CHAPTER 1

Flow Cytometry Instrumentation

INTRODUCTION

T he purpose of this chapter is to acquaint readers with the instrumentation utilizedin flow cytometry by describing various elements of the technology, and to provide

detailed information on specific methods of using the instruments. Introducing the chapteris UNIT 1.1, an overview of the instrumentation involved in flow cytometry. This unitdescribes the various parts of a flow cytometer, explains the basics of how each componentworks, and presents a brief history of the development of flow cytometry instrumentation.

Later units in the chapter describe specific aspects of the instrumentation. The fluidicssystem of a flow cytometer transports objects through the instrument, positioning themfor accurate measurement. The various aspects of this system—from the sample tube tothe interaction with the illumination beam, including flow analyzers and flow sorters—are described in UNIT 1.2. Calibration or standardization of an instrument for stability inmeasurement results (such that the same objects yield the same results day after day)requires frequent use of standard particles. It is necessary to characterize instrumentperformance if results are to be compared among different laboratories. Performancecharacterization is also used to determine whether a particular instrument is able toprovide reliable results for an application. Calibration and standardization methods andsources of particles are described in UNIT 1.3; this unit also includes a discussion of thedifference between “standardization” and “calibration” and the need for both. Methodsfor establishing and maintaining the linearity of the system in order to make reproducibleand accurate measurements are presented in UNIT 1.4. This unit includes discussion of themethods both for instruments that use logarithmic amplifiers and instruments designedfor wide dynamic range linear measurements.

Another significant aspect of a flow cytometry system is the optical filters. Flow cytome-ters and flow sorters use light beams to excite fluorescent dyes; optical filters are usedto separate the fluorescent light from the incident light. They are especially importantwith the current emphasis on multicolor analysis, which requires the simultaneous useof several dyes and light sources. UNIT 1.5 describes the basic principles of optical filtersand discusses how to select a filter for a specific measurement in a cytometer. Most flowcytometers and sorters utilize laser beams as the excitation source; UNIT 1.6 discusses thebasic optical properties of laser beams, the various lasers that are in use, and the variousmethods for shaping the final focus and delivering a laser beam to the objects beingmeasured.

A particularly valuable aspect of flow cytometry is its capacity for analyzing thousandsof cells per second. For the detection and sorting of rare cells, special versions of themachines (high-speed sorters) have been developed. UNIT 1.7 discusses the principles andpractices of operating a cell sorter to maximize its sorting rate, including the tradeoffsbetween speed and resolution. All commercial flow cytometers have a built-in capabilityfor “gating,” a feature required for cell sorting. UNIT 1.8 describes the principles underwhich data are processed in real time and off-line to select subpopulations of cells (orcell organelles) with specific characteristics.

Current Protocols in Cytometry 1.0.1-1.0.3, April 2010Published online April 2010 in Wiley Interscience (www.interscience.wiley.com).DOI: 10.1002/0471142956.cy0100s52Copyright C© 2010 John Wiley & Sons, Inc.

Flow CytometryInstrumentation

1.0.1

Supplement 52

Introduction

1.0.2

Supplement 52 Current Protocols in Cytometry

Most flow cytometers utilize lasers as their illumination source. UNIT 1.9 describes thewide variety of laser types available and explains how they work. Important performancecharacteristics, such as power and noise of the various types of lasers, are discussed.The unit covers both gas-filled lasers, such as argon-ion, used in older flow cytometerdesigns, and solid-state lasers, used in recent generations of flow cytometers.

To ensure that high-quality, accurate data are obtained, flow instruments are checked forsystem performance each time they are used. UNIT 1.10 describes two levels of systemalignment—routine alignment checks and complete alignment—that can be applied to avariety of instrumental configurations.

The measurement of microbes is becoming an increasingly important application in flowcytometry. The small size of these organisms can create special difficulties that mustbe carefully addressed. UNIT 1.11 describes the problems encountered in making suchmeasurements, the instrument requirements, and some approaches that can be taken tooptimize the instruments for such measurements. This material is essential backgroundfor the protocols found in Chapter 11, Microbiological Applications.

Fluorescence resonance energy transfer (FRET) is a technique that helps make possiblethe simultaneous use of three or more fluorescent dyes for the structural analysis of pro-teins in biological membranes. UNIT 1.12 describes the theory behind FRET, characterizesavailable parameters and instruments, discusses the method’s limitations, and presents afew examples of its applications.

