Remote Fluorescence Imaging of Dynamic Concentration Profiles with

Nov 5, 2004 - Christian Amatore,† Arnaud Chovin,‡ Patrick Garrigue,‡ Laurent Servant,§ Neso Sojic,*,‡. Sabine Szunerits,‡ and Laurent Thoui...
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Anal. Chem. 2004, 76, 7202-7210

Remote Fluorescence Imaging of Dynamic Concentration Profiles with Micrometer Resolution Using a Coherent Optical Fiber Bundle Christian Amatore,† Arnaud Chovin,‡ Patrick Garrigue,‡ Laurent Servant,§ Neso Sojic,*,‡ Sabine Szunerits,‡ and Laurent Thouin†

De´ partement de Chimie, Ecole Normale Supe´ rieure, UMR CNRS 8640 ‘PASTEUR’, 24 rue Lhomond, 75231 Paris Cedex 05, France, and Laboratoire d’Analyse Chimique par Reconnaissance Mole´ culaire, Universite´ Bordeaux I, ENSCPB, 16 avenue Pey-Berland, 33607 Pessac, France, and Laboratoire de Physico-Chimie Mole´ culaire, Universite´ Bordeaux I, 351 Cours de la Libe´ ration 33405 Talence, France

Dynamic concentration profiles within the diffusion layer of an electrode were imaged in situ using fluorescence detection through a multichannel imaging fiber. In this work, a coherent optical fiber bundle is positioned orthogonal to the surface of an electrode and is used to report spatial and temporal micrometric changes in the fluorescence intensity of an initial fluorescent species. The fluorescence signal is directly related to the local concentration of a redox fluorescent reagent, which is electrochemically modulated by the electrode. Fluorescence images are collected through the optical fiber bundle during the oxidation of tris(2,2′-bipyridine)ruthenium(II) to ruthenium(III) at a diffusion-limited rate and allow the concentration profiles of Ru(II) reagent to be monitored in situ as a function of time. Tris(2,2′-bipyridine)ruthenium(II) is excited at 485 nm and emits fluorescence at 605 nm, whereas the Ru(III) oxidation state is not fluorescent. Our experiments emphasize the influence of two parameters on the micrometer spatial resolution: the numerical aperture of optical fibers within the bundle and the Ru(II) bulk concentration. The extent of the volume probed by each individual fiber of the bundle is discussed qualitatively in terms of a primary inner-filter effect and refractive index gradient. Experimentally measured fluorescence intensity profiles were found to be in very good agreement with concentration profiles predicted upon considering planar diffusion and thus validate the concept of this new application of imaging fibers. The originality of this remote approach is to provide a global view of the entire diffusion layer at a given time through one single image and to allow the time expansion of the diffusion layer to be followed quantitatively in real time. A wide range of processes in biology and chemistry are controlled by direction, shape, and extension of concentration gradients. For example, concentration gradients of chemoattrac* To whom correspondence should be addressed. E-mail: [email protected]. † Ecole Normale Supe´rieure. ‡ Laboratoire d’Analyse Chimique par Reconnaissance Mole´culaire, Universite´ Bordeaux I. § Laboratoire de Physico-Chimie Mole´culaire, Universite´ Bordeaux I.

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tant or chemorepellent substances (e.g., nerve growth factor, folic acid, cAMP, Ca2+) influence the guiding of axons to appropriate targets during the development of the nervous system.1,2 Corrosion, phase-transfer reactions, bacterial chemotaxis, and synaptic transmission illustrate other important aspects of the role played by mass transport in various areas. Concentration profiles of electroactive species are also of fundamental importance in electrochemical studies since they determine the current. However, complete concentration profiles provide more information far away from the electrode than measured currents since these reflect only the concentration gradient adjacent at the electrode surface. Monitoring the concentration profiles of electroactive species offers therefore the opportunity to discriminate between ambiguous reaction mechanisms, which are not accessible otherwise by simple current measurements.3 A variety of complementary analytical methods has thus been developed to map localized flux of electroactive species. The majority of reported detection strategies for concentration profiles are based on scanning techniques. The principle of local sampling techniques is to analyze a microenvironment with electrochemical or optical probes. To construct the global concentration profiles, measurements have to be repeated at different locations by scanning the entire diffusion layer. Microelectrode probes in potentiometric or amperometric modes have been applied to monitor concentration profiles near various interfaces.4-8 Scanning electrochemical microscopy was applied to a wide range of studies in numerous electrochemical and biological systems.9 Ultramicroelectrode probes have also been employed to study simple and complex mechanistic situations involving conpropor(1) Mueller, B. K. Annu. Rev. Neurosci. 1999, 22, 351-388. (2) Song, H. J.; Poo, M. M. Curr. Opin. Neurobiol. 1999, 9, 355-363. (3) Amatore, C.; Szunerits, S.; Thouin, L.; Warkocz, J.-S. Electroanalysis 2001, 13, 646-652. (4) Engstrom, R. C.; Weber, M.; Wunder, D. J.; Burgess, R.; Winquist, S. Anal. Chem. 1986, 58, 844-848. (5) Engstrom, R. C.; Meaney, T.; Tople, R.; Wightman, R. M. Anal. Chem. 1987, 59, 2005-2010. (6) Kupper, M.; Schultze, J. W. Electrochim. Acta 1997, 42, 3023-3031. (7) Wei, C.; Bard, A. J.; Nagy, G.; Toth, K. Anal. Chem. 1995, 67, 1346-1356. (8) Slevin, C. J.; Unwin, P. R. Langmuir 1997, 13, 4799-4803. (9) Bard, A. J.; Fan, F.-R. F.; Mirkin, M. V. In Electroanalytical Chemistry; Bard, A. J., Rubinstein, I., Eds.; Marcel Dekker: New York, 1994; Vol. 18. 10.1021/ac049017g CCC: $27.50

