© 2003 The Royal Microscopical Society

Journal of Microscopy, Vol. 211, Pt 3 September 2003, pp. 262–265

Blackwell Publishing Ltd.

Book Reviews

Fluorescence Probes in Oncology

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By Elli Kohen, ReneSantus and Joseph G. Hirschberg. Published by Imperial CollegePress, London, 2002. Hardback, £87.00. ISBN 1-86094-150-8.

The preface to this volume illuminates some of the historybehind its conception and explains how the authors firstbegan to search for what they describe as their ‘golden fleece’of fluorescent probes in the mid-1990s. At this time theyfelt that the technology and instrumentation for imagingand analysing fluorescently labelled cells and tissues far out-stripped the sophistication of the fluorescent probes thenavailable. The current volume describes in depth the verydifferent state of the art almost a decade later.

The book opens with chapters on ‘The nature of light’ and‘Introduction to photophysics’. Here the often complex theoryunderlying fluorescence is explored. This is followed by a sec-tion in which the various types of chemical and biolumines-cence, including chemiluminescence, bioluminescence and greenfluorescent protein (GFP), are covered. Chapter IV describesmethods and instrumentation relevant to use of fluorescentprobes. Spectrophotometers and cell sorters, different types ofdetector and approaches to analysis of data are described.Tools such as Forster resonance energy transfer (FRET), multi-probe fluorescence

in situ

hybridization (FISH), photodiodearray fluorescence spectroscopy, confocal microscopy, fluores-cence lifetime imaging (FLIM) and fluorescence recovery afterphotobleaching (FRAP) are outlined, along with examplesof studies in which they have been used. A further chapteroutlines what are described as new techniques, such as totalinternal reflection fluorescence (TIRF), scanning near-fieldoptical microscopy (SNOM), Fourier interferometric stimula-tion (FIS), light fluorescence quenching (LQ) and stimulatedemission (SE). This chapter also includes detailed descriptionof fluorescence

in situ

hybridization (FISH) and related tech-niques such as comparative genomic hybridization (CGH),multifluor FISH, spectral imaging and spectral karyotyping(SKY), rather mainstream fluorescence techniques in oncol-ogy that I found surprising were included in a chapter entitled‘New methods’. Chapter VI entitled ‘Fluorescent probes’, atover 300 pages in length, represents almost half of the book. Itis an authoritative and exhaustive catalogue of the fluorescentprobes currently available with referenced examples of studiesinvolving their use. The final chapter is entitled ‘Applicationsof fluorescence techniques to processes in normal andpathological cells’, and whilst not claiming to be comprehen-sive in coverage, it describes a number of interesting studies.Each small section in the book is referenced, giving the readerimmediate access to the relevant research literature.

This book is not an easy read, but rather a reference text. Inview of this, it is a pity that the ordering of material appearsillogical in places, and the indexing is extremely limited, mak-ing specific information difficult to locate. The contents pagestoo – 17 of them in all – are too sprawling to be helpful. Ifelt throughout the book that it was a disappointment thatthe glory of this particular subject area, the astoundinglybeautiful and informative photomicrographs yielded by labell-ing with fluorescent probes, were largely rendered in mono-chrome and that the coloured plates that were included wereeither ill-chosen (for example, graphs and flow diagrams thatwould be fine in black and white), or poor quality (many platesappear to be reproductions of rather fuzzy conference slides)or, sadly, most usually, both. I would, however, support theclaim made on the cover that this book is unique. I certainlyam unaware of another like it. It provides detailed and com-prehensive descriptions of the extraordinary array of fluores-cent probes and approaches to their application and has beenwritten by pioneers in this specialist field. It appears to be aculmination of their many years of careful research and, asstated in the introduction, represents a landmark in the reali-zation of their long-held dreams. It will provide a referenceguide for those requiring such specialist information. The title‘Fluorescence probes in oncology’ is misleading, in that thebulk of the volume is not directed specifically at oncology, and,indeed, most of the examples supporting the text are drawnfrom studies unrelated to cancer biology. It will be of interest tothose outside the field of oncology and perhaps ‘Fluorescenceprobes in cell biology’ might be a more accurate description.

S

usan

B

rooks

Oxford Brookes University

? 2003212?Book reviewsBOOK REVIEWSBOOK REVIEWSBook reviews

Microprobe Characterizations of Optoelectronic Materials

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Edited by Juan Jimenez. Volume 17 of the Series ‘OptoelectronicProperties of Semiconductors and Superlattices. Taylor and Francis,2002. Hardback, £175.00, 816 pages, ISBN 1-56032-941-6

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The title of this book promises rather more than it actually delivers.It provides a good ‘snapshot’ of some microprobe and micro-structure characterization performed on semiconductors before2000, especially on quantum well and superlattice materials.In a sense, this is very much in keeping with the intention of thisvery good series, which has provided such a good source of ref-erence and service for workers in this topical and fruitful area,but it is strange that what appears to be a delay of over two yearstook place between completion of manuscripts and publication.

