Laboratory of Bioelectronics, School of Biological Science and Medical Engineering, Southeast University, Nanjing, China Ting Cao • College of Engineering, Wayne State University, Detroit, MI, USA Stewart M. Carnally • Department of Pharmacology, University of Cambridge, Cambridge, UK Calvin Chu • Miller School of Medicine, University of Miami, Miami, FL, USA Kevin D. Costa • Cardiovascular Research Center, Mount Sinai School of Medicine, New York, NY, USA Ibis Cruz • Department of Physiology, Laboratory of Andrology, University of Los Andes, Merida, Venezuela Wanderley de Souza • Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Ilha do Fundão, Rio de Janeiro, Brazil; Diretoria de Programas, Instituto Nacional de Metrologia, Normalização e Qualidade Industrial – INMETRO, Rio Comprido, Rio de Janeiro, Brazil Andreas Ebner • Institute for Biophysics, University of Linz, Linz, Austria J. Michael Edwardson • Department of Pharmacology, University of Cambridge, Cambridge, UK
- 17. xii Contributors Andreas Engel • Maurice E. Müller Institute for Structural Biology, Biozentrum, University of Basel, Basel, Switzerland; Department of Pharmacology, Case Western Reserve University, Cleveland, OH, USA Clemens M. Franz • DFG-Center for Functional Nanostructures, Karlsruhe Institute of Technology, Karlsruhe, Germany Carmen Kar Man Fung • Department of Electrical and Computer Engineering, Michigan State University, East Lansing, MI, USA Maria Gaczynska • Department of Molecular Medicine, Institute of Biotechnology, University of Texas Health Science Center at San Antonio, San Antonio, TX, USA Alessandra Gliozzi • Department of Physics, University of Genoa, Genoa, Italy Massimo Grattarola • Dipartimento di Ingegneria Biofisica ed Elettronica, University of Genoa, Genoa, Italy Hermann Gruber • Institute for Biophysics, University of Linz, Linz, Austria Peter Hinterdorfer • Institute for Biophysics, University of Linz, Linz, Austria Osamu Hoshi • Division of Microscopic Anatomy and Bio-Imaging, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan Narahari V. Joshi • Department of Physiology, University of Los Andes, Merida, Venezuela Miklós S.Z. Kellermayer • Department of Biophysics and Radiation Biology, Semmelweis University, Budapest, Hungary Ferry Kienberger • Institute for Biophysics, University of Linz, Linz, Austria; Agilent Technologies Austria GmbH, Linz, Austria Yurii G. Kuznetsov • Department of Molecular Biology and Biochemistry, University of California, Irvine, CA, USA King Wai Chiu Lai • Department of Electrical and Computer Engineering, Michigan State University, East Lansing, MI, USA Bradley E. Layton • Applied Computing and Electronics, The University of Montana College of Technology, Missoula, MT, USA Guangyong Li • Department of Electrical and Computer Engineering, Michigan State University, East Lansing, MI, USA Xuemei Liang • College of Engineering, Wayne State University, Detroit, MI, USA Lianqing Liu • Department of Electrical and Computer Engineering, Michigan State University, East Lansing, MI, USA Zuhong Lu • State Key Laboratory of Bioelectronics, School of Biological Science and Medical Engineering, Southeast University, Nanjing, China Alexander McPherson • Department of Molecular Biology and Biochemistry, University of California, Irvine, CA, USA Gerald A. Meininger • Department of Medical Pharmacology and Physiology, Dalton Cardiovascular Research Center, University of Missouri-Columbia, Columbia, MO, USA
- 18. xiii Contributors Kildare Miranda • Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Ilha do Fundão, Rio de Janeiro, Brazil; Diretoria de Programas, Instituto Nacional de Metrologia, Normalização e Qualidade Industrial – INMETRO, Rio Comprido, Rio de Janeiro, Brazil Vincent T. Moy • Miller School of Medicine, University of Miami, Miami, FL, USA Daniel J. Muller • Department of Biosystems Science and Engineering, ETH Zurich, Basel, Switzerland Isabel Neundlinger • Institute for Biophysics, University of Linz, Linz, Austria K.Y. Simon Ng • College of Engineering, Wayne State University, Detroit, MI, USA Pawel A. Osmulski • Department of Molecular Medicine, Institute of Biotechnology, University of Texas Health Science Center at San Antonio, San Antonio, TX, USA Jesus A. Osuna • Department of Physiology, Laboratory of Andrology, University of Los Andes, Merida, Venezuela Christian Rankl • Institute for Biophysics, University of Linz, Linz, Austria; Agilent Technologies Austria GmbH, Linz, Austria Annalisa Relini • Department of Physics, University of Genoa, Genoa, Italy Davide Ricci • Robotics, Brain and Cognitive Sciences Department, Italian Institute of Technology, Genoa, Italy; Department of Biophysical Electronic Engineering, University of Genoa, Genoa, Italy Félix Rico • Centre de Recherche, Institut Curie, UMR168-CNRS, Paris, France Gustavo M. Rocha • Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Ilha do Fundão, Rio de Janeiro, Brazil; Diretoria de Programas, Instituto Nacional de Metrologia, Normalização e Qualidade Industrial – INMETRO, Rio Comprido, Rio de Janeiro, Brazil Steven O. Salley • College of Engineering, Wayne State University, Detroit, MI, USA Sergio Santos • School of Physics and Astronomy, University of Leeds, Leeds, UK Hermann Schillers • Institut fur Physiologie II, University Munster, Munster, Germany Kristina Seiffert-Sinha • Department of Electrical and Computer Engineering, Michigan State University, East Lansing, MI, USA Christine Selhuber-Unkel • Institute for Materials Science, University of Kiel, Kiel, Germany Animesh A. Sinha • Department of Electrical and Computer Engineering, Michigan State University, East Lansing, MI, USA Zhe Sun • Dalton Cardiovascular Research Center, University of Missouri-Columbia, Columbia, MO, USA Haiying Tang • College of Engineering, Wayne State University, Detroit, MI, USA
- 19. xiv Contributors Mariateresa Tedesco • Dipartimento di Ingegneria Biofisica ed Elettronica, University of Genoa, Genoa, Italy Neil H. Thomson • School of Physics and Astronomy, University of Leeds, Leeds, UK Bruno Torre • Italian Institute of Technology, Genoa, Italy Takayuki Uchihashi • Department of Physics, Kanazawa University, Kanazawa, Japan; CREST, JST, Tokyo, Japan Tatsuo Ushiki • Division of Microscopic Anatomy and Bio-Imaging, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan Normand Voyer • Département de chimie, Université Laval, Quebec, QC, Canada Anfeng Wang • College of Engineering, Wayne State University, Detroit, MI, USA Donna H. Wang • Department of Electrical and Computer Engineering, Michigan State University, East Lansing, MI, USA Gilberto Weissmuller • Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Ilha do Fundão, Rio de Janeiro, Brazil Linda Wildling • Institute for Biophysics, University of Linz, Linz, Austria Ning Xi • Department of Electrical and Computer Engineering, Michigan State University, East Lansing, MI, USA Zhongdang Xiao • State Key Laboratory of Bioelectronics, School of Biological Science and Medical Engineering, Southeast University, Nanjing, China Ruiguo Yang • Department of Electrical and Computer Engineering, Michigan State University, East Lansing, MI, USA Dan Zhu • State Key Laboratory of Bioelectronics, School of Biological Science and Medical Engineering, Southeast University, Nanjing, China
- 20. Part I The Basics of Atomic Force Microscopy
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- 22. 3 Pier Carlo Braga and Davide Ricci (eds.), Atomic Force Microscopy in Biomedical Research: Methods and Protocols, Methods in Molecular Biology, vol. 736, DOI 10.1007/978-1-61779-105-5_1, © Springer Science+Business Media, LLC 2011 Chapter 1 How the Atomic Force Microscope Works? Bruno Torre, Davide Ricci, and Pier Carlo Braga Abstract This chapter aims at giving a quick but precise introduction of the atomic force microscope from the working principle point of view. It is intended to provide a useful starting point to those who first approach the instrument giving a general sketch of the working principles and technical implementations as well as last improvements. Subheading 1 is introductory: it gives an overview of what the instrument does and why it has been developed. Subheading 2 is focused on measurement ranges and on the com- parison with scanning electron microscope (SEM) and transmission electron microscope (TEM) which have similar ranges and resolutions but different sample interactions and applications. Subheading 3 gives an overview of the working principles and the most diffused technical implementations on which most of the commercial microscopes rely, as we think it gives the useful base knowledge to understand possible applications, instrument capabilities, and results. In particular, technical improvements taking place over the past few years are highlighted. Despite of the simple and not very technical approach, it has a key importance in understanding concepts at the base of Chapter 3, which is, on the other side, useful for beginners and experienced users as well. Subheading 4 compares different instrument architectures and can, therefore, be useful for those who are going to choose an instrument having clear final applications. Latest solutions are once more highlighted. Subheading 5 gives an overview and some suggestions to start working, both in air and in liquid. Following the general philosophy of the book, it follows more an “how to do” concept than a general theoretical approach. Subheading 6 contains the future develop- ments of the techniques. Key words: Introduction to AFM, AFM working principles, AFM basics Microscopes have always been one of the essential instruments for research in the biomedical field. The capability of optical microscopes to magnify and resolve details well below 1 mm has soon reached its intrinsic physical limit due to the well-known “diffraction limit”: when radiation hits obstacles of size compa- rable with its wavelength (visible light: 380–750 nm), diffraction 1. Introduction
