Jaromir Ryzicka and Walter Lindberg Department of Chemistry, BG-10 University of Washington Seattle, WA 98195
Many advances in understanding cell structure and function have been made possible by the availability of progressively refined instrumental techniques. This process, which started with t h e invention of the compound microscope by Zacharias Jansen in 1590, later enabled Antony van Leeuwenhoek to describe living cells. Thus began a n e r a during which generations of famous “microbe hunters’’ ( I ) laid the foundations of modern cell biology. The tremendous advances in understanding cell morphology and physiology have been further enhanced by quantitative imaging microscopy and electron microscopy. Today, t h e use of fluorescence spectroscopic techniques to investigate chemical and morphological details of cellular processes allows us to address fundamental problems in the life sciences. In this area, three principal technologies are merging: flu0 rescent probe chemistry, which is be ing developed; video fluorescence microscopy; a n d flow cytometry. Such a combination of techniques provides a powerful means of probing cellular processes. However, t h i s merger of refined i n s t r u m e n t a l methods with advanced solution chemistry has a serious flaw: Insufficient provision is made to control the kinetics of interaction between the 0003-2700/92/0364 -537A/$02.50/0 0 1992 American Chemical Society
cells and the stimulating reagent. Because life processes are dynamic, the information thus obtained is incomplete and perhaps even, in some cases, misleading. We believe that the interactions of probes and stimulants with cells and their subcomponents can be controlled by the flow injection (FI) technique while being observed and quantified by fluorescence spectroscopy. In this REPORT, we will review the pivotal role of fluorescent probes in cytochemical analysis. Two common instrumental techniques used to study living cells, fluorescence microscopy and flow cytometry, will be discussed, along with two novel
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methods for extending the capabili ties of these techniques: flow injection fluorescence microscopy (FIFM) and flow injection cytometry (FIC).
Fluorescent probes Determining the location of molecules in the cellular and subcellular structures and assaying them quantitatively are two important goals of modern cytochemical analysis. At tainment of these goals offers insight into the complex processes of metabolism, the regulation and control of materials essential for cell function. Because the molecules of interest (e.g., carbohydrates, lipids, nucleic acids, and proteins) are optically invisible, they must be derivatized so that they can be observed. When ob-
servations were made by transmission microscopy, color -forming r e agents were developed and used for cell staining (e.g., the Feulgen reaction for detection of DNA). Within the past 10 years, the availability of fluorescent probes has made a dramatic impact on the methods of cytoanalysis. These probes have enabled researchers to use fluorescence imaging microscopy and flow cytometry to selectively localize and quantify minute amounts of essential molecules (2-4). All probes have two areas of functionality: One region of the probe binds selectively to the target; the other has a conjugated aromatic structure that is fluorescent. These regions may be distinctly separate in the molecule, or they may be coupled. The interaction of the selective binding region with its target can affect the fluorescence by enhancing it or changing its spectral properties. In the case of fluorescent antibodies, there is no effect on the fluorescence, a n d t h e unbound probe m u s t be physically separated from the cells before a measurement can be made. Currently available probes associate noncovalently with cell structures containing RNA, DNA, redox pairs, H’, Ca2+, K+, and lipids. Propidium iodide, DAPI (4‘,6-diamidino2-phenylindole HCl), and Hoechst Dye 33342 a r e three examples of probes that bind selectively to nucleic acids. They are frequently used for cancer diagnosis by flow cytometry because they allow for the determination of the number of chromosome sets in a cell (cell ploidy). Because
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REPORT propidium iodide and DAPI cannot cross the membranes of living cells, a detergent or an organic solvent is necessary for membrane permeation in the staining process. In contrast, Hoechst Dye 33342 crosses the membrane and thus allows analysis and sorting of living cells based on their DNA content and cell-cycle position (5-7). Cell staining is a highly dynamic process, particularly in living cells (8). An instrument that facilitates control of the DNA staining process would allow improved differentiation of cell subpopulations and cells in different stages of growth. Transmembrane distribution of cationic dyes (e.g., cyanine) or a n ionic dyes (e.g., oxonol) allows estimation of transmembrane potentials or localization of electrically charged components or structures within the cell interior (5, 9, 10). Depolarization of the cell membrane causes the dyes to leak out; healthy cells with a n intact membrane retain the dye or leak it out a t a much slower rate than dead cells. A rough estimate of the membrane functionality can be obtained by manual staining; deeper insight into the kinetics of membrane transport requires better control of the contact time between the probe and the cell surface. Because a potential difference across the cell membrane is involved in the transport of material in and out of the cell, a more accurate measurement of changes in the potential difference during cell stimulation is of great interest to cell biologists. Within the cell itself, the difference in electrical potential and the pH gradient within the mitochondria is coupled to the energy release during electron transport within the ATP/ ADP cycle-a highly dynamic process. External stimulation of cells affects cell autofluorescence and the fluorescence of internalized molecular probes, but very likely a t different rates. This temporal difference in fluorescence emission, if properly induced and observed, may lead to elucidation of the physiological changes that cause it. Cation probes, which change fluorescence a s a result of changes in ionic activities (e.g., pH, pK, pNa, and especially pCa), are widely used, and their selectivity and spectral properties a r e continuously being improved ( 5 , 6, 10). Remarkable progress has been made over the past 10 years, especially in the design of Ca ion probes with improved characteristics. This progress is due mainly to researchers’ intense interest in measuring intracellular calcium lev-
