Table of Contents

CONTENTS

LIST OF TABLES AND

FIGURES

PREFACE TO THE FOURTH EDITION: WHY YOU SHOULD READ THIS BOOK - OR NOT

FOREWORD TO THE THIRD EDITION

by Leonard

A.

Herzenberg

PREFACE TO THE THIRD EDITION

PREFACE TO THE SECOND EDITION

FOREWORD TO THE FIRST EDITION

by Louis

A.

Kamenrsky

PREFACE TO THE FIRST EDITION

1. OVERTURE

1.1 What (And What Good) Is Flow Cytometry?

Tasks and Techniques of Cyrometry

Some Notable Applications

What is Measured: Parameters and Probes

1.2 Beginnings: Microscopy And Cytometry

A Little Light Music

Making Mountains out of Molehills: Microscopy

Why Cyrometry? Motivation and Machinery

Flow Cyrometry and Sorting: Why and How

Fluorescence and Flow: Love ar First Light..

Conflict: Resolution

1.3 Problem Number One: Finding The Cell(s)

Flow Cytornetry: Quick on the Trigger

The Main Event

The Pulse Quickens, the Plot Thickens

1.4 Flow Cytometry: Problems, Parameters, Probes, and Principles

Counting Cells: Precision I (Mean, S.D., CV)

Poisson Statistics and Precision in Counting

Rare Event Analysis: The Fundamental Things Apply as Cells Go By

Count Constant Numbers for Constant Precision

Alternative Counting Aids: The Venerable Bead

And Now to

See with Eye Serene the Very Pulse of the Machine: Display, Digitization, and Distributions

DNA Content Analysis: Precision II (Variance)

The Normal Distribution: Does the Word "Gaussian" Ring a Bell?

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1.4 Flow

Cytomerry: Problems, Parameters, Probes, and Principles (continued)

Binned Data: Navigating the Channels

DNA Content: Problem, Parameter, Probes

·

One-Parameter Displays: Pulse Height Distributions

Mathematical Analysis of DNA Histograms: If It's Worth Doing, It's Worth Doing Well

Linear Thinking

Lineage Thinking: Sperm Sorting

Two-Parameter Displays: Dot Plots and Histograms

Mulripararneter Analysis Without Computers: Gates Before Gates

Two-Paramerer Histograms: Enter the Computer

Modern Multiparameter Analysis: List Mode

Three-Dimensional Displays: Can We Look at Clouds from Both Sides? No

Identifying Cells in Heterogeneous Populations: Lift Up Your Heads, Oh Ye Gates!

Cluster Headaches

Painting and White- (or Gray-) Washing Gates

The Quad Rant: Are You Positive? Negative!

Deals With the Devil: Logarithmic Amplifiers and Fluorescence Compensation

Evils of Axes: Truth in Labeling Cells and Plots

When Bad Flow Happens to Good

J oumals

Sorting Sorting Out

Parameters and Probes II: What is Measured and Why

Probes versus Labels

Living and Dyeing: Stains, Vital and Otherwise

Nucleic Acid (DNA and RNA) Stains

Fluorescence and Fluorescent Labels

Binary Fishin': Tracking Dyes Through Generations

Membrane Perturbation: A Matter of Life and Death?

Cytoplasmic/Mitochondrial Membrane Potential

Indicators of Cytoplasmic [Ca

H

] :

Advantages of Ratiometric Measurements

Finding Antigen-Specific Cells Using Tetramers

Hip, Hip Arrays: Multiplexing on Slides and in Bead Suspensions

GFP and its Relatives: Mild Mannered Reporters

Beyond Positive and Negative; Purring the -Metry in Cytornetry

1.5 What's In the Box: Flow Cytometer Anatomy, Physiology, and Pathology

Light Sources for Microscopy and Flow Cytornetry

Instrument Configurations: The Orthogonal Geometry

Laser Beam Geometry and Illumination Optics

Flow Chamber and Forward Scatter Collection Optics

Fluorescence and Orthogonal Scatter Optics

Optical Filters for Spectral Separation

Multisrarion Flow Cytometers

Photomultipliers and Detector Electronics

Purring the Flow in Flow Cytornerry

Signal Processing Electronics

Is It Bigger than a Breadbox?

Flow Cytometer Pathology and Diagnostics

1.6 Alternatives to Flow Cytornetry: Cytometer Ecology

1.7 The Rest Of The Book

Lis(z)t Mode

2. LEARNING FLOW CYTOMETRY

Learning from History: Take One

Who Should Read this Book?

2.1 Information Sources and Resources

Books on Flow Cytometry in General

Books on Flow Cytornetric Methodology and Protocols

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2.1 Information Sources and Resources (continued)

Clinical Flow Cytornerry Books

Other Flow Cytornerry Books

Flow's Golden Oldies

2.2 The Reader's Guide To Periodical Literature

2.3 Resources And Courses

Flow Cytometer Manufacturers

The International Society for Analytical Cytology

The Clinical Cytornerry Society

The National Flow Cytornetry Resource

"The Annual Courses" and Others

Other Societies and Programs

The Purdue Mailing List, Web Site, and CD-ROMs

2.4 Exploring The Foundations

Optics and Microscopy

Electronics 69

Computers: Hardware and Software

Digital Signal Processing

Data Presentation and Display

Spectroscopy, Fluorescence and Dye Chemistry

Cell and Molecular Biology and Immunology

2.5 Alternatives To Flow Cytometry

3. HISTORY

3.1 Ancient History

Flow Cytometry: Conception and Birth

Staining Before and After Paul Ehrlich

Origins of Modern Microscopy

Making Cytology Quantitative: Caspersson et al

Origins of Cancer Cytology: The Pap Smear

The Fluorescent Antibody Method

Blood Cell Counting: Theory and Practice

Video and Electron Microscopy

Optical Cell Counters and the Coulter Orifice

3.2 Classical History

Analytical Cytology in the 1950's

The

Cytoanalyzer

Acridine Orange as an RNA Stain: Round One

How I Got Into this Mess

The Rise of Computers

Computers in Diagnosis: A Central Problem

Diagnosis and Classification: Statistical Methods

Cytology Automation in the 1960·s

First Steps toward Automated Differentials

Pattern Recognition Tasks in Cell Identification

Differential Leukocyte Counting: An Early Flow Systems Approach

Kamentsky's Rapid Cell Spectrophotometer

Fulwyler's Cell Sorter

3.3 Modern History

Cell Cycle Analysis: Scanning versus Flow Systems

Cancer Cytology: Scanning versus Flow Cytometry

Early Commercial Flow Cytometers

Not Quite Commercial: The Block Projects

The Evolution of Flow Cytorneters in the 1970's

Dog Days: The Genesis of Cytomutrs

The 1980's: Little Things Mean a Lot

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3.3 Modern History (continued)

