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Articles
Optical Nanosensors for Chemical Analysis inside Single Living Cells. 1. Fabrication, Characterization, and Methods for Intracellular Delivery of PEBBLE Sensors Heather A. Clark,† Marion Hoyer,‡ Martin A. Philbert,‡ and Raoul Kopelman*,†
Department of Chemistry and Department of Environmental Health Sciences, University of Michigan, Ann Arbor, Michigan 48109-1055
Spherical optical nanosensors, or PEBBLEs (probes encapsulated by biologically localized embedding), have been produced in sizes including 20 and 200 nm in diameter. These sensors are fabricated in a microemulsion and consist of fluorescent indicators entrapped in a polyacrylamide matrix. A generalized polymerization method has been developed that permits production of sensors containing any hydrophilic dye or combination of dyes in the matrix. The PEBBLE matrix protects the fluorescent dye from interference by proteins, allowing reliable in vivo calibrations of dyes. Sensor response times are less than 1 ms. Cell viability assays indicate that the PEBBLEs are biocompatible, with negligible biological effects compared to control conditions. Several sensor delivery methods have been studied, including liposomal delivery, gene gun bombardment, and picoinjection into single living cells. Optical fluorescence methods for analyzing intracellular ion concentrations have become widespread biological tools.1-4 Injection of fluorescent indicator dyes into a cell combined with dualwavelength (ratiometric) imaging, confocal microscopy, twophoton fluorescence, or fluorescence lifetime imaging has provided important insights into the concentration and spatial location of ions throughout single cells. One challenge for these optical methods has been that each dye must be chosen carefully and assessed for its ability to provide accurate and reliable information from within a cell. Factors such as toxicity to the cell, intracellular sequestration, protein binding, and leakage of the dye from the cell frequently complicate the interpretation of the results and †
Department of Chemistry. Department of Environmental Health Sciences. (1) Slavik, J. Fluorescent Probes in Cellular and Molecular Biology; CRC Press: Boca Raton, FL, 1994. (2) Mason, W. T. In Biological Techniques; Sattelle, D. B., Ed.; Academic Press: San Diego, CA, 1993; p 433. (3) Nuccitelli, R. In Methods in Cell Biology; Wilson, L., Matsudaira, P., Eds.; Academic Press: San Diego, CA, 1994; Vol. 40, p 368. (4) Herman, B. Fluorescence Microscopy, 2 ed.; Springer: New York, 1998. ‡
10.1021/ac990629o CCC: $18.00 Published on Web 10/01/1999
© 1999 American Chemical Society
must be considered.5-8 While there are indicator dyes, such as fura-2, that work very well within various cells, many fluorescent probes suffer from the problems listed above, thus limiting the number of dyes available for reliable intracellular measurements. For example, calibration of calcium probes in a cuvette can be unreliable,9 and pH indicators such as carboxyfluorescein can be affected by self-quenching and protein binding.5,10 Optodes, such as fiber-optic sensors, minimize many of the undesired interactions between fluorescent probes and cells, because the indicator dyes are entrapped within a protective polymer matrix.11-16 The matrix allows ions to diffuse easily and bind with the indicator but prevents release of indicator dyes into the cell, thus preventing unwanted interactions between the dyes and the cellular contents. Historically, optodes have been bulky and impractical for routine measurements inside living cells, due to the connecting fibers, which take up excessive space inside the cell and cause significant biological perturbations. Even with the advent of fiber-tip nanosensors,12 making the measurement of a single type of ion inside a single cell possible,17 the ability to measure multiple analytes is severely limited by the number and (5) Graber, M. L.; DiLillo, D. C.; Friedman, B. L.; Pastoriza-Munoz, E. Anal. Biochem. 1986, 156, 202-212. (6) CohenKashi, M.; Deutsch, M.; Tirosh, R.; Rachmani, H.; Weinreb, A. Spectrochim. Acta Part A 1997, 53, 1655-1661. (7) Overly, C. C.; Lee, K. D.; Berthiaume, E.; Hollenbeck, P. J. Proc. Natl. Acad. Sci. U.S.A. 1995, 92, 3156-3160. (8) Morelle, B.; Salmon, J. M.; Vigo, J.; Viallet, P. Cell Biol. Toxicol. 1994, 10, 339-344. (9) Ross, W. N. Biophys. J. 1993, 64, 1655-1656. (10) Srivastava, A.; Krishnamoorthy, G. Anal. Biochem. 1997, 249, 140-146. (11) Barker, S. L. R.; Thorsrud, B. A.; Kopelman, R. Anal. Chem. 1998, 70, 100104. (12) Tan, W.; Shi, Z.-Y.; Smith, S.; Birnbaum, D.; Kopelman, R. Science 1992, 258, 778-781. (13) Healey, B. G.; Li, L.; Walt, D. R. Biosens. Bioelectron.s 1997, 12, 521-529. (14) Panova, A. A.; Pantano, P.; Walt, D. R. Anal. Chem. 1997, 69, 1635-1641. (15) Conway, V. L.; Hassen, K. P.; Zhang, L.; Seitz, W. R.; Gross, T. S. Sens. Actuators B 1997, 45, 1-9. (16) Zhang, L.; Langmuir, M. E.; Bai, M. Q.; Seitz, W. R. Talanta 1997, 44, 1691-1698. (17) Kopelman, R.; Miller, M. T.; Brasuel, M.; Clark, H. A.; Hoyer, M.; Philbert, M. A. SPIE Proceedings 1999, 3540, 206-209.