- 23. 4 Torre, Ricci, and Braga and interference became important, and smaller details cannot be distinguished. Historically, two solutions have been found to image samples with few nanometers resolution or better: the first one is to shorten radiation wavelength, using ultraviolet, X-ray, or electron- based microscopes, to push the diffraction limit from hundreds to few nanometers scale. Radiation-based microscopes (such as the light microscope and the electron microscope) have become trustworthy companions in the laboratory and have contributed greatly to our scientific knowledge. However, short-wavelength radiation can induce sample damaging because high-energy inter- action can be involved; moreover, measurements often require special sample preparation or controlled (vacuum) conditions, that can often be incompatible with physiological environment or in vivo measurements. A second strategy relies on a completely different system: a very sharp tip is set in (weak) interaction with the sample and rastered on it while interaction is measured and controlled. In this way, the tip tracks surface morphology while its XYZ position is registered by the electronics to compose a 3D map of the sample surface. Since the interaction can be controlled and limited to very low values, this kind of imaging is usually nondestructive. Depending on the type of interaction measured, scanning probe microscopes (SPMs) take different names, such as scanning tun- neling (STM; current between tip and samples), scanning near field optical (SNOM; optical coupling), atomic force (AFM; force between last part of the tip and sample) (1), etc. Historically, AFM has been invented after STM to allow measurements on insulating samples: very soon it was clear that it could archive nanometer resolution working in different environments – air, liquid, or vacuum – regardless of conductive or optical properties of the sample. Moreover, it measures (and controls) tip–sample interaction forces and, therefore, it allows to probe (nano) mechanical properties of the specimen applying pressure or pull- ing the sample. Hereafter, we refer mainly to AFM for its wide range of applicability in biological field. After using it for the first time, three things can be noticed: Despite of a rather high-sounding name, imaging with the ● ● AFM can be quite simple: no special sample preparation is required, and images with unexpected resolution can be obtained on the very first time; Images are real 3D ones: height is measured with even higher ● ● resolution, and subnanometer steps are commonly resolved; questions on surface corrugation and feature height can be easily answered. At first use, imaging appears quite slow: one image can take ● ● some minutes to acquire and video rate measurements are not possible with commonly used instruments: this is a
- 24. 5 How the Atomic Force Microscope Works? common feature for all the mechanical scanning techniques. Nevertheless, a few minutes (without preparation needed) time interval is compatible with most experiments even for biological applications. After some experience, one learns that in some cases it is possible to push resolution to the “atomic” level (2–4) and that images do contain details not observable with any other instrument. A noteworthy feature is that imaging is only one of the exper- iments that can be performed with the instrument: the tip can be pushed on the sample, pulled out, used to make scratches, func- tionalized to bind to specific chemical groups, electrically con- nected to detect currents or potentials, and used to induce catalytic reactions or for lithography purpose. The number of experiments that can be redesigned on the nanometer scale seems to be limited just by applicant imagination: this capability gave rise to a new definition for AFM applications, “lab on tip.” These are, anyway, advanced techniques and are not described in this work: readers interested in the topic are referred to specialized literature. AFM images show significant information about surface features with unprecedented clarity. The AFM can perform nondestruc- tive examinations on any sufficiently “rigid” surface either in air or in liquid, regardless if the specimen is insulating, conductive, transparent, or opaque. Modern instruments can be endowed with temperature control stages and closed chamber for environ- mental control; some of them are especially designed to be cou- pled with an optical microscope for simultaneous imaging through advanced optical techniques so that a huge variety of complemen- tary information can be archived. The field of view can vary from the atomic and molecular scale up to sizes larger than 100 mm so that data can be coupled with other information obtained with lower resolution – and wider field of view – techniques. The AFM can also examine rough surfaces with (sub)nanometer resolution on the vertical range up to more than 10 mm; large samples can be fitted directly in the microscope without cutting. With stand-alone instruments, any area on flat or nearly flat specimens can be investigated. Compared with the SEM, AFM provides topographic con- trast of surface features with quantitative height information. Moreover, as the sample need not be electrically conductive, no metallic coating of the sample is required. Hence, no dehydration of the sample is necessary as with SEM, and samples may be imaged in their hydrated state. This eliminates the shrinkage of biofilm associated with SEM imaging, yielding a nondestructive 2. Performance Range of AFM
- 25. 6 Torre, Ricci, and Braga technique. The resolution of AFM is higher than that of environ- mental SEM, where hydrated images can also be obtained and extracellular polymeric substances may not be imaged. Compared with transmission electron microscopes, where the electron beam gives a planar projection of the sample by flow- ing through it, AFM images give information on 3D properties of the surface: in this sense, these two techniques can be regarded as the most complementary ones, since one (TEM) provides con- trast on inner structures of the sample, but it is intrinsically 2D, while the other one (AFM) gives real 3D images with similar res- olution, but it can only access to the exposed surface. Finally, it can be commented that with respect to TEM, no expensive and destructive (cross-sectioning) sample preparation is needed. Moreover, image contrast is quantitative and can be expressed in nanometer units by default and this is a pretty unique characteris- tic, allowing direct comparison between different samples. In the following subheadings, we give a brief outline of how the AFM works followed by a description of the parts that can be added to the basic instrument. Our overview has no pretense of completeness but aims at simplicity. For a more thorough descrip- tion of the physical principles involved in the operation of these instruments, we refer you to the specialized literature. In Fig. 1, a schematic diagram of the AFM working principle is shown (1, 5). In principle, AFM can remind one of those old style record players, but it incorporates a number of technical solutions that allow to detect atomic-scale corrugation: very sharp tips at the end of flexible cantilevers and a sensitive deflection sensing system capable of controlling with high accuracy the tip–sample relative position are used. A basic configuration is made up as follows: A 3D positioning system, called scanner, to adjust tip–sample ● ● relative position: if the tip is attached to the scanner, the con- figuration is called scanning probe; otherwise (as marked with 1 in Fig. 1a), if the tip is fixed and the sample is moved it is called scanning sample. A sample holder where the specimen can be placed in a stable ● ● configuration (Fig. 1b). A sharp tip at the end of a flexible cantilever (marker 3 in ● ● Fig. 1a and b). A deflection detecting system: in Fig. ● ● 1a and c (marker 4), the widely used optical beam deflection (OBD) configuration is 3. The Microscope
- 26. 7 How the Atomic Force Microscope Works? shown; in this configuration, a tiny tip displacement is detected by a laser beam, amplifying the deflection of the cantilever hold- ing the tip. Laser light, reflected from the rear of the cantilever, is centered on a (usually four vector) photodiode by means of mirrors placed on the optical path. This method allows good signal amplification and it is of rather simple use ; therefore, it is employed on almost all commercial instruments. Some signal conditioning and preamplifying stage: in case of ● ● OBD system, signals from sector A, B, C, and D are used to calculate overall power SUM=A+B+C+D, normal deflec- tion N=(A+B−C−D)/SUM, and lateral deflection as L=(A+C−B−D)/SUM. A digital control system to control tip–sample position on the ● ● basis of collected signals. The following sections contain further details on single components and on the working principle. Fig. 1. Schematic diagram of a scanned-sample AFM, based on five quadrant piezo scanner configuration (see below). In the case of scanned probe, it is the tip that is scanned instead of the sample. (a) The piezoelectric scanner (1) is the (nano)positioning element allowing movement: it works by applying opposite voltages to ±X and ±Y sectors to move the sample in X and Y directions, respectively; an additional Z sector moves the sample in the vertical direction. The sample (2) is positioned on the scanner; (3) cantilever; (4) optical beam deflection system (OBD) to detect tip displacement; (5) position-sensitive photodetector (PSD) and preamplifier; (6) electronics. (b) A magnification of tip and sample. (c) A detail of the OBD system.