© 2004 American Chemical Society Published on Web 11/05/2004

Figure 1. (A) Fluorescence image of a coherent optical fiber bundle (270-µm diameter) immersed in 30 mM Ru(bpy)32+ and 0.6 M Na2SO4. The CCD exposure time was 10 ms. (B) Schematic representation of the experimental setup (see text and Experimental Section for details). The white box represents a typical region of interest from which the fluorescence intensity was acquired. The z-axis represents the distance from the electrode.

tionation reaction or redox catalysis.3,10,11 All these electrochemical methods are very powerful as they provide spatial and temporal resolutions, respectively, in the micrometer and millisecond range depending on the size of the probe and techniques applied. Confocal resonance Raman microspectroscopy12,13 and fluorescence confocal laser scanning microscopy14 are examples of scanning optical techniques that supply spatially resolved information on the concentration profiles at microelectrode surfaces. Beside the scanning techniques, the complete imaging of the diffusion layer in a single experiment is another approach to obtain the same information. McCreery et al. have reported an absorption method, which provides global concentration versus distance profiles without scanning the sample. Using a collimated laser (10) Amatore, C.; Pebay, C.; Scialdone, O.; Szunerits, S.; Thouin, L. Chem. Eur. J. 2001, 7, 2939. (11) Baltes, N.; Thouin, L.; Amatore, C.; Heinze, J. Angew. Chem., Int. Ed. 2004, 43, 1431-1435. (12) Amatore, C.; Bonhomme, F.; Bruneel, J.-L.; Servant, L.; Thouin, L. J. Electroanal. Chem. 2000, 484, 1-17. (13) Rey, I.; Bruneel, J.-L.; Grondin, J.; Servant, L.; Lasse`gues, J.-C. J. Electrochem. Soc. 1998, 145, 3034-3042. (14) Cannan, S.; Macklam, I. D.; Unwin, P. R. Electrochem. Commun. 2002, 4, 886-892.

beam passing parallel to a planar electrode, the image of the diffusion layer is projected onto a photodiode array detector.15-17 Engstrom et al. have used luminescence imaging of fluorescent18,19 or electrochemiluminescent (ECL) species20 to visualize various interfacial processes. ECL imaging has also been applied to monitor the spatial distribution of current density.21,22 We wish to show here that diffusion layers can be dynamically imaged in a single experiment using an optical fiber bundle. An imaging fiber (Figure 1A) is a bundle comprising thousands of individually cladded 3-4-µm-diameter optical fibers (also called cores). The coherent architecture of the fiber array allows (15) Pruiksma, R.; McCreery, R. L. Anal. Chem. 1979, 51, 2253-2257. (16) Jan, C.-C.; McCreery, L. R.; Gamble, F. T. Anal. Chem. 1985, 57, 17631765. (17) Wu, H. P.; McCreery, R. L. Anal. Chem. 1989, 61, 2347-2352. (18) Engstrom, R. C.; Ghaffari, S.; Qu, H. Anal. Chem. 1992, 64, 2525-2529. (19) Vitt, J. E.; Engstrom, R. C. Anal. Chem. 1997, 69, 1070-1076. (20) Engstrom, R. C.; Johnson, K. W.; DesJarlais, S. Anal. Chem. 1987, 59, 670673. (21) Engstrom, R. C.; Pharr, C. M.; Koppang, M. D. J. Electroanal. Chem. 1987, 221, 251-255. (22) Pharr, C. M.; Engstrom, R. C.; Tople, R. A.; Bee, T. K.; Unzelman, P. L. J. Electroanal. Chem. 1990, 278, 119-128.