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size of fibers that can be inserted into a cell without destroying it. Probes encapsulated by biologically localized embedding (PEBBLEs) combine the strengths of optodes with those of free fluorescent indicator dyes. For instance, while PEBBLEs have a protective polymer matrix (like optodes), the total volume is negligible compared to that of the cell (like free dye). PEBBLE sensors are spherical, submicrometer polymer spheres, containing indicator molecules throughout the matrix. One type of PEBBLE consists of an indicator dye or pair of indicator dyes entrapped in the pores of a hydrogel. These nanosensors use commercially available fluorescent indicators for analytes such as pH, calcium, and oxygen. Also, multiple dyes can be combined in one PEBBLE to allow dual-wavelength measurements. As demonstrated below, the polymer matrix is not only biocompatible, but, as in a fiberoptic sensor, comprises a chemically and physically controlled microenvironment. The matrix also significantly reduces interactions between proteins and the indicator, as well as intracellular sequestration/leakage and toxicity of the dye. Still, the entire device is sufficiently small so as to be minimally invasive and permit rapid response times. Beads have been used previously for sensing in a variety of ways. For instance, a recent elegant optical array18 uses colored beads that act as an “artificial nose” for organic vapors in a fiber bundle assay. Most uses of sensor beads have been in combination with an optical fiber or capillary, such as originated by Peterson et al.,19 who used micrometer-sized acrylamide beads as packing material in a millimeter-sized glass capillary. To date, techniques utilizing beads in concert with optical fibers or capillaries have not been miniaturized enough, or intended, for intracellular use. Furthermore, we do not just attach indicator molecules to the surface of commercial beads, microspheres, or nanoparticles, as such a strategy would not provide the indicator molecule with the protective and controlled environment provided by the PEBBLE matrix. Optodes were invented to provide such a protective environment (see above) and in this sense the PEBBLE sensor is a genuine nano-optode. Microemulsion polymerization is a simple and convenient method for the nanofabrication of sensors. Unlike other methods of immobilizing dye molecules into polymer spheres (such as covalent binding of dyes to the outside of polystyrene beads), the acrylamide matrix noncovalently traps the fluorescent indicator into pores inside the matrix. Thus, to incorporate a new dye, the process can easily be modified by the addition of the dye into the monomer mixture. Another advantage is that the dye is trapped in the pores of the matrix during the polymerization process, rather than after the polymerization is complete. Thus the polymer does not have to swell to incorporate the dye, which minimizes the leaching out of the dye from the pores during sensor use. As has been shown previously, the pore size can be adjusted to be larger or smaller, by varying the amount of cross-linker used during polymerization.20 The pores could then be modified to accommodate dyes of differing sizes, or even larger species such as enzymes and proteins. (18) Dickenson, T. A.; Michael, K. L.; Kauer, J. S.; Walt, D. R. Anal. Chem. 1999, 71, 2192-2198. (19) Peterson, J. I.; Goldstein, S. R.; Fitzgerald, R. V.; Buckhold, D. K. Anal. Chem. 1980, 52, 864-869. (20) Ruchel, R.; Steere, R. L.; Erbe, E. F. J. Chromatogr. 1978, 166, 563-575.
