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The advent of microbeam analytical methods has opened up new possibilities of studies of biological systems at the cellular level. In situ techniques enable analysis of living cells in vitro and even in vivo. Electron microprobes and proton microprobes can be used for such studies, but thermal damage to the sample precludes their extensive use. Synchrotron radiation-based microbeams provide virtually an ideal tool for this area of study, offering sensitivity for detection of elements at the parts-per-million level comparable to proton microprobes, but without the adverse effect of sample damage. Although the information is limited to only elemental composition and not the actual compounds, SR also offers the additional advantage of enabling chemical state analysis at trace levels. In this chapter a number of applications of SR microbeam analysis at the cellular level will be presented to illustrate the power of the method. [ABSTRACT FROM AUTHOR]

When an X-ray beam passes through an absorbing medium several important processes take place, namely scattering, ionization, excitation, and the heating and breaking of molecular bonds. The four major processes are photo absorption, coherent scattering (Rayleigh scattering), incoherent scattering (Compton scattering), and pair production. Consequently, the initial beam is absorbed, reflected or transmitted through the matter. The absorption of energy, on the other hand, is directly related to excitation of atoms or molecules of the matter, and emission of characteristic X-rays from the irradiated material. [ABSTRACT FROM AUTHOR]

Synchrotron radiation (SR) was observed for the first time in April 1947 at General Electric in an advanced type of accelerator, an electron synchrotron [1]. While initially it was considered a nuisance, in the 1950s it became clear that the source of energy loss and annoyance for accelerator designers might become a very useful source of X-rays with potential applications in material science [2]. [ABSTRACT FROM AUTHOR]

A scanning capacitance microscope (SCM) combines an atomic force microscope (AFM) with a 1-GHz tuned inductance-capacitance-resistance (LCR) circuit to measure the capacitance between a conducting tip and sample. When applied to a semiconductor sample, an ac voltage at around 10 kHz is used to induce a depletion region within the semiconductor. The resulting differential capacitance is measured with a lock-in amplifier. The SCM contrast is proportional to this differential capacitance, which in turn is proportional to the inverse square root of the dopant concentration in the semiconductor beneath the tip. In this way dopant gradients in semiconductors, either natural or process induced, can be imaged. The differential capacitance measured by the SCM is also dependent on the properties of any native oxide or deposited dielectric film on the semiconductor surface, and the carrier mobility, which may be degraded near defects. This chapter will first review the history, principles, and modes of operation of the SCM. The remainder of the chapter will discuss major applications of scanning capacitance microscopy (SCM) for the electrical characterization of semiconductors and dielectric films, including qualitative characterization for integrated circuit failure analysis, quantitative dopant profiling and models for interpreting SCM images as dopant profiles, applications to semiconductors other than silicon, characterization of dielectric films, and optical pumping for carrier mobility measurements. [ABSTRACT FROM AUTHOR]

We define probe storage in a broad sense, which includes most of the mechanically addressing storage devices such as hard disk drives and optical disk drives. Its history is briefly discussed from the era of inscription, which leads to the application of scanning probe microscopy (SPM), to probe storage devices. Most of the current activities regarding the SPM based probe storage device are reviewed with special emphasis on resistive probe storage and related methods that use ferroelectric materials as information media. We present the principle of the read/write mechanism using the resistive probe accompanied by a servo/tracking concept. Such resistive probes can be implemented into probe storage devices with ferroelectric media development, which shows promise for the terabit era. [ABSTRACT FROM AUTHOR]

Scanning probes have been used to induce a variety of localized changes in the composition of self-assembled monolayers (SAMs). SAMs are useful in this regard because they offer a means to present various chemical functionalities on a surface in a relatively well organized way. This chapter reviews a diverse set of methodologies. However, they all can be generally characterized as either additive (adding molecules to the surface), subtractive (removing molecules from the surface), or exchange (a replacement of one type of molecule with another on the surface. The efficacy and utility of these processes will be emphasized. [ABSTRACT FROM AUTHOR]

The effects of domain orientation on the properties of ferroelectric surfaces are examined for single crystal BaTiO3 (100). Domain specific adsorption determined from surface potential variations suggests a mechanism for a new lithographic process. Three approaches (contact electrode, SPM, and e-beam) for patterning surface domains in the absence of a device metal electrode are explored. In particular the interaction of an electron beam with a ferroelectric surface is quantified and it is shown that it can cause both negative and positive surface charge depending on conditions. The domain specific reactivity and domain patterning are combined into a fabrication process that is demonstrated for several classes of nanostructures. [ABSTRACT FROM AUTHOR]

