Coupling Electrochemistry with Fluorescence Confocal Microscopy To

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Coupling Electrochemistry with Fluorescence Confocal Microscopy To Investigate Electrochemical Reactivity: A Case Study with the Resazurin-Resorufin Fluorogenic Couple Thomas Doneux,*,† Laurent Bouffier,‡,§ Bertrand Goudeau,‡,§ and Stéphane Arbault‡,§ †

Chimie Analytique et Chimie des Interfaces, Faculté des Sciences, Université libre de Bruxelles (ULB), Boulevard du Triomphe, 2, CP 255, B-1050 Bruxelles, Belgium ‡ Univ. Bordeaux, ISM, UMR 5255, F-33400 Talence, France § CNRS, ISM, UMR 5255, F-33400 Talence, France S Supporting Information *

ABSTRACT: The redox couple resazurin-resorufin exhibits electrofluorochromic properties which are investigated herein by absorption and fluorescence spectroelectrochemistry and by electrochemically coupledfluorescence confocal laser scanning microscopy (EC-CLSM). At pH 10, the highly fluorescent resorufin dye is generated at the electrode surface by the electrochemical reduction of the poorly fluorescent resazurin. Performing EC-CLSM at electrode surfaces allows to monitor spatially resolved electrochemical processes in situ and in real time. Using a small (315 μm diameter) cylindrical electrode, a steady-state diffusion layer builds up under potentiostatic conditions at −0.45 V vs Ag|AgCl. Mapping the fluorescence intensity in 3D by CLSM enables us to reconstruct the relative concentration profile of resorufin around the electrode. The comparison of the experimental diffusion-profile with theoretical predictions demonstrates that spontaneous convection has a direct influence on the actual thickness of the diffusion layer, which is smaller than the value predicted for a purely diffusional transport. This study shows that combining fluorescence CLSM with electrochemistry is a powerful tool to study electrochemical reactivity at a spatially resolved level.

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is in the same order as that of conventional SECM (notwithstanding recent advances both in optical microscopies1 and in SECM2,3 that provide much better spatial resolutions). This spatial scale is nevertheless interesting for electrochemical experiments, because the micrometer scale is the typical spatial dimension of diffusion phenomena. In addition, optical microscopy enables one to visualize at once the entire electrode surface, in contrast to STM which always samples only small selected areas, and within a time scale which is much faster than that required for probe scanning experiments, including STM, AFM and SECM. The present work is devoted to the coupling between fluorescence microscopy, performed in the confocal laser scanning microscopy (CLSM) mode, and electrochemistry (hereafter EC-CLSM). The early contributions in this field came from Engstrom and co-workers,4−6 who devised in the 1990s a combination between electrochemistry and wide-field (i.e., not confocal) fluorescence microscopy. Using this setup, they showed that it is possible to monitor the local electrode activity toward reactions consuming or producing protons, such

lectrochemical reactivity studies are usually performed by conventional electrochemical measurements carried out with a single electrode, whether of macroscopic (∼millimeter) or microscopic (∼micrometer) dimensions. Such kind of measurements provides information which can be timeresolved down to very short time scales, but which is typically averaged over the entire electrode area. These common techniques are thus insensitive to spatially heterogeneous reactive processes. As electrode materials employed for instance in electrocatalysis, bioelectrochemistry or electroanalysis become more and more diverse and complex, there is a great interest to characterize the electrochemical activity at a local level. For this purpose, electrochemical methods should be coupled with microscopy, which conveys spatially resolved information. In this respect, scanning probe microscopy such as atomic force microscopy (AFM), scanning tunneling microscopy (STM), and scanning electrochemical microscopy (SECM), have become very popular due to their very good spatial resolution (for the two former) and high versatility. Although less frequently employed, optical microscopy coupled with electrochemistry is also attractive to monitor electrochemical reactivity. The typical lateral resolution of an optical microscope, in the near submicrometer range, is not comparable to the nanometer resolution of STM and AFM but © XXXX American Chemical Society

Received: February 3, 2016 Accepted: May 20, 2016

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DOI: 10.1021/acs.analchem.6b00477 Anal. Chem. XXXX, XXX, XXX−XXX

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Analytical Chemistry Scheme 1. Structures of the Resazurin (RZ), Resorufin (RF), and Dihydroresorufin (DH) Moleculesa