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General techniques for fabricating PEBBLE sensors are presented in this paper, as well as the characterization of properties that applies to any PEBBLE sensors, regardless of the dye entrapped within the matrix. These properties include sensor size, response time, batch to batch consistency, and biocompatibility. Methods for inserting PEBBLE sensors into single viable cells are also discussed. In the companion paper, sensors specific for pH and calcium are presented, as well as calibration, reversibility, photobleaching, leaching, and application data for these sensors. EXPERIMENTAL METHODS Reagents. All reagents and solvents were purchased from Aldrich (Milwaukee, WI). Fluorescent dyes and indicators were purchased from Molecular Probes (Eugene, OR). Biological reagents, including buffers and media, were purchased from BRL Life Sciences (Gaithersburg, MD). Preparation of Acrylamide PEBBLEs (pH, Oxygen, and Calcium). The polymerization solution consisted of 50 µg of fluorescent indicator, 27% acrylamide, and 3% N,N-methylenebis(acrylamide), in 1 mL of 10 mM phosphate buffer, pH 7.4. The polymerization solution was then added to a solution containing 20 mL of hexane, 1.8 mmol of dioctyl sulfosuccinate sodium salt, and 4.24 mmol of Brij 30 (4 lauryl ether), and the two solutions were emulsified by stirring. The polymerization was initiated with 50 µL of a 10% sodium bisulfite solution or 24 µL of a 10% ammonium persulfate solution and 12 µL of N,N,N′,N′-tetramethylethylenediamine (TEMED), and the solution was stirred at room temperature for 2 h. Hexane was removed by rotary evaporation and then the sensors were precipitated by the addition of ethanol. Excess surfactant and dye were removed by rinsing with ethanol, to yield a product consisting of 20- and 200-nm probes. TEM Imaging. PEBBLEs fixed with OsO4 were dispersed onto a Formvar-coated TEM grid, stained with lead citrate, counterstained with uranyl acetate, and visualized using a JEOL transmission electron microscope. Response Time Measurements. Response time was determined using an Olympus IX50 inverted microscope equipped with a mercury arc lamp and a PMT. Calcium-selective probes were premixed with a caged calcium ion (cage was DM-nitrophen, Calbiochem, La Jolla, CA), and this solution was inserted into a quartz capillary. The calcium was uncaged with a pulse of UV light from a Quanta-Ray 10-ns Nd:YAG laser (Quanta-Ray, Mountainview, CA) equipped with a frequency tripler and coupled into an optical fiber positioned over the capillary. Instrumentation. Spectroscopic measurements were taken on a FluoroMax-2 spectrofluorometer (ISA Jobin Yvon-Spex, Edison, NJ), slits set to 5 nm for both the emission and excitation. Neuroblastoma Cells (Cell Line SH-SY5Y). Human SY5Y neuroblastoma cells were cultured in Dulbecco’s modified Eagle medium (DMEM) containing 400 mg/L D-glucose, 2 mM Lglutamine, and 20% (C6 glioma) or 10% (SY5Y neuroblastoma) fetal bovine serum. Cells were released from culture dishes by trypsin treatment 1 day prior to experiments and plated on uncoated 22-mm glass cover slips in 35-mm culture dishes. Gene Gun Delivery. A Biolistic PDS-1000/He system (benchtop model) from BioRad (Hercules, CA) with grade 5 helium (Cryogenics, Detroit, MI) was used to inject cells with sensor probes. Sample preparation for the particle delivery system
required dispersion of the PEBBLEs in water and the careful application of a thin film of PEBBLEs onto the target membrane. Low firing pressures were used, best results being obtained at 900 psig, with a vacuum of 25 Torr on the system. Culture medium (DMEM) was removed from the cells by pipet before delivery. Following biolistic delivery of PEBBLEs, neuroblastoma cells were rinsed three times with Dulbecco’s phosphate-buffered saline (DPBS) and incubated with DPBS during analysis. Picoinjection. A glass capillary was pulled using a standard pipet puller into the shape of a microneedle. The capillary was filled with a solution of PEBBLEs in buffer via capillary action and then mounted onto micromanipulators. A microscope was used to view the capillary as it was guided into a cell, and then positive pressure was