Practical flow cytometry:
Gespeichert in:
| Format: | Elektronisch E-Book |
|---|---|
| Sprache: | English |
| Veröffentlicht: |
Hoboken, NJ
Wiley-Liss
2003
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| Ausgabe: | 4. ed. |
| Schlagworte: | |
| Online-Zugang: | FRO01 TUM01 UBM01 UBR01 Volltext Inhaltsverzeichnis |
| Beschreibung: | 1 Online-Ressource |
| ISBN: | 0471411256 9780471411253 9780471722731 |
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Datensatz im Suchindex
| _version_ | 1804135760549380096 |
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| adam_text | CONTENTS
LIST OF TABLES AND FIGURES xxvii
PREFACE TO THE FOURTH EDITION: WHY YOU SHOULD READ THIS BOOK OR NOT xxxiii
FOREWORD TO THE THIRD EDITION by Leonard A. Herzenberg xxxix
PREFACE TO THE THIRD EDITION xli
PREFACE TO THE SECOND EDITION xlv
FOREWORD TO THE FIRST EDITION by Louis A. Kamentsky xlvii
PREFACE TO THE FIRST EDITION xlix
1. OVERTURE 1
1.1 What (And What Good) Is Flow Cytometry? l
Tasks and Techniques of Cytometry 1
Some Notable Applications 1
What is Measured: Parameters and Probes 2
1.2 Beginnings: Microscopy And Cytometry 2
A Little Light Music 4
Making Mountains out of Molehills: Microscopy 6
Why Cytometry? Motivation and Machinery 9
Flow Cytometry and Sorting: Why and How 10
Fluorescence and Flow: Love at First Light 11
Conflict: Resolution 12
1.3 Problem Number One: Finding The CeII(s) 14
Flow Cytometry: Quick on the Trigger 16
The Main Event 17
The Pulse Quickens, the Plot Thickens 17
1.4 Flow Cytometry: Problems, Parameters, Probes, and Principles 18
Counting Cells: Precision I (Mean, S.D., CV) 18
Poisson Statistics and Precision in Counting 19
Rare Event Analysis: The Fundamental Things Apply as Cells Go By 19
Count Constant Numbers for Constant Precision 20
Alternative Counting Aids: The Venerable Bead 20
And Now to See with Eye Serene the Very Pulse
of the Machine: Display, Digitization, and Distributions 21
DNA Content Analysis: Precision II (Variance) 21
The Normal Distribution: Does the Word Gaussian Ring a Bell? 22
vii
viii / Contents
1.4 Flow Cytometry: Problems, Parameters, Probes, and Principles (continued)
Binned Data: Navigating the Channels 22
DNA Content: Problem, Parameter, Probes 23
One Parameter Displays: Pulse Height Distributions 24
Mathematical Analysis of DNA Histograms: If It s Worth Doing, It s Worth Doing Well 25
Linear Thinking 26
Lineage Thinking: Sperm Sorting 26
Two Parameter Displays: Dot Plots and Histograms 26
Multiparameter Analysis Without Computers: Gates Before Gates 27
Two Parameter Histograms: Enter the Computer 29
Modern Multiparameter Analysis: List Mode 30
Three Dimensional Displays: Can We Look at Clouds from Both Sides? No 32
Identifying Cells in Heterogeneous Populations: Lift Up Your Heads, Oh Ye Gates! 33
Cluster Headaches 34
Painting and White (or Gray ) Washing Gates 34
The Quad Rant: Are You Positive? Negative! 35
Deals With the Devil: Logarithmic Amplifiers and Fluorescence Compensation 35
Evils of Axes: Truth in Labeling Cells and Plots 38
When Bad Flow Happens to Good Journals 40
Sorting Sorting Out 40
Parameters and Probes II: What is Measured and Why 42
Probes versus Labels 42
Living and Dyeing: Stains, Vital and Otherwise 43
Nucleic Acid (DNA and RNA) Stains 43
Fluorescence and Fluorescent Labels 44
Binary Fishin : Tracking Dyes Through Generations 45
Membrane Perturbation: A Matter of Life and Death? 46
Cytoplasmic/Mitochondrial Membrane Potential 46
Indicators of Cytoplasmic [Ca**]: Advantages of Ratiometric Measurements 47
Finding Antigen Specific Cells Using Tetramers 47
Hip, Hip Arrays: Multiplexing on Slides and in Bead Suspensions 48
GFP and its Relatives: Mild Mannered Reporters 48
Beyond Positive and Negative; Putting the Metry in Cytometry 48
1.5 What s In the Box: Flow Cytometer Anatomy, Physiology, and Pathology 49
Light Sources for Microscopy and Flow Cytometry 49
Instrument Configurations: The Orthogonal Geometry 50
Laser Beam Geometry and Illumination Optics 50
Flow Chamber and Forward Scatter Collection Optics 51
Fluorescence and Orthogonal Scatter Optics 52
Optical Filters for Spectral Separation 52
Multistation Flow Cytometers 54
Photomultipliers and Detector Electronics 54
Putting the Flow in Flow Cytometry 55
Signal Processing Electronics 57
Is It Bigger than a Breadbox? 57
Flow Cytometer Pathology and Diagnostics 58
1.6 Alternatives to Flow Cytometry; Cytometer Ecology 59
1.7 The Rest Of The Book 60
Lis(z)t Mode 60
2. LEARNING FLOW CYTOMETRY 61
Learning from History: Take One 61
Who Should Read this Book? 62
2.1 Information Sources and Resources 62
Books on Flow Cytometry in General 62
Books on Flow Cytometric Methodology and Protocols 62
Contents / ix
2.1 Information Sources and Resources (continued)
Clinical Flow Cytometry Books 63
Other Flow Cytometry Books 63
Flow s Golden Oldies 63
2.2 The Reader s Guide To Periodical Literature 64
2.3 Resources And Courses 66
Flow Cytometer Manufacturers 66
The International Society for Analytical Cytology 66
The Clinical Cytometry Society 66
The National Flow Cytometry Resource 66
The Annual Courses and Others 67
Other Societies and Programs 67
The Purdue Mailing List, Web Site, and CD ROMs 68
2.4 Exploring The Foundations 68
Optics and Microscopy 68
Electronics 69
Computers: Hardware and Software 69
Digital Signal Processing 70
Data Presentation and Display 70
Spectroscopy, Fluorescence and Dye Chemistry 71
Cell and Molecular Biology and Immunology 71
2.5 Alternatives To Flow Cytometry 71
3. HISTORY 73
3.1 Ancient History 73
Flow Cytometry: Conception and Birth 73
Staining Before and After Paul Ehrlich 74
Origins of Modern Microscopy 75
Making Cytology Quantitative: Caspersson et al 75
Origins of Cancer Cytology: The Pap Smear 76
The Fluorescent Antibody Method 77
Blood Cell Counting: Theory and Practice 77
Video and Electron Microscopy 78
Optical Cell Counters and the Coulter Orifice 78
3.2 Classical History 79
Analytical Cytology in the 1950 s 79
The Cytoanalyzer 79
Acridine Orange as an RNA Stain: Round One 79
How I Got Into this Mess 79
The Rise of Computers 80
Computers in Diagnosis: A Central Problem 80
Diagnosis and Classification: Statistical Methods 80
Cytology Automation in the 1960 s 81
First Steps toward Automated Differentials 81
Pattern Recognition Tasks in Cell Identification 82
Differential Leukocyte Counting: An Early Flow Systems Approach 83
Kamentsky s Rapid Cell Spectrophotometer 84
Fulwyler s Cell Sorter 85
3.3 Modern History 85
Cell Cycle Analysis: Scanning versus Flow Systems 85
Cancer Cytology: Scanning versus Flow Cytometry 86
Early Commercial Flow Cytometers 87
Not Quite Commercial: The Block Projects 89
The Evolution of Flow Cytometers in the 1970 s 90
Dog Days: The Genesis of Cytomutts 93
The 1980 s: Little Things Mean a Lot 94
x / Contents
3.3 Modern History (continued)
Measurements in the Main Stream 95
Immunofluorescence Comes of Age 95
Developments in DNA Content Analysis 96
Flow Cytometry of RNA Content 96
Measurements of Functional Parameters 97
Clinical Uses of Fluorescence Flow Cytometry 98
The End of History? 99
4. HOW FLOW CYTOMETERS WORK 101
4.1 Light and Matter 101
Introduction 101
Photometry versus Radiometry: What s in a Name? 101
Physical Measurement Units 101
Light in Different Lights 102
It s All Done With Photons 102
A Few Warm Bodies 103
Polarization and Phase; Interference 104
Light Meets Matter: Rayleigh and Mie Scattering 105
A Time for Reflection and Refraction: Snell s Law 107
Polarization by Reflection; Brewster s Angle 107
Dispersion: Glass Walls May Well a Prism Make 108
Interference in Thin Films 108
Interference and Diffraction; Gratings 108
Optical Activity and Birefringence 109
Matter Eats Light: Absorption 109
Absorption: Counting the Calories 110
A Selective Diet 110
The Chance of a Lifetime 110
Spinning a Tale of Degeneracy Ill
Facing Extinction: Cross Section and Optical Density Ill
Unexciting Times: Emigrating from the Excited States 112
Fluorescence: Working the Stokes Shift 112
Phosphorescence 113
Fluorescence Polarization 114
Stimulated Emission 114
Resonance Energy Transfer 115
Quenching, Bleaching, and Photon Saturation 115
Quantum Flotsam and Jetsam 118
Inelastic Scattering and Doppler Measurements 118
Raman Scattering 118
Nonlinear Optics and Harmonic Generation 118
Two Photon and Multiphoton Excitation 118
4.2 Optical Systems 119
Light Propagation and Vergence 119
Image Formation by Optical Systems: Magnification 119
Lens Types and Lens Aberrations 120
Numerical Aperture and Lens Performance 121
Gradient Index, Fresnel, and Cylindrical Lenses 122
The Helmholtz Invariant and Throughput 123
Photons in Lenses: See How They Run 123
Aperture and Field Stops: The f Number 124
Depth of Field and Focus and Resolution of Lenses 124
4.3 Light Sources 124
The Best and the Brightest 124
Contents / xi
4.3 Light Sources (continued)
Hare, Hare, the Arc! 126
Quartz Halogen Lamps 127
Light Emitting Diodes (LEDs) 127
Illumination Optics for Lamps and LEDs 127
Arc Source Epiillumination for Flow Cytometry 128
Lasers as Light Sources for Flow Cytometers 129
Laser Illumination: Going to Spot 130
Shedding Light on Cells: Lasers, Lamps, and LEDs 131
Lasers: The Basic Physics 133
Einstein on the Beam: Stimulated Emission 133
Look, Ma, One Cavity: Optical Resonators 133
Laser Action a la Mode..... 134
Pumping Ions 135
Laser Efficiency: Your Mileage May Vary 135
Mirrors and Prisms for Wavelength Selection 135
Brewster Windows for Polarized Output 135
Laser Power Regulation: Current and Light Control 136
Beam Profiles and Beam Quality 136
Puttin on My Top Hat? 138
Harmonic Generation and Modulation 138
Lasers Used and Usable in Cytometry 138
Argon and Krypton Ion Lasers 138
Dye Lasers Hi
Helium Neon Lasers 141
Helium Cadmium and Helium Selenium Lasers 142
Diode Lasers: Red, Infrared, Violet, and UV 142
Solid State Lasers: Like, YAG Me! 145
Laser and Light Source Noise and Noise Compensation 147
Fifty Ways to Lose Your Laser 148
Danger!!! Laser!!! Hazards and Haze 148
4.4 Light Collection 149
Microscope Objectives 149
Looking at the Observation Point 150
Stops versus Blockers 150
Signal versus Noise: To See or Not to See 150
Spectral Selection: Monochromators versus Filters 152
Monochromators and Polychromatic Detection 152
Interference Filters: Coatings of Many Colors 153
Absorptive Filters versus Interference Filters 153
Filter Transmission Characteristics 154
Dichroics 155
Neutral Density Filters 156
Beamsplitters; Ghosts and Ghostbusters 156
Optics for Polarization Measurements 156
Tunable Filters 157
Fiber Optics and Optical Waveguides 157
Through a Glass Darkly: Light Lost (and Found) in Optical Components 158
Collection Optics for Forward Scatter Signals 159
4.5 Detectors 160
Silicon Photodiodes 160
Photomultipliet Tubes (PMTs) 161
Sensitivity Training: Photodiode versus PMT 163
Single Photon Counting 164
Avalanche Photodiodes (APDs) 164
PMTs: Picking a Winner 165
xii / Contents
4.5 Detectors (continued)
Photomukipliers: Inexact Science 166
Charge Transfer Devices: CCDs, CIDs, Etc 166
4.6 Flow Systems 166
Flow System Basics 167
Gently Down the Stream: Laminar Flow 167
Flow Chambers; Backflushes, Boosts, and Burps 169
Cuvettes versus Streams for Analysis and Sorting 170
Light Collection from Streams and Cuvettes 171
When You ve a Jet 174
Core and Sheath: Practical Details 175
Grace Under Pressure: Driving die Sheadi and Core 175
Perfect Timing: Fluidics for Kinetic Experiments 177
Oriented and Disoriented Cells 178
Matchmaker, Matchmaker, Make Me a(n) Index Match! 178
Flow Unsheathed 178
Flow Systems: Garbage In, Garbage Out 178
4.7 Electronic Measurements 180
Electricity and Electronics 101 180
Charge Separation, Electric Fields, and Current 180
Resistance, Voltage, and Power; Ohm s Law 181
Alternating and Direct Current; Magnetism 181
Inductance, Reactance, Capacitance, Impedance 182
The Coulter Principle: Electronic Cell Sizing 182
Electrical Opacity: AC Impedance Measurement 183
4.8 Analog Signal Processing 183
Beam Geometry and Pulse Characteristics 183
Electronics 102: Real Live Circuits 184
Circuits: Current Sources and Loads 184
Ground Rules 185
Couplings, Casual and Otherwise; Transformers 186
Power Supplies 187
Active Electronics: Tubes, Transistors, ICs 188
Analog Nirvana: Operational Amplifiers 189
Detector Preamplifiers and Baseline Restoration 190
Analog Pulse Processing: Front Ends and Triggering 191
Peak Detectors 192
Pulse Integral or Area Measurements 194
Pulse Width Measurement Circuits 195
Analog Pulse Processing: The Bottom Line 195
Dead Times, Doublets, and Problem Pulses 196
Trigger Happy? 196
Analog Linear, Log and Ratio Circuits 197
Linear Circuits; Fluorescence Compensation 197
Logarithmic Amplifiers and Dynamic Range 199
Twin Peaks: Distributions on Linear and Log Scales 200
Falling Off a Log: Log Amps Behaving Badly 201
Limits to Dynamic Range 202
Ratio Circuits 204
4.9 Digital Signal Processing 204
Analog to Digital Conversion 204
Free Samples? Hold it! 205
Quantization: When Are Two Bits Worth a Nickel? 205
Analog to Digital Converters (ADCs) (and Digital to Analog Converters (DACs)) 208
Digital Pulse Processing and DSP Chips 209
Contents / xiii
4.9 Digital Signal Processing (continued)
The Screwy Decibel System 211
Pulse Slicing: Deja Vu All Over Again 212
In Defense of de Fence 213
Digitization: Tying it All Together 214
4.10 Performance: Precision, Sensitivity, and Accuracy 214
Precision; Coefficient of Variation (CV) 214
Sensitivity I: Minimum Detectable Signal 215
Sensitivity II: MESF Units 216
Accuracy I: Linearity and Nonlinearity 217
Sensitivity III: What s All the Noise About? 217
Sensitivity IV: More Photons Give Better Precision 218
Sensitivity V: Background Effects 218
Sensitivity VI: Electrons Have Statistics, Too 218
Source Noise Fluctuations and Performance 219
I Blurred It Through the Baseline 219
Restoration Comedy: The Case of the Disappearing Leukocytes 220
Top 40 Noise Sources 221
Sensitivity 007: Q and B (Dye Another Day?) 221
5. DATA ANALYSIS 225
5.1 Goals and Methods in Data Analysis 225
Cell Counting 225
Characterization of Pure Cell Populations 226
Identification of Cells in Mixed Populations 226
Characterization of Cell Subpopulations 226
Data Analysis Hardware and Software Evolve 226
5.2 Computer Systems for Flow Cytometry 227
The Beginning 227
The End of the Beginning 227
Data Rates and Data Acquisition Systems 228
PC Data Acquisition Boards 229
Preprocessors for Data Acquisition 230
5.3 Primary Data: Frequency Distributions 231
You Say You Want a Distribution 231
Gauss Out of Uniform 231
About Binomial Theorem, I m Teeming With a Lot o News 232
Distributions Have Their Moments 233
Statistical versus Cytometric Parameters 233
Mean, Variance, and Standard Deviation 233
Widi Many Cheerful Facts About the Square of the Hypotenuse: Euclidean Distance 234
Higher Moments; Skewness and Kurtosis 234
Some Features of the Normal Distribution 234
Measures of Central Tendency: Arithmetic and Geometric Means, Median, and Mode 235
Measures of Dispersion: Variance, Standard Deviation, CV, and Interquartile Range 235
Robustness in Statistics; the Robust CV 235
Box and Whiskers Plots of Distributions 236
Calculating and Displaying Histograms 236
Bivariate and Multivariate Distributions and Displays 237
Dot Plots; Correlation and Covariance 237
Linear Regression; Least Squares Fits 237
Breaking off Undiplomatic Correlations 238
Multivariate Measures of Central Tendency and Dispersion 238
Beyond Dot Plots: Two Parameter Histograms 238