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The opening chapter on photoluminescence microprobesis excellent, with a great deal of practical detail that is usefulbeyond the field of the main families of materials of the series.The powers and limitations of microscopy and topographyvariants of photoluminescence are clearly illustrated byexamples. The power of micro-Raman measurements is alsowell illustrated in the second chapter with examples of itsuse to investigate perturbations to structure, chemicalcomposition, doping, strain and temperature distributions indevices.

The chapters on near-field scanning microscopy (NSOM)and scanning tunnelling microscopy (STM), whilst good fromthe point of view of the potential users drawn from the low-dimensional epitaxial semiconductor community, do not gointo the physics underlying the techniques in enough detail. Itis a little disappointing not to see more discussion about thechoice of metallization of the tapered fibres used in NSOM andthe contrast mechanisms in STM at different bias conditions.Examples abound, all drawn from the very restricted ‘epitaxial’area, and there is nothing in the entire book related to free-standing quantum wires or dots. Another chapter does go abit deeper into the luminescence produced in STM and com-paring it with conventional cathodoluminescence but, again,this uses epitaxial quantum dots grown by Stranski–Krastanowmethods, rather than the much better defined materials thatcan be produced by colloidal routes.

There are some good illustrations of interfaces in superlatticematerial in the chapter dealing with transmission electron mic-roscopy but this is rather let down by a lack of critical descriptionof image formation. A similar remark has to be made about anotherwise good chapter that deals with X-ray topography. It issad to see that no mention is made in that chapter about X-raydiffraction and modelling of the superlattice structures.

The final chapter of the book deals with selective etchingand complementary techniques and this does go a bit deeperinto the physics and chemistry, and contains many interestingand intriguing results, probably illustrating the power of usingmore than one technique to probe a material. However,overall, the impression remains that this is not the ideal bookto recommend for learning about microprobe techniques, buta rather useful book for the semiconductor epitaxial layerfraternity.

P

eter

D

obson

University of Oxford

? 2003212?Book reviewsBOOK REVIEWSBOOK REVIEWS

Introduction to Conventional Transmission Electron Micros-copy

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By Marc de Graef. Cambridge University Press, 2003.Paperback, 740 pages, ISBN 0521629950, £39.95; hardback,ISBN 0-52162-006-6, $130.00

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Electron microscopy is still advancing rapidly, I’m glad to say.What then is ‘conventional’? It might mean ‘well-established’

or it might mean ‘staid’ as opposed to ‘cutting edge’. Professorde Graef does not appear to imply either of these things – hemeans that his book deals with imaging and diffraction, butnot analysis, and with TEM (with imaging lenses) rather thanSTEM. It also, as it happens, deals only with inorganic crystal-line materials. Within these clearly defined boundaries thecoverage is both wide and deep and de Graef ’s erudition shinesclearly throughout the text.

There are many excellent aspects of this substantial volume,most of which obviously arise from de Graef ’s experience ofteaching courses on TEM. However, I found myself wonderingat times for whom the book is intended. It calls itself an intro-duction, which implies that there is a lot more to learn, yet Ifound many treatments of topics at a depth that I have notneeded to deal with in almost 40 years as a microscopist andteacher of microscopy. At times I wondered whether the erudi-tion obscured the education. For example, I found 37 namedequations or algorithms, most of which are not householdphrases (Burlisch–Stoer algorithm? Hurter–Driffield curve?).What are these for? How do they help the reader?

My own answer is that the book is intended for physicistswho are intrigued by the fundamental interactions betweenelectrons and crystals. There are several clues that point in thisdirection. I loved (but strongly disagree with) the statementthat ‘An observation in a TEM is essentially an experimentin relativistic quantum mechanics’. My own alternativewould be that ‘An observation in a TEM is essentially anattempt to reveal the structure (micro-, ultra- or nano-),usually the defect structure, of a solid’. Another pointer is thenature of the exercises at the end of each chapter – one exam-ple will suffice: ‘Derive the components of the lens transfermatrix’. A further give-away (again I loved this for its ironicvalue): Chapter 4, starting on page 235, is entitled ‘Gettingstarted’. I don’t know many people who would be preparedto go through 234 pages and 257 numbered equations in an‘Introduction’ before getting started! However, enough of myown prejudices.

