Dual Imaging of Topography and Photosynthetic Activity of a Single

Sep 18, 1999 - plast (radius, 25 µm). At the tip of type A, the two disks were on the same plane. Two disk planes of electrode type. B formed an angl...
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Anal. Chem. 1999, 71, 4637-4641

Dual Imaging of Topography and Photosynthetic Activity of a Single Protoplast by Scanning Electrochemical Microscopy Tomoyuki Yasukawa, Takatoshi Kaya, and Tomokazu Matsue*

Graduate School of Engineering, Tohoku University, Aramaki 07, Aoba, Sendai 980-8579, Japan

Two types (A and B; see Figure 1 in the text) of dual-Pt microdisk electrodes (disk radius, 0.5-4.0 µm; whole tip radius, 5.0-15 µm) were fabricated to simultaneously detect two electroactive species near a single algal protoplast (radius, 25 µm). At the tip of type A, the two disks were on the same plane. Two disk planes of electrode type B formed an angle of ∼135° at the tip. Cyclic voltammetric investigation indicated that, compared with type A, one disk in type B only slightly collected the species electrogenerated at the other disk. These dual-microdisk electrodes were applied to electrochemical measurements of a single protoplast in artificial seawater. The topographic profiles of the single protoplast were obtained from the variation of oxidation current for 1.0 mM Fe(CN)64- at one disk. The photosynthetic oxygen generation from the protoplast was also monitored by detecting the reduction current for oxygen at another disk. The electrode of type B was used as a probe for scanning electrochemical microscopy (SECM) to obtain dual-SECM images of topography and activity of a single protoplast. Electrochemical measurements using microelectrodes have been extensively applied to the characterization of localized biological phenomena.1-10 The microelectrodes fabricated so far have been roughly divided into two types. One is the fiber electrode (e.g., microdisk or microring electrode) in which a metal wire (Au or Pt) or carbon fiber is encased into an insulating material such as glass and epoxy resin. Another is the planar electrode, fabricated on a silicon wafer or glass substrate by using microfabrication techniques developed in semiconductor engi* Corresponding author: (fax) +81-22-217-7209; (tel) +81-22-217-7209; (e-mail) [email protected]. (1) Adams, R. N. Anal. Chem. 1976, 48, 1126A-1138A. (2) Wightman, R. M.; May, L. J.; Michael, A. C. Anal. Chem. 1988, 60, 769A779A. (3) Wightman, R. M.; Hochstetler, S.; Michael, D.; Travis. E. Electrochem. Soc. Interface Fall. 1996, 22-26. (4) Abe, T.; Lau, Y. Y.; Ewing, A. G. J. Am. Chem. Soc. 1991, 113, 7421-7423. (5) Lau, Y. Y.; Abe, T.; Ewing, A. G. Anal. Chem. 1992, 64, 1702-1705. (6) Arbault, S.; Pantano, P.; Jankowski, J. A.; Vuillaume, M.; Amatore, C. Anal. Chem. 1995. 67, 3382-3390. (7) Malinski, T.; Taha. Z. Nature 1992. 358, 676-678. (8) Matsue, T.; Koike, S.; Abe, T.; Itabashi, T.; Uchida, I. Biochim. Biophys. Acta 1992, 1101, 69-72. (9) Matsue, T.; Koike, S.; Uchida, I. Biochem. Biophys. Res. Commun. 1993, 197, 1283-1287. (10) Yasukawa, T.; Uchida, I. Matsue, T. Biochim. Biophys. Acta 1998, 1369, 152-158. 10.1021/ac9903104 CCC: $18.00 Published on Web 09/18/1999