els, because calcium appears to provide a major pathway of communication in the cell. To measure changes of cytoplasmic calcium ion activity, the probe must cross the cell membrane. Loading the cells with dye is commonly achieved by incubating them with the acetoxymethyl ester of the dye, which is a membrane permeant. After the ester-dye complex crosses t h e membrane, t h e ester linkage is cleaved by cellular esterases, and the active probe is liberated. The charged probe molecule remains in the cytoplasm where it can complex with calcium. Two commonly used calcium probes are FURA-2 and INDO-1 (11, 12). The chelating group of both of these reagents is EGTA, but their a r omatic conjugated structures differ, and these differences yield characteristic fluorescence properties (5, 10, 1 1 ) . Although t h e s e probes a r e widely used in conjunction with flow cytometry and fluorescence microscopy, their application for systematic study of cell functions is far from trivial. As we have noted, loading probes into the cells is a complex process that depends on factors such as cell/ dye ratio, contact time, temperature, membrane permeability, and cell viability. Because all four carboxylate groups of the probe must be released by cellular enzymes, kinetic factors and the concentration of reacting species within the cytoplasm further complicate the process. Thus, obtaining cells uniformly loaded with an activated probe is difficult, and estimating the active fraction of the probe is arduous. Also, if the cells are overloaded, the high concentration of the fluorescent probe will affect cell function (13). Enzyme s u b s t r a t e s have been made fluorescent by labeling them with fluorescent tags through covalent binding. Isothiocyanates of fluorescein, eosin, or rhodamine are, along with chlorotriazinyl derivatives, the most frequently used derivatives that allow covalent binding of the fluorotag to primary amino groups of proteins (5, 12). Assays of proteases, e s t e r a s e s , a n d phosphatases have been accomplished by measuring an increase in the fluorescence of a substrate caused by enzymatic cleavage of an amide bond between the tag and the substrate ( 4 ) . Although assays of extracellular enzymatic activity are now routine, estimation of intracellular enzymatic activity is difficult. Antibodies, lectins, hormones, macromolecules, and lipids have also
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been tagged with fluorescent groups (5).Of these, antibodies are the most important ligands used in cytoanalysis because of the specificity of their binding to antigens. This specificity is the reason immunocytochemistry has emerged within the past 10 years as an important technique for locating and quantifying cellular constituents. Although the selectivity of antibodies is high, cross-reactions are known to occur, and the result is a decrease in morphological as well as quantitative information. Such nonspecific binding may occur at a different rate, and thus better control of the binding process may yield higher selectivity and more precise localization of cellular constituents. Stimulants of cell functions (e.g., growth hormones) can be observed and quantified indirectly by triggeri n g i n t r a c e l l u l a r processes. The availability of reversible calcium probes such as FURA-2 and INDO- 1 allows identification of these stimulants because resting cells loaded with a fluorescent probe exhibit low fluorescence resulting from low intracellular calcium activity (pCa is less than 7 for resting cells). If an active receptor coupled to the calcium signaling system binds a stimulant, intracellular processes will be triggered, calcium ions will be released from the inner reservoirs (lowering the pCa = 51, and the spectral properties of the fluorescent probe will change. Through the use of quantitative imaging microscopy, such measurements are now routinely performed, even on a single cell (13). State of the art of cytochemical analysis These examples represent only a fraction of the many possibilities that fluorescent probe techniques offer for analysis of cell chemistry, structure, function, cycle, and health (2, 12-1 7). Recent literature shows that predominantly static methods of cytochemical analysis (e.g., equilibrium cell staining) are rapidly being abandoned for kinetic methods, which yield temporal information that reflects active processes in living organisms. The major obstacles to such methods can be divided into two categories: cell population heterogeneity (stemming from differences in origin, genetic variations, condition of growth, and age) and the irreproducibility and imperfection of experimental conditions under which kinetic studies of cells take place. The problems posed by the irreproducibility of experimental conditions, which result from lack of kinetic con-
trol of solution handling, may be addressed. At present, the lack of reproducibility with many manual techniques of cell preparation and solution handling limits the full use of sophisticated spectroscopic instrumentation used for measurement of cell properties. By introducing two important principles of flow injection, controlled mixing and reproducible timing, using instrumentation under computer control, the interaction of the reacting components can be carried out under strictly repeatable conditions (18).Some of the inherent difficulties of cytochemical studies, which are caused by heterogeneity of cell populations, can be minimized by introducing the principles of FI to fluorescence microscopy of adherent cells.