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transmitting an image through the bundle. The spatial resolution of the image is dependent on the diameter of each individual fiber in the bundle. Such a device has already been used in a “combined imaging and chemical sensing” approach to simultaneously visualize a sample and probe local concentration fluctuations.23,24 The distal face of the fiber was then modified by a thin sensing layer. Walt et al. have employed such an imaging fiber platform to study localized corrosion,25 to perform parallel multianalyte detection,26 and to develop an artificial nose27 and DNA sensor array.28 Pantano et al. have reported the development of a pHsensitive nanotip array29 and also oxygen consumption measurements from the intact beating mouse heart.30 Optical fibers have been employed many times for remote chemical detection using fluorescence or Raman signals of species of interest, captured with single-fiber or multifiber configurations.31-41 In the single optical fiber design, light is guided to and from the sample with the same fiber. In the multifiber arrangement, one fiber carries the excitation light while several fibers are used for collection of the optical signal. Such designs have been evaluated theoretically and experimentally.31-33,37,40-42 While the optical spatial resolution of imaging fibers has been characterized in detail, the chemical spatial resolution has not been studied to our knowledge for an optical fiber bundle containing thousands of individual cores in a bulk configuration (i.e., when the fluorescent species is in solution and not immobilized on the fiber’s surface). Danowski and Pantano have evaluated the chemical spatial resolution of imaging fiber chemical sensors in a completely different approach. Fluorescent dye-embedded microbeads were deposited inside individual microwells that were etched across an imaging fiber’s face.43 The fraction of the dye’s fluorescence collected by the principal core and by the neighboring cores was investigated. As mentioned, we wish to report here a new remote approach for in situ imaging of dynamic concentration profiles based on coherent optical fiber bundles. The aim is to position a multi(23) Pantano, P.; Walt, D. R. Anal. Chem. 1995, 67, 481A-487A. (24) Walt, D. R. Acc. Chem. Res. 1998, 31, 267-278. (25) Szunerits, S.; Walt, D. R. Anal. Chem. 2002, 74, 886-894. (26) Barnard, S. M.; Walt, D. R. Nature 1991, 353, 338-340. (27) Dickinson, T. A.; Michael, K. L.; Kauer, J. S.; Walt, D. R. Anal. Chem. 1999, 71, 2192-2198. (28) Healey, B. G.; Matson, R. S.; Walt, D. R. Anal. Biochem. 1997, 251, 270279. (29) Liu, Y.-H.; Dam, T. H.; Pantano, P. Anal. Chim. Acta 2000, 419, 215-225. (30) Zhao, Y.; Richman, A.; Storey, C.; Radford, N. B.; Pantano, P. Anal. Chem. 1999, 71, 3887-3893. (31) Schwab, S. D.; McCreery, R. L. Anal. Chem. 1984, 56, 2199-2204. (32) Schwab, S. D.; McCreery, R. L.; Gamble, F. T. Anal. Chem. 1986, 58, 24862492. (33) Plaza, P.; Dao, N. Q.; Jouan, M.; Fevrier, H.; Saisse, H. Appl. Opt. 1986, 25, 3448-3454. (34) Louch, J.; Ingle, J. D. Anal. Chem. 1988, 60, 2537-2540. (35) Chong, C. K.; Shen, C.; Fong, Y.; Zhu, J.; Yan, F.-X.; Brush, S.; Mann, C. K.; Vickers, T. J. Vib. Spectrosc. 1992, 3, 35-45. (36) Myrick, M. L.; Angel, S. M.; Desiderio, R. Appl. Opt. 1990, 29, 13331344. (37) Zhu, Z. Y.; Yappert, M. C. Appl. Spectrosc. 1992, 46, 919-924. (38) Greek, L. S.; Schulze, H. G.; Haynes, C. A.; Blades, M. W.; Turner, R. F. B. Appl. Opt. 1996, 35, 4086-4095. (39) Lewis, I. R.; Griffiths, P. R. Appl. Spectrosc. 1996, 50, 12A-30A. (40) Lin, J.; Hart, S. J.; Kenny, J. E. Anal. Chem. 1996, 68, 3098-3103. (41) Wright, A. O.; Pepper, J. W.; Kenny, J. E. Anal. Chem. 1999, 71, 1, 25822585. (42) Zhu, Z. Y.; Yappert, M. C. Appl. Spectrosc. 1992, 46, 912-918. (43) Danowski, K. L.; Pantano, P. Microchem. J. 2001, 70, 51-61.

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channel imaging fiber (overall diameter, 270 µm) perpendicularly to the electrode surface (Figure 1B) so that each individual optical fiber of the bundle can probe local decreasing concentration of fluorescent electroactive species without interfering with the diffusion field, which is parallel to the probing surface. To examine the performance of our method, we investigated electrochemically formed concentration profiles at millimetric electrode corresponding to planar diffusion. A 3-mm-diameter disk electrode was used to oxidize tris(2,2′-bipyridine)ruthenium(II) into -ruthenium(III) in aqueous solution. The 270-µm-diameter imaging fiber was positioned perpendicularly to the electrode surface and used also to excite fluorescence emission of tris(2,2′-bipyridine)ruthenium(II) solution. This transition metal complex shows strong luminescence in solution at room temperature. A fraction of the isotropically emitted fluorescence was collected through the same fiber bundle. Therefore, each individual optical fiber of the bundle can sample a discrete volume located at a particular distance normal to the electrode surface. When an adequate potential step is applied to the electrode, Ru(II) is oxidized at a diffusion-limited rate into Ru(III), which is a nonfluorescent species.44 Imaging the change of the fluorescence signal with distance was performed with a charge-coupled device (CCD) camera. Fluorescence images were acquired as a function of time, which allowed the time development of the diffusion layer to be monitored, i.e., the simultaneous decrease of fluorescence with space and time due to the corresponding decrease of Ru(II) concentration. EXPERIMENTAL SECTION Materials. Ammonium fluoride (99.99%), hydrofluoric acid (48 wt % in water, 99.99%), and tris(2,2′-bipyridine)ruthenium(II) (Ru(bpy)3Cl2) were obtained from Aldrich. Potassium nitrate (99%) was purchased from Labosi; sodium sulfate (99%) was from Prolabo. Solutions were prepared from ultrapure water ((Milli-Q Plus 185, Millipore). The epoxy resin used was Plexcil C6 and A6 purchased from Escil. Apparatus and Procedures. The potentiostat used was a PGSTAT 12 Autolab (EcoChemie). The working electrode was a commercial glassy carbon electrode of 3-mm diameter (Bioanalytical Systems, BAS). It was chosen sufficiently large to study planar diffusion over the center of the electrode without showing any interference from edge effects.21 The quasi-reference and counter electrodes were 2-mm-diameter silver and 10-mm-diameter carbon wires, respectively. The three electrodes were embedded in an epoxy resin. The surface of the assembly was then polished with 30-, 15-, 3-, and 0.3-µm lapping films (Thorlabs Inc.) and finally on a microcloth (Buehler) with a deagglomerated 0.05-µm alumina suspension (Buehler). Silica imaging fibers of 50-cm length with a total diameter of ∼350 µm comprised 6000 individually cladded 3-4-µm-diameter optical fibers. Imaging fibers of 0.35 and 1.0 numerical apertures (NAs) are commercially available and were purchased respectively from Sumitomo Electric Industries and Collimated Holes Inc. Imaging fibers of 0.2 NA were specially made by Lot-Oriel and were the lowest NA fibers available. Note that numerical apertures are commercially defined versus air. The height of the imaging area of the coherent fiber-optic bundles was 270 µm. Bundles are (44) Woltman, S. J.; Even, W. R.; Weber, S. G. Anal. Chem. 1999, 71, 15041512.