The ultrahigh vacuum scanning tunneling microscope (UHV-STM) offers intriguing opportunities to explore the integration of novel nanotechnologies with existing semiconductor platforms. This chapter describes the development of the atomic-resolution hydrogen resist technique and its application to the templated self-assembly of molecular systems on silicon. The observation of a giant isotope effect in STM hydrogen desorption experiments has led to the use of deuterium to retard hot-carrier degradation in CMOS transistor technology. We have also explored the integration of carbon nanotubes with silicon and the III-V compound semiconductors. This has been facilitated by the development of the dry contact transfer (DCT) technique that enables atomically clean nanotube/substrate systems to be achieved, even for highly reactive surfaces like atomically clean silicon. [ABSTRACT FROM AUTHOR]

Local oxidation of metal, semiconductor, and insulating surfaces by a scanning probe microscope (SPM) is a promising approach for nanoelectronics device prototyping. Voltage applied between a conductive SPM tip and (positively biased) substrate, results in the formation of a highly non-uniform electric field. The electric field attracts a stable water meniscus to the tip-sample junction, creates oxyanions from water molecules, and transports these oxyanions through the growing oxide film. This leads to oxidation of the substrate on a scale determined by the dimensions of the water meniscus. Experimental studies have shown that almost every material oxidizes under the extremely high electric-field conditions at the tip-substrate junction, including some noble metals, diamond, and nitrides of silicon, titanium, and zirconium. This chapter organizes fundamental relationships between oxide volume growth, current flowduring exposure, and the resulting electrical and structural properties of the oxide. The underlying transport and reaction kinetics within the electrochemical nanocell is presented in a self-consistent manner in terms of dispersive kinetic theory through time-dependent rate constants. Key signatures of scanning probe oxidation, extreme simplicity and flexibility, become apparent when local oxidation is combined with standard device processing techniques. Nanodevices fabricated using this method include superconducting quantum interference devices, single-electron tunneling transistors, and antidote lattices in a wide variety of materials, such as titanium, Nb/NbN, GaAs/AlGaAs 2-D electron gas, SrTiO3, and SiGe thin films. Examples of some of these devices will be reviewed in terms of the kinetic and materials insights revealed by fundamental studies. [ABSTRACT FROM AUTHOR]

Scanning probe microscopes (SPM) have been envisaged and applied from the beginning as tools both to image surfaces with unprecedented resolution and to interact with surfaces like an extension of the operator's fingertips. The prototype for electrical SPM certainly was the scanning tunneling microscope (STM). For the purpose of this chapter we do not consider mechanical nano-machining, i.e., scratching with the tunneling-tip, as a form of electrical SPM, despite the mechanical contact between tip and specimen surface being induced by low tunneling voltages and high current set-points. Applying a voltage of a few 10 V to the tip, McCord and Pease [1] succeeded in writing lines of contamination on bare silicon that protected the substrate during the subsequent etch. Line widths below 50 nm were achieved. Contamination lines were already observed before on metallic glass [2]. Later, Okawa and Aono [3] were able to induce the formation of polymeric nanowires on a graphite substrate covered by a monolayer of a diacetylene compound by applying voltage pulses to the STM tip. Positioning single xenon atoms on a nickel (110) surface at cryogenic temperatures, as demonstrated by Eigler [4], or removing a single atom from a MoS2 crystal to create, according to the Guinness World Records book, the smallest hole in the world, as shown by Heckl, mark the ultimate forms of nanofabrication possible with the STM. So far, however, most application- or device-directed nanofabrication by SPM takes place on a scale of molecules to several dozen nanometers. [ABSTRACT FROM AUTHOR]

Conducting and semiconducting organic materials have long been known [1], [2], but recent advances in chemical synthesis [3], [4] have enabled organic materials to begin delivering on the promise of mass-produced economical electronic devices. Organic electronic materials are better suited for constructing high-efficiency light-emitting diodes [5]-[8], solar cells [9], [10], and cheap solution-processable thin-film transistors [6], [11]-[18] than are crystalline inorganic semiconductors such as silicon and gallium arsenide. The electronic/optical properties and solubility of organic materials can be tuned independently by chemical synthesis [4]. Since they can be processed and patterned at ambient temperature, organic electronic materials are compatible with flexible large-area substrates [19]. [ABSTRACT FROM AUTHOR]

In 1994, individual molecules in alkanethiol self-assembled monolayers (SAMs) were imaged using scanning tunneling microscopy (STM). Since this breakthrough, the electric conduction of individual molecules has been estimated using scanning probe methods. In this chapter, we describe methods for evaluating the electric properties of conjugated molecules embedded in alkanethiol SAMs. First, the electrical conduction and barrier height measurements of SAMs and single molecules using STM are described. Here we discuss a method for estimating the molecular resistance based upon analysis of STM cross-sectional profiles. Secondly, the electrical conduction measurements of SAMs using conductive probe AFM (CP-AFM) are described. Finally, we describe related methods that enable applications like molecular devices. [ABSTRACT FROM AUTHOR]