Bivariate Distributions: Display s The Thing! 238
Displaying by the Numbers 239
xiv / Contents
5.3 Primary Data: Frequency Distributions (continued)
Economies of Scale 240
How Green Were My (Peaks and) Valleys 241
Clouds on the Horizon: 3 Dimensional Displays 241
5.4 Compensating Without Decompensating 242
5.5 Dealing With the Data 244
Comparing and Analyzing Univariate Histograms 244
The K S Test, Clonal Excess, and % Tests 245
Nonparametric Histogram Comparison 245
Cumulative (Overton) Subtraction 245
Constant CV Analysis 245
Another Approach to Histogram Comparison 246
Deconvoluting Single Parameter Histograms 246
Analysis of Two Parameter Data 246
Two Parameter Gating, Bitmap and Otherwise 246
Rectangles and Quadrilaterals: Hardware and Software Gating: 246
Ellipses and Beyond: Advantages of Bitmaps 246
Storage Requirements for Bitmaps 247
Analysis of Two Parameter Distributions 247
See You Around the Quad 247
Bivariate Cell Kinetic Analysis 247
Bivariate Karyotype Analysis 247
Smooth(ing) Operators: When are Filters Cool? 248
Bivariate Analysis in Hematology Analyzers 248
5.6 Multiparameter Data Analysis 248
Multiparameter versus Multivariate Analysis 248
Multiparameter Analysis of Leukocyte Types: 1974 248
Automated Differentials via Discriminants 249
Interactive Analysis: Finding the Training Set 249
Multiparameter Analysis of Leukocyte Types: 2002 250
Procedures for Automated Classification 250
Discriminant Functions and How They Work 250
Principal Component Analysis 252
Cluster Analysis 252
Neural Network Analysis 252
Genetic Algorithms 253
5.7 Analysis of Collected Data: How Much Is Enough/Too Much? 253
5.8 Data Analysis Odds and Ends 254
Data Storage 254
The Flow Cytometry Standard (FCS) File Format 254
Magnetic/ Optical Tumors in the Digital Attic 255
Linear and Log Scales and Ratios: Proceed with Care! 255
Ratios Only Help if Variables are Well Correlated 256
6. FLOW SORTING 257
6.1 Sort Control (Decision) Logic 257
6.2 Preselected Count Circuits and Single Cell Sorting 258
6.3 Droplet Sorting, High Speed and Low 258
Droplet Generation 259
Drop Charging and Deflection 260
Drop Deflection Test Patterns 260
Two and Four Way Sorts: How Much Voltage? 260
How Many Drops Should be Charged? 261
Guilty as Charged? 261
Determining Droplet Delay Settings 262
Fractional Droplet Delays 262
Contents / xv
6.3 Droplet Sorting (continued)
Transducers and Transducer Drive Signals 263
Improving Droplet Sorting 263
Sorting Large Objects with Droplet Sorters 263
6.4 Fluidic Switching Cell Sorters 264
Sorting Large Objects Using Fluidic Switching 265
Sorting Very Small Objects: Microfluidic Switching 266
6.5 Cell Manipulation By Optical Trapping 266
6.6 Cell Damage Cell Selection ( Cell Zapping ) 266
Photodamage Cell Selection 266
Sorting (Zapping) Without Flow (Gasp!) 267
Electrodamage Cell Selection in Flow 267
6.7 Measures of Cell Sorter Performance: Purity, Recovery (Yield), and Efficiency 267
Coincidence Effects on Performance 267
6.8 Other Considerations 268
Doing the Math 268
Speed Limits: The Reynolds Rap 269
Instrument Utilization 269
Monitoring versus Sorting for Cell Preparation 269
Collection Techniques: Life and Death Decisions 269
Dilutions of Grandeur 270
Can Getting Sorted Be Hazardous to Cells Health? 270
6.9 Biohazard Control and Biosafety in Flow Cytometers and Sorters 271
6.10 Conclusions 271
7. PARAMETERS AND PROBES 273
7.1 Physical Parameters and Their Uses 273
Electrical Parameters 273
DC Impedance (Coulter Volume) 273
AC Impedance (Electrical Opacity); Capacitance 273
Acoustic Measurements of Cells in Flow 274
Optical Parameters: Light Scattering 274
Scattering: The Mueller Matrix Model 274
Forward Light Scattering and Cell Size 275
Forward Scatter and Viability 276
Side Scatter and Cytoplasmic Granularity 276
Lymphocyte Gating: Forward Scatter Aside 276
Other Applications of Side Scatter 277
What is the Right Angle for Right Angle Scatter? 278
Does Side Scatter = Total Protein? 278
Optimizing Side Scatter: Not as Easy as It Looks 270
Polarized 90° Scatter Reveal Eosinophils and Malaria
Pigment Containing Monocytes 278
Multiple Wavelength Scattering Measurements 279
From Russia with Lobes 279
Optical Parameters: Absorption 281
Absorption Effects on Light Scattering 281
Optical Parameters: Extinction 282
Other Transmitted Light Measurements 282
Interference and Phase Measurements 282
Optical Parameters: Fluorescence 283
Fluorescence Lifetime Measurements 283
Fluorescence Polarization Measurements 283
Energy Transfer Measurements: Something to FRET About 283
Quenching and Energy Transfer 284
Measuring Fluorescence Spectra in Flow 284
xvi / Contents
Optical Parameters: Fluorescence
Two Photon Fluorescence Excitation in Flow 284
Bioluminescence detection in Flow? 284
7.2 Intrinsic Cellular Parameters 285
Cell Size 285
Mean Cell Volume: The Cellocrit as Gold Standard 285
Cell Volume, Area, and Diameter 285
Cell Sizing: Slit Scans and Pulse Widths 285
Size Measurements in the Submicron Range 288
Other Size Measurement Techniques 289
Cell Shape and Doublet Discrimination 289
Measurement of Intrinsic Parameters Using Absorption and Extinction Signals 290
Fluorescence Measurement of Intrinsic Parameters 290
Autofluorescence: Pyridine and Flavin Nudeotides 290
Pyridine and Flavin Nucleotides and Redox State 291
Pyridine and Flavin Nucleotides and Cancer 291
Bacterial Autofluorescence Measurements 291
Bacterial Autofluorescence Measurements 291
Porphyrin Fluorescence in Erythroid Cells 292
Other Pigments 292
Chlorophyll and Phycobiliproteins 292
Infrared Spectra and Cancer Diagnosis 293
7.3 Probes, Labels, and [Not] Protocols for Extrinsic Parameter Measurements 293
Probes, Labels, and Dyes 293
Dyes and Quality Control: Gorillas in the NIST 294
The Dyes are Cast: An Overview 295
Mechanisms of Staining by Fluorescent Dyes 298
Environmental Sensitivity 298
Metachromasia 298
Spectral Changes and Ratiometric Measurements 299
Internal Energy Transfer in Probes and Labels 299
Vital Staining 299
Staining and Viability 299
Intact versus Live Cells 299
Getting Dyes Into and Out of Intact Cells 300
Permeancy and Permeability 300
Vital Dye Toxicity and Photosensitization of Cells 301
Fixation Why and How 302
Fixation for Biohazard Control 302
Fixation Mechanisms 302
Permeabilization versus Fixation 302
Fixative Effects on Scatter Signals 303
Fixation for Surface Antigen Measurements 303
Fixatives: Coming Out of Aldehyding Places 304
Fixation for Intracellular Antigen Measurements 305
Fixation for DNA Content Determination: Getting DNA (and Antigens) Out of a Tight Fix 305
Catch the Wave: Fixation by the (Cook) Book 305
Fixation Artifacts 305
Red Blood Cell Lysis: The Distilled Essence 306
7.4 Nucleic Acid Dyes and Their Uses 306
DNA Content Measurement 306
Feulgen Staining for DNA Content 306
DNA Staining with Ethidium and Propidium 306
Erhidium Propidium: Ionic Strength Effects 307
DNA Content: Sample Preparation and Standards 307
Chromomycin A3, Mithramycin, and Olivomycin 307
Contents / xvii
Mithramycin Plus Ethidium: Do s and Don ts 308
DNA Content Measurement (continued)
The Hoechst Dyes (33258, 33342, 34580?) 308
Detecting BrUdR by Hoechst Dye Quenching 308
Hoechst Dyes Have an A T Base Preference 308
Hoechst Dyes In And Out Of Living Cells: The Drug Efflux Pump Discovered 309
Hoechst Dye Staining Mechanisms 309
Hoechst 34580: Violet Time? 310
DAPI (and DIPI): Dyes Known for Precision 310
Determinants of High Precision in DNA Analysis 311
7 Aminoactinomycin D (7 AAD) 311
Acridine Orange 312
StyrylDyes;LDS 751 312
Cyanine Dyes I: Thiazole Orange, etc 312
Cyanine Dyes II: TOTO and YOYO a GoGo 314
Cyanine Dyes III: Alphabet Soup 315
Seeing Red: LD700, Oxazine 750, Rhodamine 800, TO PRO3, and DRAQ5 as DNA Stains 315
Miscellaneous DNA Selective Dyes 316
What Do DNA Stains Stain? 316
DNA Ploidy and Aneuploidy: The DNA Index 317
Sample Preparation for DNA Content Analysis 317
DNA Base Composition 317
Chromatin Structure; Identifying Cells in Mitosis 319
Chromatin Structure Identifies Mitotic Cells 320
RNA Content 320
RNA/DNA Staining with Acridine Orange (AO) 320
Cell Cycle Compartments Defined on the Basis of RNA and DNA Content 320
AO: Problems and Some Solutions 321
Pyronin Y, Oxazine 1, and Other Tricyclic Heteroaromatic Dyes as RNA Stains 322
What Does Pyronin Y Stain? Double Stranded (Ribosomal) RNA and Sometimes Mitochondria 323
Surviving Vital Staining with Pyronin Y 324
Other DNA Dyes Usable with Pyronin Y 324
Tips on Tricyclics (Don t Get Depressed) 325
Tricydics Gag on Mucopolysaccharides 325
Reticulocyte Counting: Cyanines Beat Tricyclics; RNA in Nucleated Cells: Cyanines Don t 326
Propidium Stains Double Stranded RNA: What of Other Dyes? 326
7.5 Fluorescent Labels and Protein Dyes 326
Estimating Total and Basic Protein Content of Cells 327
Fluorescein Isothiocyanate (FITC) 327
Sulforhodamine 101 (SR101) 327
Hematoporphyrin (HP) as a Protein Stain 328
Rhodamine 101 (or 640) as a Vital Protein Stain 328
Staining to Demonstrate Basic Protein 328
Covalent Labels for Antibodies and Other Molecules 328
Fluorescein Isothiocyanate (FITC) as a Label 329
Labeling with Lissamine Rhodamine B and Tetramethylrhodamine Isothiocyanate (TRITC) 329
Multicolor Fluorescence I: FITC and TRITC 329
Multicolor Fluorescence II: Rhodamine 101 Dyes 330
Early Problems with Multicolor Fluorescence 331
Phycobiliproteins to the Rescue! 331
Phycoerythrins: R PE, B PE and Others 332
Allophycocyanin (APC) and APC B 332
Phycocyanins 332
Phycobiliprotein Tandem Conjugates: PE APC, PE Texas Red,
PE Cy5, PE Cy5.5, PE Cy7, etc 333
Allophycocyanin Tandem Conjugates: APC Cy7 and APC Cy5.5 333
xviii / Contents
Phycobiliproteins to the Rescue! (continued)
Mercy Me! PerCP! 333
Phycobiliproteins and Tandems: Dirty Little Secrets 334
Future Tandems: Heterocycles Built for Two? 335
Cyanine Dye Labels: From Cy Fi to Hi5 for Cy5 336
Blue Notes: AMCA and Cascade Blue 337
Hey, BODIPY! 337
Alexa Dyes: Some Thoughts on Dyemographics 338
Other Organic Fluorescent Labels: A Dye Named Joe, etc 338
Quantum Dots 339
Getting Labels Onto Molecules of Interest 340
7.6 Improving Signals from Labels: Amplification and Other Techniques 340
Limits to Sensitivity: Autofluorescence 341
Improving Sensitivity: The New Wave(length) 342
Correcting and Quenching Autofluorescence 342
Raman Scattering Effects on Sensitivity 342
Increasing Sensitivity: Amplification Techniques 343
Amplification by Indirect Staining 343
Amplification Using Labeled Particles 343
Amplification Using Enzymes as Labels: Playing the Hole CARD 344
Amplifying the Analyte: The Polymerase Chain Reaction (PCR) 344
Amplification Techniques: Pros and Cons; Fluorescent versus Nonfluorescent Labels 344
Improving Sensitivity: Time Resolved Fluorescence 345
7.7 Measuring Cell Surface and Intracellular Antigens 345
History and Background 345
Monoclonal Antibodies for the Uninitiated 346
Cell Surface Antigens: Structure versus Function 347
Moving Toward Multicolor Immunofluorescence 347
Antibody Reagents and Staining Procedures 348
Antibody Fragments versus Antibodies as Reagents 348
Engineered Antibodies: Phage Display and scFvs 348
Molecular Probes Zenon Antibody Labeling 348
Antibody Shelf Life and Quality Control 349
Direct Staining Using Monoclonal Antibodies for 2, 3, and More Colors Using 488 nm Excitation. 349
Multicolor Work Using Multiple Lasers; Biotin Avidin Labeling 349
Mixing Colors: Do s and Don ts 349
Cocktails for Five: Multiplex Immunofluorescence 350
Cocktail Staining Helps Identify Rare Cells 352
Immunofluorescence Staining Procedures 352
Automated Sample Preparation 353
Fluorescence Measurements: Lurching Toward Quantitation 353
Calibration and Controls: Round One 353
Quantitative Fluorescence Cytometry: Definitions 354
Calibration Particles for QFCM 354
Defining a Window of Analysis 356
Type IIA versus Type IIIB and IIIC Standards 357
Other Aspects of Fluorescence Quantitation 358
What is Positive ? What is Negative ? 358
Making Weakly Fluorescent Beads and Cells: Do Try This Trick at Home! 358
Correlating Cytometry and Biochemistry: Studies of Antibody Binding Chemistry 359
Correlating Cytometry and Biochemistry: Intracellular Antigen Measurements 359
Analyzing Immunofluorescence Data 360
Estimating Antigen or Receptor Surface Density 360
Quantitative Fluorescence: Problems and Prospects 361
7.8 Nucleic Acid Sequence Detection 361
Peptide Nucleic Acid (PNA) Probes 362
Contents / xix
7.9 Probes for Various Cell Constituents 362
Surface Sugars (Lectin Binding Sites 362
Analysis of Total Carbohydrate Content 363
Specific Detection of Cellulose 363
A Probe for Cell Surface Aldehydes 363
Probes for Lipids and Cholesterol 364
Nile Red 364
Filipin 364
Lipid Droplet Detection Using Scatter Signals 364
Probes for Cytoskeletal Organization/Actins 364
7.10 Time as a Parameter: Kinetic Measurements 364
Sample Handling for Kinetic Measurements 365
Time as a Quality Control Parameter 366
Slooowww Flooowww 366
7.11 Labeled Ligand Binding 366
Labeling Strategies 367
Formal Analysis of Ligand binding 367
Labeled Ligands versus Anti Receptor Antibodies 368
Ligand Binding Detected by Functional Changes 368
Fluorescent Ligand Binding: Some Examples 368
7.12 Functional Parameters 1 369
Cell Surface Charge 369
Cell Membrane Characteristics 369
Membrane Integrity versus Viability : Dye Exclusion Tests 369
Detecting Dead Cells in Fixed Samples 369
Membrane Fusion and Turnover; Cell Tracking 371
Cell Proliferation Analyzed Using Tracking Dyes 371
Membrane Organization and Fluidity/Viscosity 374
Lipid Packing Assessed with Merocyanine 540 374
Membrane Fluidity and Microviscosity: Assessment Using Fluorescence Polarization 374
Lipid Peroxidation 375
Membrane Permeability to Dyes and Drugs: The Drug Efflux Pump Revisited 376
Endocytosis of Macromolecules and Particles 377
Enzyme Activity 378
Indicators of Oxidative Metabolism I: Tetrazolium Dye Reduction 379
Indicators of Oxidative Metabolism II: 2,7 Dichlorofluorescin Diacetate (DCFH DA), etc 379
Indicators of Oxidative Metabolism III: Hydroethidine (Dihydroethidium) 379
Indicators of Oxidative Metabolism IV: Dihydrorhodamine 123 379
Indicators of Oxidative Metabolism V: Detection of Hypoxic Cells 380
Detection of Caspase Activity 380
Other Enzymes 380
Detection of Enzymes and Products by Antibodies 380
Enzyme Kinetics in Single Cells 380
Sulfhydryl (Thiol) Groups; Glutathione 381
7.13 Functional Probes II: Indicators of Cell Activation 381
Introduction 381
Changes in the Cellular Ionic Environment Following Activation by
Ligand Interaction with Cell Surface Receptors 382
Structuredness of Cytoplasmic Matrix (SCM) and the Cercek Test for Cancer 383
Optical Probes of Cell Membrane Potential 385
Membrane Potential and Its Physicochemical Bases 385
A4* Measurement Using Microelectrodes 386
Single Cell Measurements with Distributional Probes 387
Oxonol Dyes as Membrane Potential Probes 390
Possible Alternatives to Distributional Probes for Flow Cytometry of Membrane Potentials 391
Ratiometric Probes for Membrane Potential 391
xx / Contents
Optical Probes of Cell Membrane Potential (continued)
Using Cyanine Dyes for Flow Cytometric A^F Estimation, In Case You re Still Interested 392
Cytoplasmic Membrane Potential: Summing Up 394
Mitochondrial Membrane Potential (A^) 394
Mitochondria! Staining with Rhodamine 123 Is Membrane Potential Dependent 394
The Search for Better A^B Probes: Round One 397
AT^JC l.andApoptosis 397
The Search for Better A*¥m Probes: Round Two 398
