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Overview of Flow Cytometry InstrumentationFlow cytometry is a technology in which a variety of measurements are made on cells, cell organelles, and other objects suspended in a liquid and flowing at rates of several thousand per second through a flow chamber. Flow sorting is an extension of this technology in which any single cell or object measured can be selectively removed from the suspension based on the measurements made. Flow cytometry is a very broadly applicable methodology. A brief list of applications that use flow cytometers includes: Disease diagnosis Chromosome karyotyping Cell function analysis Cancer therapy monitoring Detecting fetal cells Cell kinetics Identifying tumor cells Cytogenetics Fundamental cell biology. In a flow cytometer, cells in suspension are made to flow one at a time through a sensing region of a flow chamber (flow cell) where measurements are made. An example of an early flow cytometer is the Coulter counter (APPENDIX 3A). In this device, cells pass through a small orifice across which an electric current is flowing. As a cell enters the orifice, the flow of current is reduced because the cells are largely nonconducting. Electronic circuits detect the decrease in current and thus the presence of the cell. In this way the device can count the number of cells per second passing through the orifice, and because the volume flow rate can be measured one can determine the number of cells per milliliter of sample. The Coulter counter has been in use since 1949 and is still a mainstay of the clinical laboratory. Under the right conditions (e.g., size and length of orifice, current magnitude), the reduction in current through the orifice is proportional to the size (volume) of the cell, as demonstrated at the Los Alamos Scientific Laboratory in 1962. In modern flow cytometers, cells flow through a light beam rather than through a Coulter orifice; a Coulter orifice can, however, be included in these devices. Many different types of measurements can be made on the cells, based on the size and shape of the light beam and on the dyes used to stain components of interest. The light beam can come from arc lamps (e.g., mercury), as in early flow cytomeContributed by Phillip N. DeanCurrent Protocols in Cytometry (1997) 1.1.1-1.1.8 Copyright 1997 by John Wiley & Sons, Inc.

UNIT 1.1

ters, or from lasers. Methods of measurement include absorption and scattering of the light beam by the cell, fluorescence of attached fluorescent dyes, and shape of the detected signal. Some of the properties and components that can be measured by a flow cytometer using these various methods are listed in Table 1.1.1. In principle, any component of a cell to which a fluorescent dye can be attached can be measured in a flow cytometer. If the binding of the dye is stoichiometric (i.e., amount of dye is proportional to amount of component) then the measurement can be quantitative and highly accurate (to within a few percent or better).Table 1.1.1 Properties and Components of Cells Measured in Flow Cytometry

Properties Cell diameter Dye distribution Internal structure Membrane potential Nuclear diameter Surface area Volume

Components DNA Nuclear antigens Enzymes Protein RNA Hormones Surface antigens

A flow cytometer is made up of several parts, as shown diagrammatically in Figure 1.1.1. All components of the system are necessary; the weakest part of the system defines its limitations. Other chapter units discuss the different parts of the system in detail. This overview describes the technology in general to give the reader a feeling for the interplay between the various parts of a flow cytometer. It also contains a brief history of the development of flow cytometry instrumentation.

CELL PREPARATIONObjects to be measured must be suspended in a liquid. This is simple for blood cells, for example, but cells from tissue must be disaggregated and removed from any noncellular material. For most tissues this can be accomplished by procedures as simple as mincing the tissue with a knife and pulling cells through a 19-gauge needle into a syringe, followed by passing the cell suspension through a 200-mesh nylon screen. Details for such procedures are

Flow Cytometry Instrumentation

1.1.1

found elsewhere in this publication in units that deal with specific measurement and analysis protocols (e.g., see UNIT 5.2 for general procedures for handling, storage, and preparing human tissues and APPENDIX 3B for procedures for disaggregating cultured cell monolayers). After a single-cell suspension is obtained, the cells

are stained with dyes that bind to the specific features that are to be measured.

FLOW CHAMBERAfter staining, cells are made to flow one at a time through the interrogating light beam; a laser beam is illustrated in Figure 1.1.2. To

light source (UNIT 1.5 )

cell preparation

fluidics control (UNIT 1.2 )

flow chamber (UNIT 1.2 )

detectors and signal processing (UNITS 1.3 &1.4 & Chapter 10 introduction)

sorter module (this unit, UNIT 1.2 )

analysis (UNIT 10.1)

display (UNIT 10.4)

Internet (UNIT 10.2 )

Figure 1.1.1 Schematic diagram of a complete flow cytometer system.

sample sheath

flow chamber

focusing lens

laser beam

Overview of Flow Cytometry Instrumentation

Figure 1.1.2 Longitudinal cross-sectional view of the flow chamber of a flow cytometer. The sample stream is surrounded by the sheath fluid which confines the cells (black dots) to the center of the chamber. The laser beam is focused onto the cell stream.

1.1.2Current Protocols in Cytometry

obtain the best resolution, every cell must flow through the middle of the beam and be exposed to the same intensity of illuminating light. However, the laser beam has a Gaussian intensity distribution (i.e., the intensity is at a maximum in the center of the beam and decreases exponentially in the radial direction), and this puts a severe constraint on the stability of the flow stream. The system includes two features to alleviate this problem. (1) The beam leaving the laser has a circular cross section, and a long-focal-length cylinder lens is used to spread the beam in the horizontal direction and to produce a large depth of focus, resulting in a relatively large region of constant intensity in the center of the flow stream. (2) A sheath stream is introduced to the flow chamber. This sheath has a higher flow rate (5 ml/min) than the sample (100 l/min), which serves to compress the sample stream and confine it to the center of the overall flow stream. This technique, called hydrodynamic focusing, is explained in more detail in UNIT 1.2. The end result is that cells are constrained to flow through an expanded laser beam in the center of the flow chamber. An additional constraint on the flow chamber is that it must be constructed of a material that will pass the excitation beam without appreciable scattering or absorption; this is usually accomplished through the use of quartz glass, which must be kept scrupulously clean. This is especially true when using ultraviolet light for excitation. The flow chamber can take many configurations. If a small orifice (e.g., sapphire jewel with a 70- to 100-m hole) is placed at the

chamber exit, the flow stream will be compressed and will leave the chamber at high velocity. If the chamber is then vibrated at high frequency (e.g., 20,000 Hz), the stream will break up into uniform droplets and the flow cytometer will become a flow sorter. In this configuration measurements on cells can still be made in the chamber, although the time interval between cell detection and sorting can be relatively long. However, it is more common to pass the laser beam through the fluid stream just below the jewel before the stream breaks up (see Fig. 1.1.6). Then the interval between cell detection and sorting is shorter. In the latter configuration, the material requirements on the chamber are considerably reduced; the chamber becomes what is often called a sorter nozzle and can be constructed of ceramic materials. Because the hydrodynamic focusing does take place in the nozzle, in some sense the nozzle is a chamber. The sorting configuration is described in more detail later (see Sorting).

DETECTORSAs a cell flows through the beam, light scattered by the cell and fluorescence light from dyes added to the cell are collected by light detectors, usually photomultipliers and photodiodes (see UNIT 1.4 for further discussion of photodetectors). These devices convert the light signal to an electrical signal that can be processed by the data processing and analysis unit. Photomultipliers, being very sensitive to light, are used where the light signal is weak (fluorescence), and photodiodes are used where the signal is strong (small-angle light scatter). The simplest flow cytometer would have per-

photomultiplier

filter pinhole photodiode collection lens laser beam flow chamber

Figure 1.1.3 Arrangement for a simple flow cytometer, containing a single fluorescence detector (photomultiplier) and a photodiode for detecting laser light scattered by a cell.

Flow Cytometry Instrumentation

1.1.3Current Protocols in Cytometry

haps one photomultiplier and one photodiode, as shown in Figure 1.1.3. With the appropriate electronics system, this permits one to make two simultaneous measurements on a cell. As a cell flows through the beam it scatters some of the incident light, and the light scattering is typically detected by the photodiode, which is less sensitive than the photomultiplier. This continues as long as the cell is within the beam. Thus, the length of time a cell is in the beam (and the width of the electrical pulse produced) is proportional to the width of the cell. If the cell is also stained with a DNA-specific dye, the photomultiplier is used to measure the amount of fluorescent light emitted by the cell while it is in the light beam, producing a signal proportional to the DNA content of the cell. In Figure 1.1.3, an optical filter is shown that passes the fluorescent light and blocks the scattered excitation (laser) light. Thus, two measurements are made simultaneously. UNIT 1.5 contains a comprehensive discussion on how optical filters for flow cytometry are made and selected. By using a dichroic mirror (beam splitter) in front of the photomultiplier and incorporating a second photomultiplier with a different filter, as illustrated in Figure 1.1.4, three measurements can be made: e.g., DNA, total protein,

and narrow-angle light scatter. A dichroic mirror is one that reflects light below a specific wavelength and passes longer-wavelength light. The requirement for using more than one dye with this configuration is that both dyes excite at the same wavelength but emit at different wavelengths. The mirror is selected to separate the two emissions. Each detector also has a filter to block scattered excitation light. Fluorescent light is always emitted at a wavelength longer than that of the excitation light. Many flow cytometers today use two laser beams operating at different wavelengths to excite four or more dyes simultaneously. Figure 1.1.5 illustrates how this is done. The laser beams are separated vertically by 200 m so that a cell flows through the two beams with a separation time of a few microseconds. Thus the two pairs of signals are separated in time, making it easier to resolve them. Each laser beam interaction point has its own pair of photomultipliers, dichroic mirror, and filter arrangement. In addition to measuring fluorescence, these detectors can be used to measure scattered light at 90. The latter signal can help to distinguish cells with different internal structures. In principle, more detectors can be added to make even more measurements on each cell, with the limitation being the number of dye combina-

photomultiplier P2 filter P1

dichroic beam splitter

pinhole

photodiode collection lens laser beam flow chamber

Overview of Flow Cytometry Instrumentation

Figure 1.1.4 Arrangement for a flow cytometer with dual fluorescence detectors and a scatter detector. Light from two fluorescent probes is separated by the dichroic mirror and optical filters. With the appropriate filters, photomultiplier P1 can also be used to measure light scattered at 90 to the laser beam.

