Contents

#### **Preface XI**

#### **Part 1 General Techniques 1**

	- Z. Remeš and B. Rezek
	- **Part 2 Biological Molecules, Proteins and Polymers 85**
	- **Part 3 DNA, Chromatin and Membranes 175**

#### **Part 4 Viral Physiology 233**


#### **Part 5 Cellular Physiology 279**


## Preface

Atomic force microscopy, AFM, is a modern technique for generating high resolution surface topography images and can image many orders of magnitude below the optical diffraction limit. It uses a principal similar to the one used by the phonograph developed by Thomas Alva Edison. Essentially, the phonograph has a sharp object which is dragged across a moving surface, and the tip is deformed by the features it encounters. The AFM uses this principal as well, with the AFM tip generating a physical deflection in the cantilever according to Hooke's law, but unlike the phonograph, the AFM can capitalize on a wider variety of forces generated between the sharp tip and the scanned surface. The AFM cantilever deflection is quantified through the use of a laser reflected off of the back of the cantilever onto an array of photodiodes. Of course, as was seen with Edison's original invention, scanning an object at a constant height presents the danger of a collision with the surface. With this problem in mind, the AFM cantilever is mounted onto a piezoelectric column commonly called a head stage, which moves the cantilever to maintain constant force according to feedback from the measured deflection. The combination of all of these components can be recorded and produces a topographical image of the surface with nanoscale resolution. This measurement technique has also been used to develop a method of force spectroscopy which measures the force between the tip and the sample as a function of distance. However, this technique is not the only measurement these devices can produce.

The AFM is not limited to only one operating mode, but has a second distinctive mode, which itself has been developed into another mode of operation. The previous mode we discussed is known as contact or static mode, and the next mode we will briefly introduce is called non-contact, or dynamic, mode. In this mode, the tip is oscillated near its natural resonance, and brought close to the surface. As the tip approaches the surface, the interaction between the surface and tip forces generates a change in the natural resonance of the cantilever. The feedback circuit is used to reestablish the original oscillation set point by changing the distance between the sample and the tip. The difference that the cantilever moves can be recorded and compiled to produce topographical images. Oscillation differences can be detected as a function of amplitude or frequency. Detections of changes in the frequency of oscillation produce very high-resolution measurements. Through the measurement of changes in amplitude, non-contact mode becomes the third main mode, known as

#### XII Preface

tapping or intermittent mode. In tapping mode, the physical distance of the amplitude of oscillation is large enough to produce brief contact with the surface. This contact leads to changes the amplitude which is measured by the feedback circuit. The head stage moves the cantilever to maintain constant amplitude, and once again can be used to generate topographical images. Changes in the phase of oscillations can also be measured in this mode and is useful in detecting differences in surface friction or between different materials.

Preface XI

After redesigning the control system for Professor Saddow's SiC reactor, I was teamed with one of his graduate students, Dr. Camilla Coletti, who was studying the surface effects of hydrogen etching on SiC in collaboration with Dr Ulrich Starke of the Max-Planck-Institut für Festkörperforschung in Stuttgart, Germany. Thanks to the graciousness of both Professor Saddow and Dr. Starke, I was able to study at the Institut for a month so that I could gather some data for Dr. Coletti. Here it was that I had my first "hands on" exposure to AFM. It was an experience I will never forget as we immediately got off the plane, drove to the institute, and after introductions to Dr. Starke's group, Prof. Saddow trained me on how to operate the AFM. In a laboratory where there were many complicated, large, ultra-high vacuum instruments, like timeof-flight secondary ion mass spectrometry (ToF-SIMS) and scanning tunneling microscopy (STM) systems, the small, blue can-like system sitting on a floating table seemed almost out of place and ineffectual for surface science. However, I was amazed to find that such a simple machine could be used to examine so many different properties. I imaged the surfaces of the SiC materials we had hydrogen etched in the CVD reactor back in Florida, and Dr. Colletti was able to answer some issues she had