Bacterial Membrane Potentials 400
Ratiometric A^P Measurement in Bacteria 401
A*F Measurement: Cautions and Conclusions 402
Optical Probes of Intracellular Calcium 402
The Bad Old Days 402
Chlortetracycline as a Probe of Membrane Bound Calcium in Cells 402
Probes for Free Cytoplasmic Calcium: Quin 2 403
Fura 2 and Indo 1: Ratiometric Ca Indicators 403
Fluo 3 and Other Visible Excited Ca Probes 404
Flow Cytometric Probes of Intracellular pH 405
The Hat Trick: Multiparameter Approaches to Ion Flux Measurements in Cell Activation 407
NOsing Around for Nitric Oxide 408
Other Ions in the Fire 408
7.14 Reporter Genes 408
Somebody Cloned My Gal: Enzymes as Reporter Genes 408
Green Fluorescent Protein (GFP) et al 409
Minority Report(er)? 410
8. BUYING FLOW CYTOMETERS 411
8.1 Introduction 411
8.2 History 411
8.3 BD Biosciences 412
Background 412
The BD FACS Vantage SE™ Cell Sorter 413
The BD FACSCalibur™ Analyzer 414
The B D™ LSRII Analyzer 416
The B D FACSAria™ Cell Sorter 417
The B D FACSCount 418
8.4 Beckman Coulter, Inc 418
Background and Signal to Background 418
The Beckman Coulter EPICS® ALTRA™ Cell Sorter 419
The Beckman Coulter Cytomics™ FC 500 Analyzer 420
The EPICS® XL and XL MCL Analyzers 422
8.5 DakoCytomation 423
Background 423
The MoFlo® Cell Sorter 423
The CyAn Flow Cytometer 424
8.6 Cytopeia 425
The InFlux Cell Sorter 425
8.7 Optoflow AS 426
Background 426
The MICROCYTE® Flow Cytometer 426
8.8 Partec GmbH 427
Background 427
The CyFlow® and CyFlow® ML Flow Cytometers 427
The PAS, PAS II, and PAS III Flow Cytometers [ ..!..... 428
PA Ploidy Analyzer and CCA Cell Counter Analyzer 429
Contents / xxi
8. BUYING FLOW CYTOMETERS (continued)
8.9 Some Other Flow Cytometer Companies 429
Advanced Analytical Technologies, Inc. (AATI) 429
Agilent Technologies, Inc 429
Apogee Flow Systems Ltd 430
Bentley Instruments 430
ChemunexSA 430
CytoBuoy b.v 430
Delta Instruments bv 431
Fluid Imaging Technologies, Inc 431
FOSS Electric A/S 431
Guava Technologies, Inc 431
Howard M. Shapiro, M.D., P.C 431
iCyt — Visionary Bioscience 431
International Remote Imaging Systems 431
Luminex Corporation 431
NPE Systems, Inc 432
Union Biometrica, Inc 432
8.10. Hematology Instruments, Etc 433
8.11 Little Orphan Analyzers (And Big Orphan Sorters) 434
Bio/Physics and Ortho: Cytofluorograf to Cytoron 434
HEKA Elektronik GMBH: The FLUVO II Analyzer 435
TheKratel Partograph 435
The ODAM ATC 3000 435
Also Among the Missing 436
Flow Cytometer Rehabilitation; Used Instruments 436
Following Suit 436
8.12 Third Party Software 437
8.13 The Selling of Flow Cytometers: Hype and Reality 437
8.14. Applying for a Grant for a Cytometer 438
9. BUILDING FLOW CYTOMETERS 441
9.1 Why Buy a Flow Cytometer? 441
9.2 Why Build a Flow Cytometer? 441
9.3 Learning to Build Your Own 442
10. USING FLOW CYTOMETERS: APPLICATIONS, EXTENSIONS, AND ALTERNATIVES 443
10.1 The Daily Grind 443
Keeping the Instrument Running: Diet and Exercise 443
Particulars: Drawing a Bead on Flow Cytometer Alignment, Calibration, and Standardization 444
Alignment Particles: Fearful Asymmetry 445
Reference and Calibration Particles 445
Compensation Standards 446
Cells and Nuclei as Alignment Particles 446
Rose Colored Glasses: Optical Filter Selection 446
Experimental Controls 447
Shake Well Before Using: When Controls Won t Help 447
10.2 Significant Events in the Lives of Cells 448
Taking the Census: Cell Counting 448
A Counting Alternative: Image Analysis 448
The Doubled Helix: Reproduction 448
The Cell Cycle and Cell Growth 448
DNA Content Analysis 448
Mathematical Models for DNA Analysis 449
Clinical Application of DNA Content Analysis 450
xxii / Contents
DNA Content Analysis(continued)
DNA Content Alternatives: Static Photometry and Scanning Laser Cytometry 451
The Mummy s GC/AT: DNA Content Analysis in Anthropology and Forensic Science 451
Haifa Genome is Better Than None: Sperm Sorting 452
The Widening Go/G, re Detecting Mutation 453
Detecting DNA Synthesis: Cell Kinetics 453
Kinetics Before Flow Cytometry: Mitotic Indices, Doubling Times, and Radiolabel Studies.... 453
Labeling Index versus DNA Content 454
Early Flow Cytometric Approaches to Labeling Using BrUdR and 3H TdR 455
Detection of Incorporated BrUdR with Hoechst Dyes and Propidium Iodide 455
Detection of BrUdR Incorporation with Anti BrUdR Antibodies 455
Cytochemical Detection of BrUdR Incorporation Using Difference and Ratio Signals 456
Breaking Up Is Easy To Do: SBIP, a Simpler Way to Detect BrUdR Incorporation into DNA 456
Anti BrUdR Antibody: Seeing the Light 457
Cytochemical Detection of BrUdR: Still Around 457
Detecting RNA Synthesis Using Bromouridine 458
Generation Gaps: Tracking Dyes and Cell Kinetics 458
Cell Cycle Related Proteins: Cyclins, Etc 458
Detecting Mitotic Cells 462
Memento Mori: Detecting Cell Death 462
Necrosis versus Apoptosis 462
Identifying Apoptotic Cells 462
Les Feuilles Mortes (Autumn Leaves) 462
Apoptosis: Getting With the Program 463
ISNT there Light at the End of the TUNEL? 463
Apoptosis: The Case Against Flow Cytometry 463
Die Another Day: Cytometry of Telomeres 464
10.3 Identification of Cells in Mixed Populations 464
Mixed Genotypes versus Mixed Phenotypes 464
No Parameter Identifies Cancer Cells 464
Many Parameters Identify Blood Cells 464
Flow Cytometric Parameters Useful for Blood Cells 464
Specific Gene Products Identify Cell Types 465
Maturation Processes and Missing Links : The Ginger Root Model 465
Practical Multiparameter Gating: Color Wars 467
Finding Rare Cells 469
One Parameter is Not Enough 469
Cocktail Staining Can Help 470
Dirt, Noise, and Rare Event Detection 470
Really, Really Rare Events: Alternatives to Flow 470
10.4 Tricks and Twists: Odd ]obs for Flow Cytometry 471
Single Molecule Detection 471
DNA Sizing, if not Sequencing, in Flow 471
Solid Phase (Bead) Assays Using Flow Cytometry 473
Cocktails for 100: Multiplexed Bead Assays 473
Cells in Gel Microdroplets and on Microspheres 474
Hanging Ten Pseudopodia? 475
10.5 Single Cell Analysis: When Flow Won t Do 475
10.6 Applications of Flow Cytometry 476
Cell Differentiation, Ab Ovo and De Novo 476
Differentiation in the Nervous System 476
Whole Embryo Sorting 476
Somatic Cell Genetics and Cell Hybridization 477
Reporter Genes Revisited 477
Isolating and Characterizing Hybrid Cells 477
Chromosome Analysis and Sorting and Flow Karyotyping 477
Contents / xxiii
10.6 Applications of Flow Cytometry (continued)
Probing Details of Cellular Structures and Inter and Intramolecular Interactions 479
Dissection of Structures Using Antibodies, Ligands, and Genetic Methods 479
Intramolecular Interactions 479
Clinical Flow Cytometry: Turf and Surf 480
Hematology 480
Clinical Application: Blood Cell Counting and Sizing 480
Red Blood Cells (Erythrocytes) 481
Clinical Application: Reticulocyte Counts 481
The Reticulocyte Maturity Index (RMI) 482
Erythrocyte Flow Cytometry: Other Clinical Uses 482
White Blood Cells (Leukocytes) 483
Clinical Application: Differential Leukocyte Counting 483
CD Characters: Leukocyte Differentiation Antigens 484
Granulocytes: Basophils 484
Basic Orange 21: The Best Basophil Stain Yet 485
Allergy Tests Using Basophil Degranulation 485
Granulocytes: Eosinophils 486
Granulocytes: Neutrophils 486
(Clinical?) Tests of Neutrophil Function 486
Neutrophil CD64 in Inflammation and Sepsis 487
When I m Sick CD64? 487
Platelets and Megakaryocytes 487
Hematopoietic Stem Cells 488
Clinical Application: Monitoring CD34+ Stem Cells According to the ISHAGE Protocol 488
Side Population (SP) Stem Cells: Plastic, Fantastic 489
Immunology 489
Immunologic Applications of Flow Cytometry: Still a Growth Industry 489
HIV Infection The Killer Application 490
Clinical Application: T Cell Subset Analysis 490
T Cell Subsets: Alternative Technologies 491
Clinical Application: Transplantation 493
Detecting Lymphocyte Activation 494
Foundations: From PHA (the Lectin) to PHA (the Pulse Height Analyzer) 495
Functional Probes for Activation 495
DNA, RNA, and Activation Antigens 497
Mitogen Response versus Antigen Response 498
Detecting Activation by CD69 Expression 499
Cytokines: Detecting Activation and More 499
Ins and Outs of Cytokine Staining 500
Tetramer Staining: Talking the Talk; Walking the Walk? 500
Tracking Dyes: Activation and Ontogeny 501
What is Early Activation (Trick Question)? 501
Cancer Biology and Clinical Oncology 502
Cancer Diagnosis: Cervical Cytology 503
DNA Content Measurements Yet Again 503
Beyond DNA Content: Antigens, Oncogenes, and Receptors, and Response to Therapy 504
Immunophenotyping in Hematopathology 504
Detecting Minimal Residual Disease 505
Biological Implications of Phenotyping Results 506
Digression: A Slight Case of Cancer 507
Analysis of Sperm 508
Isolating Fetal Cells from the Maternal Circulation for Prenatal Diagnosis 509
The March of Time: Orcadian Rhythms, Aging, and Atherosclerosis 510
Clinical Application: Urine Analysis 510
The Animal Kingdom 510
xxiv / Contents
The Animal Kingdom (continued)
Lions and Pumas and Clams, Oh, My! 510
Fish Story; FISH Story 511
Flow Cytometry: For the Birds? 512
Big Stuff, Vegetable, Animal or Mineral 512
Flow Cytometry of Plant Cells and Chromosomes 512
Measurement of Plant Cell DNA Content 513
Plant Chromosome Analysis and Sorting 514
Other Flow Cytometric Applications in Plants 514
Microbiology, Parasitology and Marine Biology 514
Measuring Microbes: Motivation 515
Measuring Microbes: Instrument Issues 515
Parameters Measured in Microorganisms 516
Flow Cytometric Gram Stains 516
Detection and Sizing: Light Scattering 517
Detection and Sizing: Electrical Impedance 517
Nucleic Acid (DNA and RNA) Staining 517
Total Protein Content: Scatter versus Stains 518
Antibodies, Etc.: Labeling Strategies 518
Ribosomal RNA Based Species Identification 518
Functional Probes in Bacteria 518
Potential, Permeability, Viability , and Metabolic Activity 519
Digression: A Therapeutic Approach Based on Transient Permeabilization 522
So Few Molecules; So Little Time 522
Applications in Marine Microbiology 524
Extensions: Cytometers for Marine Applications 527
References: Flow Cytometry and Oceanography 527
General Microbiology 527
Previously Noted 527
Cell Cycles and Cell Division 528
Fluorescent Protein Methods in Microbes 528
Microbial Communities: Will Flow Work? 528
Bad Guys Don t All Wear Black Hats: Microbial Detection/Identification
in Health Related Contexts 528
The Basic Questions 528
Detection: Intrinsic Parameters are Not Enough 529
Detection: Fluorescence Improves Accuracy 529
Detection: When the Tough Get Going 529
Identification: Too Many Broths 530
Identification: Can Multiplexing Help? 531
Environmental and Sanitary Microbiology 531
Water That Made Milwaukee (and Sydney) Infamous 531
Food Microbiology 532
Bioterrorism and Bioopportunism 532
Viruses and Other Intracellular Pathogens 532
Clinical Microbiology 533
Antimicrobial Susceptibility Testing: One Size Does Not Fit All 534
Bacteria: Confusion Reigns 535
Mycobacteria: Down for the Count 535
Antifungal Susceptibility: Flow Does the Job 535
Antiviral Susceptibility by Flow Cytometry 536
Cytometry in Vaccine Development 536
Microbiology Odds and Ends 536
Parasitology 535
Pharmacology and Toxicology 537
Drugs and the Life and Death of Cells 538
Contents / xxv
Pharmacology and Toxicology (continued)
Erythrocyte Micronucleus Assays 538
Toxic Waste and B Cell Proliferation 538
Radiation Dosimetry 538
Food Science 538
Somatic Cell Counts in Milk 538
Brewhaha 539
A Loaf of Bread, A Jug of Wine 539
Seeing the Blight 539
Major Food Group: Chocolate 539
Biotechniques and Biotechnology 539
Protein and Gene Expression on Cells and Beads 540
Getting Big Molecules into Small Cells 540
Staying Alive, Staying Alive 540
Et Cetera 540
Alternatives: Microfluidic Cytometers, Flow and Static 541
Cytometry Afield 541
The Lymphocytes of the Long Distance Runner 541
War and Peace 541
Blood, Sweat and Tears? 542
Pulp Nonfiction 542
Flow Cytometry On the Rocks 542
To Boldly Go Where No Cytometer Has Gone Before 542
11. SOURCES OF SUPPLY 543
11.1 Resources, Societies, Journals 543
11.2 Optical Supply Houses 543
11.3 Probes and Reagents 544
11.4 Calibration Particles/Cytometry Controls 548
11.5 Flow Cytometers 549
Hematology Instruments 551
11.6 Data Analysis Software/Systems 551
Hardware and Software 551
Commercial Software Sources 552
Noncommercial Software Sources 552
11.7 Cytometer Rehabilitation/Add ons 553
11.8 Flow Cytometer Parts 553
Flow System Plumbing 553
Photodetectors 554
DC DC Converter Modules for HV Power Supplies 555
Power Supplies (Low Voltage) 555
Other Electronics 555
11.9 Lasers 555
Laser Trade Publications 555
Laser Manufacturers 555
11.10 Optical Filters 556
Color Glass Filters 556
Interference Filters 557
Neutral Density Filters 557
Polarizing Filters and Optics 557
Tunable Filters 557
11.11 Aids to Troubleshooting Flow Cytometers When All Else Fails 557
11.12 Proficiency Testing 557
11.13 Sex Selection 557
11.14 Alternative Technology 558
xxvi / Contents
12. AFTERWORD 561
12.1 Dotting i s and Crossing t s 561
12.2 Late Breaking News 561
New Book 561
New Protein Stain 561
Caveat on Fluorescent Caspase Inhibitors 561
Polyamide Probes 561
Tearing Down the (Picket) Fences 562
New Instrument: The BD FACSArray™ 563
Science Special Section: Biological Imaging 563
Cytomics in Predictive Medicine: a Clinical Cytometry Special Issue and Other
Recent Citings and Sightings 563
12.3 Analytical Biology, Such as it Isn t: Is This Any Way to Run a Science? 563
12.4 Colophon 564
12.5 Unfinished Business 565
AIDS and Infectious Disease in the Third World 565
A Center for Microbial Cytometry 565
A Nobel Prize for Herzenberg and Kamentsky? 566
12.6 Flow and the Human Condition 566
There s No Business Like Flow Business 566
12.7 One More Thing 566
Contents / xxvii
TABLES AND FIGURES
TABLES
1 1. Some parameters measurable by cytomeuy 3
3 LA brief outline of flow cytometric history (1945 2000) 100
4 1. SI units and prefixes 102
4 2. Emission wavelengths of lasers 139
4 3. Cathode quantum efficiencies of diode and PMT detectors between 300 and 800 nm 165
4 4. Logarithmic Amplifiers: What goes in, what comes out 203
4 5. Characteristics of analog to digital converters 206
5 1. Some landmarks of the normal or Gaussian distribution 234
5 2. Equations (1 4) that must be solved to permit 4 color fluorescence compensation 242
5 3. Header and text portion of an FCS2.0 data file 254
6 1. Safe speed limits for sorting based on Reynolds number calculations 269
7 1. Some cellular parameters measurable by cytometry 286
7 2. Fluorescence spectral properties of a selection of reagents usable for common cytometric tasks 297
7 3. Tricydic heteroaromatic compounds usable for staining DNA and/or RNA 323
7 4. Raman emission from water 343
xxviii / Contents
FIGURES
1 1. Interaction of light with a cell 4
1 2. Transmitted light and dark field images of an unstained suspension of human peripheral blood leuokocytes 7
1 3. Transmitted light microscope images of an unstained smear of human peripheral blood 8
1 4. Schematic of a fluorescence microscope 9
1 5. Scanned images of Feulgen stained lymphoblastoid cells 15
1 6. One dimensional scanning of cells deposited in a narrow line 16
1 7 Idealized plot of signal amplitude vs. time 16
1 8. Ideal and real DNA content distributions 22
1 9. Single parameter histogram displays from a multichannel pulse height analyzer 24
1 10. Use of a mathematical model to determine fractions of DNA aneuploid breast cancer cells 25