Measurements in the Main Stream

Immunofluorescence Comes ofAge

Developments in DNA Content Analysis

Flow Cytometry of RNA Content

Measurements of Functional Parameters

Clinical Uses of Fluorescence Flow Cytometry

The End of History?

4. HOW FLOW CYTOMETERS WORK

4.1 Light and Matter

Introduction

Photometry versus Radiometry: What's in a Name?

Physical Measurement Units

Light in Different Lights

It's All Done With Photons

A Few Warm Bodies

Polarization and Phase; Interference

Light Meets Matter: Rayleigh and Mie Scattering

A Time for Reflection - and Refraction: Snell's Law

Polarization by Reflection; Brewster's Angle

Dispersion: Glass Walls May Well a Prism Make

Interference in Thin Films

Interference and Diffraction; Gratings

Optical Activity and Birefringence

Matter Eats Light: Absorption

Absorption: Counting the Calories

A Selective Diet

The Chance of a Lifetime

Spinning a Tale of Degeneracy

Facing Extinction: Cross Section and Optical Density

Unexciting Times: Emigrating from the Excited States

Fluorescence: Working the Stokes Shift

Phosphorescence

Fluorescence Polarization

Stimulated Emission

Resonance Energy Transfer

Quenching, Bleaching, and Photon Saturation

Quantum Flotsam and Jetsam

Inelastic Scattering and Doppler Measurements

Raman Scattering

Nonlinear Optics and Harmonic Generation

Two-Photon and Mulriphoron Excitation

4.2 Optical Systems

Light Propagation and Vergence

Image Formation by Optical Systems: Magnification

Lens Types and Lens Aberrations.....

Numerical Aperture and Lens Performance

Gradient Index, Fresnel, and Cylindrical Lenses

The Helmholtz Invariant and Throughput

Photons in Lenses: See How They Run

Aperture and Field Stops: The f

Number

Depth ofField and Focus and Resolution of Lenses

4.3 Light Sources

The Best and the Brightest

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4.3 Light Sources (continued)

Hare, Hare, the Arc!

Quartz Halogen Lamps

Light Emitting Diodes (LEDs)

Illumination Optics for Lamps and LEDs

Arc Source Epiillumination for Flow Cytometry

Lasers as Light Sources for Flow Cytomerers

Laser Illumination: Going to Spot

Shedding Light on Cells: Lasers, Lamps, and LEDs

Lasers: The Basic Physics

Einstein on the Beam: Stimulated Emission

Look, Ma, One Cavity: Optical Resonators

Laser Action 11 la Mode

Pumping Ions

Laser Efficiency: Your Mileage May Vary

Mirrors and Prisms for Wavelength Selection

Brewster Windows for Polarized Output

Laser Power Regulation: Current and Light Comro!...

Beam Profiles and Beam Quality

Puttin' on My Top Hat?

Harmonic Generation and Modulation

Lasers Used and Usable in Cytometry

Argon and Krypton Ion Lasers

Dye Lasers

Helium-Neon Lasers

Helium-Cadmium and Helium-Selenium Lasers

Diode Lasers: Red, Infrared, Violet, and UV

Solid-State Lasers: Like, YAG Me!

Laser and Light Source Noise and Noise Compensation

Fifty Ways to Lose Your Laser

Danger!!! Laser!!! Hazards and Haze

4.4 Light Collection

Microscope Objectives

Looking at the Observation Point

Stops versus Blockers

Signal versus Noise: To See or Not to

See

Spectral Selection: Monochromators versus Filters

Monochromators and Polychromatic Detection

Interference Filters: Coatings of Many Colors

Absorptive Filters versus Interference Filters

Filter Transmission Characteristics

Dichroics

Neutral Density Filters

Beamsplitters; Ghosts and Ghostbusters

Optics for Polarization Measurements

Tunable Filters

Fiber Optics and Optical Waveguides

Through a Glass Darkly: Light Lost (and Found) in Optical Components

Collection Optics for Forward Scatter Signals

4.5 Detectors

Silicon Photodiodes

Photomultiplier Tubes (PMTs)

Sensitivity Training: Photodiode versus PMT

Single Photon Counting

Avalanche Photodiodes (APDs)

PMTs: Picking a Winner

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4.5 Detectors (continued)

Photomultipliers: Inexact Science

Charge Transfer Devices: CCDs, CIDs, Etc.........

4.6 Flow

Systems

Flow System Basics

Gently Down the Stream: Laminar Flow

Flow Chambers; Backflushes, Boosts, and Burps

Cuvettes versus Streams for Analysis and Sorting

Light Collection from Streams and Cuvettes

Whenyou'veaJet

Core and Sheath: Practical Details

Grace Under Pressure: Driving the Sheath and Core

Perfect Timing: Fluidics for Kinetic Experiments

Oriented and Disoriented Cells

Matchmaker, Matchmaker, Make Me a(n) Index Match!