- 27. 8 Torre, Ricci, and Braga High-resolution (nanometer) positioning can be performed using piezoelectric ceramic materials. These materials undergo a revers- ible deformation when an external (high) bias voltage is applied across two opposite faces of it: in a first approximation, such defor- mation can be considered to depend linearly on the applied voltage. A widely used scanner configuration relies on piezoelectric tubes made up by four or five sectors (Fig. 2). A differential bias (with respect to the inner part of the tube, grounded) applied to opposite electrodes induces a bending of the tube in one of the two main directions, while common mode voltage induces a contraction or elongation in the vertical direction. The same happens for the other two electrodes so that differential signals can be used for X and Y movement and common bias for Z movement and four electrodes are sufficient to provide a complete 3D positioning. Anyway, for technical reasons, it is preferable to decouple the Z movement from the XY one, by adding a fifth dedicated electrode (see Figs. 1a and 2) so that a common voltage to side electrodes is no more needed. As shown in Fig. 2, this configuration gives an undesired parabolic component to the motion, therefore this type of scanners are usu- ally endowed with embedded positioning sensors that allow distor- tion compensation and linearization. This kind of distortion (commonly referred as bow) is mostly relevant for high scan ranges (above some micrometers) and becomes less important for smaller regions, that is to say in case of high resolution: for this reason, some instruments allow to operate also in open-loop mode (i.e., without sensor compensation) for high-resolution imaging, to fur- ther reduce electrical noise of readout circuitry. Modern microscopes use a slightly different configuration: single linear piezoelectric elements are embedded in a metallic frame machined by electroerosion to be easily deformable in 3.1. The Scanner Fig. 2.Working principle of a five quadrant piezo tube: right image shows how deflection occurs upon differential biasing of two opposite sectors, here −X and +X; the Z sector is visible just below the sample holder on the upper part of the tube. Figure is not in scale and deflection is intentionally exaggerated to highlight the effect.
- 28. 9 How the Atomic Force Microscope Works? predefined directions (flexure system); for each axis, a deformation occurs easily in the parallel direction to the piezoelectric strain so that the three directions are efficiently decoupled on the three axes. The frames usually incorporate low noise, often capacitive or inductive positioning sensors, and therefore are good candidates for metrology purposes. In some configurations, one of the axes (vertical one) is also mechanically and physically decoupled from the other two. A few words can be spent on the topic of positioning sensors to detect displacements on the nanometer scale. Neglecting for the moment interferometric solutions, that are often used for metrological standards but are not very easy to be integrated, three different accurate positioning sensors are commercially available: Strain gauge (resistive) sensors can be easily integrated even ● ● on piezoelectric tubes by simply gluing them: upon deforma- tion, sensors change their electrical resistance that can be directly read by the electronics. Anyway, since resistors are intrinsically thermal noise generators, this kind of sensors is noisy and resolution is usually limited to a few nanometers. Capacitive sensors: basically they are made up by a capacitor ● ● with one plate coupled with the moving part and one fixed to a standing position; a change in the relative position of the two plates implies a change in the relative capacitance that is electrically detected. This type of sensors has very low noise and commonly allows sensitivities of the order of tens of nanometers or better. Integration of these sensors in a scan- ner is more difficult than for the strain-based ones, and paral- lelism between faces is often an issue, so they are more often found on flexure scanners than on piezo tubes motors. Inductive or eddy current sensors: recently, some commer- ● ● cially available microscopes have successfully employed induc- tive sensors for embedded position detection. These have reported resolutions of few tens of nanometer. Readers interested on this topic can find further details in ref. 6. For our purpose, it is sufficient to keep in mind that in some lower cost microscope, where this compensation is not implemented, a postprocessing software correction can always be performed. The tip, which is mounted at the end of a small cantilever, is the heart of the instrument because it is brought in closest contact with the sample and gives rise to images though its force interac- tions with the surface. When the first AFM was made, a very small diamond fragment was carefully glued to one end of a tiny piece of gold foil. Today, the tip–cantilever assembly typically is 3.2. Tip and Cantilever
- 29. 10 Torre, Ricci, and Braga fabricated from silicon or silicon nitride and, using technology similar to that applied to integrated circuit fabrication, allows a good uniformity of characteristics and reproducibility of results (7, 8). The essential parameters to consider are the sharpness of the apex, measured by the radius of curvature (spherical approxi- mation), and the aspect ratio of the whole tip (Fig. 3). Nowadays, a variety of cantilevers are commercially available: in addition to standard pyramid tips, usually 3 mm tall with approximately 30-nm apex radius, also tetragonal, high aspect ratio and conical tips can be found. The tip can end with dia- mond-like carbon spikes, carbon nanotubes, or whiskers for low curvature radius, and they can also be further machined by means of focused electron beam (FEB) or focused ion beam (FIB) to obtain even higher aspect ratios. Commercial tips commonly end with curvature radius smaller than 10 nm, but ultrasharp tips with R2 nm or even 1 nm are commercially available. Moreover, tips can be coated with metal films to enhance conductivity in the contact area, or to obtain magnetic properties; low electrical resis- tance doped silicon tips are also available. Chemically functional- ized tips can be also purchased for applications involving specific bindings with biochemical species on the sample surface. Although it would seem that sharper tips should yield more detailed images, this may not occur with all samples: in fact, quite often, the so-called atomic resolution on crystals is obtained best with standard silicon nitride tips. This is because reducing apex radius has the drawback of increasing tip fragility. Moreover, the measurement load rises quickly due to contact area reduction, and this can lead to quick tip erosion or to sample damage. Fig. 3. The essential parameters in a tip are the radius of curvature (R) and the aspect ratio (ratio of H to W ).
- 30. 11 How the Atomic Force Microscope Works? The cantilever carrying the tip is attached to a small glass “chip” that allows easy handling and positioning of the instru- ment. There are essentially two designs for cantilevers, the “V” shaped and the single-arm kind (Fig. 4), which have different tor- sional properties. The length, width, and thickness of the beam(s) determine the mechanical properties of the cantilever and have to be chosen depending on mode of operation needed and on the sample to be investigated. Cantilevers are essentially classified by their force (or spring) constant and resonance frequency: soft and low-resonance frequency cantilevers are more suitable for imag- ing in contact and resonance mode in liquid, whereas stiff and high-resonance frequency cantilevers are more appropriate for resonance mode in air (9). AFMs can generally measure the vertical deflection of the cantile- ver with picometer resolution. To achieve this, most AFMs today use the optical lever or OBD method that achieves resolution comparable to an interferometer while remaining inexpensive and easy to use. 3.3. Deflection Sensor Fig.4.SEM image of triangular (A) and single-beam (B) cantilevers (MLCT silicon nitride probe,Veeco) (courtesy S.Marras). The mechanical properties, such as the force constant and resonant frequency, depend on the values of width (W ), length (L), and thickness (T ). Bottom right image shows pyramidal tip.
- 31. 12 Torre, Ricci, and Braga In this system, a laser beam is reflected on the backside of the cantilever (often coated by a thin metal layer to enhance reflectiv- ity) onto a position-sensitive photodetector (PSD), consisting of two (more often four, as in Fig. 1) side-by-side mounted photo- diodes. In this arrangement, a small deflection of the cantilever tilts the reflected beam and changes the position of the light spot on the photodetector. The signal difference between the different sections of the photodiode indicates the position of the laser spot on the detector, and thus the deflection of the cantilever. Because the distance between cantilever and detector is gen- erally three orders of magnitude greater than the length of the cantilever (millimeters compared to micrometers), the optical lever greatly magnifies motions of the tip giving rise to an extremely high sensitivity. Images are formed by recording the effects of the interaction forces between tip and surface as the cantilever is scanned over the sample. The scanner and the electronic feedback circuit, together with sample, cantilever, and optical lever form a feed- back loop set up for the purpose. The presence of a feedback loop is a key difference between AFM and older stylus-based instru- ments so that AFM not only measures the force on the sample, but also controls it, allowing acquisition of images at very low tip- to-sample forces (5, 10). The scanner is an extremely accurate positioning stage used to move the tip over the sample (or the sample under the tip) to form an image. The AFM electronics drives the scanner across the first line of the scan and back. It then steps in the perpendicular direction to the second scan line, moves across it and back, then to the third line, and so forth (Fig. 5). Usually, both forth and back traces (trace and retrace) are recorded, giving two images that ideally should overlap: if images differ at some point, this can 3.4. Image Formation Fig. 5. Raster scan for image acquisition.The AFM electronics drive the scanner across the first line of the scan and back. The scanner then steps in the perpendicular direction to the second scan line, moves across it and back, then to the third line, and so forth.
