Instrumentation
Dan Pinkel Lawrence Livermore 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
Sample • Sheath Fluid
Pulse Profile
Pulse Area Pulse Width
Flow Chamber
Time
Laser Beam Optical Detectors
Signal Property
Figure 1. Flow cytometric instrumentation Vibrating the flow chamber causes the liquid jet to break up into regular drops. Electrical charging of the appropriate drops allows sorting of individual particles based on their measured parameters
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ANALYTICAL CHEMISTRY, VOL. 54, NO. 3, MARCH 1982 • 503 A
(ο)
(a)
(b) Gi Phase ! S ; Phase j G2 and M Phases
Cell Cycle Phase
G2and M
DNA Content
DNA Content
Figure 2. The cell cycle (a) DNA content of a cell doubles before it divides, (b) Distribution of DNA content of a population of cells randomly spaced through the division cycle, (c) Com puter analysis of flow cytometric measurements allows quantification of the fraction of cells in the various stages of the cycle. The dashed line separates the contribution of S-phase cells from those of the other phases
in this area, and its more than 70 con tributors indicate the scope of the in ternational effort. Many of the articles related to new developments in flow instrumentation and applications are published in the journal Cytometry. Instrumentation 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 es tablishing a sheath of nonturbulent fluid centered around a small-diame ter sample injection tube (Figure 1). The sample stream exits from the in jection tube, the stream diameter de creasing further as the sample passes through the nozzle of the flow cham ber. In typical instruments, the sheath exit orifice is on the order of 200 μπι in diameter while the sample stream (core) diameter is several microme ters. The use of "hydrodynamic focus ing" avoids the problems of clogging and velocity gradients across the sam ple stream that would occur if control of the particle trajectories were at tempted 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 atmo sphere pressure. The Reynolds num ber 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 di rection 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 with
stand passage through the instrument and be viable for further study. CW lasers with outputs ranging from milliwatts to a few watts in a sin gle line are usually used for cell illumi nation, although mercury lamps also work well. To maximize illumination of the particles, the light source is fo cused 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 illumi nation 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 uni versally used for fluorescence detec tion, and photodiodes are suitable for the larger signals of forward scatter ing. Illustrated in the upper right of Figure 1 is the hypothetical output of a detector as a function of time for the passage of a single object through the beam, which typically takes 1 0 - 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 fluo rescence intensity is given by the con volution of the laser beam intensity profile with the distribution of stain along the particle. Characteristics of the pulse that might be measured are
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area, width, height, and details of the profile. The desired pulse parameter is digitized and measured by a multi channel analyzer. The data is dis played as a histogram that shows the number of particles vs. the value of the measured parameter. Two differ ent-sized particles are shown in the sample stream in Figure 1. Each popu lation is responsible for one of the peaks in the resulting histogram. Individual objects can be sorted based on the measurements. Mechani cally, 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 regu lar droplets as illustrated in Figure 1. Typical droplet formation frequencies are in the tens of kHz range with jet diameters under 100 μπι. If, on the basis of the cytometric measurements, a cell meets preset criteria, a voltage is applied to the sheath (which is a con ducting 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 microsec onds 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 con ductivity of the flow chamber orifice to be measured. Developed by Wallace
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Coulter, the de 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 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 at specific points. One cell constituent that can be 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 cont e n t is plotted as a function of time
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D N A Distribution
Cell Cycle Distribution Oh
Gi
G2/M
2.9h
Gi
G2/M 5.7 h
Gi
G2/M
Figure 3. Computer simulation of a synchronized cell population The distribution of cells through the cell cycle is reflected in the DNA content distribution
during one cycle of division. The initial period with l x DNA content is called Growthi (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 Growtli2 (G2) 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. T h e distribution of DNA cont e n t of a population of cells t h a t 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. T h e 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 be 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 fluorescence distribution is a drawing of the corresponding chromosome type
The action of drugs that perturb the cycling of cells can be monitored flow cytometrically 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 Gi phase, 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 same 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
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example, are blocked and then released at the same time, there may be a later time at which the ratio of the killing of the tumor to normal cells will he 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 chromosome type if the resolution is high enough. Peak widths below a 2% coefficient of variation are obtainable on chromosomes with 1 0 - 1 3 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
Figure 5. Dual parameter analysis of human chromosomes Perspective view and corresponding contour plot of the fluorescence distribution of the smaller human chromosomes, numbers 9 to 22 and Y
of the distributions obtained from dif ferent individuals to understand nor mal variations and those that may be characteristic of genetic diseases. While most Chinese hamster chro mosomes 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 homo geneous molecule, and some of its more subtle properties can be exploit ed to improve the discrimination. The genetic information in DNA is encoded in the sequence of its constituent bases, A (adenine), Τ (thymine), G (guanine), and C (cytosine), which al ways occur in pairs, A associated with Τ and G with C. The ratio of A-T to G-C pairs in each chromosome is close to 1.5, but slight differences occur. These differences can be detected by using two dyes, one of which preferen tially 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 usu ally overlap in the emissions, so some light from each of the dyes must be fil tered out to discriminate between the two signals. This is undesirable, since in many cases the measurement preci sion is limited by photon statistics. It is better to use dyes with differing ex citation bands, which requires two ex citation 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 histo gram (8). The chromosomes have been double stained with Hoechst 33258
(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 reso lution 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 be distin guished. These measurements also yield information on the dye-DNA in teraction and some details of the base sequences of the chromosomes.
