Dan Pinkei
Instrumentation
Lawrence Liverme National Laboratory Biomedical Sciences Division University of California P.O. Box 5507 L-452 Livermore. Calif. 94550
Flow Cytometry and Sorting The ability to rapidly obtain quantitative information on cells or other particles of biological interest is of major importance to medicine and biology. Classification and sorting of individual chromosome types, detection of malignant cells by determining their protein and DNA content, and searching a large population of cells for rare mutants require rapid automated techniques. The development of flow cytometry, in which a stream of suspended particles is made to flow on a controlled trajectory past optical or electrical sensors, has been motivated by these needs. Work over the last 15 years has
resulted in instruments capable of measuring several parameters of each particle at a rate of thousands of particles per second, with the resulting data sets stored and analyzed by computer. In addition, the computer can make decisions based on the measurements rapidly enough to allow sorting of desired particles from the general population for further study. Measurements of such parameters as fluorescence (both intrinsic and of cellcomponent-specific stains), fluorescence depolarization, phosphorescence, light scatter, absorbance, and electrical conductivity and capacitance are possible.
Flow cytometry has found widespread application, with several hundred installations in use worldwide. Three commercial manufacturers supply general-purpose flow cytometers and sorters, and several others build dedicated clinical instruments. Many laboratories choose to build their own for specific applications or exploration of advanced techniques. This article describes the.basic technology and capability of the field, drawing examples from work being done at Lawrence Livermore National Laboratory. Much more detail is contained in References 1,2,and 3. Reference 4 is a comprehensive source of information
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Figure 1. Flow cytometric Instrumentation Vibratinglhe flow dumber caus8s bIiquM jed to break up into regular drops. Elenlcal charging of the appqxiate drqos allows swting 01 individualpanicles based on b i r measured parameters W03-2700/82/035 1-503AS01.OO/O Q 1982 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 54, NO. 3, MARCH 1982
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INA Content
Figure 2. The cell cycle (a) M A cartent d a call doubles befae It divides. (b) Distributionof ONA mn(enl d a Wpulatlon of cells m m l y spaced though the division cycle. (c) Corn puter anaiwls of flow cytomeb’lc measurementsallows quantlflcetlm of VNI IraCfion of cells in the various stases of the cycle. The dashed line separams VNI ConblbuIlon of Sphase cells from VIoae of the other phases
in this area, and its more than 70 contributors indicate the scope of the international effort. Many of the articles related to new developments in flow instrumentation and applications are published in the journal Cytometry.
lnstrumeniatlon To accurately measure suspended particles, the flow must be controlled so that each particle follows nearly the same trajectory through the analysis sensors. This is accomplished by establishing a sheath of nonturbulent fluid centered around a small-diameter sample injection tube (Figure 1). The sample stream exits from the injection tube, the stream diameter decreasing further as the sample passes through the nozzle of the flow chamber. In typical instruments, the sheath exit orifice is on the order of 200 pm in diameter while the sample stream (core) diameter is several micrometers. The use of “hydrodynamic focusing” avoids the problems of clogging and velocity gradients across the sample stream that would occur if control of the particle trajedaries were attempted using an orifice of the core diameter dimension. Fluid velocity in the orifice is typically 10 m/s, attained by applying a fraction of an atmosphere pressure. The Reynolds number of the flow is low enough so that it is laminar and extremely stable. The flow field orients elongated objects with their long axis parallel to the direction of flow, and by special shaping of the orifice and sample injection tube, even the orientation of flat cells about the flow axis can be controlled. It has been found that cells can with504A
