Microscopy techniques


Methods in plant electron microscopy and cytochemistry

Ed. by William V. Dashek. xi + 300 pages. Totawa, NJ, USA: Humana Press, 2000. US$ 89.50; £67.50 p/b. ISBN 089603 589 1

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There is certainly scope for more books on microscopic techniques for studying plants, because most of the ‘standard’ methods were developed for animal tissues and often give poor results with plant tissues. A new book on plant techniques could be useful to everyone from schoolteachers planning plant biology classes to senior researchers needing to acquire specific new skills. Does this book fulfil this potential? The answer, as so often, is yes and no. Yes, because of the several excellent chapters which give all the information required for using the methods described. But on balance, no. It appears that the hard-pressed editor, valiantly trying to meet publication deadlines but failing to extract the promised chapters from a number of colleagues, was forced to step in late to make up the shortfall: he is author or coauthor of almost half the main chapters and coverage of the subject is very uneven.

There are some startling omissions. Open up any issue of a plant journal which has a molecular slant, and every second or third paper features illustrations of in situ hybridizations of riboprobe to transcript, a key technique in the study of tissue specificity of gene expression. Both hybridization to tissue sections followed by light microscopy, and tissue print analysis, have been used extensively. But except for fleeting mentions, RNA detection isn’t covered at all in the light microscopy section, and is given only a brief note in the final chapter, ‘EM and molecular biology’, insufficient to enable anyone to use the method. Moreover, there’s more on detecting bacterial surface antigens than on plant RNA.

Another microscopy technique which has revolutionized plant cytology is the application of fluorescence in situ hybridization (FISH) to plant chromosomes, using either total genomic DNA (GISH) or specific probes. Again, nothing in this book: studies on chromatin are limited to a brief description of 4,6-diamidino-2-phenylindole dihydrochloride (DAPI) staining for nuclei, and a couple of pages outlining SEM observation of chromosomes, visualization of DNA helix and nucleosomes, and determination of chromatin loop size. While the first method is described for Vicia faba, the latter two are methods developed for chicken erythrocytes and human leukaemia cells, respectively. Will they work for plant (or fungal) material? We aren’t told. There’s nothing at all on cytological/microscopic techniques for looking at meiosis.

Now I come to my biggest criticism. Three of my colleagues (plant biochemist, physiologist and molecular biologist) independently considered the defining features of a plant (as opposed to other eukaryote) cell to be, in different orders, chloroplasts/plastids, cell walls, and vacuoles/tonoplasts. Except in the introductory chapter, there is hardly a mention of any of these compartments. The chapters on topics such as fluorescence microscopy of aniline blue stained pistils, dark-field microscopy and its application to pollen tube culture, and isolation and characterization of endoplasmic reticulum from mulberry cortical parenchyma cells are more suited to a methodological journal or the proceedings of a methods workshop: someone wanting to use dark-field microscopy to study pollen tubes wouldn’t buy this book just for the relevant chapter, and those of us who don’t work on pollen tubes aren’t going to gain much (except a couple of attractive pictures). I imagine that some people who assumed that they were investing in a work devoted to higher plants might feel similarly about ‘Scanning electron microscopy – preparations for diatoms’, although diatoms are so breathtakingly beautiful that I can’t see enough of them. Some equivalents from higher plants would have been welcome too – how about SEM studies of meristem/floral development, a technique which is often very informative? More to the point, I expected chapters on the study of plant cell walls, vacuoles/tonoplasts, and, most importantly, plastids. In the otherwise useful ‘Identification of isolated organelles’, a table of marker enzymes for identifying particular organelles includes Golgi apparatus, plasma membranes, ER, secretory vesicles, intact vacuoles and tonoplast, but omits mitochondria and plastids.

A few other gripes before I come to the positive points. Several errors have slipped through at the proof stage. Some of the diagrams, which have been reproduced from other publications, are confusing because the text doesn’t tie in with them very well. But more worrying are safety issues, especially important in a book which might be used in schools and in the design of undergraduate practicals. In several chapters coverage was reasonable, but I was somewhat taken aback by a figure showing the method for preparation of stripping-film autoradiographs, in which a microscope slide is manoeuvred under water to pick up a piece of stripping film. Although this slide would carry specimens radiolabelled with tritium, 14C or even a higher-energy isotope, the worker’s hand is ungloved. The radiation hazard apart, the ribonucleases on the worker’s skin would damage the specimen if RNA were the macromolecule of interest. Still on safety, I was surprised to read that radioactive waste must be ‘further broken down into solid waste, liquid waste and animal carcasses to aid in its proper disposal’. How many radioactive animal carcasses does the average plant or fungal research lab produce? Flies which have fallen into the stock isotope solution, maybe? Or graduate students who have failed to observe proper safety precautions?

So what’s good about this book? Many of the chapters are excellent. The first gives a good general introduction to plant cells and tissues – useful to an animal biologist or hardline molecular biologist needing to get up to speed on plant structure and composition. Chapters 2–4 contain a great deal of information about localization of chemicals in plant tissues, radioautography, and fluorescence methods. ‘A short introduction to immunocytochemistry and a protocol for immunovisualization of proteins with alkaline phosphatase’ could certainly have been longer, but would be extremely helpful to those wanting to implement the methods in their own labs. It is well illustrated and gives sensible tips about choice of controls and how to get started (choose a simple method; consult your colleagues; ideally try the favourite method of a nearby lab or Hospital). My only criticism is that monoclonal antibodies are recommended for overcoming the problems presented by polyclonals. Often, because each monoclonal by definition recognizes only one epitope, the epitope in question, exposed in the denatured protein used to raise the antibody, is inaccessible in the tissue section being challenged. Hence a monoclonal will sometimes work well with western blots but not in immunolocalization. The reader could do with a warning about this. Several of the electron microscopy sections are also useful: the one about immunogold localization for EM is particularly good on controls to avoid artefacts, although protocols should be more detailed. Since I didn’t know much about microanalysis, I found the coverage of the principles and applications of the different types of analytical instrument (electron probe microanalysis, particle induced X-ray emission (PIXE), laser microprobe mass analyzer (LAMMA), electron energy loss spectroscopy (EELS), secondary ion mass spectrometry (SIMS)) informative. Although the title of Chapter 15 is ‘Methods for atomic force and scanning tunneling microscopies’ it is actually an introduction to the principles and a summary of some recent applications, which direct the reader to further information.

Finally, my favourite chapter, on computer-aided microphotometry (CAM), is a systematic guide to everything you need to know about using a photometer attached to a light microscope for computer-aided image analysis and quantification of cell components, whether you’ve just acquired a ready-made commercial kit or want to construct your own. I loved the thought that a teenage nerd might be inspired to turn her, or his, school’s ancient microscope and a mid-range PC into a fairly sophisticated analytical facility. The components of a generic CAM system, with or without epifluorescence and polarized-light capability on the camera; illumination, shutters (real or virtual), photometers and monochromators; choice of interface and software are all covered. Programming is described using BASIC, and although this language might not be many people’s choice, the concepts transfer well enough. The section on image analysis is excellent. I thought the description and illustrations of image matrix smoothing using a kernel should be required reading for anyone working with computer images. At the end of the chapter is a troubleshooting guide, something which would have been useful elsewhere in the book. Throughout, the style is clear, readable and witty, and I like the author’s attitude, summed up in his remark, ‘For those with minimal funding, this is a way to have some fun and do good science’.

Do I recommend this book? The value of individual chapters would make it a useful addition to an Institutional library, but I can’t recommend it for individual purchase because it doesn’t cover all the main areas of plant electron microscopy and cytochemistry by a long way.