Anal. Chem. 1992, 64, 2657-2660
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The Fountain Cell: A Tool for Flow-Based Spectroscopies Kurt M. Scudder, Cy H. Pollema, and Jaromir Ruzicka* Department of Chemistry, BG-IO, University of Washington, Seattle, Washington 98195
INTRODUCTION The design of a flow cell for optical measurements in flowing liquid streams involves a compromise between the flow characteristics of the cell and the detection optics. Desirable fluid-flow properties in a flow cell are low or controlled dispersion, low dead volume, and minimal transitions from one flow cross section to Optical considerations include proper illumination of the sample, sufficient path length, and efficient light collection. Typical flow cells for fluorescence have the further complication that the emitted light must be collected with high optical efficiency a t some angle other than collinear with the excitation light. We have developed a novel flow cell geometry in which the flow characteristics are emphasized. I t should be well suited for reflectance, chemiluminescence, fluorescence by epiillumination, or radiometric measurements since the fluid element forms a thin flat volume which can be physically matched to planar detectors. It has been primarily designed as a chamber for fluorescence microscopy of living cells which would allow rapid, reproducible changes in the media perfusing the cells. In this design (Figures 1and 2), the fluid entering via a central inlet is directed normal to a flat optical surface. The resulting fountain-like flow pattern is forced into a thin space between the optical surface and a parallel rear plate. The fluid ultimately collects in a ring-shaped well which is at all points equidistant from the point of inlet. The fluid-flow advantages to this geometry are numerous. First, the flow becomes spatially resolved in a flat image plane, which can be viewed by an array detector, or placed directly against the sensing area of a detector for luminescence or radiometric measurements. Second, this fluid geometry is ideally suited to microscopy, since it forces the flow into a thin plane which can be evenly illuminated and focused. This allowsthe study of cells either transported by the liquid stream or fixed within the observation area. Third, the cell geometry gives a smooth flow transition from the inlet tubing to the detection region with minimal unswept volume and no wall effects. The absence of wall effects preserves the shape of the injected zone and enables its rapid removal from the flow cell by flow reversal. Finally, the fountain cell has an unusual feature which may be used to advantage: the linear flow velocity varies inversely with the distance from the inlet.
EXPERIMENTAL SECTION The body of the fountain cell (Figure 1) cell is made from a cylinder of Plexiglas or Teflon 30 mm in diameter and 10 mm thick. The top is formed by a 22-mm-round microscope cover slip (no. 1 thickness) which is separated from the cell body by a 0.5-mm Teflon spacer. This sandwich is held together by a ring which is screwed to the cell body. In later versions, the sides of the body were extended to allow the ring to thread into the cell body. The inlet is a 0.5-mm-i.d. tube, and the outlet is a 1.0-mm-i.d. tube which is placed at the bottom of a ring-shaped well. The well has an i.d. of 16mm, an 0.d. of 20 mm, and a depth of 2 mm. The fluid enters through the inlet in the center, radiates outward through the thin flow path defined by the cell body and (1)Ruzicka, J.; Hansen, E. H. Anal. Chim. Acta 1985,173, 3-21. (2) Jeppensen, M. T.; Hansen, E. H. Anal. Chim.Acta 1988,214,147159. (3) Ruzicka, J.; Hansen, E. H. Anal. Chim.Acta 1988,214, 1-27. (4) Pavon, J. L. P.; Gonzalo, E. R.; Christian, G. D.; Ruzicka, J. Anal. Chem. 1992,64, 923-929.
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Flgure 1. Top view and cross section of the fountain cell. The cell consists of a circular Teflon base plate (BP) containingan inlet channel surrounded by a ring-shaped outlet well with a drain. The flow path is formed by a thin circular (0.5") Teflon spacer (S) covered by a cover glass (CG) (22-mm-round1no. 1). The cover glass is secured in place with a ring (R).
