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Flow cytometry: First principles
MISCELLANEA
,
use of fluorescence microscopes and video cameras, and the analysis of images to obtain dynamic information from the observation of living specimens, as some of the most important techniques in modern cell biology. The book intends to give a survey of this technology and to describe the kinds of applications and in. terpretations that can be addressed using optical microscopy. It also tries to give us an idea of the future potential of optical microscopy. The basic issues are well presented and in sufficient detail for newcomers to the field. The book also provides those scientists already using some Ernst H. K. of the many different microscopy Stelzer techniques with new views and some EMBL, Light ideas on how to improve their Microscopy instrumentation. The first two chapters introduce Croup, Posffach 10.2209, new fluorescent probes and the hot Meyerhofstrasse issue of caged photosensitive com1, D-69012 pounds. Chapters 3, 5 and 6 explain Heidelberg, ratio imaging in great detail. Since Germany. their information overlaps, the edi-
A starter for flow cytometry... Flow Cytometry: First Principles by Alice Longobardi Givan, Wiley. Liss, 1992. £30.50 (xiv + 202 pages) ISBN 0 471 56095 2
With this book, the field of flow cytometry - an intriguing and chal. lenging technique that encompasses ever-widening areas of application has been given the introductory textbook it deserves. Although the book has fewer than 200 pages, it covers much more than just first principles of flow cytometry. It is a complete introduction to the field, emphasizing the necessity to understand the principles and giving a wealth of practical knowledge. The book starts off with two perspective chapters that put the reader right in the middle of the world of the flow cytometrist. These serve appetizers from the history of the development of this successful tech-
320
tors should have been more precise with instructions to the authors to avoid duplication. Chapter 5 contains a lengthy discussion of computers and operating systems that will make the book look outdated in a year. However, the discussion of software issues is well worth reading since they are treated in general terms. Chapter 8 describes the advantages of simultaneously observing DIC and low-light-level images. The instrumental requirements are well explained; problems are pointed out and an application is presented. However, an unnecessary complication for any reader is the presentation of the DIC and fluorescence images inverted top to bottom. Chapter 11 on total internal reflectance fluorescence microscopy is well written, and presents the subject in a comprehensible way. Some of the aspects are of general interest to those working with three-dimensional light microscopy. The chapters on confocal microscopy of living cells, intravital microscopy and time-
resolved fluorescence lifetime imaging can be regarded as descriptions of potential new directions. Ap. plications are presented, but these methods have not yet spread as much as, say, ratio imaging. The idea of performing cell biology experiments inside the cheek of a living hamster or in the proximal tibia of a rabbit is fascinating and is not yet being pursued in many cell biology laboratories, but I think it gives us an idea of what could be done in the future. Combine this technique with confocal fluorescence microscopy, and the observation of dynamic systems under physiologically relevant conditions in three dimensions is a feasible project. It is not fair to expect a single book to cover the development of a technique that has been growing for decades. I think the editors have put a lot of effort into selecting some of the more important techniques and showing us current interests and future directions.