Electrical measurements on perovskite materials were reported as early as 1930 by [1]. Since that time research has changed its focus from bulk ceramic and single crystals to thick and thin films. Within this trend to nanotechnology, the characterization techniques have changed to investigate the nanosized material properties. From the various methods that exist to characterize electrically perovskite materials, we concentrate in this chapter on the local electrical measurements of ferroelectric hysteresis, small signal capacitance, and local conductivity distributions. In these methods an atomic force microscope (AFM) tip is brought into contact with a nanosized top electrode or with the surface of the sample. Table 1 describes the interplay between the requirements for characterization on the nanoscale and the measurement limitations imposed by the setup or the recording system for different sample configurations and characterization methods. Exploring nanosized properties ranging over many orders of magnitude poses high demands on the measuring equipment. Current values from femto- to microamperes, deformations from pico- to micrometers, and potentials from hundreds of volts down to microvolts have to be measured. In order to investigate these properties, a thorough understanding of the measuring equipment is vital. Therefore, the principal setup of the measuring systems used is presented and their possibilities and drawbacks are discussed. Reliable results on small structures can only be obtained if parasitic effects are taken into account. In this chapter, we show ferroelectric as well as dielectric measurements on small capacitors. [ABSTRACT FROM AUTHOR]

In this chapter, a perspective is given on some aspects of electron flow through molecular bridges. The advent of molecular electronics, drive to understand charge transfer in biological systems, and functioning of devices such as organic lightemitting diodes and dye-sensitized solar cells all require deeper understanding of this topic. Application of SPM to such studies has opened the possibility of true single—molecule measurements, but has also introduced a number of artifacts and complications into the work, notably perturbations due to the force and local electric field applied by the tip. Additional chapters in this book provide specific studies of electron flow in thiolated hydrocarbons and of electrical and electromechanical measurements on biomolecules. Here, SPM measurements on two specific systems—electron flow through DNA, and STM measurements of isolated molecules on a semiconductor surface, are developed in detail. These studies are presented in the general context of electron flow measurements through single molecules, and are compared with parallel, non-SPM techniques. The strength of SPM to combine imaging with the electrical measurement is emphasized. [ABSTRACT FROM AUTHOR]

Recent interest in the technological potential of nitride-based semiconductors has led to the development and commercialization of a wide range of electronic devices. The nanoscale investigation of the electric properties of III-nitride thin films, bulk crystals, and heterostructures is of considerable interest for determining how interfaces, defects, and inversion domain boundaries affect device performance. The pyroelectric nature of wurtzitic III-nitrides is characterized by a spontaneous polarization that exists without the presence of an external field and by a polarization-bound surface charge. Scanning probe-based measurements of surface contact potentials and surface band bending in these materials, which are of crucial importance to device design, are summarized in this review chapter. In addition, the role of charged defects on device performance is explored by scanning probe techniques. Lastly, the measurement of polarity and of the screening mechanism of III-nitrides, which are fundamental issues for the fabrication of devices based on polarity effects, are discussed. This chapter provides a critical analysis of the contributions made by scanning probe-based techniques toward a deeper understanding of the III-nitride material system. [ABSTRACT FROM AUTHOR]

Due to their technological importance, III-V compound semiconductors have been widely studied. While extensive work has been done on their geometric and electronic structure, Kelvin probe force microscopy (KPFM) in ultrahigh vacuum (UHV) creates the possibility to study the electronic structure of the surfaces on a nanometer scale [1]. The work function is one of the most important values characterizing the property of a surface. Chemical and physical phenomena taking place at the surface are strongly affected by the work function. In turn, the work function variation reflects physical and chemical changes of surface conditions [2]. For example, due to a localized dipole at atomic steps, the averaged work function on a metal surface decreases in proportion to the step density [3]. If molecules or atoms are adsorbed on a surface, the work function changes depending on the magnitude of the electric dipole formed by the adsorbates [2]. Although the work function is defined as a macroscopic concept, it is necessary to consider its microscopic local variations in understanding the details of the formation of semiconductor interfaces and device behavior. [ABSTRACT FROM AUTHOR]

Though most widely used as a two-dimensional (2D) dopant-profiling tool, scanning capacitance microscopy (SCM) has a diverse array of applications in the semiconductor industry. This chapter highlights three emerging applications of SCM: failure analysis of semiconductor devices, imaging of device operation, and the visualization of radiation effects. Each application is described in detail, providing practical experimental details and a number of examples. [ABSTRACT FROM AUTHOR]