Flow Unsheathed

Flow Systems: Garbage In, Garbage Out

4.7 Electronic Measurements

Electricity and Electronics 101

Charge Separation, Electric Fields, and Current

Resistance, Voltage, and Power; Ohm's Law

Alternating and Direct Current; Magnetism

Inductance, Reactance, Capacitance, Impedance

The Coulter Principle: Electronic Cell Sizing

Electrical Opacity: AC Impedance Measurement

4.8 Analog Signal Processing

Beam Geometry and Pulse Characteristics

Electronics 102: Real Live Circuits

Circuits: Current Sources and Loads

Ground Rules

Couplings, Casual and Otherwise; Transformers

Power Supplies

Active Electronics: Tubes, Transistors, ICs

Analog Nirvana: Operational Amplifiers

Detector Preamplifiers and Baseline Restoration

Analog Pulse Processing: Front Ends and Triggering

Peak Detectors

Pulse Integral or Area Measurements

Pulse Width Measurement Circuits

Analog Pulse Processing: The Bottom Line

Dead Times, Doublets, and Problem Pulses

Trigger Happy?

Analog Linear, Log and Ratio Circuits

Linear Circuits; Fluorescence Compensation

Logarithmic Amplifiers and Dynamic Range

Twin Peaks: Distributions on Linear and Log Scales

Falling Off a Log: Log Amps Behaving Badly

Limits to Dynamic Range

Ratio Circuits

4.9 Digital Signal Processing

Analog-to-Digital Conversion

Free Samples? Hold it!

Quantization: When Are Two Bits Worth a Nickel?

Analog-to-Digital Converters (ADCs) (and Digital-to-Analog Converters (DACs))

Digital Pulse Processing and DSP Chips

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Digital Signal Processing (continued)

The Screwy Decibel System

Pulse Slicing: Deja Vu All Over Again

In Defense of de Fence

4.10

Digitization: Tying it All Together

Performance: Precision, Sensitivity, and Accuracy

Precision; Coefficient of Variation (CV)

Sensitivity I: Minimum Detectable Signal

Sensitivity II: MESF Units

Accuracy I: Linearity and Nonlinearity

Sensitivity III: What's All the Noise About?

Sensitivity IV: More Photons Give Better Precision

Sensitivity V: Background Effects

Sensitivity VI: Electrons Have Statistics, Too

Source Noise Fluctuations and Performance

I Blurred It Through the Baseline

Restoration Comedy: The Case of the Disappearing Leukocytes

Top 40 Noise Sources

Sensitivity 007:

Q and B (Dye Another Day?)

5. DATA ANAL

YSIS

5.1

Goals and Methods in Data Analysis..

Cell Counting

Characterization of Pure Cell Populations

Identification of Cells in Mixed Populations

Characterization of Cell Subpopulations

Data Analysis Hardware and Software Evolve

5.2 Computer Systems for Flow Cytometry

The Beginning

The End of the Beginning

Data Rates and Data Acquisition Systems

PC Data Acquisition Boards

Preprocessors for Data Acquisition

5.3 Primary Data: Frequency Distributions

You Say You Want a Distribution

Gauss Out of Uniform

About Binomial Theorem, I'm Teeming With a Lot

0'

News

Distributions Have Their Moments

Statistical versus Cytornerric Parameters

Mean, Variance, and Standard Deviation

With Many Cheerful Facts About the Square of the Hypotenuse: Euclidean Distance

Higher Moments; Skewness and Kurtosis

Some Features of the Normal Distribution

Measures of Central Tendency: Arithmetic and Geometric Means, Median, and Mode

Measures of Dispersion: Variance, Standard Deviation, CV, and Interquartile Range

Robustness in Statistics; the Robust CV

"Box-and-Whiskers" Plots of Distributions

Calculating and Displaying Histograms

Bivariate and Multivariate Distributions and Displays

Dot Plots; Correlation and Covariance

Linear Regression; Least-Squares Fits

Breaking off Undiplomatic Correlations

Multivariate Measures of Central Tendency and Dispersion

Beyond Dot Plots: Two-Parameter Histograms

Bivariate Distributions: Display's The Thing!

Displaying by the Numbers

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5.3 Primary Data: Frequency Distributions

(continued)

Economies of Scale

How Green Were My (Peaks and) Valleys

Clouds on the Horizon: 3-Dimensional Displays

5.4 Compensating Without

Decompensating

5.5 Dealing With the Data

Comparing and Analyzing Univariate Histograms

The K-S Test, Clonal Excess, and X' Tests

"Nonparametric" Histogram Comparison

Cumulative (Overton) Subtraction

Constant CV Analysis

Another Approach to Histogram Comparison

Deconvoluting Single-Parameter Histograms

Analysis of Two-Parameter Data

Two-Parameter Gating, Bitmap and Otherwise

Rectangles and Quadrilaterals: Hardware and Software Gating:

Ellipses and Beyond: Advantages of Bitmaps

Storage Requirements for Bitmaps

Analysis of Two-Parameter Distributions

See You Around the Quad

Bivariate Cell Kinetic Analysis

Bivariate Karyotype Analysis

Smooth(ing) Operators: When are Filters Cool?

Bivariate Analysis in Hematology Analyzers

5.6 Multiparameter Data

Analysis

Multiparameter versus Multivariate Analysis

Multiparameter Analysis of Leukocyte Types: 1974

Automated Differentials via Discriminants

Interactive Analysis: Finding the Training Set

Multiparamerer Analysis of Leukocyte Types: 2002

Procedures for Automated Classification

Discriminant Functions and How They Work

Principal Component Analysis

Cluster Analysis

Neural Network Analysis

Genetic Algorithms

5.7 Analysis of Collected Data: How Much Is Enough/Too Much?

5.8 Data Analysis Odds and

Ends

Data Storage

The Flow Cyrornetry Standard (FCS) File Format

Magnetic! Optical Tumors in the Digital Attic

Linear and Log Scales and Ratios: Proceed with Care!

Ratios Only Help if Variables are Well Correlated

6. FLOW SORTING

6.1 Sort Control (Decision) logic

6.2 Preselected Count Circuits and Single Cell Sorting

6.3 Droplet Sorting, High-Speed and Low

Droplet Generation

Drop Charging and Deflection

Drop Deflection Test Patterns

Two- and Four-Way Sorts: How Much Voltage?

