Langmuir 2006, 22, 10299-10306
10299
Articles Topographic, Electrochemical, and Optical Images Captured Using Standing Approach Mode Scanning Electrochemical/Optical Microscopy† Yasufumi Takahashi,‡ Yu Hirano,‡ Tomoyuki Yasukawa,‡ Hitoshi Shiku,‡ Hiroshi Yamada,§ and Tomokazu Matsue*,‡ Graduate School of EnVironmental Studies, Tohoku UniVersity, Aramaki Aoba 6-6-11, Sendai 980-8579, and Department of Applied Chemistry, National Defense Academy, Kanagawa 239-8686 ReceiVed April 29, 2006. In Final Form: July 9, 2006 We developed a high-resolution scanning electrochemical microscope (SECM) for the characterization of various biological materials. Electrode probes were fabricated by Ti/Pt sputtering followed by parylene C-vapor deposition polymerization on the pulled optical fiber or glass capillary. The effective electrode radius estimated from the cyclic voltammogram of ferrocyanide was found to be 35 nm. The optical aperture size was less than 170 nm, which was confirmed from the cross section of the near-field scanning optical microscope (NSOM) image of the quantum dot (QD) particles with diameters in the range of 10-15 nm. The feedback mechanism controlling the probe-sample distance was improved by vertically moving the probe by 0.1-3 µm to reduce the damage to the samples. This feedback mode, defined as “standing approach (STA) mode” (Yamada, H.; Fukumoto, H.; Yokoyama, T.; Koike, T. Anal. Chem. 2005, 77, 1785-1790), has allowed the simultaneous electrochemical and topographic imaging of the axons and cell body of a single PC12 cell under physiological conditions for the first time. STA-mode feedback imaging functions better than tip-sample regulation by the conventionally available AFM. For example, polystyrene beads (diameter ∼6 µm) was imaged using the STA-mode SECM, whereas imaging was not possible using a conventional AFM instrument.
Introduction Scanning electrochemical microscopy (SECM) has been successfully used to investigate various biological systems including membranes,1-3 cells,4-15 enzymes,16-23 and DNA24-29 †
Part of the Electrochemistry special issue. * Corresponding author. E-mail:
[email protected]. Tel and Fax: +81-22-795-7209. ‡ Tohoku University. § National Defense Academy. (1) Amemiya, S.; Ding, Z.; Zhou, J.; Bard, A. J. J. Electroanal. Chem. 2000, 483, 7-17. (2) Wilburn, J. P.; Wright, D. W.; Cliffel, D. E. Analyst 2006, 131, 311-316. (3) Yamada, H.; Matsue, T.; Uchida, I. Biochem. Biophys. Res. Commun. 1991, 180, 1330-1334. (4) Yasukawa, T.; Kaya, T.; Matsue T. Chem. Lett. 1999, 9, 975-976. (5) Yasukawa, T.; Kaya, T.; Matsue T. Anal. Chem. 1999, 71, 4637-4641. (6) Torisawa, Y.; Kaya, T.; Takii, Y.; Oyamatsu, D.; Nishizawa, M.; Matsue, T. Anal. Chem. 2003, 75, 2154-2158. (7) Takii, Y.; Takoh, K.; Nishizawa, M.; Matsue T. Electrochim. Acta 2003, 48, 3381-3385. (8) Nishizawa, M.; Takoh, K.; Matsue, T. Langmuir 2002, 18, 3645-3649. (9) Takoh, K.; Ishibashi, T.; Matsue, T.; Nishizawa, M. Sens. Actuators, B 2005, 108, 683-687. (10) Feng, W.; Rotenberg, S. A.; Mirkin M. V. Anal. Chem. 2003, 75, 41484154. (11) Mauzeroll, J.; Bard, A. J.; Omeed, O.; Monks, T. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 17582-17587. (12) Hengstenberg, A.; Blo¨chl, A.; Dietzel, I. D.; Schuhmann, W. Angew. Chem., Int. Ed. 2001, 40, 905-908. (13) Bauermann, L. P.; Schuhmann, W.; Schulte, A. Phys. Chem. Chem. Phys. 2004, 6, 4003-4008. (14) Liebetrau, J. M.; Miller, H. M.; Baur, J. E.; Takacs, S. A.; Anupunpisit, V.; Garris, P. A.; Wipf, D. O. Anal. Chem. 2003, 75, 563-571. (15) Kurulugama, R. T.; Wipf, D. O.; Takacs, S. A.; Pongmayteegul, S.; Garris, P. A.; Baur, J. E. Anal. Chem. 2005, 77, 1111-1117. (16) Wittstock, G. Fresenius J. Anal.Chem. 2001, 370, 303-315. (17) Wilherm, T.; Wittstock, G. Langmuir 2002, 18, 9485-9493. (18) Shiku, H.; Uchida, I.; Matsue, T. Langmuir 1997, 13, 7239-7244.