1.1.4Current Protocols in Cytometry

tions that can be used. The combinations of excitation and emission spectra must be significantly different (see UNIT 1.5).

ANALYSISAll modern flow cytometers incorporate computers to monitor and in some cases to control the instrument, and to provide a capability for on-line analysis of instrument data. The computers are mostly Macintosh and IBMcompatible personal computers, which now have the power to perform virtually any kind of analysis desired. As the method of analysis required is not always known during an experiment, the computers are also used for off-line analysis. Software packages are available from the instrument manufacturers and from independent software companies. For more details on data processing and analysis, see Chapter 10. Flow cytometers are capable of producing enormous quantities of data very rapidly. This presents a challenge to the user, who must provide a means for storing the data in such a fashion that they can be recalled on demand. Because most data are stored in listmode, data files can be very large. Listmode means that every measurement on every cell is stored in a list. Thus, if five measurements are made on each of 50,000 cells, with a maximum value of 1024 per measurement (2 bytes), space has to be found for 500,000 bytes of information per sample. With the current development of

ever larger and less expensive storage devices such as read/write optical disk cartridges, this is not a major problem. A data file standard has been developed for the storage of flow cytometry data to make it possible for different laboratories to share data. This topic is discussed in UNIT 10.2. Sharing of data and the results of data analysis has become an important part of research; access to the Internet has become a desirable attribute of flow cytometer systems. To accomplish this one needs an Internet service provider and a browser, a computer program that provides access to other sites on the network. There are several browsers available, notably Mosaic, Netscape, and Internet Explorer. Many flow cytometry laboratories throughout the world have established sites on the Internet and made them available to other researchers in the field. A convenient location to begin a journey through the Internet is the home page of the International Society for Analytical Cytology (http://nucleus.immunol.washington.edu/ISAC. html), which contains links to most of these sites as well as to other sources dealing with both flow and image cytometry.

SORTING PrinciplesA flow sorter is a cytometer with the additional capability of selectively removing from

beam 1 photomultiplier P1 beam 2 filter P2 beam splitter

P3

flow chamber

P4

half mirror

Figure 1.1.5 Flow cytometer with two excitation beams (lasers) that are separated vertically by 200 m. A half mirror is used to direct fluorescent light from each beam interaction to a different pair of photomultipliers, each of which has a beam splitter and filter arrangement as in Figure 1.1.4. A photodiode could be added for each beam to permit a total of six measurements per cell.

Flow Cytometry Instrumentation

1.1.5Current Protocols in Cytometry

the suspension of cells any selected cell flowing through it. The physical arrangement of a sorter is illustrated in Figure 1.1.6; the detailed fluidics are discussed in UNIT 1.2. Basically, the fluid containing the cells passes through a narrow orifice (100 m in diameter) into the air. At the same time, the flow chamber is vibrated by the attached piezoelectric crystal, at frequencies on the order of 20,000 Hz. The vibration produces a disturbance in the ejected stream. The disturbance grows very rapidly, and the stream eventually breaks up into drops (i.e., 20,000 drops per second in the example given). The steady vibration causes the drops to be very uniform in size and spacing. Each cell that flows through the system will end up in a drop.

Measurements are made on the cells while they are either in the flow chamber or in the stream just below the orifice, before the disturbance of the stream has grown significantly. If the measurement result indicates that the cell is to be sorted, a voltage is applied to the stream just as the cell of choice reaches the end of the stream and a droplet is forming. When the drop separates from the stream it will carry electrical charge. The voltage on the stream is immediately reduced to zero so other drops will not be charged. Charges can be negative or positive, leading to the possibility of sorting two categories of cells simultaneously. As the drops continue to move downward they pass between two metal plates charged to a high voltage. Because

nozzle sapphire jewel laser beam

droplets

deflection plates

charged droplets

collection beakers

Overview of Flow Cytometry Instrumentation

Figure 1.1.6 Diagram illustrating the principle of cell sorting. Cells flowing through the system are represented by small black dots. As cells to be sorted approach the end of the solid stream, a charge is applied to the stream. As the drop carrying the cell separates from the stream, the drop carries the charge. Passing between the high-voltage plates, charged drops containing desired cells are deflected into separate collection beakers. Deflection can be left or right, allowing for the simultaneous sorting of two classes of cells. In this illustration, two drops are sorted for each cell.

1.1.6Current Protocols in Cytometry

the drops containing the selected cells are charged, they are deflected from the main stream of drops and collected in tubes or onto microscope slides for visual examination. Drops containing undesired cells are not charged and go directly into a waste tube.

Sort PurityEfficient and accurate sorting requires that the charge be applied just as the cell reaches the end of the stream. To compensate for variation in the flow velocity of the stream and to be certain the desired cell is sorted, typically two or three drops are sorted for each cell; Figure 1.1.6 shows two drops per cell. The sorting electronics are capable of detecting other cells

in the vicinity of the desired cell; if an unwanted cell might be sorted along with the desired cell, the sort is aborted. In some cases, particularly in the detection of very rare cells, one might want to accept some impurity in the sorted cell population to guarantee collection of the wanted cells. In that case, the sort purity requirement can be eased and the abort circuit disabled.

CHRONOLOGY OF FLOW CYTOMETRY DEVELOPMENTTable 1.1.2 is a list of significant events in the development of flow cytometry instrumentation. It is by no means comprehensive but illustrates the long history of the field. For a

Table 1.1.2

Development of Flow Cytometry

Year 1934 1941 1947 1949 1953

Event Moldovan measures red blood cells in microscope with capillary flow and photodetector Kielland patents device like that developed by Moldovan Gucker uses Reynolds work to design laminar flow system for aerosols Coulter files for patent, Means for Counting Particles Suspended in a Fluid: the Coulter counter Crosland-Taylor designs system for aqueous suspension of cells flowing with a sheath: hydrodynamic focusing Parker and Horst apply for patent on device to do blood cell differentials using absorption of two colors of light Model A Coulter counter introduced Van Dilla uses Coulter counter to measure cell volume distribution Kamentsky measures UV absorbance and visible light scatter (500 cells/sec), generates bivariate plots Fulwyler develops electrostatic cell sorter, based on volume measurement Kamentsky develops fluidic switch cell sorter Van Dilla et al. introduce orthogonal measurements and laser excitation; prove DNA measurements were accurate; first to show discrete G1, S, and G2/M phase populations; show the possibility of quantitative cell kinetics studies Dittrich and Gohde patent microscope-based flow system with flow parallel to optic axis Hulett et al. introduce sorting based on cell fluorescence Wheeless et al. patent cell classification combining size and fluorescence Dittrich and Gohde introduce dual staining for DNA/protein, using ethidium bromide/FITC stains Wheeless et al. patent slit-scan cytofluorometry for automated cell recognition Gray et al. introduce flow karyotyping Various investigators develop indexed sorting, high-speed sortingFlow Cytometry Instrumentation

1956 1962 1965

1966

1968 1969 1970 1971 1972 1975 1975 and beyond

1.1.7Current Protocols in Cytometry

more complete discussion of the history, the interested reader is directed to Melamed et al. (1990).

Melamed, Lindmo, and Mendelsohn (eds.) 1990. See above. Second edition with all-new papers, producing true update of the first edition. Also contains extensive history of the field. Shapiro, H.M. 1995. Practical Flow Cytometry, 3rd ed. Wiley-Liss, New York. Comprises users reference manual for the laboratory. Also includes extensive list of current literature and list of key suppliers of instruments, parts, and reagents. Van Dilla, M.A., Dean, P.N., Laerum, O.D., and Melamed, M.R. 1985. Flow Cytometry: Instrumentation and Data Analysis. Academic Press, London. Contains papers by leading experts in the field on both subjects.

SUMMARYFlow cytometry as a technology is still developing. New instruments with new or improved capabilities are constantly being introduced. This overview will be updated frequently to keep the reader apprised of developments in flow cytometry. A number of books provide excellent summaries of flow cytometry instrumentation (see Key References). For more details on particular techniques, the reader is referred to the articles in these books and to their extensive lists of references.

