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## Über dieses Buch

This book features reviews by leading experts on the methods and applications of modern forms of microscopy. The recent awards of Nobel Prizes awarded for super-resolution optical microscopy and cryo-electron microscopy have demonstrated the rich scientific opportunities for research in novel microscopies. Earlier Nobel Prizes for electron microscopy (the instrument itself and applications to biology), scanning probe microscopy and holography are a reminder of the central role of microscopy in modern science, from the study of nanostructures in materials science, physics and chemistry to structural biology.

Separate chapters are devoted to confocal, fluorescent and related novel optical microscopies, coherent diffractive imaging, scanning probe microscopy, transmission electron microscopy in all its modes from aberration corrected and analytical to in-situ and time-resolved, low energy electron microscopy, photoelectron microscopy, cryo-electron microscopy in biology, and also ion microscopy.

In addition to serving as an essential reference for researchers and teachers in the fields such as materials science, condensed matter physics, solid-state chemistry, structural biology and the molecular sciences generally, the Springer Handbook of Microscopy is a unified, coherent and pedagogically attractive text for advanced students who need an authoritative yet accessible guide to the science and practice of microscopy.

## Inhaltsverzeichnis

### 1. Atomic Resolution Transmission Electron Microscopy

This chapter provides an overview of the essential theory and instrumentation relevant to high-resolution imaging in the transmission electron microscope together with selected application examples. It begins with a brief historical overview of the field. Subsequently, the theory of image formation and resolution limits are discussed. We then discuss the effects of the objective lens through the wave aberration function and coherence of the electron source. In the third section, the key instrument components important for HRTEM imaging are discussed; namely, the objective lens, electron sources and monochromators, energy filters and detectors. The theory and experimental implementation of exit wavefunction reconstruction from HRTEM images is detailed in the fourth section, including examples taken from studies of complex oxides. The final section treats the simulation of HRTEM images with particular reference to the widely adopted multislice method.

Angus I. Kirkland, Shery L.-Y. Chang, John L. Hutchison

### 2. Scanning Transmission Electron Microscopy

The scanning transmission electron microscope ( scanning transmission electron microscopy (STEM) ) has become one of the preeminent instruments for high spatial resolution imaging and spectroscopy of materials, most notably at atomic resolution. The principle of STEM is quite straightforward. A beam of electrons is focused by electron optics to form a small illuminating probe that is raster-scanned across a sample. The sample is thinned such that the vast majority of electrons are transmitted, and the scattered electrons detected using some geometry of detector. The intensity as a function of probe position forms an image. It is the wide variety of possible detectors, and therefore imaging and spectroscopy modes, that gives STEM its strength. The purpose of this chapter is to describe what the STEM is, to highlight some of the types of experiment that can be performed using a STEM, to explain the principles behind the common modes of operation, to illustrate the features of typical STEM instrumentation, and to discuss some of the limiting factors in its performance.

Peter D. Nellist

### 3. In Situ Transmission Electron Microscopy

In situ transmission electron microscopy provides dynamic observations of the physical behavior of materials in response to external stimuli such as temperature, gas environment, stress, and electric or magnetic fields. The goal of these experiments is to provide insight into atomic or nanoscale phenomena that are not easily accessed by other methods to inform our understanding of materials properties. In this chapter, we aim to demonstrate how in situ microscopy has enhanced our understanding of dynamic phenomena associated with phase transformations, catalysis, crystal growth, liquid-phase processes, electrical and mechanical properties, magnetism, and ferroelectricity. We also address dynamic processes induced by the electron beam, whether intentional or not. In many cases, in situ research is driven by developments in novel instrumentation and testing methodologies. We discuss how advances in aberration correction, sample preparation and manipulation, detector development, and data analysis enable new types of in situ experiments and provide fundamental insight into dynamic materials phenomena.

Frances M. Ross, Andrew M. Minor

### 4. Cryo-Electron Tomography

Classical structural biology approaches rely on highly purified molecules which are isolated from their neighbors far from the complex macromolecular interaction network of the cell (ex situ). We have seen breathtaking results of such isolated molecular structures at atomic or near-atomic resolution obtained by single-particle cryo-electron microscopy (cryo-EM). However, many supra- and macromolecular complexes involved in key cellular processes cannot be studied in isolation; their function is so deeply rooted in their cellular context that it is impossible to isolate them without compromising their structural integrity. The challenge now is to apply cryo-EM to protein complexes and other biological objects in their natural environment, namely cells. Cryo-electron tomography (cryo-ET) offers this opportunity, and in this chapter we provide an overview of recent advances in sample preparation, data acquisition and data processing, including technology for focused ion beam milling, correlative light and electron microscopy, phase-plate imaging and direct electron detection. We show that these developments can be used synergistically to generate 3-D images of cells of unprecedented quality, enabling direct visualization of macromolecular complexes and their spatial coordination in undisturbed eukaryotic cell environments (in situ).