because a localized chemical reaction under physiological conditions can be quantitatively characterized in situ in a noninvasive manner. In SECM imaging, the distance between the microelectrode and sample is a crucial parameter.30 Furthermore, significant efforts have been made to improve the lateral resolution and quality of SECM images by incorporating the distance control mechanisms of AFM,19,31-35 shear (19) Kueng, A.; Kranz, C.; Lugstein, A.; Bertagnolli, E.; Mizaikoff, B. Angew. Chem., Int. Ed. 2005, 44, 3419-3422. (20) Oyamatsu, D.; Hirano, Y.; Kanaya, N.; Mase, Y.; Nishizawa, M.; Matsue, T. Bioelectrochemistry 2003, 60, 115-121. (21) Yamada, H.; Fukumoto, H.; Yokoyama, T.; Koike, T. Anal. Chem. 2005, 77, 1785-1790. (22) Horrocks, B. R.; Schmidtke, D.; Heller, A.; Bard, A. J. Anal. Chem. 1993, 65, 3605-3614. (23) Suzuki, M.; Yasukawa, T.; Mase, Y.; Oyamatsu, D.; Shiku, H.; Matsue, T. Langmuir 2004, 20, 11005-11011. (24) Yamashita, K.; Takagi, M.; Uchida, K.; Kondo, H.; Takwnak, S. Analyst 2001, 126, 1210-1211. (25) Liu, B.; Bard, A. J.; Li, Chen-Zhong.; Kraatz, Heinz-Bernhard. J. Phys. Chem. B 2005, 109, 5193-5198. (26) Turcu, F.; Schulte, A.; Hartwich, G.; Schuhmann, W. Angew. Chem., Int. Ed. 2004, 43, 3482-3485. (27) Fortin, E.; Mailley, P.; Lacroix, L.; Szunerits, S. Analyst 2006, 131, 186193. (28) Wang, J.; Song, F.; Zhon, F. Langmuir 2002, 18, 6653-6658. (29) Guckenberger, R.; Heim, M.; Cevc, G.; Knapp, H. F.; Wiegrabe, W.; Hillebrand, A. Science 1994, 266, 1538-1540. (30) Wittstock, G.; Emons, H.; Ridgway, T. H.; Blubaugh, E. A.; Heineman, W. R. Anal. Chem. Acta 1994, 298, 285-302 (31) Hirata, Y.; Yabuki, S.; Mizutani, F. Bioelectrochemistry 2004, 63, 217224. (32) Gardner, C. E.; Unwin, P. R.; Macpherson, J. V. Electrochem. Commun. 2005, 7, 612-618. (33) Dobson, P. S.; Weaver, J. M. R.; Hplder, M. N.; Unwin, P. R.; Macpherson, J. V. Anal. Chem. 2005, 77, 424-434. (34) Macpherson, J. V.; Unwin, P. R.; Hiller, A. C.; Bard, A. J. J. Am. Chem. Soc. 1996, 118, 6445-6452.
10.1021/la0611763 CCC: $33.50 © 2006 American Chemical Society Published on Web 08/11/2006
10300 Langmuir, Vol. 22, No. 25, 2006
force,12,13,20,21,36-46 Faradaic current,15,47,48 and impedance.15,22,49-52 Shear force-based tip-sample distance control is a successful feedback mechanism for obtaining high-resolution images; in particular, the feedback mechanism utilizing a tuning fork37-41 has become the standard system to control the probe position with relatively simple, low-cost system design. The fabrication of a nanometer-sized electrode has been a long-standing objective for electrochemists, and various methods have been introduced during the last 20 years.53-69 Geometries of the electrode probes such as an island disk,53 recessed,53 nanopore,54,55 and conical-shaped56-58 electrodes have been studied. Furthermore, the diffusion and migration effects in the double layer should be considered to solve the diffusion equations for the nanoelectrodes with a 10-nm-level radius,59,70 although these effects were not observed for the electrodes used in the present study. Applications of the nanoelectrodes as SECM probes have been challenging, and the SECM probe should be small at the entire tip including the insulating part. Glass capillary,59-61 Apiezon wax,59 polyimide,59,62 and nail varnish59,63 have been used as the insulating materials, and the fabrication using them facilitated making the nanodes. However, it is still difficult to (35) Fasching, R. J.; Tao, Y.; Prinz, F. B. Sens. Actuators, B 2005, 108, 964972. (36) Betzig, E.; Trautman, J. K.; Wolfe, R.; Gyorgy, E. M.; Finn, P. L.; Kryder, M. H.; Chang, C.-H. Appl. Phys. Lett. 1992, 61, 142-144. (37) Karrai, K.; Grober, R. D.; Appl. Phys. Lett. 1995, 66, 1842-1844. (38) Karrai, K.; Grober, R. D. Ultramicroscopy 1995, 61, 197-205. (39) Koopman, M.; Cambib, A.; Bakkera, B. I.; Joostenb, B.; Figdorb, C. G.; Van Hulst, N. F.; Garcia-Parajoa M. F. FEBS Lett. 2004, 573, 6-10. (40) Koopman, M.; Bakkera, B. I.; Garcia-Parajoa M. F.; Van Hulst, N. F. Appl. Phys. Lett. 2003, 83, 5083-5085. (41) Rensen, W. H. J.; Van Hulst, N. F.; Ruiter, A. G. T.; West, P. E. Appl. Phys. Lett. 1999, 75, 1640-1642. (42) Luwig, M.; Kranz, C.; Schuhmann, W.; Gaub, H. E. ReV. Sci. Instrum. 1995, 66, 2857-2860. (43) Katemann, B. B.; Schulte, A.; Schuhmann, W. Chem.sEur. J. 2003, 9, 2025-2033. (44) James, P. I.; Garfias-Mesias, L. F.; Moyer, P. J.; Smyrl, W. H. J. Electrochem. Soc. 1998, 145, L64-L66. (45) Bu¨chler, M.; Kelley, S. C.; Smyrl, W. H. Electrochem. Solid-State Lett. 2000, 3, 35-38. (46) Garay, M. F.; Ufheil, J.; Borgwarth, K.; Heinze, J. Phys. Chem. Chem. Phys. 2004, 6, 4028-4033. (47) Wipf, D. O.; Bard, A. J.; Tallman, D. E. Anal. Chem. 1993, 65, 13731377. (48) Fan, F.-R. F.; Bard, A. J. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 1422214227. (49) Mario, A.; Alpuche-Aviles. Wipf, D. O. Anal. Chem. 2001, 73, 48734881. (50) Etienne, M.; Schulte, A.; Schuhmann, W. Electrochem. Commun. 