coated with a ∼40-µm-thick silica jacket and a ∼40-µm silicon resin to preserve mechanical strength. To position the imaging area of the bundle directly at the electrode surface, the protective coatings were removed near the bundle tip. The insulating silicon resin layer was dissolved with acetone and sonicated in water for 30 s to remove any residuals. Silica jacket was etched using a HF solution prepared by mixing 500 µL of 40% (w/w) aqueous NH4F solution, 100 µL of a 48% HF solution, and 100 µL of deionized water. (Caution: HF etching solutions are extremely corrosive!). The distal face was placed horizontally into the HF etching solution and left for 3 h. The fiber bundle was then sonicated in water and polished before final use with 30-, 15-, 3-, and 0.3-µm lapping films. The distal face of the resulting bare 270-µm diameter imaging fiber was precisely positioned normal to and directly in contact with the working electrode surface (Figure 1B) with a three-axis submicrometer manipulator (MDT616, Thorlabs) and a goniometer (Melles Griot). This crucial step is monitored with a stereomicroscope (Stemi 2000, Zeiss). To image planar diffusion and to avoid any interferences from edge diffusion,21 the imaging fiber was placed at the center of the working disk electrode. The three-electrode assembly and the imaging fiber were then covered by Ru(II) solution containing Na2SO4 or KNO3 as the supporting electrolyte. Fluorescence images were acquired through the imaging fiber as a function of time as the working electrode potential was stepped from 0 to 1.2 V/Ag. Because any object positioned at the electrode surface could interfere with the concentration profiles if its symmetry is not compatible with that of the electrode, a model system of planar diffusion was selected in order to minimize the effect of the probe intrusion into the diffusion layer. Indeed, positioning the fiber bundle along a plane orthogonal to the disk electrode diameter (Figure 1B) respects the symmetry requirement. The instrument used for fluorescence imaging was a modified epifluorescence microscope (BX-30, Olympus) similar to the one described previously.45 Light from a 75-W xenon arc lamp was optimized on the region of interest (ROI). Another experimental key point is to adjust appropriately the light distribution over the ROI. Light was then collimated, passed through a 485 ( 11 nm excitation filter (Omega Optical) corresponding to the Ru(II) excitation wavelength. Light intensity was adjusted with a neutral density filter. The selected wavelength was reflected at 90° by a 540-nm dichroic mirror and focused onto the proximal end of the imaging fiber with a 20× microscope objective. Excitation light was transmitted through the optical fiber bundle and induced fluorescence emission of Ru(II). The fluorescence intensity reported by each fiber of the bundle is proportional to the local Ru(II) concentration observed by the fiber (see text and Figure 4). A portion of the emitted light is collected by the same optical fiber bundle, passed through the same microscope objective, and transmitted through the dichroic mirror. Sample emission was filtered by a band-pass filter (605 ( 25 nm) to ensure that only Ru(II) fluorescence was observed. The fluorescence images were acquired by a CCD camera (Roper Scientific), which was fitted with a back-illuminated chip (Marconi 47-10) that has 1024 × 1024 pixels. Maximum pixel readout rate is 1 MHz at 16 bits. The camera head cools the chip thermoelectrically to -40 °C and has (45) Bronk, K. S.; Michael, K. L.; Pantano, P.; Walt, D. R. Anal. Chem. 1995, 67, 2750-2757.