nology, and describe the typical setup of flow cytometry today. A careful tutorial about the most important technical aspects of flow cytometry is given in the next three chapters. The emphasis here is on understanding the important things correctly, thereby preventing misconceptions from taking root. Thus, the reader !earns that the big lasers often found in sorting flow cy. tometers are not there because 'bigger is better' but mostly to compensate for the inefficiency of light collection that is typical of many sorting instruments. Altogether, there is little emphasis on cell sorting. The main principles and the practical aspects such as cell.sorting speed (or lack of it) are we!l explained. However, some important points, such as accurate setting of sort delay and subsequent effects on sort recovery and purity, are not mentioned. I find it understandable that the author spend~ much more time explaining in depth the principles, practices and intricacies of gating, a technique that is of fundamental importance in flow cytomerry, and that most novice users find difficult to understand. From teaching flow cytometry courses, I have found that students often have difficulties grasping the full extent of the information contained in histo-
grams. The author explains the relationship between histograms based on linear and logarithmic scales, but the various aspects of information content to be read from histograms could have been presented in more detail. A clear understanding of how measurement precision can influence a DNA histogram, for example, and the ability to extract the under. iying biol,~gical information from histograms having, say, very different coefficients of variance, may be a good test of the student's degree of understanding. The most important applications of flow cytometry are well documented in the next chapters, with the emphasis on immunophenotyping, DNA measurements for ploidy, and cell proliferation studies. The technique of dead.ceU discrimination based on uptake of propidium iodide stain by dead (membrane.ruptured) cells is found in the chapter on DNA and molecular biology, presumably because it u.ses a DNA stain. This procedure might, however, be more appropriately discussed with other techniques that are used to ensure proper immunofluorescence measurements, which are well described in the previous chapter on immunophenotyping. The following chapter discusses routine clinical applications of flow cytometry, which have
TRENDS IN CELLBIOLOGYVOL. 3 SEPTEMBER1993
MISCELLANEA III
become increasingly numerous as more user-friendly instruments have become available. Important aspects such as quality control, mass data handling, and safe and qualified sample handling, which are critically important for the successful application of flow cytometry in routine environments, are well covered in the book. The last two chapters give the reader some f~eelfor emerging applications of flow cytometry, some of which are clearly linked to instrument performance and fiuorochrome technology (such as microbiology), whereas others depend only on the imagination of the researcher. Personally, I agree with the author's speculation that flow cytometry is about to enter the biochemistry laboratory, where flow cytometric reading of particle-based immunoassays offers several advantages over conventional ELISA assays in microtitre plates. At the end of each chapter the reader gets a summary of what has been learnt so far, and an annotated list of further reading. At the end of the book there are listings of general references such as books, cataIogues, and other useful information, all carefully commented on by the author. Finally there is a glossary of >100 words. Throughout the book, the reader's interest and appetite for knowledge are well catered for by the inviting language and good humour that shine through the text on every page. This book will be a most suitable text for those who are on their way into flow cytometry and perhaps have had some colleagues explain the instrument to them, but nevertheless (or because of that!) need a more systematic tutorial. The book will also be ideal as a textbook for introductory flow cytometry courses; here the students will find most of what the teacher has tried to explain, or should have tried to explain. The author has managed to write a book that introduces the reader to the whole field of flow cytometry, with its history, attitudes, possibilities and problems, as well as its scientific knowledge. It is a book that will entice newcomers into becoming as interested in and as fond of this scientific tool as the 'oldtimers' are, and we are therefore very glad to add this book to the literature of the field and recommend its use.
...and a guide for further analysis Flow Cytometry Data Analysis: Basic Concepts and Statistics by James V. Watson, Cambridge University Press, 1992. £35.00 (viii + 288 pages) ISBN 0 521 41545 4 This book brings together statistics and flow cytometric data characteristics, enabling researchers to understand and perform meaningful statistical determinations based on flow cytometric data. It is written principally for biologists, and assumes little knowledge of statistics or mathematics. The text is in a colloquial style, and the author's 'story-telling' approach, filled with practical detail, spiritual digressions and interesting comments, should serve well to keep the reader's attention and also get him or her to swallow and digest some 'heavy' statistics. The necessary background to statistics is given in the first four chapters, using concrete examples to explain the statistical concepts that underly the rest of the book. Before going into flow cytometric data analysis, a chapter explains in detail the various sources of variability that give rise to the stochastic nature of flow cytometric data: even if all particles (or cells) measured were exactly equal, the measurements recorded by the flow cytometer would be displayed as histograms of a certain width or dispersion, due to random sources of measurement variability. There is also a discussion of other sources of error of a more systematic nature that may influence the results, such as debris, coincidences, colour overlap and so on. Building on these chapters, the main subject matter of the book is dealt with in two chapters, on 'lmmunofluorescence data' and 'DNA histogram analysis'. The chapter on immunofluorescence is especially important since immunofluorescence results have often not been subjected to advanced data analysis. The underlying mathematical concept is that a measured flow cytometric histogram is a combination of the true biological (or theoretical) distribution and a broadening function due to the vari-