1 mL/min perfuses the cells, and the fluorescence is continuously moni tored. The reagent zone, which typically has a volume of 20 pL, is injected upstream by a valve and passes rapidly through the chamber, thus allowing a well-defined contact in terms of time and concentration to occur between the cells and the injected reagent. In this way, a selected group of cells (or even a single cell) can repeatedly be exposed to a predetermined concentration of a stimulant with a high degree of reproducibility over a well - defined period of time. An FI apparatus suitable for this purpose comprises a pump, an injection or selector valve, and a computer with appropriate software (18, 19). This setup allows precise synchronization of the valve and pump action and recording of the fluorescence signal from a photomultiplier tube attached to the microscope. These components can be arranged in either the classical FI configuration (Figure 2a) or the sequential injection (SI) mode (Figure 2b). The classical FI
Flow injection fluorescence microscopy The principle of the FIFM technique is to expose cells to a well-defined zone of a reagent while the cells are observed under a microscope. The reagent may be either a fluorescent probe that is permanently taken up by the cell (Figures la, lb, and IC)or a stimulant that triggers a cell reaction and results in transient fluorescence (Figures l a , lb, and Id). In the simplest case, adherent cells grown on a coverslip are mounted in a perfusion chamber with a well-defined straight flow path, 2 mm wide and 0.2 mm deep, formed by a spacer sandwiched between two parallel planar coverslips. A carrier stream propelled by the pump at a rate of
Figure 2. FIFM apparatus.
1
Figure 1. Principle of flow injection fluorescence. (a) Adherent cells illuminated by excitation light prior to arrival of reagent zone exhibit a low (background) fluorescence. (b) The fluorescent probe (or a stimulant) contained within the injected zone causes cells to fluoresce intensely. (c) The emission of intense fluorescence continues because the fluorescent probe has been trapped within the cells or (d) Removing the zone of stimulant causes the cells to return to the resting state with a decrease in fluorescence.
(a) In the classical FI configuration, reagent (R) is initially aspirated into the valve loop and, following switching of the valve, injected as a zone by the forward movement of the carrier stream (C) into the perfusion chamber, where it comes into contact with the adherent cells. The cells are continuously monitored by a fluorescence microscope equipped with a detector (D) or by a video imaging system. (b) The sequential injection system operates with a directional valve and pump capable of a precise reverse or forward movement of the carrier stream (C). Reverse pump motion aspirates a selected volume of reagent ( R l ) into a holding conduit (HC) and, following switching of the valve, the reagent zone is injected by a forward motion of the pump through the transfer line (TL) into the perfusion chamber. W is the waste container into which the reagent zone is ultimately discharged, AW is the auxiliary waste container, and R2 and R3 are optional ports for additional reagents.
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REPORT technique has the advantage of allowing continuous perfusion of the cells with buffer even when no analysis is performed. With FI, however, using more than one reagent is difficult. In contrast, the SI technique allows greater flexibility in selecting the number, amount, and sequence of reagents perfusing the cells at the expense of providing continuous per fusion. The FI apparatus can be used to stain cells “digitally”; t h a t is, the cells are repeatedly stained by a zone of a fixed profile as outlined in Figures l a , lb, and IC.The dye is retained in the cells and continues to fluoresce after the zone has passed through. By changing the contact time and dye concentration, it is possible to determine the degree of cell loading, kinetics of uptake and, with video microscopy, differentiation of cells in the population. A repeated injection of the same reagent (Figure 3a) results in incremental staining, thus allowing a choice of optimal loading conditions without running the risk of cell poisoning. If the flow is stopped while the reagent zone is in contact with
the cells, the temporal increase of the signal reflects the rate of staining (Figure 3b). An example of this technique is the staining of baby hamster kidney (BHK) cells by DAPI, which binds to DNA in the cell nucleus (2,5). By using the FI system shown in Figure 2a, adherent BHK cells were repeatedly exposed to a zone obtained by injecting 25 yL of a 10 pg/mL solution of DAPI in tris-buffered saline containing Nonidet -40 detergent, into a continuously flowing carrier stream of phosphate- buffered saline (PBS). Because the reaction of DAPI with DNA results in a 40-fold fluorescence enhancement, the intensity of the fluorescence emitted by the stained cells increases dramatically. This increase is seen on the resulting FI fluorograms (Figures 4a and 4b) obtained by continuous monitor ing of blue fluorescence excited at 365 nm. The amount of DAPI retained by the cells can be evaluated by comparing the baseline before and after the zone has passed through the perfusion chamber. As would be expected, each repeated injection of DAPI increases