1 11. Dotplot (cytogram) of Hoechst 33342 and fluorescein fluorescence in CCRF CEM cells 26
1 12. Gating regions for counting or sorting set electronically, drawn on an oscilloscope display 27
1 13. Histogram of 90° (side) scatter from leukocytes in lysed whole blood 30
1 14. Bivariate distribution of anti CD3 antibody fluorescence intensity vs. large angle scatter in leukocytes 31
1 15. The bivariate distribution of Figure 1 14 shown as an isometric or peak and valley plot 32
1 16. The two parameter histogram of Figures 1 14 and 1 15 displayed as a contour plot 32
1 17. Identification of human peripheral blood T lymphocytes bearing CD4 and CD8 antigens 34
1 18. Why fluorescence compensation is necessary 37
1 19. How compensation gets data to fit into quadrants 38
1 20. Fluorescence intensities of antibody stained cells and beads bearing known amounts of antibody 49
1 21. Schematic of die optical system of a fluorescence flow cytometer 51
1 22. A typical flow chamber design 56
1 23. FACScan Analyzer (Becton Dickinson) 58
1 24. MoFlo High Speed Sorter (Cytomation) 58
1 25. Microcyte Cytometer (Optoflow) 58
1 26. Estimated numbers of fluorescence flow cytometers in use worldwide, 1975 92 59
2 1. Growth of the flow cytometry literature, 1987 93 64
3 1. The first working flow cytometer (Gucker particle counter) 74
3 2. Digitized image of a neutrophil polymorphonudear leukocyte 82
3 3. Refined and prototype versions of Kamentsky s Rapid Cell Spectrophotometer (RCS) 84
3 4. A two parameter histogram of blood cells analyzed in the RCS 84
3 5. Louis Kamentsky and the Bio/Physics Systems Cytofluorograf 87
3 6. The Technicon Hemalog D Differential Leukocyte Counter 88
3 7. Leonard Herzenberg with B D s first commercial version of the Fluorescence Activated Cell Sorter (FACS) 88
3 8. A two parameter display from die Block differential counter showing five leukocyte clusters 89
3 9. The author with Cytomutt and Cerberus 94
3 10. Cell cycle phases defined by DNA content and by DNA/RNA content 97
4 1. Radian andsteradian 101
4 2. Light as an electromagnetic wave 104
4 3. Constructive and destructive interference 105
4 4. Circularly polarized light 105
4 5. Reflection and refraction of light at a surface 106
4 6. The Jablonski diagram of electronic energy levels, or states, and transitions 112
4 7. Fluorescence spectrum of fluorescein 113
4 8. Light from a point source at the focal length of a lens is collimated 119
4 9. Rays from a point source can be focused to an image point 119
4 10. Rays from many points of an object add up to make an image 120
4 11. Elements of a typical microscope lens, showing die half angle that defines the acceptance cone and N.A 120
4 12. Showing die effect of N.A. on light collection 121
4 13. Multiple views of magnification 123
Contents / xxix
FIGURES (continued)
4 14. Output characteristics of arc, quartz halogen, and deuterium lamps 125
4 15. Kohler and critical illumination 128
4 16. Optics for arc source epiillumination for fluorescence microscopy or flow cytometry 129
4 17. Use of crossed cylindrical lenses to focus a laser beam to an elliptical spot on the core stream 131
4 18. Energy levels involved in laser action 133
4 19. Schematic of a laser 134
4 20. Laser transverse excitation modes 134
4 21. Measured beam intensity profiles of diode, CO2, and He Ne lasers 136
4 22. Intensity profiles of a focused and defocused violet laser diode beam 136
4 23. Sizes, power requirements, and approximate costs of some smaller lasers used for cytometry 145
4 24. Effect of noise compensation circuitry on precision of fluorescence measurements 147
4 25. Looking at the observation point 150
4 26. Maximizing light collection: Not always a good way to do things 151
4 27. A prism monochromator 153
4 28. Light transmission characteristics of bandpass, short pass, and long pass interference filters 154
4 29. Transmission of several wavelength regions through different dichroic configurations 155
4 30. Cube and plate beamsplitters 156
4 31. Total internal reflection 157
4 32. A fiber optical waveguide 157
4 33. Optical arrangements for collection of forward scattered light 159
4 34. Elements of a photomultiplier tube (PMT) 160
4 35. PMT electrode voltage supply circuits: dynode chain and Cockcroft Walton voltage multiplier 162
4 36. Detectors and housings 163
4 37. Fluid flow in a flow cytometer 167
4 38. Laminar flow profile illustrated by diatoms in the Flow CAM imaging flow cytometer 167
4 39. Flow chamber designs 169
4 40. New angles on light collection in flow 171
4 41. Flow chamber designs used with arc source flow cytometers 173
4 42. Minimizing turbulence generated at the sheath inlet to flow chambers 175
4 43. Sheath fluid supply plumbing 175
4 44. The Coulter orifice 183
4 45. Effect of beam geometry on pulse shape 184
4 46. Some circuit elements 185
4 47. A line powered DC power supply 187
4 48. Basic operational amplifier circuits 190
4 49. A photodetector preamplifier circuit 191
4 50. Waveforms in preamplifier, front end electronics, and peak detector circuits 192
4 51. Schematic diagram of a peak detector 192
4 52. Preamp and peak detector outputs (oscilloscope traces) 193
4 53. Telling two cells from one gets harder as the cells go through closer together 196
4 54. Uncompensated and compensated fluorescence signals from FL and PE labeled beads 198
4 55. One side of a two color compensation circuit 198
4 56. Signals from PE labeled antibody bound to beads, on linear and 4 decade logarithmic scales 199
4 57. Response curves of different types of log amps as determined according to Parks, Bigos, and Moore 202
4 58. Continuous and sampled signals 205
4 59. Digital pulse processing: slicing a slightly noisy Gaussian pulse with a baseline 209
4 60. MESF threshold sensitivity determination using fluorescein labeled beads 216
4 61. Fluorescence distributions for bead sets measured with progressively lower values of Q 222
4 62. Separation (or lack thereof) of CD4+ lymphocytes and unstained cells at various values of Q and B 223
5 1. Distributions of sums of uniformly distributed random numbers approach the normal distribution 231
5 2. Pascal s triangle 232
5 3. Binomial distributions for n = 2, 4, 8, and 16, with/) = q = 0.5 232
5 4. Binomial distributions for n = 16 and/» = 0.5, 0.25, and 0.125; they are skewed when/ * 0.5 233
xxx / Contents
FIGURES (continued)
5 5. Euclidean distance between two points, with an assist from Pythagoras 234
5 6. Box and whiskers plots showing medians and interquartile ranges of distributions, after Tukey and Tufte 236
5 7. Histogram display formats 237
5 8. Dot plots of computer generated data showing various degrees of correlation 237
5 9. Varieties of two parameter data display (cover figure) 239
5 10. Density plot showing a scale indicating numbers of events 240
5 11. 3 Dimensional cloud plots of CD3, CD4, and CD8 antigens on blood lymphocytes and thymocytes 241
5 12. How compensation gets data to not quite fit into quadrants 243
5 13. Two generated near normal distributions and their cumulative distributions, or integrals 245
5 14. Quadrant statistics 247
5 15. Multiparameter analysis of peripheral blood leukocyte types: partitioning in two dimensional subspaces 249
5 16. Linear transformation of data to separate clusters, as is done in linear discriminant analysis 251
5 17. Calculation is not always necessary to reduce the dimensionality of data 254
5 18. Linear and log scales revisited 255
6 1. Droplet sorting 259
6 2. A droplet sorter test stream pattern 261
6 3. Mack Fulwyler widi his islet sorter 264
6 4. Fluidic sorter designs 264
6 5. A microfluidic flow sorter for bacteria 265
7 1. Forward scatter does not measure particle size 275
7 2. Depolarized 90° scatter signals can be used to identify eosinophil granulocytes 278
7 3. Indicatrices (plots of intensity vs. scattering angle) of particles and cells 280
7 4. Erythrocytes scatter less light at a wavelength at which hemoglobin exhibits strong absorption 281
7 5. Side scatter signals from T2 bacteriophages 288
7 6. The principle of doublet discrimination using pulse height and pulse integral measurements 290
7 7. Fluorescence spectra of some materials implicated in mammalian cell autofluorescence 291
7 8. Zinc protoporphyrin fluorescence in human red cells 292
7 9. Probe fluorescence spectra and source emission wavelengths 296
7 10. A rogues gallery of nucleic acid dyes 301
7 11. DNA content distributions in sperm from a normal ram and a ram bearing a translocation 311
7 12. Structure of symmetric cyanine dyes given die formula DiYCn(1(2m + 1) by Sims et al 313
7 13. Flow karyotype of human chromosomes stained with Hoechst 33258 and chromomycin A, 318
7 14. Hoechst/chromomycin fluorescence signatures of bacteria with different DNA base compositions 318
7 15. Flow cytometry of chromatin structure identifies cells in mitosis 320
7 16. DNA and RNA content analysis of mitogen stimulated lymphocytes 321
7 17. Chemical structures of some reactive labels 327
7 18. Multiplex immunofluorescent labeling to demonstrate multiple cell types in blood 351
7 19. Quantitative determination of CD4 epitopes on peripheral blood lymphocytes and monocytes 356
7 20. Flow cytometric detection of HIV 1 nucleic acids in cells after in situ PCR 361
7 21. Plot of cytoplasmic [Ca ] as indicated by indo 1 fluorescence ratio versus time 365
7 22. Time used as a quality control parameter in DNA analysis (after Watson) 366
7 23. Cell proliferation indicated by dilution of fluorescence of the tracking dye PKH26 372
7 24. Cell proliferation indicated by dilution of fluorescence of CFSE labeled CD4+ lymphocytes 373
7 25. Specific staining of glutathione in cells by monobromobimane (MBB) 381
7 26. Valinomycin induced changes in fluorescence intensity of cyanine dye loaded red cell suspensions 387
7 27. Distributions of the fluorescence of DiOC6(3) in CCRF CEM T lymphoblasts 388
7 28. Distributions of membrane potential in cells suspended in NaCL, KC1 and a mixture of the two 389
7 29. Structures of two membrane potential probes, the cyanine DiOC5(3) and the oxonol DiBAC4(3) 390
7 30. Two parameter analyses of lectin stimulated lymphocytes: DNA vs. RNA and membrane potential 396
7 31. Demonstration of apoptotic HL 60 cells with deenergized mitochondria by JC 1 staining 399
7 32. Measurement of membrane potential of Staphylococcus aureus using a ratiometric technique 400
7 33. Emission spectra of indo 1 in solutions of increasing free calcium ion concentration 404
Contents / xxxi
FIGURES (continued)
7 34. Emission spectra of fluo 3 in solutions of increasing free Ca concentration 405
7 35. Estimation of cytoplasmic pH in human lymphocytes from carboxyfluorescein fluorescence ratio 406
7 36. The pH dependent emission spectra of carboxy SNARF 1 excited at 488 nm 407
8 1. The BD FACSVantage cell sorter 413
8 2. The BD FACSCalibur benchtop cell sorter 415
8 3. The BD LSR multi beam benchtop analyzer 416
8 4. BD s Octagon collection optics 416
8 5. The BD FACSAria cell sorter 417
8 6. The Beckman Coulter EPICS Altra sorter 419
8 7. The Beckman Coulter Cytomics FC 500 analyzer 421
8 8. The Beckman Coulter EPICS XL MCL analyzer 422
8 9. The CyAn benchtop flow cytometer [DakoCytomation] 425
8 10. Cytopeia s InFlux cell sorter platform 426
8 11. Partec s CyFlow flow cytometer 427
8 12. The Partec PAS cytometer 429
10 1. Using DRAQ5 stained nuclei as alignment particles 446
10 2. Equal opportunity and unequal opportunity staining of T cells 447
10 3. Separation of X and Y bull sperm 453
10 4. Detection of BrUdR incorporation using anti BrUdR antibody and the SBIP method 457
10 5. Use of a combination of propidium and TO PRO 3 to detect bromodeoxyuridine incorporation 458
10 6. DNA content vs. expression of cydins B and E in exponentially growing MOLT 4 cells 459
10 7. DNA content vs. RNA content and CD71 expression in activated CD4 cells 460
10 8. Identification of mitotic cells by antibody to phosphorylated histone H3 462
10 9. Subsetting of human T cells into memory classes and measurement of kinases using 11 color fluorescence 468
10 10. Analysis of bacteriophage lambda DNA and fragments from a digest of lambda DNA in a slow flow system....472
10 11. Clusters representing 100 different color coded beads used with Luminex s system for multiplexed analysis 473
10 12. Growth of an encapsulated Gram positive marine bacterium in gel microdroplets 474
10 13. Identification of occupied and unoccupied gel microdroplets 475
10 14. Univariate flow karyotype of human chromosomes stained with propidium iodide 477
10 15. Bivariate karyotypes of RPET001 and Daudi human cell lines 478
10 16. Clusters of peripheral blood leukocyte types in two dimensional displays from a hematology analyzer 483
10 17. Side population (SP) stem cells identified by blue vs, red Hoechst 33342 fluorescence 489
10 18. Time course of events in T lymphocyte activation and probes for their cytometric detection 494
10 19. Intracellular cytokine staining 500
10 20. ERK1/2 kinase phosphorylation in T cells exposed to various activation stimuli 501
10 21. Gating scheme for detection of minimal residual disease in chronic lymphocytic leukemia 505
10 22. T cell subset analysis in lion and puma peripheral lymphocytes 511
10 23. Analysis of clam cells in a hematology analyzer 511
10 24. Determination of nuclear genome size in diploid banana 513
10 25. DNA content of cactus nuclei showing endopolyploidy 513
10 26. Flow karyotype of a translocation line of broad bean 514
10 27. rRNA probes show differences in enteric flora between breast fed infants and infants fed reconstituted milk ...518
10 28. Membrane potential in dormant and resuscitated cultures of Micrococcus luteus 519
10 29. Combined rRNA probe and CTC staining of a genetically and metabolically complex cell mixture 520
10 30. Functional states of Salmonella typhimurium shown by staining with DiBAC (3), ethidium, and propidium ...521
10 31. Effects of amoxicillin on [ratiometric] membrane potential and permeability of Staphylococcus aureus 521
10 32. Fluorescence profiles of viruses stained with SYBR Green 1 523
10 33. Forward scatter, Hoechst 33342, and chlorophyll fluorescence of the marine bacterium Prochlorococcus 524
10 34. Side scatter and fluorescence signatures of four viruses shown in Figure 10 32 525
10 35. Side scatter and fluorescence signatures of viruses and bacteria in a water sample from a small Alpine pond 525
10 36. Size and DNA content of bacteria in seawater from Prince William Sound, Alaska 526
10 37. Size and DNA content of Oligobacterium RBI from Resurrection Bay, Alaska 526
xxxii / Contents
FIGURES (continued)
10 38. Flow cytometry in vivo 542
12 1. Tearing down the picket fence and reuniting the negatives using a BiExponential data transform 562
|
| adam_txt |
CONTENTS
LIST OF TABLES AND FIGURES xxvii
PREFACE TO THE FOURTH EDITION: WHY YOU SHOULD READ THIS BOOK OR NOT xxxiii
FOREWORD TO THE THIRD EDITION by Leonard A. Herzenberg xxxix
PREFACE TO THE THIRD EDITION xli
PREFACE TO THE SECOND EDITION xlv
FOREWORD TO THE FIRST EDITION by Louis A. Kamentsky xlvii
PREFACE TO THE FIRST EDITION xlix
1. OVERTURE 1
1.1 What (And What Good) Is Flow Cytometry? l
Tasks and Techniques of Cytometry 1
Some Notable Applications 1
What is Measured: Parameters and Probes 2
1.2 Beginnings: Microscopy And Cytometry 2
A Little Light Music 4
Making Mountains out of Molehills: Microscopy 6
Why Cytometry? Motivation and Machinery 9
Flow Cytometry and Sorting: Why and How 10
Fluorescence and Flow: Love at First Light 11