How Many Drops Should be Charged?

Guilty as Charged?

Determining Droplet Delay Settings

Fractional Droplet Delays

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6.3 Droplet Sorting (continued)

Transducers and Transducer Drive Signals

Improving Droplet Sorting

Sorting Large Objects with Droplet Sorters

6.4 Fluidic Switching Cell Sorters

Sorting Large Objects Using Fluidic Switching

Sorting Very Small Objects: Microfluidic Switching

6.5 Cell Manipulation By Optical Trapping

6.6 Cell Damage Cell Selection ("Cell Zapping").

Photodamage Cell Selection

Sorting (Zapping) Without Flow (Gasp!)

Electrodamage Cell Selection in Flow

6.7 Measures of Cell Sorter Performance: Purity, Recovery (Yield), and Efficiency

Coincidence Effects on Performance

6.8 Other Considerations

Doing the Math

Speed Limits: The Reynolds Rap

Instrument Utilization

Monitoring versus Sorting for Cell Preparation

Collection Techniques: Life and Death Decisions

Dilutions of Grandeur

Can Getting Sorted Be Hazardous to

Cells' Health?

6.9 Biohazard Control and Biosafety in Flow Cytometers and SOrters

6.10 Conclusions

7. PARAMETERS AND PROBES

7.1 Physical Parameters and Their Uses

Electrical Parameters

DC Impedance (Coulter Volume)

AC Impedance (Electrical Opacity); Capacitance

Acoustic Measurements of Cells in Flow

Optical Parameters: Light Scattering

Scattering: The Mueller Matrix Model

Forward Light Scattering and Cell Size

Forward Scatter and "Viability"

Side Scatter and Cytoplasmic Granulariry

Lymphocyte Gating: Forward Scatter Aside

Other Applications of Side Scatter

What is the Right Angle for "Right Angle" Scatter?

Does Side Scatter

=

Total Protein?

Optimizing Side Scatter: Not as Easy as It Looks

Polarized 90° Scatter Reveal Eosinophils and Malaria

Pigment-Containing Monocytes

Multiple Wavelength Scattering Measurements

From Russia with Lobes

Optical Parameters: Absorption

Absorption Effects on Light Scattering

Optical Parameters: Extinction

Other Transmitted Light Measurements

Interference and Phase Measurements

Optical Parameters: Fluorescence

Fluorescence Lifetime Measurements

Fluorescence Polarization Measurements

Energy Transfer Measurements: Something to FRET About

Quenching and Energy Transfer

Measuring Fluorescence Spectra in Flow

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Optical Parameters: Fluorescence

Two-Photon Fluorescence Excitation in Flow

Bioluminescence detection in Flow?

7.2 Intrinsic Cellular

Parameters

Cell Size

Mean Cell Volume: The Cellocrit as Gold Standard

Cell Volume, Area, and Diameter

Cell Sizing: Slit Scans and Pulse Widths

Size Measurements in the Submicron Range

Other Size Measurement Techniques

Cell Shape and Doublet Discrimination

Measurement of Intrinsic Parameters Using Absorption and Extinction Signals

Fluorescence Measurement of Intrinsic Parameters

Autofluorescence: Pyridine and Flavin Nucleotides

Pyridine and Flavin Nucleotides and Redox State

Pyridine and Flavin N uc1eotides and Cancer

Bacterial Autofluorescence Measurements

Bacterial Autofluorescence Measurements

Porphyrin Fluorescence in Erythroid Cells

Other Pigments

Chlorophyll and Phycobiliproteins

Infrared Spectra and Cancer Diagnosis

7.3 Probes, labels, and [Not] Protocols for Extrinsic Parameter

Probes, Labels, and Dyes

Dyes and Quality Control: Gorillas in the NIST

The Dyes are Cast: An Overview

Mechanisms of Staining by Fluorescent Dyes

Environmental Sensitivity

Metachromasia

Spectral Changes and Ratiomerric Measurements

Internal Energy Transfer in Probes and Labels

"Vital" Staining

Staining and "Viability"

"Intact" versus "Live" Cells

Getting Dyes Into - and Out of - Intact Cells

Permeancy and Permeability

Vital Dye Toxicity and Photosensitization of Cells

Fixation - Why and How

Measurements

Fixation for Biohazard Control

Fixation Mechanisms

Permeabilization versus Fixation

Fixative Effects on Scatter Signals

Fixation for Surface Antigen Measurements

Fixatives: Coming Our ofAldehyding Places

Fixation for Intracellular Antigen Measurements

Fixation for DNA Content Determination: Getting DNA (and Antigens) Out of a Tight Fix

Catch the Wave: Fixation by the (Cook) Book

Fixation Artifacts '"

Red Blood Cell Lysis: The Distilled Essence

7.4 Nucleic Acid Dyes and Their Uses

DNA Content Measurement

Feulgen Staining for DNA Content

DNA Staining with Erhidium and Propidium

Ethidium & Propidium: Ionic Strength Effects

DNA Content: Sample Preparation and Standards

Chromomycin

A."

Mithramycin, and Olivomycin

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Mithrarnycin Plus Ethidium: Do's and Don'ts

DNA Content Measurement (continued)

The Hoechst Dyes (33258, 33342, 34580?)

Detecting BrUdR by Hoechst Dye Quenching

Hoechst Dyes Have an A-T Base Preference

Hoechst Dyes In - And Out Of - Living Cells: The Drug Efflux Pump Discovered

Hoechst Dye Staining Mechanisms

Hoechst 34580: Violet Time?

DAPI (and DIP!): Dyes Known for Precision

Determinants of High Precision in DNA Analysis

7-Aminoactinomycin D (7-AAD)

Acridine Orange

Styryl Dyes; LDS-751

Cyanine Dyes I: Thiazole Orange, etc

Cyanine Dyes II: TOTO and YOYO a

GoGo

Cyanine Dyes III: Alphabet Soup

Seeing Red: LD700, Oxazine 750, Rhodamine 800, TO-PR03, and DRAQ5 as DNA Stains

Miscellaneous DNA-Selective Dyes

What Do DNA Stains Stain?

