Posted on 08 August 2011

Book Review

This Handbook is clearly written, comprehensive in its discussion, illustrated with clear colored figures, and contains a critical analysis of the limitations, artifacts, and assumptions in both the experimental protocols and the theoretical analysis of the data. The contributions are from laboratories that cover a range of research interests and are located in Asia, Europe, and America. Perhaps a second edition could be augmented by including the spectacular advances in theory and experimental technique that are occurring in the field of materials science.

The Handbook appeals to a wide variety of readers from such formerly disparate, but now increasingly integrated fields as chemistry, biology, and biophysics. I concur that the suggested audience consists of advanced undergraduates and graduate students.

Why study single molecules? Researchers have used spectroscopic studies to investigate atoms and molecules in the gas phase, in solutions, in glasses at low temperature, and in two- and three-dimensional lattices. Each of these phases contained large numbers of molecules, each with different velocities, orientations, rotational, vibrational and electronic states, molecular configuration, intermolecular and intramolecular interactions, and solvent-molecular interactions. What was studied was an ensemble average over all of these states during the time of data acquisition. As the editors state in their preface, "through the removal of ensemble averaging, distributions and fluctuations of molecular properties can be characterized, transient intermediates identified; and catalytic mechanism elucidated....direct records of the system's fluctuations provide dynamic and statistical information that is often hidden in ensemble-averaged results."

The Handbook's single-molecule content can be divided into several broad areas of research investigations: particle tracking, imaging in live cells, Förster resonance energy transfer (FRET), enzymology, rotatory molecular motors, and their manipulation with optical traps, magnetic tweezers, and atomic force microscopy. While the end of chapter references point the reader to further insights, and the index is useful to locate specific subjects, the utility of this book rests on the high pedagogical and production quality of the individual chapters. The content of the Handbook is uniformly high. The chapter of stochastic optical reconstruction microscopy is clearly written, and it is augmented by the inclusion of experimental protocols. What is missing from the Handbook is a comprehensive chapter that compares and contrasts all the techniques that achieve superresolution microscopy.

Perusal of the Handbook will demonstrate the clear exposition of the concepts, the instrumentation, the theory, the analytical techniques, the limitations of both theory and techniques, the validity of the results, and the strong integration of high-quality figures that are exceptionally well designed and clearly explained in the figure legends. Another exemplary feature of the volume is the experimental detail that is explicitly described in the text of each chapter. For the reader, this is a superior approach as compared with a cursory description with references to publications for further details. Again, the utility of this volume is in the details that are contained in each chapter. The chapters contain a critical account of both the theory, methods of analysis, and assumptions and the experimental difficulties and constraints of the methodology. The reader of the chapters emerges with an appropriate sense of caution in the performance of the measurements, in the analysis of the experimental results, and in the interpretation of the data.

Single-molecule studies are advancing our experimental and measurement techniques and instruments. There are many confounding processes that can lead to failure and an invalid interpretation of the data. These are indeed difficult experimental techniques, and a high degree of experimental skill is required in order to obtain valid experimental results and to have a high degree of confidence in the interpretation of the results. My reading of the Handbook demonstrates that these confounding processes are discussed in detail and adequately considered in the experimental design and the control experiments.

Experimental techniques to detect the fluorescence of single molecules are limited by the signal-to-noise ratio and the measurements are confounded by background fluorescence and stray light. In fact there are numerous confounding aspects of the instrumentation, the data acquisition, the data analysis, and finally the interpretation of the data that permeate this active field of research. Fortunately, the Handbook is replete with prudent cautions and suggestions to reduce artifacts in both imaging and in the interpretation of experimental results. For example on page 5 the authors suggest the use of β-mercaptoethanol and dithiothreitol to mitigate blinking in dye molecules. The physical-chemical explanation is presented; in this case the dye molecule is trapped in the triplet state, and the relaxation to the ground state results in the blinking. In another more complex example from the first chapter on single-molecule fluorescent particle tracking there is an excellent discussion of the problem of rotational movement, specifically the ambiguity of angular degeneracy. The problem is clearly defined in terms of molecular specificity, and experimental protocols are devised to minimize this ambiguity in the interpretation of the data. In another good example the chapter on precise measurements of diffusion by fluorescence correlations spectroscopy (FCS) is in my opinion a unique exposition of the potential optical and physical artifacts that plague the technique and how dual-focus FCS can mitigate or circumvent them. Similarly, the chapter on single-molecule manipulation using optical traps contains a rigorous discussion of practical experimental considerations to understand the sources of error, how to compare optical trapping results with results from other methods, and how to ensure that "real" signals are measured. In summary, among the strengths of this Handbook are the multiple discussions of artifacts and errors that can compromise the validity of the investigations and protocols and techniques that mitigate them.

The field of single-molecule biophysics is highly integrated with and is strongly dependent on four other disciplines: molecular spectroscopy, photodetectors (both point detectors and wide-field detectors), digital signal processing, and probe chemistry and photochemistry. This is why the field requires a team of researchers and engineers that cover this skill set and the capability to work in an interdisciplinary environment to advance the field. In order to further the development of the field it is necessary to make advances in all of these disciplines as they impact single-molecule biophysics. A corollary to this statement is that each of these disciplines can introduce artifacts into the experimental data and images as well as their interpretation. This book performs an excellent task of focusing on the latter in order to mitigate these problems. The chapter on single-molecule imaging in live cells is a good exemplar with its comprehensive analysis of probe construction, expression, and photochemistry.

I encourage the reader to peruse the highly recommended Handbook and extract a highly cautious and skeptical approach to the experiments, the data, and the interpretation in this fascinating field of research.