the amount of retained dye, which causes an elevation of the baseline. Two flow modes can be used: continuous flow (Figure 4a) and stopped flow (Figure 4b) (20). Even in the continuous-flow mode, when the cells are in contact with DAPI for a brief (4 s) period of time, a considerable amount of dye is taken up and retained, yet the cell capacity allows more dye to be taken up during each subsequent zone passage. The dye loss resulting from leaching or photobleaching is mini mal. In a stopped-flow experiment the rate of dye uptake can be followed continuously d u r i n g t h e stopped-flow period when the dye uptake is enhanced by an increased contact time. Interestingly, there is a noticeable difference in the shape of the dye uptake curves for stoppedand continuous-flow mode staining, which indicates different mechanisms of DAPI uptake. Analysis of these rate curves is likely to reveal the sequence and number of rate - de termining steps involved in this staining process. Stimulation of cells can be performed by the sequence outlined in Figures l a , lb, and Id. Cells adhered to a coverslip and preloaded with a fluorescent probe are activated by a
Tir
Figure 3. Typical FI fluorograms.
Figure 4. FI fluorograms of BHK cells.
(a) Repetitive cell staining in the continuous-flow mode, whereby a fluorescent probe becomes trapped within the cells; (b) cell staining in a stopped-flow mode, showing staining rate and an elevated baseline resulting from the probe entrapment; (c) transient stimulation of adherent cells in the continuous-flow mode, with no hysteresis; and (d) transient stimulation of adherent cells in a stopped-flow mode, showing rate of stimulation and return to baseline in absence of hysteresis. The start of the experiment as initiated by zone injection is denoted by I.
Incremental staining by DAPI is monitored by blue fluorescence. (a) Results for 15 consecutive injections in the continuous-flow mode. (b) Results for nine consecutive injections in the stopped-flow mode programmed to allow sustained contact of cells with stimulant for 30 s.
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zone of stimulant. FURA-2, a frequently used probe, has a specific emission spectrum at the resting calcium concentration in the cells (Figure la). Contact with a zone of stimulant triggers a marked change in the fluorescence (Figure lb), which decays after the zone has been carried away (Figure Id). By using the FIFM format, such stimulation can be achieved in either the continuous(Figure 3c) or stopped-flow mode (Figure 3d). The continuous-flow mode allows only a rough estimate of receptor kinetics by comparing the width of the response peak to the width of the stimulant zone (shaded area, Figure 3). The stopped-flow mode allows the stimulation rate to be continuously monitored and evaluated. If required, very narrow zones (< 0.5 s) of stimulant can be generated by flow reversal in an SI apparatus. Figure 5 illustrates the response of FURA-2-loaded BHK cells to ionomycin. The cells were perfused with HEPES - buffered balanced saline so lution (pH 7.4), enriched with 5 mM glucose and 1mM CaCl,, a t a flow rate of 0.5 mL/min; the injected zone was 2.5 pM ionomycin. Ionomycin transports calcium across the cell membrane and allows its release from intracellular stores (10,13-17). The response of five individual cells selected from the video image (Figure 5a) and integrated by the computer was plotted as a function of time (Figure 5b). Before injection, the fluorescence signal corresponded to a resting calcium level in the cells. On contact with the injected zone, a rapid increase in fluorescence occurred as the calcium level in the cytoplasm rose sharply. This rise in calcium level was sustained during t h e stopped-flow period and decreased soon after the zone of ionomycin was washed out by the carrier stream. A second injection of a zone with a n identical Ca2+/ionomycin content resulted in a lower response. A third injection showed the same result as the second injection. This technique allows the cells to recover from stimulation, which is not possible in a batch-mode experiment. FIFM versus conventional fluorescence microscopy Most staining and fluorescent probing of cells is done manually in a Petri dish or in small wells, with reagents added manually. The need for improved solution handling has led to numerous perfusion chamber designs. These chambers are typically fed by gravity from a hanging re-
agent bag. The more recent perfusion chambers are ingenious pieces of engineering (21-23), and the design is typically optimized for maximum viewing area. These chamber geometries, often circular, allow the formation of eddies, which results in poor washout. Only a few perfusion chambers are designed for streamlined, nondispersed flow (22, 23), a necessary condition for FI applications. Even the most advanced perfusion studies described thus far have been executed in a stepwise mode, simply by switching from a buffer to a stimulant solution (22, 23). Although this approach allows for the study of kinetics at the leading edge of the stepwise concentration change, this is typically not done. In addition, no
Figure 5. Response of five individual cells to ionomycin. (a) Video image of adherent BHK cells loaded with FURA-2. The ratioed spectra reflect cell fluorescence and changes in intracellular calcium activity. The cells selected for continuous monitoring are boxed in. (b) Five traces corresponding to the selected cells in (a) showing baseline fluorescence at the beginning of cell perfusion, a sharp rise during the passage of the stimulant zone, decrease in fluorescence approaching the baseline when the Ca2+/ionomycinzone had been flushed away, and an increase followed by a decrease of fluorescence as the second and third injected zones of stimulant passed across the cells. A 30-sstopped-flow period was programmed to allow sustained contact of the cells with the Ca*+/ionomycin zone. The times of injection are indicated by arrows, and the stopped-flow periods are marked with horizontal bars.