DNA Ploidy and Aneuploidy: The DNA Index

Sample Preparation for DNA Content Analysis

DNA Base Composition

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Chromatin Structure; Identifying Cells in Mitosis

Chromatin Structure Identifies Mitotic Cells

RNA Content

RNNDNA

Staining with Acridine Orange (AO)

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Cell Cycle Compartments Defined on the Basis of RNA and DNA Content

AO: Problems and Some Solutions

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Pyronin Y, Oxazine 1, and Other Tricyclic Heteroaromatic Dyes as RNA Stains 322

What Does Pyronin Y Stain? Double-Stranded (Ribosomal) RNA and Sometimes Mitochondria 323

Surviving Vital Staining with Pyronin Y 324

Other DNA Dyes Usable with Pyronin

Y 324

Tips on Tricyclics (Don't Get Depressed)

Tricyclics Gag on Mucopolysaccharides

Reticulocyte Counting: Cyanines Beat Tricyclics; RNA in Nucleated Cells: Cyanines Don't

Propidium Stains Double-Stranded RNA: What of Other Dyes?

7.5 Fluorescent Labels and Protein Dyes

Estimating Total and Basic Protein Content of Cells

Fluorescein Isorhiocyanate (FITC)

Sulforhodamine 101 (SR101)

Hematoporphyrin (HP) as a Protein Stain

Rhodamine 101 (or 640) as a Vital Protein Stain

Staining to Demonstrate Basic Protein

Covalent Labels for Antibodies and Other Molecules

Fluorescein Isothiocyanate (FITC) as a Label

1.

Labeling with Lissamine Rhodamine B and Tetramerhylrhodarnine Isothiocyaila:e (TRITC)

Multicolor Fluorescence I: FITC and TRITC

Multicolor Fluorescence II: Rhodamine 101 Dyes

Early Problems with Multicolor Fluorescence

Phycobiliproteins to the Rescue!

Phycoeryrhrins: R-PE, B-PE and Others

Allophycocyanin (APC) and APC-B

Phycocyanins

Phycobiliprotein Tandem Conjugates: PE-APC, PE-Texas Red,

PE-Cy5, PE-Cy5.5, PE-Cy7, etc

Allophycocyanin Tandem Conjugates: APC-Cy7 and APC-Cy5.5

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J

Contents

Phycobiliproteins to the Rescue! (continued)

Mercy Me! PetCPl

Phycobiliproteins and Tandems: Dirty Little Secrets

Future Tandems: Heterocycles Built for Two?

Cyanine Dye Labels: From Cy-Fi to

Hi5 for Cy5

Blue Notes: AMCA and Cascade Blue

Hey, BODIPY!

Alexa Dyes: Some Thoughts on Dyemographics

Other Organic Fluorescent Labels: A Dye Named Joe, etc

Quantum Dots

Getting Labels Onto Molecules of Interest ,

7.6 Improving Signals from Labels: Amplification and Other

Techniques

Limits to

Sensitivity:Autofluorescence

Improving Sensitivity: The New Wave(length)

Correcting and Quenching Autofluorescence

Raman Scattering Effects on Sensitivity

Increasing Sensitivity: Amplification Techniques

Amplification by Indirect Staining

Amplification Using Labeled Particles

Amplification Using Enzymes as Labels: Playing the Hole CARD

Amplifying the Analyte: The Polymerase Chain Reaction (peR)

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Amplification Techniques: Pros and Cons; Fluorescent versus Nonfluorescent Labels

Improving Sensitivity: Time-Resolved Fluorescence

7.7 Measuring Cell Surface and Intracellular Antigens

History and Background

MonoeIonal Antibodies for the Uninitiated

Cell Surface Antigens: Structure versus Function

Moving Toward Multicolor Immunofluorescence

Antibody Reagents and Staining Procedures

Antibody Fragments versus Antibodies as Reagents

Engineered Antibodies: Phage Display and scFvs

Molecular Probes' Zenon Antibody Labeling

Antibody Shelf Life and Quality Conrrol..

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Direct Staining Using Monoclonal Antibodies for 2, 3, and More Colors Using 488 nm Excitation. 349

Mulricolor Work Using Multiple Lasers; Biotin-Avidin Labeling

Mixing Colors: Do's and Don'ts

Cocktails for Five: Multiplex Immunofluorescence

Cocktail Staining Helps Identify Rare Cells

Immunofluorescence Staining Procedures

Automated Sample Preparation

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Fluorescence Measurements: Lurching Toward Quanritation

Calibration and Controls: Round One

Quantitative Fluorescence Cytomerry: Definitions

Calibration Particles for QFCM

Defining a Window of Analysis

Type IIA versus Type IlIB and mc

Standards

Other Aspects of Fluorescence Quantiration

What is "Positive"? What is "Negative"?

Making Weakly Fluorescent Beads and Cells: Do

Try

···

This Trick at Home!

Correlating Cytometry and Biochemistry: Studies ofAntibody Binding Chemistry

Correlating Cytornetry and Biochemistry: Intracellular Antigen Measurements

Analyzing Immunofluorescence Data

Estimating Antigen or Receptor Surface Density

Quantitative Fluorescence: Problems and Prospects

7.8 Nucleic Acid Sequence

Detection

Peptide Nucleic Acid (PNA) Probes

····

···· ·..·..·..··..·

·.. ······ ·

····

·..·

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7.9 Probes for Various Cell Constituents

Surface Sugars (Lectin Binding Sites

Analysis of Total Carbohydrate Content

Specific Detection of Cellulose

A Probe for Cell Surface Aldehydes

Probes for Lipids and Cholesterol

Nile Red

Filipin

Lipid Droplet Detection Using Scatter Signals

Probes for Cytoskeletal Organization/Actins

7.10 Time as a Parameter: Kinetic Measurements

Sample Handling for Kinetic Measurements

Time as a Quality Control Parameter

Slooowww Flooowww

7.11 Labeled Ligand Binding

Labeling Strategies

Formal Analysis of Ligand binding

Labeled Ligands versus Anti-Receptor Antibodies

Ligand Binding Detected by Functional Changes

Fluorescent Ligand Binding: Some Examples

7.12 Functional Parameters

I

Cell Surface Charge '"

