Imaging in Neuroscience: A Laboratory Manual
Fritjof Helmchen, Arthur Konnerth, Editors
|Reviewed by Barry R. Masters, Visiting Scientist, Department of Biological Engineering, Massachusetts Institute of Technology. Fellow of AAAS, OSA, and SPIE.|
Imaging in Neuroscience: A Laboratory Manual is a highly recommended book for graduate students and advanced undergraduates who require detailed protocols for optical imaging of the nervous system. To put the book's content in perspective and to elucidate and amplify some of the historical aspects that are lacking in the volume, I briefly summarize the seminal advances of the past in neuroscience. What is intriguing is the modern reappearance of older techniques and the resurfacing of previous problems of artifacts that can obfuscate the correct interpretation of the experiments. With the pioneering works of Camillo Golgi on sparse staining of neurological tissue, Santiago Ramón y Cajal, Wilhelm His, and Charles Sherrington formulated the Neuron Theory. The early pioneers had a prescient understanding of the importance of sparse staining of the cortex. The history of staining neural tissue, from Golgi stains, to methylene blue, to the modern panoply of probes and sensors, runs parallel to that of neuroimaging. The development of the electron microscope together with new methods of specimen preparation visualized the synapse and synaptic vesicles. Biophysical studies of neurons increased our understanding of the electrical and chemical mechanism of neuronal excitation, culminating with the seminal work on the molecular structure of channels and the invention of the patch clamp technique. Imaging in Neuroscience is about optical techniques to investigate the nervous system; and it is built on many previous studies, many of which are not mentioned in this book (for example, see Ungar's Excitation [Charles C. Thomas Co., 1963]).
The book's organization is based on the increasing spatial level of complexity of neural organization: molecules, synapses, cells, tissues, circuits, brain area, as well as studies on whole organisms including development, neuronal plasticity, organism behavior, and brain pathology. What is not covered are the imaging and mechanical slicing techniques to image fixed whole brains, as well as functional magnetic resonance imaging (fMRI) and positron emission tomography (PET).
A variety of imaging techniques are clearly and concisely described in the protocols. Each described technique is associated with its own limitations, artifacts, and problems, which are typically carefully addressed in the book. As every experimenter knows there are experimental trade-offs in instrument design: it is extremely difficult to optimize multiple perimeters; thus there are a variety of imaging techniques in use. Many of the described techniques involve some form of an optical microscope, and thus the questions of spatial (x, y, and z) and temporal resolution must be addressed. While this field is rapidly progressing, image acquisition time can confound temporal kinetics, and the signal-to-noise ratio is a critical factor. An often overlooked and certainly understated problem is the effect of the incident light on the biological specimen; while this is generally acknowledged, there is a paucity of control experiments for each imaging technique to measure this effect and to understand its significance in the interpretation of the results. Protocols are described for imaging studies, from a variety of types of specimens from cells in culture, brain slices, to in vivo organisms, including those studies with anesthesia and those performed in awake, moving animals. The advantages and the disadvantages of performing the studies in each specimen are clearly described in the book.
To derive a consistent understanding of the nervous system in a various organisms, there must be an eventual convergence of structural, functional, and behavioral studies. Perusal of this book demonstrates that the interdisciplinary approaches (chemistry, molecular biology, probe design and insertion, microscope design, behavioral studies, digital signal processing, neurophysiology, as well as clinical studies of brain pathology) all are converging to improve our understanding of the nervous system. The use of correlative techniques, i.e., light and electron microscopy, is an important research approach described in the book.
The production quality of the volume is excellent. There are very clear illustrations, many of which are in color. Most of the protocols include a good selection of references. The references do not always cite the key works of other groups. It would be helpful to have annotated references that are separated into the following categories: peer-reviewed original research articles, review articles, and conference proceedings. The volume is supplemented by short movie clips that are freely available online; they could be better documented and longer in order to enhance their pedagogical utility. As with the other volumes in this series, there are useful appendices. Beside the standard introduction to microscopes and a glossary of imaging terms, there is a very important appendix on cautions. These cautions are not exhaustive, as the reader is warned; nevertheless they are very useful, and before beginning any protocol the reader should be familiar with them. A detailed index is included.
The book is a laboratory manual, and in that function it succeeds very well. The protocols are clearly described, and the sections on troubleshooting and cautions and animal preparations are exemplary. The applications usually demonstrate proof of principle. Methods developments are crucial to the advancement of neuroscience, but more is needed. What would be useful is a clear statement of the aims of the work and the specific questions that the investigator posed. This should be followed by a discussion of what specific questions the specific technical development actually answered, and if possible a comparison of the results obtains with other techniques used by other groups.
A careful reading of the 92 chapters points out the necessity to apply caution to the performance, analysis, and finally the interpretation of the results. Throughout the book authors stress the need to comply with federal, local, and institutional guidelines for the care and use of animals. Many of the protocols also pose significant dangers to the investigators, and in those cases clear warnings are provided. There are excellent and multiple discussions of the advantages and disadvantages of the following techniques: intrinsic versus extrinsic optical imaging, anesthetized animals versus live moving animals, and thinned skull preparation versus imaging through an chronic cranial window. The discussion of a chapter on two-photon imaging provides a clear warning that the investigators must validate the potential effect of two-photon imaging on tissue physiology; often this aspect is ignored or marginalized.
Imaging in Neuroscience: A Laboratory Manual provides a useful guide to explore these exciting advances in imaging techniques.