There are some really good ideas in this book. de Graef con-centrates on just four ‘study materials’ from which almostall of the micrographs are taken. They are a metal, an alloy, asemiconductor and a ceramic, covering several crystal struc-ture types. He describes how to prepare a thin foil from eachmaterial, and his micrographs and diffraction patterns arestunning and plentiful. He also includes lots of computedimages, together with access, via his website, to the routinesthat are used to calculate them (and the code). He includesphotographs of TEM components from the sectioned columnhe has prepared at Carnegie Mellon. This practical slant iscontinued with a section entitled ‘A typical microscope session’,which will be particularly valuable to a new practitioner.The coverage of diffraction phenomena is much wider than inmost general TEM books and the bibliography is extensive.

This is a difficult book to summarize. It is tempting todescribe it as a curate’s egg, but that implies that bits of it are

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bad. They are not – it is just that different sections of the bookhave quite different target readerships. If you are just startingas an electron microscopist, you need the 65 pages (and onlyeight equations) of Chapter 4. If you are trying to interpret apuzzling diffraction pattern you need the same number ofpages in Chapter 9. The rest of the book is largely for those whowant or need to know the physics that lies behind the imagesimulations, the lens design or the gun brightness. For most ofus the programs have already been written, the lenses arealready in the microscope and we are glad to get what gunbrightness we can at the highest kV our lab could afford. Forthese microscopists this is a reference book – reassuring tohave on the shelf, but I bet you won’t read much of it!

P

eter

G

oodhew

University of Liverpool

? 2003212?Book reviewsBOOK REVIEWSBOOK REVIEWS

Scanning Electron Microscopy and X-ray Microanalysis(3rd edn)

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By J. Goldstein, D. Newbury, D. Joy, C. Lyman, P. Echlin,E. Lifshin, L. Sawyer and J. Michael. Kluwer Academic, 2003.Hardback, 689 pages,

+

76.50/$75.00/£48.00. ISBN 0-30647-292-9

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This is the latest edition of a book that owes its origin to thewell-known Microscopy Summer School run at Lehigh Universityeach year, the book forming the text of the short course inBasic Scanning Electron Microscopy and X-ray Microanalysis.It is almost 700 pages in length with approximately 40% cov-erage devoted to SEM, a similar amount to X-ray microanalysisand the remaining 20% to specimen preparation techniques.Included with the book is a compact disc that supplementsthe information contained within most of the book’s 15chapters.

The introduction outlines how the techniques of scanningelectron microscopy and X-ray microanalysis evolved andthen illustrates the various types of information: topo-graphical, compositional, structural, etc., that can be extractedfrom a specimen with a modern and well-equipped analyticalSEM.

Chapters 2–5 take the reader through the principles ofoperation of the SEM and describe electron beam generation,the different types of electron gun, focusing the beam with elec-tromagnetic lenses, the interaction of energetic electrons withthe specimen, the factors governing the size of the interactionvolume and how electron emissions from the specimen areused to form images. Chapter 4, dealing with electron detec-tors and the construction and interpretation of images, isextremely comprehensive and I found the discussion on therole of the detector and specimen in contrast formation to beparticularly informative. Chapter 5, on special topics, incorpo-rates high-resolution imaging, stereomicroscopy and mag-netic contrast as well as two relatively recent techniques,namely high-pressure SEM (variable pressure and ESEM)

for the examination of hydrated and non-conducting samples,and electron backscatter diffraction (EBSD) for localizedstructural determinations.

The next five chapters cover X-ray microanalysis: the firstbeginning with the generation of characteristic and contin-uum X-rays, leading onwards to the principles of operation ofenergy-dispersive (ED) and wavelength-dispersive (WD) spec-trometers and then to a performance comparison between thetwo spectrometer types. Within Chapter 7 is a brief mention ofsome of the latest detector technologies, encompassing silicondrift detectors, parallel X-ray optical systems and X-ray micro-calorimeters. Chapter 8 explains how one goes about correctlyidentifying characteristic X-ray peaks in ED and WD spectra inorder to perform a qualitative compositional analysis; it dealsalso with methods of background removal and peak overlap,and is very practically orientated. Next, in Chapter 9, the originsof matrix effects in quantitative X-ray analysis are addressedand some examples of the size of the correction factors inmetal and ceramic specimens are provided using ZAF andphi-rho-z modelling. Also included here are standardlessanalysis methods, the Bence–Albee approach for the analysesof geological samples and a discussion of the factors affectingthe precision and sensitivity of compositional measurements.Chapter 10 is on special topics in X-ray microanalysis, encom-passing the analysis of thin surface films, rough surfaces,particles, beam-sensitive materials and light elementstogether with X-ray mapping techniques and low-voltagemicroanalysis.