TRENDS IN CELLBIOLOGYVOL. 3 SEPTEMBER1993
ability introduced by the measuring process (Gauss broadening). These concepts have for a long time been accepted as the basis for interpretation of DNA histograms, and are implemented in slightly different ways in the well-established meth. ads of DNA histogram analysis, such as multiple gaussian, polynomial fitting and rectilinear integration. These methods are all adequately described, but I feel it would have been better to introduce them in the opposite order, i.e. discuss DNA histogram analysis before immunofluorescence data analysis. The remaining chapters of the book discuss cell cycle kinetics and dynamic cellular events. Some of these are not typical cytometric data, and some are not even measured by flow cytometers, but they represent experimental data that flow cytometrists are likely to encounter, and therefore widen the scope of the book in a worthwhile manner. The last chapter introduces the concept of multivariable analysis, which will be increasingly used for analysis of multiparameter data. The book has nine appendices full of useful statistical and mathematical information. With the advent of user-friendly flow cytometers, insight into the mathematical and statistical tools for analysis of flow cytometric data is necessary to ensure its correct use and interpretation. Biophysicists, like myself, would probably prefer more standard textbooks where the formal mathematical and statistical parts are presented more rigorously. However, this book will prove valuable to biologists and medical doctors interested in flow cytometry and the correct interpretation of the vast amount of data it generates. As such, it serves an important purpose, since flow cytometric data are about to influence diagnostic decisions and therapeutic regimens.
Tore Llndmo BiophysicsGroup, Departmentof Physics,University of Trondheim, NTH, N-7034 Trondheim, Norway.
Please mention
Trends in Cell Biology when replying to advertisements.
321
,
use of fluorescence microscopes and video cameras, and the analysis of images to obtain dynamic information from the observation of living specimens, as some of the most important techniques in modern cell biology. The book intends to give a survey of this technology and to describe the kinds of applications and in. terpretations that can be addressed using optical microscopy. It also tries to give us an idea of the future potential of optical microscopy. The basic issues are well presented and in sufficient detail for newcomers to the field. The book also provides those scientists already using some Ernst H. K. of the many different microscopy Stelzer techniques with new views and some EMBL, Light ideas on how to improve their Microscopy instrumentation. The first two chapters introduce Croup, Posffach 10.2209, new fluorescent probes and the hot Meyerhofstrasse issue of caged photosensitive com1, D-69012 pounds. Chapters 3, 5 and 6 explain Heidelberg, ratio imaging in great detail. Since Germany. their information overlaps, the edi-
A starter for flow cytometry... Flow Cytometry: First Principles by Alice Longobardi Givan, Wiley. Liss, 1992. £30.50 (xiv + 202 pages) ISBN 0 471 56095 2
With this book, the field of flow cytometry - an intriguing and chal. lenging technique that encompasses ever-widening areas of application has been given the introductory textbook it deserves. Although the book has fewer than 200 pages, it covers much more than just first principles of flow cytometry. It is a complete introduction to the field, emphasizing the necessity to understand the principles and giving a wealth of practical knowledge. The book starts off with two perspective chapters that put the reader right in the middle of the world of the flow cytometrist. These serve appetizers from the history of the development of this successful tech-
320
tors should have been more precise with instructions to the authors to avoid duplication. Chapter 5 contains a lengthy discussion of computers and operating systems that will make the book look outdated in a year. However, the discussion of software issues is well worth reading since they are treated in general terms. Chapter 8 describes the advantages of simultaneously observing DIC and low-light-level images. The instrumental requirements are well explained; problems are pointed out and an application is presented. However, an unnecessary complication for any reader is the presentation of the DIC and fluorescence images inverted top to bottom. Chapter 11 on total internal reflectance fluorescence microscopy is well written, and presents the subject in a comprehensible way. Some of the aspects are of general interest to those working with three-dimensional light microscopy. The chapters on confocal microscopy of living cells, intravital microscopy and time-
resolved fluorescence lifetime imaging can be regarded as descriptions of potential new directions. Ap. plications are presented, but these methods have not yet spread as much as, say, ratio imaging. The idea of performing cell biology experiments inside the cheek of a living hamster or in the proximal tibia of a rabbit is fascinating and is not yet being pursued in many cell biology laboratories, but I think it gives us an idea of what could be done in the future. Combine this technique with confocal fluorescence microscopy, and the observation of dynamic systems under physiologically relevant conditions in three dimensions is a feasible project. It is not fair to expect a single book to cover the development of a technique that has been growing for decades. I think the editors have put a lot of effort into selecting some of the more important techniques and showing us current interests and future directions.