Cell Membrane Characteristics

Membrane Integrity versus "Viability": Dye Exclusion Tests

Detecting "Dead" Cells in Fixed Samples

Membrane Fusion and Turnover; Cell Tracking

Cell Proliferation Analyzed Using Tracking Dyes

Membrane Organization and Fluidity/Viscosity

Lipid Packing Assessedwith Merocyanine 540

Membrane Fluidity and Microviscosity: Assessment Using Fluorescence Polarization

Lipid Peroxidation

Membrane Permeability to Dyes and Drugs: The Drug Effiux Pump Revisited

Endocytosis of Macromolecules and Particles

Enzyme Activity

Indicators of Oxidative Metabolism I: Tetrazolium Dye Reduction

Indicators of Oxidative Metabolism II: 2,7-Dichlorofluorescin Diacetare (DCFH-DA), etc

Indicators of Oxidative Metabolism III: Hydroethidine (Dihydroethidium)

Indicators of Oxidative Metabolism IV: Dihydrorhodamine 123

Indicators of Oxidative Metabolism V: Detection of Hypoxic Cells

Detection of Caspase Activity

Other Enzymes

Detection of Enzymes and Products by Antibodies

Enzyme Kinetics in Single Cells

Sulfhydryl (Thiol) Groups; Glutathione

7.13 Functional Probes II: Indicators of Cell Activation

Introduction

Changes in the Cellular Ionic Environment Following Activation by

Ligand Interaction with Cell Surface Receptors

"Structuredness of Cytoplasmic Matrix" (SCM) and the Cercek Test for Cancer

Optical Probes of Cell Membrane Potential

Membrane Potential and Its Physicochemical Bases

~\f

Measuremenr Using Microe1ectrodes

Single Cell Measurements with Distributional Probes

Oxonol Dyes as Membrane Potential Probes

Possible Alternatives to Distributional Probes for Flow Cytornetry of Membrane Potentials

Ratiometric Probes for Membrane Potential

Contents

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xx

I

Contents

Optical Probes of Cell Membrane Potential (continued)

Using Cyanine Dyes for Flow Cyrornerric A'l' Estimation, In Case You're Still Interested

Cytoplasmic Membrane Potential: Summing Up

Mitochondrial Membrane Potential (1'1':1'01)

Mitochondrial Staining with Rhodamine 123 Is Membrane Potential-Dependent

The Search for Better A':I'm Probes: Round One

A':I'01,JC-I, and Apoptosis

The Search for Better A':I'm Probes: Round Two

Bacterial Membrane Potentials '"

Ratiometric A':I' Measurement in Bacteria

A':I'Measurement: Cautions and Conclusions

Optical Probes of Intracellular Calcium

The Bad Old Days

Chlortetracycline as a Probe of "Membrane-Bound" Calcium in Cells

Probes for Free Cytoplasmic Calcium: Quin-Z

Fura-2 and Indo-I: Rariometric

Ca"

Indicators

Fluo-3 and Other Visible-Excited Ca' Probes

Flow Cytometric Probes of Intracellular pH

The Hat Trick: Multiparameter Approaches to Ion Flux Measurements in Cell Activation

NOsing Around for Nitric Oxide

7.14

Other Ions in the Fire

Reporter Genes

Somebody Cloned My Gal: Enzymes as Reporter Genes

Green Fluorescent Protein (GFP) et

aI

Minority Reportter)?

8.

BUYING

FLOW

CYTOMETERS

8.1

Introduction

8.2

History

8.3

BD Biosciences

Background '"

The BD FACS Vantage SE™ Cell Sorter

The BD FACSCalibur™ Analyzer

The B_D™ LSR II Analyzer

The B-D FACSAria™ Cell Sorter

The B-D FACSCount

8.4

Beckman Coulter, Inc

Background - and Signal-to-Background

The Beckman Coulter EPICS® ALTRA™ Cell Sorter

8.5

The Beckman Coulter Cyromics ' FC 500 Analyzer

®

The EPICS XL and XL-MCL Analyzers

DakoCytomation

Background

The MoFlo ®Cell Sorter ·..· ·

The CyAn™ Flow Cytometer

8.6

Cytopela

The InFlux Cell Sorter

8.7

Optoflow

AS

Background

®

The MICROCYTE Flow Cytometer

8.8

Partee GmbH

Background ,

········

The CyFloW® and CyFloW® ML Flow Cytometers

The PAS, PAS II, and PAS III Flow Cyt0meters

PA Ploidy Analyzer and CCA Cell Counter Analyzer

,

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Contents / xxi

8. BUYING FLOW CYTOMETERS

(continued)

8.9 Some Other Flow Cytometer Companies

Advanced Analytical Technologies, Inc. (AATI)

Agilent Technologies, Inc

Apogee Flow Systems Ltd

Bentley Instruments

Chemunex SA

CytoBuoy b.v

Delta Instruments bv

Fluid Imaging Technologies, Inc

FOSS Electric

NS

Guava Technologies, Inc

Howard M. Shapiro, M.D., P.C

iCyt - Visionary Bioscience

International Remote Imaging Systems

Luminex Corporation

NPE Systems, Inc

Union Biornerrica, Inc

8.10. Hematology Instruments, Etc.

8.11 little Orphan Analyzers (And Big Orphan SOrters)

Bio/Physics and Ortho: Cytofluorograf to Cytoron

HEKA Elektronik GMBH: The FLUVO II Analyzer

The Kratel Partograph

The ODAM ATC 3000

Also Among the Missing

Flow Cytometer Rehabilitation; Used Instruments

Following Suit

8.12 Third Party Software

8.13 The Selling of Flow Cytometers: Hype and Reality

8.14. Applying for a Grant for a Cytometer

9. BUILDING FLOW

CYTOMETERS

9.1 Why Buy a Flow Cytometer?

9.2 Why Build a Flow Cytometer?

9.3 Learning to Build Your Own

;