Figure 2. The flow pattern within the fountain cell as visualized by the injectionof a zone of dye into the cell. (A, Top) Sample bolus emerges from the central inlet. (B, Middle)A ring-shaped zone radiates outward over the detection area. (C, Bottom) The zone reaches the outer edges of the disk and exits into the outer well.
the cover slip, collects in the ring-shaped well, and exits through the outlet tube. In all experiments described here, the total volume of the thin layer within the disk-shapeddetection region 0 1992 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 64, NO. 21, NOVEMBER 1, 1992
was 100 pL. The fountain cell was mounted horizontally with the inlet coming from below. The microscope system used for flow characterization was a Zeiss Universal with a type 111-RSepi-fluorescenceattachment (Carl Zeiss, Oberkochen, Germany). Excitation light was provided by an HBO 50-W mercury arc source or a 30-W tungsten lamp. The excitation filter was a 450-490-nm bandpass filter. The emission filter was a 520-nm long pass filter. Both 1OX (Zeiss Plan 10/0.22) and 2.5X (Zeiss Plan 2.5/0.08) dry planarapochromatic lenses were used. Single-channelfluorescence signals were measured with a Nikon P1 microscope photometry system (Nikon, Tokyo, Japan). For visual observation and recording of the flow characteristics, a Sony XC-57 CCD camera connected to a HQ VHS videocassette recorder was used. The flow injection systemconsistedof an Alitea C-4Vperistaltic pump (Alitea USA, Medina, WA) and a Valco 10-port injection valve with an electrical acuator (Valco Instrument Co., Inc., Houston, TX) configured as a six-port injection valve. A 50-pL loop volume was used for most experiments. The pump and valve were computer-controlled. Data acquisition and control were performed with a Genesis Systems 80286-based computer (Trinity Technology, Inc., Seattle, WA) equipped with a Real Time Devices ADA1100-2 analog-digital interface card (Real Time Devices,State College,PA). Softwarewas written in-house using MATLAB (The Mathworks, South Natick, MA). The fountain cell was characterized by injecting a bolus of a fluorescent tracer into the carrier stream, which was propelled at a flow rate of 1mL/min. Either 1.0 mg/L fluorescein (Sigma) or Fluoresbrite latex beads (1.5- and 6.0-pm diameter, Polysciences,Inc., Warrington, PA) emitting green fluorescencewere used. Both were prepared in 0.01 M sodium borate (J.T. Baker). The microscopesystem used for observation of living cells was a Nikon Diaphot inverted microscope equipped with a mercury arc lamp and a 40X Nikon Fluar dry objective lens. A computeractuated filter wheel allowed excitation at 340 and 380 nm. Images were collected at 500 nm using a Dage-MTI 72 CCD camera equipped with a Genesis I1 image intensifier. Image acquisition and control were performed on a Sun SPARC I1 workstation running Inovision (Research Triangle Park, NC). The fountain cell used with this system was constructed using a clear Plexiglas base to allow visual observation of the cells by transmitted light. A Toshiba T3100 laptop computer equipped with the same software and interface as the Genesis Systems computer was used to control a sequential injection system which consisted of the same pump described above and a Valco dead stop 10-port selector valve.
RESULTS AND DISCUSSION Initial visual observations of the flow patterns in the fountain cell were made by injecting zones of colored dye into a clear carrier stream. These observations, along with subsequent video imaging, revealed radially symmetric concentration gradients as the dye zone entered the fountain cell (Figure 2). On occasion, asymmetry in the spatial distribution of the zone was observed. This was initially thought to be a result of not having the flow cell in a horizontal position. However, this phenomenon was traced to the position and orientation of the conduit connecting the valve to the cell. Bends in this conduit resulted in radial asymmetry of the leading edge of the dye zone, which translated into distortion of the circular shape of the zone in the detection region. Since knitted reactors are known to randomize radial asymmetry in flowing streams? several tight knots were made in the conduit cloae to the entrance to the cell. This eliminated the sensitivity of the circular pattern to the position of the conduit. The orientation of the cell itself had no observable effect on the radial symmetry of the zone. Next, the dispersion characteristics of the fountain cell were studied by measuring the concentration profile of a constant injected volume (50pL)of fluorescentdye a t discrete (5) Ruzicka, J.; Hansen, E. H. Flow Injection Analysis, 2nd ed.; Wiley-Interecience: New York, 1988.