a shutter. The exposure time was set at 10 ms. To increase the CCD camera dynamics and thus to obtain a better temporal resolution, a ROI was selected from the complete image (white box in Figure 1A). The readout takes less time if only the ROI instead of the entire CCD image is digitized. We have also customized the chip and used a process called on-chip charge binning to speed up the readout time (70 ms) of the CCD camera, i.e., the delay between two consecutive captured images. Each image, which represents the 2-D distribution of the fluorescence intensity, is normalized to the image measured before the potential step (Figure 1A), in other words, corresponding to the fluorescence signal of the bulk Ru(II) concentration (C°). The fluorescence intensity profiles are calculated by averaging the intensity of the pixels perpendicular to the z-axis in the ROI (Figure 1A). Since the fluorescence intensity is directly related to the concentration of the fluorophore, this procedure allows obtaining the normalized concentration (C/C°) in adequate experimental conditions (see below). The diffusion coefficient D of Ru(II) was determined from the steady-state current measured with a Au disk ultramicroelectrode (20-µm diameter) and from the Randles-Sevcik equation46 with a glassy carbon electrode (3-mm diameter) in a solution containing 1 M KNO3 or 0.6 M Na2SO4 as supporting electrolytes. The determined value was 5.9 × 10-6 cm2 s-1 in very good agreement with the previously reported value.47 Molar extinction coefficients  were evaluated on the absorbance measurements made with a Perkin-Elmer Lambda-2 spectrometer. The values for Ru(II) and Ru(III) dissolved in water are respectively 4200 and 600 M-1 cm-1 at 485 nm (λexc). At 605 nm (λem), absorbance of Ru(II) is negligible and the molar extinction coefficient of Ru(III) is 200 M-1 cm-1. Ru(III) solution were prepared by electrolysis of Ru(II) in a H2SO4 solution. RESULTS AND DISCUSSION Epifluorescence Imaging through a Coherent Optical Fiber Bundle. A coherent optical micrometric fiber bundle was positioned at the surface of a millimetric disk electrode perpendicular to its diameter (Figure 1B) to monitor micrometric changes in the fluorescence intensity during the oxidation of Ru(II) into Ru(III) at the electrode (Figure 2). As mentioned above, the fluorescence intensity is related to the local concentration of Ru(II) since Ru(II) is fluorescent, whereas the Ru(III) oxidation state is not. Therefore, each individual fiber of the bundle acts as a local sensor probing only a discrete volume limited to a particular distance normal to the electrode surface. Direct imaging of the expanding diffusion layer was performed by acquiring successive fluorescence images (Figure 2) at several selected times after the electrode was stepped to a potential ensuring a diffusion-limited transfer rate. Figure 2 shows a sequence of normalized fluorescence images collected through the coherent optical fiber bundle. Each captured image results from a fluorescence profile averaged during 10 ms, which corresponds to the minimum exposure time allowed by our device. It gives a very accurate representation of the fluorescence intensity as a function of the distance from the electrode at a given time. Fluorescence images (Figure 2) normalized by the image monitored in the initial (46) Bard, A. J.; Faulkner, L. R. Electrochemical Methods; Wiley: New York, 2001. (47) Zu, Y.; Ding, Z.; Zhou, J.; Lee, Y.; Bard, A. J. Anal. Chem. 2001, 73, 21532156.

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Figure 2. Sequential fluorescence images collected with a coherent optical fiber bundle (0.2 NA) at different times (0, 0.05, 0.1, 0.2, 0.4, and 0.8 s; from left to right) after applying a potential step from 0 to 1.2 V/Ag at a glassy carbon electrode immersed in a solution containing 30 mM Ru(bpy)32+ and 0.6 M Na2SO4. The fluorescence images were normalized with respect of the initial bulk Ru(II) concentration. The CCD exposure time was 10 ms; each frame represents a ROI of 50 × 150 µm. All the images were coded according to the same color scale. Black symbolizes low normalized fluorescence intensities.

bulk solution (Figure 1A) reflect at first glance the concentration profiles of Ru(II). As expected, the fluorescence decreases near the electrode where Ru(II) is consumed, and this alteration propagates with time toward the solution. Figure 2 shows that, upon using an optical fiber bundle, fluorescence profiles could be monitored directly in one single image taken at a series of selected times, thus providing information about the building-up of diffusion layers at electrode. Since the exposure time to acquire one image is 10 ms and the time delay between two successive images is 70 ms, it allows us to monitor dynamic processes occurring at the micrometer scale like the time expansion of diffusion layers at millimetric electrodes in the usual solvents. Influence of Numerical Aperture and Ru(II) Concentration on the Spatial Resolution. The first objective of this work was to find the experimental conditions to establish from the fluorescence measurements the concentration profiles generated at an electrode with the appropriate spatial resolution. This resolution depends not only on the diameter of a single fiber in the bundle but as well on the optical fiber characteristics and physicochemical properties of the sample. Beside the diameter and number of individual optical fibers in the bundle, the parameters that set the spatial resolution are the geometrical arrangement of the fibers, the NA, the distance between neighboring cores, the refractive index, and the absorbance of the sample at the excitation and emission wavelength.42,43 Indeed, light exits and enters the fiber within an acceptance cone of angle θacceptance with respect to the normal to the fiber end face. θacceptance is given by38

NA ) sin θacceptance )

(

)

n2co - n2cl n2m

Figure 3. (A) Normalized fluorescence intensity (I/I°) as a function of distance from the electrode 100 ms after a potential step for imaging fibers of different NA in 5 mM Ru(II) solution. (B) Influence of Ru(II) concentrations on fluorescence intensity profiles measured with a 0.2 NA imaging fiber. The supporting electrolyte was 0.6 M Na2SO4; the CCD exposure time was 10 ms. Solid lines represent theoretical concentration profiles (C/C°) calculated at 100 ms from eq 2.

