Book Review
Microscopy Microanalysis AND
doi:10.1017/S1431927609990936
Š MICROSCOPY SOCIETY OF AMERICA 2009
Handbook of Cryo-Preparation Methods for Electron Microscopy. Annie Cavalier, Danielle Spehner, and Bruno M. Humbel ~Eds.!. CRC Press, Boca Raton, FL; 2009, 682 pages. ISBN 978-0-8493-7227-8
One indication that electron microscopy of whole cells and tissues is enjoying a renaissance is the number of new books being published on the subject ~Kuo, 2007; McIntosh, 2007; Allen, 2008!. This is at least the fourth methodological book in the last two years to focus on biological electron microscopy of cells and tissues. While each of the other books includes chapters on cryotechniques, the distinction of this book is that it is almost entirely about cryo-preparation methods. The book is divided into five subsections: Part 1, Cryo-Fixation Methods; Part II, Cryo-Electron Microscopy; Part III, Low-Temperature Embedding; Part IV, FreezeFracture and Metal Shadowing; Part V, Analysis. This review will first consider some overall aspects of the volume such as the format or layout for the chapters, the illustrations, and references to other sources of information. I will then discuss each subsection in more detail, and finally I will consider whether this book and those like it are really the best approach to use for this kind of learning. The chapter format in the book is a good one, much superior to the usual methods-oriented book. Each page is divided into two columns: the left column is used for text and images and the right column is for figure legends and additional comments about the text. The illustrations are usually ample for most chapters and are a combination of photos, micrographs, line drawings, tables, and computergenerated images. While the idea is good, the execution is not always so. In general, the quality of the photo reproduction is poor, resulting in somewhat dark, low-contrast, low-magnification images that make it difficult to see what is being illustrated. A few of the images such as Figures 2.2 and 2.4 are so grossly out of focus that it makes you wonder how they passed editorial review. The editors have provided each author with a chapter outline or template so there would be some consistency of form from chapter to chapter. The contents of each chapter include the following subtopics: Principles of @chapter subject#, Summary of the Different Steps, Materials and Preparation, Protocol, Advantages/Disadvantages, Why and When to Use a Specific Method, Observed Results, and References. This worked reasonably well, though differences in writing style still emerge with some authors writing in a clear narrative style and others simply making a list of facts and observations. The latter style is fine for the purely descriptive chapter subtopics but is not so helpful in the Introduction and Why and When to Use a Specific Method. With a
few exceptions these sections of each chapter were disappointing. In what seems likely to have been an editorial decision, there was very little effort to indicate other sources of information about these techniques from the literature. I realize that these chapters are not literature reviews, but still, I think you owe it to your readers to point out a half dozen or so of the most recent and/or important writings on the subject that are available elsewhere. In the end this style of referencing makes many of these chapters much less useful than they might have been. What you learn from many chapters of this book is how a particular lab does a particular technique on a particular type of sample. You have no idea how many papers are out there on the same topic but with a different point of view or for different types of samples. A little guidance from these experts on other sources of information would have been useful. Part I on Cryofixation Methods includes a chapter on slam-freezing, two chapters on plunge-freezing ~one for manual methods and the other for the Vitribot device!, and two chapters on high-pressure freezing ~one for the Bal-Tec HPM 010 machine and the other on the Leica EM PACT!. The chapter by DeCarlo on plunge-freezing may be the most generally useful because it is not tied to a particular commercial instrument. If you happen to own a Leica MM80 impact freezer, a Vitribot, Bal-Tec HPM 010, or Leica EMPACT high-pressure freezer then the other chapters will be of interest. It is curious that no one saw fit to mention the Wohlwend HPF 01 Compact high-pressure freezer, a commercially available model very similar to the Bal-Tec instrument that uses all the same types of specimen holders. On the other hand, this is typical of the narrow focus of many chapters. Part II is on Cryo-Electron Microscopy, or the observation of frozen samples in a cooled electron microscope. Some of the chapters in this section are a difficult fit for the theme of the book because their subject matter is so complicated that learning the methods would require far more space than is available. For example, Leis et al. wrote about Cryo-Electron Tomography, but the only cryopreparation technique they describe is putting fiducial markers on the samples. Likewise, the chapter on vitreous cryosectioning contains a lot of useful information about the process but almost certainly not enough detail to actually get cryosections. One cannot really blame the authors because it would simply take a lot more space than they were probably allotted. 469
