Preface

Among the more ambitious goals in modern biology is our quest to find a link between (patho-) physiological state of an organism and the role that key molecules play in it, be that the process of tissue and organ development, the differentiation of individual cells, or the manifestation of disease.

However, biological systems are inherently complex with many components needed to sustain cellular function. In order to understand any complex system, one must determine its composition, i.e., identify the components it is made of, how each of these components carry out their specific task, and how they interact with one another to function together as an assembly of components. In other words, we need to determine the specific mechanism of function and its regulation for each relevant individual macromolecule, but we must also determine its interaction with other macromolecules. Therefore, detailed information is needed at different levels of scale and complexity, ranging from three-dimensional (3D) structure and function of individual macromolecules, which allows the determination of the reaction or interaction mechanisms at atomic scale, over the structural organization of such individual components into macromolecular and/or supramolecular complexes, as well as their spatiotemporal distribution and kinetics within organelles, cells, and tissues.

Furthermore, it is important to see how genetic modification or pharmacological intervention alters such macromolecular function, macromolecular interactions, and/or spatiotemporal distribution, and thus the overall supramolecular structural organization of organelles, cells, and tissues. Only when we understand holistically 3D structure, function, kinetics, and regulation of such macromolecules, molecular machines, and supramolecular complexes in their organelle-, cell-, and tissue-context will we be able to understand how such changes lead to physiological cell and tissue function and/or pathogenesis/disease progression.

The list of systems components can be determined by the various "-omics" approaches, with genomics and transcriptomics determining the nucleic acid sequences of genes and their transcribed messenger RNA, respectively, and thus link gene sequences and expression profiles to cellular behavior. Proteomics probes the translated gene products, i.e., proteins along with possible posttranslational modifications. Lipidomics studies cellular lipids and is a subset of metabolomics that aims to study all small molecules that are synthesized or broken down in any of the primary and secondary metabolic pathways of a cell. The presence and abundance of inorganic, small organic, and macromolecular components in cells and tissues can be detected by mass spectrometry approaches. Many of these aforementioned approaches are easily scalable, and in many cases the information can be obtained through the service of large regional or national centers, enabled by commercial vendors.

The "-omics" approaches often are nontargeted, collecting a vast amount of information about all present components. Targeted approaches that focus on the presence of a small subset of components are typically based on affinity probes with a reporter molecule that can be easily read out, e.g., with an optical light detector. Therefore, data collection and analysis is straightforward and lends itself to automation of the process and hence high throughput.

Not too surprisingly, the aforementioned tools of "-omics" for identification of components are widely used and can provide a large amount of information about a biological system, resulting in an ever-increasing parts list. However, knowing what components constitute such complex systems only gets one so far in understanding the inner workings of the system. A deeper understanding requires knowledge on how the parts fit together, in other words what their spatial and temporal relationship is, which components interact with other, and whether there are additional organizational levels that transcend the individual components. As an analogy, any complex machinery such as a car is more than the sum of all its constituent components (e.g., engine, wheels, brakes, a steering wheel, and power transmission). Thus, higher-level 3D organization of individual components can lead to new function, and 3D organization manifests itself at different levels. Naturally, it is not enough to understand the combustion engine; we also need to understand how this engine is connected to the wheels. Hence, we need to understand organization at different levels of scale and complexity, ranging from pistons and valves in the engine to the level of the entire car.

The biological equivalent is the anatomy of a living organism, which describes the system at the organ level, whereas histology examines the various tissue types that constitute the organs, and cell biology examines the complex system at the level of individual cells (and possibly a small group of individual cells), subcellular level of organelles, and supramolecular assemblies like the cytoskeleton. Determining the 3D structure of individual macromolecules (e.g., proteins and nucleic acids) and macromolecular machines (e.g., ribosomes and transcription machinery) typically falls into the repertoire of structural biology, which aims for atomic-resolution insight into the protein architecture of, e.g., an enzyme, and thus a chemical understanding of its enzymatic activity. There are good arguments to consider structural biology a part of bioimaging, and therefore the major techniques for structure determination are discussed in this volume, but a purist may point out that strictly speaking, techniques like NMR-spectroscopy and X-ray crystallography (and other scattering techniques) are not imaging techniques, at least not in a stricter sense of the word. Similarly, mass spectrometry in itself is not an imaging technique; however, images can be created from the information that is being recorded.

Layout of the Book

The layout of this book is guided by the idea that basic concepts, which typically have their home in disciplines like physics, (bio-)chemistry, biology, or computer science/mathematics need to be conquered first to truly appreciate how the different imaging disciplines are built on these basic concepts. We then examine how these basics concepts are used for each imaging approach.

A desired outcome would be for the reader to realize the many commonalities of different imaging techniques, which are often only separated from each other by the type of radiation that is used (e.g., particle radiation versus photons) or by which narrow band of the electromagnetic spectrum is being used for imaging. Ideally, the reader would also realize the common challenges that many imaging techniques are trying to overcome in their own way, be that with respect to signal detection, scale, complexity, and resolution.

Another important aspect in the layout of this book is the realization that different imaging techniques have substantially different goals. There are imaging techniques that provide information about the exact shape of individual macromolecules and supramolecular assemblies, where we aim for high-resolution 3D structures, whereas other approaches may not at all be interested in shape. Instead, they may be interested in spatiotemporal organization and macromolecular vicinity of such macromolecules.

Also, different levels of complexity exist: the most fundamental level of cellular function consists of small inorganic and organic molecules (e.g., metabolites) and macromolecules (e.g., proteins, nucleic acids, carbohydrates, and lipids). Thus, knowledge about their identity, presence, abundance, location, spatiotemporal distribution, and kinetics as well as regulation stands at the center of focus. 3D structural information may yield atomic-level insight about mechanisms of function.

The next higher level of organization are macromolecular machines and supramolecular complexes that typically underlie defined cellular function such as metabolism, replication, transcription and translation of genetic information, cell division, cell differentiation, signaling, and cell death, to name a few important cell biological phenomena.

Yet another level up; such processes often occur in defined cellular compartments. Hence, such processes ideally need to be studied in their native subcellular region and organelle context. However, such environments are often ill-defined with respect to the presence, abundance, and/or location of other macromolecules. For this reason, much progress of such cell biological processes has been made in reconstituted in vitro systems, where different parameters (such as composition and concentration) can be more easily controlled.

For some processes, e.g., cell-cell adhesion (to name an example), one may assume the presence of certain macromolecules based on the cell biological models that have been established for cell adhesion, but the presence of certain macromolecules known to be involved adhesion does not guarantee an intact, functional cell-cell adhesion complex. Hence, both the spatiotemporal localization of suspected cell-cell adhesion proteins to cell-cell adhesion sites and the actual ultrastructural organization of a cell-cell adhesion site need to be studied.

Therefore, regarding the layout of this book, the sequence of the chapters discussing the different imaging techniques also reflects differences in complexity when going from the level of macromolecular complexes to the levels of cellular organelles,...