applied to the capillary for the injection. It was estimated that picoliter volumes of PEBBLE solution were injected into each cell. Liposomal Delivery. PEBBLEs were incubated with cationic lipids, ESCORT (Sigma, St. Louis, MO) and DMEM for 15 min and then the mixture was introduced into neuroblastoma cells. The cells were maintained in a 5% CO2 atmosphere in a 37 °C incubator for 5 h before removal of excess PEBBLE/liposome mixture from the cells. Neuroblastoma cells were incubated in fresh medium overnight and then rinsed before analysis. Trypan Blue Viability Assay. Cells were rinsed with Dulbecco’s phosphate-buffered saline and incubated with 0.2% Trypan Blue, and stained cells were counted. Triplicate cultures were analyzed for each control (no PEBBLEs) and each treated group (PEBBLEs injected using the gene gun), in the two tests analyzed. RESULTS AND DISCUSSION Several initiators were studied for the microemulsion polymerization of the PEBBLE sensors, including TEMED/ammonium persulfate, 2,2-azobisisobutyronitrile (AIBN), and sodium bisulfite. The polymerization reaction of the acrylamide sensors can be initiated using a combination of TEMED and ammonium persulfate21,22 which are the initiators historically used for acrylamide polymerization, to make not only nanoparticles but also slab gels used in electrophoresis. Many of our PEBBLE sensors have been produced using these initiators23,24 with good success and no degradation of the dyes entrapped in the pores of the polymer matrix. It was found that in some cases the initiator reacted with the dye that was being incorporated into the matrix, producing a nonfluorescent species. Dyes such as sulforhodamine 101 (which is often used as a reference dye in the PEBBLE sensors) appear to react with ammonium persulfate. Other initiators, such as AIBN, did not initiate the polymerization in the microemulsion. However, in contrast to the ammonium persulfate/ TEMED combination, sodium bisulfite initiated the polymerization reaction without reacting with the dye. Bisulfite has proven to be a generally useful initiator and many dyes (such as sulforhodamine 101) and fluorescent indicators (such as Calcium Green) have now been successfully immobilized into PEBBLE sensors. Not only did sodium bisulfite not react with sulforhodamine but it did not (21) Daubresse, C.; Grandfils, C.; Jerome, R.; Teyssie, P. J. Colloid Interface Sci. 1994, 168, 222-229. (22) Leong, Y. S.; Candau, F. J. Phys. Chem. 1982, 86, 2269-2271. (23) Clark, H. A.; Hoyer, M.; Parus, S.; Philbert, M. A.; Kopelman, R. Mikrochim. Acta 1999, 131, 121-128. (24) Clark, H. A.; Kopelman, R.; et al. Sens. Actuators B 1998, 51, 12-16.
Figure 1. Spectra of a pH-sensitive PEBBLE containing an internal standard. Both dyes are effectively excited at 488 nm, and the fluorescein derivative responds to changes in pH while the internal standard remains nearly constant.
Figure 2. Transmission electron micrograph of a single 250-nm PEBBLE sensor dried on a Formvar grid.
react with other dyes that have been immobilized. Figure 1 illustrates spectra from a pH-sensitive PEBBLE that contains a fluorescein derivative and an internal standard, sulforhodamine 101. Both dyes are still fluorescent in the PEBBLEs, and the pH dye remains sensitive to changes in pH. Calibration of such sensors will be discussed in detail in the companion paper.25 Transmission electron microscopy (TEM) was used to determine the size and the shape of the PEBBLE sensors (Figure 2). It was determined that the microemulsion polymerization produces a bimodal size distribution of 20- and 200-nm sensors. This size distribution is a result of the properties of the micellar formation and aggregation process and has been studied previously.22 The predominant size produced is the 20-nm PEBBLE, constituting about 98% of the number of sensors formed (this is based on number of particles; therefore, considering the volume differences between the two sizes of sensors, the bulk of the dye molecules reside in 200-nm PEBBLEs). Previous studies have determined that the size of the acrylamide nanospheres can be (25) Clark, H. A.; Tjalkens, R.; Philbert, M. A.; Kopelman, R. Anal.Chem. 1999, 71, 4837-4843 (following paper).