provision has been made to study cell recovery because in the step-flow mode the cells are continuously perfused with the stimulant or dye. In contrast, the impulse-response mode of FI allows the contact time and reagent concentration to be selected at will. Because FI is based on the injection of a well-defined zone of a reagent and the controlled motion of that reagent by forward or reverse pump action, this technique permits unique control of cell loading or repetitive stimulation of cells. Contact times ranging from a fraction of a second to as long as several hours are achievable. The cells may also be stimulated repeatedly with increasing, decreasing, or constant levels of reagents or stimulants, all under strict computer control. If the system is configured in the SI mode, cells can be targeted by several zones of different solutions in a preprogrammed sequence under computer control. In addition to automation, a rigorous protocol of reproducible cell treatments eliminates imprecision resulting from manual operations. Rates of physical processes (diffusion or membrane perfusion) and chemical reactions (enzymatic activity) can be studied by using the stopped-flow technique. At present, the following processes are possible: controlled loading of cells with a well-defined quantity of fluorescent probe measurement of the rate of cellular uptake of a probe repeated treatment of the same group of adherent cells with a compound in a highly reproducible manner stimulation of individual cells by a series of well-defined zones Repeated stimulation of a group of cells or a n individual cell without the risk of overloading or poisoning is unique. This technique circumvents the problem of biological variability of cells, which makes “one-shot” experiments difficult to compare. Indeed, the repeatability of stimulation, coupled with the high reproducibility of t h e obtained t r a n s i e n t signals and stable baselines, allows corrections to be made for unwanted experimental variables such as the slow leakage of fluorescent probe from the cells, photobleaching, and variability of cell loading. Because “the strongest case for quantitative imaging of cells can be made when changes of a particular parameter can be measured over time as a function of stimulation” (13), we believe that FIFM may be the missing link
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REPORT for making optical imaging techniques useful i n cell biology. The ability to monitor cell behavior and distinguish between a reversible and partially reversible recovery should allow a comparison between the impact of different stimulants on cell properties and functions such as calcium pumps, receptor kinetics, and membrane integrity. The use of precisely selected doses of stimulant should make automated evaluation of drugs and stimulants more convenient and reliable. Poration (14) of adherent cells by reagents such as surfactants, ionomycin, or ATP4- using FI is conceivable. Because the concentration and time of contact of the porating reagent determines the size and number of pores, the FI technique would be well suited for this process. Loading of cells with fluorescent probes is limited by membrane permeability. Temporarily porated cells, however, can be loaded with probes or even with charged proteins that would not cross a n uninterrupted lipid membrane. In this way, FIFM may become a well-controlled preparative technique, because sequential automated poration and cell loading could be accomplished not only with better reproducibility than that allowed by the manual approach, but also under continuous monitoring conditions. Flow injection cytometry A flow cytometer is a specialized flow - through spectrometer in which each cell is analyzed individually as it passes through a n optical system. The instrument is designed to yield cell count as well as fluorescence and scatter of individual cells with a fair degree of spectral resolution (3, 24). These results are achieved as cells, loaded with fluorescent probes and carried by a microscopic jet of water, are passed one by one through one or more intense beams of light, which become scattered and excite fluoro phores within the cells. Individual cells produce short flashes of scattered light and fluorescence that are counted and quantified by the instrument. The resulting cytogram shows the distribution of intensities for a given population of cells, which is a measure of the cell function of interest. In contrast to macro- and microfluorometry, which yield flu0 rescence averages collected simulta neously over a number of cells, flow cytometry measures a large number of cells sequentially. Thus flow cytometry can distinguish between subpopulations of cells, such as cells in different phases of growth. 542 A