- 32. 13 How the Atomic Force Microscope Works? be due to tip contamination, sample modification, or to an improper choice of measurement parameters, and therefore sug- gests that something needs to be adjusted. As the probe is scanned over the surface, a topographic image is obtained storing the ver- tical control signals sent by the feedback circuit to the scanner moving it up and down to follow the surface morphology while keeping the interaction forces constant. The image data are sam- pled digitally at equally spaced intervals, up to some thousands points per line. The number of lines is usually chosen to be equal to the number of data points per line, obtaining at the end a square grid of data points each corresponding to the relative X, Y, and Z coordinates in space of the sample surface (11). Usually, during scanning, data are represented by gray scale or RGB images, in which the brightness of points can range from black to white across 256 levels, corresponding to the informa- tion acquired by the microscope (that can be height, force, phase, and so on); anyway, data are usually collected with higher resolution, since they are digitalized as 16 bit data (65,536 levels) or better, therefore the available information is by far more than what is displayed on the screen. Usually, microscopes are endowed with software solutions allowing statistical analysis and quantitative mathematical parameterizations of collected data. A number of free software programs, compatible with the more widespread AFM file formats, can also be downloaded from the Web (12). The first instruments introduced on the market had all very simi- lar features and range of applications. They had scanners with small range, limited optical access, and could accommodate only small samples. Essentially, they were built to make very high- resolution imaging on flat samples in a dry environment. With the development of new technical solutions, fields of application grew very rapidly, and now it is possible to find instrument add-ons and architectures allowing to perform very sophisticated measurements in different fields: we can now find instruments that are specifically designed for large samples, such as silicon wafers, that have metrological capabilities, utilize closed- loop scanners that are optimized for liquid and electrochemistry operation and can be mounted on an inverted microscope for biological investigations. Usually, one single instrument can have different options to extend its capabilities to a wide range of applications and a huge variety of experiments are easily software controllable so that even nonexperts can relatively quickly per- form advanced experiments; anyway, it is still true that instru- ments are designed keeping in mind some particular application 4. Instruments, Architectures, and Options
- 33. 14 Torre, Ricci, and Braga so that they are optimized for a specific purpose. For this reason, it is necessary to have clearly in mind the main features that are desired in an instrument before its purchase, understanding at the same time that a loss of performance in other applications may be possible. One can still distinguish, to clarify this point, essentially between two main instrumentation categories, even if intermedi- ate or hybrid configurations are possible: scanned-sample and scanned-tip microscopes. With reference to the widely used piezo tube configuration, we give a brief description of the advantages of one system with respect to the other and of their limitations; finally, we are going to clarify how these problems are minimized, thanks to the introduction of modern flexure scanners. This scanned-sample AFM is the oldest design in which the sam- ple is attached to the scanner and moved under the tip. Depending on how the cantilever holder, laser, and photodetector are assem- bled, it can easily accommodate an overhead microscope provided that long focal length objectives are used. A clear view of where the tip is landing is usually possible, speeding up the time it takes to get a meaningful image of the sample. Since in this configuration scanner and optical lever are physi- cally independent, it is less packed, allowing an easier optical access to the tip and to the scanner. Scanners with wide X, Y, and Z range are usually available and closed-loop control feedback is more easily implemented in this scheme, and often a lower mechanical noise level can be obtained allowing higher ultimate resolution. There are quite a few drawbacks. First of all, the size and weight of the sample have to be limited because it is sitting on the scanner and may change its behavior. For the same reason, opera- tion in liquid is impaired because liquid cells tend to be small and difficult to seal, and liquid flow or temperature control are more complicated to implement. Notwithstanding these difficulties, excellent results can be obtained on typical biomedical science specimens by ingeniously adapting them to the instrument’s characteristics. In the scanned-tip method of operation, the sample stays still and it is the cantilever, attached to the scanner, which is moved across the surface. Although for scanning tunneling microscopes this was one of the first solutions applied, to build a scanned-tip AFM requires overcoming some difficulties, essentially related to adapt- ing the beam bounce detection scheme to a moving cantilever. In fact, because of its weight, it is not possible to mount all the opti- cal setup on the moving assembly and some position-related undesired effects can often occur. Some design couple correction lenses moving with the tip to partially compensate for these 4.1. Scanned Sample 4.2. Scanned Tip
- 34. 15 How the Atomic Force Microscope Works? effects. One advantage of this configuration is that no limitations in sample weight or size occur: since the scanner is loaded with always the same weight and the sample is still, its mechanical per- formance is independent from the sample changes. Instrument operation can be easily automated, by coarse positioning the sam- ple below the scanner using a micrometric motor, and some mod- els are endowed with three supports that enable them to scan the surface of any object under their probe. More recently, special- ized instruments were developed, capable of being coupled or even integrated into inverted optical microscopes for biological applications. With respect to the scanned-sample models, scanned-tip instruments can be more easily equipped with temperature- controlled stages, open or closed liquid cells, liquid flow systems, electrochemistry cells, and controlled atmosphere chambers. Concerning limitations, one could say that what is gained on one side is lost on the other. For example, often the overall noise level is higher, limiting ultimate resolution. Large scan areas are more difficult to perform because tracking systems have to be used to keep the laser spot on the back of the cantilever. A top view of samples is obstructed by the scanner assembly: special hollow tubes have been developed recently, but even so on-axis micro- scopes, which are useful on nontransparent samples, still have limited resolution and lateral field of view. As anticipated, these limitations, occurring in both cases, have been greatly reduced by the introduction of flexure technol- ogy for scanners: the rigid metallic frame is by far less sensitive to loads so that heavier samples can be measured without affecting microscope capabilities (if the sample is scanned) or the whole optical lever can be moved with the tip (scanned-tip configura- tion), eliminating undesired tip-lased displacements. Some inter- mediate configurations scan the sample in the XY direction, while moving the tip (and the optical lever) in Z direction to physically decouple raster from retroactive movements. Moreover, they can be easily endowed with closed liquid cells, external cir- cuitries, and coarse movements for advanced experiments or easy repositioning. The choice of configurations in most cases is more influenced by the possibility to couple the instrument with inverted or top-view optical microscopes, than from real techni- cal limitations. Even if modern flexure stage microscopes prevents most of the architecture-related problems, it is still true that the greater the mechanical paths (big frame for large scanning, “open” architecture for easy optical access), the higher are the effects of mechanical vibrations or thermal expansions so that high (atomic) resolution instruments still rely on a very packed design and usually do not allow scanning frames larger then few micrometers.
- 35. 16 Torre, Ricci, and Braga Samples to be viewed in atmospheric environment are often simply glued to a sample holder, usually a metal disk to be magnetically positioned or a microscope glass. An essential feature is that the sample has to adhere firmly adherent to the sample holder; other- wise, very poor imaging is achieved. For this reason, one has to be careful in the choice of the glue or sticky tape: slow drying glue or thick sticky tape should be avoided. A drawback is that after use in the AFM, the sample is difficult to take off without damage. Some systems, usually scanned-tip ones, can accept samples directly, securing them with a metal clip or springs. This method allows sample recovery without damage for further use in other experiments, but it can be less stable and needs special care for high-resolution work. Sometimes, because of the ease of use of the AFM, one for- gets to be careful while handling the sample: fingerprints, dust, or scratches contaminate sample and affect all measurements, there- fore one should avoid touching the surface in any case. To remove some dust on the surface, one can try to gently flush dry gas, e.g., nitrogen, on the sample, obtaining, in some cases, benefits. As a general rule, it is better to wear (powder free) gloves and to use tweezers and clean tools to handle the specimen (for these reason toolsets are provided with most microscopes). Also, it is best to keep a reserved area of the laboratory free from contaminants for the operations of sample and cantilever mounting. One of the main reasons for the success of AFM in biomedical investigations is its ability to scan samples in physiological condi- tion, that is, immersed in liquid solutions (13, 14). Just to make an example, scanned-tip systems can often be directly used to image cells into a standard Petri dish. Each manufacturer has its own design of liquid cells, sometimes different ones depending on the application, and users may decide to make their own to fit specific needs. A few additional things that have to be taken care of when imaging in liquid are the temperature of the solution (eventually added during imaging; ref. 15), maintenance of the liquid cell, and cantilever holder assembly. Because the cantilever is extremely sensitive to temperature changes, it is important to let the system equilibrate before taking images. For example, in the case of contact-mode imaging with silicon nitride cantilevers and tips, a large variation in time of the signal on the photodetec- tor corresponding to cantilever deflection can be observed in the presence of a temperature change (16). If temperature is not sta- ble prior to approach of the tip to the sample and one starts tak- ing images, after some time the applied force could be quite different than at the beginning of the imaging session. 5. Loading a Sample in the Microscope 5.1. Imaging Dry Samples 5.2. Imaging in Liquid
- 36. 17 How the Atomic Force Microscope Works? If liquid has to be refilled or replaced, a good rule is to avoid abrupt temperature changes to the cantilever and to the sample: if the microscope operates in a closed acoustic box, we suggest keeping some small amount of spare liquid inside so that it stays at the same temperature of the system. Another problem can arise from bubble formations. Quite often, some air remains trapped below the tip or above the sam- ple: in these cases, the cantilever is bent toward the surface and landing on the sample is not possible. This effect can be easily recognized because the deflection signal does not give reasonable values and sometimes the optical lever cannot be aligned. This effect occurs very often at first landing and during liquid refilling, mostly if the added liquid has a different temperature from the measurement bath. In these cases, the best solution is to remove the cantilever holder, dry the sensor with some gentle nitrogen flux, and remount the setup. Usually, wetting the cantilever with a droplet of buffer liquid before starting the experiment helps to prevent gas trapping. Once finished using the microscope for imaging in liquid, it is essential to immediately clean thoroughly all parts that have been in contact with the solution to avoid contamination of future experiments. Usually, it should be possible to disassemble and sonicate all vital parts of the liquid cell and the cantilever holder. The AFM is part of a family of SPMs that has a great growth potential. It is a fact that the majority of novel applications and techniques developed in SPMs in the last years are related to the life sciences. There is still much room for technical improvement: electronics, scanners, and tips are constantly improving. Scan speed limitations, sample accessibility, and ease of use have been addressed and can be still improved. As more and more biomedi- cal researchers will be involved in the use of AFM, with their experience they will be able to contribute in developing an instru- ment less related to the physical sciences (its origin) and more tailored to our specific needs. References 6. Future Developments 1. Binnig, G., Quate, C. F., and Gerber, Ch. (1986) Atomic force microscope. Phys. Rev. Lett. 56, 930–933. 2. Binnig, G., Gerber, C., Stoll, E., Albrecht, T. R., and Quate, C. F. (1987) Atomic resolu- tion with the atomic force microscope. Europhys. Lett. 3, 1281–1286. 3. Hug, H. J., Lantz, M. A., Abdurixit, A., et al. (2001) Subatomic features in atomic force microscopy images. Science 291, 2509. 4. Jarvis, M. R., Perez, R., and Payne, M. C. (2001) Can atomic force microscopy achieve atomic resolution in contact mode? Phys. Rev. Lett. 86, 1287–1290.