LITERATURE CITEDMelamed, M.R., Lindmo, T., and Mendelsohn, M.L. (eds.) 1990. Flow Cytometry and Cell Sorting, 2nd ed. Wiley-Liss, New York.

INTERNET RESOURCEShttp://nucleus.immunol.washington.edu/ISAC.html Homepage of the International Society for Analytical Cytology (ISAC).

KEY REFERENCESMelamed, M.R., Mullaney, P.F., and Mendelsohn, M.L. (eds.) 1979. Flow Cytometry and Cell Sorting, 1st ed. John Wiley & Sons, New York. First overall summary of the field, with many authors describing the state of the art as of 1979. Covers applications of the technology as well as instrumentation.

Contributed by Phillip N. Dean Livermore, California

Overview of Flow Cytometry Instrumentation

1.1.8Current Protocols in Cytometry

FluidicsFlow cytometry is so named because in this technique cells or subcellular particles suspended in a fluid are made to flow past sensors that take measurements from the cells. The primary function of the fluidics of a flow cytometric instrument is to deliver cells to the sensing area in single file and well aligned with the sensors. In a sorting flow cytometer, the fluidics must additionally be able to physically isolate cells chosen on the basis of measurements made by the instrument. namic focusing to control the position of particles in the flow (Fig. 1.2.2). In this technique, a flow of carrier fluid, called the sheath fluid, is established in the cytometer. The sheath fluid, which is usually normal physiological saline with perhaps a few additives, originates from a supply tank under pressure and flows through tubing to a sensing region where the detectors are located. Just before its arrival at the sensing region, the sheath fluid flows into a chamber of relatively large diameter, then out through a tapered conical section that reduces the diameter of the flow to the dimensions of the sensing region. The sample-containing fluid is introduced into the middle of this chamber through a tube positioned on the central axis of the sheath flow. Under laminar flow conditions, the sample and sheath fluids do not mix but join together to form a coaxial flow. This combined flow then passes through the tapered section, which reduces the diameter (and increases the velocity) of both the sheath and sample flows simultaneously before they reach the sensing region. This technique confines the cells to a very narrow central core so that the path cells follow through the sensing region is very consistent. The central area of combined flow that originated from the sample flow is called the sample core. It should be noted that the size of the sample

UNIT 1.2

PRIMARY FLUIDIC FUNCTIONS Sample and Sheath FlowFigure 1.2.1 illustrates the main fluidic elements of a flow cytometer. Two primary lines feed fluid to the sensing region: the sample flow line and the sheath flow line. The sample flow line delivers the cell sample to the sensing region, and the sheath flow line provides a carrier fluid that helps position the sample flow and ultimately cells in the sensing region.

Hydrodynamic FocusingMost flow cytometry measurements are made optically, and it is important to keep particles well positioned in the flow stream in order to make accurate optical measurements. Flow cytometers use a fluidic method called hydrody-

samplesheath pressure

sheath fluid

sample pressure

hydrodynamic focusing sectionsensing region

sample

sheath tank

sample tube

Figure 1.2.1 The basic flow system of a flow cytometer consists of sample and sheath forced under pressure through tubing to a hydrodynamic focusing section where the flows are combined in a coaxial flow prior to arrival at the sensing region. Flow Cytometry Instrumentation Contributed by Richard StovelCurrent Protocols in Cytometry (1997) 1.2.1-1.2.7 Copyright 1997 by John Wiley & Sons, Inc.

1.2.1Supplement 1

sample sheath fluid sheath fluid

Figure 1.2.2 Flow cytometers use the principle of hydrodynamic focusing to align cells (represented here as black dots) in the center of the flow prior to their passage through the sensing region. The sample is injected into the middle of a sheath flow and the combined flow is reduced in diameter in a tapered section, forcing the cell into the center of the stream as shown.

to sensing region

core in relation to the diameter of the combined flow at the sensing area is a function of the relative sample and sheath flow rates, not the relative diameters of the sample and sheath introduction tubes. The effects of increasing or decreasing sample flow rate relative to sheath flow rate are shown in Figure 1.2.3. The reason that this point must be stressed is that the size of the sample core can be important for achieving data consistency. It is difficult optically to achieve spatially uniform illumination across the sensing region, so variation in particle position will result in variation of illumination intensity and therefore more variation in measurements due to position. Sample flow rates must be kept low relative to the sheath flow rate to maintain a narrow sample core and high data consistency.

diameter narrows at the entrance to the orifice. The jet emerges from the orifice with the cells confined to the center of the jet. A light source, usually a laser, is focused onto the jet near the orifice, and optical elements collect the light scattered or emitted by cells as they pass through the light source and transmit this light to detectors, which convert it to an electronic signal. The jet-in-air configuration is well suited to droplet sorting (see discussion of Sorting).

Flow CellA second sensing configuration is the flow cell, in which the hydrodynamically focused coaxial flow passes through a transparent closed chamber wherein excitation light is brought into the chamber, and scattered and emitted light from the cells passes back out of the chamber to the detectors (Fig. 1.2.5). Generally, the inner diameters of flow cells are larger than the nozzles of jet-in-air systems, and the particles flow more slowly and spend more time in the sensing region. Higher-quality optical resolution can be achieved with flow-cell designs; moreover, the closed-flow system is advantageous when biohazardous samples are being handled.

TYPES OF SENSING AREAFor most flow cytometers, the fluidic configuration of the sensing region can be classified as belonging to one of two types: jet-in-air or flow cell. Each type has its advantages and disadvantages. Some cytometers allow either type to be installed, and hybrids of the two types exist.

Jet-in-AirIn a jet-in-air cytometer, the sheath and sample flow are combined in a nozzle, which tapers down to an orifice. The fluid in the nozzle is under enough pressure to form a continuous cylinder of fluid, or jet, as it emerges from the orifice (Fig. 1.2.4). Although some hydrodynamic focusing occurs in the tapered section, further focusing takes place as the fluid flow

HybridSome cytometers combine advantages of both configuration types by sensing within a flow cell, then passing the flow through a jetforming orifice for droplet sorting. In this combination the exit section shown in Figure 1.2.5 is replaced by the orifice shown in Figure 1.2.4. This configuration provides a combination of high-quality optical measurement and droplet-

Fluidics

1.2.2Supplement 1 Current Protocols in Cytometry

A

sample sheath fluid sheath fluid

B

sample sheath fluid sheath fluid

broad sample core

narrow sample core

Figure 1.2.3 Diameter of the sample core is not dependent on the relative diameters of the sheath and sample tubes, but rather on the relative flow rates of the two fluids. (A) A sample at relatively high flow bulges after leaving the sample injection tube and is focused to a relatively broad sample core in the sensing region. (B) Low flow produces a narrow sample core that results in more accurate positioning of the cells in the sensing region.

sorting capabilities, but the sorting is less precise due to the longer transit time of the cells between sensing and sorting, and the biohazard containment advantage of a closed-flow system is lost.

Other ConfigurationsIn some cases flow cytometry techniques have been adapted to existing microscope technology by devising various flow configurations that use hydrodynamic focusing and pass cells through the focal plane of a microscope objective. One cytometer designed on these principles uses a jet-forming orifice to squirt the flow onto a microscope slide. The jet forms a continuous sheet of fluid on the slide, and flow conditions can be kept stable enough for cytometric measurements to be made through the microscope. Another type uses flow along the optical axis of the microscope; cells are measured as they pass axially through the focal plane of the microscope objective.

the sample through the sample tubing to be combined with the sheath flow in the flow cell or nozzle. The pressure in the sample line at the point where the sample is injected into the sheath flow need be only marginally higher than the pressure in the sheath flow. Actual pressures applied at the sheath supply tank and the sample tube may differ by a larger amount due to pressure drops in the tubing and filters and differences in the height of the sample tube and sheath tank.

Alternative TechniquesOther methods and devices, such as syringe pumps, are sometimes used to introduce samples into the cytometer. Automated delivery devices that can pick up and deliver samples sequentially from a rack of many samples are available. These are usually based on motordriven syringe pumps. There are also semiautomated pickup devices that are designed to pick up and deliver small-volume samples from microtiter plates. Special sample chambers are also available for certain specific purposes, such as mixing reagents with samples just prior to delivery for timed-reaction experiments.