1/2

(1)

where nco, ncl, and nm are respectively the refraction indexes of the core, the cladding (nco > ncl), and the medium. The two first refraction indexes, nco and ncl, are imposed by the characteristics of the fibers whereas the last one, nm, is related to the local physicochemical properties of the sample. 7206 Analytical Chemistry, Vol. 76, No. 24, December 15, 2004

Since NA defines the extent of the acceptance cone (eq 1) and thus the spatial resolution, we first investigated the influence of this optical fiber characteristic on the actual size of the probed volume. From normalized fluorescence images, a series of fluorescence profiles were established (Figure 3A) with three imaging fibers of different NA (0.2, 0.35, and 1.0) 100 ms after a potential step was imposed at the electrode. The fluorescence images were normalized by the image captured at t ) 0, i.e., in

bulk solution before the composition of the solution was altered by the electrochemical reaction. The resulting profiles are compared to the theoretical concentration profiles predicted at 100 ms considering planar diffusion (Figure 3A):46

C(z,t)/C° ) erf[z/2(Dt)1/2]

(2)

where C is the concentration at distance z, C° the concentration in bulk solution, and D the diffusion coefficient of the species consumed at the electrode. Figure 3A shows clearly that the bundle characteristics (i.e., number, size, and distances between cores) allow the fluorescence intensity above the electrode surface to be mapped. However, one observes that the experimental intensities do not match the theoretical concentration profiles whatever the NA values. In comparison, the intensities are lower than the theoretical concentrations far from the electrode whereas they are much higher close to the electrode surface. The deviation of the experimental intensities versus the predicted ones is more pronounced with fibers of higher NA. The effect of NA on the probed volume can be explained as follows. With the highest NA value the theoretical half-acceptance angle of the fiber is 90°, so that in principle, all of the light falling on its surface is transmitted to the CCD detector regardless of angle. Therefore, a given core collects fluorescence light originating also from excitation by neighboring cores. In other words, an individual optical fiber probes a large volume defined by the acceptance cone. This is therefore not related to the Ru(II) concentration existing at a specific distance from the electrode surface, but it integrates this concentration over a large domain (Figure 4A). Thus, the overlapping of acceptance cones and the effectively probed volume worsen the global spatial resolution. With lower NA fibers, the sampled volume is decreased (Figure 4B) and thus the locally measured intensity is more “sensitive” to its microenvironment. Best results were obtained using the coherent optical fiber bundle of the lowest NA. Therefore, all subsequent experiments were performed with the custom-made 0.2 NA bundle. The second parameter affecting the spatial resolution is the absorbance of the solution. We tested its influence using a similar procedure by plotting the fluorescence intensity as a function of the distance from the electrode for several Ru(II) concentrations. Figure 3B demonstrates that when the concentration of Ru(II) is large, the determined fluorescence profile approaches better the theoretical one. With the 30 mM solution, a rather good agreement is observed between theory and measurements. This result can be explained by considering the effective penetration depth of the excitation light into the solution. It is related to the local absorbance of the solution, which is inversely proportional to the local concentration of the light-absorbing species. The effects of the absorption on the excitation and emission light are well known in fluorescence measurements and are termed respectively primary and secondary inner-filter effects.48,49 As the excitation light propagates away from the fiber tip, its intensity (i.e., the fluence expressed in energy/area) decreases because of both the light absorption at excitation wavelength by the fluorescent reagent and the expansion of the illuminated cone. In the same

way, the fraction of fluorescence collected depends on the absorption at emission wavelength and on a term proportional to the square of the distance, related to the isotropically expanding sphere of emitted light.31-33,37,42 In other words, the higher the distance between the fluorescent Ru(II) and the optical fiber surface, the less these species contribute to the detected fluorescence intensity. Yuan and Walt have reported the different mechanisms of fluorescence modulation by absorbing species, such as static and dynamic quenching and inner-filter effects.49 Applying their model to the single optical fiber configuration (i.e., the same fiber excites and also detects fluorescence), they showed the importance of the concentration of the light-absorbing species, which finally determines the effective path length of the incident probing light. The local absorbance of the solution depends on the Ru(II) and Ru(III) local concentrations and of theirs respective molar extinction coefficients at the excitation and emission wavelengths.37,42 Ru(II) absorbs strongly the excitation light ( ) 4200 M-1 cm-1 at 485 nm) whereas the molar extinction of the oxidized form is clearly inferior ( ) 600 M-1 cm-1 at 485 nm).50 In addition, at the emission wavelength, absorbance of Ru(II) is negligible and the molar extinction coefficient of Ru(III) is 200 M-1 cm-1. Consequently, we assume that the secondary inner-

(48) Leese, R. A.; Wehry, E. L. Anal. Chem. 1978, 50, 1193-1197. (49) Yuan, P.; Walt, D. R. Anal. Chem. 1987, 59, 2391-2394.

(50) Kalyansundaram, K. Coord. Chem. Rev. 1982, 46, 159-244.

Figure 4. Schematic representation of the volumes probed with the imaging fiber under different experimental conditions. (A) The hatched zone represents the overlap of the acceptance cones of two adjacent individual fibers. (B) Influence of the NA (θ1 > θ2) of the imaging fiber on the intersection of the cones. (C) Influence of the effective penetration depth (p1 > p2) of excitation light on the spatial resolution.

