Edited by Brian Matsumoto Academic Press (2002) 507 pages. ISBN 0-12-580445-8 £62.50/£89.95 (paperback)
If motorists drove their cars the way many cell biologists used their confocal microscopes, our highways would be littered with twisted wrecks of automobiles and the mangled corpses of run-over pedestrians. Everyone recognizes that driving a car is a tricky procedure that requires months of training and practice as well as a formal licensing system to verify that the required skills have been attained. In contrast, many cell biologists seem to think they can just sit down in front of a microscope and start pressing buttons without necessarily knowing what they are doing, in spite of the fact that a confocal microscope is probably at least as complicated to operate as a car. Open any cell biology journal, and you will see the grisly evidence:grainy, speckly images in which cellular structures can sometimes be discerned, but only if you squint. As with poor driving, the keys to solving this problem are education and enforcement. Since we do not have Cell Biology Police to arrest people who misuse their microscopes, education remains the best option, and Cell Biological Applications of Confocal Microscopy,edited by Brian Matsumoto, is an attempt to educate the cell biology community in the proper use of a confocal microscope for their purposes.
This is a not a trivial task, because the challenges that arise in using confocal microscopy to image cells are serious. Confocal microscopy attains its high resolution and 3D imaging by excluding out-of-focus light by using a pinhole, thus essentially throwing away many of the photons generated by the fluorescent sample. Moreover, most current confocal systems still rely on photomultiplier tubes, which are very poor detectors in terms of quantum efficiency (that is, the fraction of photons that are actually converted into a useful signal). For a huge, screaming-bright sample like a Drosophila embryo, imaged at low magnification, these problems are very minor because the sample emits so many photons, and any idiot can obtain a nice confocal image from such samples. Cells are another story. Because cells are so tiny, there are only so many fluorescent molecules that one can put into each one (either by immunofluorescence or by in vivo GFP labeling). Consequently, the amount of light emitted by a cell is strictly limited. With a pinhole excluding half of the light, and the photomultiplier throwing out the other half, one ends up with only a small number of photons per pixel in the final image. High resolution is achieved at the expense of incredibly high statistical noise in the image. The result: a grainy speckly image that doesn't tell us much about the sample and is actually less informative than a standard non-confocal fluorescence image.
Because confocal imaging of cells, particularly of living cells, forces one to face these severe trade-offs between resolution and photon statistics, it is essential to understand how the instrument operates. Chapter 1 of this book provides a general overview of confocal instrumentation as well as detailed descriptions of many different commercially available confocal systems. In addition, chapter 4 addresses some of the theoretical aspects of confocal imaging that are of critical importance in cell biology applications; chapter 5 discusses fluorophores and laser light sources, and chapter 6 discusses selection of objective lenses. These chapters are the most useful in the whole book because they are very general. A slightly more specialized instrumentation topic is tackled in chapter 3, which covers two-photon microscopy, a technique rising in popularity (although mainly used for large thick samples and therefore of unclear relevance for most cell biologists). Another specialized type of instrumentation is described in chapter 2, namely,spinning-disk confocals that incorporate microlenses. These new microscopes promise to revolutionize confocal imaging in live cells by allowing a highly efficient CCD camera to detect the light. Taken together, the chapters on instrumentation are thorough and extremely useful, and even if the book didn't contain any other information, these chapters alone make it well worth owning.
The next part of the book covers the special issues that arise when imaging specific cell types. This is a good idea but unfortunately was not carried out in an entirely systematic way. Only a few randomly chosen organisms are covered, namely, vertebrate histological sections, Tetrahymena,Drosophila embryos and Xenopus oocytes. What about cell culture?What about plants? What about yeast or worms or zebrafish? I was disappointed by the spotty coverage of cell types, mainly because these chapters are otherwise excellent. The details given are extremely explicit and thorough, so it would have been nice to see similarly thorough discussions of a wider range of cell types. Perhaps having complete coverage of all model organisms would have made the book too long. In any case, for those potential readers who are interested in the model systems covered, these chapters will be extremely practical and useful, since they cover all aspects of microscopy including sample preparation and mounting.
The book also contains three chapters on using confocal microscopy to make biophysical measurements within cells, using indicator dyes for pH, calcium and membrane potential. These topics are a bit specialized, but for someone who wanted to do this type of imaging it is handy to have them discussed all together in one place. A final chapter covers the practical concerns that face someone running a shared user imaging facility.
Overall, Cell Biological Applications of Confocal Microscopyshould be considered required reading for all cell biologists using confocal microscopy. It is the most up-to-date work on the subject currently available,and its availability in a relatively low cost paperback edition is a big plus.