TYPES OF SAMPLE DELIVERY Standard SetupSamples of suspended cells are typically loaded into the flow cytometer in test tubes which are pushed on over an uptake tube that reaches to the bottom of the sample tube (see Fig. 1.2.1). The test tube pushes against a seal, and air pressure is applied through a port that projects through the seal. The pressure forces

POTENTIAL PROBLEMS AFFECTING SAMPLE FLOW Transient Flow EffectsLiquid flowing through small tubing tends to travel slowly near the edges of the tubing,Flow Cytometry Instrumentation

1.2.3Current Protocols in Cytometry

Figure 1.2.4 A jet-in-air flow cytometer forces the coaxial flow of sheath and sample fluid through an orifice under sufficient pressure to form a jet. Illumination and detection take place in the jet after it has left the orifice.

orifice illumination

jet

due to drag by the walls, and faster in the center. The fluid develops a parabolic velocity profile in which the center of the flow travels twice as fast as the average flow. When a flow cytometric sample enters the sample tubing, the tubing is already filled with the sheath fluid that has been used to backflush the sample line. Because the outer fluid travels more slowly than the inner, the interface between sample and sheath fluid does not remain flat, but rather forms a curved surface; therefore, the first part of the sample to reach the sensing area will be what traveled in the center of the sample tubing, not the outside. This can lead to unusual effects. The first cells arriving at the sensing area may be more accurately positioned than following cells. Also, the first cells through have an opportunity to mix with and become diluted by the sheath fluid that was already in the tube. In situations where fluorescent dye is loosely associated with cells and the amount attached to cells depends on an equilibrium with the amount of free dye in the sample, this first-cell-through effect can cause measurements to vary over time until a stable sample flow that completely fills the sample tubing is achieved.

The adherence properties, or stickiness, of cells influence their tendency to adhere to tubing walls or to each other. Differential settling of cells with different properties, in either the sample tube or the delivery tubing, can lead to changes in the distribution of cells reaching the sensing area.

CloggingClogging of the small-diameter sensing region is frequently a problem in flow cytometry, especially in the case of jet-in-air systems, which have small jet-forming orifices; to avoid this problem, filtration of the cell sample is often advisable. Instruments with closed-flow sensing and no sorting orifice are more forgiving, because flow cells typically have a larger inner diameter than does the orifice of a sorting machine and therefore do not clog as easily. Filters are typically installed in the sheath fluid line both to guard against particulates that might clog the orifice or flow cell and to sterilize the fluid. Cell samples may or may not require filtration as they are introduced into the flow system of the cytometer, depending on the reliability of the sample preparation and the tendency of the cells to aggregate. If a filter is used, it can be a source of cross-contamination between samples; where high purity is needed, such as during reanalysis of sorted samples, the filter should be changed between samples.

SettlingCells will tend to settle out of suspension at a rate that varies with cell size and density. A typical settling rate for 10-mm-diameter lymphocytes is ~1 cm/hr. Thus, cells in a tube mounted on a cytometer for long periods require occasional resuspension. If the flow rate is very low, settling in the sample delivery tube can also be a concern.

Random Cell ArrivalIn a well-mixed cell suspension, the cells are, in principle, randomly distributed throughout the sample. Although it would be advanta-

Fluidics

1.2.4Current Protocols in Cytometry

Figure 1.2.5 In a flow cell, the sensing region is contained within a chamber with transparent sides. Although not shown here, the cell usually has a square or rectangular cross section. The flow cell optical configuration may be combined with jet-in-air sorting by replacing the exit section with a jet-forming orifice.

illumination

transparent flow cell

geous to control the interval at which cells arrive at the sensing region, there is no way to do so, and in fact cell arrival time tends to follow a random distribution called the Poisson distribution. A number of effects may cause the actual arrival rate to deviate from the theoretical Poisson distribution: for instance, cells in the sample tube may not be mixed adequately, or may adhere to each other and tend to clump. The random arrival of cells at the sensing area presents problems for the signal processing electronics of the cytometer. A cell may arrive too soon after the previous cell so that the cytometer is not ready to measure it, or even worse, two cells may be so close together that the cytometer sees them as one. Inevitably, a certain percentage of measurements cannot be made satisfactorily. Such problems are alleviated if the average cell arrival rate is kept low, but this means that samples take longer to process.

Electrostatic Drop DeflectionA liquid jet in air emerges from its orifice as a column of fluid, but surface tension eventually causes it to break up into drops. If the jet is allowed to break up on its own, the sizes of the resulting drops will vary randomly over some range. In a sorting instrument, this breakup is brought under precise control by applying a periodic vibration at the orifice: this causes the breakup to occur in a very regular way so that the stream of drops is very uniform. The vibration is produced by means of a piezoelectric transducer attached to the nozzle: a periodic electrical signal applied to the transducer causes a small periodic variation in the diameter of the jet. Surface tension amplifies this variation as the jet progresses, and eventually the wave grows big enough to sever the jet, creating drops. The vibration may be applied either to the nozzle as a whole or to the fluid inside the nozzle. The main features of a sorting jet are illustrated in Figure 1.2.6. If the cell-sensing region of the cytometer is in the jet, a band of light is reflected and scattered perpendicular to the jet. The presence of a small surface wave on the jet causes this light band to be deflected up or down periodically by an amount that depends on the strength of the vibration that is applied to the jet. This effect causes additional optical noise that must be blocked in the measurement optical system usually accomplished with an obscuration bar. The vibration amplitude required to initi-

SORTINGThe utility of flow cytometry in scientific experimentation is greatly enhanced by the ability of the instruments to isolate, or sort, cells on the basis of measurements made by the device. Some flow cytometers have this added capability and others do not. Most sorting flow cytometers use the electrostatic drop deflection method, which employs a jet-in-air configuration; some use other fluidic methods.

Flow Cytometry Instrumentation

1.2.5Current Protocols in Cytometry

illumination

Figure 1.2.6 A jet-in-air sorter generates drops by vibrating the jet at a suitable frequency. Drops form with a uniform separation distance, called the drop wavelength.

satellite drop

drop wavelength

Fluidics

ate breakup of the jet by surface tension is quite small, however, and the optical noise problem is manageable. After measurements are made on a cell passing through the sensing region, the cytometer makes a decision on the basis of these measurements whether or not to sort the cell. The cytometer keeps track of the timing of desired cells, and when a desired cell arrives in the breakoff region of the jet, an electrical charging pulse is applied to the jet (which is an electrically conducting fluid) in such a way that the drop that contains the cell becomes charged and surrounding drops do not. The drop stream then passes through a strong, steady electric field that deflects the charged drops containing desired cells out of the stream of drops and into a collecting vessel. A drop may be either positively or negatively charged, allowing two populations to be sorted simultaneously, one to each side of the uncharged drop stream. In order to isolate chosen cells successfully, the rate at which cells arrive at the sensing region must be kept well below the drop formation rate. Otherwise, the probability of an unwanted cell being in the same drop as a wanted cell would be unacceptably high. Be-

cause sorting speed often limits the scope of experiments that can be performed with sorted cells, it is advantageous to generate drops at as high a frequency as possible. Drops are produced at the rate of the imposed vibration, but physical principles limit the size of the drops that can be produced by a jet of a particular diameter. For a cylinder of fluid, such as a sorting jet, the shortest section of fluid that can be made to form drops is a wavelength of about three times the diameter of the jet. A wavelength of 4.5 times the diameter is most favorable for drop formation. The frequency of the applied vibration must remain within these bounds. Given this limitation, there are only two ways to increase the rate of drop formation: to use a smaller-diameter jet (smaller orifice size) or to increase the velocity of the jet (higher nozzle pressure). Orifice size is limited by the tendency of small orifices to become clogged and by the size of the objects being sorted. Objects with diameters approaching that of the orifice will interfere with sorting in two ways: they will interfere with the regular formation of the surface wave at the orifice, and with the regular breakup of the jet at the breakoff point. Jet velocity is limited by the ability of cells to

1.2.6Current Protocols in Cytometry

withstand the mechanical rigors of the sorting process and by the ability of the fluidic plumbing to withstand the higher pressures required. For historical reasons, commercial sorters have been limited to operating pressures of ~1 atm (15 psi) and nozzle sizes of ~70 to 100 m, which meant that drop frequencies have been limited to ~25,00035,000/sec. More recently, sorters have become available that operate at significantly higher pressure and correspondingly higher jet velocity and drop frequency. Sorting jets have the interesting property of forming a satellite drop between the main drops. Normally this smaller drop soon merges with an adjacent larger drop and is of little consequence. Under some circumstances, however, it may not merge, and a separate stream of smaller satellite drops may be formed; this can have the undesirable effects of interfering with sorting and causing an additional biohazard.

Closed-System Mechanical SortingAn alternative sorting method available in some commercial machines employs a mechanical actuator situated downstream from the sensing region of a closed-system flow cell. The actuator moves a collection tube into the flow of particles and picks off desired cells as they flow by. This method has the significant advantage of maintaining a closed flow path, which makes it more suitable for biohazardous experiments than jet-in-air methods. Its disadvantages include a slower sorting rate and a more dilute sorted fraction.

Another function that is often provided is a fill function, which allows the fluidic system to be filled more quickly after it has been drained. To do this, an extra port near the sensing region is opened to allow fluid from the sheath tank to enter the system at a much faster rate than if the sheath were flowing normally through the small-diameter sensing region. It is important to backflush the sample line between samples to remove all trace of the previous sample from the fluid system before introducing the next one. To do this, the uptake end of the sample line is left open to the atmosphere while the sheath flow and pressure is maintained at the sensing end. The resulting pressure differential causes sheath fluid to flow backward through the sample line, clearing out the remaining sample. Waste removal capability is provided to receive the sample after it has passed the sensing region and to remove fluid during backflushing. Waste fluids may drain into a waste tank under gravity or may be sucked into the waste lines by means of a vacuum applied to the waste tank. Jet-in-air systems usually have an aspirator to catch the unsorted droplet stream.