nology, and describe the typical setup of flow cytometry today. A careful tutorial about the most important technical aspects of flow cytometry is given in the next three chapters. The emphasis here is on understanding the important things correctly, thereby preventing misconceptions from taking root. Thus, the reader !earns that the big lasers often found in sorting flow cy. tometers are not there because 'bigger is better' but mostly to compensate for the inefficiency of light collection that is typical of many sorting instruments. Altogether, there is little emphasis on cell sorting. The main principles and the practical aspects such as cell.sorting speed (or lack of it) are we!l explained. However, some important points, such as accurate setting of sort delay and subsequent effects on sort recovery and purity, are not mentioned. I find it understandable that the author spend~ much more time explaining in depth the principles, practices and intricacies of gating, a technique that is of fundamental importance in flow cytomerry, and that most novice users find difficult to understand. From teaching flow cytometry courses, I have found that students often have difficulties grasping the full extent of the information contained in histo-
grams. The author explains the relationship between histograms based on linear and logarithmic scales, but the various aspects of information content to be read from histograms could have been presented in more detail. A clear understanding of how measurement precision can influence a DNA histogram, for example, and the ability to extract the under. iying biol,~gical information from histograms having, say, very different coefficients of variance, may be a good test of the student's degree of understanding. The most important applications of flow cytometry are well documented in the next chapters, with the emphasis on immunophenotyping, DNA measurements for ploidy, and cell proliferation studies. The technique of dead.ceU discrimination based on uptake of propidium iodide stain by dead (membrane.ruptured) cells is found in the chapter on DNA and molecular biology, presumably because it u.ses a DNA stain. This procedure might, however, be more appropriately discussed with other techniques that are used to ensure proper immunofluorescence measurements, which are well described in the previous chapter on immunophenotyping. The following chapter discusses routine clinical applications of flow cytometry, which have
TRENDS IN CELLBIOLOGYVOL. 3 SEPTEMBER1993
MISCELLANEA III
become increasingly numerous as more user-friendly instruments have become available. Important aspects such as quality control, mass data handling, and safe and qualified sample handling, which are critically important for the successful application of flow cytometry in routine environments, are well covered in the book. The last two chapters give the reader some f~eelfor emerging applications of flow cytometry, some of which are clearly linked to instrument performance and fiuorochrome technology (such as microbiology), whereas others depend only on the imagination of the researcher. Personally, I agree with the author's speculation that flow cytometry is about to enter the biochemistry laboratory, where flow cytometric reading of particle-based immunoassays offers several advantages over conventional ELISA assays in microtitre plates. At the end of each chapter the reader gets a summary of what has been learnt so far, and an annotated list of further reading. At the end of the book there are listings of general references such as books, cataIogues, and other useful information, all carefully commented on by the author. Finally there is a glossary of >100 words. Throughout the book, the reader's interest and appetite for knowledge are well catered for by the inviting language and good humour that shine through the text on every page. This book will be a most suitable text for those who are on their way into flow cytometry and perhaps have had some colleagues explain the instrument to them, but nevertheless (or because of that!) need a more systematic tutorial. The book will also be ideal as a textbook for introductory flow cytometry courses; here the students will find most of what the teacher has tried to explain, or should have tried to explain. The author has managed to write a book that introduces the reader to the whole field of flow cytometry, with its history, attitudes, possibilities and problems, as well as its scientific knowledge. It is a book that will entice newcomers into becoming as interested in and as fond of this scientific tool as the 'oldtimers' are, and we are therefore very glad to add this book to the literature of the field and recommend its use.