2004, 6, 288-293. (51) Gabrielli, C.; Huet, F.; Keddam, M.; Rousseau, P.; Vivier, V. J. Phys. Chem. B. 2004, 108, 11620-11626. (52) Osbourn, D. M.; Sanger, R. H.; Smith, P. J. S. Anal. Chem. 2005, 77, 6999-7004. (53) Shao, Y.; M. Mirkin, V. G.; Kokotov, Fish, S.; Palanker, S.; Lewis, A. Anal. Chem. 1997, 69, 1627-1634. (54) Zhang, B.; Zhang, Y.; White, H. S. Anal. Chem. 2004, 76, 6229-6238. (55) Lemay, S. G.; Van den Broel, D. M.; Storm, A. J.; Krapf, D.; Smeets, R. M. M.; Heering, H. A.; Dekker: C. Anal. Chem. 2005, 77, 1911-1915. (56) Mirkin, M. V.; Fan, F.-R. F.; Bard, A. J. J. Electroanal. Chem. 1992, 328, 47-62. (57) Zoski, C. G.; Mirkin, M. V. Anal. Chem. 2002, 74, 1986-1992. (58) Xiong, X.; Guo, J.; Kurihara, K.; Amemiya, S. Electrochem. Commun. 2004, 6, 615-620. (59) Arrigan, D. W. M. Analyst 2004, 129, 1157-1165. (60) Katemann, B. B.; Schuhmann, W. Electroanalysis 2002, 14, 22-28. (61) Ufheil, J.; Hess, C.; Borgwarth, K.; Heinze, J. Phys. Chem. Chem. Phys. 2005, 7, 3185-3190. (62) Sun, P.; Zhang, Z.; Guo, J.; Shao, Y. Anal. Chem. 2001, 73, 5346-5351. (63) Woo, D.-H.; Kang, H.; Park, S.-M. Anal. Chem. 2003, 75, 6732-6736. (64) Slevin, C. J.; J. G. Nicola.; Macpherson, J. V.; Webb, M. A.; Unwin, P. R. Electrochem. Commun. 1999, 1, 282-288. (65) Zu, Y.; Ding, Z.; Zhou, J.; Lee, Y.; Bard, A. J. Anal. Chem. 2001, 73, 2153-2156. (66) Lee, Y.; Bard, A. J. Anal. Chem. 2002, 74, 3626-3633. (67) Lee, Y.; Ding, Z.; Bard, A. J. Anal. Chem. 2002, 74, 3634-3643. (68) Chen, S.; Kucernak, A. J. Phys. Chem. B 2002, 106, 9396-9404. (69) Watkins, J. J.; Chen, J.; White, H. S.; Abruna, H. D.; Maisonhaute, E.; Amatore, C. Anal. Chem. 2003, 75, 3962-3971.
Takahashi et al.
make the nanoprobe, including the insulating part. The use of electrophoretic paints,59,64-69 or parylene C,19 is appropriate to minimize the whole tip size with relatively higher yields. The use of a combination of topographic, electrochemical, and optical information during scanning microscopy has recently been proposed.65-67,71-74 Smyrl et al. reported an SECM/optical microscope (OM) study in which a microelectrode probe that employs a gold-sputtered optical fiber coated with an insulating polymer was used to investigate the electrochemical/photochemical properties of TiO2/Ti surfaces.71,72 Bard et al. observed simultaneous topographic, electrochemical, and optical signals on an interdigitated array (IDA) using an optical fiber electrode combined with a shear force feedback distance control mechanism. 67 Despite remarkable improvements,75, 76 the imaging of fragile biological samples such as living mammalian cells under physiological conditions is still difficult for SECM and the other scanning probe techniques. The characterization of living cells by SECM has been the focus of attention for a long time; however, most of these characterizations were carried out in constantheight mode. Although several groups have successfully accomplished the constant-distance mode of an SECM system, the simultaneous imaging of the topographic and biofunctional information of individual living cells has not been successfully performed in any study. In this article, we describe the fabrication of a conical electrode probe comprising an optical fiber or a glass capillary and an improved probe-sample distance control mechanism. To achieve constant-distance imaging, the probe was moved vertically by 0.1-3 µm to reduce the damage to the samples at each data point.21 This feedback mode, defined as the standing approach (STA) mode, permits extensive applications of the probe to samples with large height differences on the surface or fragile biomaterials including microbeads or living cells. Experimental Section Materials. Horseradish peroxidase (HRP, EC 1.11.1.7)-labeled antimouse IgG was purchased from ICN Pharmaceuticals, Inc. Ferrocenylmethanol (FcMetOH) was purchased from Aldrich Chemicals. Potassium hexacyanoferrate (III), D-(+)-glucose, NaCl, KCl, Na2SO4‚10H2O, and glutaraldehyde (GA) were purchased from Wako Pure Chemicals (Osaka, Japan) and used without further purification. Calcein AM and MitoTracker Green FM were purchased from Molecular Probes and used for fluorescence imaging. All of the solutions were prepared using purified water from a Milli-Q II system (Millipore). PBS was prepared from 7.2 mM Na2HPO4‚ 12H2O, 2.8 mM KH2PO4, and 150 mM NaCl (pH 7.0). RPMI-1640 was purchased from Gibco. Amino-coated beads and fluorescent beads with diameters of 6 µm and 500 nm, respectively, were purchased from Polyscience Inc. Q dots (with diameters of 10-15 nm, Qdot705) were purchased from Quantum Dot Corporation. Measurement System. The configuration of the SECM/OM system with feedback control of the probe-sample distance was basically the same as that reported previously by Oyamatsu20 with the addition of optical components in the former. Electrochemical (70) Rubinstein, I. Physical Electrochemistry: Principles, Methods, and Applications; Marcel Dekker: New York, 1995. (71) James, P.; Casillas, N.; Smyrl, W. H. J. Electrochem. Soc. 1996, 143, 3853-3865. (72) Shi, G.; Garfias-Mesias, L. F.; Smyrl, W. H. J. Electrochem. Soc. 1998, 145, 2011-2016. (73) Szunerits, S.; Walt, D. R. ChemPhysChem. 2003, 4, 186-192. (74) Maruyama, K.; Ohkawa, H.; Ogawa, S.; Ueda, A.; Niwa, O.; Suzuki, K. Anal. Chem. 2006, 78, 1904-1912. (75) Korchev, Y. E.; Raval, M.; Lab, M. J.; Gorelik, J.; Edwards, C. R. W.; Rayment, T.; Klenerman, D. Biophys. J. 2000, 78, 2675-2679. (76) Gorelik, J.; Zhang, YJ.; Sanchez, D.; Shevchuk, A.; Frolenkov, G.; Lab, M.; Klenerman, D.; Edwards, C.; Korchev, Y. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 15000-15005.