Jürgen Plitzko, Wolfgang P. Baumeister

### 5. Scanning Electron Microscopy

This chapter provides an overview of the concepts of scanning electron microscopy ( scanning electron microscopy (SEM) ) from a theoretical as well as practical operational perspective. The theory section begins with the basics of image formation followed by an explanation of the interaction of the electron beam with the sample. A description of the different types of electron guns is also included. The concepts involved with image formation from a rastered (or scanned) electron beam on a surface is explained along with the mechanisms of contrast generation from sample surface topography and sample composition. The different SEM detectors are also explained including a description of the practical application of detectors under various sample conditions. Numerous diagrams and figures in this chapter illustrate imaging geometries and possible SEM system configurations. Included in the chapter is an explanation of the various instrument operation parameters for different samples as well as a discussion of the effects of electron-beam accelerating voltages on sample imaging, contrast, and resolution.More advanced topics are also included such as the use of beam deceleration and in-lens imaging and detectors. Analytical SEM techniques are also explained with the explanation of the use of energy-dispersive x-ray energy-dispersive x-ray (EDX) detectors (EDS) used to measure sample composition as well as provide compositional maps of a sample. Application of SEM to a variety of materials systems under varying conditions are discussed with multiple examples and illustrations given.

Natasha Erdman, David C. Bell, Rudolf Reichelt

### 6. Variable Pressure Scanning Electron Microscopy

This chapter outlines the basic design modifications and operational considerations of variable-pressure SEM's relative to their high-vacuum counterparts. As the physics of electron–solid interactions are central to understanding the operation of these instruments and optimal selection of operating parameters, an introduction to that topic is provided. That section is divided into high-energy interactions, covering scattering of electrons from the primary beam, and low-energy interactions, regarding the interactions of secondary electrons with gas molecules. The latter topic leads into the discussion of the gas ionization cascade, which is the process by which secondary electron emissions can be amplified for detection. The background in this section forms the basis for a discussion of the wide variety of secondary electron signal detection strategies that have been developed. The operational principles and signal composition of several detector classes are discussed.The gas ionization cascade also generates positive gaseous ions, which enable uncoated insulators to be imaged without resorting to the use of conductive coatings or low-voltage imaging. The principle of charge neutralization will be discussed, along with some of the imaging artifacts that can result from the positive ions. As a consequence of the charge neutralization process, some dynamic contrast mechanisms can be observed in some dielectric specimens. These effects will be described using a model for time-dependent charge decay.Another primary use case for the VPSEM is in the examination of hydrated specimens in their native state, or more generally, water-containing specimens. The considerations for conducting experiments under humid conditions are discussed, along with the principles governing dynamic experiments such as hydration and dehydration. Particular attention is paid to the role of dissolved species in determining the thermodynamic activity of water in solutions.Finally, the considerations for performing electron beam microanalysis under variable-pressure conditions are presented, along with various strategies for minimizing the uncertainties for quantification.

### 7. Analytical Electron Microscopy

Analytical electron microscopy ( analytical electron microscopy (AEM) ) refers to a collection of spectroscopic techniques that are capable of providing structural, compositional, and bonding information about samples probed by an electron beam, typically inside a transmission electron microscope ( transmission electron microscopy (TEM) ). Several AEM techniques are covered with particular attention given to the energy-dispersive x-ray spectroscopy (EDXS) (energy-dispersive x-ray spectroscopy) microanalysis and electron energy-loss spectroscopy (EELS) (electron energy-loss spectroscopy) techniques. First, the different AEM techniques available in TEMs are surveyed and a parallel between EELS and EDXS is drawn. A fundamental description of the elastic and inelastic scattering events responsible for these signals is presented. The practical challenges related to electron optics and instrumentation capabilities are then discussed. Technical advances that have affected the performance of these AEM techniques are outlined, including successive generations and technologies of energy filters, monochromators, aberration correctors, and advanced energy-dispersive x-ray spectrometers. The different approaches of spectroscopic imaging with x-rays and energy-loss spectroscopy, the resolution limits, and the effects of electron-beam propagation are also described along with the types of information that can be extracted with electron-energy-loss near-edge structures. After a review of dielectric theory and low-loss spectroscopy, examples of plasmonic imaging are presented. The review also draws attention to the many efforts to extend the limits of spatial resolution and the atomic-level chemical analyses of materials. Some important progress in the statistical analysis of signals and associated numerical methods is mentioned. The review also presents some novel developments in image capture, such as the pixelated detectors. Finally, the realm of phonon spectroscopy made possible through the latest instrumentation is also discussed.