Conflict: Resolution 12
1.3 Problem Number One: Finding The CeII(s) 14
Flow Cytometry: Quick on the Trigger 16
The Main Event 17
The Pulse Quickens, the Plot Thickens 17
1.4 Flow Cytometry: Problems, Parameters, Probes, and Principles 18
Counting Cells: Precision I (Mean, S.D., CV) 18
Poisson Statistics and Precision in Counting 19
Rare Event Analysis: The Fundamental Things Apply as Cells Go By 19
Count Constant Numbers for Constant Precision 20
Alternative Counting Aids: The Venerable Bead 20
And Now to See with Eye Serene the Very Pulse
of the Machine: Display, Digitization, and Distributions 21
DNA Content Analysis: Precision II (Variance) 21
The Normal Distribution: Does the Word "Gaussian" Ring a Bell? 22
vii
viii / Contents
1.4 Flow Cytometry: Problems, Parameters, Probes, and Principles (continued)
Binned Data: Navigating the Channels 22
DNA Content: Problem, Parameter, Probes 23
One Parameter Displays: Pulse Height Distributions 24
Mathematical Analysis of DNA Histograms: If It's Worth Doing, It's Worth Doing Well 25
Linear Thinking 26
Lineage Thinking: Sperm Sorting 26
Two Parameter Displays: Dot Plots and Histograms 26
Multiparameter Analysis Without Computers: Gates Before Gates 27
Two Parameter Histograms: Enter the Computer 29
Modern Multiparameter Analysis: List Mode 30
Three Dimensional Displays: Can We Look at Clouds from Both Sides? No 32
Identifying Cells in Heterogeneous Populations: Lift Up Your Heads, Oh Ye Gates! 33
Cluster Headaches 34
Painting and White (or Gray ) Washing Gates 34
The Quad Rant: Are You Positive? Negative! 35
Deals With the Devil: Logarithmic Amplifiers and Fluorescence Compensation 35
Evils of Axes: Truth in Labeling Cells and Plots 38
When Bad Flow Happens to Good Journals 40
Sorting Sorting Out 40
Parameters and Probes II: What is Measured and Why 42
Probes versus Labels 42
Living and Dyeing: Stains, Vital and Otherwise 43
Nucleic Acid (DNA and RNA) Stains 43
Fluorescence and Fluorescent Labels 44
Binary Fishin': Tracking Dyes Through Generations 45
Membrane Perturbation: A Matter of Life and Death? 46
Cytoplasmic/Mitochondrial Membrane Potential 46
Indicators of Cytoplasmic [Ca**]: Advantages of Ratiometric Measurements 47
Finding Antigen Specific Cells Using Tetramers 47
Hip, Hip Arrays: Multiplexing on Slides and in Bead Suspensions 48
GFP and its Relatives: Mild Mannered Reporters 48
Beyond Positive and Negative; Putting the Metry in Cytometry 48
1.5 What's In the Box: Flow Cytometer Anatomy, Physiology, and Pathology 49
Light Sources for Microscopy and Flow Cytometry 49
Instrument Configurations: The Orthogonal Geometry 50
Laser Beam Geometry and Illumination Optics 50
Flow Chamber and Forward Scatter Collection Optics 51
Fluorescence and Orthogonal Scatter Optics 52
Optical Filters for Spectral Separation 52
Multistation Flow Cytometers 54
Photomultipliers and Detector Electronics 54
Putting the Flow in Flow Cytometry 55
Signal Processing Electronics 57
Is It Bigger than a Breadbox? 57
Flow Cytometer Pathology and Diagnostics 58
1.6 Alternatives to Flow Cytometry; Cytometer Ecology 59
1.7 The Rest Of The Book 60
Lis(z)t Mode 60
2. LEARNING FLOW CYTOMETRY 61
Learning from History: Take One 61
Who Should Read this Book? 62
2.1 Information Sources and Resources 62
Books on Flow Cytometry in General 62
Books on Flow Cytometric Methodology and Protocols 62
Contents / ix
2.1 Information Sources and Resources (continued)
Clinical Flow Cytometry Books 63
Other Flow Cytometry Books 63
Flow's Golden Oldies 63
2.2 The Reader's Guide To Periodical Literature 64
2.3 Resources And Courses 66
Flow Cytometer Manufacturers 66
The International Society for Analytical Cytology 66
The Clinical Cytometry Society 66
The National Flow Cytometry Resource 66
"The Annual Courses" and Others 67
Other Societies and Programs 67
The Purdue Mailing List, Web Site, and CD ROMs 68
2.4 Exploring The Foundations 68
Optics and Microscopy 68
Electronics 69
Computers: Hardware and Software 69
Digital Signal Processing 70
Data Presentation and Display 70
Spectroscopy, Fluorescence and Dye Chemistry 71
Cell and Molecular Biology and Immunology 71
2.5 Alternatives To Flow Cytometry 71
3. HISTORY 73
3.1 Ancient History 73
Flow Cytometry: Conception and Birth 73
Staining Before and After Paul Ehrlich 74
Origins of Modern Microscopy 75
Making Cytology Quantitative: Caspersson et al 75
Origins of Cancer Cytology: The Pap Smear 76
The Fluorescent Antibody Method 77
Blood Cell Counting: Theory and Practice 77
Video and Electron Microscopy 78
Optical Cell Counters and the Coulter Orifice 78
3.2 Classical History 79
Analytical Cytology in the 1950's 79
The Cytoanalyzer 79
Acridine Orange as an RNA Stain: Round One 79
How I Got Into this Mess 79
The Rise of Computers 80
Computers in Diagnosis: A Central Problem 80
Diagnosis and Classification: Statistical Methods 80
Cytology Automation in the 1960's 81
First Steps toward Automated Differentials 81
Pattern Recognition Tasks in Cell Identification 82
Differential Leukocyte Counting: An Early Flow Systems Approach 83
Kamentsky's Rapid Cell Spectrophotometer 84
Fulwyler's Cell Sorter 85
3.3 Modern History 85
Cell Cycle Analysis: Scanning versus Flow Systems 85
Cancer Cytology: Scanning versus Flow Cytometry 86
Early Commercial Flow Cytometers 87
Not Quite Commercial: The Block Projects 89
The Evolution of Flow Cytometers in the 1970's 90
Dog Days: The Genesis of Cytomutts 93
The 1980's: Little Things Mean a Lot 94
x / Contents
3.3 Modern History (continued)
Measurements in the Main Stream 95
Immunofluorescence Comes of Age 95
Developments in DNA Content Analysis 96
Flow Cytometry of RNA Content 96
Measurements of Functional Parameters 97
Clinical Uses of Fluorescence Flow Cytometry 98
The End of History? 99
4. HOW FLOW CYTOMETERS WORK 101
4.1 Light and Matter 101
Introduction 101
Photometry versus Radiometry: What's in a Name? 101
Physical Measurement Units 101
Light in Different Lights 102
It's All Done With Photons 102
A Few Warm Bodies 103
Polarization and Phase; Interference 104
Light Meets Matter: Rayleigh and Mie Scattering 105
A Time for Reflection and Refraction: Snell's Law 107
Polarization by Reflection; Brewster's Angle 107
Dispersion: Glass Walls May Well a Prism Make 108
Interference in Thin Films 108
Interference and Diffraction; Gratings 108
Optical Activity and Birefringence 109
Matter Eats Light: Absorption 109
Absorption: Counting the Calories 110
A Selective Diet 110
The Chance of a Lifetime 110
Spinning a Tale of Degeneracy Ill
Facing Extinction: Cross Section and Optical Density Ill
Unexciting Times: Emigrating from the Excited States 112
Fluorescence: Working the Stokes Shift 112
Phosphorescence 113
Fluorescence Polarization 114
Stimulated Emission 114
Resonance Energy Transfer 115
Quenching, Bleaching, and Photon Saturation 115
Quantum Flotsam and Jetsam 118
Inelastic Scattering and Doppler Measurements 118
Raman Scattering 118
Nonlinear Optics and Harmonic Generation 118
Two Photon and Multiphoton Excitation 118
4.2 Optical Systems 119
Light Propagation and Vergence 119
Image Formation by Optical Systems: Magnification 119
Lens Types and Lens Aberrations 120
Numerical Aperture and Lens Performance 121
Gradient Index, Fresnel, and Cylindrical Lenses 122
The Helmholtz Invariant and Throughput 123
Photons in Lenses: See How They Run 123
Aperture and Field Stops: The f Number 124
Depth of Field and Focus and Resolution of Lenses 124
4.3 Light Sources 124
The Best and the Brightest 124
Contents / xi
4.3 Light Sources (continued)
Hare, Hare, the Arc! 126
Quartz Halogen Lamps 127
Light Emitting Diodes (LEDs) 127
Illumination Optics for Lamps and LEDs 127
Arc Source Epiillumination for Flow Cytometry 128
Lasers as Light Sources for Flow Cytometers 129
Laser Illumination: Going to Spot 130
Shedding Light on Cells: Lasers, Lamps, and LEDs 131
Lasers: The Basic Physics 133
Einstein on the Beam: Stimulated Emission 133
Look, Ma, One Cavity: Optical Resonators 133
Laser Action a la Mode. 134
Pumping Ions 135
Laser Efficiency: Your Mileage May Vary 135
Mirrors and Prisms for Wavelength Selection 135
Brewster Windows for Polarized Output 135
Laser Power Regulation: Current and Light Control 136
Beam Profiles and Beam Quality 136
Puttin' on My Top Hat? 138
Harmonic Generation and Modulation 138
Lasers Used and Usable in Cytometry 138
Argon and Krypton Ion Lasers 138
Dye Lasers Hi
Helium Neon Lasers 141
Helium Cadmium and Helium Selenium Lasers 142
Diode Lasers: Red, Infrared, Violet, and UV 142
Solid State Lasers: Like, YAG Me! 145
Laser and Light Source Noise and Noise Compensation 147
Fifty Ways to Lose Your Laser 148
Danger!!! Laser!!! Hazards and Haze 148
4.4 Light Collection 149
Microscope Objectives 149
Looking at the Observation Point 150
Stops versus Blockers 150
Signal versus Noise: To See or Not to See 150
Spectral Selection: Monochromators versus Filters 152
Monochromators and Polychromatic Detection 152
Interference Filters: Coatings of Many Colors 153
Absorptive Filters versus Interference Filters 153
Filter Transmission Characteristics 154
Dichroics 155
Neutral Density Filters 156
Beamsplitters; Ghosts and Ghostbusters 156
Optics for Polarization Measurements 156
Tunable Filters 157
Fiber Optics and Optical Waveguides 157
Through a Glass Darkly: Light Lost (and Found) in Optical Components 158
Collection Optics for Forward Scatter Signals 159
4.5 Detectors 160
Silicon Photodiodes 160
Photomultipliet Tubes (PMTs) 161
Sensitivity Training: Photodiode versus PMT 163
Single Photon Counting 164
Avalanche Photodiodes (APDs) 164
PMTs: Picking a Winner 165
xii / Contents
4.5 Detectors (continued)
Photomukipliers: Inexact Science 166
Charge Transfer Devices: CCDs, CIDs, Etc 166
4.6 Flow Systems 166
Flow System Basics 167
Gently Down the Stream: Laminar Flow 167
Flow Chambers; Backflushes, Boosts, and Burps 169
Cuvettes versus Streams for Analysis and Sorting 170
Light Collection from Streams and Cuvettes 171
When You've a Jet 174
Core and Sheath: Practical Details 175
Grace Under Pressure: Driving die Sheadi and Core 175
Perfect Timing: Fluidics for Kinetic Experiments 177
Oriented and Disoriented Cells 178
Matchmaker, Matchmaker, Make Me a(n) Index Match! 178
Flow Unsheathed 178
Flow Systems: Garbage In, Garbage Out 178
4.7 Electronic Measurements 180
Electricity and Electronics 101 180
Charge Separation, Electric Fields, and Current 180
Resistance, Voltage, and Power; Ohm's Law 181
Alternating and Direct Current; Magnetism 181
Inductance, Reactance, Capacitance, Impedance 182
The Coulter Principle: Electronic Cell Sizing 182
Electrical Opacity: AC Impedance Measurement 183
4.8 Analog Signal Processing 183
Beam Geometry and Pulse Characteristics 183
Electronics 102: Real Live Circuits 184
Circuits: Current Sources and Loads 184
Ground Rules 185
Couplings, Casual and Otherwise; Transformers 186
Power Supplies 187
Active Electronics: Tubes, Transistors, ICs 188
Analog Nirvana: Operational Amplifiers 189
Detector Preamplifiers and Baseline Restoration 190
Analog Pulse Processing: Front Ends and Triggering 191
Peak Detectors 192
Pulse Integral or Area Measurements 194
Pulse Width Measurement Circuits 195
Analog Pulse Processing: The Bottom Line 195
Dead Times, Doublets, and Problem Pulses 196
Trigger Happy? 196
Analog Linear, Log and Ratio Circuits 197
Linear Circuits; Fluorescence Compensation 197
Logarithmic Amplifiers and Dynamic Range 199
Twin Peaks: Distributions on Linear and Log Scales 200
Falling Off a Log: Log Amps Behaving Badly 201
Limits to Dynamic Range 202
Ratio Circuits 204
4.9 Digital Signal Processing 204
Analog to Digital Conversion 204
Free Samples? Hold it! 205
Quantization: When Are Two Bits Worth a Nickel? 205
Analog to Digital Converters (ADCs) (and Digital to Analog Converters (DACs)) 208
Digital Pulse Processing and DSP Chips 209
Contents / xiii
4.9 Digital Signal Processing (continued)
The Screwy Decibel System 211
Pulse Slicing: Deja Vu All Over Again 212
In Defense of de Fence 213
Digitization: Tying it All Together 214
4.10 Performance: Precision, Sensitivity, and Accuracy 214
Precision; Coefficient of Variation (CV) 214
Sensitivity I: Minimum Detectable Signal 215
Sensitivity II: MESF Units 216
Accuracy I: Linearity and Nonlinearity 217
Sensitivity III: What's All the Noise About? 217
Sensitivity IV: More Photons Give Better Precision 218
Sensitivity V: Background Effects 218
Sensitivity VI: Electrons Have Statistics, Too 218
Source Noise Fluctuations and Performance 219
I Blurred It Through the Baseline 219
Restoration Comedy: The Case of the Disappearing Leukocytes 220
Top 40 Noise Sources 221
Sensitivity 007: Q and B (Dye Another Day?) 221
5. DATA ANALYSIS 225
5.1 Goals and Methods in Data Analysis 225
Cell Counting 225
Characterization of Pure Cell Populations 226
Identification of Cells in Mixed Populations 226
Characterization of Cell Subpopulations 226
Data Analysis Hardware and Software Evolve 226
5.2 Computer Systems for Flow Cytometry 227
The Beginning 227
The End of the Beginning 227
Data Rates and Data Acquisition Systems 228
PC Data Acquisition Boards 229
Preprocessors for Data Acquisition 230
5.3 Primary Data: Frequency Distributions 231
You Say You Want a Distribution 231
Gauss Out of Uniform 231
About Binomial Theorem, I'm Teeming With a Lot o' News 232
Distributions Have Their Moments 233
Statistical versus Cytometric Parameters 233
Mean, Variance, and Standard Deviation 233
Widi Many Cheerful Facts About the Square of the Hypotenuse: Euclidean Distance 234
Higher Moments; Skewness and Kurtosis 234
Some Features of the Normal Distribution 234
Measures of Central Tendency: Arithmetic and Geometric Means, Median, and Mode 235
Measures of Dispersion: Variance, Standard Deviation, CV, and Interquartile Range 235
Robustness in Statistics; the Robust CV 235
"Box and Whiskers" Plots of Distributions 236
Calculating and Displaying Histograms 236
Bivariate and Multivariate Distributions and Displays 237
Dot Plots; Correlation and Covariance 237
Linear Regression; Least Squares Fits 237
Breaking off Undiplomatic Correlations 238
Multivariate Measures of Central Tendency and Dispersion 238
Beyond Dot Plots: Two Parameter Histograms 238
Bivariate Distributions: Display's The Thing! 238
Displaying by the Numbers 239
xiv / Contents
5.3 Primary Data: Frequency Distributions (continued)
Economies of Scale 240
How Green Were My (Peaks and) Valleys 241
Clouds on the Horizon: 3 Dimensional Displays 241
5.4 Compensating Without Decompensating 242
5.5 Dealing With the Data 244
Comparing and Analyzing Univariate Histograms 244
The K S Test, Clonal Excess, and % Tests 245
"Nonparametric" Histogram Comparison 245
Cumulative (Overton) Subtraction 245
Constant CV Analysis 245
Another Approach to Histogram Comparison 246
Deconvoluting Single Parameter Histograms 246
Analysis of Two Parameter Data 246
Two Parameter Gating, Bitmap and Otherwise 246
Rectangles and Quadrilaterals: Hardware and Software Gating: 246
Ellipses and Beyond: Advantages of Bitmaps 246
Storage Requirements for Bitmaps 247
Analysis of Two Parameter Distributions 247
See You Around the Quad 247
Bivariate Cell Kinetic Analysis 247
Bivariate Karyotype Analysis 247
Smooth(ing) Operators: When are Filters Cool? 248
Bivariate Analysis in Hematology Analyzers 248
5.6 Multiparameter Data Analysis 248
Multiparameter versus Multivariate Analysis 248
Multiparameter Analysis of Leukocyte Types: 1974 248
Automated Differentials via Discriminants 249
Interactive Analysis: Finding the Training Set 249