The final five chapters are concerned with preparingvarious types of specimen for imaging and X-ray microanalysis.The first treats hard materials, e.g. metals and ceramics, butalso deals with these materials in non-standard forms as inparticulates, fibres or fabricated devices such as microelec-tronic circuits. Chapter 12 is on surface preparation ofpolymers and covers polishing, microtomy, fracturing, staining,etching, freeze drying, critical point drying and replicationof surface features. Chapters 13 and 14 are concerned withbiological specimen preparation. In Chapter 13 considerationis given to preparing single cells, organic particles, organicfibres and soft biological tissue for structural study and forX-ray analysis. Chapter 14 is devoted to low-temperaturetechniques and incorporates rapid methods of cooling, pre-paring the surfaces of frozen specimens, handling the frozenliquids within the specimen and the observation and analysisof samples at low temperature. In the final chapter ways ofovercoming charging problems in non-conducting samplesare addressed.

The reference lists are grouped at the end of each chapter, amethod which sometimes leads to needless repetition of com-mon references. However, it seems to have worked well on thisoccasion, and I was impressed by the quality of the lists. It isvery easy to overwhelm readers by referencing every possiblepaper on a subject, but here the authors seem to have achieveda very balanced result by being selective, ensuring that the list

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is up to date but including only those references that are mostimportant whether they be new or old.

I have very mixed feelings about the CD. The authors’ inten-tion was to make the book itself more readable by movingdetailed tabulations and more complex material for theadvanced student to the CD. Additionally, they were able toadd high-quality images such as stereo pairs on the CD. Thishas worked in part. The database of David Joy fits in well hereand his interactive Monte Carlo program is a very valuableaddition for users to experiment with (it is only guaranteedto work under Windows 95 or 98 but should run under 3.1). Inaddition, useful background information on polymers andmuch of the detailed quantitative microanalysis material wasneatly incorporated. However, whereas the contents of thebook were well integrated, each author seems to have donehis or her own thing on the CD. The layout differed dependingon the chapter, and the list of contents was very poor. Anotherproblem I had was that figures and images were kept separatefrom the text, which made for very tortuous reading if viewingdirectly from the computer monitor. In fact, I recommend thatanyone using the CD for purposes other than Monte Carlosimulations or referencing the tabulations works from a down-loaded hard copy. One should be aware also that to read thefiles on the CD a copy of Microsoft Office is required and thatwhen using it not all image files will be visible unless ‘All Files’rather than the default ‘Office Files’ is selected as the file type.

Compared with the second edition the third is slightly shorter(689 pages as opposed to 820) but this is more than compen-sated for by the contents of the CD. The major changes thathave taken place between the two editions are that a separatechapter on polymer preparation has been introduced, dueweight has been given to newer techniques such as ESEM,EBSD, field-emission SEMs, quantitative digital mappingand low-voltage operation. Also, there have been substantialupdates on quantitative microanalysis particularly for com-plex structures. To gain a better idea of how much materialwas new, I glanced quickly through the second and thirdeditions looking for figures that were common to both. Inmost chapters I found that more than 50% had been changed,which emphasizes the scale of the rewrite involved in thisthird edition.

I have only two criticisms of the book itself and both arerelatively minor ones. I felt it would have been useful to havehad the contents of the CD clearly listed within the book sothat readers might readily establish whether the informationthey required was on the CD or not. Also, although the indexwas otherwise comprehensive, the cross-referencing could beimproved in places. For example, Beam and Electron beam – ifone needs information on stability this comes under ‘Beam’whereas interaction volume is under ‘Electron beam’ andsimilar inconsistencies apply to ‘Sample’ and ‘Specimen’.

Overall though, I found the book to be very well writtenwith a uniformity of style not always evident in multi-authored books. It is one of only a few books in which a seriousattempt is made to provide a meaningful balance betweenthe requirements of physical scientists and engineers on theone hand and life scientists on the other. Organization of thematerial was good and coverage of the subject matter appearedcomprehensive to me. I liked the layout too, with the space lefton the right-hand margin to indicate the chapter title givingan uncluttered feel. Illustrations are used effectively through-out and the quality of the diagrams and photomicrographsare almost uniformly excellent. However, the single most import-ant thing that impressed me about the book, as indeed withprevious editions, was its very practical nature. Attentionhas been focused on the basic factors of importance in SEMand microanalysis and the information has been presented tothe reader simply and clearly. The experience of the authorsin teaching the subject over many years is apparent and theresult is a book that is easy and enjoyable to read. It is alsoexcellent value for money, retailing in the UK at less than £50for a hardback copy. The book is too detailed for an undergrad-uate text but I have no hesitation in recommending it to allpost-graduates, scientists and engineers who are involvedin the practice of scanning electron microscopy and/or X-raymicroanalysis. Indeed a copy of this book should be availableas a source of reference in all SEM laboratories. Everybodycan learn something new about the subject from reading it – Icertainly have.

G

lyn

L

ove

University of Bath