The flow chambers and optics of flow cytometers a r e remarkable pieces of engineering. Cells are carried through the observation field at a typical flow rate of 50 p l l m i n and at a linear flow velocity of 10 m/s, corresponding to a r a t e of 5000 cells/s. At this flow rate, only 1ps is available for detection of a single 10 -pm cell, which typically contains lo4 or fewer fluorophore molecules. Optical and hydrodynamic focusing in the flow chambers have been the subjects of detailed studies (24-26) that have resulted in sophisticated flow designs whereby the cells enter the optical system precisely aligned within a thin core stream. In contrast, surprisingly little at tention has been paid to handling of the cell suspension before its entry into the nozzle of the instrument. Most frequently, compressed air is used to propel both the core (sample) and the sheath fluid (3,24).Syringeor peristaltic pump- driven systems are rare (27, 28), and although some of these systems allow a stepwise sample injection, no better provision has been made to perform automated sample - handling procedures or t o study the kinetics of staining. In addition to being labor intensive, manual staining can be imprecise, introducing intersample variation that can obscure relevant information. An example of the present state of the art of handling suspended cells is the work by Omann et al. (29),who constructed a sample introduction device that fits the Becton Dickinson FACS cytometer. This attachment is manually operated and consists of a container with three reagent feed lines connected on-line to the core stream of the instrument. Recently Kelley (30) developed a similar attachment for the addition of one reagent, with emphasis on resolution of fast kinetic events, where measurements of events occurring 1 s after reagent addition could be made. Pennings et al. (28)developed a system based on continuous pumping of cells and reagents with a peristaltic pump. In this system, the cell suspension, staining reagent (acridine orange), and a detergent are brought to confluence and pumped to the flow cytometer. Although this design is a definite step toward full automation, it wastes reagent and sample. Because this design allows only a 3-s fixed contact time between sample staining and measurement, it yields only a single point of kinetic information. The principle of flow injection cytometry is shown in Figure 6, and a
ANALYTICAL CHEMISTRY, VOL. 64, NO. 9, MAY 1, 1992
schematic drawing of the apparatus is shown in Figure 7. The heart of the system is a mixing chamber contained in the injection loop of the valve. This interface has two functions (31):to bring together the cell suspension and staining reagent in a reproducible m a n n e r for a well defined period of time and to interface the FI system, for which a typical flow rate is 1-2 mL/min, with the core stream of a cytometer, which flows 20 times slower. In the first stage (Figures 6a and 7a), well-defined volumes of reagent (stain, R1) and sample (cells, S) are sequentially aspirated into a holding conduit (HC) by reverse pump motion. Then, after the selector valve has been switched to the transfer line (TL), the stacked zones are injected into a micromixing chamber by forward pump motion, where rapid mixing of stain and cells is initiated. The mixing chamber is in the loop of a n injector valve, which interfaces the flow injection instrument with the core stream of the cytometer. In the second stage (Figures 6b and 7b), the injector valve is turned, and the carrier transports the homogenized mixture of cells and stain into the cytometer. The apparatus constructed
Figure 6. Principle of flow injection cytometry. (a) Cells, stacked in the transfer line, are injected via a carrier stream into the micromixing chamber filled with staining reagent. (b) The measurement period is initiated when the valve is switched and the stained cells are transported into the cytometer by the core stream.
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for this purpose (Figure 7) operates in the SI mode (19, 32). Fluorescence and appearance time are measured for each cell. The collected data can be displayed in either a three-dimensional (isometric) or a composite plot. A composite flow injection cytogram (Figure 8) has three panels: (a) cell count as a function of time and fluorescence intensity, (b) number of cells versus fluorescence intensity, and (c) cell count versus time. Panels b and c can be viewed as two-dimensional projections of the three-dimensional dot plot seen in a. Panel b shows only a single peak, a result that is consistent with a homogeneous population. A heterogeneous cell population, such a s one containing aneuploid cells (cells having a n abnormal number of chromosomes), would show two or more peaks in this type of plot because the cells containing more DNA would take up more dye and thus fluoresce more intensely. This FI cytogram was obtained by staining a homogeneous population of trout erythrocytes with DAPI (31). To unravel the kinetics of staining, the hydrodynamics of the FI system must be considered. If the cells and the reagent are brought together a t