Dicentric
The differences in ratios of the two fluorescence signals for each chromo some are greater than the differences in their AT-GC ratios and thus en hance the separation of the peaks (9). Two factors contribute to this en hancement. First it appears that Hoechst binds preferentially to three sequential A-T base pairs while chro momycin has its highest affinity for G-C triplets. The probability of find ing such binding sites changes nonlinearly with changes in the average base composition of the DNA, and this ex aggerates the changes in the two fluo rescence signals. In addition, the dye concentration on the chromosomes is sufficiently high so that some Hoechst (continued on page 517 A)
Metacentric
Figure 6. Slit scanning of chromosomes Photographs and corresponding flow scans of two chromosomes; normal (right) and abnormal (left)
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and chromomycin molecules are close enough for the Hoechst excited state to transfer its energy to the chrom omycin before it can be radiated. Vari ations in energy transfer efficiency among chromosome types also en hance observed fluorescence differ ences. The measurements just discussed dealt with properties characteristic of the whole chromosome. Other tech niques allow measurement of some as pects 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 ana lyzed {10,11). 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 μβ, approximately 50 points 20 ns apart being sampled for a typical 10-jum-long chromosome. The centro mere can be resolved in chromosomes as small as 3.5 μια 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 rejoin ing breaks caused by the radiation, and the result is a dicentric chromo some with two centromeres. Chromo some aberrations, like the dicentrics, can be used as biological dosimeters, since their frequency of occurrence de pends on the dose received. This is particularly useful in cases of inadver tent human exposure, where no other dosimeters are present. Searching for aberrations visually is time-consum ing, since they are rare for low expo sures. 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 classifi cation 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 re quire the ability to sort subpopula tions for further study or use. Applica tions outside of biology are waiting to be explored. References (1) Herzenberg, L. Α.; Sweet, R. G.; Herzenberg, L. A. Sci. Am. 1976, 234 (3), 108. (continued on page 519 A)
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(2) Horan, P . Κ.; Wheeless, L. L. Science 1977,198, 149. (3) Arndt-Jovan, D. J.; Jovin, T. M. In "Annual Review of Biophysics and Bioengineering"; Mullins, L. J.; Hagins, W. Α.; Newton, C ; Weber, G., Eds.; An nual Review Inc.: Palo Alto, Calif., 1978; Vol. 7. (4) Melamed, M. R.; Mullaney, P. F.; Men delsohn, M. L., Eds. "Flow Cytometry and Sorting"; John Wiley and Sons: New York, Chichester, Brisbane, and Toron to, 1979. (5) Gray, J. W.; Carrano, A. V.; Steinmetz, L. L.; Van Dilla, Μ. Α.; Moore, D. H. II; Mayall, B. H.; Mendelsohn, M. L. Proc. Nat. Acad. Sci. USA 1975, 72 (4), 1231. (6) Sillar, R.; Young, B. J. Histochem. Cytochem. 1981,29,74. (7) Lebo, R. V.; Carrano, A. V.; B u r k h a r t Schultz, K.; Dozy, A. M.; Yu, L.-C; Kan, Y. W. Proc. Nat. Acad. Sci. USA 1979, 76 (11), 5804. (8) Gray, J. W.; Langlois, R.; Carrano, A. 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.; Merrill, J. T.; Martin, R.; Van Dilla, M. A. J. Histo chem. Cytochem. 1979,27 (1), 441. (11) Cram, L. S.; Arndt-Jovin, D. J.; Grimwade, B.; Jovin, T. M. J. Histochem. Cy tochem. 1979,27, 445.
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Dan Pinkel received his Β A degree in physics from the University of Michi gan, and MS and PhD degrees in solid-state physics from the Universi ty 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 Na tional Laboratory. Current research interests include instrumentation de velopment and high-precision mea surements on mammalian sperm.
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