Standing Approach Mode Optical Microscopy measurements were performed using a two-electrode configuration with a Ag/AgCl (sat. KCl) reference electrode. The current signal was amplified with a high-gain current amplifier (Keithley, model 427) and converted to digital data using a 16-bit D/A board (Interface, PCI-3310). For optical measurements, a laser beam from a solidstate laser unit (λ ) 473 nm, Shimadzu HK-5510) was introduced through the optical fiber. The laser light transmitted through the sample and the fluorescence emitted from the sample were collected using an objective lens (CFI Fluor 20×, 0.45 NA, Nikon), detected by a PMT (Hamamatsu H5920-01), and converted to digital data using a pulse counter board (Interface PCI-6202). The PMT was attached to the side port of the microscope. For the probe-sample distance control, we used a tuning fork. The properties of the tuning fork are almost the same as those reported by Karrai.37,38 The probe was attached to one of the legs of a tuning fork and vibrated using a piezoelectric buzzer to drive the tuning fork into the resonance state. The resonance frequency of the unprocessed tuning fork was 32 768 (215) Hz. The buzzer was driven by an AC signal (0.1-10 mV p-p, sine wave) from a reference function generator equipped with a digital lock-in amplifier (NF Corp., LI-5640). When the probe was positioned away from the substrate, the vibration from the buzzer induced a voltage signal in the tuning fork due to the piezoelectric effect. The amplitude of the induced signal was 10-100 µV, and its frequency was the same as that of the driving signal. The induced signal was detected by the digital lock-in amplifier. As the distance between the probe and the substrate became less than 100 nm, the shear force between them decreased the magnitude of the vibration of the tuning fork. A decrease in the induced signal was digitized and acquired by a 16-bit A/D converter (Interface, PCI-3178) equipped in the PCI bus of an IBMcompatible PC. Feedback Control Using STA Mode. The STA mode features repetitive approaches to the surface and retractions of the probe for measurement at each point. Namely, a vertical motion of the probe, which is similar to the “engage” function for the commercial AFM produced by Digital Instruments, has been performed at each (x, y) data point during imaging. The STA-mode program used in this work is basically the same as that developed by Yamada,21 but we adopted PI (proportional-integral) feedback control20 in the approach process. The PI feedback was adapted to the vertical approach process in this work, whereas it was adapted to the lateral scan in ref 20. Details of STA-mode programming were described in Supporting Information. The probe approached the surface until the tuning fork was damped to 5.0-7.5% of the original amplitude. The probe was retraced 0.1-3 µm upward to avoid contacting the surface at each data point. Sample Preparation. (a) HRP-Immobilized Beads on (3Aminopropyl)triethoxysilane (APS)-Coated Glass. An APS-coated glass slide (Matsunami Glass Ind., Ltd.) was dipped into a 1% (v/v) glutaraldehyde (GA) aqueous solution for 60 min. Amino-coated beads conjugate (100 µL) (Fluoresbrite Carboxylate Microspheres Polyscience, Inc.; 2.1 × 108 particles mL-1 and a mean diameter of 6.0 µm) was incubated with 50 µL of a PBS solution containing 600 µg mL-1 HRP-labeled antimouse IgG for 60 min and was rinsed three times with the buffer solution. The bead suspension was dropped onto the GA/APS-coated glass slide and dried after incubating for 60 min. (b) Cell Culture. Stock PC12 cells were donated by the Cell Resource Center for Biomedical Research, Tohoku University, and placed in an RPMI-1640 medium (Gibco) supplemented with 10% heat-inactivated fetal bovine serum (Gibco) and 1% penicillinstreptomycin (Gibco) in a 5% CO2, 100% humidity atmosphere at 37 °C. The nerve growth factor (7s NGF, Gibco) from a mouse was added (50 ng mL-1) to the medium to differentiate the neuronal PC12 cells. After 3 days of cultivation, the PC12 cells attached to polystyrene dishes (Falcon) were coated with collagen (Research Institute for the Functional Peptides) and differentiated to extend their axons. NGF promotes the differentiation of the PC12. Collagen coating facilitates cell adhesion to the dish. (c) E. coli Expressing GFP. Escherichia coli BL21 competent cells were purchased from TAKARA Bio Inc. These cells were
Langmuir, Vol. 22, No. 25, 2006 10301