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Figure 3. Effect of protein on sensors. Adding as little as 0.01% albumin to a solution of CNF dye molecules causes an almost 90% change in the fluorescence intensity ratio of this pH-sensitive dye, even though the pH of the solution remained constant (as measured by a standard pH electrode). The error is equivalent to a change of an entire pH unit, rendering this highly stable dye useless for intracellular measurements. Under the same conditions, the PEBBLEs containing the CNF dye are not affected by the addition of albumin, with an error of only a hundredth of a pH unit, even at higher concentrations of albumin.
varied by changing the amount of surfactant, solvent, or monomer or the temperature of the reaction.22 It has been our experience that surfactant concentration plays the largest role in the control of sensor size achieved in the emulsion. The use of two types of surfactant (Brij 30 and AOT) in high concentration keeps the initial monomer micellar size very small.22 This small initial size prevents the sphere from becoming too large during polymerization, and submicrometer sensors are produced. By reducing the concentration of surfactant used, larger spheres were made, with sizes up to 1 µm attained by reducing the concentration of surfactant by 1 order of magnitude. However, larger particles, e.g., 1 µm spheres, tend to be less uniform in size. We have found that the polymer matrix of PEBBLE sensors prevents macromolecules, such as proteins, from diffusing through the matrix. The matrix thus protects the indicator dyes from their intracellular environments, preventing interference with the fluorescent properties of the dyes.5 Without this shielding of a dye, its fluorescence would behave unpredictably inside a given cell, making calibration of even ratiometric dyes difficult or impossible. CNF is one example of a dye that is not commonly used for intracellular measurements, due to its propensity for interactions with proteins. Even though CNF is a highly photostable, ratiometric dye, incubation of the free dye with as little as 0.01% albumin induces alterations in the emission ratios of almost 90% (while the pH remained constant, as measured with a standard electrode). This error (Figure 3) is equivalent to approximately 1 pH unit. However, when protected by the microenvironment of the PEBBLE, the same dye shows minimal interference, with a resulting error of only about a hundredth of a pH unit. The matrix protection allows the use of any given pH dye for intracellular use, without being limited by intracellular interference. Furthermore, the calibration of PEBBLEs in a cuvette, therefore, remains valid in the intracellular environment. The response time of the PEBBLE sensors is very short due to their small size, which allows for rapid diffusion of the ions through the polymer matrix. To measure the true response time of the PEBBLE sensors, the solution of sensors was premixed 4834 Analytical Chemistry, Vol. 71, No. 21, November 1, 1999
Figure 4. Response time of PEBBLE sensors. Calcium was released using a single 10-ns UV pulse from a Nd:YAG laser, which photolyzed the cage, releasing free calcium into a solution of PEBBLEs. The observed response time, less than 1 ms, was indiscernible from that of the corresponding dye not entrapped in a polymer matrix.
Figure 5. Batch to batch consistency of PEBBLE sensors. Three separate batches of PEBBLE sensors, each containing a pH-sensitive dye and an internal standard, were produced and calibrated three times each. The average of the calibrations is plotted, and the error bars on the graph indicate the standard deviation of the results.
with a caged ion (in this case calcium). The cage was then removed with a 10-ns pulse of UV light that photolyzed the cage. The response time of the PEBBLE sensors was compared to that of the free dye (no polymer matrix), to separate the diffusion time through solution from the diffusion time through the matrix. As can be seen in Figure 4, the 90% response time of the PEBBLE sensor to the increase in free calcium is on the order of 1 ms. It should be noted that the response time of the free dye (no polymer) was also measured, and no measurable difference in response times was noted. The diffusion time of calcium through the solution is measured by this technique, with the diffusion time through the polymer being at most 100 µs. Theoretically, with an approximate diffusion constant of 10-6 cm2/s, the average diffusion time should be about 100 µs for a 100-nm-radius sensor and 1 µs for a 10-nm radius sensor. The batch to batch consistency of the PEBBLE sensors is good. In one example, three batches of dual-wavelength pH
Figure 6. Transmission electron micrographs of PEBBLE sensors embedded biolistically (via gene gun), at 900 psig, into the cytoplasm of neuroblastoma cells: (a) two 200-nm PEBBLEs, near or inside the cell nucleus; (b) one 20-nm PEBBLE next to a primary lysosome in the cell cytoplasm. Original magnification is indicated on the figure and the inset.
Figure 7. Calcium PEBBLEs delivered to neuroblastoma cells by liposomal delivery: (a) Nomarski illumination, (b) fluorescence image with excitation at 590 nm. Note that the PEBBLEs remain in the cytoplasm of the cells and are not contained in the nucleus.