- 37. 18 Torre, Ricci, and Braga 5. Alexander, S., Hellemans, L., Marti, O., et al. (1989) An atomic-resolution atomic-force microscope implemented using an optical lever. J. Appl. Phys. 65, 164–167. 6. Kwon, J., Hong, J., Kim,Y.S., Lee, D.Y., Lee K., Lee, S., Park,S.,(2003) Atomic force microscope with improved scan accuracy, scan speed,and optical vision Rev. Sci. Inst. V 74, N 10, 4378–4383. 7. Albrecht, T. R., Akamine, S., Carver, T.E., and Quate, C. F. (1990) Microfabrication of cantilever styli for the atomic force micro- scope. J. Vac. Sci. Technol. A 8, 3386–3396. 8. Tortonese, M. (1997). Cantilevers and tips for atomic force microscopy. IEEE Engl. Med. Biol. Mag. 16, 28–33. 9. Cleveland, J. P., Manne, S., Bocek, D., and Hansma, P. K. (1993) A non-destructive method for determining the spring constant of cantilevers for scanning force microscopy. Rev. Sci. Instrum. 64, 403–405. 10. Meyer, G. and Amer, N. M. (1988) Novel approach to atomic force microscopy. Appl. Plrys. Lett. 53, 1045–1047. 11. Baselt, D. R., Clark, S. M., Youngquist, M. G., Spence, C. F., and Baldeschwieler, mJ. D. (1993) Digital signal control of scanned probe microscopes. Rev. Sci. Instrum. 64, 1874–1882. 12. I. Horcas, R. Fernandez, J.M. Gomez- Rodriguez, J. Colchero, J. Gomez-Herrero, and A.M. Baro, Review of Scientific Instruments 78, 013705 (2007). 13. Wade, T., Garst, J. F., and Stickney, J. L. (1999). A simple modification of a commer- cial atomic force microscopy liquid cell for in situ imaging in organic, reactive or air sensi- tive environments. Rev. Sci. Instr. 70, 121–124. 14. Lehenkari, P. P., Charras, G. T., Nykanen, A., and Horton, M. A. (2000) Adapting atomic force microscopy for cell biology. Ultramicroscopy 82, 289–295. 15. Workman, R. K. and Manne, S. (2000) Variable temperature fluid stage for atomic force microscopy. Rev. Sci. Instrum. 71, 431–436. 16. Radmacher, M., Cleveland, J. P., and Hansma, P. K. (1995) Improvement of thermally induced bending of cantilevers used for atomic force microscopy. Scanning 17, 117–121.
- 38. 19 Pier Carlo Braga and Davide Ricci (eds.), Atomic Force Microscopy in Biomedical Research: Methods and Protocols, Methods in Molecular Biology, vol. 736, DOI 10.1007/978-1-61779-105-5_2, © Springer Science+Business Media, LLC 2011 Chapter 2 Measurement Methods in Atomic Force Microscopy Bruno Torre, Claudio Canale, Davide Ricci, and Pier Carlo Braga Abstract This chapter is introductory to the measurements: it explains different measurement techniques both for imaging and for force spectroscopy, on which most of the AFM experiments rely. It gives a general over- view of the different techniques and of the output expected from the instrument; therefore it is, at a basic level, a good tool to properly start a new experiment. Concepts introduced in this chapter give the base for understanding the applications shown in the following chapters. Subheading 1 introduces the distinc- tion between spectroscopy and imaging experiments and, within the last ones, between DC and AC mode. Subheading 2 is focused on DC mode (contact), explaining the topography and the lateral force channel. Subheading 3 introduces AC mode, both in noncontact and intermittent contact case. Phase imaging and force modulation are also discussed. Subheading 4 explains how the AFM can be used to measure local mechanical and adhesive properties of specimens by means of force spectroscopy tech- nique. An overview on the state of the art and future trends in this field is also given. Key words: AFM imaging modes, Contact mode, Noncontact mode, Intermittent contact mode, Phase imaging, Force modulation, Force spectroscopy Different kinds of measurements and advanced experiments can be grouped under two main categories: imaging and spectroscopy. In the first case, the tip is scanned over the surface to com- pose a topographic map of the sample and depending on the operation mode and on the parameters under control, the imag- ing mode can take different names (i.e., contact, noncontact, constant height, amplitude modulation, frequency modulation, etc.); in all these cases, data is organized to compose an image that is representative of surface morphology and/or related to some of its properties (e.g., composition). 1. Introduction
- 39. 20 Torre et al. In general, one can easily distinguish between two imaging modes depending on which tip–sample interaction is detected: usually, interaction can be detected by looking at static deflection of the cantilever (force measurements) or by forcing it into reso- nance and measuring the changes in its oscillation due to the presence of an interaction force. The first case is usually called static mode, or DC mode, because it records the static deflection of the cantilever, whereas the second takes a variety of names (some patented) among which we may point out the resonant or AC mode. In this case, the feedback loop tries to keep at a set value not the deflection but one of the oscillation parameters, usually the amplitude, of the cantilever while scanning the sur- face. To do this, more complicated electronics are necessary in the detection circuit, including a lock-in or a phase-locked loop amplifier, and also some actuator in the cantilever holder to induce the oscillatory excitation; anyway, almost all modern instruments have all these capabilities already implemented and software con- trollable as default so that inducing cantilever resonance and tun- ing measurements parameters can be easily done. From a physical point of view, one can make a distinction between the two imaging modes depending on the sign of the forces involved in the interaction between tip and sample, that is, by whether the forces there are purely repulsive or also account for attractive contributions (1). In Fig. 1, an idealized plot of the forces between tip and sample is shown, highlighting where typi- cal imaging modes are operated. intermittent Fig. 1. Idealized plot of the forces between tip and sample, highlighting where typical imaging modes are operative.