Hierarchical structure of connective and calcified tissues from the macro- to nanoscale level determines the mechanical and biological functionality of biological materials and has been the focus of numerous recent studies. Further progress in this field requires development of microscopic techniques capable of probing materials properties, including local composition, crystallographic orientation, and mechanical properties on the nanometer-length scale. Here, we describe a piezoresponse force microscopy (PFM) approach to high-resolution imaging of biological systems, based on detection of the local piezoelectric response. Samples include human tooth, femoral cartilage, deer antler, and butterfly wing scales. PFM allows differentiation between organic and mineral components and provides additional important information on materials microstructure. We also demonstrate the PFM capability of studying the internal structure and orientation of protein microfibrils with a spatial resolution of several nanometers. Future potential of the PFM approach for biological imaging is discussed. [ABSTRACT FROM AUTHOR]

Electrostatic properties play an important role in the interactions and functions of biomolecules. Electrical scanning probe microscopy (SPM) is a unique tool for the study individual biomolecules. Variations of the electrostatic probe technique such as Kelvin probe force microscopy (KFM), scanning surface potential microscopy (SSPM), and scanning Maxwell stress microscopy (SMM), have been developed. These instruments obtain similar electrostatic images but with a different theoretical basis and electronic instrumentation. The experimental technique and substrate preparation are similar to those of other kinds of SPM but with more stringent measurement requirements, which are needed to avoid static charges in air and on the substrate. Detailed experimental techniques and specific examples using Photosystem I (PSI) reaction centers, DNA, protein microarrays on surfaces, and PSI at the air-liquid interface are discussed. The electrical SPM techniques provide a method to study biomolecules in a label-free fashion with nanometer resolution. In the future, with improved resolution, it will be possible to image the charge distribution and polarization of individual biomolecular reaction sites, which will provide insights into biological function at the molecular level. [ABSTRACT FROM AUTHOR]

Scanning voltage microscopy and scanning differential resistance microscopy analyses on diode lasers are presented: the direct observation of the current blocking breakdown in a buried heterostructure laser; the effect of current spreading inside actively biased ridge waveguide lasers; origins of anomalously high series resistance encountered in ridge lasers; parasitic series resistance power dissipation; and electron overbarrier leakage. Applications to emerging fields of nanotechnology and nanoelectronics are suggested. Specific sources of artifacts (such as circuit time constants and photocurrent) are described and evaluated for optimal performance. [ABSTRACT FROM AUTHOR]

The quest to unravel the properties of manganites has fueled several important concepts in contemporary solid-state physics, involving intricately coupled degrees of freedom (lattice, charge, spin and orbital) and electronic inhomogeneity or phase separation between states of different electronic character. With simultaneous spatial and spectral resolution, scanning tunneling microscopy (STM) can provide direct information on each of these states and on their spatial extent. Here we highlight some recent advances and outline the potential for further STM studies of manganites. [ABSTRACT FROM AUTHOR]

Dynamic force microscopy (DFM) operated in ultrahigh vacuum, which is often also called noncontact atomic force microscopy (NC-AFM), is able to image the atomic structure of surfaces, including observation of point defects independent from the sample's conductivity. Within the last ten years, a variety of materials, including conductors, semiconductors, and insulators, have been imaged down to the atomic scale. Atomic arrangements and individual defects such as vacancies or impurity atoms have been observed, and chemically different species have been distinguished due to their different energy dissipation properties in DFM. In these atomic-scale images, the contrast is mainly due to short-range interatomic bonding forces, repulsive atom—atom forces, or van der Waals forces. However, in many experiments, electrostatic forces play a significant role in contrast formation, and electrostatic charge distributions can be imaged with high resolution. For example, the charge distribution around charged monoatomic vacancies can be mapped, as well as the charge distribution around individual doping atoms in semiconductors. At higher doping levels, the charge clouds around individual doping atoms overlap, and DFM images the resulting modulation in the surface potential of the sample. Finally, using dynamic force spectroscopy, the relative contributions of long-ranged electrostatic and van der Waals forces as opposed to short-ranged chemical bonding forces can be distinguished and analyzed site-specifically. [ABSTRACT FROM AUTHOR]

A multi-tip scanning tunneling microscope (STM) can be used as a very versatile tool for electrical transport measurements at the nanometer scale. The STM tips are employed as current sources, voltage pick-up probes, and field-gate electrodes. This tool is very useful not only for fundamental physics research, but also for testing electrical characteristics of nanometer-scale electronic devices. Several groups are developing different types of multi-tip STM's [1]-[7]. Some companies have begun to deliver products combining multi-probe scanning probe microscopes with conductive atomic force microscopes [8], but the apparatus and operating system are not yet fully developed and still have much room for evolution in many aspects. [ABSTRACT FROM AUTHOR]

One of the central difficulties of devising new nanostructures is to monitor characterization and manipulation processes at extreme dimensions. In that respect, scanning probe microscopes (SPMs) constitute an important class of experimental tools for imaging the local atomic, mechanical, electronic, and transport properties of samples at dimensions ranging from the mesoscale to the nanoscale. [ABSTRACT FROM AUTHOR]