stand passage through the instrument and be viable for further study. CW lasers with outputs ranging from milliwatts to a few watts in a single line are usually used for cell illumination, although mercury lamps also work well. To maximize illumination of the particles, the light source is focused on the sample stream. Focus across the core (perpendicular to the flow direction) is limited by the need .to have uniform illumination over the range of particle trajectories allowed by the sample stream diameter. Focus along the stream is frequently much tighter, to shorten the time of illumination or to extract one-dimensional morphologic information by scanning individual particles as they pass through the beam. Collection optics focused on the laser beam-sample stream intersection point pick up the signals and transmit them to photodetectors. Photomultiplier tubes are universally used for fluorescence detection, and photodiodes are suitable for the larger signals of forward scattering. Illustrated in the upper right of Figure 1is the hypothetical output of a detector as a function of time for the passage of a single object through the beam, which typically takes 10-6 s. The dimensions have been chosen such that the length of the object, shown oriented as discussed above, is larger than the laser beam. If internal optical properties of the cells can be neglected, the time course of the fluorescence intensity is given by the convolution of the laser beam intensity profile with the distribution of stain along the particle. Characteristics of the pulse that might be measured are
ANALYTICAL CHEMISTRY, VOL. 54. NO. 3, MARCH 1982
area, width, height, and details of the profile. The desired pulse parameter is digitized and measured by a multichannel analyzer. The data is displayed as a histogram that shows the number of particles vs. the value of the measured parameter. Two different-sized particles are shown in the sample stream in Figure 1.Each population is responsible for one of the peaks in the resulting histogram. Individual objects can be sorted based on the measurements. Mechanically, sorting is usually accomplished by vibrating the flow chamber with a piezoelectric crystal while allowing the fluid to exit into the air. The vibration causes the fluid jet to break into regular droplets as illustrated in Figure 1. Typical droplet formation frequencies are in the tens of kHz range with jet diameters under 100 pm. If, on the basis of the cytometric measurements, a cell meets preset criteria, a voltage is applied to the sheath (which is a conducting fluid such as saline) with the appropriate delay and duration so that the drop containing the desired cell is electrically charged when it breaks off. Several hundred microseconds are available for the electronics to make the sorting decision. The droplets pass between a pair of charged plates, and those with a charge are deflected into the collection vessel. The more sophisticated instruments can measure several properties of each particle. The use of a conducting sheath fluid allows the effect of the presence of a cell on the ac or dc conductivity of the flow chamber orifice to be measured. Developed by Wallace
New programmable UV-100detector makes the 5000 LC even better
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Rellabie. fully integrated. and simple to workwith. theVarian Model 5000 is widely recognized as the best liquid chromatographyou can buy. Now, the best LC is even better. We've built-ina fullyautomated.variable wavelength detector, the sensitive. fast new UV-100. Now, everything in the 5000 system is automated and it's ail programmed and controlled through the same heipful interactive CRT and keyboard. Time program wavelength selection, detector au1ozero and range The UV-100 allows you to select as many wavelengths as you need to optimize detection of all components in a run. v,a simple keyboard entries, you program any wavelengths you want, in increments as small as 1 nm. from 190nm to 370 nm. at exactly the timeS you want. youget optlmum detection 01 each component as it elutes from your LC Your wavelength entries are part of the complete LC program stored in memory and displayed on the CRT Over 100 unattended waveiength changes are possible for overnight operation or automated methods development. In addition, to improve the accuracy of your data and the quality of your report, you can attenuate peaks and rezero the baseline at any point in a ruii and as often as you like. Also, through program linking, you can change the 3 time constants up to 9 times during an analysis. Patented' flow cell and selectable time constants for best detection We designed the UV-100for conventional as well as high speed LC applications. With 15 to 30cm analytical columns at normal fiow rates, the 4.5 microliter fiow cell and 0.5 sec time constant give you
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ANALYTICAL CHEMISTRY, VOL. 54, NO. 3, MARCH 1982
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Major shortcomings of most LC columns currently available have included inadequate reproducibility of retention, selectivity and performance (efficiency, permeability, peak symmetry), plus poor chemical and mechanical stability (short lifetime). NOW! ULTRASPHERE'" columns represent a series of advances in modern column technology which significantly reduces these problems. Advanced Packing The Reversed Phase ODS (C-18), Octyl (C-8) and IP (Ion Pairing) columns feature a maximum coverage, endcapped bonded si Iica-based material. These columns are packed with 5 ,um spherical particles having extremely uniform particle and pore size distributions for high efficiencies and lower pressure drop.