© 1999 American Chemical Society

neering including photolithography, etching, chemical vapor deposition, etc. Planar electrodes, such as interdigitated microarray electrodes,11-19 microdisk array electrodes,20-25 interdigitated ring array electrodes,26-29 and independent band electrodes,30-32 have been used as detectors of flow analysis and in vitro biosensors. The most remarkable aspect of such planar electrodes is that multiple electrodes can be arranged at any position on the substrate. Fiber electrodes have been used in amperometric in vivo and single-cell measurements because their tips can be miniaturized on a cell-sized scale or less, thereby being able to approach a single cell using a three-dimensional micromanipulator. To clarify biological functions it is important to detect multiple species simultaneously in a localized space. Cahill and Wightman reported a simultaneous amperometric measurement of ascorbate and catecholamine secreted from a single cell by using two microelectrodes.33 Dual-disk microelectrodes with two working electrodes were prepared34,35 to detect two electroactive species. (11) Aoki, A.; Matsue, T.; Uchida, I. Anal. Chem. 1990, 62, 2206-2210. (12) Niwa, O.; Morita, M.; Tabei, H. Anal. Chem. 1990, 62, 447-457. (13) Takahashi, M.; Morita, M.; Niwa, O.; Tabei, H. J. Electroanal. Chem. 1992, 335, 253-263. (14) Takahashi, M.; Morita, M.; Niwa, O.; Tabei, H. Sens. Actuators B 1993, 13, 336-339. (15) Aoki, K.; Tokuda, K.; Matsuda, H. J. Electroanal. Chem. 1977, 79, 49. (16) Aoki, K.; Morita, M.; Niwa, O.; Tabei, H. J. Electroanal. Chem. 1988, 256, 269-282. (17) Fosdick, L. E.; Anderson, J. L. Anal. Chem. 1986, 58, 2481-2485. (18) Sanderson, D. G.; Anderson, J. L. Anal. Chem. 1985, 57, 2388. (19) Matsue, T. Trends Anal. Chem. 1993, 12, 100-108. (20) Caudill, W. L.; Howell, J. O.; Wightman, R. M. Anal. Chem. 1982, 54, 25322535. (21) Thormann, W.; Bosch, P.; Bond, A. M. Anal. Chem. 1985, 57, 2764. (22) Strohben, W. E.; Smith, D. K.; Evans, D. H. Anal. Chem. 1990, 62, 17091712. (23) Horiuchi, T.; Niwa, O.; Morita, M.; Tabei, H. J. Electroanal. Chem. 1990, 256, 25. (24) Hoogvliet, J. C.; Reijn, J. M.; Bennekom, W. P. Anal. Chem. 1991, 63, 24182423. (25) Kasai, N.; Matsue, T.; Uchida, I.; Niwa, O.; Horiuchi, T.; Morita, M. Denki Kagaku 1996, 64, 1269-1271. (26) Niwa, O.; Morita, M. Anal. Chem. 1996, 68, 355-359. (27) Niwa, O.; Morita, M.; Solomon, B. P.; Kissinger, P. T. Electroanalysis 1996, 8, 427-433. (28) Hoogvliet, J. C.; Elferink, F.; Poel, C. J.; Bennekom, W. P. Anal. Chim. Acta 1983, 53, 149-159. (29) Chen, D.-T.; Chandran, R. J. Electrochem. Soc. 1981, 128, 1904-1912. (30) Kasai, N.; Matsue, T.; Uchida, I. Electroanalysis 1996, 8, 748-752. (31) Aoki, A.; Matsue, T.; Uchida, I. Anal. Chem. 1992, 64, 44-49. (32) Matsue, T.; Aoki, A.; Ando, E.; Uchida, I. Anal. Chem. 1990, 62, 407-409. (33) Cahill, P. S.; Wightman, R. M. Anal. Chem. 1995, 67, 2599-2605. (34) Matysik; Michael, F. Electrochim. Acta 1997, 42, 3113-3116.