Conducting scanning probe microscopy provides a powerful tool for measuring electric transport through small surface features. In this chapter, carbon nanotubes and carbon nanotube networks are analyzed with a scanning probe microscopy method that employs solid metal tips to provide improved electrical contact to the nanotubes. The study reveals paths of electrical transport through carbon nanotubes and provides a means to differentiate between semiconducting and metallic nanotubes. Finally, high-resolution images provide insight into the conductance decay around nanotube junctions. [ABSTRACT FROM AUTHOR]

Since the discovery of carbon nanotubes, considerable insight into their electronic properties has been gained by electrical transport characterization of individual nanotube devices. At the same time, both experimental and theoretical investigations found that many of the phenomena observed in transport studies of these devices are not due to bulk properties of nanotubes, but depend instead on local atomic-scale details including defects and impurities within the nanotube itself and interfaces between the nanotube and microfabricated electrodes. This prompted application of a number of scanning probe microscopies (SPM), where local probes are used to both stimulate the nanotube and its response to localized electrostatic potentials. In this chapter, we review the state of the art in several methods of SPM, including scanning gate microscopy (SGM), electrostatic force microscopy (EFM), and current-sensitive and thermal SPM, as they are applied to study physical phenomena in carbon nanotube devices at both room and low temperatures. [ABSTRACT FROM AUTHOR]

The ability to control the placement of molecules is essential for the patterning and fabrication of nanoscale electronic devices. We apply selective chemistry and self-assembly to reach higher resolution, greater precision, and chemical versatility in the nanostructures that we create. These methods demonstrate the possibilities of patterning films by exploiting the intrinsic properties and interactions of the molecules. We employ self-assembled monolayers as a means to isolate molecules with various electronic properties to determine the fundamental transport mechanisms, and the relationships between molecular structure, environment, connection, coupling, and function. Using self-assembly techniques in combination with scanning tunneling microscopy (STM), we are able to study candidate molecular switches individually and in small bundles. Alkanethiolate self-assembled mono-layers on gold are used as a host two-dimensional matrix to isolate and to insulate electrically the molecular switches. We then individually address and electronically probe each molecule using STM. The conjugated molecules exhibit reversible conductance switching, manifested as a change in apparent topographic height in STM images. The origins of switching and the relevant aspects of the molecular structure and environment required are discussed below. [ABSTRACT FROM AUTHOR]

We present an overview of spin-polarized scanning tunneling microscopy (Sp-STM). As in STM, the electron density near the sample surface can be imaged. In addition, Sp-STM allows us to map the spin polarization. Thus, information on the magnetic configuration of the sample surface can be gathered. Three imaging modes are currently being used: the constant-current mode, the spectroscopic mode, and the differential magnetic mode. The principles of the three modes are explained, and their advantages and limitations are discussed in the framework of imaging ferromagnetic and antiferromagnetic surfaces of bulk materials and thin film systems. Further, two approaches for controlling the spin direction of the tip apex, i.e., the sensitive spin component, are discussed. Surface or interface magnetic anisotropies at the tip apex may be used to align the axis of sensitivity or alternatively, the shape anisotropy of the whole tip may determine the spin direction. Finally, it is demonstrated that Sp-STM can be used beyond magnetic imaging. Valuable information on the spin resolved electronic structure or on the fundamental processes of spin-polarized tunneling may be obtained. [ABSTRACT FROM AUTHOR]

Since the discovery of the quantum Hall effect [1], a lot of insight into the behavior of quasi-free electron systems housed in III-V-semiconductors has been gained by macroscopic measurements such as transport, magnetization, or optics [2], [3]. However, theoretical descriptions of the observed phenomena mostly rely on a distinct local arrangement of the corresponding electron phases [4]-[8]. For example, it is believed that the quantum Hall plateaus require localization of the electrons everywhere within a sample except close to the edge. The electrons inside the sample should reside at equipotential lines in valleys and at hills of the potential disorder. This specific description, as well as many others explaining other effects, triggered the effort to observe the local arrangement of the electrons in real space by scanning probe techniques. In this chapter the current state of the art of the different scanning probe methods as applied to low-dimensional III-V semiconductors is reviewed and the specific advantages and drawbacks are discussed. [ABSTRACT FROM AUTHOR]

We review a range of near-field probes that have been used for measuring the localized high-frequency electromagnetic properties of materials. First, near-field microscopy is briefly discussed, thereby establishing a background for describing near-field probing experiments. The basic dielectric properties of metals, insulators, and dielectrics are briefly reviewed since these provide the basis for image contrast. Finally, a wide range of near-field probes are described, including optical, infrared, microwave, etc. The literature on high-frequency near-field probes is surveyed throughout the text. [ABSTRACT FROM AUTHOR]