I found that AFM was an invaluable tool in not only examining etched SiC surfaces, but also in the development of an improved heteroepitaxial growth process for cubic silicon carbide (3C-SiC) on silicon substrates, as many of the defects at the heteroepitaxial interface are transferred to the 3C-SiC film surface. AFM proved to be a fast and efficient method, especially when compared to instruments like transmission electron microscopy (TEM) and X-ray Diffraction (XRD), to perform quick analysis of the crystal film so one could determine what growth parameters needed to be adjusted for the reduction of defects. AFM reduced the amount of empirical experimentation involved in SiC heteroepitaxial growth, and reduced the analysis workload by allowing us to only analyze our best materials using more time and preparation

Of course, this is not a book about the use of AFM in materials science, as it is a mainstay in that field, but about the use of AFM in applications within biological sciences. Personally, the experience once again began with Professor Saddow, who saw the potential of SiC for use in medical technology. His group began in earnest to examine SiC biocompatibility using the AFM, starting with Dr. Coletti, continuing with myself, and now with Alexandra Oliveros, a current PhD candidate in his group, to this day. Dr. Saddow purchased a Park Systems XE-100 AFM system because it not only could examine dry materials, but with a removable liquid cell would allow us to examine the interaction of living cells on our materials. During my early graduate experience, I was exposed to many biological science techniques through Dr. Coletti. Her methods included chemical assays to examine cellular proliferation, fluorescent microscopy to examine cellular morphology, and AFM to examine fixed cells on our novel materials. During my Ph.D., I came across a difficulty that stemmed from many optical microscopes used for biology. Although 3C-SiC is translucent, our films are on silicon, which is opaque. The fluorescent optical microscopes I had available to me at

concerning the etching process.

intensive methods, like TEM and XRD.

The AFM is a very dynamic measurement tool and provides many methods to quantitatively measure a wide range of physical, electromagnetic, and atomic forces. However, the one aspect of this device is that puts it in a class by itself among nanoscale microscopy is the fact that AFM can be used in almost any environment. Vacuum, air, and liquid are not a barrier for this measurement style, and because of this, the AFM lends itself very well for biological investigations where environmental factors can influence biological reactions. This book was developed to showcase the growing use of AFM techniques and methodologies in the investigation of many different aspects of biological and medical sciences. Another, less known advantage for the use of biological AFM is it can be used to investigate structures as large as a whole cell down to the very proteins which constitute the cells themselves. Another advantage is this measurement technique does not require complicated, invasive, and often permanent sample preparation like that required for other microscopy techniques.

Personally, I was unknowledgeable about this fantastic device until I was almost a senior in college. Even further from my expectations is that I never thought I would have used AFM as a tool to investigate biology, specifically neurological cells and their properties. After taking undergraduate physics, and influenced greatly by my childhood idol Nikola Tesla, I wanted to explore the mysteries of electricity and magnetism, so I chose electrical engineering. As with most students, I entered college with preconceptions as to what I would be exposed to within this field, but I found that my expectations were not in line with reality. I thought that I would learn about electrical circuits, power transfer, and communications and then go out into the world and work in some job designing these things, but in the beginning of my junior year, I was fortunate to be able to join, Professor Stephen E. Saddow, in his silicon carbide (SiC) laboratory. Little did I know it at the time, but Dr. Saddow would become my Ph.D. mentor. At first, like many undergraduates, I was just amazed to be in a real research laboratory, but became slightly disillusioned with research due to my preconceptions. My new lab was focused on materials research, which seemed more like chemistry and only seemed connected to electrical engineering due to the fact that crystalline SiC is a semiconductor. As time moved on, I slowly began to understand the importance of this research as educational maturity set in. Finally I was able to look past the chemistry and see a material which could withstand almost every base or acid compound, conduct large amounts of electricity, dissipate heat as well as copper, and even emit visible light from yellow to blue. I began to become intrigued with the possibilities, and wanted to build electrical devices with this wonderful material.