Gianluigi Botton, Sagar Prabhudev

### 8. High-Speed Electron Microscopy

High-speed electron microscopy has emerged as a well-established in situ transmission electron microscopy ( transmission electron microscopy (TEM) ) capability that can provide observations and measurements of complex, transient materials phenomena with high spatial and temporal resolutions. The development and advancement of the two categories of high-speed electron microscopy, each optimized for specific regimes of spatial and temporal resolutions—single-shot dynamic TEM ( dynamic transmission electron microscopy (DTEM) ) and stroboscopic or ultrafast TEM ( ultrafast TEM (UTEM) )—are reviewed and the technologies employed in both techniques are described. Limitations of the techniques are described and example applications are provided. Finally, an outlook for future development of time-resolved electron microscopy is provided, offering potential directions for new levels of performance and flexibility.

Geoffrey H. Campbell, Joseph T. McKeown, Melissa K. Santala

### 9. LEEM, SPLEEM and SPELEEM

This chapter discusses some of the most important imaging methods with low-energy electrons, including: low-energy electron microscopy ( low-energy electron microscopy (LEEM) ), its extension to spin-polarized low-energy electron microscopy ( spin-polarized low-energy electron microscopy (SPLEEM) ), and its combination with spectroscopic photoemission and low-energy electron microscopy ( spectroscopic photoemission and low-energy electron microscopy (SPELEEM) ). Other imaging methods mentioned only briefly in the chapter include ultraviolet photoemission electron microscopy ( ultraviolet (UV) photoemission electron microscopy (UVPEEM) ), mirror electron microscopy () mirror electron microscopy (MEM) , low-electron energy loss microscopy ( low-electron energy loss microscopy (LEELM) ), and Auger electron emission microscopy ( Auger electron (AE) emission microscopy (AEEM) ). The instruments used in these imaging methods allow imaging not only in real space but also in reciprocal space, such as low-energy electron diffraction ( low-energy electron diffraction (LEED) ) and angle-resolved photoelectron spectroscopy (ARPES angle-resolved photoelectron spectroscopy (ARPES) in SPELEEM). The combination of these methods with complementary high-lateral-resolution methods renders imaging with low-energy electrons a comprehensive surface analysis tool.

Ernst Bauer

### 10. Photoemission Electron Microscopy

Photoemission electron microscopy (PEEM) is a cathode lens electron microscopy technique. This specialized electron microscopy technique excels in studying the morphology, electronic and chemical properties and the magnetic structure of surfaces and thin film materials with nanometer-scale spatial resolution. In this chapter, we describe X-PEEM instrumentation and a typical X-PEEM optical system, discuss aberrations that limit the optical performance of X-PEEM microscopes, describe contrast mechanisms, and present several examples that cover some of the common use cases for X-PEEM, in particular the magnetic and time-resolved microscopy of nanostructures.

Jun Feng, Andreas Scholl

### 11. Spectroscopy with the Low Energy Electron Microscope

Photo electron emission microscopy (PEEM), going back to the earliest days of electron microscopy, and low-energy electron microscopy (LEEM), successfully deployed since the late 1980s, are examples of cathode lens microscopy in which the sample itself is an integral part of the image forming system. While applications have naturally gravitated towards the acquisition of images to elucidate structure and structural evolution, recent years have also seen a rapidly expanding range of spectroscopic capabilities. These address, for example, the occupied and unoccupied electronic band structures of materials, electrical transport in 2-D systems, crystal growth and 2-D strain, inelastic electron energy loss mechanisms, as well as radiation damage in organic materials during low-energy electron irradiation. In this chapter, we discuss applications of these new spectroscopic methods, as well as recent instrumental developments that further expand the potential uses of cathode lens microscopy.

Rudolf Tromp

### 12. Model-Based Electron Microscopy

The growing interest in materials design and control of nanostructures explains the need for precise determination of the atomic arrangement of non-periodic structures. This includes, for example, locating atomic column positions with a precision precision in the picometer range, a precise determination of the chemical composition of materials, and counting the number of atoms with single atom sensitivity. In order to extract these quantitative measurements from atomic resolution (scanning) transmission electron microscopy ( scanning transmission electron microscopy (STEM) ) images, statistical analysis methods are needed. For this purpose, statistical parameter estimation statistical parameter estimation theory has been shown to provide reliable results. In this theory, observations are purely considered as data planes, from which structure parameters have to be determined using a parametric model describing the images. This chapter summarizes the underlying theory and highlights some of the recent applications of quantitative model-based (S)TEM.