The application of scanning probe microscopy (SPM) techniques to the study of electrochemical interfaces is discussed in this review. Experimental requirements for performing electrochemical scanning probe microscopy (EC-SPM) experiments are briefly discussed as well as their successful merger with conventional scanning probe microscopy instrumentation. Methods highlighted include electrochemical-scanning tunneling microscopy (EC-STM), electrochemicalatomic force microscopy (EC-AFM), and hybrid variants of AFM, including scanning electrochemical microscopy-atomic force microscopy (SECM-AFM) and local electrochemical impedance spectroscopy-atomic force microscopy (LEISAFM). Applications of these modern EC-SPM techniques in emerging areas of energy storage and conversion, corrosion, catalysis, and electrochemical deposition processes are underscored to emphasize the resolving power of these methods. [ABSTRACT FROM AUTHOR]

Over the last two decades scanning near-field optical microscopy (SNOM) has demonstrated its ability to provide optical resolution significantly better than the diffraction limit (<20 nm). The general principle of SNOM relies on the approach of a nanometer-sized object in the optical near-field of a sample to be studied. This nano-object (NO) is usually the extremity of a probe. Regardless of the nature of the observed SNOM signal (inelastic scattering, fluorescence, etc.), the detection of the light is achieved in the far-field regime where the NO acts as a mediator between the optical near-field and the detector. Figure 1 is a schematic illustration of the SNOM principle. [ABSTRACT FROM AUTHOR]

Near-field microwave microscopy is concerned with quantitative measurement of the microwave electrodynamic response of materials on length scales far shorter than the free-space wavelength of the radiation. Here we review the basic concepts of near-field interactions between a source and sample, present an historical introduction to work in the field, and discuss a novel quantitative modeling approach to interpreting near-field microwave images. We discuss the spatial resolution and a number of concrete applications of near-field microwave microscopy to materials property measurements, as well as future prospects for new types of microscopy. [ABSTRACT FROM AUTHOR]

This chapter describes the principles, theoretical background, recent developments, and applications of a local probe-based technique for nondestructive high-resolution ferroelectric domain imaging and manipulation—piezoresponse force microscopy (PFM). This technique has proven to be a powerful tool for the characterization of ferroelectric thin films, ceramics, and single crystals. Recent advances in application of PFM for studying a mechanism of polarization reversal at the nanoscale, domain dynamics, degradation effects, and size-dependent phenomena in ferroelectrics are reviewed in detail. Examples of using PFM for the characterization of various polar materials such as ferroelectric films, piezoelectric semiconductors, and ferroelectric relaxors are given. [ABSTRACT FROM AUTHOR]

Frequency-dependent transport measurement, including impedance spectroscopy and capacitance-voltage measurement, is a key tool for materials and device characterization and failure analysis. The rapid development of nanomaterials and nanoscale devices necessitates extending our fundamental understanding of transport phenomena to the nanoscale as well. The ability to conduct nanoscale impedance measurements provides a promising route to extend our understanding of nanoscale transport physics and device operation. In this chapter, we describe two paradigms for scanning probe microscopy (SPM)-based impedance measurement [1-5]. In a first configuration, the SPM tip is used as a current probe in a manner similar to conventional impedance spectroscopy (nanoimpedance microscopy). In a second configuration, the SPM tip is used as a moving voltage electrode in a manner similar to four probe impedance measurements (scanning impedance microscopy). Applying these two configurations, nanometer scale measurement and visualization of impedance is demonstrated for a wide variety of materials systems, including solid electrolytes, semiconductors, electroceramics, corrosion research, and fuel cell systems. Future prospects for SPM-based impedance measurement are discussed. [ABSTRACT FROM AUTHOR]

In this chapter we describe and discuss Kelvin probe force microscopy (KPFM), a scanning probe microscopy technique designed to obtain laterally resolved work function images by measuring the electrostatic forces between probe and sample surface. By operating the microscope in ultrahigh vacuum, even absolute work function measurements with very high lateral and energy resolution can be realized. [ABSTRACT FROM AUTHOR]

As the downscaling in semiconductor industry continues, the correct operation of devices becomes critically dependent on the precise location and activation of the dopants in two dimensions. For Si technology, Duane had already determined in 1996 that, in the 0.25-μm CMOS technology, a 10-nm decrease in the channel length was responsible for a more than 10% increase in the gate-to-drain overlap capacitance [1]. In the 65-nm devices processed today, effects of shifts in lateral position of a few nanometers or of variations in concentration of a few percent in the channel are drastically more pronounced. [ABSTRACT FROM AUTHOR]