After redesigning the control system for Professor Saddow's SiC reactor, I was teamed with one of his graduate students, Dr. Camilla Coletti, who was studying the surface effects of hydrogen etching on SiC in collaboration with Dr Ulrich Starke of the Max-Planck-Institut für Festkörperforschung in Stuttgart, Germany. Thanks to the graciousness of both Professor Saddow and Dr. Starke, I was able to study at the Institut for a month so that I could gather some data for Dr. Coletti. Here it was that I had my first "hands on" exposure to AFM. It was an experience I will never forget as we immediately got off the plane, drove to the institute, and after introductions to Dr. Starke's group, Prof. Saddow trained me on how to operate the AFM. In a laboratory where there were many complicated, large, ultra-high vacuum instruments, like timeof-flight secondary ion mass spectrometry (ToF-SIMS) and scanning tunneling microscopy (STM) systems, the small, blue can-like system sitting on a floating table seemed almost out of place and ineffectual for surface science. However, I was amazed to find that such a simple machine could be used to examine so many different properties. I imaged the surfaces of the SiC materials we had hydrogen etched in the CVD reactor back in Florida, and Dr. Colletti was able to answer some issues she had concerning the etching process.

X Preface

between different materials.

techniques.

tapping or intermittent mode. In tapping mode, the physical distance of the amplitude of oscillation is large enough to produce brief contact with the surface. This contact leads to changes the amplitude which is measured by the feedback circuit. The head stage moves the cantilever to maintain constant amplitude, and once again can be used to generate topographical images. Changes in the phase of oscillations can also be measured in this mode and is useful in detecting differences in surface friction or

The AFM is a very dynamic measurement tool and provides many methods to quantitatively measure a wide range of physical, electromagnetic, and atomic forces. However, the one aspect of this device is that puts it in a class by itself among nanoscale microscopy is the fact that AFM can be used in almost any environment. Vacuum, air, and liquid are not a barrier for this measurement style, and because of this, the AFM lends itself very well for biological investigations where environmental factors can influence biological reactions. This book was developed to showcase the growing use of AFM techniques and methodologies in the investigation of many different aspects of biological and medical sciences. Another, less known advantage for the use of biological AFM is it can be used to investigate structures as large as a whole cell down to the very proteins which constitute the cells themselves. Another advantage is this measurement technique does not require complicated, invasive, and often permanent sample preparation like that required for other microscopy

Personally, I was unknowledgeable about this fantastic device until I was almost a senior in college. Even further from my expectations is that I never thought I would have used AFM as a tool to investigate biology, specifically neurological cells and their properties. After taking undergraduate physics, and influenced greatly by my childhood idol Nikola Tesla, I wanted to explore the mysteries of electricity and magnetism, so I chose electrical engineering. As with most students, I entered college with preconceptions as to what I would be exposed to within this field, but I found that my expectations were not in line with reality. I thought that I would learn about electrical circuits, power transfer, and communications and then go out into the world and work in some job designing these things, but in the beginning of my junior year, I was fortunate to be able to join, Professor Stephen E. Saddow, in his silicon carbide (SiC) laboratory. Little did I know it at the time, but Dr. Saddow would become my Ph.D. mentor. At first, like many undergraduates, I was just amazed to be in a real research laboratory, but became slightly disillusioned with research due to my preconceptions. My new lab was focused on materials research, which seemed more like chemistry and only seemed connected to electrical engineering due to the fact that crystalline SiC is a semiconductor. As time moved on, I slowly began to understand the importance of this research as educational maturity set in. Finally I was able to look past the chemistry and see a material which could withstand almost every base or acid compound, conduct large amounts of electricity, dissipate heat as well as copper, and even emit visible light from yellow to blue. I began to become intrigued with the possibilities, and wanted to build electrical devices with this wonderful material.

I found that AFM was an invaluable tool in not only examining etched SiC surfaces, but also in the development of an improved heteroepitaxial growth process for cubic silicon carbide (3C-SiC) on silicon substrates, as many of the defects at the heteroepitaxial interface are transferred to the 3C-SiC film surface. AFM proved to be a fast and efficient method, especially when compared to instruments like transmission electron microscopy (TEM) and X-ray Diffraction (XRD), to perform quick analysis of the crystal film so one could determine what growth parameters needed to be adjusted for the reduction of defects. AFM reduced the amount of empirical experimentation involved in SiC heteroepitaxial growth, and reduced the analysis workload by allowing us to only analyze our best materials using more time and preparation intensive methods, like TEM and XRD.