Sandra Van Aert

### 13. Aberration Correctors, Monochromators, Spectrometers

After four decades of attempts to correct the primary spherical and chromatic aberrations of electron lenses that led to no improvement in resolution, success was at last achieved in the 1990s with both quadrupole-octupole and sextupole correctors. The successful correctors focused on three aspects of aberration correction: primary aberrations, parasitic aberrations aberration parasitic parasitic aberration , and overall stability. They quickly demonstrated resolution improvement in the microscopes they were built into, and in the early 2000s, they advanced the attainable resolution to $$<{\mathrm{1}}\,{\mathrm{\AA{}}}$$ < 1 Å —a level not achievable by uncorrected electron microscopes. Subsequent generations of correctors included further multipoles and corrected aberrations up to the fifth order, enabling resolution of better than $${\mathrm{0.5}}\,{\mathrm{\AA{}}}$$ 0.5 Å to be reached at $${\mathrm{300}}\,{\mathrm{kV}}$$ 300 kV primary voltage, and around $${\mathrm{1}}\,{\mathrm{\AA{}}}$$ 1 Å at $${\mathrm{30}}\,{\mathrm{kV}}$$ 30 kV . The effect of chromatic aberration was reduced by the use of hybrid quadrupoles or by incorporating a monochromator in the microscope column.After a brief summary of the optics of multipoles, the various types of correctors are examined in detail: quadrupole–octopole correctors quadrupole –octopole corrector , which first improved the performance of a scanning electron microscope and, soon after, that of scanning transmission electron microscopes; and sextupole correctors sextupole corrector , which first increased the resolving power of conventional (fixed-beam) transmission electron microscopes, and were later used in scanning transmission electron microscopes as well. Ways of combating chromatic aberration are then described, including mirror correctors mirror corrector employed in low-energy-electron and photoemission microscopes ( low-energy electron microscopy (LEEM) and PEEM photoelectron emission microscopy (PEEM) ). A section is devoted to studies of aberrations beyond the third order and of parasitic aberrations.Electron spectrometers and imaging filters are routine accessories of electron microscopes, and they too must be carefully designed, especially when attached to aberration-corrected instruments. A section covers these devices, and much of the reasoning also applies to monochromators. Separate paragraphs are devoted to post-column and in-column spectrometers and monochromators, and the attainable energy resolution is discussed. Practical aspects of the correction process are described, notably autotuning and aberration measurement. We conclude with a survey of current performance limits and comments on the problems to be overcome if further progress is to be made.

Peter W. Hawkes, Ondrej L. Krivanek

### 14. Ion Microscopy

Helium ion microscopy ( helium ion microscopy (HIM) ) is a relatively young imaging and nanofabrication technique, which is based on a gas field ion source ( gas field ion source (GFIS) ). It rasters a narrow beam of helium ions across the surface of the specimen, to obtain high-resolution surface-sensitive images. Usually, secondary particles such as electrons are collected for image formation but also photons, backscattered atoms or sputtered sample atoms can be used for image formation. Thanks to the very high brightness of the source, a lateral resolution of $${\mathrm{0.5}}\,{\mathrm{nm}}$$ 0.5 nm can be achieved. The method is in particular suitable for obtaining high-resolution images of insulating samples (such as ceramic materials and biological samples) as the built-in charge compensation allows us to observe such specimens without any additional conductive coatings. In this chapter, I will introduce the method and briefly sketch the underlying physics. In the remainder of the chapter, a number of imaging modes will be discussed and selected examples will be presented. Finally, an outlook is presented on the ongoing efforts to add analytical capabilities to the method.

Gregor Hlawacek

### 15. Atom-Probe Tomography

This chapter provides an overview of the current state of atom-probe tomography ( atom probe tomography (APT) atom probe tomography (APT) history ). The history of APT is recounted so that the reader may put the many modern developments in context. It is noted that atom-probe tomography has the highest spatial resolution among analytical techniques ( $${\mathrm{0.2}}\,{\mathrm{nm}}$$ 0.2 nm ), and it has the highest absolute analytical sensitivity (single atoms), a unique combination. The fundamentals of APT, including the operative physics, performance metrics, and hardware configurations, are discussed. Before examining the many benefits that may be realized in APT, however, its limitations such as image distortions and specimen failures are discussed in full. Specimen preparation procedures for most materials are explained. A comprehensive overview of the many materials applications including metals, ceramics, semiconductors, and organics is provided. Finally, there is a look toward the future to see where the technique is headed.