The introduction of scanning tunneling microscopy (STM) followed by scanning tunneling spectroscopy (STS) opened experimental access to the geometric and electronic structure of materials on an atomic scale and essentially ushered in the modern field of nanoscience. The goal of scanning tunneling potentiometry (STP) is to adapt the scanning tunneling probe to measure electrical transport on the same length scale. The approach is to establish a current laterally in the sample, then to map the voltage locally by determining the tip bias that produces no tunneling at each point. The technique is similar to the macroscopic four-probe method commonly adopted for measuring electrical transport, with the inner two probes replaced by a scanning tunneling tip. This chapter describes principles, reviews approaches, and illustrates capabilities of STP. [ABSTRACT FROM AUTHOR]

Progress in modern science is impossible without reliable tools for characterization of structural, physical, and chemical properties of materials and devices at the micro-, nano-, and atomic scale levels. While structural information can be obtained by such established techniques as scanning and transmission electron microscopy, high-resolution examination of local electronic structure, electric potential and chemical functionality is a much more daunting problem. Local electronic properties became accessible after the development of Scanning Tunneling Microscopy by G. Binnig and H. Rohrer in 1981 at IBM Zurich 25 years ago—an invention that earned its authors the Nobel Prize in Physics five years later [1]. Based on the quantum mechanical tunneling between an atomically sharp metallic tip and conductive surface, scanning tunneling microscopy (STM) has become the first instrument to generate real-space images of surfaces with atomic resolution and has triggered development of new classes of STM-related techniques. In 1986, Binnig, Quate, and Gerber demonstrated atomic force microscope (AFM) based on the mechanical detection of the Van der Waals forces between the tip and the surface using a pliable cantilever [2]. It was almost immediately realized that AFM could be extended to map forces of various types, such as magnetic and electrostatic forces, as well as for probing chemical interactions. This dual capability of probing currents and forces at the nanometer and atomic level has led to a rapid growth of a variety of scanning probe microscopy (SPM) techniques over the last two decades. Techniques such as AFM, magnetic force microscopy (MFM), electrostatic force microscopy (EFM), scanning capacitance microscopy (SCM), near-field scanning optical microscopy (NSOM), and others have emerged to provide capability to access local electrical, magnetic, chemical, mechanical, optical, and thermal properties of materials on the nanometer scale. It has been demonstrated that the SPM approach allows not only imaging, but also control and modification of the local structure and material functionality at the nano- and atomic level. As a consequence, the last two decades have witnessed an explosive growth in application of SPM techniques in a wide spectrum of fields of science, ranging from condensed matter physics, chemistry and materials science to medicine and biology. It will not be an exaggeration to say that the rapid development of nanoscience and nanotechnology in the last two decades was strongly stimulated by the availability of SPM techniques, and in turn constantly stimulates development of novel SPM probes. [ABSTRACT FROM AUTHOR]

Intracellular structures in Pichia pastoris can be visualized by the complementary methods of fluorescence microscopy and electron microscopy. An improved immunofluorescence protocol yields better optics and more reliable antigen preservation than conventional methods. As an alternative to immunofluorescence, if a protein of interest is fused to GFP or another fluorescent tag, the cells can be fixed and viewed directly. For higher-resolution studies of organelle morphology, thin-section electron microscopy of permanganate-fixed cells yields good preservation of intracellular membranes. [ABSTRACT FROM AUTHOR]

This chapter describes the different methods used for localization of proteins and organelles in Pichia pastoris. A series of plasmids and a modified immunofluorescence protocol for localization and co-localization of proteins and organelles are described. Also included are protocols for the labeling of different subcellular organelles with vital stains. [ABSTRACT FROM AUTHOR]

With the approaching completion of the Pichia pastoris genome, a greater emphasis will have to be placed on the proteome and the protein-protein interactions between its constituents. This chapter discusses methods that have been used for the study of such interactions among both soluble and membrane-associated proteins in peroxisome biogenesis. The procedures are equally applicable to other cellular processes. [ABSTRACT FROM AUTHOR]

Pichia pastoris has proven to be a valuable model for examining the molecular events of the selective degradation of peroxisomes by a process called pexophagy. We have developed a protocol to rapidly identify genes essential for glucose-induced pexophagy. This method utilizes the random integration of a Zeocin resistance cassette vector into the genomic DNA thereby disrupting gene expression. Transformed yeast are selected by growth on Zeocin and pexophagy mutants identified by their inability to degrade the peroxisomal enzyme alcohol oxidase. The Zeocin vector along with flanking genomic DNA is then isolated from the mutants and the disrupted genes identified by sequencing. We have been able to isolate 59 mutants and identify 8 unique genes. The identification of 24 genes in P. pastoris and 7 genes in H. polymorpha have been reported using this approach which has been referred to as restriction-mediated integration. [ABSTRACT FROM AUTHOR]