Of course, this is not a book about the use of AFM in materials science, as it is a mainstay in that field, but about the use of AFM in applications within biological sciences. Personally, the experience once again began with Professor Saddow, who saw the potential of SiC for use in medical technology. His group began in earnest to examine SiC biocompatibility using the AFM, starting with Dr. Coletti, continuing with myself, and now with Alexandra Oliveros, a current PhD candidate in his group, to this day. Dr. Saddow purchased a Park Systems XE-100 AFM system because it not only could examine dry materials, but with a removable liquid cell would allow us to examine the interaction of living cells on our materials. During my early graduate experience, I was exposed to many biological science techniques through Dr. Coletti. Her methods included chemical assays to examine cellular proliferation, fluorescent microscopy to examine cellular morphology, and AFM to examine fixed cells on our novel materials. During my Ph.D., I came across a difficulty that stemmed from many optical microscopes used for biology. Although 3C-SiC is translucent, our films are on silicon, which is opaque. The fluorescent optical microscopes I had available to me at

#### XIV Preface

the time were all inverted; illuminating the sample from the base and collecting the transmitted light from optics placed above the sample, making live examination of the cells difficult at best. The AFM provided the ideal solution in that we could look at the interaction between our novel materials and living cell concurrently. I also found that AFM easily visualized the protrusions of many motile cells, the lamellipodia and filopodia, and we monitored changes in these protrusions to get an insight into surface permissiveness. AFM also was able to image in great detail paraformaldehyde fixed cells. The AFM is a crucial tool in the exploration of both materials properties as well as biological processes on novel materials in Professor Saddow's research. He is just one of many researchers the world over who are finding out that this tool can assist them with specific biological issues.

Preface XIII

various natural and artificial amyloid fibers, and provides as a bonus a detailed

The biological complexity increases within the third section of the book, which is dedicated to the AFM methodologies for examining deoxyribonucleic acid (DNA), chromatin, and biological membranes. Professor Yong Jiang and Yuan Yin have generated AFM methods to quantitatively identify and examine DNA, and substantiate their results using gel electrophoresis. The next chapter, by Dr. Yamini Dalal, *et al*., increases the level of complexity and examines chromatins and related DNA transcriptional pathways through functionalized AFM tips. This chapter also provides an interesting comparison between many traditional biological analytical methodologies. Dr. György Váró and Zsolt Szegletes detail in the final chapter of the section the process for generating artificial and natural lipid membranes to allow for the examination of individual membrane proteins, like bacteriorhodopsin, with AFM

The next section leaves the realm of the nanostructure and examines AFM for virology. Dr. Benjamín Pineda, *et al*., details virology basics and how AFM can be used to identify virus particles efficiently and quickly during the initial stages of an epidemic. The second chapter of this last section, by M.Sc. Luis Cuellar Jose, *et al*., uses the AFM in force spectroscopy mode to quantify and model viral shells and capsids as well as investigate viral receptor pathways through the functionalization of an AFM

The final section of the book focuses on cellular components and processes, either through examination of different parts of a cell as a whole, or using the AFM to measure a whole cell and its functionality. Although the first chapter of the section does not examine cells per se, it exemplifies the spirit of the section in that Dr. David J. Burritt, *et al*. are fractionalizing plant cell walls in order to study their structure and composite materials. Their development AFM force mapping simulation is very interesting and could be applied to study structures *in situ*. The next section, by Professor Stephen E. Saddow, *et al*., combines both principals of using the AFM to examine novel materials and whole cell AFM to investigate aspects of neural permissiveness on a substrate as well as the effects of self assembled monolayers on cellular behavior. Dr. Irina V. Ogneva and Igor B. Ushakov end the section and the book by demonstrating how the AFM can be used to quantitatively measure the material properties of transversal stiffness and Young's modulus in rat and gerbil

In closing I would like to give each of the authors and the people that assisted them in gathering the data and analysis for each of these chapters a great deal of thanks. These chapters show that there is not only a broad range of research that the AFM can be used for, but there are a lot of creative people who are moving the edge of science forward. The use of AFM in biology is clearly growing by leaps and bounds, as is seen by companies like Bruker and Park Systems making specific systems which combine

explanation of the method of using AFM as a force spectroscope.

techniques.

tip.

skeletal muscle fibers.