Figure 1. Optical (a) and SEM (b) images of glass capillary electrode probes. (c) Cyclic voltammogram of a glass capillary electrode probe in 4 mM K4Fe(CN)6, 0.1 M KCl at a scan rate of 10 mV/s. transformed using a plasmid pQBI T7-GFP (Wako). A 900 µL SOC medium (Invitrogen) was added, and the culture was stirred at 37 °C for 60 min. It was then scattered in agar an medium contained in ampicillin and incubated. Fabrication of an Optical Fiber Electrode. In the present study, we used four types of conical probes: optical fiber electrode, optical fiber, glass capillary electrode, and glass capillary. The optical fiber electrode probes were fabricated as follows. A single-mode optical fiber (New Port, F-SA) was pulled using a carbon dioxide laser puller (Sutter Instruments, model P-2000). This optical fiber was then coated by Ti/Pt sputtering (Anelva, L-332S-FH, RF200, thickness of 280 nm) and subsequently insulated with a 900 nm layer of xylene polymer (parylene C, Daisan Kasei) via vapor deposition polymerization (PDS-2010, Parylene Japan). To expose an electroactive area on the SECM tip, the insulating layer of parylene C was removed by blowing hot air (at 450 °C) vertically onto the probe tip using a hot-air heater (PJ-208A). The optical fiber electrode probe was required for both electrochemical and optical measurements. An optical aperture was formed by repeatedly tapping the probe onto the glass substrate under shear force feedback regulation until an optical signal was observed. The fabrication of an optical fiber electrode probe requires high skill, and ca. 20% of this type of probe is successfully fabricated. Therefore, such probes were used only for simultaneous topographic, electrochemical, and optical imaging. For dual imaging with topography and optical observations, these probes without the parylene C coating were used. For electrochemical measurements, glass capillary electrode probes were fabricated as follows. A glass capillary (World Precision Instruments, Inc., PG10165-4) was pulled using a capillary puller (Narishige PE-21), equipped with a Pt ribbon heater. The processes for metal deposition and parylene C coating were the same as those used for the optical fiber electrode probes. The pulled glass capillary probes without metal and parylene C coatings were used for the topography measurements. The tip of the probe was investigated with an SEM (JEOL, JSM-5310LV, accelerating voltage of 10 kV). Because the insulator (parylene C) is transparent, the Pt film deposited on the glass capillary surface was observed.
10302 Langmuir, Vol. 22, No. 25, 2006
Figure 2. Optical and topographic images of the deposited Pt pattern (a) and the cross section (b). The white line in part a indicates the position of the cross section. Topography of a red blood cell fixed with glutaraldehyde (c) and microbeads with a diameter of 2 µm (d) captured in STA mode using capillary probes. The following scan ranges were used: 5 µm × 2.5 µm (a), 20 µm × 20 µm (c), and 20 µm × 20 µm (d).
Results and Discussion Characterization of the Conical Electrode Probe. Part a and b of Figure 1 show the optical and SEM images of the glass capillary electrode probe, respectively. According to the SEM image, the radius of curvature of the tip including the insulator part was ca. 250 nm, and the intense brightness near the apex was due to the charging effect with parylene C. The probe tip angle was less than 10°, which allows extensive applications of the probe to samples with a relatively larger height difference (greater than 10 µm), including microbeads or living cells and tissues. The border of the xylene polymer and the electroactive area are not observed in the image. Figure 1c shows a cyclic voltammogram for the glass capillary electrode probe observed in 4 mM K4Fe(CN)6, 0.1 M KCl at a scanning rate of 10 mV/s. The apparent electrode radius was found to be 35 nm as estimated from the steady-state current (35 pA). This effective electrode radius is reasonably smaller than the radius of curvature of the tip including the insulator part. Thus, it is difficult to determine the electroactive area in the SEM image. The probe fabrication process used in the present study is a relatively simple process. In this process, polishing the tip end to expose the conducting area and applying an anodic pulse to form an insulating layer are not required. Metal deposition was performed only once in this process; however, in a conventional process, multistep sputtering or horizontal rotation is required at the stage where the probes are attached during deposition60-64 in order to obtain a homogeneous metal layer. Improved Topographic Images Captured Using the Conical Probe and STA Mode. The advantages of our STA mode of the SECM/OM system employing the pulled optical fiber and glass capillary probes can be directly observed in the topographic images captured using the capillary probes in STA mode in air. Figure 2a and b shows the optical, topographic images and cross section of the deposited Pt pattern with a thickness of 70 nm. The cross section was taken along with the white line in the topography image. This STA-mode system allows a precise
Takahashi et al.