Light scatter was the first parameter measured in a flow cytometer. This highly usefulparameter is probably the most widely used and least well understood by laboratoriestoday. UNIT 1.13 describes in general terms the interaction of light with small particles, howthe light is measured, and some current applications of this parameter in flow cytometry.

In general, fluorescent dyes have relatively broad excitation and emission spectra, andwhen two or more are used simultaneously, the emission spectra will usually overlap tosome extent. The measured fluorescence for a given dye can actually contain a significantcontribution from any or all of the other dyes. To obtain accurate flow results fromindividual dyes, it becomes necessary to compensate (correct) for this spectral overlap.UNIT 1.14 explains why compensation is necessary, how it is accomplished, and how itaffects the visualization of data. The author addresses popular misconceptions that resultin incorrect compensation and provides extensive discussion of suitable controls—cells,beads, and gates.

The measurement of time-resolved fluorescence is a relatively new technique still underdevelopment in both flow and image cytometry, and promises to add a new dimension tomultiparameter cytometry. Properly used, this technique can provide information aboutfluorophore/cell-interactions at the molecular level. UNIT 1.15 describes the theory behindfluorescence lifetime measurements and presents some applications.

The simultaneous measurement of several fluorescent proteins allows one to monitorsuch things as gene expression and the intracellular localization of proteins. UNIT 1.16

presents a protocol for using a single laser operating at 458 nm to detect three fluorescentproteins simultaneously in a single cell. This is accomplished using a simple opticalconfiguration and real-time compensation.

Rapid screening of large combinatorial libraries, for instance of potentially valuable com-pounds in drug discovery tests, requires reliable automated sample-handling techniques.UNIT 1.17 describes one such technique, “plug flow cytometry,” in which precisely definedvolumes of sample suspensions are injected at regular intervals into the flow stream.

Flow CytometryInstrumentation

1.0.3

Current Protocols in Cytometry Supplement 52

Many functions of living cells, as well as molecular interactions, are temperature depen-dent. UNIT 1.18 presents a protocol for using a Peltier module to control the temperatureof a sample being processed in a flow cytometer/cell sorter.

UNIT 1.19 covers in fine detail the photophysical and chemical properties of many commonprobes routinely employed in current flow cytometry. The spectral characteristics anduse of example fluorescent dyes, probes, and labels for various functional assays andapplications are described.

The ability to make accurate measurements on dimly fluorescent cells is important inmany flow cytometry applications. Fluorescence sensitivity, the ability to resolve dimlyfluorescent populations, is limited by instrument factors that can be characterized andmeasured. The primary factors limiting measurement of dim fluorescence are opticaldetection efficiency and optical background. Characterization of fluorescence sensitivityis discussed in UNIT 1.20, and a detailed protocol is provided for measuring detectionefficiency and background.

The separation index (SI) is a measure of fluorescence sensitivity that takes into accountboth the difference in mean fluorescence between stained and unstained populationsand the width of the unstained population. UNIT 1.21 presents the concept of SI anddemonstrates its use for assessing the effect of changes in flow cytometer configurationand in measuring the broadening of unstained populations due to spectral overlap offluorochromes.

Acoustic energy can be used to affect the position of particles in a flowing stream. Undercertain conditions, particles can be forced to the center of the stream, which produces thesame effect as hydrodynamic focusing—but without the need for sheath flow. Acousticcytometry may also enhance flow cytometry with additional capabilities. The fundamen-tal aspects and potential applications of the emerging technology of acoustic cytometryare discussed in UNIT 1.22.

Forward light scatter is often used in flow cytometry to discriminate particles basedon their apparent size. But forward scatter depends on several factors besides size, andthe factors interact in complex ways. Pulse width from fluorescence or light scatter is analternative and often more accurate particle size measurement. UNIT 1.23 provides examplesof pulse-width measurements for particle discrimination and sizing. General methods forcalibrating the pulse-width measurement and determining the useful operating range aredescribed. An approach for accurately measuring the diameter of fluorescent particles isalso described.

The current generation of cell sorters is able to analyze and sort cells at high speeds.But to make best use of this capability requires attention to details ranging from samplepreparation to selection of the optimal sort conditions for a specific application. UNIT 1.24

addresses important practical aspects of high speed cell sorting, including instrumentsetup, sorting strategies, cell preparation and viability, and safety.

Robert A. Hoffman