Improved Retention and Selectivity Based on the carbon content and surface area, the ULTRASPHERE columns possess a maximum surface coverage of the bonded moiety (C-18 and C-8). The remaining accessible silanols are endcapped with a second silanization to minimize the undesirable influence of unreacted accessible silanol groups on solute retention. The absence of micropores (as found in some silicas) also minimizes unbonded silanols. This ensures good peak shape with excellent reproducibility in column-to-column (batch-tobatch) retention and selectivity. Extended Column Lifetime Column stability is further enhanced by a new proprietary packing process. The result is a column with high chemical and mechanical stability
which resists troublesome headspace formation that will degrade column performance. We believe these columns represent state-of-the-art technology and should enable you to attain the best possible results from your liquid chromatograph. To ensure maximum column performance, each and every column is individually packed and tested. Sample test mixture, test chromatogram and conditions are supplied with each column. For more information, write for Beckman Catalog 7358. Or ask your local Sales Representative. Beckman Instruments Inc., Scientific Instruments Division, 1780 Fourth Street, Berkeley, CA 94710
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ANALYTICAL CHEMISTRY, VOL. 54, NO. 3. M A R C H 1982
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Can Your GC Analyze It Can
Pyroprobe: The Pyroprobe expands the capabilities of your GC, GUMS or GC/IR by allowing you to analyze most solids including synthetic polymers, geo-polymers and biopolymers. And that's not all. A Series 120 Pyroprobe can perform: 0 Analytical Pyrolysis 0 Headspace Analysis of Volatile Organics 0 Simulated Combustion Studies 0 Simulated Thermal Analysis
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Coulter, the dc conductivity measurement provides information on cell volume. Cytometers with an electrical sensor plus one or more excitation beams spaced along the flow path have been developed. Since the flow is stable, signals from the same particle can be associated by their fixed separations in time. Collection, manipulation, analysis, and display of this multiparameter data require computer systems. To facilitate sorting, dedicated microprocessors are coming into widespread use. Sample preparation is often a major task. If the study involves solid tissue, dispersal of the cells into a dilute single particle suspension without altering the characteristics of interest is required. While in some applications size information obtained electrically or by light scatter is sufficient, most of the time fluorescent stains (probes) are employed. Probes specific for DNA, RNA, proteins, and membranes are available, and fluorescently tagged antibodies for specific cellular components are frequently used. Dyes sensitive to pH or membrane potential give information on the functional state of the cells. Changes in the local environment of dyes in the membrane and cytoplasm can be monitored by fluorescence depolarization measurements. Energy transfer between different fluorescent molecules has been used to obtain information on the mutual distribution of their binding sites. In what follows I will describe some applications of this technology to measurements on cells and chromosomes drawn from work at Lawrence Livermore National Laboratory. I wish to emphasize that work in these and many other areas is under way in a large number of other laboratories.
Cell Cycle Kinetics
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Cell reproduction follows a series of distinct stages whose detailed characterization is of significance to both fundamental biology and clinical medicine. Flow cytometry has made major contributions to these studies since probes for various biochemical constituents, such as DNA and proteins, which vary throughout the division cycle, allow monitoring of the stage of each cell. Much of this work is directed toward understanding and improving cancer chemotherapeutic agents, which may interrupt the cell cycle or have maximum effectiveness a t specific points. One cell constituent that can he used to monitor the progress of a cell through the cell cycle is DNA. The DNA content of a cell doubles before division so that each of the daughter cells has a complete copy of the genetic information. In Figure 2a, DNA content is plotted as a function of time NO. 3, MARCH 1982
DNA
Distribution
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Figure 3. Computer simulation of a synchronized cell population The distribution 01 cells mrWgh lhe cell cycle is reflected in the DNA COntent distribution
during one cycle of division. The initial period with l x DNA content is called Growthl (GI). During this period, the biochemistry appropriate for beginning DNA synthesis is carried out. During the DNA synthesis (S) phase, the DNA content continuously increases. After the completion of the doubling, the phase called Growth2 (Gz)is reached, where preparation for cell division, labeled M for mitosis, is made. Two daughter cells in the GI phase of the cell cycle result from the division. The distribution of DNA content of a population of cells that are randomly distributed in their cycles is shown in Figure 2b. If the cells are stained fluorescently for DNA and measured with a flow cytometer, a distribution such as Figure 2c results. The features of the fluorescence distribution are not as sharp as that of the actual DNA distribution due to slight variability in stain uptake, nonspecific staining of cellular components besides the DNA, and instrumental factors. The analysis of these fluorescence histograms using computer models allows determination of the fraction of cells in the various compartments of the cell cycle. Dyes such as Hoechst 33342 do not interfere with cell viability, so living cells can he sorted based on their DNA content and then grown in culture.
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Relative Fluorescence
Figure 4. Flow cytometric analysis of Chinese hamster chromosomes Above each peak in the flwxeacence distribution is a drawing of !he conespanding chomo6ome type
The action of drugs that perturb the cycling of cells can be monitored flow eytometrically by following changes in DNA distribution after drug administration. Figure 3 is a computer simulation of three such serial distributions. Initially all of the cells are concentrated in GIphase, as might exist after the administration of a drug that halts their progress through the cell cycle at this point. This state of the cell population yields a single peak in the DNA distribution. When the block is removed the cells resume cycling, but not all at exactly the m e rate. By 2.9 h many of the cells have progressed into S phase, which is indicated by the presence of cells with higher DNA content. After several passes through the cycle, the dispersion in cell cycle times will randomize the distribution of the cells. Drug therapy for cancer is complicated by, among other things, the fact that cancer cells differ only slightly from normal cells. Thus, it is difficult to find biochemical differences that can be exploited to give preferential killing of a tumor in the midst of normal cells. Differences in the cell cycle offer an alternate possibility. This approach is based on the fact that some drugs kill by interfering with a cellular process at a particular part of the cycle, S phase for example. If such an S-phase-specific drug were administered to an unsynchronized cell population characterized by the DNA content distribution of Figure 2, less than half of the cells would be killed. However, if it were given 5.7 h after the release of the block in Figure 3, most of the cells would die. If a mixed population of two kinds of cells with different cycling rates, tumor and normal for
ANALYTICAL CHEMISTRY, VOL. 54, NO. 3, MARCH 1982
example, are blocked and then released at the same time, there may he a later time at which the ratio of the killing of the tumor to normal cells will be maximized. Complicated studies of drug action on normal and tumor cells in whole animals, in which flow cytometric techniques play a major role in monitoring the effect on the cells, are now under way to improve our understanding of this area.
