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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?.............................................................................................1

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 Cell(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)tMode ...........................................................................................................................................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 1980V. Little Things Mean a Lot........................................................................................................94

χ / 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 .............................................................................................................. I l l

Facing Extinction: Cross Section and Optical Density............................................................................. I l l

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.....................................................................................................................................141

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

Photomultiplier 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)

Photomultipliers: 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 the Sheath 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 OfFa 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: DejaVu 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 Β (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

With 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 χ

2

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

Datastorage ........................................................................................................................................ 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 Nucleotides..........................................................................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

Ethidium & Propidium: Ionic Strength Effects ...................................................................... 307

DNA Content: Sample Preparation and Standards ......................................................................... 307

Chromomycin A

3

, 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 Υ Stain? Double-Stranded (Ribosomal) RNA and Sometimes Mitochondria.......323

Surviving Vital Staining with Pyronin Υ..........................................................................................324

Other DNA Dyes Usable with Pyronin Υ........................................................................................324

Tips on Tricyclics (Don't Get Depressed) ....................................................................................... 325

Tricyclics 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 Β 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 CercekTest for Cancer ....................................... 383

Optical Probes of Cell Membrane Potential ............................................................................................ 385

Membrane Potential and Its Physicochemical Bases ......................................................................... 385

ΔΨ 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 ΔΨ Estimation, In Case You're Still Interested ................392

Cytoplasmic Membrane Potential: Summing Up ...........................................................................394

Mitochondrial Membrane Potential (ΔΨ^ .....................................................................................394

Mitochondrial Staining with Rhodamine 123 Is Membrane Potential-Dependent....................394

The Search for Better A

A v

F m

x

¥ m

Probes: Round One........................................................................397

,JC-l,andApoptosis.......................................................................................................397

The Search for Better ΔΨ^, Probes: Round Two......................................................................398

Bacterial Membrane Potentials......................................................................................................400

Ratiometric ΔΨ Measurement in Bacteria .............................................................................401

ΔΨ 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

++

Fluo-3 and Other Visible-Excited Ca

Indicators ................................................................................403

++

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

83 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

TheCyAn™ Flow Cytometer................................................................................................................. 424

8.6Cytopeia ............................................................................................................................................ 425

The InFlux Cell Sorter .......................................................................................................................... 425

8.7OptofIow AS......................................................................................................................................... 426

Background .......................................................................................................................................... 426

The MICROCYTE® Flow Cytometer ................................................................................................... 426

8.8 PartecGmbH......................................................................................................................................... 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

The Kratel 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 G

0

/Gj 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

3

H-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

An ti-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 ofTelomeres.............................................................................................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 Jobs 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 Ονο 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: Τ Cell Subset Analysis.............................................................................490

Τ 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: Circadian Rhythms, Aging, and Atherosclerosis ....................................................... 510

Clinical Application: Urine Analysis ....................................................................................................... 510

The Animal Kingdom ............................................................................................................................ 510

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 ..................................................................................................................................536

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 Β 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 cytometry............................................................................................................ 3

3-1. A 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. Tricyclic 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. Dot plot (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 the 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 polymorphonuclear 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 the 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 and steradian ...................................................................................................................................... 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 the half angle that defines the acceptance cone and N.A............ 120

4-12. Showing the 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, CO

2

, 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 Β...................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 η = 2, 4, 8, and 16, with/? = q = 0.5 ...........................................................................232

5-4. Binomial distributions for η = 16 and/> = 0.5, 0.25, and 0.125; they are skewed when/) Φ 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 with 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 the formula "DiYC n+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 DiOC

6

(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 DiOC

5

(3) and the oxonol DiBAC

4

(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 / xxx

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 BDFACSVantage 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 ofX- 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 cyclins Β and Ε 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

4

(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 Pro ch low coccus.............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 Οligobacterium 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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