...and a guide for further analysis Flow Cytometry Data Analysis: Basic Concepts and Statistics by James V. Watson, Cambridge University Press, 1992. £35.00 (viii + 288 pages) ISBN 0 521 41545 4 This book brings together statistics and flow cytometric data characteristics, enabling researchers to understand and perform meaningful statistical determinations based on flow cytometric data. It is written principally for biologists, and assumes little knowledge of statistics or mathematics. The text is in a colloquial style, and the author's 'story-telling' approach, filled with practical detail, spiritual digressions and interesting comments, should serve well to keep the reader's attention and also get him or her to swallow and digest some 'heavy' statistics. The necessary background to statistics is given in the first four chapters, using concrete examples to explain the statistical concepts that underly the rest of the book. Before going into flow cytometric data analysis, a chapter explains in detail the various sources of variability that give rise to the stochastic nature of flow cytometric data: even if all particles (or cells) measured were exactly equal, the measurements recorded by the flow cytometer would be displayed as histograms of a certain width or dispersion, due to random sources of measurement variability. There is also a discussion of other sources of error of a more systematic nature that may influence the results, such as debris, coincidences, colour overlap and so on. Building on these chapters, the main subject matter of the book is dealt with in two chapters, on 'lmmunofluorescence data' and 'DNA histogram analysis'. The chapter on immunofluorescence is especially important since immunofluorescence results have often not been subjected to advanced data analysis. The underlying mathematical concept is that a measured flow cytometric histogram is a combination of the true biological (or theoretical) distribution and a broadening function due to the vari-
TRENDS IN CELLBIOLOGYVOL. 3 SEPTEMBER1993
ability introduced by the measuring process (Gauss broadening). These concepts have for a long time been accepted as the basis for interpretation of DNA histograms, and are implemented in slightly different ways in the well-established meth. ads of DNA histogram analysis, such as multiple gaussian, polynomial fitting and rectilinear integration. These methods are all adequately described, but I feel it would have been better to introduce them in the opposite order, i.e. discuss DNA histogram analysis before immunofluorescence data analysis. The remaining chapters of the book discuss cell cycle kinetics and dynamic cellular events. Some of these are not typical cytometric data, and some are not even measured by flow cytometers, but they represent experimental data that flow cytometrists are likely to encounter, and therefore widen the scope of the book in a worthwhile manner. The last chapter introduces the concept of multivariable analysis, which will be increasingly used for analysis of multiparameter data. The book has nine appendices full of useful statistical and mathematical information. With the advent of user-friendly flow cytometers, insight into the mathematical and statistical tools for analysis of flow cytometric data is necessary to ensure its correct use and interpretation. Biophysicists, like myself, would probably prefer more standard textbooks where the formal mathematical and statistical parts are presented more rigorously. However, this book will prove valuable to biologists and medical doctors interested in flow cytometry and the correct interpretation of the vast amount of data it generates. As such, it serves an important purpose, since flow cytometric data are about to influence diagnostic decisions and therapeutic regimens.
Tore Llndmo BiophysicsGroup, Departmentof Physics,University of Trondheim, NTH, N-7034 Trondheim, Norway.
Please mention
Trends in Cell Biology when replying to advertisements.
321