10. USING FLOW CYTOMETERS: APPLICATIONS, EXTENSIONS, AND ALTERNATIVES

10.I The Daily Grind

Keeping the Instrument Running: Diet and Exercise

Particulars: Drawing a Bead on Flow Cytometer Alignment, Calibration, and Standardization

Alignment Particles: Fearful Asymmetry

Reference and Calibration Particles

Compensation Standards

Cells and Nuclei as Alignment Particles

Rose Colored Glasses: Optical Filter Selection

Experimental Controls

Shake Well Before Using: When Controls Won't Help

10.2 Significant Events in the lives of Cells

Taking the Census: Cell Counting

A Counting Alternative: Image Analysis

The Doubled Helix: Reproduction

The Cell Cycle and Cell Growth

DNA Content Analysis

Mathematical Models for DNA Analysis

Clinical Application of DNA Content Analysis

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I

Contents

DNA Content Analysis(continued)

DNA Content Alternatives: Static Photometry and Scanning Laser Cytornetry

The Mummy's

GC/AT:

DNA Content Analysis in Anthropology and Forensic Science

Half a Genome is Better Than None: Sperm Sorting

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The Widening

Go/G]

re Detecting Mutation

Detecting DNA

Synthesis:

Cell Kinetics

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453

Kinetics Before Flow Cytometry: Mitotic Indices, Doubling

Times,

and Radiolabel Studies

453

Labeling Index versus DNA Content

Early Flow Cyromerric Approaches to Labeling Using BrUdR and 3H-TdR

Detection of Incorporated BrUdR with Hoechst Dyes and Propidium Iodide

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455

Detection of BrUdR Incorporation with Anti-BrUdR Antibodies

Cytochemical Detection ofBrUdR Incorporation Using Difference and Ratio Signals

Breaking Up Is Easy To Do: SBIP, a Simpler Way to Detect BrUdR Incorporation into DNA 456

Anti-BrUdR Antibody: Seeing the Light

Cytochemical Detection of BrUdR: Still Around

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Detecting RNA Synthesis Using Bromouridine

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456

Generation Gaps: Tracking Dyes and Cell Kinetics

Cell-Cycle Related Proteins: Cyclins, Etc

Detecting Mitotic Cells

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Memento Mori: Detecting Cell Death

Necrosis

versus Apoptosis

Identifying Apoptotic Cells

Les Feuilles Mortes (Autumn Leaves)

Apoptosis: Getting With the Program

ISNT there Light at the End of the TUNEL?

Apoptosis: The Case Against Flow Cytornetry

Die Another Day: Cytometry ofTelomeres

10.3 Identification of Cells in Mixed

Populations

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Mixed Genotypes versus Mixed Phenotypes

No Parameter Identifies Cancer Cells

Many Parameters Identify Blood Cells

Flow Cytornetric Parameters Useful for Blood Cells

Specific Gene Products Identify Cell Types

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Maturation Processes and "Missing Links": The "Ginger Root" Model

Practical Multiparameter Gating: Color Wars

Finding Rare Cells

One Parameter is Not Enough

Cocktail Staining Can Help

Dirt,

Noise,

and Rare Event Detection

Really, Really Rare Events: Alternatives to Flow

10.4 Tricks and Twists: Odd Jobs for Flow

Cytometry

Single Molecule Detection

DNA Sizing, if not Sequencing, in Flow

Solid Phase (Bead) Assays Using Flow Cytometry

Cocktails for 100: Multiplexed Bead Assays

Cells in Gel Microdroplets and on Microspheres

Hanging Ten Pseudopodia?

10.5 Single Cell Analysis: When Flow Won't Do

10.6 Applications of Flow Cytometry

Cell Differentiation,

Ab Ovo

and

De Novo

Differentiation in the Nervous System

Whole Embryo Sorting

Somatic Cell Genetics and Cell Hybridization

Reporter Genes Revisited

Isolating and Characterizing Hybrid Cells

Chromosome Analysis and Sorting and Flow Karyotyping

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Contents / xxiii

10.6 Applications of Flow Cytometry (continued)

Probing Details of Cellular Structures and Inter- and Intramolecular Interactions

Dissection of Structures Using Antibodies, Ligands, and Genetic Methods

Intramolecular Interactions

Clinical Flow Cytornetry: Turf and Surf

Hematology

Clinical Application: Blood Cell Counting and Sizing

Red Blood Cells (Erythrocytes)

Clinical Application: Reticulocyte Counts

The Reticulocyte Maturity Index (RMI)

Erythrocyte Flow Cytometry: Other Clinical Uses

White Blood Cells (Leukocytes)

Clinical Application: Differential Leukocyte Counting

CD Characters: Leukocyte Differentiation Antigens

Granulocytes: Basophils

Basic Orange 21: The Best Basophil Stain Yet..

Allergy Tests Using Basophil Degranulation

Granulocytes: Eosinophils

Granulocytes: Neutrophils

(Clinical?) Tests of Neutrophil Function

Neutrophil

CD64

in Inflammation and Sepsis

When I'm Sick - CD64?

Platelets and Megakaryocytes

Hematopoietic Stem Cells

Clinical Application: Monitoring

488

CD34+

Stem Cells According to the ISHAGE Protocol .488

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Side Population (SP) Stem Cells: Plastic, Fantastic

Immunology

Immunologic Applications of Flow Cyrometry: Still a Growth Industry

HIV Infection - The "Killer Application"

Clinical Application: T Cell Subset Analysis

T Cell Subsets: Alternative Technologies

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Clinical Application: Transplantation

Detecting Lymphocyte Activation

Foundations: From PHA (the Lectin) to PHA (the Pulse Height Analyzer)

Functional Probes for Activation

DNA, RNA, and Activation Antigens

Mitogen Response versus Antigen Response

Detecting Activation by

CD69

Expression

Cytokines: Detecting Activation and More

Ins and Outs of Cytokine Staining

Tetramer Staining: Talking the Talk; Walking the Walk?