Thomas F. Kelly

### 16. Electron Holography

Electron holography is a powerful technique that allows the phase shift of a high-energy electron wave that has passed through a specimen in the transmission electron microscope to be measured directly. The phase shift can then be used to provide quantitative information about local variations in magnetic field and electrostatic potential both within and surrounding the specimen. This chapter begins with an outline of the experimental procedures and theoretical background that are needed to obtain phase information from electron holograms. It then presents recent examples of the application of electron holography to the characterization of magnetic domain structures and electrostatic fields in nanoscale materials and working devices, including arrangements of closely spaced nanocrystals, patterned elements and nanowires, and electrostatic fields in field emitters and doped semiconductors. The advantages of using digital approaches to record and analyze electron holograms are highlighted. Finally, high-resolution electron holography, alternative modes of electron holography and future prospects for the development of the technique are briefly outlined.

Rafal E. Dunin-Borkowski, András Kovács, Takeshi Kasama, Martha R. McCartney, David J. Smith

### 17. Ptychography

Ptychography ptychography is a computational imaging technique. A detector records an extensive data set consisting of many inference patterns obtained as an object is displaced to various positions relative to an illumination field. A computer algorithm of some type is then used to invert these data into an image. It has three key advantages: it does not depend upon a good-quality lens, or indeed on using any lens at all; it can obtain the image wave in phase as well as in intensity; and it can self-calibrate in the sense that errors that arise in the experimental set up can be accounted for and their effects removed. Its transfer function is in theory perfect, with resolution being wavelength limited. Although the main concepts of ptychography were developed many years ago, it has only recently (over the last 10 years) become widely adopted. This chapter surveys visible light, x-ray, electron, and EUV ptychography as applied to microscopic imaging. It describes the principal experimental arrangements used at these various wavelengths. It reviews the most common inversion algorithms that are nowadays employed, giving examples of meta code to implement these. It describes, for those new to the field, how to avoid the most common pitfalls in obtaining good quality reconstructions. It also discusses more advanced techniques such as modal decomposition and strategies to cope with three-dimensional () multiple scattering.

John Rodenburg, Andrew Maiden

### 18. Electron Nanodiffraction

This chapter introduces the practice and theory of electron nanodiffraction. After a brief introduction, the chapter provides a comprehensive description of electron diffraction techniques and their use for nanodiffraction. This is followed by discussions on electron probe properties, electron energy filtering and electron diffraction data analysis. Throughout the chapter, we emphasize different electron nanoprobes that can be formed inside an electron microscope, from a focused beam to parallel illumination, and how these probes can be used to extract structural information from different materials. For this purpose, we outline the electron diffraction theories based on both kinematic approximation and dynamic diffraction, which serve as the basis for the interpretation of electron nanodiffraction patterns. The principles and applications of scanning electron nanodiffraction and coherent diffraction imaging are covered in detail with applications for orientation mapping, imaging strain, 3-D nanostructure determination, and study of defects.

Jian-Min Zuo

### 19. High-Energy Time-Resolved Electron Diffraction

This chapter reviews the application of relativistic energy ultrashort electron beams to the direct investigation of structural changes in matter at atomic length scale with sub-picosecond (sub-ps) resolution by time-resolved electron diffraction ( electron diffraction (ED) ) time-resolved electron diffraction . There are many benefits of using higher-energy electron beams for time-resolved electron scattering instrumentation, due mainly to the space charge force suppression at relativistic energies, which enables more intense and shorter electron bunches. Speed-of-light probes, higher penetration, and shorter de Broglie wavelength are other advantages associated with MeV electron energy.The use of MeV beams for electron scattering demands excellent beam quality in both the transverse and longitudinal phase spaces, i. e., an exquisitely high six-dimensional ( six-dimensional (6-D) ) brightness. Expertise from the low-energy microscopy and diffraction community has converged with advances made in relativistic electron sources for high-energy particle accelerators and fourth-generation synchrotron light sources in the effort to create and deliver electron beams with these characteristics.