A significant advantage of Pichia pastoris as an experimental system is the ability to readily bring to bear both classical and molecular genetic approaches to a research problem. Although the advent of yeast molecular genetics has introduced new and exciting capabilities, classical genetics remains the approach of choice in many instances. These include the generation of mutations in previously unidentified genes (mutagenesis), the removal of unwanted secondary mutations (backcrossing), the assignment of mutations to specific genes (complementation analysis), and the construction of strains with new combinations of mutant alleles. This chapter describes these genetic manipulation methods for P. pastoris. In addition, certain yeast genes are essential for survival of the organism. However, determining whether a newly cloned gene is essential or not can be difficult with P. pastoris. In this chapter, we also describe a series of experiments to investigate the potential essential nature of a cloned gene in this yeast. [ABSTRACT FROM AUTHOR]

Labeling proteins with stable isotopes is important for many analytical and structural techniques, including NMR spectroscopy and mass spectrometry. Nonselective labeling, which uniformly labels all amino acids in the protein, may be accomplished with readily available wild-type expression hosts. However, there are often advantages to labeling a specific amino acid, and residue-selective labeling generally requires the use of an expression strain that is auxotrophic for the amino acid in order to efficiently incorporate the isotopic label. The behavior of an auxotrophic strain may be complicated by the regulatory properties of the biosynthetic pathway, by secondary nutritional requirements resulting from disruption of a biosynthetic pathway, and from acquired sensitivity to environmental factors resulting from build-up of metabolic intermediates. As a result, it is important to characterize the phenotype of the each auxotrophic strain in order to optimize its performance as an expression host for selective labeling of proteins. The application of aromatic auxotroph strains of Pichia pastoris to labeling tyrosines in a recombinant protein (galactose oxidase) will be used to illustrate selective-labeling methods. [ABSTRACT FROM AUTHOR]

Selenomethionine incorporation is a standard method for determining the phases in protein crystallography by single- or multiwavelength anomalous dispersion. Recombinant expression of selenomethionine-containing protein in non-auxotrophic Pichia pastoris strains yield an incorporation of about 50%. The expression of a mutated variant of Penicillium minioluteum dextranase in P. pastoris is used to illustrate the method utilized to obtain selenomethionyl-substituted protein and to show the phasing power of the acquired anomalous signal. The dextranase structure was solved using the anomalous signal achieved from 50% selenomethionine incorporation. [ABSTRACT FROM AUTHOR]

Because of the cost of isotopic chemicals and heterologous proteins to produce, an economical 15N/13C isotopic labeling method is critically needed. Four protocols have been tested for the expression of Ovine interferon-τ in Pichia pastoris. 13C-glucose in place of 13C-glycerol as well as the need for 15N/13C-sources were evaluated during the growth phase. Sequential addition of 15NH4Cl and 13C-methanol were also evaluated at different ratio. Our results demonstrate that 15N/13C isotopes are not required throughout the initial growth period but are necessary at low concentration a few hours prior to the methanol induction period. We have evaluated the cost of the use of isotopes 15NH4Cl, 13C-glucose and 13C-methanol in our optimised P4 protocol conditions. The cost was one-third that of the standard method using 15NH4Cl and 13C-glucose throughout the entire growth period and was even lower using 13C-glycerol. [ABSTRACT FROM AUTHOR]

Our laboratory has focused on the re-engineered of the secretory pathway of Pichia pastoris to perform glycosylation reactions that mimic processing of N-glycans in humans and other higher mammals (1,2). A reporter protein with a single N-linked glycosylation site, a His-tagged Kringle 3 domain of human plasminogen (K3), was used to identify combinations of optimal leader/catalytic domain(s) to recreate human N-glycan processing in the Pichia system. In this chapter we describe detailed protocols for high-throughput purification of K3, enzymatic release of N-glycans, matrix-assisted laser desorption ionization time-of-flight and high-performance liquid chromatography analysis of the released N-glycans. The developed protocols can be adapted to the characterization of N-glycans from any purified protein expressed in P. pastoris. [ABSTRACT FROM AUTHOR]

Glycosylation is an important issue in heterologous protein production for therapeutic applications. Glycoproteins produced in Pichia pastoris contain high mannose glycan structures that can hamper downstream processing, might be immunogenic, and cause rapid clearance from the circulation. This chapter describes a method that helps solving these glycosylation-related problems by inactivation of OCH1, overexpression of an HDEL-tagged mannosidase, and overexpression of a Kre2/GlcNAc-transferase I chimeric enzyme. Different plasmids are described as well as glycan analysis methods. [ABSTRACT FROM AUTHOR]

Recombinant and native proteins of Pichia pastoris can be O-mannosylated on serine and threonine residues, allowing further elongation reactions to generate short O-linked oligosaccharides of mannose. Methods for release from the protein with alkaline β-elimination with or without reduction of the released saccharides, and for subsequent chromatographic and enzymatic characterization of these saccharides are described. [ABSTRACT FROM AUTHOR]