Tracking Dyes: Activation and Ontogeny

What is "Early" Activation (Trick Question)?

Cancer Biology and Clinical Oncology

Cancer Diagnosis: Cervical Cytology

DNA Content Measurements Yet Again

Beyond DNA Content: Antigens, Oncogenes, and Receptors, and Response to Therapy

Immunophenotyping in Hematopathology

Detecting Minimal Residual Disease

Biological Implications of Phenoryping Results

Digression: A Slight Case of Cancer

Analysis of Sperm

Isolating Fetal Cells from the Maternal Circulation for Prenatal Diagnosis

The March of Time: Circadian Rhythms, Aging, and Atherosclerosis

Clinical Application: Urine Analysis

The Animal Kingdom

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xxiv / Contents

The Animal Kingdom (continued)

Lions and Pumas and Clams, Oh, My!

Fish

Story;

FISH Story

Flow Cytometry: For the Birds?

Big Stuff, Vegetable, Animal or Mineral

Flow Cytornetry of Plant Cells and Chromosomes

Measurement of Plant Cell DNA Content

Plant Chromosome Analysis and Sorting

Other Flow Cytornetric Applications in Plants

Microbiology, Parasitology and Marine Biology

Measuring Microbes: Motivation

Measuring Microbes: Instrument Issues

Parameters Measured in Microorganisms

Flow Cytornerric "Gram Stains"

Detection and Sizing: Light Scattering

Detection and Sizing: Electrical Impedance

N udeic Acid (DNA and RNA) Staining

Total Protein Content: Scatter versus Stains

Antibodies, Etc.: Labeling Strategies

Ribosomal RNA-Based Species Identification

Functional Probes in Bacteria

Potential, Permeability, "Viability", and Metabolic Activity

Digression: A Therapeutic Approach Based on Transient Permeabilization

So Few Molecules; So Little Time

Applications in Marine Microbiology

Extensions: Cytometers for Marine Applications

References: Flow Cytometry and Oceanography

General Microbiology

Previously Noted

Cell Cycles and Cell Division

Fluorescent Protein Methods in Microbes

Microbial Communities: Will Flow Work?

Bad Guys Don't All Wear Black Hats: Microbial Detection/Identification in Health-Related Contexts

The Basic Questions

Detection: Intrinsic Parameters are Not Enough

Detection: Fluorescence Improves Accuracy

Detection: When the Tough Get Going

Identification: Too Many Broths

Identification: Can Multiplexing Help?

Environmental and Sanitary Microbiology

Water That Made Milwaukee (and Sydney) Infamous

Food Microbiology

Biorerrorism and Bioopportunisrn

Viruses and Other Intracellular Pathogens

Clinical Microbiology

Antimicrobial Susceptibility Testing: One Size Does Not Fit All

Bacteria: Confusion Reigns

Mycobacteria: Down for the Count

Antifungal Susceptibility: Flow Does the ]ob

Antiviral Susceptibility by Flow Cytometry

Cytornetry in Vaccine Development

Microbiology Odds and Ends

Parasitology

Pharmacology and Toxicology

Drugs and the Life and Death of Cells

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Pharmacology and Toxicology (continued)

Erythrocyte Micronucleus Assays

Toxic Waste and B Cell Proliferation

Radiation Dosimetry

Food Science

Somatic Cell Counts in Milk

Brewhaha

A Loaf of Bread, A]ug of Wine

Seeing the Blight

Major Food Group: Chocolate

Biotechniques and Biotechnology

Protein and Gene Expression on Cells and Beads

Getting Big Molecules into Small Cells

Staying Alive, Staying Alive

Et Cetera

Alternatives: Microfluidic Cytometers, Flow and Static

Cytornerry Afield

The Lymphocytes of the Long Distance Runner

War and Peace

Blood, Sweat and Tears?

Pulp Nonfiction

Flow Cytometry On the Rocks

To Boldly Go Where No Cytometer Has Gone Before

ll.

SOURCES OF

SUPPLy

11.1 Resources, Societies, Journals

ll.2

Optical Supply Houses

ll.3

Probes and Reagents

ll.4

Calibration Particles/Cytometry Controls

ll.S

Flow Cytometers

Hematology Instruments

11.6 Data Analysis Software/Systems

Hardware and Software

Commercial Software Sources

Noncommercial Software Sources

ll.7

Cytometer Rehabilitation / Add-ons

ll.8

Flow Cytometer Parts

Flow System Plumbing

Photodetectors

DC-DC Converter Modules for HV Power Supplies

Power Supplies (Low Voltage)

Other Electronics

ll.9

Lasers

Laser Trade Publications

Laser Manufacturers

11.10 Optical Filters

Color Glass Filters

Interference Filters

Neutral Density Filters

Polarizing Filters and Optics

Tunable Filters

11.11 Aids to Troubleshooting Flow Cytometers When All Else Fails

11.12 Proficiency Testing

11.13 Sex Selection

11.14 Alternative Technology

Contents /

xxv

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xxvi

I

Contents

12. AFTERWORD

12.1 Dotting i's and Crossing t's

12.2 Late Breaking News

New Book

New Protein Stain

Caveat on Fluorescent Caspase Inhibitors

Polyamide Probes

Tearing Down the (Picket) Fences

New Instrument: The BD FACSArrayTM

Science

Special Section: Biological Imaging

Cytornics in Predictive Medicine: a

Clinical Cytometry

Special Issue and Other

Recent Citings and Sightings

12.3 Analytical Biology, Such as it Isn't: Is This Any Way to Run a Science?

12.4 Colophon

12.5 Unfinished Business

AIDS and Infectious Disease in the Third World

A Center for Microbial Cytometry

A Nobel Prize for Herzenberg and Kamentsky?

12.6 Flow and the Human

Condition

There's No Business Like Flow Business

12.7 One More Thing

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