Multiparameter Analysis of Leukocyte Types: 2002 250
Procedures for Automated Classification 250
Discriminant Functions and How They Work 250
Principal Component Analysis 252
Cluster Analysis 252
Neural Network Analysis 252
Genetic Algorithms 253
5.7 Analysis of Collected Data: How Much Is Enough/Too Much? 253
5.8 Data Analysis Odds and Ends 254
Data Storage 254
The Flow Cytometry Standard (FCS) File Format 254
Magnetic/ Optical Tumors in the Digital Attic 255
Linear and Log Scales and Ratios: Proceed with Care! 255
Ratios Only Help if Variables are Well Correlated 256
6. FLOW SORTING 257
6.1 Sort Control (Decision) Logic 257
6.2 Preselected Count Circuits and Single Cell Sorting 258
6.3 Droplet Sorting, High Speed and Low 258
Droplet Generation 259
Drop Charging and Deflection 260
Drop Deflection Test Patterns 260
Two and Four Way Sorts: How Much Voltage? 260
How Many Drops Should be Charged? 261
Guilty as Charged? 261
Determining Droplet Delay Settings 262
Fractional Droplet Delays 262
Contents / xv
6.3 Droplet Sorting (continued)
Transducers and Transducer Drive Signals 263
Improving Droplet Sorting 263
Sorting Large Objects with Droplet Sorters 263
6.4 Fluidic Switching Cell Sorters 264
Sorting Large Objects Using Fluidic Switching 265
Sorting Very Small Objects: Microfluidic Switching 266
6.5 Cell Manipulation By Optical Trapping 266
6.6 Cell Damage Cell Selection ("Cell Zapping") 266
Photodamage Cell Selection 266
Sorting (Zapping) Without Flow (Gasp!) 267
Electrodamage Cell Selection in Flow 267
6.7 Measures of Cell Sorter Performance: Purity, Recovery (Yield), and Efficiency 267
Coincidence Effects on Performance 267
6.8 Other Considerations 268
Doing the Math 268
Speed Limits: The Reynolds Rap 269
Instrument Utilization 269
Monitoring versus Sorting for Cell Preparation 269
Collection Techniques: Life and Death Decisions 269
Dilutions of Grandeur 270
Can Getting Sorted Be Hazardous to Cells' Health? 270
6.9 Biohazard Control and Biosafety in Flow Cytometers and Sorters 271
6.10 Conclusions 271
7. PARAMETERS AND PROBES 273
7.1 Physical Parameters and Their Uses 273
Electrical Parameters 273
DC Impedance (Coulter Volume) 273
AC Impedance (Electrical Opacity); Capacitance 273
Acoustic Measurements of Cells in Flow 274
Optical Parameters: Light Scattering 274
Scattering: The Mueller Matrix Model 274
Forward Light Scattering and Cell Size 275
Forward Scatter and "Viability" 276
Side Scatter and Cytoplasmic Granularity 276
Lymphocyte Gating: Forward Scatter Aside 276
Other Applications of Side Scatter 277
What is the Right Angle for "Right Angle" Scatter? 278
Does Side Scatter = Total Protein? 278
Optimizing Side Scatter: Not as Easy as It Looks 270
Polarized 90° Scatter Reveal Eosinophils and Malaria
Pigment Containing Monocytes 278
Multiple Wavelength Scattering Measurements 279
From Russia with Lobes 279
Optical Parameters: Absorption 281
Absorption Effects on Light Scattering 281
Optical Parameters: Extinction 282
Other Transmitted Light Measurements 282
Interference and Phase Measurements 282
Optical Parameters: Fluorescence 283
Fluorescence Lifetime Measurements 283
Fluorescence Polarization Measurements 283
Energy Transfer Measurements: Something to FRET About 283
Quenching and Energy Transfer 284
Measuring Fluorescence Spectra in Flow 284
xvi / Contents
Optical Parameters: Fluorescence
Two Photon Fluorescence Excitation in Flow 284
Bioluminescence detection in Flow? 284
7.2 Intrinsic Cellular Parameters 285
Cell Size 285
Mean Cell Volume: The Cellocrit as Gold Standard 285
Cell Volume, Area, and Diameter 285
Cell Sizing: Slit Scans and Pulse Widths 285
Size Measurements in the Submicron Range 288
Other Size Measurement Techniques 289
Cell Shape and Doublet Discrimination 289
Measurement of Intrinsic Parameters Using Absorption and Extinction Signals 290
Fluorescence Measurement of Intrinsic Parameters 290
Autofluorescence: Pyridine and Flavin Nudeotides 290
Pyridine and Flavin Nucleotides and Redox State 291
Pyridine and Flavin Nucleotides and Cancer 291
Bacterial Autofluorescence Measurements 291
Bacterial Autofluorescence Measurements 291
Porphyrin Fluorescence in Erythroid Cells 292
Other Pigments 292
Chlorophyll and Phycobiliproteins 292
Infrared Spectra and Cancer Diagnosis 293
7.3 Probes, Labels, and [Not] Protocols for Extrinsic Parameter Measurements 293
Probes, Labels, and Dyes 293
Dyes and Quality Control: Gorillas in the NIST 294
The Dyes are Cast: An Overview 295
Mechanisms of Staining by Fluorescent Dyes 298
Environmental Sensitivity 298
Metachromasia 298
Spectral Changes and Ratiometric Measurements 299
Internal Energy Transfer in Probes and Labels 299
"Vital" Staining 299
Staining and "Viability" 299
"Intact" versus "Live" Cells 299
Getting Dyes Into and Out of Intact Cells 300
Permeancy and Permeability 300
Vital Dye Toxicity and Photosensitization of Cells 301
Fixation Why and How 302
Fixation for Biohazard Control 302
Fixation Mechanisms 302
Permeabilization versus Fixation 302
Fixative Effects on Scatter Signals 303
Fixation for Surface Antigen Measurements 303
Fixatives: Coming Out of Aldehyding Places 304
Fixation for Intracellular Antigen Measurements 305
Fixation for DNA Content Determination: Getting DNA (and Antigens) Out of a Tight Fix 305
Catch the Wave: Fixation by the (Cook) Book 305
Fixation Artifacts 305
Red Blood Cell Lysis: The Distilled Essence 306
7.4 Nucleic Acid Dyes and Their Uses 306
DNA Content Measurement 306
Feulgen Staining for DNA Content 306
DNA Staining with Ethidium and Propidium 306
Erhidium Propidium: Ionic Strength Effects 307
DNA Content: Sample Preparation and Standards 307
Chromomycin A3, Mithramycin, and Olivomycin 307
Contents / xvii
Mithramycin Plus Ethidium: Do's and Don'ts 308
DNA Content Measurement (continued)
The Hoechst Dyes (33258, 33342, 34580?) 308
Detecting BrUdR by Hoechst Dye Quenching 308
Hoechst Dyes Have an A T Base Preference 308
Hoechst Dyes In And Out Of Living Cells: The Drug Efflux Pump Discovered 309
Hoechst Dye Staining Mechanisms 309
Hoechst 34580: Violet Time? 310
DAPI (and DIPI): Dyes Known for Precision 310
Determinants of High Precision in DNA Analysis 311
7 Aminoactinomycin D (7 AAD) 311
Acridine Orange 312
StyrylDyes;LDS 751 312
Cyanine Dyes I: Thiazole Orange, etc 312
Cyanine Dyes II: TOTO and YOYO a GoGo 314
Cyanine Dyes III: Alphabet Soup 315
Seeing Red: LD700, Oxazine 750, Rhodamine 800, TO PRO3, and DRAQ5 as DNA Stains 315
Miscellaneous DNA Selective Dyes 316
What Do DNA Stains Stain? 316
DNA Ploidy and Aneuploidy: The DNA Index 317
Sample Preparation for DNA Content Analysis 317
DNA Base Composition 317
Chromatin Structure; Identifying Cells in Mitosis 319
Chromatin Structure Identifies Mitotic Cells 320
RNA Content 320
RNA/DNA Staining with Acridine Orange (AO) 320
Cell Cycle Compartments Defined on the Basis of RNA and DNA Content 320
AO: Problems and Some Solutions 321
Pyronin Y, Oxazine 1, and Other Tricyclic Heteroaromatic Dyes as RNA Stains 322
What Does Pyronin Y Stain? Double Stranded (Ribosomal) RNA and Sometimes Mitochondria 323
Surviving Vital Staining with Pyronin Y 324
Other DNA Dyes Usable with Pyronin Y 324
Tips on Tricyclics (Don't Get Depressed) 325
Tricydics Gag on Mucopolysaccharides 325
Reticulocyte Counting: Cyanines Beat Tricyclics; RNA in Nucleated Cells: Cyanines Don't 326
Propidium Stains Double Stranded RNA: What of Other Dyes? 326
7.5 Fluorescent Labels and Protein Dyes 326
Estimating Total and Basic Protein Content of Cells 327
Fluorescein Isothiocyanate (FITC) 327
Sulforhodamine 101 (SR101) 327
Hematoporphyrin (HP) as a Protein Stain 328
Rhodamine 101 (or 640) as a Vital Protein Stain 328
Staining to Demonstrate Basic Protein 328
Covalent Labels for Antibodies and Other Molecules 328
Fluorescein Isothiocyanate (FITC) as a Label 329
Labeling with Lissamine Rhodamine B and Tetramethylrhodamine Isothiocyanate (TRITC) 329
Multicolor Fluorescence I: FITC and TRITC 329
Multicolor Fluorescence II: Rhodamine 101 Dyes 330
Early Problems with Multicolor Fluorescence 331
Phycobiliproteins to the Rescue! 331
Phycoerythrins: R PE, B PE and Others 332
Allophycocyanin (APC) and APC B 332
Phycocyanins 332
Phycobiliprotein Tandem Conjugates: PE APC, PE Texas Red,
PE Cy5, PE Cy5.5, PE Cy7, etc 333
Allophycocyanin Tandem Conjugates: APC Cy7 and APC Cy5.5 333
xviii / Contents
Phycobiliproteins to the Rescue! (continued)
Mercy Me! PerCP! 333
Phycobiliproteins and Tandems: Dirty Little Secrets 334
Future Tandems: Heterocycles Built for Two? 335
Cyanine Dye Labels: From Cy Fi to Hi5 for Cy5 336
Blue Notes: AMCA and Cascade Blue 337
Hey, BODIPY! 337
Alexa Dyes: Some Thoughts on Dyemographics 338
Other Organic Fluorescent Labels: A Dye Named Joe, etc 338
Quantum Dots 339
Getting Labels Onto Molecules of Interest 340
7.6 Improving Signals from Labels: Amplification and Other Techniques 340
Limits to Sensitivity: Autofluorescence 341
Improving Sensitivity: The New Wave(length) 342
Correcting and Quenching Autofluorescence 342
Raman Scattering Effects on Sensitivity 342
Increasing Sensitivity: Amplification Techniques 343
Amplification by Indirect Staining 343
Amplification Using Labeled Particles 343
Amplification Using Enzymes as Labels: Playing the Hole CARD 344
Amplifying the Analyte: The Polymerase Chain Reaction (PCR) 344
Amplification Techniques: Pros and Cons; Fluorescent versus Nonfluorescent Labels 344
Improving Sensitivity: Time Resolved Fluorescence 345
7.7 Measuring Cell Surface and Intracellular Antigens 345
History and Background 345
Monoclonal Antibodies for the Uninitiated 346
Cell Surface Antigens: Structure versus Function 347
Moving Toward Multicolor Immunofluorescence 347
Antibody Reagents and Staining Procedures 348
Antibody Fragments versus Antibodies as Reagents 348
Engineered Antibodies: Phage Display and scFvs 348
Molecular Probes' Zenon Antibody Labeling 348
Antibody Shelf Life and Quality Control 349
Direct Staining Using Monoclonal Antibodies for 2, 3, and More Colors Using 488 nm Excitation. 349
Multicolor Work Using Multiple Lasers; Biotin Avidin Labeling 349
Mixing Colors: Do's and Don'ts 349
Cocktails for Five: Multiplex Immunofluorescence 350
Cocktail Staining Helps Identify Rare Cells 352
Immunofluorescence Staining Procedures 352
Automated Sample Preparation 353
Fluorescence Measurements: Lurching Toward Quantitation 353
Calibration and Controls: Round One 353
Quantitative Fluorescence Cytometry: Definitions 354
Calibration Particles for QFCM 354
Defining a Window of Analysis 356
Type IIA versus Type IIIB and IIIC Standards 357
Other Aspects of Fluorescence Quantitation 358
What is "Positive"? What is "Negative"? 358
Making Weakly Fluorescent Beads and Cells: Do Try This Trick at Home! 358
Correlating Cytometry and Biochemistry: Studies of Antibody Binding Chemistry 359
Correlating Cytometry and Biochemistry: Intracellular Antigen Measurements 359
Analyzing Immunofluorescence Data 360
Estimating Antigen or Receptor Surface Density 360
Quantitative Fluorescence: Problems and Prospects 361
7.8 Nucleic Acid Sequence Detection 361
Peptide Nucleic Acid (PNA) Probes 362
Contents / xix
7.9 Probes for Various Cell Constituents 362
Surface Sugars (Lectin Binding Sites 362
Analysis of Total Carbohydrate Content 363
Specific Detection of Cellulose 363
A Probe for Cell Surface Aldehydes 363
Probes for Lipids and Cholesterol 364
Nile Red 364
Filipin 364
Lipid Droplet Detection Using Scatter Signals 364
Probes for Cytoskeletal Organization/Actins 364
7.10 Time as a Parameter: Kinetic Measurements 364
Sample Handling for Kinetic Measurements 365
Time as a Quality Control Parameter 366
Slooowww Flooowww 366
7.11 Labeled Ligand Binding 366
Labeling Strategies 367
Formal Analysis of Ligand binding 367
Labeled Ligands versus Anti Receptor Antibodies 368
Ligand Binding Detected by Functional Changes 368
Fluorescent Ligand Binding: Some Examples 368
7.12 Functional Parameters 1 369
Cell Surface Charge 369
Cell Membrane Characteristics 369
Membrane Integrity versus "Viability": Dye Exclusion Tests 369
Detecting "Dead" Cells in Fixed Samples 369
Membrane Fusion and Turnover; Cell Tracking 371
Cell Proliferation Analyzed Using Tracking Dyes 371
Membrane Organization and Fluidity/Viscosity 374
Lipid Packing Assessed with Merocyanine 540 374
Membrane Fluidity and Microviscosity: Assessment Using Fluorescence Polarization 374
Lipid Peroxidation 375
Membrane Permeability to Dyes and Drugs: The Drug Efflux Pump Revisited 376
Endocytosis of Macromolecules and Particles 377
Enzyme Activity 378
Indicators of Oxidative Metabolism I: Tetrazolium Dye Reduction 379
Indicators of Oxidative Metabolism II: 2,7 Dichlorofluorescin Diacetate (DCFH DA), etc 379
Indicators of Oxidative Metabolism III: Hydroethidine (Dihydroethidium) 379
Indicators of Oxidative Metabolism IV: Dihydrorhodamine 123 379
Indicators of Oxidative Metabolism V: Detection of Hypoxic Cells 380
Detection of Caspase Activity 380
Other Enzymes 380
Detection of Enzymes and Products by Antibodies 380
Enzyme Kinetics in Single Cells 380
Sulfhydryl (Thiol) Groups; Glutathione 381
7.13 Functional Probes II: Indicators of Cell Activation 381
Introduction 381
Changes in the Cellular Ionic Environment Following Activation by
Ligand Interaction with Cell Surface Receptors 382
"Structuredness of Cytoplasmic Matrix" (SCM) and the Cercek Test for Cancer 383
Optical Probes of Cell Membrane Potential 385
Membrane Potential and Its Physicochemical Bases 385
A4* Measurement Using Microelectrodes 386
Single Cell Measurements with Distributional Probes 387
Oxonol Dyes as Membrane Potential Probes 390
Possible Alternatives to Distributional Probes for Flow Cytometry of Membrane Potentials 391
Ratiometric Probes for Membrane Potential 391
xx / Contents
Optical Probes of Cell Membrane Potential (continued)
Using Cyanine Dyes for Flow Cytometric A^F Estimation, In Case You're Still Interested 392
Cytoplasmic Membrane Potential: Summing Up 394
Mitochondrial Membrane Potential (A^) 394
Mitochondria! Staining with Rhodamine 123 Is Membrane Potential Dependent 394
The Search for Better A^B Probes: Round One 397
AT^JC l.andApoptosis 397
The Search for Better A*¥m Probes: Round Two 398
Bacterial Membrane Potentials 400
Ratiometric A^P Measurement in Bacteria 401
A*F Measurement: Cautions and Conclusions 402
Optical Probes of Intracellular Calcium 402
The Bad Old Days 402
Chlortetracycline as a Probe of "Membrane Bound" Calcium in Cells 402
Probes for Free Cytoplasmic Calcium: Quin 2 403
Fura 2 and Indo 1: Ratiometric Ca" Indicators 403
Fluo 3 and Other Visible Excited Ca" Probes 404
Flow Cytometric Probes of Intracellular pH 405
The Hat Trick: Multiparameter Approaches to Ion Flux Measurements in Cell Activation 407
NOsing Around for Nitric Oxide 408
Other Ions in the Fire 408
7.14 Reporter Genes 408
Somebody Cloned My Gal: Enzymes as Reporter Genes 408
Green Fluorescent Protein (GFP) et al 409
Minority Report(er)? 410
8. BUYING FLOW CYTOMETERS 411
8.1 Introduction 411
8.2 History 411
8.3 BD Biosciences 412
Background 412
The BD FACS Vantage SE™ Cell Sorter 413
The BD FACSCalibur™ Analyzer 414
The B D™ LSRII Analyzer 416
The B D FACSAria™ Cell Sorter 417
The B D FACSCount 418
8.4 Beckman Coulter, Inc 418
Background and Signal to Background 418
The Beckman Coulter EPICS® ALTRA™ Cell Sorter 419
The Beckman Coulter Cytomics™ FC 500 Analyzer 420
The EPICS® XL and XL MCL Analyzers 422
8.5 DakoCytomation 423
Background 423
The MoFlo® Cell Sorter 423
The CyAn Flow Cytometer 424