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However, these electrodes were too large to apply to single-cell measurements. Recently, a dual microsensor based on electrochemistry and fluorescence measurements was fabricated to simultaneously detect Ca2+ and catecholamines following their secretion from single cells.36 In this paper, we report the fabrication and characterization of a dual-microdisk electrode which can be applied to single-cell measurements. The dual-microdisk electrode consists of two Ptbased microwires with 0.5-4-µm radii sealed into a θ-type glass capillary. The basic electrochemical behavior of the dual-microdisk electrodes was investigated by cyclic voltammetry. The steadystate diffusion layers formed at two disks slightly overlapping each other to show the electrochemical cross-talking influence, i.e., one disk collected the electrogenerated species from another disk. To prevent the cross-talking influence, we fabricated dual-microdisk electrodes with open-L-shaped tips. These dual-microdisk electrodes were used as probes for scanning electrochemical microscopy (SECM) to characterize a single cell. SECM has been introduced as a new tool to obtain topographic images37-41 of various surfaces and concentration profiles at various interfaces. Dual-SECM imaging of the topography and concentration of a chemical species, however, has some limitations. To obtain topographic information, one has to add a redox species, which might induce an undesired reaction with the chemical species of interest. Electrochemical cross-talk between the two disks might cause deformation of the images. Recently, topographic images of a single cell or images of cellular functions were studied.42-44 We simultaneously detect two electroactive species by using dualmicrodisk electrodes to obtain dual-SECM images of the topography and photosynthesis activity of a single, living algal protoplast. EXPERIMENTAL SECTION Reagents. K4Fe(CN)6 were purchased from Wako Pure Chemicals (Osaka, Japan) and used without further purification. All the solutions were prepared from distilled and deionized water by Aquarius GS-200 (Advantec) and Milli-Q Jr. (Millipore). Fabrication of Dual-Microdisk Electrodes. Dual-microdisk electrodes with Pt-Rh (10%) wires were used in this work. The dual electrode was fabricated as follows. A θ-type glass tube (World Precision Instruments, Tokyo, Japan) was pulled using a capillary puller (model PD-5, Narishige, Tokyo, Japan) to make a capillary with two orifices of 0.05-0.5-µm radii. A Pt-Rh wire was etched electrochemically (300 Hz, ac voltage with 5-10 V peakto-peak) in a NaNO3-saturated aqueous solution. Two etched Pt(35) Zhong, M.; Zhou, J.; Lunte, S. M. Anal. Chem. 1996, 68, 203-207. (36) Xin, Q.; Wightman, R. M. Anal. Chem. 1998, 70, 1677-1681. (37) Engstrom, R. C.; Weber, M.; Wunder, D. J.; Burgess, R.; Winquist, S. Anal. Chem. 1986, 58, 844-848. (38) Liu, H.-Y.; Fan, F.-R. F.; Lin, C. W.; Bard, A. J. J. Am. Chem. Soc. 1986, 108, 3838-3839. (39) Bard, A. J.; Fan, F.-R. F.; Kwak, J.; Lev, O. Anal. Chem. 1989, 61, 132138. (40) Mirkin, M. V.; Arca, M.; Bard, A. J. Science 1992, 257, 364-366. (41) Bard, A. J.; Fan, F.-R. F.; Pierce, D. T.; Unwin, P. R.; Wipf, D. O.; Zhou, F. Science 1991, 254, 68-74. (42) Lee, C.; Kwak, J.; Bard, A. J. Proc. Natl. Acad. Sci. U.S.A. 1990, 87, 17401743. (43) Tsionsky, M.; Gardon, Z. G.; Bard, A. J.; Jackson, R. B. Plant Physiol. 1997, 113, 895-901. (44) Yasukawa, T.; Kondo, Y.; Uchida, I. Matsue, T. Chem. Lett. 1998, 320, 767768.

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Figure 1. (Top) Pt dual-microdisk electrodes with different tip shapes (types A and B). Horizontal (middle) and vertical (bottom) microscopic images of dual-microdisk electrodes.