KEY REFERENCESKachel,V., Fellner-Feldegg, H., and Menke, E. 1990. Hydrodynamic properties of flow cytometry instruments. In Flow Cytometry and Sorting (M. R. Melamed, T. Lindmo, and M.L. Mendelsohn, eds.) pp. 27-44. Wiley-Liss, New York. Excellent review of published work in fluidics relevant to flow cytometry. Pinkel, D. and Stovel, R. 1985. Flow chambers and sample handling. In Flow Cytometry: Instrumentation and Data Analysis (M. A. Van Dilla, P.N. Dean, O.D. Laerum, and M.R. Melamed, eds.) pp. 77-128. Academic Press, London. Detailed treatment of both theoretical and practical flow cytometric fluidics. Shapiro, H.M. 1995. Practical Flow Cytometry, 3rd ed. Wiley-Liss, New York. Includes thorough description of fluidics as well as many other aspects of flow cytometry.

OTHER FLUIDIC FUNCTIONSIn addition to its primary functions of delivering samples to the sensing region and combining sample and sheath flow for hydrodynamic focusing, the fluidic system of a flow cytometer generally has secondary functions built in. A boost function advances the sample from the sample tube to the sensing region at high speed at the start of a sample run to reduce the waiting time before the leading edge of the sample reaches the sensing region. This is done by switching the sample pressure momentarily to a higher level. Depending on the cytometer, this may be done either automatically or by the operator by means of a boost air-switch on the flow-control panel.

Contributed by Richard Stovel Stanford University Stanford, California

Flow Cytometry Instrumentation

1.2.7Current Protocols in Cytometry

Standardization, Calibration, and Control in Flow CytometryStandardization, control, and calibration provide different degrees of certainty about the data acquired with an instrument. Each process is aimed at assuring that results from the instrument have the quality required for the intended purpose (Horan et al., 1990; NCCLS, 1992; Muirhead, 1993a,b; Schwartz and FernandezRepollet, 1993; Owens and Loken, 1995; Schwartz et al., 1996). The purpose may be an individual research experiment or a clinical result that determines the course of patient treatment. In the terminology used in this commentary, an instrument is standardized at certain time points and subsequently operated under quality control conditions (see UNITS 3.1-3.2). These processes maintain the instrument within predetermined bounds and assure that results will vary only within certain limits. If a result is also calibrated when the instrument is standardized, then future results can be objectively and quantitatively compared with those from other laboratories. Quantitation of results should be considered. Most results from flow cytometers are expressed either in terms of percent positive or in qualitative terms such as dim or bright. These terms are relative: what is considered negative, dim, and bright in one laboratory may be quite different in another laboratory. When visualizing fluorescence using a fluorescence microscope, such relative terms are necessary. Flow cytometers can measure the amount of fluorescence and provide more objective criteria for expressing results. As flow cytometers are designed to measure particle characteristics (see UNIT 1.1 for an overview of flow cytometry), particles are the most common materials used to calibrate, control, and standardize the instruments. This commentary describes how various types of particles are used for these purposes. It also briefly reviews the status of standardization and quality control for flow cytometry (see Chapter 3 for further discussion of quality control). UNIT 1.4 covers calibration of detection system components (e.g., linear and logarithmic amplifiers) to ensure linearity of the flow cytometer response. The first section of this unit focuses on how the term standard has been used in flow cytometry (see Standards, Standardization, and Jargon). The intent is to alert readers of flowContributed by Robert A. HoffmanCurrent Protocols in Cytometry (1997) 1.3.1-1.3.19 Copyright 1997 by John Wiley & Sons, Inc.

UNIT 1.3

cytometry literature that they must always interpret critically how standard is being used in a particular context. The next section defines terms and also includes comments to put the term in context or to highlight issues (see Definitions). After providing extensive background on particle types and cautions (see Overview of Standardization in Flow Cytometry), this unit describes practical aspects of methods to standardize and calibrate flow cytometers (e.g., in terms of optical alignment, fluorescence and light scatter resolution, and sensitivity; see Standardization and Calibration section). Finally, suggestions are given for analyzing particles used as calibrators, including how to assign to fluorescent beads a value for molecules of equivalent soluble fluorochrome (MESF) and how to determine the inherent fluorescence coefficient of variation (CV) of a dim bead sample (see Characterizing Particles for Calibration and Control of a Flow Cytometer).

STANDARDS, STANDARDIZATION, AND JARGONIt is common in flow cytometry to combine words that describe use of a particle with the word standard. Examples are calibration standard and alignment standard (Horan et al., 1990; Schwartz and Fernandez-Repollet, 1993; Shapiro, 1995; Schwartz et al., 1996). Rarely is there any indication of who has set the standard and by what authority or consensus. There can be many levels of standards, depending on the size and authority of the group that establishes them. For example, an individual laboratory or investigator may have standard practices or materials. A large clinical or research study may have standard practices and materials that are agreed to by all investigators involved in the study. A professional organization may establish standard methods or identify standard materials for specific purposes. If the word standard is not modified by a term such as laboratory, clinical trial, or study XYZ, it may imply something that is generally and widely accepted by acknowledged authorities. In that authoritative sense, however, there are few standards in flow cytometry.

Flow Cytometry Instrumentation

1.3.1

Clear and common understanding of what is meant by a term is important, especially as flow cytometry is used by increasing numbers of investigators. The verb standardize means to cause to be without variation. Early use of the noun standard in flow cytometry seems to have been in the sense of a particle used to standardize (make consistent) one instrument in one laboratory (Fulwyler, 1979). This is much different from the authoritative sense of standard. In this commentary, other terms are used to describe more specifically what type of particle or material is being used for a particular purpose. For example, calibration particle or calibrator is used instead of calibration standard, and alignment particle rather than alignment standard.

DEFINITIONSConcern with terminology and its evolution is not just semantics, but reflects what has been important in flow cytometer technology and how the technology has grown and changed. More precise and generally accepted terminology should clarify communication and understanding among flow cytometrists as well as scientists in other fields. The definitions here should be considered a reasonable point along the way toward authoritative and broadly accepted and understood terminology. Some definitions include comments and references that may help put them in context. Accuracy: degree to which a measurement agrees with the true or expected value. Alignment particle: particle with uniform size, fluorescence, and light scatter characteristics that is used to check the alignment (or, in some instruments, adjust the alignment) of the excitation and emission optics in the flow cytometer. It is desirable that the alignment particle emits fluorescence in all detector channels, as this allows all channels to be checked simultaneously. Alignment of the optics is optimal when signals from the particles have maximum intensity and minimum variation or CV. The more uniform the particles, the better the degree to which small deviations from optimal alignment can be detected. Optimal alignment is most critical for measuring DNA, because of the very low inherent variation in DNA content from cell to cell. Antibody binding capacity (ABC): number of antibodies of a particular type that can bind to a cell under saturating staining conditions. Autofluorescence: inherent fluorescence from a cell or particle to which no stain or fluorochrome has been added. Manufactured

Standardization, Calibration, and Control in Flow Cytometry

particles (such as plastic beads) can be prepared to have nearly the same autofluorescence as lymphocytes. Background (noise, fluorescence, scatter): signal present when no particles are flowing in the sample stream. Background noise is one factor that limits the sensitivity of fluorescence detection (see definitions of fluorescence sensitivity and light scatter sensitivity below). Depending on how low the signals are that one is trying to detect in the sample, different factors are dominant contributors to the background. When no light is coming from the flow cell (e.g., lasers turned off), detector noise is the background limit. For photomultiplier tubes (PMTs), the detector background noise is called dark current and is due to random emission of electrons from the photocathode. For photodiodes and other solid-state detectors, which have no or low signal amplification, the limiting factor under best conditions is noise from the amplifier required to raise the signal to a useful level. Sources of fluorescence noise include Raman scatter from water and optical components; fluorescence from unbound fluorochrome, reagent, or contaminants in the sample or sheath stream; and fluorescence from optical components. Calibration: process of adjusting an instrument so that the analytical result is accurately expressed in some physical measure. Calibrator: material that has been manufactured or assayed to have known, measured values of one or more characteristics. The assayed values are provided with the material. Fluorescent manufactured particles can be assayed for diameter or for the amount of fluorescence they produce. A practical measure of particle fluorescence is the number of fluorochrome molecules in solution that produce the same amount of fluorescence as one bead (see definition of MESF). Coefficient of variation (CV): statistical measurement of the broadness of a distribution of values, usually defined as CV = /, where 1 the standard deviation = [(xi )2/(N 1)] 2, with the sum over N measurements of xi (where xi is the ith measurement of variable x), and the mean = ( xi)/N. Shapiro (1995) gives an excellent discussion of CV and other, more robust statistics for flow cytometry. Another excellent reference for statistical methods is Bevington (1969). Control particle or material: stable material (e.g., sample of manufactured particles) that gives reproducible results when analyzed. Particles used to set up a flow cytometer are used