- 40. 21 Measurement Methods in Atomic Force Microscopy An alternative method, the spectroscopy mode, is more related to the evaluation of the forces shown in Fig. 1 than to the reconstruct ion of image morphology. It consist of sweeping one of the measurement parameters S while the tip is not scanning the surface (point mode) and recording cantilever response R at the same time: in this way, a curve R=f(S) is obtained. A very well- established technique consists in monitoring cantilever deflection D (i.e., force, after spring constant calibration) while changing the tip–sample distance Z to bring the tip into contact with the sample, starting very far from the surface. This technique is referred as force spectroscopy, and D=f(Z) curves are called force–distance curves: data contain quantitative information on tip–sample interaction and on sample mechanical properties, such as elasticity and plasticity, therefore, this kind of spectroscopy is very popular. The spectroscopy mode is a general technique and other experiments can be performed to measure sample proper- ties, such as conductivity, piezoelectric response, or dynamic mechanical response. Spectroscopy curves can be acquired on an N×M grid of points, obtaining a three-dimensional map in which each pixel is a spectroscopy curve containing information about the interaction. In the following chapter and subheadings, we briefly describe the DC and AC imaging modes and the basics of spectroscopy operation for relevant applications in the biomedical field. Also called constant force mode, the contact mode is the most direct AFM mode, where the tip is brought in contact with the surface and the cantilever deflection is kept constant during scan- ning by the feedback loop. Image contrast depends on the applied force which again depends on the cantilever spring constant (Fig. 2). Softer cantilevers are used for softer samples. It can be employed easily also in liquids, allowing to considerably reduce the capillary forces between tip and sample and, hence, limit the damage to the surface (Fig. 3; refs. 2, 3). Because the tip is per- manently in contact with the surface while scanning, a consider- able shear force can be generated, causing sample damage, especially on very soft specimens, such as biomolecules or living cells (4). Since share forces depend on load, recently very soft (spring constant 10–100 times smaller than usual contact-mode cantilever), small, low-noise silicon nitride cantilever has been specifically designed to work in contact mode on biological sam- ples, particularly in liquid environment, allowing stable imaging: for these reasons, many authors prefer to work in contact mode even on very soft biological samples. 2. DC Modes 2.1. Contact Mode
- 41. 22 Torre et al. In some cases, especially on rough and relatively rigid samples, the error signal (i.e., the difference between the set point and the effective deflection of the cantilever that occurs during scanning as a result of the finite time response of the feedback loop) is used to record images. By turning down on purpose the feedback gain, the cantilever will press harder on asperities and less on depressions, giving rise to images that contain high-frequency information oth- erwise not visible (5). This method has been extensively used to image submembrane features in living cells. The same method is also often used to record high-resolution images on crystals. In this case (a variation of standard contact mode), not only the vertical deflection of the cantilever, but also the lateral deflection (torsion) is measured by the photodetector assembly, which in 2.2. Deflection or Error Mode 2.3. Lateral Force Microscopy Fig. 2. In contact mode, the tip follows directly the topography of the surface while it is scanned. Fig. 3. In contact mode, capillary forces caused by a thin water layer can considerably increase the total force between sample and tip.
- 42. 23 Measurement Methods in Atomic Force Microscopy this case has four photodiodes instead of two (Fig. 4). The degree of torsion of the cantilever supporting the probe is a relative mea- sure of the surface friction caused by the lateral force exerted on the scanning probe (6). This method has been used to discrimi- nate between areas of the sample that have the same height (i.e., that are on a same plane), but that present different frictional properties due to absorption of different chemical properties. All AC modes require setting the cantilever in oscillation using an additional driving signal. This can be accomplished by inducing oscillations in the cantilever with a piezoelectric motor (acoustic mode) or, as developed more recently, by directly driving using external coils a probe coated with a magnetic layer (magnetic mode). By using this second method interesting results have been obtained, especially in liquid, as it allows better control of the oscillation dynamics and has inherently less noise (7, 8). An oscillating probe is brought into proximity of (but without touching) the surface of the sample and senses the van der Waals attractive forces that induce a frequency shift in the resonant frequency of a stiff cantilever (Fig. 5; ref. 9). Images are taken by keeping a constant frequency shift during scanning, and usu- ally this is performed by monitoring the amplitude of the cantile- ver oscillation at a fixed frequency and feeding the corresponding value to the feedback loop exactly as for the DC modes. 3. AC Modes 3.1. Noncontact Mode Fig. 4. Using a four-section photodetector, it is possible to measure also the torsion of the cantilever during contact mode AFM scanning. The torsion of the cantilever reflects changes in the surface chemical composition.
- 43. 24 Torre et al. The tip–sample interactions are very small in noncontact mode, and good vertical resolutions can be achieved, whereas lateral resolution is lower than in other operating modes. The greatest drawback is that it cannot be used in liquid environment, but only on dry samples. Also, even on dry samples, if a thick con- tamination or water layer is present, as the oscillation amplitude is small, the tip can sometimes get trapped as it does not have sufficient energy to detach from the sample. The general scheme is similar to that of noncontact mode, but in this case, during oscillation, the tip is brought into contact with the sample surface causing a dampening of the oscillation ampli- tude by the same repulsive forces that are present in contact mode (Fig. 6). Usually, in the intermittent contact mode, the cantilever oscillation amplitude is larger than the one used for noncontact. There are several advantages that have made this mode of opera- tion quite popular. The vertical resolution is very good together with the lateral resolution, there are less interactions with the sample compared to contact mode (especially lateral forces are greatly reduced), and it can be used in liquid environment (10–14). This mode of operation is the most generally used for imaging biological samples and is still under constant improvement, thanks to additional features, such as Q-control (12) or magnetically driven tips (7, 8). 3.2. Intermittent Contact Mode Fig. 5. In the noncontact operation mode, a vibrating tip is brought near the sample surface, sensing the attractive forces. This induces a frequency shift in the resonance peak of the cantilever that is then used to operate the feedback.
- 44. 25 Measurement Methods in Atomic Force Microscopy If the phase lag of the cantilever oscillation relative to the driving signal is recorded in a second acquisition channel dur- ing imaging in intermittent contact mode, noteworthy infor- mation on local properties that are not revealed by other AFM techniques (15), such as stiffness, viscosity, and adhesion, can be detected. In fact, it is good practice to always acquire both the amplitude and phase signals simultaneously during inter- mittent contact operation, as the physical information is entwined and all the data are necessary to interpret the images obtained (16–20). In this case, a low-frequency oscillation is induced (usually to the sample) and the corresponding cantilever deflection recorded while the tip is kept in contact with the sample (Fig. 6). The varying stiffness of surface features induces a corresponding dampening of the cantilever oscillation so that local relative vis- coelastic properties can be imaged. 3.3. Phase Imaging Mode 3.4. Force Modulation Fig. 6. In intermittent contact mode, the free oscillation of a vibrating cantilever is dampened when the tip touches the sample surface at each cycle. The image is performed keeping constant the oscillation amplitude decrease while scanning.
- 45. 26 Torre et al. The AFM can provide much more information than simply taking images of the surface of the sample. The instrument can be used to record the amount of force felt by the cantilever as the probe tip is brought close to a sample surface, eventually indent the surface and then pulled away. By doing this, the long-range attrac- tive or repulsive forces between the probe tip and the sample sur- face can be studied, local chemical and mechanical properties like adhesion and elasticity may be investigated, and even the bonding forces between molecules may be directly measured (21–23). By acquiring a series of force curves, one at each point of a square grid, it is possible to acquire the so-called force vs. volume map that allows the user to compute images representing local mechan- ical properties of the sample observed. Force curves typically show the deflection of the cantilever, as the probe is brought vertically toward and then away from the sample surface using the vertical motion of the scanner driven by a triangular wave (Fig. 7). By controlling the amplitude and fre- quency of the vertical movement of the scanner, it is possible to change the distance and speed that the AFM probe travels during the force measurement. Conceptually, what happens during a 4. Beyond Topography Using Force Curves Fig. 7. From positions A to B, the tip is approaching the surface, and at position B contact is made (if an attractive or repulsive force is active before contact, the portion of the force curve will reflect it).After position B, the cantilever bends until it reaches the specified force limit that is to be applied (S). Depending on the relative stiffness of the cantilever with respect to the sample, during this portion of the curve the tip can indent the surface. The tip is then withdrawn toward positions C and D.At position D under application of the retraction force, the tip detaches from the sample (often referred to as “snap off”). Between positions D and A, the cantilever returns to its resting position and is ready for another measurement.