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The reduction of RZ to RF is chemically irreversible, while the reduction of RF to DH is reversible.

as water reduction,4,5 oxygen reduction,4,5 oxygen evolution6 or oxide formation.6 While the electrochemical reactivity depended on the electrode material and applied potential, the local information was obtained by fluorescence imaging, using an indicator whose fluorescence is pH-sensitive. Quinine was used to monitor the reaction consuming hydroxide ions (oxygen evolution and oxide formation), 6 because its fluorescence increases when the pH decreases. Conversely, the quantum yield of fluorescein is enhanced in basic conditions and was used for reaction consuming protons (oxygen or water reduction).4,5 Employing the same strategy of using a pH-sensitive fluorescent indicator, the establishment of pH-gradients induced by electrochemical reactions was investigated in further details by Boldt et al.7 and by Unwin and colleagues.8,9 The latter group combined electrochemistry with CLSM, which provides an additional resolution in the axial direction and enabled them to reconstruct 3D maps of the pH profile surrounding the electrode surface. Beyond confirming the usefulness of the coupling to monitor local reactivity, they confronted their fluorescence results with voltammetric data and theoretical simulations to demonstrate that EC-CLSM imaging is quantitative, meaning that the measured fluorescence intensity is indeed correlated with the proton concentration. Since many electrochemical reactions are coupled to proton production or consumption, the use of pH-sensitive fluorophores is very versatile and we have for example recently employed such a strategy to image redox reactivity at bipolar electrodes.10 In the above works, the electrochemical reactivity is monitored in an indirect way, through the fluorescence modulation arising from an indicator dissolved in solution. However, as noted by Engstrom and colleagues, an ideal method “should be extendable to [...] any reaction that produces a product which is, itself, fluorescent”.5 More generally, it can be extended to any electrochemical reaction where the measured fluorescence is dependent on the applied potential, including reactions involving electrofluorochromic molecules (i.e., electrochemically switchable fluorophores11).12,13 For instance, the group of Bizzotto14,15 has investigated the reductive desorption of self-assembled monolayers by EC-fluorescence measurements, providing in a single experiments a reactivity map of gold single crystals in the complete stereographic triangle,15 while Miomandre and colleagues11,16,17 have made significant contributions toward establishing a coupled electrochemical-fluorescence microscopy as a powerful tool to investigate fluorescent redox switches. Concerning electrofluorochromic molecules, Sojic and coworkers18 developed an in situ fluorescence setup to image in real time the buildup of the diffusion concentration profile resulting from the oxidation of the fluorescent tris(2,2′bipyridine)ruthenium(II) into the nonfluorescent tris(2,2′bipyridine)ruthenium(III). Very recently, the group of Zhang

has significantly expanded the potentialities of the coupling for the study of electrochemical reactivity, in an ongoing series of publications.19−21 The group is developing an imaging tool, called “fluorescence enabled electrochemical microscopy (FEEM)″ whose principle is to couple a redox reaction to a fluorogenic reporter reaction through a closed bipolar electrode. The electrochemical reaction of interest takes place at one pole of the bipolar electrode, whereas a fluorescent species is generated and imaged at the other pole of the electrode. The authors have extensively characterized the performance of the setup in terms of spatial (2D) and temporal resolution and of electrochemical kinetics. They showed that the method can evidence electrochemical heterogeneity using microelectrode arrays, and demonstrated the applicability of the method to electrocatalyst screening. The fluorogenic reporter used by Zhang and colleagues was resorufin (RF, 7-Hydroxy3H-phenoxazin-3-one, Scheme 1), a highly fluorescent phenoxazine (Φ = 0.41 in aqueous solution at pH 1022). Interestingly, this species can be generated either by reduction of the parent N-oxide resazurin (RZ, 7-Hydroxy-3H-phenoxazin-3-one-10-oxide, Scheme 1), or by oxidation of the dihydroresorufin (DH, 3,7-Dihydroxy-phenoxazine, Scheme 1), which are poorly fluorescent (Φ = 0.11 in aqueous solution at pH 1022) and not fluorescent, respectively. Thus, the same reporter RF can be used in electrochemical oxidation or reduction reactions. Taking advantage of the microelectrode array configuration, the same group was able to use the images recorded at the “fluorescent pole” of the closed bipolar electrode to reconstruct the 3D diffusion profile of an electrochemical reaction taking place at a microelectrode placed in a precisely controlled distance from the edge of the bipolar electrode.21 Along the same line, Xu et al. fabricated a device comprising an interdigitated electrode array and two microfluidic channels in a closed bipolar electrode configuration, enabling them to monitor independently the proton concentration (using fluorescein) and electron transfer processes (using resazurin/resorufin).23 This literature survey demonstrates that coupling fluorescence microscopy to electrochemical methods can provide spatially resolved data regarding the electrochemical reactivity, in real time. Most of these works are however rather “indirect”, in the sense that the fluorescence is somehow decoupled from the electrochemical reaction of interest. In the case of Rudd et al.,9 the fluorescence is correlated to the electrochemical reactivity through the pH gradient resulting from the electrode reaction. Although it is very versatile, because many redox reactions are coupled to proton transfer, all the experimental factors affecting the local pH (buffered versus nonbuffered conditions, presence of proton donors/acceptors,...) can influence the measured fluorescence intensity. In the approach of Zhang and co-workers,19−21 the correlation between the reaction of interest and the measured fluorescence occurs through the bipolar electrode. Here again, the setup is very B