The book is divided into five sections. It begins with a section which gives an overview of the AFM, its measurement modes, and techniques associated with the device. This section would be a good starting point for readers unfamiliar with this type of microscopy, but it also contains a good deal of information on image artifacts and difficulties associated with the AFM. The next four sections are divided by the objects being investigated. The second section investigates measurements concerning the smallest biological particles, proteins and amino acid molecules, with each subsequent section growing in biological complexity. The book ends with a section dedicated to the examination of cells, starting with sections of cells, then whole cells, then matrices of several fibers from cells.

The first section, General Techniques, begins with an overview by Dr. Michele Giocondo, *et al*., which details AFM functional modes, the physics behind AFM functionality, and general examples of AFM applications for biological materials. This is followed by an excellent chapter by Dr. Egor Ukraintsev, *et al*. which details something that has plagued me personally, and I expect many others who have performed AFM at one time or another have seen as well: AFM artifacts. The final part of the section, by Dr. Nadine Mourougou-Candoni, not only examines the use of the tapping mode in liquid, but also gives an excellent assessment of the methods used to prepare biological materials for AFM investigation.

Section two of the book investigates small (10-9 m) particles which either are components for larger, more complex structures, or are engineered by man to interact or visualize biological processes at this scale. Prof. Loredana Latterini and L. Tarpani report their utilization of AFM to examine interactions between engineered and industrial particulates with proteins and DNA to gain a better understanding of toxicity related to the workplace. The second chapter, by Dr. Taiji Ikawa, details his process to capture biological molecules within a matrix of azobenzene-containing polymers isomirized through photo-illumination so they can be more easily examined with the AFM. Professor Borut Božič, *et al*. detail the increasingly complex stages of their *in vitro* study which models interactions between a phospholipid membrane, proteins, and antibody-antigen interactions and is measured through the use of AFM. The last chapter in the section, by Dr. Dong Mingdong, *et al*., details investigation of various natural and artificial amyloid fibers, and provides as a bonus a detailed explanation of the method of using AFM as a force spectroscope.

XII Preface

them with specific biological issues.

of several fibers from cells.

prepare biological materials for AFM investigation.

the time were all inverted; illuminating the sample from the base and collecting the transmitted light from optics placed above the sample, making live examination of the cells difficult at best. The AFM provided the ideal solution in that we could look at the interaction between our novel materials and living cell concurrently. I also found that AFM easily visualized the protrusions of many motile cells, the lamellipodia and filopodia, and we monitored changes in these protrusions to get an insight into surface permissiveness. AFM also was able to image in great detail paraformaldehyde fixed cells. The AFM is a crucial tool in the exploration of both materials properties as well as biological processes on novel materials in Professor Saddow's research. He is just one of many researchers the world over who are finding out that this tool can assist

The book is divided into five sections. It begins with a section which gives an overview of the AFM, its measurement modes, and techniques associated with the device. This section would be a good starting point for readers unfamiliar with this type of microscopy, but it also contains a good deal of information on image artifacts and difficulties associated with the AFM. The next four sections are divided by the objects being investigated. The second section investigates measurements concerning the smallest biological particles, proteins and amino acid molecules, with each subsequent section growing in biological complexity. The book ends with a section dedicated to the examination of cells, starting with sections of cells, then whole cells, then matrices