1.3.2Current Protocols in Cytometry

as a control even if they do not have an assayed value assigned to a physical characteristic. Controls can be used to monitor the stability of an instrument and determine whether it is acceptably within calibration. A calibrator can be used as a control material, but a control material does not have to have an assigned value for a characteristic. Control sample: sample prepared in the same or nearly same way as a test or unknown sample and which should give an expected, predetermined result. In immunofluorescence analysis a positive control sample may use known cells (characterized for reactivity to a panel of antibodies) and the same antibody reagents as the test sample. A negative control sample may use the test cells but without antibody reagent or with an irrelevant antibody reagent. Fluorescence sensitivity: In flow cytometry there are two different aspects to the notion of sensitivity: threshold and resolution. The first has to do with the smallest amount of light that can be detected (Wood, 1993; Owens and Loken, 1995; Shapiro, 1995; Schwartz et al., 1996). This notion has also been given the name detection threshold (Schwartz et al., 1996). The second has to do with the ability to resolve dimly stained cells from unstained cells in a mixture (Brown et al., 1986; Horan et al., 1990; Shapiro, 1995). These concepts do not measure the same thing. The second notion incorporates a measure of the broadness of the fluorescence distributions for dim and unstained particles, not just the average fluorescence. Two instruments can have the same detection threshold but differ significantly in ability to resolve a dimly stained population. This is illustrated by example later (see Standardization and Calibration section). 1. Degree to which a flow cytometer can measure dimly stained particles and distinguish them from a particle-free background (threshold). Threshold is important when the mean fluorescence of a dimly fluorescent population is measured. The greater the number of particles analyzed, the more accurately and precisely will the mean fluorescence be measured. 2. Degree to which a flow cytometer can distinguish unstained and dimly stained populations in a mixture of particles (resolution). Resolution is important for immunofluorescence analysis of subpopulations and is strongly affected by the measurement CVs for dim and unstained particles. Inherent sample CV: actual variability in the characteristics of a sample; for example, the

actual variation in the amount of fluorochrome per bead in a sample of beads. Because the measurement process is not perfect and itself adds variation, the CV of the measured fluorescence will be greater than the inherent sample CV. The inherent CV of a sample can be estimated within a small uncertainty if the measurement variability added by the flow cytometer is well characterized (see Determining Inherent Fluorescence CV of a Dim Particle Sample). Light scatter sensitivity: degree to which small particles can be detected above particlefree fluid. In practice, forward scatter sensitivity is usually limited by optical noise caused by the excitation source, and side scatter sensitivity is usually limited by submicron particles in the sheath fluid. Manufactured particles (beads, plastic beads, latex particles, microspheres, microbeads): particles made of synthetic polymers (plastics). Sizes range from submicron to over 100 m, which generally covers the range of cells analyzed in flow cytometry. Most manufactured particles are made by bulk polymerization, but very uniform beads can be made employing the same droplet generation principle used for flow cytometric cell sorting (Fulwyler et al., 1973). Colored or fluorescent particles can be made by staining the beads with dyes or fluorochromes. Nonfluorescent beads, as well as many fluorescently stained beads, seem to be stable for many years. Two methods, namely, solvent (or hard) dying and surface staining, are used to stain particles. In solvent staining, non-water-soluble dyes are mixed with the particles in an organic solvent. The particles take up the dye and are then suspended in aqueous solution. The dye is trapped in the beads, which essentially become a hard-dyed plastic material. In some cases, hard-dyed particles can be synthesized directly using fluorescent monomers (Rembaum, 1979). As most dyes or fluorochromes used to stain cells are water soluble, solvent staining cannot generally be used for them. When solvent staining is possible for water-soluble fluorochromes, the spectral characteristics can differ significantly from those of fluorochrome in aqueous solution. Surface staining allows many common fluorochromesespecially those used as tags on fluorescent antibodiesto be used for particle staining. In this case a chemical group on the particle surface (e.g., amino group) is covalently bound to a reactive group on the fluorochrome. MESF (molecules of equivalent soluble fluorochrome): measure of particle fluores-

Flow Cytometry Instrumentation

1.3.3Current Protocols in Cytometry

Standardization, Calibration, and Control in Flow Cytometry

cence in which the signal from a fluorescent particle is equal to that from a known number of molecules in solution. This is a practical measure because a known concentration of particles can be compared directly with a solution of fluorochrome in a spectrofluorometer (see Calibrating Particle Fluorescence in MESF). Nonfluorescent particle: particle whose fluorescence distribution is the same as that of a particle-free sample. In practice, the concept of nonfluorescence is dependent on the sensitivity of the instrument making the measurement. A particle that is not measurably fluorescent in one instrument may be so in a more sensitive instrument. Fluorescence (or other luminescence or Raman scatter) from otherwise unstained manufactured particles depends on the material and treatment with which the beads are made. With all other factors equal, the fluorescent signal from microbeads will be proportional to the volume of a single bead. Precision or reproducibility: degree to which repeated measurements of the same thing agree with each other. In flow cytometry, precision of a measurement is estimated by the CV obtained when measuring a sample of particles (biological or nonbiological) with very uniform characteristics. Resolution: degree to which a flow cytometry measurement parameter can distinguish two populations in a mixture of particles that differ in mean signal intensity. Fluorescence sensitivity (see above) can be considered a special case of fluorescence resolution for which the signals are very dim. Note that the resolution will appear different when data are acquired and/or displayed on a logarithmic rather than linear intensity scale. Depending on the maximum number of channels into which the signal intensity is acquired (e.g., 256 or 1024), a logarithmic display of the data may not have sufficient resolution to display populations that can actually be resolved by the instrument. Standard: 1. noun. a. acknowledged measure of comparison for quantitative or qualitative value. b. something recognized as correct by common consent or by those most competent to decide. 2. adj. a. serving as a standard of measurement or value. b. commonly used and accepted as an authority. Standardize: a. cause to conform to a given standard. b. cause to be without variation. Test pulsetriggered background fluorescence: measurement of background fluorescence in a flow cytometer by using an electronic

pulse to trigger the pulse detection electronics and acquire data from the fluorescence detector(s) (see UNIT 1.4). As no particle is present to emit light, the fluorescence signals acquired are due only to instrument background light and noise and thus establish the lowest signal that can be measured. The duration of a test pulse usually simulates a signal from a particle of typical size. Larger particles would have signals of longer duration and produce more background signal and noise. If equipped with a test pulse function, the flow cytometer can provide a measurement equivalent to running a sample of truly nonfluorescent particles. The background fluorescence distribution produced by a test pulse should provide a measure of the detection threshold described by Schwartz et al. (1996). In many instruments the test pulse signal produces a pulse of light from a lightemitting diode that is detected and processed by one detector. When the test pulse signal is applied only to the forward scatter detector, the response of all other detectors to background light and noise can be measured. When a sample is run under normal conditions, any signal from particles above this background and noise level actually comes from the particles. There is no guarantee, however, that the particle signal is from particle fluorescence; for example, light scattered by the particles may not be totally blocked by the optical filters, or in some cases the light scatter may actually induce the filter to fluoresce. The possibility of scatter-induced fluorescence signal can be checked by running unstained cells and looking for a signal in the fluorescence channel. Because such a signal can also come from autofluorescence, one should also look at the side scatter versus fluorescence histogram.

OVERVIEW OF STANDARDIZATION IN FLOW CYTOMETRYStandardization (see Definitions) is the foundation of flow cytometry and allows investigators to have confidence in instrument performance. This section surveys characteristics of particles used in flow cytometry, for example, to standardize immunofluorescence and to check alignment and measurement precision (see Types of Particles). Specific types of particles are compared. Standardization can be complicated, however, by factors other than particle type (see General Cautions for Using Particles in Standardization and Calibration; see What the Instrument Cannot Control: Sample, Reagent, and Data Analysis), but prospects

1.3.4Current Protocols in Cytometry

for formalizing flow cytometry standards are encouraging (see Standard-Setting Organizations). The next section (see Standardization and Calibration) reviews various parameters of flow cytometers that can be standardized, such as resolution and sensitivity, and the final section (see Characterizing Particles for Calibration and Control of a Flow Cytometer) describes procedures and cautions for characterizing particles.