t h e same time that t h e valve is turned (Figure 6b), cell staining is initiated simultaneously with the start of the chamber outflow, which occurs a t the rate of the core stream flow. Because t h e volume of t h e chamber dominates the flow system, a single mixed-tank model applies, and the material outflow follows an exponential decrease function. The concentration of the chamber material C decreases in time as
C = C,e-kt where k = V / Q (Vis the chamber volume; Q is the flow rate). By defining tl12as the time span during which C decreases by one-half, then tl12= 0.693 V/Q. At some point on the exponential decay curve, the suspension becomes so dilute that the distribution no longer can be accurately determined, a condition limiting the experimental run time. For this experiment, the experimental run time was arbitrarily chosen to be 3t1,,. At this point, only 12.5% of the original cell material remains in the chamber. The choice of Q is limited because most cytometers operate best at flow rates between 25 and 100 pL/ min; the remaining variable is the chamber volume, which should not be larger than 300 pL. In addition to the kinetic control offered by flow injection cytometry, the ratio of reactants is better controlled by this technique than by tra-
ditional manual staining. The SI system allows sampling of the same number of cells from a stirred suspension with good reproducibility. Typically, 6000 cells can be repeatedly injected and counted with an RSD of 5%. Because selected volumes of stain or stimulant can be injected into the mixing chamber as well, kinetic studies of cell interactions with various concentrations of stains and stimulants can be automated. This automation allows optimization of experimental conditions under which the fluorescent probes used do not inhibit the physiological processes under investigation. Conclusion The principal difference between FIFM and FIC is the same as that between classical fluorescence microscopy a n d flow c y t o m e t r y , namely, continuous observation of a single cell or a group of cells versus sequential one - time measurements of a large number of individual cells. Also, FIFM requires adherent cells, and FIC requires individual suspended cells. FIFM might conceivably be used to study suspended cells, if the cells could be temporarily immobilized in the focal plane of the microscope. With the aid of video microscopy, the cells could be differentiated according to their kinetic behavior. Indeed, preliminary experiments with fluorescent particles confirm
I
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through a transfer line (TL) into the micromixing chamber. During the measuring cycle the core stream carries the cells into the flow cytometer.
(a) Dot plot showing cell distribution as a function of fluorescence intensity and time, (b) cell count versus fluorescence intensity, and (c) cell count versus time. The measurement was obtained by on-line staining of trout erythrocytes with DAPI. ANALYTICAL CHEMISTRY, VOL. 64, NO. 9, MAY 1,1992
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REPORT that a heterogeneous zone can be reproducibly stopped in the observation field of a microscope, thus allowing fluorescence measurements. The idea of FIC was conceived three years ago in cooperation with Peter Rabinovitch, and the concept of FIFM was considered a year later. In the year since the project started, the concepts of FIG (32) and FIFM (20) have been verified and developed further. As work continues, novel aspects of the use of FI in cell biology are coming to light. It is hoped that this development will parallel the previous pattern in which FI successfully enhanced many techniques of instrumental analysis (33). We express our gratitude to Peter S. Rabinovitch, Department of Pathology, University of Washington, for stimulating discussions on many aspects of flow cytometry and to Ole Thastrup and Rudi Pedersen, ZymoGenetics, Inc., for their assistance and advice on cell biology and video microscopy. Stimulating discussions with Gary D. Christian and Kurt M. Scudder are also much appreciated. This work was supported by NIH (grant no. SSS-3 (5) R01 GM 45260-2).
References (1) The Microbe Hunters; de k i f , P., Ed.; Harcourt, Brace & World: New York, 1926. (2) Applications of Fluorescence in the Biomedical Sciences; Taylor, D. L.; Wa goner, A. S.; Murphy, R. F.; Lanni, Birge, R. R., Eds.; A. R. Liss: New York, 1986. (3) Flow Cytometry and Sorting, 2nd ed.; Melamed, M. R.; Lindmo, T.; Mendelsohn, M. L., Eds.; Wiley-Liss: New York, 1990. (4) Methods in Cell Biology; Tartakoff, A. M., Ed.; Academic Press: New York, 1989; Vol. 31. (5) Waggoner, A. S. In Applications of Fluorescence in the Biomedical Sciences; Taylor, D. L.; Waggoner, A. S.; Murphy, R. F.; Lanni, F.; Birge, R. R., Eds.; A. R. Liss: New York, 1986; pp. 3-28. (6) Muirhead, K.; Horan, P. K.; Poste, G. Bio/Tech. 1985, 3, 337. (7) Arndt-Jovin, D. J.; Jovin, T. M. In Methods in Cell Biology. Fluorescence Microscopy of Living Cells in Culture; Taylor, D. L.; Wang, Y. L., Eds.; Academic Press: San Diego, 1989; Vol. 30, Part B. (8)Krishan, A. Cytometry 1987, 8, 642-
#.;
45.