Chromosomes The DNA of higher life forms is packaged in compact units called chromosomes. Normal humans have two each of 22 different chromosomes (one from the mother and one from the father) and either two X chromosomes (female) or an X and a Y (male). It is the chromosomes that duplicate during the S phase of the cell cycle, and by the M phase they can be visualized (Figures 4 and 6), in general, as pairs of parallel rods joined at a narrow region called the centromere. During cell division they separate, and one goes to each of the daughter cells. Figure 4 shows the fluorescence distribution of chromosomes isolated from Chinese hamster cells and stained for their DNA content (5,6). Since each chromosome type contains a specific amount of DNA, the flow measurements result in a series of peaks, each representing a single chro mosome type if the resolution is high enough. Peak widths below a 2% coefficient of variation are ohtainable on chromosomes with g of DNA. Major applications of flow measurements on chromosomes include the sorting of purified single chromosome fractions for biochemical and gene mapping (7)studies, and comparison
Chromatograph ic Adsorbents for TLC and CC.
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PARTICLE SIZE SIGNIFICANCE IN TLC I
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Figure 5. Dual parameter analysis of human chromosomes Pmpmive view and mnespondingmtour plot 01 me tiuwe~~ence distribution of the smiier human dromo8omes, numbers 9 to 22 and Y
of the distributions obtained from different individuals to understand normal variations and those that may be characteristic of genetic diseases. While most Chinese hamster chromosomes can be resolved, this is not the case with human chromosomes, many of which are too similar in DNA content. However, DNA is not a homogeneous molecule, and some of its more subtle properties can he exploited to improve the discrimination. The genetic information in DNA is encoded in the sequence of its constituent bases, A (adenine), T (thymine), G (guanine), and C (cytosine), which always occur in pairs, A associated with T and G with C. The ratio of A-T to G-C paira in each chromosome is close to 1.5, hut slight differences occur. These differences can he detected by using two dyes, one of which preferentially binds to A-T and the other to G-C. If a single excitation wavelength is used, separation of the signals from the two dyes must rely on differences in the emission spectra. There is usually overlap in the emissions, 80 some light from each of the dyes must he fil tered out to discriminate between the two signals. This is undesirahle, since in many cases the measurement precision is limited by photon statistics. It is better to use dyes with differing excitation bands, which requires two excitation sources that intersect the sample stream at different points. An instrument of this type is used for the following measurements. Figure 5 shows two presentations of the same human chromosome histogram (8).The chromosomes have been double stained with Hoechst 33258 512A
(A-T binding preference), excited with 351 363 nm wavelength light, and chromomycin A-3 (G-C binding preference), which is excited at 458 nm. One illustration of the value of the double stain procedure is the resolution of the peaks corresponding to chromosomes 14,15,16, and 17. They appear at the same chromomycin brightness, so this stain alone would not resolve them. However, they are separated along the Hoechst axis. With Hoechst alone, chromosome peaks 16 and 18 would not he distinguished. These measurements also yield information on the dye-DNA interaction and some details of the base sequences of the chromosomes.
+
The differences in ratios of the two fluorescence signals for each chromosome are greater than the differences in their AT-GC ratios and thus enhance the separation of the peaks (9). Two factors contribute to this enhancement. First it appears that Hoechst binds preferentially to three sequential A-T base pairs while chromomycin has its highest affinity for G-C triplets. The probability of finding such binding sites changes nonlinearly with changes in the average base composition of the UNA, and this en. aggerates the changes in the two fluorescence signals. In addition, the dye concentration on the chromosomes is sufficiently high 80 that some Hoechst (mntinued on page 517 A )
Flgure 6. Slit scanning of chromosomes photographa and colresponding tiow scans of two chromomes: nwmai (rigM) and sbnOrmal (iefl)
ANALYTICAL CHEMISTRY, VOL. 54, NO. 3. MARCH 1982
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TORBAL Principle The fundamental parts of the EA-1 weighing mechanism are shown below. Instead of the pointer and fixed index as used in a conventional balance, the EA-1 uses a sensor called an The E-core, through an amplifier, senses the rotation of the beam and produces a meter deflection proportional to this deflection. All the weighing operations on the EA-1 are performed to return the meter needle to the original point of equilibrium or zero.