- 46. 27 Measurement Methods in Atomic Force Microscopy force curve is not much different from what happens between tip and sample during intermittent contact imaging. The differences are in the frequency used, much lower for force curves, and the probe, much smaller in intermittent contact. In a force curve, many data points are acquired during the motion so that very small forces can be detected and interpreted by fitting the force curve according to theoretical models. In order to obtain quantitative data from force vs. distance curves, two technical details need special care. The position- sensitive photodetector signal has to be calibrated so to measure accurately the cantilever deflection, and after calibration it is essential that the laser alignment is left unchanged. Usually, the AFM software has a routine for such calibration, performed by taking a force curve on a hard sample and using the scanner’s vertical movement as reference (which means that the scanner also has to be accurately calibrated). At this point, the curve we are plotting is not yet a force curve but a calibrated deflection curve. The next step is to convert it to a force curve using the force constant of the cantilever we are using. Manufacturers usu- ally specify this value, but for each cantilever there can be quite large variations so that for accurate work direct determination becomes necessary. There are different ways to measure the force constant, some requiring external equipment for measuring reso- nant frequency (such as spectrum analyzers) and others making use of reference cantilevers (24, 25). From the point of view of biomedical applications, interesting experiments can be performed by coating the tip with a ligand and approaching through a force curve a surface where receptor molecules can be found. In this case, the portion of the curve before snap-off has a different shape, reflecting the elongation of the bond between ligand and receptor before dissociation: from the shape the curve, it is possible to derive quantitative informa- tion on the binding forces (26–28). If a force curve is taken at each point of an N×N grid, it is possible derive images that are directly correlated to a physical property of the surface of the sample. For example, if the approach portion of each curve after contact is fitted using indentation the- ory, a map of the sample stiffness can be calculated. This data can be represented by an image in which the level of gray of each pixel, instead of representing the height of the sample, corre- sponds to the elasticity modulus. Similar images can be calculated for adhesion, binding, electrostatic forces, and so forth (29, 30). If the same operation is done while dithering the cantilever close to its resonance frequency, tip–sample interaction is probed in dynamic mode (dynamic force spectroscopy), and several para meters can be measured as a function of distance (such as static deflection, amplitude, phase, higher harmonics, frequency, etc.) 4.1. State of the Art and Future Challenges: Dynamic Spectroscopy
- 47. 28 Torre et al. containing a larger amount of data: valuable information about local interactions can be extracted or reconstructed, revealing material properties such as short- and long-range forces (31), fric- tion (32), plasticity (33), chemical composition (34), and so on. Quantitative interpretation of all the interactions at the base of spectroscopy data is still under development and involve the inversion of dynamic parameters to reconstruct interaction forces: historically, they have been first inverted to reconstruct tip– sample interaction forces in the case of FM–AFM by Durig using Hamilton–Jacobi perturbation theory in the large amplitude oscillation – or short range forces – case (35), and then general- ized by Sader et al. (36, 37) considering first resonance. Durig also investigated dynamic behavior by considering amplitude and phase of higher harmonics, using the Chebyshev polynomial expansion method (38). Under particular oscillating regimes, also subharmonic and chaotic cantilever dynamics, fingerprints of tip–sample interac- tions have been found (39). Each of these advanced spectroscopy methods imply a huge amount of data that require very high computational power to reconstruct physically valuable parameters from comparison with contact models (40); as a result, a fast and easy analysis relying on these dynamic methods is still far to be routinely implemented for spectroscopy maps or it is limited to a subset of information. References 1. Israelachvili, J. N. (1985) Intermolecular and Surface Forces. Academic Press, London. 2. Weisenhorn A. L., Maivald, P., Butt, H. J., and Hansma, P. K. (1992) Measuring adhesion, attraction, and repulsion between surfaces in liquids with an atomic-force microscope. Phys. Rev. B. 45, 11,226–11,232. 3. Weisenhorn A.L., Hansma, P. K., Albrecht T. R., and Quate, C. F. (1989) Forces in atomic force microscopy in air and water. Appl. Phys. Lett. 54, 2651–2653. 4. Butt, H.-J., Siedle, P., Seifert, K., et al. (1993) Scan speed limit in atomic force microscopy. J. Microsc. 169, 75–84. 5. Putman, C. A., van der Werf, K. O., de Grooth, B. G., van Hulst, N. F., and Greve, J. (1992) New imaging mode in atomic-force microscopy based on the error signal. SPIE Proceedings 1639, 198–204. 6. Gibson, C. T., Watson, G. S., and Myhra, S. (1997) Lateral force microscopy–a quantita- tive approach. Wear 213, 72–79. 7. Han, W. and Lindsay, S. M. (1998) Precision interfacial molecular force measurements with a MAC mode atomic force microscope. Appl. Phys. Lett. 72, 1656–1658. 8. Han, W., Lindsay, S. M., and Jing, T. (1996) A magnetically-driven oscillating probe micro- scope for operation in liquids. Appl. Phys. Lett. 69, 4111–4113. 9. Garcia, R. and San Paulo, A. (2000) Amplitude curves and operating regimes in dynamic atomic force microscopy. Ultramicroscopy 82, 79–83. 10. Hansma, P. K., Cleveland, J. P., Radmacher, M., et al. (1994) Tapping mode atomic force microscopy in liquids. Appl. Phys. Lett. 64, 1738–1740. 11. Lantz, M., Liu, Y. Z., Cui, X. D., Tokumoto, H., and Lindsay, S. M. (1999) Dynamic force microscopy in fluid. Surface Interface Anal. 27, 354–360. 12. Tamayo, J., Humphris, A. D., Owen, R. J., and Miles, M. J. (2001) High-Q dynamic force microscopy in liquid and its application to living cells. Biophys. J. 81, 526–537. 13. Burnham, N. A., Behrend, O. P., Oulevey, F., et al. (1997) How does a tip tap? Nano technology 8, 67–75.
- 48. 29 Measurement Methods in Atomic Force Microscopy 14. Behrend, O. P., Oulevey, F., Gourdon, D., et al. (1998) Intermittent contact: Tapping or hammering? Appl. Phys. A66, S219–S221. 15. Magonov, S. N., Elings, V., and Whangbo, M.-H. (1997) Phase imaging and stiffness in tapping mode AFM. Surface Sci. 375, L385–L391. 16. Bar, G., Delineau, L., Brandsch, R., Bruch, M., and Whangbo, M.-H. (1999) Importance of the indentation depth in tapping-mode atomic force microscopy study of compliant materials. Appl. Phys. Lett. 75, 4198–4200. 17. Bar, G. and Brandsch, R. (1998) Effect of vis- coelastic properties of polymers on the phase shift in tapping mode atomic force micros- copy. Langmuir. 14, 7343–7347. 18. Cleveland, J. P., Anczykowski, B., Schmid, A. E., and Elings, V. B. (1998) Energy dissipa- tion in tappingmode atomic force microscopy. Appl. Phys. Lett. 72, 2613–2615. 19. Chen, X., Davies, M. C., Roberts, C. J., Tendler, S. J. B., and Williams, P. M. (2000) Optimizing phase imaging via dynamic force curves. Surface Sci 460, 292–300. 20. Pang, G. K., Baba-Kishi, K. Z., and Patel, A. (2000) Topographic and phase-contrast imag- inginatomicforcemicroscopy.Ultramicroscopy 81(2), 35–40. 21. Butt, H-J. (1991) Measuring electrostatic, van der Waals, and hydration forces in electro- lyte solutions with an atomic force micro- scope. Biophys. J. 60, 1438–1444. 22. Vinckier, A. and Semenza, G. (1998) Measuring elasticity of biological materials by atomic force microscopy. FEBS Lett. 430, 12–16. 23. Hutter Jeffrey L. and John Bechhoefer (1994) Measurement and manipulation of Van der Waals forces in atomic force microscopy. J. Vacuum Sci. Technol. B, 12, 2251–2253. 24. Cleveland, J. P., Manne, S., Bocek, D., and Hansma, P. K. (1993) A non-destructive method for determining the spring constant of cantilevers for scanning force microscopy. Rev. Sci. Instrum. 64, 403–405. 25. D’Costa, N. P. and Hoh, J. H. (1995) Calibration of optical lever sensitivity for atomic force microscopy. Rev. Sci. Instrum. 66, 5096–5097. 26. Hoh, I., Cleveland, J. P., Prater, C. B., Revel, J.-P., and Hansma, P. K. (1992) Quantized adhesion detected with the atomic force microscope. J. Am. Chem. Soc. 4917–4918. 27. Mckendry, R. A., Theoclitou, M., Rayment, T., and Abell, C. (1998) Chiral discrimination by chemical force microscopy. Nature 14, 2846–2849. 28. Okabe, Y., Furugori, M., Tani, Y., Akiba, U., and Fujihira, M. (2000) Chemical force microscopy of microcontact-printed self- assembled monolayers by pulsedforce-mode atomic force microscopy. Ultramicroscopy 82, 203–212. 29. Willemsen, O. H., Snel, M. M., van Noort, S. J., et al. (1999) Optimization of adhesion mode atomic force microscopy resolves indi- vidual molecules in topography and adhesion. Ultramicroscopy 80, 133–144. 30. Thundat, T., Oden, P. I., and Warmack, R. J. (1997) Chemical, physical, and biological detection using microcantilevers. Electrochem. Society Proc. 97, 179–187. 31. F.J. Giessibl.1997.Forces and frequency shifts in atomic-resolution dynamic force micros- copy” Phys. Rev. B 56: 16010–16015. 32. Holscher H, Schwarz U D, Zworner O and Wiesendanger R.1998.Consequences of the stick-slip movement for the scanning force microscopy imaging of graphite. Phys. Rev. B 57: 2477–81. 33. Butt HJ, Cappella B and Kappl M. 2005. Force measurements with the atomic force microscope: Technique, interpretation and applications. Surf. Sci. Rep. 59:1–152. 34. Magonov S N, Elings V and Papkov V S. 1997. AFM study of thermotropic structural transitions in poly(diethylsiloxane). Polymer 38: 297–307. 35. Durig U. 2000. Extracting interaction forces and complementary observables in dynamic probe microscopy. Appl. Phys. Lett. 76: 1203–1205. 36. Sader J E and Jarvis S P.2004. Accurate for- mulas for interaction force and energy in fre- quency modulation force spectroscopy. Appl. Phys. Lett. 84:1801–1803. 37. Sader J E, Uchihashi T, Higgins MJ, Farrell A, Nakayama Y and Jarvis S P. 2005. Quantitative force measurements using frequency modula- tion atomic force microscopy—theoretical foundations. Nanotechnology 16:S94–S101. 38. Durig U. 1999. Relations between interaction force and frequency shift in large-amplitude dynamic force microscopy. Appl. Phys. Lett. 75:433–435. 39. F Jamitzky, M Stark, W Bunk, WM Heckl and R W Stark, 2006. Chaos in dynamic atomic force microscopy.Nanotechnology 17: S213–S220. 40. W. N. Unertl “Implications of contact mechanics models for mechanical properties measurements using scanning force micros- copy”, J. Vac. Sci. Technol. A 17, 1779 (Jul/ Aug 1999).