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filter effect is negligible in this study and we just consider the absorbance of Ru(II) at the excitation wavelength. Figure 4C shows the effects of penetration depth on the size of the probed volume. At low concentrations, the absorbance of the solution is weak and the penetration length of excitation light into the solution is significant. It induces the overlapping of probing neighboring acceptance cones and results in an overestimation of local Ru(II) concentration when this is low, viz., close to the electrode surface. Indeed, the fluorescence signal is then multiply detected by overlapping probing cones. This situation leads to a decrease of the spatial resolution. On the contrary, increasing the concentration of the light-absorbing species to 30 mM results in a decrease of the effective incident light penetration depth, so that excitation light exiting from each individual fiber probes only the contents of an adequate micrometric volume immediately adjacent to the fiber (Figure 4C). As expected, close to the electrode surface, the fluorescence intensities are the lowest according to the depletion of Ru(II) in the diffusion layer. However, significant deviations, which are reproducible between experimental and theoretical data, are still observed for distances lower than 10 µm even for a 30 mM Ru(II) solution. The experimental value of C/C° deduced from fluorescence measurements appears clearly overestimated close to the electrode. Various optical effects may induce a deviation from the theory or alter the fluorescence intensity: reflection of light at the electrode surface, primary inner-filter effect, effects related to refractive index gradient, static and dynamic quenching, resonance energy transfer (Fo¨rster transfer), etc.49 Although carbon is a very absorbing material for visible light, we tested the first hypothesis by comparing the fluorescence intensities measured when the imaging fiber is positioned on the carbon surface and far from the electrode in the bulk solution (data not shown) in the absence of electrochemical perturbation. No noticeable difference was detected even in the vicinity of the electrode surface. Therefore, the reflection of the light at the electrode surface is negligible in this front-illumination geometry. Another optical phenomenon that may contribute to some deviations is the primary inner-filter effect described above. Indeed, the concentration profiles of Ru(II) and Ru(III) after electrochemical polarization of the electrode may induce strong local variations of absorbance. Since Ru(II) concentration diminishes near the electrode (cf. eq 2), the local absorbance of the solution adjacent to the optical fiber decreases and the penetration length of excitation light increases. Such an increase of the penetration depth tends to modify the probed volume and also leads to the overlapping of neighboring acceptance cones (Figure 4C) resulting in a global overestimation of the local concentration. This innerfilter effect finally should alter noticeably the spatial resolution in the 5-10-µm-distance range from of the electrode surface where most of the concentration gradients take place. Besides these local variations of sampling volume induced by absorbance gradients, as mentioned above, another optical phenomenon that could explain these discrepancies can be related to refractive indexes. Indeed, it is well known that light does not propagate along a straight line in the presence of refractive index variations normal to the propagation direction. The deviation θ of the light trajectory by a spatially nonuniform distribution of species in solution is proportional to the magnitude of the 7208

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concentration gradient and to the path length L through the concentration gradient:

θ)

L ∂n L ∂n ∂C ) nm ∂z nm ∂C ∂z

( )

( )( )

(3)

where nm is the refractive index of the unperturbed medium. Such mirage effects are well documented and have been used in electrochemistry for analytical purposes.51-54 For example, at a distance z from the electrode surface after time t following a potential step, the resulting refractive index gradient generated above the electrode can be estimated by51

[

] ( )

∂n(z,t) dn C° dn - z2 exp ) ∂z 4Dt xπDt dCRu(III) dCRu(II)

(4)

The difference dn/dCRu(III) - dn/dCRu(II) is expected to be significantly smaller than each of the individual terms, whose values are ∼10-2 M-1.51,53 At short time, and close to the electrode, the refractive index gradient is expected to be important, especially for solutions where C° is high. Interestingly, significant deviations from theory are observed close to the electrode surface and might be also ascribed to deflection effects caused by refractive index gradient on the light emitted by fluorescent Ru(II) in the direction of the bundle. In the case of a one-dimensional refractive index profile with planes of constant refractive index parallel to the electrode surface, the angular light deflection, caused by a refractive index gradient normal to the propagation direction, is an increasing function of the light path length (eq 3).53 Such effects are expected to be large close to the electrode surface, where the penetration depth of the exciting light into the solution is high and the path length of the emitted light is large. But, as discussed above, the contribution to the monitored fluorescence intensity of light emitted far from the optical fiber surface decreased extremely fast with distance. In other words, when the path length of light emitted by fluorescence is long, then the mirage effects are more pronounced but the fluorescence emission is less initiated by the excitation light and is less collected by the optical fiber bundle. In addition, it is important to notice that local variations of the refractive index (nm) may also slightly modify the acceptance cone of fibers comprising the bundle (see eq 1). Since refractive index gradient effects are expected to be the most pronounced where the concentration gradient is the largest (i.e., close to the electrode), all these optical distortions may concur to a divergence between experimental and theoretical data as observed in the vicinity of the electrode surface. Based only on the experimental data taken at 100 ms, we are not able to discriminate between the respective importance of the optical distortions induced by the penetration depth gradient, these due to the refractive index variations and the other optical phenomena cited above. However, exploitation of data taken at a series of selected times seems to indicate that the main distortion arises (51) Pawliszyn, J. Anal. Chem. 1988, 60, 1751-1758. (52) Pawliszyn, J. Anal. Chem. 1992, 64, 1552-1555. (53) Muller, R. H. Advances in Electrochemistry and Electrochemical Engineering; Wiley: New York, 1973. (54) Vieil, E.; Lopez, C. J. Electroanal. Chem. 1999, 466, 218-233.