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and chromomycin molecules are close enough for the Hoechst excited state to transfer its energy to the chromomycin before it can be radiated. Variations in energy transfer efficiency among chromosome types also enhance observed fluorescence differences. The measurements just discussed dealt with properties characteristic of the whole chromosome. Other techniques allow measurement of some aspects of chromosome morphology. Using a narrowly focused excitation beam, the distribution of dye along a chromosome can be scanned as it flows past and the pulse profile analyzed ( 1 0 , l l ) .Measurements with scanning microscope systems have shown that there is less DNA content per unit length at the centromere than at other points. Figure 6 shows the profiles of two chromosomes obtained in flow, and the centromeric dips are clearly visible. The time for the scan is about 1 ps, approximately 50 points 20 ns apart being sampled for a typical 10-pm-long chromosome. The centromere can be resolved in chromosomes as small as 3.5 pm in length. On the right is a normal chromosome with a single centromere. On the left is a chromosome from a cell exposed to X-rays. An error was made in rejoining breaks caused by the radiation, and the result is a dicentric chromosome with two centromeres. Chromosome aberrations, like the dicentrics, can be used as Diological dosimeters, since their frequency of occurrence depends on the dose received. This is particularly useful in cases of inadvertent human exposure, where no other dosimeters are present. Searching for aberrations visually is time-consuming, since they are rare for low exposures. The high analysis rates possible in flow may find application in speeding this process. Conclusion Among the additional applications of flow cytometry are blood analysis, bacterial classification, tumor classification and diagnosis, monitoring of cancer therapy, and immunological studies. All of these exploit the high speed and accuracy of the technique, which allow collection of information on a sufficient number of individual particles to describe the distribution of characteristics in a population with good statistical precision. Many require the ability to sort subpopulations for further study or use. Applications outside of biology are waiting to be explored. References (1) Herzenberg, L,. A.; Sweet, R. G.; Herzenberg, L. A. Scz Am 1976,234 (3),
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(2) norm, Y . K.; Wheeleas, L.L. Science 1977,198,149.
(3) AmdMovan, D. J.; Jonn, T. M. In “Annual Review of Biophysics and Bioengineering”; Mullins, L. J.; Hagins, W. A,; Newton, C.; Weber, G.,Eds.; Annual Review Inc.: Palo Alto, Calif,, 1978;
Vnl . ~ .7... (4) Melamed, M. R.; Mullaney, P. F.;Men-
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delsahn, M. L., Eds. “Flow Cytornetry and S o r t i n p John Wiley and Sons: New York, Chic eater, Brishane, and Toronto, 1979. (5) Gray, J. W.; Carrano, A. V.; Steinmetz, L. L.; Van Dilla, M. A,; Moore, D. H. 11; Mayall, B. H.; Mendelsohn, M. L. Proc. Nat. Acod. Sei. USA 1975.72 (4). 1231. (6) Sillar, R.; Young, B. J. Histochem. Cy-
tochem. 1981,29,74.
(7) Lebo, R. V.; Carrano, A. V.; B u r k h a h
Schultz,K.;Dozy.A. M.;Yu,L.-C.;Kan, Y. W. Proc. Not. Acad. Sei. USA 1979,
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V.; Burkhart-Schultz. K.; Van Dilla, M. A. Chromosoma 1979,73,9. (9) Langlois, R.; Carrano, A. V.; Gray, J. W.; Van Dilla, M. A. Chromosoma 1980,77,229. (10) Gray, J. W.; Peters, D.;MerriII,
J.T.; Martin, R.; Van Dilla, M. A. J. Histochem. Cytochem. 1979.27 (I),441.
(11) Cram, L. S.;ArndMovin, D. J.; Grim-
wade, B.; Jovin, T. M. J. Histoehem. Cv-
toehem. 1979,27,445.
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Dan Pinkel received his BA degree in physics from the University of Michigan, and MS and PhD degrees in solid-state physics from the Universit y of California at San Diego. After a postdoc at UCLA in medical physics, where he first came into contact with flow cytometry, he went to his present position in the Cytophysics Section of the Biomedical Sciences Division at Lawrence Livermore National Laboratory. Current research interests include instrumentation development and high-precision measurements on mammalian sperm.
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