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- 50. 31 Pier Carlo Braga and Davide Ricci (eds.), Atomic Force Microscopy in Biomedical Research: Methods and Protocols, Methods in Molecular Biology, vol. 736, DOI 10.1007/978-1-61779-105-5_3, © Springer Science+Business Media, LLC 2011 Chapter 3 Recognizing and Avoiding Artifacts in Atomic Force Microscopy Imaging Claudio Canale, Bruno Torre, Davide Ricci, and Pier Carlo Braga Abstract Atomic force microscopy (AFM) measurements could be affected by different kinds of artifacts; some of them derive from the improper use of the instrument and can be avoided by setting the correct experi- mental parameters and conditions. In other cases, distortions of the images acquired by AFM are intrinsi- cally related to the operating principle of the instrument itself and to the kind of interactions taken into account for the reconstruction of the sample topography. A perfect knowledge of all the artifacts that can perturb AFM measurements is fundamental to avoid misleading interpretations of the results. In this chapter, all the most common sources of artifact are presented, and strategies to avoid them are proposed. Subheading 1 is a brief introduction to the chapter. In Subheading 2, the artifacts due to the interac- tions between the sample and the AFM tip are presented. Subheading 3 is focused on the deformations due to the AFM scanner nonlinear movements. The interaction with the environment surrounding the instrument can affect the quality of the AFM results and the environmental instability are discussed in Subheading 4. Subheading 5 shows the effects of an incorrect setting of the feedback gains or other parameters. Subheading 6 aims on the artifacts that can be produced by the improper use of the image processing software. Subheading 7 is a short guide on the test that can be done to easily recognize some of the artifacts previously described. Key words: AFM, Tip artifacts, Nonlinearity, Instability, Creep Images and other information obtained by using atomic force microscopy (AFM) are derived from the physical interaction between the AFM probe and the sample. The different working principles of the SPMs with respect to the conventional micro- scopes are responsible for a new series of artifacts that affect 1. Introduction
- 51. 32 Canale et al. images and are not easily recognizable by inexpert users. Since we are addressing novices in this field, we would like to give an idea of what can happen while taking images with the AFM, how one can recognize the source of the artifact, and then try to avoid it or minimize it. Sources of artifacts in AFM images are essentially the tip, the scanner, the environment, the control system elec- tronics, and the image-processing software. The AFM tip plays a fundamental role in the generation of the AFM image: it explores the sample surface while the cantilever bends under the action of the complex force field established between the sample and the tip itself. The geometrical shape of the tip always affects the AFM images acquired using it. The images result as the convolution between the sample and the tip shape; intuitively, as long as the tip is sharper than the feature under observation, the profile resembles closely the true shape of the sample. The choice of the optimal probe is important to minimize the artifacts due to tips: the smaller the size of the object, the sharper the tip. A notable exception arises in the case of high-resolution imaging on ordered crystals, where often better images are obtained with standard tips. This can be explained by realizing that at this dimensional scale the measurable radius of curvature of the tip is not in fact involved in the imaging process, but instead smaller local protrusions on the apex of the probe perform as the real tip (or tips) effectively taking the image. Further details on AFM tip properties and related artifacts can be gathered from the vast literature on the subject, together with a variety of methods for their correction (1–9). Specific arti- facts, depending on the mode of operation, have been investi- gated and explanations have been proposed (10–14). Since we are now interested in showing a general overview of the subject for beginners in the field, we shall have a look at the main tip artifacts in a very simple way. Different profiles were obtained using a dull or a sharp tip (Fig. 1). Depending on the lateral size and height of the feature to be imaged, both the sharpness of the apex and the sidewall angle of the tip become important. In general, when scanning rough sam- ples, the tall features are displayed as a mirror image of the tip sidewall (Fig. 2). In addition to sharpness, the geometrical shape of the tip also is important, as the level of broadening in a particu- lar direction depends on the tip geometrical symmetry. In par- ticular, a conical tip affect the lateral size of the sample features 2. Tip Artifacts 2.1. Tip Broadening Effect on Protruding Features
- 52. 33 Recognizing and Avoiding Artifacts in Atomic Force Microscopy Imaging symmetrically in all the directions while the level of broadening due to a pyramidal tip is dependent on the scan angle; the dis- tance between two faces of the pyramid is significantly smaller with respect to the distance between two opposite edges. Very small features, such as nanoparticles, nanotubes, proteins, and DNA strands, ideally interact only with the tip apex; therefore, the images result as the convolution between the sample features and the hemisphere approximating the apex of the tip. Due to tip image broadening, the measured lateral size should be taken as an upper limit for the true size of the objects imaged by AFM. Note that in all these cases, the measured height of the sample is reported accurately. Fig. 1. Line profiles obtained using two tips with different aspect ratio. The shape of the object is better approximated using tip with a sharper profile. In spite of this, tip broad- ening ever affects AFM images. Fig. 2. Three-dimensional view obtained from an AFM image showing part of a neuron. In particular, the soma (the taller part of the sample) is strongly affected by the tip and it appears as a mirror image of the tip sidewall itself, while the neuritis structure, although broadened by the tip, is clearly displayed.
- 53. 34 Canale et al. Multiple protrusions at the AFM tip apex can be present as a result of damage or contamination. Due to the interaction of the multiple tip apexes with the sample features, repetitive patterns may appear in an image (Fig. 3). Images affected by this artifact are often called “double image”; actually, sample particles can be replicated several times in the AFM image, depending on the number of apical protrusion interacting with the sample surface. Furthermore, spherical nanoparticles or small molecules may assume an elongated or triangular shape, reflecting an asymmetri- cal geometry of the apex of the tip (Fig. 3). The finite size of the tip has an effect also in the visualization of features that are below the surface mean level, such as a hole. The lateral size of small holes at the sample surface is underestimated. Furthermore, the tip may not be able to reach the bottom of a hole, resulting on a lack of physical depth in the AFM image. AFM scanners are made of piezoelectric ceramic, a material that undergoes a change of its shape under the effect of an applied voltage. Piezoelectric scanners can provide subnanometric posi- tioning of a probe, and they have been one of the breakthroughs that made AFM possible. In spite of this, a number of artifacts arise from their physical and mechanical properties, even though their design has been constantly improved and some of the 2.2. Tip-Induced Deformations and “Double Image” 2.3. Flattening of Pits and Holes 3. Scanner Artifacts Fig. 3. Different artifacts arise from the use of deformed tips. Single amyloid fibrils are displayed as two separate adjacent structures due to the use of a “double tip” (a). Data scale 3×3 mm2 ; Z-range 12 nm. The asymmetrical geometry of a contaminated tip affected the shape of globular protein aggregates: all the features on the mica substrate displayed a similar elongated shape (b). Data scale 2×2 mm2 , Z-range 13 nm.
- 54. 35 Recognizing and Avoiding Artifacts in Atomic Force Microscopy Imaging artifacts have been removed, or at least minimized, in the newest instruments. One point that must not be neglected is that scanner proper- ties change with time and use. In fact, the piezoelectric material changes its sensitivity to driving signals. If it is used often, it will become slightly more sensitive; if left idle, it will depolarize and become less sensitive. The best thing to do is to periodically cali- brate the scanner according the manufacturer’s instructions. Piezoelectric scanners are inherently nonlinear: if the extension of the scanner in any one direction is plotted as a function of the driving signal, the plot will not be a straight line but it will appear a curve similar to the one shown in Fig. 4. The nonlinear relation- ship between the applied voltage and the displacement of a piezo- actuator contributes to positioning error (15). Nonlinear effects are more pronounced for large scans while they can be neglected for small scans. In this case, we refer to large scans when they are more than 70% of the full scale displacement of the piezoelectric scanner. The nonlinearity may be expressed as a percentage (describing the deviation from linear behavior), and it typically ranges from 2 to 25%, depending on the driving signal applied and the scanner construction. The effects are present both in the plane and in the vertical directions. An AFM image of a calibration grid with periodic structures, such as squares, appears severely distorted, with inhomogeneous spacing and anomalous curvature of features, typically appearing smaller on one side of the image than on the other (Fig. 5). 3.1. Effects of Intrinsic Nonlinearity Fig. 4. Plot of the scanner extension vs. driving signal. Notice the large deviation from linearity.
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