Figure 3. Topography (a) and electrochemical (b) images of HRPimmobilized polystyrene microbeads with a diameter of 6 µm imaged in STA mode using a capillary electrode probe. Electrochemical imaging (b) was performed at a probe potential of +50 mV (vs Ag/AgCl) in 0.5 mM FcMetOH, 0.5 mM H2O2, and 0.2 M PBS with a scan range of 18 µm × 18 µm and a step size of 1.0 µm. (c) Cross sections of the topographic and electrochemical images. The white lines in a and b indicate the positions of the cross section.
Figure 4. (a) Topography of a single PC12 cell in culture medium RPMI-1640 in STA mode using a capillary probe. The scan range was 40 µm × 40 µm, and the step size was 1.0 µm. Optical (b) and fluorescence (c) images of the sample PC12 cells. Calcein AM was introduced into the culture dish after STA-mode scanning, as shown in part a, and washed three times with PBS before the optical and fluorescence images were obtained. The squares shown in b and c indicate the exact scanning area.
vertical dimension resulting in exactly the same thickness as that obtained by a conventional AFM (Veeco, Dimension3000). Probes with a narrow tip angle are important for imaging sample surfaces with relatively larger height differences, such as a fixed red blood cell (Figure 2c) and polystyrene microbeads with a diameter of 2 µm (Figure 2d). The STA-mode system based on the shear force feedback distance control mechanism21,23 can successfully trace a relatively larger height difference (∼10 µm), which is difficult to image using a conventional AFM. This can be attributed to a difference in the amplitude of the vertical oscillations required to approach the sample surface. In our STAmode SECM system, the width of the vertical motion at each data point was 0.1-3 µm, whereas it is 50 nm in a conventional tapping-mode AFM.
Standing Approach Mode Optical Microscopy
Langmuir, Vol. 22, No. 25, 2006 10303
Figure 5. Topography (a) and electrochemical (b) images of PC12 in a PBS solution in STA mode using a capillary electrode probe. Electrochemical imaging was performed at a probe potential of -0.50 V (vs Ag/AgCl). The cross sections of the cell body (c) and axons (d) were shown for the topographic and electrochemical signals. The scan range was 47 µm × 47 µm, and the step size was 1.0 µm. The white lines in a and b indicate the position of the cross section.
A major disadvantage of the STA mode is that the acquisition time for imaging is long compared to the conventional shear force feedback mode.20 It sometimes caused solution evaporation. For the conventional shear force detection in solution, probe immersion of less than 1 mm is allowable to prevent a decrease in the tuning fork Q factor. We circumvent this problem by advancing the tuning fork Q factor. The probe protruded 4 to 5 mm out of the prong’s end, which was longer than the probe that was conventionally fabricated (∼1 mm). The probe is notably lightweight and thin compared to the conventional electrode. The glass capillary electrode probe has been found to be less affected by the viscosity resistance of the solution. Namely, the shift between in-air and in-liquid resonance frequencies was much smaller than that for the conventional electrode probe. The Q factors for the glass capillary electrode probe in air and in liquid were about 800-1200 and 400, respectively. In liquid, the probe was dipped 2 mm into the measuring solution. HRP-immobilized polystyrene microbeads with a diameter of 6 µm were imaged using the STA-mode SECM with a capillary electrode probe, as observed in Figure 3. The measuring solution contains 0.5 mM FcMetOH, 0.5 mM H2O2, and 0.2 M PBS. H2O2 at 0.5 mM is the optimum concentration for HRP imaging with SECM.77 The potential of the probe electrode was set at 50 mV (vs Ag/AgCl). The topographic image in Figure 3a indicates that the bead height is 5.9 µm, which is in accord with the actual size. The HRP catalyzes the reduction of H2O2 and the oxidation of a suitable donor molecule. In the present system, FcMetOH was oxidized to [FcMetOH]+ by HRP catalysis at the microbead (77) Limoges, B.; Saveant, J.-M.; Yazidi, D. J. Am. Chem. Soc. 2003, 125, 9192-9203.
Figure 6. Optical (a) and fluorescence (b) images of PC12 cells stained with MitoTracker Green FM obtained using confocal laser microscopy. Fluorescence images c and d were obtained in constantheight mode with an optical fiber probe. The height was 15 µm. The excitation and emission wavelengths were 490 and 516 nm. The scan ranges were 80 µm × 80 µm (c) and 30 µm × 18 µm (d).
surface. [FcMetOH]+ was reduced to FcMetOH at the microelectrode probe. The reduction current profile for [FcMetOH]+
10304 Langmuir, Vol. 22, No. 25, 2006
Takahashi et al.