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Flgure 3. Plot of peaks recorded by fluorescence mlcroscopy at locations with lncreaslng radii (1-6 mm, A-F) from the Inlet channel. Data collected using the 1OX objectbe lens and 50-pL Injections of 1 mg/L fluorescein In sodium borate buffer at a flow rate of 1 mL/mln. The same pattern can be observed for all radial dlrectlons, thus confirmlng the symmetry of the circular zones Illustrated In Flgure 2.
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Figure 4. Plot of the peaks obtalned in contlnuous-flow (A) and stopped-flow mode (B) at a fixed location (4 mm from the inlet) by
fluorescencemlcroscopy. Altogether three runs of the stopped flow and three runs of contlnuous flow are shown in the overley, demonstratlng the hlgh reproduclbllltyof zone formatlon and the ability of the flow Injection system to stop the zone quickly. points along the radial axis. The fluorescence intensity of a small area (0.345 mm X 0.436 mm) was measured at six locations in increasing 1-mm increments from the center (as indicated by arrows in Figure 1). The results of this experiment are shown in Figure 3. The sample zone progressively disperses,and the position of peak maximum shifts toward longer times as the zone travels away from the cell center. The zone is most disperse at the edge of the disk (Figure 3, curve F),and the fluorescence returns to the baseline within 20 s of injection. Next, the stability of the concentration gradient in time was investigated by positioning the observation field of the microscope 4 mm from the inlet and stopping the flow of the carrier stream 4.5 s after injection of the zone. This allowed monitoring of a small section of the steep leading edge of the dye zone. The stop flow characteristics of this system are quite remarkable as seen in Figure 4, curve B. The mean RSD of the fluorescence intensity during the stop flow period was 0.6% for the three runs. This illustrates the ability of this system to perform reliable stop flow even on the leading edge of the peak, i.e., the same element of fluid can be
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Flgure 5. Responses for a 25-pL Injected volume wlth the dlrectlon of flow reversed at 1.O, 1.5, 1.7, 2.0, and 2.5 s after Injection. Each run was carried out in trlpllcate. These results show the reproduclbllity as well as the speed wlth which a zone may be moved In and out of the chamber by flow reversal.
repeatedly captured even in the steepest section of the concentration gradient. Indeed, both continuous-flowmeasurements (Figure4, curve A) and stopped-flow measurements can be performed with the same degree of reproducibility. This ability to manipulate concentration gradients with high reproducibility in the time frame from seconds to minutes is of central importance to both classical flow injection analysis5 as well as to the novel technique of flow injection fluorescence micro~copy.6~~ To further investigate the flow characteristics of the fountain cell, fluorescent beads were injected into the flow stream and imaged via a CCD camera. Using a 2.5X objective, the observation field had an area of 1.34 mm2. This allowed observation of a large number of beads. Using a l00x objective, the observation field had an area of 34 pm2, which allowed the resolution of single beads in the flow stream. These observations may be summarized as follows: (1)The fluid emerges from the inlet conduit, with the flow rapidly transitioning to a smooth radial pattern. This transition occurs within approximately 100 pm, leaving a negligible unswept volume. (2) During stop flow, by examining the fluid with a l00X objective, the only change in position of a single bead in solution was caused by Brownian motion. There was no observed drift after the flow had been stopped. Since the 1.5-pm fluorescent bead used in this experiment is smaller than most cells studied by fluorescence microscopy, it is possible that the fountain cell coupled to a flow injection system will become a useful tool for the study of non-adherent cells. The system was also investigated with respect to the precision of control over the fluid direction. These results reflect the potential for using flow reversals for added mixing or repeated introduction of a zone into the detection region. In addition, it may be used to expose adherent cells to short impulsesof reagent. Figure 5 shows the results obtained from 25-pL injections which underwent a single flow reversal. The zone appeared just prior to 1.0 s after injection, and flow reversals were carried out at 1.0, 1.5, 1.7,2.0, and 2.5 s after injection. Each run was carried out in triplicate. This figure shows the fast exchange times possible with reproducible results as a zone is brought into and removed from the detection region of the fountain cell. ~
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(6) Scudder, K.M.;Christian, G. D.; Ruzicka, J., unpublished results.. (7) Ruzicka, J.; Lindberg, W. Anal. Chem. 1992,64, 537A-545A.