Table 1. Trypan Blue Viability Assay: Untreated Controls and Neuroblastoma Cells Bombarded with PEBBLEs at 900 psig, Using a Gene Gun for Delivery of the PEBBLEs into the Cellsa
a
experiment
test 1 (%)
test 2 (%)
controls with PEBBLEs
98.8 ( 0.2 97.4 ( 0.1
98.8 ( 0.6 96.2 ( 0.7
Variations of three results shown as mean ( standard deviation.
sensors (containing carboxydimethyl fluorescein as the pHsensitive dye and sulforhodamine 101 as the internal standard) were produced and calibrated. Each batch of sensors was calibrated three times and the results of the calibrations were averaged to find the standard deviation. Even with two separate dyes in the matrix, the ratio remained relatively constant, and small differences can be compensated for in a normalized plot (Figure 5). In practice, each new batch of sensors is recalibrated before intracellular measurements are begun. Nevertheless, even without recalibration, the dye behaves consistently within the polymer matrix from batch to batch, and the dynamic range and calibration do not change with a new batch of sensors. The biocompatibility of PEBBLE sensors was investigated by assessing the viability of cells in a culture, using a variety of
histological and biochemical techniques, e.g., Trypan Blue exclusion (Table 1), lactate dehydrogenase leakage, and energy charge. The viability of cultured neuronal cells containing PEBBLE sensors was indistinguishable from that of control samples or cells containing other often-used nanoparticles, such as gold colloids and latex spheres. As the PEBBLE sensors were designed for intracellular measurements, methods were explored for inserting these sensors noninvasively into living, viable cells. Techniques such as bead loading, scrape loading, and pinocytosis were explored as PEBBLE delivery methods, but were not practical due to the severely reduced viability of the cells or minimal loading of the sensors into the cell. Three methods, gene gun bombardment, picoinjection, and liposomal delivery, were found to be effective at inserting the PEBBLEs into cells, while at the same time allowing the cells to remain viable. The gene gun uses a burst of helium to fire the PEBBLEs into cells. The intracellular distribution of PEBBLEs was confirmed using transmission electron microscopy (Figure 6). Due to the light mass of the PEBBLE sensors (compared to gold colloids, which are commonly used with the gene gun to insert DNA into cells), only a few sensors are inserted into cells. While many cells are inserted with PEBBLEs with a single injection, no more than Analytical Chemistry, Vol. 71, No. 21, November 1, 1999
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one or two PEBBLEs can be inserted into any individual cell. Thus, the gene gun is effective for injecting single PEBBLEs into cells and could be used for spatially resolved measurements of microdomains in a cell. The disadvantage is that there is no way to direct the sensor to a particular region of the cell, since the PEBBLEs are shot in a random pattern. However, in a culture of many cells, appropriate cases can be selected. Picoinjection employs a glass capillary that is pulled into a small (micrometer)-sized tip and inserted into a single cell. A buffered aqueous solution containing PEBBLE sensors is then injected into the cell. The technique is effective for inserting many PEBBLEs into a single cell, improving the signal that can be attained from a cell, and allowing several regions of the cell to be imaged at once. A distinct advantage is that the injection capillary can be positioned in a cell and a specific location (such as the nucleus) can be selectively injected with sensors. This area can then be imaged optically without interference from signal that may come from sensors in other regions of the cell, such as the cytoplasm. The disadvantage of picoinjection is the need to inject each cell separately, requiring considerable time and effort. Liposomal delivery of PEBBLEs into cells makes use of commercially available lipid vesicles. Preprepared, commercially available, cationic lipids are mixed with PEBBLEs, which interact with them spontaneously. When incubated with living cells, the
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liposomes fuse with the cells and empty the PEBBLEs into the cytoplasm. As can be seen from Figure 7, the PEBBLEs stay selectively in the cytoplasm of the cells, and the nucleus remains free of the sensor. Any signal measured is therefore solely attributed to the cytoplasm of the cell and is not a combination of intra- and extranuclear signals. This selectivity is important, because with many techniques of dye loading, it is difficult to contain the dye in one location of the cell. Liposomal delivery of PEBBLEs has become the current method of choice, due to its ease of use and effective loading, and is utilized in our laboratory for routine intracellular measurements (e.g., following paper). ACKNOWLEDGMENT The authors thank Rhonda Lightle for TEM imaging, Eric Monson for response time measurements, and Steve Parus and Susan Barker for technical assistance. The authors acknowledge support from the Defense Advanced Research Projects Agency (DARPA) MDA972-97-1-006 and the U.S. National Institutes of Health (NIH) GM503000-04 and ES08845.
Received for review June 11, 1999. Accepted August 11, 1999. AC990629O