Figure 7. (a) Fluorescence image of the fluorescent beads with a diameter of 500 nm. Topographic (b) and fluorescence (c) images of GFP-E. coli. The scan ranges are 10 µm × 10 µm (a) and 20 µm × 20 µm (b and c).
respectively. These values are in good agreement with the height obtained by constant-current imaging.15 The reduction currents for oxygen at the center and the exterior of the cell were -1.5 and -3.0 pA, respectively. If we assume that the shape of the cell is hemispherical, then the respiration rate by a single PC12 cell can be calculated on the basis of the hemispherical diffusion theory expressed by
F ) 2πrsD∆C
Figure 8. Cross section of the fluorescence image of the Q dots with diameters in the range of 10-15 nm.
(Figure 3b) was in good agreement with the microbead topography (Figure 3c) because of the conical probe. Topographic and Electrochemical Imaging of a Single Living Cell under Physiological Conditions. Before we discuss the simultaneous topographic and electrochemical imaging of living cells using a capillary electrode probe, the damage to living cells during imaging in STA mode should be mentioned. Figure 4a shows the topography of a single PC12 cell in a culture medium (RPMI-1640) in STA mode using a glass capillary probe. This experiment required a total of 60 min for a scan range of 40 × 40 µm2 with 1600 data points. The topography of the PC12 cell shows a lower lateral resolution; however, the axons and the cell body are still distinguishable. The height of the PC12 cell was found to be ∼8 µm. The topographic image is noisy as a result of the viscosity of the medium. The voltage applied to the piezoelectric buzzer was set to be very low (0.6 mV) in order to reduce the force interactions with the sample surface. The set-point value was increased by a small amount (7.5% of the original amplitude), and the response signal amplitude from the tuning fork was set to approximately 15 µV. In this case, the probe-sample interaction force was estimated to be less than 10 nN. 37-40 After STA-mode scanning, calcein AM was introduced into the culture dish with a final concentration of 0.5 µM and stained for 15 min. Parts b and c of Figures 4 show the optical and fluorescence images of the resulting PC12 cells, respectively. The squares in the images indicate the scanning area for topographic measurement. All of the cells in the image emit green fluorescence at an identical level, indicating that all of the cells have no obvious damage due to the probe-sample interaction during scanning. Figure 5 shows topography and oxygen reduction current images simultaneously obtained for a single PC12 cell in a PBS solution. The potential of the probe electrode was set at -0.50 V (vs Ag/AgCl) to reduce oxygen. The other measurement conditions were basically similar to those shown in Figure 4. The heights of the axon and the cell body are 3.2 and 8.8 µm,
where rs is the radius of a single cell (12 µm), D is the diffusion constant for oxygen at room temperature (2.1 × 10-5 cm2 s-1), and ∆C is the difference in oxygen concentration between the surface and out of the cell. Because the ∆C value is determined from the current response to be 105 µM, the F value is found to the 1.7 × 10-14 mol s-1, which is in good agreement with that previously reported ((0.3-1.0) × 10-14 mol s-1).8 It is noteworthy that the respiration activity of a single cell reported here and that of the others were obtained by using probe electrodes of different sizes. In the present case, the probe electrode is extremely small, and the oxygen reduction current (Itip) observed at the microelectrode tip was ca. 3.0 pA. The oxygen consumed by the probe electrode was 7.8 × 10-18 mol s-1 (Ftip ) Itip/nF), which was negligibly smaller than the estimated respiration rate of a single cell. The respiration rate of a single cell can also be estimated from the cell suspension or the assembled cell mass, ranging from 10-17 to 10-16 mol s-1,78,79 which is considerably different from that obtained in the present study. Although the oxygen reduction current could fluctuate during STA-mode imaging because the tip-sample distance is very small (less than 100 nm), it is possible that the oxygen reduction current reflects the local oxygen concentration. Furthermore, because the cellular membrane is highly oxygen-permeable, the oxygen reduction current should be greater at the cell surface than at the culture dish surface during constant-distance tip regulations. Thus, we conclude that the oxygen reduction current can be attributed to cell respiration. In Figure 5, the axons and cell body can be observed in both the topographic and electrochemical images, which have been visualized for the first time in the present study in the STA-mode system using the conical capillary electrode probe. The mitochondria in the PC12 cell have been fluorescently labeled using MitoTracker Green FM, a mitochondria-selective dye that is retained in living cells. Parts a and b of Figure 6 show the optical and fluorescence images simultaneously obtained using confocal laser microscopy, respectively. The mitochondria are located in both the axons and cell body. Constant-height mode imaging (Figure 6c and d) with the present system also (78) Torisawa, Y.; Shiku, H.; Yasukawa, T.; Nishizawa, M.; Matsue, T. Sens. Actuators, B 2005, 108, 654-659. (79) Torisawa, Y.; Takagi, A.; Shiku, H.; Yasukawa, T.; Matsue, T. Oncol. Rep. 2005, 13, 1107-1112.