The first section, General Techniques, begins with an overview by Dr. Michele Giocondo, *et al*., which details AFM functional modes, the physics behind AFM functionality, and general examples of AFM applications for biological materials. This is followed by an excellent chapter by Dr. Egor Ukraintsev, *et al*. which details something that has plagued me personally, and I expect many others who have performed AFM at one time or another have seen as well: AFM artifacts. The final part of the section, by Dr. Nadine Mourougou-Candoni, not only examines the use of the tapping mode in liquid, but also gives an excellent assessment of the methods used to

Section two of the book investigates small (10-9 m) particles which either are components for larger, more complex structures, or are engineered by man to interact or visualize biological processes at this scale. Prof. Loredana Latterini and L. Tarpani report their utilization of AFM to examine interactions between engineered and industrial particulates with proteins and DNA to gain a better understanding of toxicity related to the workplace. The second chapter, by Dr. Taiji Ikawa, details his process to capture biological molecules within a matrix of azobenzene-containing polymers isomirized through photo-illumination so they can be more easily examined with the AFM. Professor Borut Božič, *et al*. detail the increasingly complex stages of their *in vitro* study which models interactions between a phospholipid membrane, proteins, and antibody-antigen interactions and is measured through the use of AFM. The last chapter in the section, by Dr. Dong Mingdong, *et al*., details investigation of

The biological complexity increases within the third section of the book, which is dedicated to the AFM methodologies for examining deoxyribonucleic acid (DNA), chromatin, and biological membranes. Professor Yong Jiang and Yuan Yin have generated AFM methods to quantitatively identify and examine DNA, and substantiate their results using gel electrophoresis. The next chapter, by Dr. Yamini Dalal, *et al*., increases the level of complexity and examines chromatins and related DNA transcriptional pathways through functionalized AFM tips. This chapter also provides an interesting comparison between many traditional biological analytical methodologies. Dr. György Váró and Zsolt Szegletes detail in the final chapter of the section the process for generating artificial and natural lipid membranes to allow for the examination of individual membrane proteins, like bacteriorhodopsin, with AFM techniques.

The next section leaves the realm of the nanostructure and examines AFM for virology. Dr. Benjamín Pineda, *et al*., details virology basics and how AFM can be used to identify virus particles efficiently and quickly during the initial stages of an epidemic. The second chapter of this last section, by M.Sc. Luis Cuellar Jose, *et al*., uses the AFM in force spectroscopy mode to quantify and model viral shells and capsids as well as investigate viral receptor pathways through the functionalization of an AFM tip.

The final section of the book focuses on cellular components and processes, either through examination of different parts of a cell as a whole, or using the AFM to measure a whole cell and its functionality. Although the first chapter of the section does not examine cells per se, it exemplifies the spirit of the section in that Dr. David J. Burritt, *et al*. are fractionalizing plant cell walls in order to study their structure and composite materials. Their development AFM force mapping simulation is very interesting and could be applied to study structures *in situ*. The next section, by Professor Stephen E. Saddow, *et al*., combines both principals of using the AFM to examine novel materials and whole cell AFM to investigate aspects of neural permissiveness on a substrate as well as the effects of self assembled monolayers on cellular behavior. Dr. Irina V. Ogneva and Igor B. Ushakov end the section and the book by demonstrating how the AFM can be used to quantitatively measure the material properties of transversal stiffness and Young's modulus in rat and gerbil skeletal muscle fibers.

In closing I would like to give each of the authors and the people that assisted them in gathering the data and analysis for each of these chapters a great deal of thanks. These chapters show that there is not only a broad range of research that the AFM can be used for, but there are a lot of creative people who are moving the edge of science forward. The use of AFM in biology is clearly growing by leaps and bounds, as is seen by companies like Bruker and Park Systems making specific systems which combine

#### XVI Preface

fluorescent or confocal microscopes with the power of AFM. The wide variety of the subjects in this book shows that in the next decade will have AFM increase in popularity and utility within biological research. It is my hope that many of the readers of this book who are unfamiliar with the AFM technique will see the utility of this measurement and will be able to use it to perform investigations of their own. Lastly, I hope that you the reader will find that this book is interesting and informative.

> **Christopher L. Frewin**  University of South Florida USA