The flow and optical properties detailed above are ideal for studying the response of living cultured cells to rapid changes in the chemical composition of their surrounding medium. Such experiments can be performed by growing the cells on a microscope cover glass, which is then assembled into the fountain cell, and mounting the fountain cell on the microscope stage. To demonstrate this application of the fountain cell, a dose-response experiment was performed on baby hamster kidney (BHK) cells which had been modified to express a surface receptor to calcitonin. Cellular response to an external stimulant is coupled to a transient rise in the intracellular calcium ion concentration.* Intracellular calcium levels can be monitored with a fluorescence microscope by loading the cells with the calcium-sensitivefluorescent dye Fura-2 and recording the ratio of the fluorescenceat 340-nm excitation to the fluorescence at 380-nm excitation.QJ0 Seven solutions of calcitonin ranging from 0.1 to 100 nM in logarithmic half-decade increments were prepared in a buffer suitable for perfusion of living cells.7 A 120-pL aliquot of each was sequentially injected into the fountain cell and stopped for 200 s. This volume allowed complete filling of the fountain cell with undiluted calcitonin solution. The trailing edge of the zone was flushed to waste without disturbing the solution in the fountain cell, and replaced with the leading edge of the next solution to be injected. This allowed complete exchange of the medium on the next injection in a matter of a few seconds. Figure 6 shows the response traces of two cells (Band C) overlaid with the time course of the calcitonin concentration (A). The significance of this experiment is that the flow injection system coupled to the fountain cell allowed a welldefined stepwise addition of the stimulant, which revealed that individual cells responded only when a minimum threshold level had been applied. Also, individual cells responded to different threshold levels, and only a small fraction of the cell population (2 out of about 40) showed any response at all. This documents not only the suitability of the fountain cell design for well-defined step input stimulation, but also its optical features which allow it to be interfaced to an imaging system whereby individual cells can (8)Tsien, R. W.; Taien, R. Y. A ~ R URev. . Cell Biol. 1990,6,715-760. (9)Tsien, R. Y. Methods Cell Biol. 1989,30, 127-156. (10)Bright, G.R.; Fisher, G. W.; Rogowska, J.; Taylor, D. L. Methods Cell Biol. f989, 30, 157-192.
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be selected and continuously monitored.
CONCLUSIONS The fountain cell is a result of our research aimed a t controlledperfusion of living cells for fluorescencemicroscopic analysis. We believe that the thin, flat, radially symmetric flow path of the fountain cell may also offer advantages for certain other spectroscopic applications. Any sub-area of the detection region may be probed, as in microscopy, or the entire surface may be observed. In conjunction with flow injection fluid handling techniques, the dispersion and mixing of adjacent zones can be precisely manipulated. The large area of the fountain cell would allow entire concentration gradients to be viewed at once, while the excellent stop-flow ability of the system should prove useful for monitoring the rates of physical processes and chemical reactions. Finally, this geometry may prove superior to the conventional serpentine
design often used for chemiluminescence detection, in which the walls of the conduit forming the serpentine flow path absorb and scatter the light, as well as occupy a substantial portion of the detection region.
ACKNOWLEDGMENT We express our gratitude to Gary D. Christian for his interest and to Ole Thastrup of ZymoGeneticsfor his valuable discussion on the needs of cell biology. We would also like to thank Bob Smith of ZymoGenetics for his gracious assistance with the measurements on living cells. This work was supported by the NIH (Grant No. SSS-3 (5) R01 GM 45260-2).
RECEIVED for review April 16, 1992. Accepted July 23, 1992.