Standing Approach Mode Optical Microscopy
Langmuir, Vol. 22, No. 25, 2006 10305
Figure 9. Electrochemical (a and d), optical (b and e), and topographic (c) images of a Au band array obtained in constant-height mode (a and b) and in STA mode (c, d, and e) using an optical fiber electrode probe. Electrochemical imaging (b and e) was performed at a probe potential of +0.50 V (vs Ag/AgCl) in 0.5 mM FcMetOH, 0.1 M KCl. In constant-height mode, the height was set at 3 µm above the glass part of the sample, and scan rate was 1 µm/s. The scan range was 40 µm × 30 µm, and the step size was 1.0 µm. The acquisition time for constant-height mode was 20 min, and that for constant-distance mode was 60 min. The approximate size of this probe was 6.5 µm.
shows the heterogeneous distribution of fluorescence emitted from mitochondria in a single cell. This contribution of mitochondria in the axons and cell body might be the cause of the heterogeneity of the oxygen concentration at the cell surface observed in STA-mode SECM imaging (Figure 5b), although a detailed correlation among the images is not possible at present. Topographic and Fluorescence Near-Field Imaging Using Optical Fiber Probes. The fabrication procedure of the conical electrode probe with metal deposition can be used for an optical fiber probe. The metal layer plays the role of the electrode as well as a light barrier to confine the light irradiation within the probe. Figure 7a shows a fluorescence image of fluorescent beads with a diameter of 500 nm. On the basis of the topographic observation (data not shown), the averaged height and width of the microbeads were 500 nm and 1.2 µm, respectively. The width of the fluorescence cross section of the single bead was found to be about 600 nm, suggesting that the horizontal resolution of fluorescence is better than that of topography. The faint optical signals observed on the left side of each bead are due to the light scattered from the probe. Parts b and c of Figure 7 show the topographic and fluorescence images of E. coli cells expressing GFP (GFP-E. coli.). Inhomogeneous fluorescence distribution within the E. coli cells was observed at the single-cell level. On the basis of the cross section of the topography, the height of E. coli was found to be 490 nm. Figure 8 shows the cross section of a fluorescence image of the Q dots with diameters in the range of 10-15 nm. The width of the averaged cross section of a single Q dot was 170 nm. The spatial resolution was defined by the half length of the maximum photon intensity. These results indicate that the lateral resolution of the optical image obtained using the conical probe has been beyond the diffraction limit (λ/2); this is indicative of a successful near-field optical measurement. Simultaneous Topographic, Electrochemical, and Optical Imaging. Finally, simultaneous topographic, electrochemical, and optical imaging was performed in STA mode using an optical fiber electrode probe. Figure 9 shows the images of an Au
Figure 10. Cross sections of current (a) and optical (b) signals of the image shown in Figure 9 obtained in constant-height and STA modes.
band array (width of 5 µm and gap of 5 µm) obtained in constantdistance mode (STA mode) (c-e) and in constant-height mode (a and b). In constant-height mode, the probe was positioned 3 µm above the sample surface (from the glass part) in order to prevent the probe tip from making contact with the sample surface during scanning. The measuring solution contained 0.5 mM FcMetOH and 0.1 M KCl. The potential of the probe electrode was set at 0.50 V (vs Ag/AgCl) to oxidize FcMetOH to [FcMetOH]+. When the probe moves above the Au band, the electrochemical signal increases as a result of redox cycling, and the optical signal decreases. The constant-distance image permits a higher contrast than the constant-height mode because the feedback effect strongly depends on the probe-sample distance. Figure 10 shows the cross sections of Figure 9. Because the probe-sample distance is very small (less than 100 nm) in the constant-distance measurements, the positive feedback effect due to redox cycling between the probe and substrate becomes dominant, resulting in the large signal gain to offer the refined contrast on the sample surface. For the optical measurement, the optical signals obtained at the Au band edge were overshot as a result of the scattering light, suggesting that the optical
10306 Langmuir, Vol. 22, No. 25, 2006
aperture was not small enough to obtain near-field lateral resolution with the probe used. The undesirable scattered light caused by the sample’s topography was suppressed in STA mode. Probe-sample distance control has been found to be necessary to carry out simultaneous topographic, current, and optical imaging.
Conclusions In the present study, conical optical fiber/glass capillary electrodes were fabricated and used as probes for SECM/OM systems in STA mode on the basis of a shear force probesample distance control mechanism. To control the shear force, we used a tuning fork and applied a very low voltage (∼0.6 mV) to the piezoelectric buzzer when the fork was at its resonance frequency. The conical probe tip vertically approached the sample surfaces in order to reduce the damage at each data point during imaging. HRP-immobilized polystyrene microbeads with a diameter of 6 µm were clearly imaged by SECM in STA mode, whereas it was difficult to image the microbeads by using a conventional AFM system because of a larger height difference in the sample surface. The simultaneous topographic and electrochemical images of a single living PC12 cell were visualized for the first time. The image clearly showed the axons
Takahashi et al.
and the cell body at the single-cell level. Fluorescence images obtained using an optical fiber probe demonstrated that the lateral resolution was greater than the diffraction limit (170 nm). Finally, simultaneous topographic, electrochemical, and optical imaging was performed in STA mode using an optical fiber electrode probe. Acknowledgment. This work was partially supported by a Grant-in-Aid for Scientific Research on Priority Areas (417) from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of the Japanese Government. It was also supported by Grants-in-Aid for Scientific Research (18101006 and 15201030) from the Ministry of Education, Science and Culture, Japan, and a research grant from the Center for Interdisciplinary Research, Tohoku University. Supporting Information Available: Experimental details, including PI feedback control, schematic of the STA-mode program, fabrication of the probe, evaluation of probe-cell interaction damage, the measurement system, and the tuning fork and fluorescence signals. This material is available free of charge via the Internet at http://pubs.acs.org. LA0611763