8.6 Cytopeia 425
The InFlux Cell Sorter 425
8.7 Optoflow AS 426
Background 426
The MICROCYTE® Flow Cytometer 426
8.8 Partec GmbH 427
Background 427
The CyFlow® and CyFlow® ML Flow Cytometers 427
The PAS, PAS II, and PAS III Flow Cytometers [ .!. 428
PA Ploidy Analyzer and CCA Cell Counter Analyzer 429
Contents / xxi
8. BUYING FLOW CYTOMETERS (continued)
8.9 Some Other Flow Cytometer Companies 429
Advanced Analytical Technologies, Inc. (AATI) 429
Agilent Technologies, Inc 429
Apogee Flow Systems Ltd 430
Bentley Instruments 430
ChemunexSA 430
CytoBuoy b.v 430
Delta Instruments bv 431
Fluid Imaging Technologies, Inc 431
FOSS Electric A/S 431
Guava Technologies, Inc 431
Howard M. Shapiro, M.D., P.C 431
iCyt — Visionary Bioscience 431
International Remote Imaging Systems 431
Luminex Corporation 431
NPE Systems, Inc 432
Union Biometrica, Inc 432
8.10. Hematology Instruments, Etc 433
8.11 Little Orphan Analyzers (And Big Orphan Sorters) 434
Bio/Physics and Ortho: Cytofluorograf to Cytoron 434
HEKA Elektronik GMBH: The FLUVO II Analyzer 435
TheKratel Partograph 435
The ODAM ATC 3000 435
Also Among the Missing 436
Flow Cytometer Rehabilitation; Used Instruments 436
Following Suit 436
8.12 Third Party Software 437
8.13 The Selling of Flow Cytometers: Hype and Reality 437
8.14. Applying for a Grant for a Cytometer 438
9. BUILDING FLOW CYTOMETERS 441
9.1 Why Buy a Flow Cytometer? 441
9.2 Why Build a Flow Cytometer? 441
9.3 Learning to Build Your Own 442
10. USING FLOW CYTOMETERS: APPLICATIONS, EXTENSIONS, AND ALTERNATIVES 443
10.1 The Daily Grind 443
Keeping the Instrument Running: Diet and Exercise 443
Particulars: Drawing a Bead on Flow Cytometer Alignment, Calibration, and Standardization 444
Alignment Particles: Fearful Asymmetry 445
Reference and Calibration Particles 445
Compensation Standards 446
Cells and Nuclei as Alignment Particles 446
Rose Colored Glasses: Optical Filter Selection 446
Experimental Controls 447
Shake Well Before Using: When Controls Won't Help 447
10.2 Significant Events in the Lives of Cells 448
Taking the Census: Cell Counting 448
A Counting Alternative: Image Analysis 448
The Doubled Helix: Reproduction 448
The Cell Cycle and Cell Growth 448
DNA Content Analysis 448
Mathematical Models for DNA Analysis 449
Clinical Application of DNA Content Analysis 450
xxii / Contents
DNA Content Analysis(continued)
DNA Content Alternatives: Static Photometry and Scanning Laser Cytometry 451
The Mummy's GC/AT: DNA Content Analysis in Anthropology and Forensic Science 451
Haifa Genome is Better Than None: Sperm Sorting 452
The Widening Go/G, re Detecting Mutation 453
Detecting DNA Synthesis: Cell Kinetics 453
Kinetics Before Flow Cytometry: Mitotic Indices, Doubling Times, and Radiolabel Studies. 453
Labeling Index versus DNA Content 454
Early Flow Cytometric Approaches to Labeling Using BrUdR and 3H TdR 455
Detection of Incorporated BrUdR with Hoechst Dyes and Propidium Iodide 455
Detection of BrUdR Incorporation with Anti BrUdR Antibodies 455
Cytochemical Detection of BrUdR Incorporation Using Difference and Ratio Signals 456
Breaking Up Is Easy To Do: SBIP, a Simpler Way to Detect BrUdR Incorporation into DNA 456
Anti BrUdR Antibody: Seeing the Light 457
Cytochemical Detection of BrUdR: Still Around 457
Detecting RNA Synthesis Using Bromouridine 458
Generation Gaps: Tracking Dyes and Cell Kinetics 458
Cell Cycle Related Proteins: Cyclins, Etc 458
Detecting Mitotic Cells 462
Memento Mori: Detecting Cell Death 462
Necrosis versus Apoptosis 462
Identifying Apoptotic Cells 462
Les Feuilles Mortes (Autumn Leaves) 462
Apoptosis: Getting With the Program 463
ISNT there Light at the End of the TUNEL? 463
Apoptosis: The Case Against Flow Cytometry 463
Die Another Day: Cytometry of Telomeres 464
10.3 Identification of Cells in Mixed Populations 464
Mixed Genotypes versus Mixed Phenotypes 464
No Parameter Identifies Cancer Cells 464
Many Parameters Identify Blood Cells 464
Flow Cytometric Parameters Useful for Blood Cells 464
Specific Gene Products Identify Cell Types 465
Maturation Processes and "Missing Links": The "Ginger Root" Model 465
Practical Multiparameter Gating: Color Wars 467
Finding Rare Cells 469
One Parameter is Not Enough 469
Cocktail Staining Can Help 470
Dirt, Noise, and Rare Event Detection 470
Really, Really Rare Events: Alternatives to Flow 470
10.4 Tricks and Twists: Odd ]obs for Flow Cytometry 471
Single Molecule Detection 471
DNA Sizing, if not Sequencing, in Flow 471
Solid Phase (Bead) Assays Using Flow Cytometry 473
Cocktails for 100: Multiplexed Bead Assays 473
Cells in Gel Microdroplets and on Microspheres 474
Hanging Ten Pseudopodia? 475
10.5 Single Cell Analysis: When Flow Won't Do 475
10.6 Applications of Flow Cytometry 476
Cell Differentiation, Ab Ovo and De Novo 476
Differentiation in the Nervous System 476
Whole Embryo Sorting 476
Somatic Cell Genetics and Cell Hybridization 477
Reporter Genes Revisited 477
Isolating and Characterizing Hybrid Cells 477
Chromosome Analysis and Sorting and Flow Karyotyping 477
Contents / xxiii
10.6 Applications of Flow Cytometry (continued)
Probing Details of Cellular Structures and Inter and Intramolecular Interactions 479
Dissection of Structures Using Antibodies, Ligands, and Genetic Methods 479
Intramolecular Interactions 479
Clinical Flow Cytometry: Turf and Surf 480
Hematology 480
Clinical Application: Blood Cell Counting and Sizing 480
Red Blood Cells (Erythrocytes) 481
Clinical Application: Reticulocyte Counts 481
The Reticulocyte Maturity Index (RMI) 482
Erythrocyte Flow Cytometry: Other Clinical Uses 482
White Blood Cells (Leukocytes) 483
Clinical Application: Differential Leukocyte Counting 483
CD Characters: Leukocyte Differentiation Antigens 484
Granulocytes: Basophils 484
Basic Orange 21: The Best Basophil Stain Yet 485
Allergy Tests Using Basophil Degranulation 485
Granulocytes: Eosinophils 486
Granulocytes: Neutrophils 486
(Clinical?) Tests of Neutrophil Function 486
Neutrophil CD64 in Inflammation and Sepsis 487
When I'm Sick CD64? 487
Platelets and Megakaryocytes 487
Hematopoietic Stem Cells 488
Clinical Application: Monitoring CD34+ Stem Cells According to the ISHAGE Protocol 488
Side Population (SP) Stem Cells: Plastic, Fantastic 489
Immunology 489
Immunologic Applications of Flow Cytometry: Still a Growth Industry 489
HIV Infection The "Killer Application" 490
Clinical Application: T Cell Subset Analysis 490
T Cell Subsets: Alternative Technologies 491
Clinical Application: Transplantation 493
Detecting Lymphocyte Activation 494
Foundations: From PHA (the Lectin) to PHA (the Pulse Height Analyzer) 495
Functional Probes for Activation 495
DNA, RNA, and Activation Antigens 497
Mitogen Response versus Antigen Response 498
Detecting Activation by CD69 Expression 499
Cytokines: Detecting Activation and More 499
Ins and Outs of Cytokine Staining 500
Tetramer Staining: Talking the Talk; Walking the Walk? 500
Tracking Dyes: Activation and Ontogeny 501
What is "Early" Activation (Trick Question)? 501
Cancer Biology and Clinical Oncology 502
Cancer Diagnosis: Cervical Cytology 503
DNA Content Measurements Yet Again 503
Beyond DNA Content: Antigens, Oncogenes, and Receptors, and Response to Therapy 504
Immunophenotyping in Hematopathology 504
Detecting Minimal Residual Disease 505
Biological Implications of Phenotyping Results 506
Digression: A Slight Case of Cancer 507
Analysis of Sperm 508
Isolating Fetal Cells from the Maternal Circulation for Prenatal Diagnosis 509
The March of Time: Orcadian Rhythms, Aging, and Atherosclerosis 510
Clinical Application: Urine Analysis 510
The Animal Kingdom 510
xxiv / Contents
The Animal Kingdom (continued)
Lions and Pumas and Clams, Oh, My! 510
Fish Story; FISH Story 511
Flow Cytometry: For the Birds? 512
Big Stuff, Vegetable, Animal or Mineral 512
Flow Cytometry of Plant Cells and Chromosomes 512
Measurement of Plant Cell DNA Content 513
Plant Chromosome Analysis and Sorting 514
Other Flow Cytometric Applications in Plants 514
Microbiology, Parasitology and Marine Biology 514
Measuring Microbes: Motivation 515
Measuring Microbes: Instrument Issues 515
Parameters Measured in Microorganisms 516
Flow Cytometric "Gram Stains" 516
Detection and Sizing: Light Scattering 517
Detection and Sizing: Electrical Impedance 517
Nucleic Acid (DNA and RNA) Staining 517
Total Protein Content: Scatter versus Stains 518
Antibodies, Etc.: Labeling Strategies 518
Ribosomal RNA Based Species Identification 518
Functional Probes in Bacteria 518
Potential, Permeability, "Viability", and Metabolic Activity 519
Digression: A Therapeutic Approach Based on Transient Permeabilization 522
So Few Molecules; So Little Time 522
Applications in Marine Microbiology 524
Extensions: Cytometers for Marine Applications 527
References: Flow Cytometry and Oceanography 527
General Microbiology 527
Previously Noted 527
Cell Cycles and Cell Division 528
Fluorescent Protein Methods in Microbes 528
Microbial Communities: Will Flow Work? 528
Bad Guys Don't All Wear Black Hats: Microbial Detection/Identification
in Health Related Contexts 528
The Basic Questions 528
Detection: Intrinsic Parameters are Not Enough 529
Detection: Fluorescence Improves Accuracy 529
Detection: When the Tough Get Going 529
Identification: Too Many Broths 530
Identification: Can Multiplexing Help? 531
Environmental and Sanitary Microbiology 531
Water That Made Milwaukee (and Sydney) Infamous 531
Food Microbiology 532
Bioterrorism and Bioopportunism 532
Viruses and Other Intracellular Pathogens 532
Clinical Microbiology 533
Antimicrobial Susceptibility Testing: One Size Does Not Fit All 534
Bacteria: Confusion Reigns 535
Mycobacteria: Down for the Count 535
Antifungal Susceptibility: Flow Does the Job 535
Antiviral Susceptibility by Flow Cytometry 536
Cytometry in Vaccine Development 536
Microbiology Odds and Ends 536
Parasitology 535
Pharmacology and Toxicology 537
Drugs and the Life and Death of Cells 538
Contents / xxv
Pharmacology and Toxicology (continued)
Erythrocyte Micronucleus Assays 538
Toxic Waste and B Cell Proliferation 538
Radiation Dosimetry 538
Food Science 538
Somatic Cell Counts in Milk 538
Brewhaha 539
A Loaf of Bread, A Jug of Wine 539
Seeing the Blight 539
Major Food Group: Chocolate 539
Biotechniques and Biotechnology 539
Protein and Gene Expression on Cells and Beads 540
Getting Big Molecules into Small Cells 540
Staying Alive, Staying Alive 540
Et Cetera 540
Alternatives: Microfluidic Cytometers, Flow and Static 541
Cytometry Afield 541
The Lymphocytes of the Long Distance Runner 541
War and Peace 541
Blood, Sweat and Tears? 542
Pulp Nonfiction 542
Flow Cytometry On the Rocks 542
To Boldly Go Where No Cytometer Has Gone Before 542
11. SOURCES OF SUPPLY 543
11.1 Resources, Societies, Journals 543
11.2 Optical Supply Houses 543
11.3 Probes and Reagents 544
11.4 Calibration Particles/Cytometry Controls 548
11.5 Flow Cytometers 549
Hematology Instruments 551
11.6 Data Analysis Software/Systems 551
Hardware and Software 551
Commercial Software Sources 552
Noncommercial Software Sources 552
11.7 Cytometer Rehabilitation/Add ons 553
11.8 Flow Cytometer Parts 553
Flow System Plumbing 553
Photodetectors 554
DC DC Converter Modules for HV Power Supplies 555
Power Supplies (Low Voltage) 555
Other Electronics 555
11.9 Lasers 555
Laser Trade Publications 555
Laser Manufacturers 555
11.10 Optical Filters 556
Color Glass Filters 556
Interference Filters 557
Neutral Density Filters 557
Polarizing Filters and Optics 557
Tunable Filters 557
11.11 Aids to Troubleshooting Flow Cytometers When All Else Fails 557
11.12 Proficiency Testing 557
11.13 Sex Selection 557
11.14 Alternative Technology 558
xxvi / Contents
12. AFTERWORD 561
12.1 Dotting i's and Crossing t's 561
12.2 Late Breaking News 561
New Book 561
New Protein Stain 561
Caveat on Fluorescent Caspase Inhibitors 561
Polyamide Probes 561
Tearing Down the (Picket) Fences 562
New Instrument: The BD FACSArray™ 563
Science Special Section: Biological Imaging 563
Cytomics in Predictive Medicine: a Clinical Cytometry Special Issue and Other
Recent Citings and Sightings 563
12.3 Analytical Biology, Such as it Isn't: Is This Any Way to Run a Science? 563
12.4 Colophon 564
12.5 Unfinished Business 565
AIDS and Infectious Disease in the Third World 565
A Center for Microbial Cytometry 565
A Nobel Prize for Herzenberg and Kamentsky? 566
12.6 Flow and the Human Condition 566
There's No Business Like Flow Business 566
12.7 One More Thing 566
Contents / xxvii
TABLES AND FIGURES
TABLES
1 1. Some parameters measurable by cytomeuy 3
3 LA brief outline of flow cytometric history (1945 2000) 100
4 1. SI units and prefixes 102
4 2. Emission wavelengths of lasers 139
4 3. Cathode quantum efficiencies of diode and PMT detectors between 300 and 800 nm 165
4 4. Logarithmic Amplifiers: What goes in, what comes out 203
4 5. Characteristics of analog to digital converters 206
5 1. Some landmarks of the normal or Gaussian distribution 234
5 2. Equations (1 4) that must be solved to permit 4 color fluorescence compensation 242
5 3. Header and text portion of an FCS2.0 data file 254
6 1. Safe speed limits for sorting based on Reynolds number calculations 269
7 1. Some cellular parameters measurable by cytometry 286
7 2. Fluorescence spectral properties of a selection of reagents usable for common cytometric tasks 297
7 3. Tricydic heteroaromatic compounds usable for staining DNA and/or RNA 323
7 4. Raman emission from water 343
xxviii / Contents
FIGURES
1 1. Interaction of light with a cell 4
1 2. Transmitted light and dark field images of an unstained suspension of human peripheral blood leuokocytes 7
1 3. Transmitted light microscope images of an unstained smear of human peripheral blood 8
1 4. Schematic of a fluorescence microscope 9
1 5. Scanned images of Feulgen stained lymphoblastoid cells 15
1 6. One dimensional scanning of cells deposited in a narrow line 16
1 7 Idealized plot of signal amplitude vs. time 16
1 8. Ideal and "real" DNA content distributions 22
1 9. Single parameter histogram displays from a multichannel pulse height analyzer 24
1 10. Use of a mathematical model to determine fractions of DNA aneuploid breast cancer cells 25
1 11. Dotplot (cytogram) of Hoechst 33342 and fluorescein fluorescence in CCRF CEM cells 26
1 12. Gating regions for counting or sorting set electronically, drawn on an oscilloscope display 27
1 13. Histogram of 90° (side) scatter from leukocytes in lysed whole blood 30
1 14. Bivariate distribution of anti CD3 antibody fluorescence intensity vs. large angle scatter in leukocytes 31
1 15. The bivariate distribution of Figure 1 14 shown as an isometric or "peak and valley" plot 32
1 16. The two parameter histogram of Figures 1 14 and 1 15 displayed as a contour plot 32
1 17. Identification of human peripheral blood T lymphocytes bearing CD4 and CD8 antigens 34
1 18. Why fluorescence compensation is necessary 37
1 19. How compensation gets data to fit into quadrants 38
1 20. Fluorescence intensities of antibody stained cells and beads bearing known amounts of antibody 49
1 21. Schematic of die optical system of a fluorescence flow cytometer 51
1 22. A typical flow chamber design 56
1 23. FACScan Analyzer (Becton Dickinson) 58
1 24. MoFlo High Speed Sorter (Cytomation) 58
1 25. Microcyte Cytometer (Optoflow) 58
1 26. Estimated numbers of fluorescence flow cytometers in use worldwide, 1975 92 59
2 1. Growth of the flow cytometry literature, 1987 93 64
3 1. The first working flow cytometer (Gucker particle counter) 74