Pietro Musumeci, Renkai Li

### 20. Diffractive Imaging of Single Particles

The computational methods and applications of diffractive diffractive imaging (lensless) lensless imaging imaging lensless imaging are reviewed. Far-field scattering (e. g., by neutrons, electrons, light, or x-rays) by a nonperiodic localized potential is detected and the phase problem solved using iterative optimization methods, which allows a three-dimensional image of the object potential to be reconstructed without the need for a lens. The history of the subject and its relationship to the crystallographic phase problem are reviewed, together with a summary of theory, algorithms, uniqueness issues, resolution limits, the constraint ratio concept, and coherence requirements. Applications, from various forms of microscopy (electron, optical, and x-ray) to snapshot x-ray laser single-particle imaging, are reviewed. The method of ptychography, which achieves a similar aim, is reviewed elsewhere in these volumes.

John C. H. Spence

### 21. Fluorescence Microscopy

In this two-part chapter, the background to confocal microscopy and two-photon fluorescence microscopy is first presented, with a detailed description of the optical setup. This is followed by a critical account of the many super-resolution techniques: coordinated stochastic fluorescence microscopy (photoactivation localization microscopy ( photoactivated localization microscopy (PALM) ), stochastic optical reconstruction microscopy ( stochastic optical reconstruction microscopy (STORM) ), point accumulation for imaging in nanoscale topography ( points accumulation for imaging in nanoscale topography (PAINT) ), coordinate targeted fluorescence microscopy (STED, reversible saturable optical fluorescence transition ( reversible saturable optical fluorescence transition (RESOLFT) )), structured illumination microscopy, expansion microscopy ( expansion microscopy (ExM) ), and liquid tunable microscopy ( liquid tunable microscopy (LIQUITOPY) ).

Alberto Diaspro, Paolo Bianchini, Francesca Cella Zanacchi, Luca Lanzanò, Giuseppe Vicidomini, Michele Oneto, Luca Pesce, Isotta Cainero

### 22. Fluorescence Microscopy with Nanometer Resolution

Nanoscale Resolution in Far-Field Fluorescence Microscopy

Throughout the twentieth century, it was widely accepted that a light microscope relying on propagating light waves and conventional optical lenses could not discern details that were much finer than about half the wavelength of light, or $$200{-}400\,{\mathrm{nm}}$$ 200 - 400 nm , due to diffraction. However, in the 1990s, the potential for overcoming the diffraction barrier was realized, and microscopy concepts were defined that now resolve fluorescent features down to molecular dimensions. This chapter discusses the simple yet powerful principles that make it possible to neutralize the resolution-limiting role of diffraction in far-field fluorescence nanoscopy methods such as STED, RESOLFT, PALM/"​"​STORM, or PAINT. In a nutshell, feature molecules residing closer than the diffraction barrier are transferred to different (quantum) states, usually a bright fluorescent state and a dark state, so that they become discernible for a brief period of detection. With nanoscopy, the interior of transparent samples, such as living cells and tissues, can be imaged at the nanoscale. A fresh look at the foundations shows that an in-depth description of the basic principles spawns powerful new concepts. Although they differ in some aspects, these concepts harness a local intensity minimum (of a doughnut-shaped or a standing wave pattern) for determining the coordinate of the fluorophore(s) to be registered. Most strikingly, by using an intensity minimum of the excitation light to establish the fluorophore position, MINFLUX nanoscopy has obtained the ultimate (super)resolution: the size of a molecule ( $$\approx{}{\mathrm{1}}\,{\mathrm{nm}}$$ ≈ 1 nm ).

Steffen J. Sahl, Andreas Schönle, Stefan W. Hell

### 23. Zone-Plate X-Ray Microscopy

Fresnel zone Fresnel zone plate plates are the most commonly used optic in x-ray microscopes. Following a short discussion of historical developments, the properties of zone plates are outlined, along with the microscope systems that employ them. A number of applications of x-ray microscopes are then surveyed, including in biology, environmental science, and materials science.

Chris Jacobsen, Malcolm Howells, Tony Warwick

### 24. Microcomputed Tomography

Since Röntgen discovered x-rays at the end of the nineteenth century and established their usefulness for medical diagnostics imaging, many technological advances have allowed for x-rays to be employed in even more powerful ways. This includes utilizing x-rays for tomographic imaging and quantification.This chapter describes the principles of microcomputed tomography ( microcomputed tomography (microCT) ) and its use in obtaining internal structural and compositional data about materials/objects of interest. The authors introduce this material with a brief history of the development of laboratory and synchrotron microCT for engineering, biology, and biomedical applications.As will be evident, microCT imaging requires many components to operate together with precision, and the standard microCT subsystems will be described. This chapter will also explain the principles behind x-ray attenuation in materials as well as common methods by which microCT image processing software may handle complex detected data to reconstruct grayscale slice images. The quality of the resulting images relies on a few key factors, including spatial resolution, noise, and contrast, and these concepts will be explained. Additionally, microCT image reconstruction and processing may produce various types of artifacts, and the most common of these artifacts will be discussed.In a typical microCT imaging workflow, the reconstructed two-dimensional () slice images can subsequently be processed to generate segmentations and three-dimensional () renderings of the material(s) of interest. Because image segmentation and quantification of the material's geometry and composition could be performed via many possible procedures, these processes will be generally discussed within this chapter.Finally, microCT forms the basis for various novel techniques that are rapidly gaining momentum for use in biology, engineering, and biomedical research applications to provide accurate, non-destructive high-resolution images and quantitative data. Some of these techniques, such as phase contrast CT, dual-energy CT, fluorescence CT, and x-ray scattering tomography, will be introduced and briefly discussed.