Rh wires were inserted into the θ-type glass capillaries at their two large openings. The tip region was thermally fused in vacuo. Then, the tip was carefully polished with a diamond grinder (No. 5000) on a turntable (model EG-6, Narishige) to produce a diskshaped electrode. We also fabricated the dual electrodes with different tip shapes (type B) by carefully polishing the tips of the electrodes of type A. At the tip of type B, half of the tip was ground until the angle of the two disk planes became ∼135° to form an open-L shape. Optical microscopic images (TE 300 and EPI 200, Nikon, Tokyo, Japan) of these dual-microdisk electrodes as given in Figure 1. These electrodes were characterized by cyclic voltammetry. Measurement System. All measurements were made using a self-made SECM system, which consisted of an inverted microscope (TE 300, Nikon), a multipotentiostat (HA-1512µM8, Hokuto Denko, Tokyo, Japan), and an XYZ stage (LM-641-2AE1, Chuo Precision Industrial Co., Tokyo, Japan). The precise position of the microelectrode tip was determined by an image processor (Argus-20, Hamamatsu Photonics, Hamamatsu, Japan) connected to a CCD video camera (C6391, Hamamatsu Photonics) mounted on the microscope. Control of electrode potential and data acquisition were performed with a notebook computer with a 16bit AD (AZI-3155, Interface, Hiroshima, Japan) and a 16-bit DA board (AZI-3310, Interface). A microelectrode was mounted on a piezoactuator (17PAZ013, Melles Griot, Tokyo, Japan) which was attached to the XYZ stage. The piezoactuator was used to control the microelectrode in direction z at the nanometer level. The piezodriver (17PCZ013, Melles Griot) and XYZ stage were controlled by a notebook computer through a 16-bit AD/DA board

(AZI-3506, Interface) and a GP-IB interface (AZI-4301, Interface), respectively. The measurements were carried out in a twoelectrode system with an Ag/AgCl (saturated KCl) as a counter/ reference electrode. Light irradiation for photosynthesis was provided by a halogen lamp attached to the microscope. Light intensity was maintained at 20 kLx for the measurements using a protoplast. All the measurements were performed at 25 °C in a shield box. Cyclic voltammetry was carried out in an aqueous solution of 4.0 mM Fe(CN)64- in the generation-collection mode; i.e., one of the disks (W1) of the dual-microdisk electrode was swept away at 0.02 V/s between -0.1 and 0.6 V, while the other (W2) was held at 0.05 V to reduce Fe(CN)63- generated at W1. Amperometric experiments above a glass substrate with a microhole or a single protoplast were carried out in air-saturated artificial seawater containing 1.0 mM Fe(CN)64-. The potential of W1 was held at 0.50 V to oxidize Fe(CN)64- and that of W2 at -0.60 V to reduce oxygen. The current-z direction profiles were recorded by moving the tip from the bulk solution to the surface of the protoplast or glass substrate. The dual-microdisk electrode scanned at a speed of 2.4 µm/s over a glass substrate or a single protoplast with a constant tip-substrate distance to obtain a profile of two redox currents simultaneously. The glass substrate with a microhole (radius, 50 µm; depth, 27 µm) was prepared by ordinary photolithography with the HF etching process. For dual-SECM imaging of a single protoplast, the dualmicrodisk electrode (type B) scanned at a speed of 9.8 µm/s over the protoplast. The topographic image of the protoplast was obtained based on the oxidation current for Fe(CN)64- at one disk, which was mounted as parallel to the substrate. The image of the photosynthetic activity was obtained by mapping of the reduction current for oxygen monitored at the other disk. The time required to obtain an image of 150 × 150 µm (spatial resolution, 10 µm) was ∼4 min. Preparation of Protoplast. Protoplasts were made from marine alga Bryopsis plumosa by the method reported previously45 in synthetic artificial seawater containing 480.2 mM NaCl, 2.3 mM NaHCO3, 11.1 mM CaCl2, and 83.3 mM MgCl2. All the amperometric measurements were started ∼20 min after the protoplast was prepared.