3 2. Digitized image of a neutrophil polymorphonudear leukocyte 82
3 3. Refined and prototype versions of Kamentsky's Rapid Cell Spectrophotometer (RCS) 84
3 4. A two parameter histogram of blood cells analyzed in the RCS 84
3 5. Louis Kamentsky and the Bio/Physics Systems Cytofluorograf 87
3 6. The Technicon Hemalog D Differential Leukocyte Counter 88
3 7. Leonard Herzenberg with B D's first commercial version of the Fluorescence Activated Cell Sorter (FACS) 88
3 8. A two parameter display from die Block differential counter showing five leukocyte clusters 89
3 9. The author with Cytomutt and Cerberus 94
3 10. Cell cycle phases defined by DNA content and by DNA/RNA content 97
4 1. Radian andsteradian 101
4 2. Light as an electromagnetic wave 104
4 3. Constructive and destructive interference 105
4 4. Circularly polarized light 105
4 5. Reflection and refraction of light at a surface 106
4 6. The Jablonski diagram of electronic energy levels, or states, and transitions 112
4 7. Fluorescence spectrum of fluorescein 113
4 8. Light from a point source at the focal length of a lens is collimated 119
4 9. Rays from a point source can be focused to an image point 119
4 10. Rays from many points of an object add up to make an image 120
4 11. Elements of a typical microscope lens, showing die half angle that defines the acceptance cone and N.A 120
4 12. Showing die effect of N.A. on light collection 121
4 13. Multiple views of magnification 123
Contents / xxix
FIGURES (continued)
4 14. Output characteristics of arc, quartz halogen, and deuterium lamps 125
4 15. Kohler and critical illumination 128
4 16. Optics for arc source epiillumination for fluorescence microscopy or flow cytometry 129
4 17. Use of crossed cylindrical lenses to focus a laser beam to an elliptical spot on the core stream 131
4 18. Energy levels involved in laser action 133
4 19. Schematic of a laser 134
4 20. Laser transverse excitation modes 134
4 21. Measured beam intensity profiles of diode, CO2, and He Ne lasers 136
4 22. Intensity profiles of a focused and defocused violet laser diode beam 136
4 23. Sizes, power requirements, and approximate costs of some smaller lasers used for cytometry 145
4 24. Effect of noise compensation circuitry on precision of fluorescence measurements 147
4 25. Looking at the observation point 150
4 26. Maximizing light collection: Not always a good way to do things 151
4 27. A prism monochromator 153
4 28. Light transmission characteristics of bandpass, short pass, and long pass interference filters 154
4 29. Transmission of several wavelength regions through different dichroic configurations 155
4 30. Cube and plate beamsplitters 156
4 31. Total internal reflection 157
4 32. A fiber optical waveguide 157
4 33. Optical arrangements for collection of forward scattered light 159
4 34. Elements of a photomultiplier tube (PMT) 160
4 35. PMT electrode voltage supply circuits: dynode chain and Cockcroft Walton voltage multiplier 162
4 36. Detectors and housings 163
4 37. Fluid flow in a flow cytometer 167
4 38. Laminar flow profile illustrated by diatoms in the "Flow CAM" imaging flow cytometer 167
4 39. Flow chamber designs 169
4 40. New angles on light collection in flow 171
4 41. Flow chamber designs used with arc source flow cytometers 173
4 42. Minimizing turbulence generated at the sheath inlet to flow chambers 175
4 43. Sheath fluid supply plumbing 175
4 44. The Coulter orifice 183
4 45. Effect of beam geometry on pulse shape 184
4 46. Some circuit elements 185
4 47. A line powered DC power supply 187
4 48. Basic operational amplifier circuits 190
4 49. A photodetector preamplifier circuit 191
4 50. Waveforms in preamplifier, front end electronics, and peak detector circuits 192
4 51. Schematic diagram of a peak detector 192
4 52. Preamp and peak detector outputs (oscilloscope traces) 193
4 53. Telling two cells from one gets harder as the cells go through closer together 196
4 54. Uncompensated and compensated fluorescence signals from FL and PE labeled beads 198
4 55. One side of a two color compensation circuit 198
4 56. Signals from PE labeled antibody bound to beads, on linear and 4 decade logarithmic scales 199
4 57. Response curves of different types of log amps as determined according to Parks, Bigos, and Moore 202
4 58. Continuous and sampled signals 205
4 59. Digital pulse processing: "slicing" a slightly noisy Gaussian pulse with a baseline 209
4 60. MESF threshold sensitivity determination using fluorescein labeled beads 216
4 61. Fluorescence distributions for bead sets measured with progressively lower values of Q 222
4 62. Separation (or lack thereof) of CD4+ lymphocytes and unstained cells at various values of Q and B 223
5 1. Distributions of sums of uniformly distributed random numbers approach the normal distribution 231
5 2. Pascal's triangle 232
5 3. Binomial distributions for n = 2, 4, 8, and 16, with/) = q = 0.5 232
5 4. Binomial distributions for n = 16 and/» = 0.5, 0.25, and 0.125; they are skewed when/ * 0.5 233
xxx / Contents
FIGURES (continued)
5 5. Euclidean distance between two points, with an assist from Pythagoras 234
5 6. "Box and whiskers" plots showing medians and interquartile ranges of distributions, after Tukey and Tufte 236
5 7. Histogram display formats 237
5 8. Dot plots of computer generated data showing various degrees of correlation 237
5 9. Varieties of two parameter data display (cover figure) 239
5 10. Density plot showing a scale indicating numbers of events 240
5 11. 3 Dimensional "cloud" plots of CD3, CD4, and CD8 antigens on blood lymphocytes and thymocytes 241
5 12. How compensation gets data to not quite fit into quadrants 243
5 13. Two generated near normal distributions and their cumulative distributions, or integrals 245
5 14. Quadrant statistics 247
5 15. Multiparameter analysis of peripheral blood leukocyte types: partitioning in two dimensional subspaces 249
5 16. Linear transformation of data to separate clusters, as is done in linear discriminant analysis 251
5 17. Calculation is not always necessary to reduce the dimensionality of data 254
5 18. Linear and log scales revisited 255
6 1. Droplet sorting 259
6 2. A droplet sorter test stream pattern 261
6 3. Mack Fulwyler widi his islet sorter 264
6 4. Fluidic sorter designs 264
6 5. A microfluidic flow sorter for bacteria 265
7 1. Forward scatter does not measure particle size 275
7 2. Depolarized 90° scatter signals can be used to identify eosinophil granulocytes 278
7 3. Indicatrices (plots of intensity vs. scattering angle) of particles and cells 280
7 4. Erythrocytes scatter less light at a wavelength at which hemoglobin exhibits strong absorption 281
7 5. Side scatter signals from T2 bacteriophages 288
7 6. The principle of doublet discrimination using pulse height and pulse integral measurements 290
7 7. Fluorescence spectra of some materials implicated in mammalian cell autofluorescence 291
7 8. Zinc protoporphyrin fluorescence in human red cells 292
7 9. Probe fluorescence spectra and source emission wavelengths 296
7 10. A "rogues' gallery" of nucleic acid dyes 301
7 11. DNA content distributions in sperm from a normal ram and a ram bearing a translocation 311
7 12. Structure of symmetric cyanine dyes given die formula "DiYCn(1(2m + 1)" by Sims et al 313
7 13. Flow karyotype of human chromosomes stained with Hoechst 33258 and chromomycin A, 318
7 14. Hoechst/chromomycin fluorescence signatures of bacteria with different DNA base compositions 318
7 15. Flow cytometry of chromatin structure identifies cells in mitosis 320
7 16. DNA and RNA content analysis of mitogen stimulated lymphocytes 321
7 17. Chemical structures of some reactive labels 327
7 18. Multiplex immunofluorescent labeling to demonstrate multiple cell types in blood 351
7 19. Quantitative determination of CD4 epitopes on peripheral blood lymphocytes and monocytes 356
7 20. Flow cytometric detection of HIV 1 nucleic acids in cells after in situ PCR 361
7 21. Plot of cytoplasmic [Ca"] as indicated by indo 1 fluorescence ratio versus time 365
7 22. Time used as a quality control parameter in DNA analysis (after Watson) 366
7 23. Cell proliferation indicated by dilution of fluorescence of the tracking dye PKH26 372
7 24. Cell proliferation indicated by dilution of fluorescence of CFSE labeled CD4+ lymphocytes 373
7 25. Specific staining of glutathione in cells by monobromobimane (MBB) 381
7 26. Valinomycin induced changes in fluorescence intensity of cyanine dye loaded red cell suspensions 387
7 27. Distributions of the fluorescence of DiOC6(3) in CCRF CEM T lymphoblasts 388
7 28. Distributions of membrane potential in cells suspended in NaCL, KC1 and a mixture of the two 389
7 29. Structures of two membrane potential probes, the cyanine DiOC5(3) and the oxonol DiBAC4(3) 390
7 30. Two parameter analyses of lectin stimulated lymphocytes: DNA vs. RNA and membrane potential 396
7 31. Demonstration of apoptotic HL 60 cells with deenergized mitochondria by JC 1 staining 399
7 32. Measurement of membrane potential of Staphylococcus aureus using a ratiometric technique 400
7 33. Emission spectra of indo 1 in solutions of increasing free calcium ion concentration 404
Contents / xxxi
FIGURES (continued)
7 34. Emission spectra of fluo 3 in solutions of increasing free Ca" concentration 405
7 35. Estimation of cytoplasmic pH in human lymphocytes from carboxyfluorescein fluorescence ratio 406
7 36. The pH dependent emission spectra of carboxy SNARF 1 excited at 488 nm 407
8 1. The BD FACSVantage cell sorter 413
8 2. The BD FACSCalibur benchtop cell sorter 415
8 3. The BD LSR multi beam benchtop analyzer 416
8 4. BD's "Octagon" collection optics 416
8 5. The BD FACSAria cell sorter 417
8 6. The Beckman Coulter EPICS Altra sorter 419
8 7. The Beckman Coulter Cytomics FC 500 analyzer 421
8 8. The Beckman Coulter EPICS XL MCL analyzer 422
8 9. The CyAn benchtop flow cytometer [DakoCytomation] 425
8 10. Cytopeia's InFlux cell sorter platform 426
8 11. Partec's CyFlow flow cytometer 427
8 12. The Partec PAS cytometer 429
10 1. Using DRAQ5 stained nuclei as alignment particles 446
10 2. "Equal opportunity" and "unequal opportunity" staining of T cells 447
10 3. Separation of X and Y bull sperm 453
10 4. Detection of BrUdR incorporation using anti BrUdR antibody and the SBIP method 457
10 5. Use of a combination of propidium and TO PRO 3 to detect bromodeoxyuridine incorporation 458
10 6. DNA content vs. expression of cydins B and E in exponentially growing MOLT 4 cells 459
10 7. DNA content vs. RNA content and CD71 expression in activated CD4 cells 460
10 8. Identification of mitotic cells by antibody to phosphorylated histone H3 462
10 9. Subsetting of human T cells into memory classes and measurement of kinases using 11 color fluorescence 468
10 10. Analysis of bacteriophage lambda DNA and fragments from a digest of lambda DNA in a slow flow system.472
10 11. Clusters representing 100 different color coded beads used with Luminex's system for multiplexed analysis 473
10 12. Growth of an encapsulated Gram positive marine bacterium in gel microdroplets 474
10 13. Identification of occupied and unoccupied gel microdroplets 475
10 14. Univariate flow karyotype of human chromosomes stained with propidium iodide 477
10 15. Bivariate karyotypes of RPET001 and Daudi human cell lines 478
10 16. Clusters of peripheral blood leukocyte types in two dimensional displays from a hematology analyzer 483
10 17. Side population (SP) stem cells identified by blue vs, red Hoechst 33342 fluorescence 489
10 18. Time course of events in T lymphocyte activation and probes for their cytometric detection 494
10 19. Intracellular cytokine staining 500
10 20. ERK1/2 kinase phosphorylation in T cells exposed to various activation stimuli 501
10 21. Gating scheme for detection of minimal residual disease in chronic lymphocytic leukemia 505
10 22. T cell subset analysis in lion and puma peripheral lymphocytes 511
10 23. Analysis of clam cells in a hematology analyzer 511
10 24. Determination of nuclear genome size in diploid banana 513
10 25. DNA content of cactus nuclei showing endopolyploidy 513
10 26. Flow karyotype of a translocation line of broad bean 514
10 27. rRNA probes show differences in enteric flora between breast fed infants and infants fed reconstituted milk .518
10 28. Membrane potential in dormant and resuscitated cultures of Micrococcus luteus 519
10 29. Combined rRNA probe and CTC staining of a genetically and metabolically complex cell mixture 520
10 30. Functional states of Salmonella typhimurium shown by staining with DiBAC (3), ethidium, and propidium .521
10 31. Effects of amoxicillin on [ratiometric] membrane potential and permeability of Staphylococcus aureus 521
10 32. Fluorescence profiles of viruses stained with SYBR Green 1 523
10 33. Forward scatter, Hoechst 33342, and chlorophyll fluorescence of the marine bacterium Prochlorococcus 524
10 34. Side scatter and fluorescence signatures of four viruses shown in Figure 10 32 525
10 35. Side scatter and fluorescence signatures of viruses and bacteria in a water sample from a small Alpine pond 525
10 36. Size and DNA content of bacteria in seawater from Prince William Sound, Alaska 526
10 37. Size and DNA content of Oligobacterium RBI from Resurrection Bay, Alaska 526
xxxii / Contents
FIGURES (continued)
10 38. Flow cytometry in vivo 542
12 1. Tearing down the "picket fence" and reuniting the negatives using a BiExponential data transform 562 |
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| discipline | Biologie Medizin |
| discipline_str_mv | Biologie Medizin |
| edition | 4. ed. |
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| index_date | 2024-07-02T15:57:11Z |
| indexdate | 2024-07-09T20:45:38Z |
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| isbn | 0471411256 9780471411253 9780471722731 |
| language | English |
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| spelling | Practical flow cytometry Howard M. Shapiro 4. ed. Hoboken, NJ Wiley-Liss 2003 1 Online-Ressource txt rdacontent c rdamedia cr rdacarrier Flow Cytometry Flow cytometry Durchflusscytometrie (DE-588)4226628-2 gnd rswk-swf Electronic books Durchflusscytometrie (DE-588)4226628-2 s b DE-604 Shapiro, Howard M. Sonstige oth https://onlinelibrary.wiley.com/doi/book/10.1002/0471722731 Verlag Volltext HBZ Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=015042652&sequence=000002&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Practical flow cytometry Flow Cytometry Flow cytometry Durchflusscytometrie (DE-588)4226628-2 gnd |
| subject_GND | (DE-588)4226628-2 |
| title | Practical flow cytometry |
| title_auth | Practical flow cytometry |
| title_exact_search | Practical flow cytometry |
| title_exact_search_txtP | Practical flow cytometry |
| title_full | Practical flow cytometry Howard M. Shapiro |
| title_fullStr | Practical flow cytometry Howard M. Shapiro |
| title_full_unstemmed | Practical flow cytometry Howard M. Shapiro |
| title_short | Practical flow cytometry |
| title_sort | practical flow cytometry |
| topic | Flow Cytometry Flow cytometry Durchflusscytometrie (DE-588)4226628-2 gnd |
| topic_facet | Flow Cytometry Flow cytometry Durchflusscytometrie Electronic books |
| url | https://onlinelibrary.wiley.com/doi/book/10.1002/0471722731 http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=015042652&sequence=000002&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
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