470
Kent McDonald
Low Temperature Embedding is the theme of Part III and includes chapters on Freeze-Substitution, Cryofixation followed by rehydration for Tokuyasu sectioning, and freezedrying and embedding of biological material. These are all cryotechniques where new information is quite welcome. The chapter on Freeze-Substitution is especially good, and not surprisingly because Bruno Humbel has been working on this topic since the mid-1980s. Furthermore, this is one of the few chapters in the book where a historical context with references is provided for the topic and the illustrations are at a magnification that is meaningful. The other two chapters are also well-written and will be useful to those interested in the Tokuyasu and freeze-drying methods. There are only two chapters in Part IV, which is entitled “Freeze-Fracture and Metal Shadowing.” One is about biological material and the other about self-assembled materials in organic solvents. They demonstrate very clearly the difficulty shared by many other chapters in this book of trying to condense a very complicated subject into a few pages. The result is a sketchy outline of the freeze-fracture process for only one type of sample. In the case of the biological sample, it is “. . . a hydrated suspension of particles ~virus, membrane, macromolecular complexes!. . .”. One would hope to see some good illustrations of these subjects to show the power of freeze-fracture, but sadly they are lacking. In the references there are only two papers more recent than 1987, and neither one of those is from this century. The final section of the book ~Part V! is about “Analysis,” but it is hard to know what the editors mean by this category. It is somewhat of a catch-all, including three chapters on cryopreparation techniques ~progressive lowering of temperature dehydration, cryosectioning according to Tokuyasu, and cryopreparation procedures for elemental imaging!, three chapters on immunolabeling, and one on electron tomography. Most of these chapters are well written and will be useful for those interested in the topics. The chapter on Electron Tomography has little room for detailed practical advice but serves as a good introduction to the general topic. Finally, the historical, sociological, and economic observations of Hellmuth Sitte in the chapter “Final Considerations” are interesting to read. One can say the same thing about the opening chapter of the book, written by Jacques Dubochet, who gives a very readable account of water, the remarkable substance that one needs to know about to understand cryopreparation techniques. This book will certainly provide an introduction to some cryomethods for beginners, and there may even be
some new information for researchers experienced in cryopreparation methods for biological electron microscopy. But with a little extra effort it could have been an even more useful reference. Overall, the book lacks a broader vision that fails to provide enough links to other sources of information on these techniques. With a few exceptions, the literature citations are generally not recent, as shown by the fact that the median value reference date for all citations is 15 years old ~1994!. Even with that, there are no references to two earlier books ~Robards & Sleytr, 1985; Echlin, 1992! that comprehensively cover low-temperature biological electron microscopy methods and are excellent guides to the earlier literature. These earlier reference books also provide basic information about the properties of materials ~thermal conductivity, specific heats, melting points, etc.! that are useful to know in thinking about cryomethods. It is difficult to say how valuable this book may be to researchers using cryotechniques. In this day and age, it would be more useful to have downloadable instructional videos for learning these techniques. Likewise, if these chapters are not available as PDF files through libraries ~see, e.g., Kuo, 2007; McIntosh, 2007; Allen, 2008!, then their impact will also be limited. In the end, this book will serve as a limited introduction to these topics, but do not expect much guidance to the vast literature that exists for some of them.
R EFER ENCES Allen, T.D. (Ed.) ~2008!. Introduction to Electron Microscopy for Biologists ~Methods in Cell Biology, Vol. 88!. San Diego, CA: Academic Press. Echlin, P. ~1992!. Low Temperature Microscopy and Analysis. New York: Plenum. Kuo, J. (Ed.) ~2007!. Electron Microscopy: Methods and Protocols ~Methods in Molecular Biology, Vol. 369!. Totowa, NJ: Humana Press. McIntosh, J.R. (Ed.) ~2007!. Cellular Electron Microscopy ~Methods in Cell Biology, Vol. 79!. San Diego, CA: Academic Press. Robards, A.W. & Sleytr, U.B. ~1985!. Low Temperature Methods in Biological Electron Microscopy ~Practical Methods in Electron Microscopy!. Amsterdam: Elsevier.
Kent McDonald Electron Microscope Lab University of California Berkeley, California