Types of ParticlesManufactured particles and biological particles may be used to standardize flow cytometers. Beads may be spectrally matched to the fluorochromes used to stain cells, or they may simply fluoresce to a useful extent in the spec-

tral range of interest. Spectrally matched beads allow standardization or even calibration across instruments that do not have exactly the same emission filters and/or excitation wavelengths. Biological particles may be stained with the same fluorochromes used in experiments to stain cells. Examples of data for some of the more common types of particles follow. Classification schemes for various types of particles used for standardization in flow cytometry have been proposed (Poon et al., 1994; Schwartz et al., 1996). Comparison of spectrally matched and unmatched fluorescent particles Figures 1.3.1 and 1.3.2 show emission spectra from three types of particles: fluorochrome-

A2.0Relative fluorescence

1.0

0.0 500650 550 600 Emission wavelength (nm)

700

FITC CaliBRITE

Rainbow beads RFP-30-5K

BioSure glutaraldehyde-fixed CRBC

BFluorescence relative to FITC CaliBRITE beads

300

200

100 0 505-525 nm 515-545 nm 512-545 nm FL1 filter bandwidth (nm) CRBC Rainbow

Figure 1.3.1 (A) Fluorescence emission spectra of FITC CaliBRITE beads (Becton Dickinson Immunocytometry), Rainbow beads RFP-30-5K (Spherotech), and glutaraldehyde-fixed chicken red blood cells (gCRBC, BioSure). Excitation at 488 nm was used. (B) Percentage of fluorescence signal through different optical filters for Rainbow beads and gCRBC normalized to signal from FITC CaliBRITE beads. Data in B are also scaled relative to the signal through the 505 to 525nm filter.

Flow Cytometry Instrumentation

1.3.5Current Protocols in Cytometry

A2.0Relative fluorescence

1.0

0.0 500

550 600 650 Emission wavelength (nm)

700

PE CaliBRITE

Rainbow beads RFP-30-5K

BioSure glutaraldehyde-fixed CRBC

BFluorescence relative to PE CaliBRITE beads

100

0 564-606 nm 562-588 nm 564-627 nm FL2 filter bandwidth (nm) CRBC Rainbow

Figure 1.3.2 (A) Fluorescence emission spectra of PE CaliBRITE beads (Becton Dickinson Immunocytometry), Rainbow beads RFP-30-5K (Spherotech), and glutaraldehyde-fixed chicken red blood cells (gCRBC, BioSure). Excitation at 488 nm was used. (B) Percentage of fluorescence signal through different optical filters for Rainbow beads and gCRBC normalized to signal from PE CaliBRITE beads. Data in B are also scaled relative to the signal through the 564- to 606-nm filter.

Standardization, Calibration, and Control in Flow Cytometry

tagged beads (CaliBRITE beads, Becton Dickinson Immunocytometry), stained with either fluorescein isothiocyanate (FITC) or phycoerythrin (PE); broad spectrum hard-dyed beads (Rainbow beads, Spherotech); and glutaraldehyde-fixed chicken red blood cells (gCRBC, BioSure). Figure 1.3.1A compares FITCstained CaliBRITE beads with Rainbow beads and gCRBC, which are not spectrally matched to FITC. Figure 1.3.2A makes the same comparison with PE-stained CaliBRITE beads. Figure 1.3.1B compares quantitatively the fluorescence signal of each particle through filters of differing spectral bandwidth placed in front of PMT1, with data being normalized to the signal from FITC CaliBRITE beads for each

filter. The gCRBC varied by about 35% over the range of filters used. Rainbow beads, however, varied by nearly 200% with the same filters. The differences in relative fluorescence with different filters should be considered when comparing different instruments. Even for a particular flow cytometer type or model, filters vary slightly due to manufacturing tolerances. Figure 1.3.2B shows a similar comparison for the relative fluorescence with different filters placed in front of PMT2. In this case, both gCRBC and Rainbow beads vary only slightly from PE-stained CaliBRITE beads. The fluorescence could be standardized with a maximum difference of 40% with any of these particles.

1.3.6Current Protocols in Cytometry

0.20Relative fluorescence

0.00 500 600 700 Emission wavelength (nm)CaliBRITE unstained beads BioSure osmium-fixed CRBC

Figure 1.3.3 Fluorescence emission spectra of unstained CaliBRITE beads (Becton Dickinson Immunocytometry) and osmium-fixed chicken red blood cells (CRBC, BioSure). Excitation at 488 nm was used, and concentrations of the two types of particles were the same. No fluorescence from the osmium-fixed CRBC could be measured above background noise in the spectrofluorometer.

Nonfluorescent and autofluorescent particles Figure 1.3.3 shows emission spectra for particles with very low fluorescence. Unstained CaliBRITE beads have fluorescence comparable to autofluorescence from lymphocytes. Osmium-fixed chicken red blood cells (CRBC) had no fluorescence detectable above background in the fluorometer. Such negative particles are useful for estimating how well low level signals can be detected, as discussed later (see Sensitivity or Signal/Noise for Dim Fluorescence). Comparison of particles for standardizing immunofluorescence analysis Figure 1.3.4 shows light scatter dot plots (panel A) and green (515 to 545 nm) fluorescence histograms (panels B-F) for several types of particles used to standardize flow cytometers for immunofluorescence analysis. Fluorescence from the stained particles is in the range observed for immunofluorescence from cellsurface markers. All data were acquired using the same instrument settings, and panels A-D were obtained from the same sample acquisition of a mixture containing (1) unstained (autofluorescence) and FITC-stained CaliBRITE beads, shown in region R1 in panel A; (2) a combination of unstained and multiple levels of stained Rainbow beads, shown in region R2 in panel A; (3) gCRBC, shown in region R3;

and (4) forward scatter (FSC) test pulses (no particle, R4 in panel A). Fluorescence histograms in Figure 1.3.4 are from unstained and FITC CaliBRITE beads (panel B), Rainbow beads (panel C), FSC test pulses and gCRBC (panel D), Quantum 24 beads (Flow Cytometry Standards; panel E), and osmium- and glutaraldehyde-fixed CRBC (panel F). Panels B, D, and F illustrate different pairs of particles or signals at the low and high ranges of a scale for immunofluorescence. The Quantum 24 beads shown in Figure 1.3.4E had calibration values for the stained beads (upper four peaks in the histogram) of 4,201, 16,936, 37,466, and 65,797 fluorescein MESF (molecules of equivalent soluble fluorochrome). Particles for aligning and checking measurement precision Figure 1.3.5 shows scatter and fluorescence data for a uniform 2.49-m-diameter fluorescent bead that is useful for checking or adjusting optical alignment. All fluorescence CVs were

10 is used with a covered specimen, the image quality will be poor. At a magnification of 10, objectives designed for use with and without cover slips may be used interchangeably for routine applications. Microscope cover glasses come in several thicknesses, as indicated by a number (e.g., 1, 1.5, or 2). Each number represents a defined

2.2.10Current Protocols in Cytometry

eyepiece integrated observation tube

intermediate image

tube lens

infinity space

parallel light beam

intermediate attachments

objective

Figure 2.2.14 Infinity space: the distance between the back of an infinity-corrected objective and the tube lens (schematic). Reproduced from Abramowitz (1994) by courtesy of Olympus America.

thickness range. For example, #1.5 cover glasses are typically 0.16 to 0.19 mm in thickness. Typical dry biological objectives are designed and optically corrected for a cover-glass thickness of precisely 0.17 mm. Dry objectives with NA greater than ~0.75 will suffer noticeable image degradation if the cover glass differs even by a few hundredths of a millimeter from the specified thickness. Because cover glass thickness may vary by several hundredths of a millimeter even within a package, high dry (40 high-NA) objectives are available with an adjustable correction collar and scale that permits them to be adjusted for different cover glass thicknesses (e.g., from 0.11 to 0.23 mm). Turning the correction collar to match the actual thickness of the individual cover glass in use prevents the introduction of spherical aberration and its consequence, image degradation. Using too much mounting medium on the tissue will create an additional cover glasslike optical layer that must be added to the thickness of the cover glass to determine the total effective cover glass thickness. If the effective cover glass thickness is different from that specified for the objective, spherical aberration will be introduced into the image. For this reason, many experienced microscopists do not rely on the correction collars numbered scale

to set the proper correction. Rather, they choose a suitable area of the specimen and repeatedly refocus the microscope while moving the correction collar to different positions, finally reaching the setting that provides the best image. On objectives used for inverted tissue culture studies with flasks or other relatively thick culture vessels, the correction collar may have a range of correction from 0 to 2 mm; on standard upright microscope objectives, the range is usually from 0.11 to 0.22 mm.

OBJECTIVES FOR OTHER MICROSCOPY APPLICATIONS Phase ContrastFor phase-contrast microscopy, an annular phase plate is installed by the manufacturer inside the back of the objective. This plate serves to speed up the undiffracted light passing through it and also to reduce its intensity. Phase specimens, such as unstained cells and tissues, are almost invisible in standard brightfield microscopy. The phase plate in the objective, when aligned with the annular opening of a phase condenser, optically renders small phase objects visible without the use of stains. Because phase-contrast observation is often done through glass or plastic culture vessels,

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2.2.11Current Protocols in Cytometry

eyepiece diaphragm plane

objective front focal plane of objective specimen

Figure 2.2.15 Objective system of finite tube length, showing the projection of the image by a finite objective to the intermediate image plane within the eyepiece tube. Reproduced from Abramowitz (1994) by courtesy of Olympus America.

some manufacturers offer interchangeable accessory lenses or caps that attach to the front lens of the objective (one set for use with plastic vessels, one set for glass vessels) to avoid distortion of images.