Figure 5. Transient concentration profiles of Ru(II) in the diffusion layer at short times (A) and at longer times (C) under the same conditions as in Figure 2. Solid lines represent theoretical concentration profiles calculated from eq 2. Correlation at short (B) and longer (D) times between experimental and theoretical concentration profiles.

from the penetration depth gradients (see Possible Origins of Optical Distortions, below). Dynamic Imaging of Transient Concentration Profiles. To investigate the dynamic of diffusion layers generated at millimetric electrodes, fluorescence images from single potential step experiments (data from Figure 2) were analyzed to establish fluorescence profiles at different times. Figure 5 compares the resulting profiles with the ones predicted by eq 2 for times ranging from 0.03 to 0.8 s. The investigation is limited to 30 ms since at shorter times the exposure time would become significant compared to the measurement time. A good agreement is observed between data at short time scale (30-110 ms, Figure 5A) as well as at longer times (0.1-0.8 s, Figure 5C). This agreement is further illustrated using an adequate representation3 of the data in Figure 5B and D. Except for the intensities measured at 30 ms, the average value of the linear correlation coefficients is equal to 0.991 ( 0.001. At times longer than 1 s (data not shown), a deviation from the theoretical behavior (eq 2) is observed far from the electrode (i.e., at distances higher than 50 µm) and this deviation is larger as time increases. This effect is ascribed to the natural convection, which alters the building up of diffusion layer far from the electrode.55 In the case of motionless solutions, deviations from eq 2 usually occurs after a few seconds. However, in our experimental conditions, convection is probably forced locally by additional vibrations induced by the air-cooled CCD camera, which is placed close to the cell. This phenomenon limited our investigations to time scales lower than 1 s. (55) Amatore, C.; Szunerits, S.; Thouin, L.; Warkocz, J.-S. J. Electroanal. Chem 2001, 500, 62-70.

The deviations mentioned previously near the electrode surface (Figure 3) are still observed whatever the concentration gradients or duration of the experiments. They are even more pronounced at shorter times. The spatial resolution in these conditions seems to be appropriate to investigate local concentrations beyond 10 µm from the electrode. Possible Origins of Optical Distortions. Considering the building-up dynamics of the diffusion layers, we have here the opportunity to estimate the respective importance of the optical effects, which can be responsible for the discrepancies observed close to the electrode surface, i.e., mainly between the primary inner-filter effect and the mirage effects on the light propagation or on the angle of acceptance cone of optical fibers. Since the influence of the refractive index gradient is directly related to the concentration gradient, the divergence between experimental and theoretical data should thus correlate with the concentration gradient. Figure 6A shows the relative difference between experimental and theoretical data as a function of the concentration gradient at different times. One can clearly observe that the deviation arises for different values of the concentration gradient, thereby illustrating that the effect of the refractive index gradient is probably negligible under our experimental conditions. The discrepancy could be then discussed in term of a dynamic innerfilter effect due to the local absorbance of Ru(II). Figure 6B represents the relative divergence between experimental and theoretical data as a function of the Ru(II) concentration at different times. This plot shows that the discrepancy occurs for a given value of Ru(II) concentration (∼12-15 mM). Consequently, the inner-filter effect seems to be the dominant process limiting Analytical Chemistry, Vol. 76, No. 24, December 15, 2004

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tions occurring in the immediate vicinity of the electrode surface. However, if the preponderance of the inner-filter effect is confirmed by future works, this relative drawback could be exploited in the future to map indirectly a nonfluorescent reagent with fluorescence.56 This complementary approach would be based on indirect fluorescence detection of a nonfluorescent species, which absorbs differentially the excitation light depending on its redox states.

Figure 6. Evolution of the divergence between experimental and theoretical normalized data with (A) the gradient of normalized theoretical concentration and with (B) the normalized theoretical concentration of Ru(II) at different times (t ) 0.03, 0.06, 0.08, 0.1, 0.15, 0.2, 0.3, 0.4, 0.5, 0.8 s). The symbols are similar to those used in Figure 5.

the performance of our experimental approach, in agreement with the previous observation in Figure 3B. However, from the dynamic data, we cannot test the possible contribution of other optical effects mentioned above. Therefore, we are not able to demonstrate definitely and unambiguously the origins of optical distor-

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CONCLUSION We have developed an approach for rapid remote in situ fluorescence imaging of local concentration gradients generated at the vicinity of polarized electrodes. We demonstrated that combining fluorescence detection with an optical fiber bundle affords the possibility to image dynamic processes such as the building up of diffusion layers generated at an electrode with a micrometer resolution and within the millisecond time scale. Micrometer spatial resolution was achieved by controlling the volume probed by each individual fibers of the bundle. We obtained adequate resolution by decreasing the acceptance angle of the fibers (i.e., lower NA) and by decreasing the penetration depth of excitation light, i.e., by increasing the concentration of absorbing species in solution. Concentration profiles deduced from fluorescence measurements were found to be in good agreement with theoretical concentration profiles given for planar diffusion. The discrepancies that were observed at short distances from the electrode surface have been discussed qualitatively in terms of distortions resulting mainly from a primary inner-filter effect. The comparison with theoretical concentration profiles allowed us to validate this new concept of imaging physicochemical processes. Received for review July 5, 2004. Accepted September 3, 2004. AC049017G (56) Tohda, K.; Lu, H.; Umezawa, Y.; Gratzl, M. Anal. Chem. 2001, 73, 20702077.