(9) Chused, T. M.; Wilson, H. A.; Selimann, B. E.; Tsien, R. In Applications of Fluorescence in the Biomedical Sciences; Taylor, D. L.; Waggoner, A. S.; Murphy, R. F.; Lanni, F.; Birge, R. R., Eds.; A. R. Liss: New York, 1986; pp. 531-44. (10) Rabinovitch, P. S.; June, C. H. In Flow Cytometry and Sorting, 2nd ed.; Melamed, M. R.; Lindmo, T.; Mendelsohn, M. L., Eds.; Wiley-Liss: New York, 1990; pp. 651-68. (11) Grynkiewicz, G.; Poenie, P.; Tsien, R. Y. J. Bzol. Chem. 1985, 260(6), 344050. (12) Haugland, R. P. Molecular Probes: Handbook of Fluorescent Probes and Research Chemicals; Molecular Probes, Inc.: Eugene, OR, 1989.
(13) Gross, D. J. In Modern Cell Biology, Non-Invasive Techniques in Cell Biology; Foskett, K. J.; Grinstein, S., Eds.; Wiley-Liss: New York, 1990; Vol. 9, pp. 21-51. (14) Steinberg, T. H.; Silverstein, S. C. In Methods incell Biology; Tartakoff, A. M., Ed.; Academic Press: New York, 1989; Vol. 31. (15) Modern Cell Biology, Non-Invasive Techniques in Cell Biology; Foskett, K. J.; Grinstein, S., Eds.; Wiley-Liss: New York, 1990; Vol. 9. (16) Methods in Cell Biology. Fluorescence Microscopy of Living Cells in Culture;Wang, Y. L.; Taylor D. L., Eds.; Academic Press: San Diego, 1989; Vol. 29, Part A. (17) Methods in Cell Biology. Fluorescence Microscopy of Living Cells in Culture; Taylor, D. L.; Wang, Y. L., Eds.; Academic Press: San Diego, 1989; Vol. 30, Part B. (18) Ruzicka, J.; Hansen, E. H. Flow Injection Analysis, 2nd ed.; Wiley: New York, 1988. (19) Ruzicka, J.; Marshall, G. D. Ana&. Chim. Acta 1990,237, 329-43. (20) Scudder, K.; Christian, G. D.; Ruzicka, J., unpublished results. (21) Finch, S.A.E.; Stier, A. Microscopy 1988, 151, 71-75. (22) Braga, P. C. Pharmacol. Methods 1989, 22, 1-6. (23) Winzek, C.; Plieninger, P.; Baumgartel, H. Histochemistry 1987, 86, 421-26. (24) Shapiro, H. M. Practical Flow Cytometry, 2nd ed.; A. R. Liss: New York, 1988. (25) Kachel, V.; Fellner-Feldeg, H.; Menke, E. In Flow Cytometry and Sorting, 2nd ed.; Melamed, M. R.; Lindmo, T.;
Mendelsohn, M. L., Eds.; Wiley-Liss: New York, 1990; pp. 27-44. (26) Kachel, V. In Flow Cytometry and Sorting, 2nd ed.; Melamed, M. R.; Lindmo, T.; Mendelsohn, M. L., Eds.; Wiley-Liss: New York, 1990; pp. 45-80. (27) Steen, H. B. In Flow Cytometry and Sorting, 2nd ed.; Melamed, M. R.; Lindmo, T.; Mendelsohn, M. L., Eds.; Wiley-Liss: New York, 1990; pp. 11-25. (28) Pennings, A.; Speth, P.; Wessels, H.; Haanen, C. Cytometry 1987, 8, 335-38. (29) Omann, G. M.; Coppersmith, W.; Finney, D. A.; Sklar, L. A. Cytometry 2985, 6,69-73. (30) Kelley, K. A. Cytometry 1991, 12, 464- 68. (31) Lindberg, W.; Ruzicka, J.; Christian, G . D., unpublished results. (32) Gbeli,. T.; Christian, G. D.; Ruzicka, J. Anal. Chem. 1991,63, 2407-13. (33) Ruzicka, J. Anal. Chim. Acta, in press.
Jaromir Ruzicka is a professor of chemistry at the University of Washington, a member of the Danish Academy of Technical Sciences, and a past president of the
Danish Society of Analytical Chemistry. He is a graduate of Charles Uniuersity^‘of Prague and holds a Ph.D. and a Doctor of Natural Science degree. His research interests include radiochemistry, chemical separations, electro-analytical chemistry, and the automation of instrumental analysis with emphasis on flow injection. The focus of his current research is bioanalysis and, within the fi-amework of the Center for Process Analytical Chemistry, continu ous monitoring of chemical processes.
Walter Lindberg is currently a visiting scientist at the University of Washington in Seattle. His research interests includeflow injection analysis,flow cytometry,and applications of chemometrics in analytical chemistry, He received his B.S. degree in chemistry and his Ph.D. in analytical chemistryfrom the University of Umei in Sweden.
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