Figure 2. Cyclic voltammograms of 4.0 mM Fe(CN)64- at Pt dualmicrodisk electrodes of types A (a, c), and B (b, d). The potential applied to one disk (W1) was scanned at 0.02 V/s and that to the other disk (W2) was fixed at 0.05 V vs Ag/AgCl (a, c). The curves (b, d) were obtained by changing the connection to W1 and W2 of the electrodes of types A and B. Radii of disks: type A, 2.6 µm for W1, 2.7 µm for W2; type B, 2.1 µm for W1, 2.9 µm for W2.

RESULTS AND DISCUSSION Characterization of Dual-Microdisk Electrodes. Figure 1 shows schematic illustrations of the tip areas of the platinum dualmicrodisk electrode, together with optical microscopic images. Two platinum fine wires are encased in glass and exposed as disk shapes. The optical microscopic image of the tip (type A) indicates that the radii of the right disk (W1) and left disk (W2) were ∼2.7 µm, respectively, and the whole tip including insulating glass was ∼14 µm. The gap between two disks was ∼16 µm. The image of the tip (type B) indicates that the angle of the two planes was ∼135°. The radii of the left disk (W1) and right (W2) disk were approximately 2.0 and 3.0 µm, respectively. The radii of the semicircle with the disks was ∼12 µm. The horizontal and vertical spacings between the two disks (W1 and W2) were approximately 14 and 9 µm, respectively (see Figure 1). Figure 2 shows the cyclic voltammograms of 4.0 mM Fe(CN)64- at the Pt dual-microdisk electrodes of types A (a,c) and B (b,d). In these measurements,

W1 was scanned for at 0.02 V/s while W2 was held at 0.05 V vs Ag/AgCl (a,c). The cyclic voltammograms at W1 disks show steplike shapes to provide steady-state currents for oxidation of Fe(CN)64- at a positive potential region. The W2 disks of the dualdisk electrodes of type A collect Fe(CN)63- generated at neighboring W1 disks; the collection efficiency, the ratio of the reduction current at W2 to the oxidation current at W1, is 6%. For type B, the collection efficiency is only 2%, indicating that the redox interference from the neighboring disk is small in type B as compared with that of type A. The voltammogram of type A showed similar behavior when the connection to W1 and W2 was switched (Figure 2c). The type B electrode also showed a similar voltammogram when the connection was switched. The radii of Pt disks of type A were determined from the steady-state oxidation currents of Fe(CN)64- in voltamograms in a single-operation mode46 and found to be 2.6 µm for the W1 disk and 2.7 µm for the W2 disk. Similarly, the radii of disks of type B were 2.1 and 2.9 µm. These values correspond well with those observed in the optical microscopic image. Profiles of Current-Z Direction Distance. Figures 3 and 4 show the variations of oxidation current for 1.0 mM Fe(CN)64and reduction current for oxygen when the dual-microdisk electrode (type A or B) approaches a glass substrate (Figure 3) or a single protoplast (Figure 4) in a stepwise fashion along the z axis in artificial seawater saturated with air. The tip surface of the type A electrode and the leveled disk surface (W1) of type B were mounted to be parallel to the substrate surface under microscopy. The leveled disk (W1) and the beveled disk (W2) of the type B electrode were used to detect Fe(CN)64- and oxygen. Numbers in these figures represent the distance of the tip-glass substrate (Figure 3) and tip-surface of the protoplast (Figure 4). The redox currents for Fe(CN)64- and oxygen depend on the position of the electrode and show similar traces synchronized with the movement of the electrode. Insets of Figures 3 and 4

(45) Tatewaki, M.; Nagata, K. J. Phycol. 1970, 4, 401-403.

(46) Wightman, R. M. Anal. Chem. 1981, 53, 1125A-1133A.