Angela S.P. Lin, Stuart R. Stock, Robert E. Guldberg

### 25. Scanning Probe Microscopy in Materials Science

The advent of scanning probe microscopy ( scanning probe microscopy (SPM) ) revolutionized surface science in the 1980s and facilitated the nanotechnology revolution in the ensuing decades. First scanning tunneling microscopy, then atomic force microscopy ( atomic force microscopy (AFM) ) and near-field optical methods, were developed and employed for fundamental and applied research in many disciplines including physics, biology, chemistry, and a wide range of engineering fields. But SPM, especially AFM, has in particular contributed to materials science due to the fact that atomic to nanoscale resolution of materials properties can be achieved. Routine and specialized SPM approaches now provide measurements and maps not just of the topography, but also of local mechanical, electronic, magnetic, optical, thermal, chemical, and coupled properties. Important recent developments include increases in imaging speed, in situ and in operando studies, advanced probes, and even tomographic AFM. This chapter describes the concepts and implementation of these various SPM methods focused on new discoveries in materials science.

Bryan D. Huey, Justin Luria, Dawn A. Bonnell

### 26. Electron Tomography in Materials Science

This chapter illustrates how electron tomography has become a technique of primary importance in the three-dimensional () microscopic analysis of materials. The foundations of tomography are set out with descriptions of the Radon transform and its inverse and its relationship to the Fourier transform and the Fourier slice theorem. The acquisition of a tilt series of images is described and how the angular sampling in the series affects the overall 3-D resolution in the tomogram. The imaging modes available in the (scanning) transmission electron microscope are explored with reference to their application in electron tomography and how each mode can provide complementary information on the structural, chemical, electronic, and magnetic properties of the material studied. The chapter also sets out in detail methods for tomographic reconstruction from backprojection and iterative methods, such as simultaneous iterative reconstruction technique ( simultaneous iterative reconstruction technique (SIRT) ) and algebraic reconstruction technique ( algebraic reconstruction technique (ART) ), through to more recent compressed sensing approaches that aim to build in prior knowledge about the specimen into the reconstruction process. The chapter concludes with a look to the future.

Rowan K. Leary, Paul A. Midgley

### 27. Scanning Tunneling Microscopy in Surface Science

This chapter discusses the use of scanning tunneling microscopy (STM) in surface science. Basic principles of STM imaging are introduced, and the imaging methodology is discussed, along with practical and instrumentation requirements. The approach taken in surface imaging by STM is illustrated by the example of silicon surfaces. An application of STM that has gained ever-increasing importance is discussed in detail: atomic-scale spectroscopy. A survey of the spectroscopic capabilities of STM is provided, and a variety of techniques for local spectroscopy and spectroscopic imaging are introduced. In addition, pathways toward obtaining chemical and element specificity at the atomic scale—traditionally a weakness of STM—are discussed. The extension of the operating conditions of STM to high and low temperatures has opened up new avenues of investigation. Here, variable-temperature STM of dynamic surface processes is discussed, as well as manipulation of atoms and molecules at cryogenic temperatures. Examples of STM imaging and spectroscopy on subsurface structures include the use of ballistic electrons to probe buried interfaces, and cross-sectional STM on cleavage faces of III/V semiconductors to image embedded nanostructures. The chapter concludes with a brief discussion of STM image simulation techniques.

Peter Sutter

### 28. Visualizing Electronic Quantum Matter

Modern quantum materials support a wide variety of exotic and unanticipated states of quantum matter and differ radically in phenomenology from conventional systems such as metals, semiconductors, band insulators, and ferromagnets. For example, quantum materials exhibit states such as electron liquid crystals, fluids of fractionalized quantum particles, quantum-entangled spin liquids, and topologically protected composite quantum particles. However, predictive theory is not fully developed for these forms of electronic quantum matter ( electronic quantum matter (EQM) ) and exploratory empirical research is required to discover and understand their properties. One of the most powerful and productive new techniques to achieve this is direct visualization of EQM at the atomic scale. For EQM, as with many highly complex systems in nature, seeing is believing and understanding. Here we describe the experimental, theoretical and analysis techniques of atomic-resolution spectroscopic imaging scanning tunneling microscopy (SI-STM) that allow such complex and enigmatic electronic/magnetic states to be directly visualized, identified, and understood.