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Analytical Chemistry

wavelength was 543 nm and the fluorescence emission spectra were collected between 575 and 750 nm at a wavelength scan rate of 240 nm min−1 (0.25 s integration time). In Situ Confocal Laser Scanning Fluorescence Microscopy. Fluorescence images were acquired at 20× magnification (Objective Leica HCX PL FLUOTAR, NA 0.4, working distance 6.9 mm) using a Leica TCS-SP5 inverted microscope equipped with a 543 nm laser source and a spectral selector module in front of the photomultiplier tube (PMT) detector. Unless otherwise stated, the wavelength bandwidth was set at 585−605 nm. The pinhole size was 1 airy disk (∼106 μm). The images, 512 × 512 pixels in size, were acquired at a 400 Hz rate. The in situ microscopy experiments were conducted in a custom-made glassware cell with a 0.25 mm-thick quartz optical window. The homemade spectroelectrochemical cell was provided by Prof. Bizzotto (University of British Columbia, Canada).14 The reference and counter electrodes were a “leakless” Ag|AgCl (eDAQ) and a coiled platinum wire, respectively. The working electrode was a gold-coated optical fiber bundle (F & T Fibers and Technology GmbH, core diameter 315 μm, ∼20 000 fibers/mm2). This type of electrode was already used for bioanalytical sensing.25 Its fabrication is made according to a previously reported method.25,26 All images were processed and analyzed using the software ImageJ.27

versatile since it can be applied to many electrochemical reactions, but the properties of the bipolar electrode (size, geometry, heterogeneous kinetics) can be the limiting factors determining the amount of collected fluorescence.24 In the present contribution, we describe a direct investigation regarding the electrochemical reactivity of an electrofluorochromic system by EC-CLSM. The RZ−RF−DH system is used as a model to explore in detail the relationship between the electrochemical behavior, at gold electrodes, and the fluorescence microscopy results. The electrochemistry and spectroelectrochemistry (absorption spectroscopy and steadystate fluorescence spectroscopy) of the system are carefully examined in the experimental conditions used in fluorescence microscopy. The EC-CLSM experiments enable us to image the electrochemical reactivity at a gold electrode and to reconstruct in 3D the diffusion layer generated at constant potential by the reduction of RZ. In particular, we directly image the impact of natural convection on the thickness of the diffusion layer.