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Figure 5. Variations of oxidation current for 1.0 mM Fe(CN)64- (a, W1) and reduction current for oxygen (b, W2) when the dual-microdisk electrode of type A scanned (2.4 µm/s) over the microhole (radius, 50 µm; depth, 27 µm) in the glass substrate. Tip-substrate distance, 1 µm. Radii of disks: 0.7 µm for W1; 1.1 µm for W2. Figure 3. Variations of oxidation current for 1.0 mM Fe(CN)64- (a, W1) and reduction current for oxygen (b, W2) at the dual-microdisk electrode of type A when the electrode approaches a glass substrate in a stepwise fashion along the z axis in artificial seawater saturated with air. Insets show the variation of the oxidation current for 1.0 mM Fe(CN)64- (a) and reduction current for oxygen (b) as a function of distance from the glass substrate. Radii of disks: 3.6 µm for W1; 3.8 µm for W2.

Figure 4. Variations of oxidation current for 1.0 mM Fe(CN)64- (a, leveled disk, W1) and reduction current for oxygen (b, beveled disk, W2) at the dual-microdisk electrode of type B when the electrode approaches a single protoplast in steps along the z axis in artificial seawater saturated with air. The oxidation current for Fe(CN)64- was obtained at one disk that was mounted parallel to a substrate under the protoplast. Insets show the variation of the oxidation current for 1.0 mM Fe(CN)64- (a) and reduction current for oxygen (b) as a function of distance from the surface of the protoplast. Light intensity; 20 kLx. The radii of leveled and beveled disks are 3.6 µm for W1 and 3.8 µm for W2.

show the variation of the oxidation current for 1.0 mM Fe(CN)64(a) and the reduction current for oxygen (b) as a function of distance from the glass substrate and the surface of the protoplast, respectively. For the glass substrate, both redox responses become small as the dual-microdisk electrode moves to the 4640 Analytical Chemistry, Vol. 71, No. 20, October 15, 1999

substrate, because the substrate acts as a barrier for Fe(CN)64and oxygen to diffuse from the bulk solution to the electrode surface. Since the hemisphere diffusion region formed at each disk has the size of a disk’s diameter, the decrease of the redox response becomes pronounced when the tip-substrate distances are less than the disk size. For the protoplast under light irradiation with 20 kLx, the oxidation current for Fe(CN)64- decreases while the reduction current for oxygen increases as the electrode approaches the protoplast. The decrease in Fe(CN)64- is due to the barrier effect. The increase of the oxygen reduction current is caused by the photosynthetic generation of oxygen from the protoplast.47 Since oxygen easily permeates the protoplast membrane, no barrier effect is found within the distance of disk size. The reduction current, therefore, reflects the localized concentration of oxygen. The inset of Figure 4b indicates that the oxygen-rich region around the protoplast expounds at least 50 µm to the solution. This difference in the responses for Fe(CN)64- and oxygen can be used for characterization of single protoplasts. Current Profiles over a Glass Substrate with a Microhole and Protoplast. Figure 5 show the variations of the oxidation current for Fe(CN)64- and the reduction current for oxygen when the dual-microdisk electrode (type A) scanned (2.4 µm/s) over a glass substrate with an etched microhole (radius, 50 µm; depth, 27 µm). The tip was positioned ∼1 µm above the glass substrate. The redox responses for Fe(CN)64- and oxygen both increase at the microhole. The space in the microhole ensures the supply of these redox species to the microelectrode by a diffusion which is partially blocked above the unetched area. Note that both current responses simultaneously synchronize with the movement of the electrode. This technique of one line scan can be applied to detection of a single protoplast. Figure 6 shows the variation of responses of the oxidation current for Fe(CN)64- (b) and the reduction current for oxygen (c) under light irradiation (20 kLx) when the dual-disk electrode (type B) scanned along these lines at a constant height (51 µm from the substrate). This figure also shows the microscopic photograph of a single protoplast (radius, 25 µm) with the tip part of a dual-microdisk electrode (type B) which appears as a dark shadow to the left. The tip scans (2.4 µm/s) along the dashed lines A, B, and C in Figure 6a. The oxidation current for Fe(CN)64(47) Yasukawa, T. Uchida, I.; Matsue, T. Denki Kagaku 1998, 66, 660-661.