Kazuhiro Fujita, Mohammad H. Hamidian, Peter O. Sprau, Stephen D. Edkins, J.C. Séamus Davis

### 29. Microscopy of Nanoporous Crystals

Nanoporous crystals are widely studied and used for applications in $$\mathrm{H_{2}}$$ H 2 storage, $$\mathrm{CO_{2}}$$ CO 2 capture, petrochemical catalysis and many other applications, yet the imaging of their atomic structure has proven difficult because of their radiation sensitivity and the small size of these microcrystals. This chapter describes the development of the new modes of electron microscopy needed to study them, and compares these with traditional methods such as x-ray diffraction. This class of materials has traditionally been dominated by the zeolites and zeotype materials, but has recently been expanded to include meso-/macroporous crystals and other new framework structures (MOFs, ZIFs COFs, etc.). Using different building blocks or units, versatile crystal structures have been synthesized for various applications. Their properties and functions are governed primarily by periodic arrangements of pores and/or cavities and their surroundings with various atomic moieties inside crystals. In this chapter, electron microscopy studies of nanoporous materials are discussed from different perspectives. Special attention is paid to the observation of fine defect structures, through careful analysis of electron diffraction, high-resolution images and spectroscopy data. The experimental conditions for imaging beam-sensitive materials, such as MOFs, are described. The contents have been divided into sections based on the types of materials and their geometric features. Examples of structure analysis of various nanoporous materials are given and discussed. New technical developments and existing challenges are described.

Yanhang Ma, Lu Han, Zheng Liu, Alvaro Mayoral, Isabel Díaz, Peter Oleynikov, Tetsu Ohsuna, Yu Han, Ming Pan, Yihan Zhu, Yasuhiro Sakamoto, Shunai Che, Osamu Terasaki

### 30. Biomedical X-Ray Phase-Contrast Imaging and Tomography

This chapter reviews the main categories of methods that have been developed for preclinical and clinical imaging applications clinical imaging application . The methods covered here are all based on geometric magnification by the projection of a cone beam emitted from a pseudopoint source, without the use of a lens or other types of focusing optics. These methods share the common goal of detecting x-ray refraction and diffraction, in order to complement the conventional attenuation contrast information in radiography and computed tomography.

Han Wen

### 31. Atomic Force Microscopy in the Life Sciences

The last decade have established AFM as a technique in life sciences applications ranging from single molecules to living cells and tissues. AFM still remains one of the few microscopy tools to offer premium resolution on bio-macromolecules at near physiological/native sample conditions. The demand for correlative immunochemical and ultrastructural characterization of macromolecular complexes and cells has made the combination of AFM and advanced optical microscopy techniques almost ubiquitous in every life sciences lab. This chapter gives an overview of the instrumentation and most common imaging modes used in AFM nowadays, as well as different preparation protocols for single molecule and cell applications. We finish with application examples that feature some recent developments in state-of-the-art AFMs as tools to study molecular and cellular dynamics with high spatial and temporal resolution.

Matthias W. Amrein, Dimitar Stamov

### 32. Microscopy in Forensic Sciences

This chapter examines the use of electron microscopy, atomic force microscopy and other analytical techniques in forensic investigation forensic investigation and research. These tools can be used to enhance examination of human remains and trace evidence to improve understanding of cause of death, victim identification, or postmortem interval.A police-designed scenario is used to highlight trace evidence such as glass, gunshot residue, and paint. The validity of forensic techniques is discussed, with reference to international standards, repeatability, and false convictions. Ballistic evidence is used to highlight the complexities in evidence interpretation, including manufacturing variability, environmental effects, and likelihood ratios.The use of scanning electron microscopy ( scanning electron microscopy (SEM) ), atomic force microscopy ( atomic force microscopy (AFM) ), and other techniques in the development of forensic research is showcased, with particular examples from the field of fingerprint analysis. Examples include improvements in the development of fingermarks from difficult surfaces, the interaction of evidence types, and added intelligence from the crime scene, such as forensic timeline forensic timeline or gender of perpetrator. entomology

Benjamin J. Jones

### Backmatter

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