Polarization TechniquesIn polarization microscopy, it is important that the objective itself not contribute to the alteration of polarization effects induced by the specimen. Because glass that is physically strained affects polarized light, microscope manufacturers carefully select objectives in which the glass elements and their mountings are strain-free. The barrel of strain-free objectives supplied with polarizing microscopes is usually marked with a P, SF, or POL and is sometimes inscribed in a color different from the usual inscription color. Differential interference contrast (DIC) microscopy is also invaluable for making small phase objects readily visible. It has further advantages in that it (1) yields a pseudo-three-

dimensional image, in which the object appears shadowedbrighter on one side and darker on the otherdisplaying elevations and depressions within the specimen; (2) permits the use of high-NA optics; and (3) makes possible optical staining and optical sectioning of the specimen. In DIC microscopy, the distance from the back focal plane of the objective to the upper Wollaston prism (a special prism positioned above the objective) is usually critical, and microscope companies may therefore designate particular objectives for use in DIC microscopy. These objectives are relatively strainfree, because interference microscopy also involves the use of polarized light, and may be labeled DIC or NIC (for Nomarski interference contrast, a particular type of DIC).

Dark-Field MicroscopyIn transmitted-light dark-field microscopy, the illumination is directed obliquely so that the specimen appears bright on a dark background. For dark-field microscopy with high-NA objec-

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2.2.12Current Protocols in Cytometry

tives (1.00), the NA of the objective must be reduced below that of the oil darkfield condenser. Manufacturers therefore provide highNA objectives with built-in iris diaphragms (see Fig. 2.2.12). For dark-field use, the diaphragm is closed down to yield an NA below 1.1. For general use, the diaphragm must be fully open or optical performance will be degraded.

Ultraviolet (UV), Fluorescence, and Infrared (IR) ApplicationsStandard glass objectives are relatively opaque to wavelengths in the lower UV range, below 380 nm. Special objectives are manufactured with special glasses to achieve greater transmission of these lower wavelengths, which are used to excite certain fluorescent dyes for measurement of intracellular ions. The cements used in complex lens elements for fluorescence microscopy are nonfluorescing, and the best fluorescence objectives are made using quartz optics. Other investigations may be carried out using longer, IR wavelengths (>750 nm), which offer poorer resolution (see Abbes equation in the discussion of Resolving Power) but greater depth of penetration into biological (and other) materials. Several companies offer objectives specially designed to more efficiently transmit wavelengths up to 1800 nm. The technical departments of the major microscope companies can provide transmission and spectral data for their objectives upon request to aid in selecting the proper objectives for special applications.

OTHER CONSIDERATIONS IN CHOOSING OBJECTIVESOther considerations may prove valuable in understanding the performance of objectives and in guiding the selection, purchase, and use of suitable objectives. Numerical aperture, the ability of the objective to capture a cone of light of wider angle, has a crucial effect on resolution. Although intuitively it may seem that resolving power should increase with increasing magnification, it can be shown that the ability to distinguish closely spaced details within a specimen is directly proportional to the twice the working NA. However, the use of objectives with higherthan-necessary magnification and NA for a given application can be detrimental not only because they are more expensive, but also because the specimen area observed within a field of view will be smaller and both the depth of

field (the vertical distance above and below the plane being observed that is still in acceptable focus) and working distance are shallower. When the finest specimen details need to be observed, high-NA objectives are required. High-NA objectives are also indicated when maximum throughput of light is needed. The light transmittance for an objective, using visible wavelengths, typically varies with the square of the NA of the objective. In reflectedlight and epifluorescence microscopy, light passes through the objective twice (first the illuminating light, and then the reflected or fluorescent signal), and so the intensity varies with the fourth power of the NA. In situations where the light level is low, NA is a critical factor in obtaining brighter images. A question often asked is why higher magnification cannot be achieved simply by using higher-magnification eyepieces with a given objective. Because of limitations due to the size of light waves themselves and the phenomenon of diffraction, higher and higher magnifications unaccompanied by increased NA will result in images that are less and less clear. The limiting factor in ensuring usable, as opposed to empty, magnification is the NA of the objective (more precisely, the average NA of the objective and the condenser). Eyepieces and accessory lenses are designed for use with certain objectives and condensers, and should not be switched to increase magnification except as recommended by the manufacturer. An oft-cited rule of thumb is that the user should limit the total optical magnification (the objective magnification multiplied by the eyepiece magnification and that of any other lenses) to between 500 and 1000 times the NA of the objective. At 1000 times the NA, the likely result is empty magnification. In the favored method of Koehler illumination, the condenser diaphragm is partially closed down, slightly lowering the overall NA in order to improve contrast. Hence, a total magnification of 750 times the NA will usually produce excellent images with satisfactory contrast. All of the foregoing discussion of objective design, features, and performance assumes that the optics (and the rest of the microscope) remain forever in the pristine state in which they presumably arrived. Proper care of the objectives, including handling, storage, and cleaning, are essential prerequisites to keeping them in proper working order. The authors have often noted that the best microscopy is not necessarily performed by those with the best equipment,

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and quite often the performance of superior optics is profoundly or subtly degraded by a lack of care in choosing and maintaining the optics.

Delly, J.G. 1988. Photography Through The Microscope. Eastman Kodak, Rochester, N.Y. Inoue, S. 1986. Video Microscopy. Plenum Press, New York. Leitz, E. 1938. The Microscope And Its Application. Ernst Leitz, Wetzlar, Germany. Mollring, F.K. 1976. Microscopy From The Very Beginning. Carl Zeiss, Oberkochen, Germany. Spencer, M. 1982. Fundamentals of Light Microscopy. Cambridge University Press, Cambridge, UK.

LITERATURE CITEDAbramowitz, M. 1994. Optics: A Primer. Olympus America Inc., New York.

KEY REFERENCESAbramowitz, M. 1985. Microscope Basics and Beyond. Olympus Corporation, New York. Abramowitz, M. 1987. Contrast Methods in Microscopy: Transmitted Light. Olympus Corporation, New York. Abramowitz, M. 1993. Fluorescence Microscopy: The Essentials. Olympus America Inc., New York. Abramowitz, 1994. See above. Bradbury, S. 1984. An Introduction to the Optical Microscope. Oxford University Press, Oxford, UK.

Contributed by Mortimer Abramowitz Olympus America Inc. Melville, New York Marc M. Friedman AccuMed International Chicago, Illinois

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2.2.14Current Protocols in Cytometry

CamerasOne of the most critical components of an image cytometer is the camera, the unit that converts the optical image into electrical form so that it can be viewed on a TV monitor, recorded, or digitized for subsequent analysis. Any image degradation introduced at this stage will affect the quality and accuracy of the systems output. Compared to those of the past, modern cameras offer an array of reasonably inexpensive alternatives. The proper choice of camera depends on the usage for which the instrument is designed. The camera should be matched both to the other system components and to the problems that will be addressed by the instrument. The camera is the eye of an image cytometer. It is called upon to convert a two-dimensional spatial distribution of light intensity into a corresponding electrical signal that is a faithful representation of the specimen. This, in turn, can be displayed, recorded, or processed. How accurately the camera can conduct this transformation affects the quality of results one can obtain from the instrument. Image sensing is a complex technology that harnesses a variety of phenomena. There are numerous sources of noise, distortion, and loss of resolution in the process. Any particular camera represents a series of design tradeoffs, and its performance will be higher in some areas and lower in others when compared with another unit. A poor choice of camera can severely limit the accuracy and usefulness of an image cytometry system. The camera with the best specifications or the highest price is not always the right one for a particular job. It is necessary to understand the fundamentals of camera phenomena in order to design an image cytometer or use it to best advantage. Fortunately, many of the image degradations that are introduced by camera shortcomings can be reduced or eliminated by subsequent image processing. Thus, the cameras performance is interwoven with that of the software. the light intensity at an individual pixel location. It requires a sampling aperture that defines the size and shape of the pixel (usually circular, square, or rectangular). Transduction is the process of converting the light intensity at a particular pixel location into a corresponding voltage. Scanning is the process of selectively addressing the picture elements in order. This creates the data stream that represents the image. If the image is to be processed digitally, it must be digitized. Quantization is the process of generating an integer that reflects the brightness of the image at a particular pixel location. Digitization is the process of sampling and quantizing an image. The degree to which a camera can reproduce small objects is its resolution. If it warps the objects in the image, this is distortion. Any undesirable additive components of the image are called noise.

UNIT 2.3

IMAGE SENSING Light SensingLight-sensing devices produce an electrical signal proportional to the intensity of light falling upon them. Different physical phenomena can be employed for this purpose, giving rise to different types of light sensors. Photoconductors, such as selenium, show a drop in their electrical resistance when exposed to light. Semiconductor devices made from pure silicon crystals generate free electrons in response to incident photons. Both these phenomena have been harnessed to sense images.

PhotometryPhotometry is the technology of quantifying light intensity, and there are many ways to do this. For example, photons of different wavelength have different energy. Thus, if incident light energy flux is measured, the spectrum of the light affects the intensity. Commonly used image sensors, however, merely count photons, so wavelength considerations do not directly affect the measured intensity. Although the sensors do have different sensitivities at different wavelengths,