Figure 6. (a) Microscopic photograph of a single protoplast (radius, 25 µm) and a tip of the dual-microdisk electrode (type B). The tip was placed over the protoplast and scanned (2.4 µm/s) along the dashed lines A, B, and C. (b) Variations of responses of oxidation current to Fe(CN)64- at the leveled disk of W1 and reduction current for oxygen at the beveled disk of W2 upon light irradiation (20 kLx) when the dual-disk electrode of type B scanned along the dashed lines. Fe(CN)64- was detected at the disk parallel to the glass substrate and oxygen at the other disk. The radii of leveled and beveled disks are 0.7 µm for W1 and 1.1 µm for W2.

was monitored at the disk aligned parallel to the glass substrate and the reduction current for oxygen at the disk with an angle of 45° to the substrate. When the tip moves over a protoplast (line A), the oxidation current for Fe(CN)64- decreases due to a blockage of Fe(CN)64- diffusion by the protoplast membrane while the reduction current for oxygen increases because of the generation of oxygen by photosynthesis. When the tip scans along line B, which deviates from the center of the protoplast, the blockage effect of the diffusion of Fe(CN)64- becomes small compared to that of line A. For line C, no variation of response was observed. The response for Fe(CN)64- is very sensitive to the tip-membrane distance, and a noticeable decrease in the response is observed only when the distance is within the size of a disk’s diameter. The response, therefore, fades rapidly as the scanning line shifts from the spherical protoplast. The variations of oxygen current, however, decrease only slightly when the scanning line deviates from the protoplast since the oxygenenriched region around the protoplast expands deeply into the solution. The simultaneous detection of the redox responses for Fe(CN)64- and oxygen affords information on both the photosynthetic activity and the topographic profile of single protoplasts. Dual-SECM Imaging. SECM images of the topography and concentration profile of oxygen can be obtained using a dualmicrodisk electrode. Figure 7 shows dual-SECM images of the topography (a) and photosynthetic activity (b) of a single, living

Figure 7. Dual-SECM images of a single, living protoplast (radius, 25 µm) based on the oxidation current for Fe(CN)64- (a, leveled disk, W1) and the reduction current for oxygen (b, beveled disk, W2). Probe, dual-microdisk electrode (type B). Scan rate: 9.8 µm/s. Concentration of Fe(CN)64-, 1.0 mM. The radii of leveled and beveled disks are 2.8 µm for W1 and 1.2 µm for W2.

protoplast (radius, 25 µm). The dark area with low oxidation currents for Fe(CN)64- (Figure 7a) coincided with the location of the protoplast in an optical microscopic image. The oxidation current decreases because of blocking of diffusion when the tip approaches the protoplast surface. Therefore, the image based on the oxidation current for Fe(CN)64- gives topographic information at the single-cell level. The SECM image based on the reduction current for oxygen (Figure 7b) shows the opposite image. The image of the cell appears as an area with a large reduction in current because of photosynthetic generation of oxygen from the single protoplast. Thus, this image affords information on the photosynthetic activity of individual cells. The above results demonstrate that images of the topography and photosynthetic activity can be obtained at the same time by the SECM system with a dual-microdisk electrode as a probe. ACKNOWLEDGMENT This work was partly supported by Grants-in-Aid for Scientific Research (10145104, 11450323) from the Ministry of Education, Science and Culture, Japan. T.Y. acknowledges a research fellowship from the Japan Society for the Promotion of Science. Received for review March 23, 1999. Accepted August 9, 1999. AC9903104 Analytical Chemistry, Vol. 71, No. 20, October 15, 1999

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