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EXPERIMENTAL SECTION Reagents. All experiments were carried out in 0.05 M carbonate buffer solutions (0.025 M NaHCO3 + 0.025 M Na2CO3, pH 10) prepared with ultrapure water (Milli-Q system from Millipore). NaHCO3 (Sigma-Aldrich, ≥95%), Na2CO3 (Alfa Aesar, 98%), Resazurin (sodium salt, Alfa Aesar, >96%), and Resorufin (Sigma-Aldrich, dye content 95%) were used without further purification. Cyclic Voltammetry. Cyclic voltammetry experiments were conducted at room temperature in a three-electrode cell using a polycrystalline gold disk as the working electrode, a large area gold wire as the counter electrode, and a Ag|AgCl| saturated KCl reference electrode, all connected to a PGSTAT30 potentiostat (Metrohm-Autolab). Before the experiments, water-saturated nitrogen was purged for ∼20 min in the solution to remove dissolved oxygen, then kept flowing over the solution during the measurements. Spectroelectrochemical Measurements. Absorption spectra were recorded on a Varian Cary 100 scan UV−vis double beam spectrophotometer. Fluorescence spectra were collected with a Varian Cary Eclipse spectrofluorimeter. In both cases, a spectroelectrochemical kit (BioLogic) comprising a 1 cm (width) × 1 mm (path length) thin layer quartz cell, a platinum gauze working electrode (80 mesh, height 5 mm), a silver|silver chloride reference electrode and a platinum counter electrode, was used to record the spectra under polarization. A μAutolab Type III potentiostat was used to control the potential. For the absorption spectroscopy experiments, the wavelength scan rate was 600 nm min−1. To minimize the artifacts originating from the presence of the platinum mesh in the beam path, the blank was recorded with the spectroelectrochemical cell positioned in the sample cell holder and filled with carbonate buffer. Meanwhile, a conventional 1 cm-path cell also filled with carbonate buffer was positioned in the reference cell holder of the double beam spectrophotometer. Despite these precautions, negative absorbance values were measured in the nonabsorbing regions of the spectra, which were thus further baseline-corrected. For the fluorescence spectroscopy experiments, the spectroelectrochemical cell was placed in such a way that the excitation beam (slit width 2.5 nm) proceeded along the small side (1 mm) of the cell and the emission (slit width 5.0 nm) collected at 90°, i.e., in the direction along the long side of the cell. The excitation

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RESULTS AND DISCUSSION Cyclic Voltammetry. The voltammetric behavior of RF and RZ is presented in parts a and b of Figure 1, respectively.

Figure 1. Cyclic voltammograms of 1 mM resorufin (a) and 1 mM resazurin (b) recorded at polycrystalline gold electrodes in 0.05 M carbonate buffer. Scan rate 10 mV s−1. The insets show the dependences on the first reduction peak currents on the scan rate.

The cyclic voltammogram (CV) of RF (Figure 1a) displays one pair of peaks centered around −0.48 V vs Ag|AgCl with a peakto-peak separation ΔEp ≈ 50 mV. This value is consistent with a fairly fast (quasi-reversible), 2 e− redox process. The cathodic peak can be ascribed to the 2 e− reduction of RF to DH, and the anodic peak to the reverse process. The linear dependence C


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was subsequently applied to the platinum mesh working electrode, and consecutive absorption spectra were recorded every 60 s. According to the CV of Figure 1a, RF is reduced to DH at such negative potentials. The evolution of the absorption spectra shows a continuous decrease of the absorbance over time, at all wavelengths, because the pink colored RF (λmax = 570 nm) is consumed at the applied potential of −0.65 V vs Ag|AgCl. However, no new peak appears during the consumption of RF, because its reduction product, DH, does not absorb in the visible range. Upon reversing the applied potential to −0.30 V vs Ag|AgCl (see Figure S-1), where RF is no longer reduced, the spectra increase toward the initial absorbance values, confirming that DH is reoxidized to RF, as previously inferred from the cyclic voltammetry measurements. A similar experiment was carried out with RZ. The reduction potential was chosen at −0.45 V vs Ag|AgCl, which is in between the two cathodic peaks observed in the CV (Figure 1b). At this potential, it is expected that RZ should be reduced to RF and that the further reduction to DH should not be thermodynamically possible. Figure 2b presents the results of this experiment. The first spectrum, recorded at open circuit potential and labeled t = 0 s, is the absorption spectrum of the blue colored RZ. It has an absorption band whose maximum is located at λmax = 600 nm. When the potential of −0.45 V vs Ag|AgCl is applied to the mesh electrode, the spectra evolve significantly, leading to the appearance of an isosbestic point at 585 nm. Above this wavelength, the absorbance decreases over time, whereas a new peak grows at lower wavelengths, its maximum being at λmax = 570 nm. At long times, the peak at 600 nm has almost vanished and the observed spectrum clearly displays the characteristic absorption features of RF. The higher absorbance of the new peak at 570 nm as compared to the absorbance of the initial peak at 600 nm is consistent with the reported values of the molar absorption coefficient of RZ (εRZ (λmax) = 42 000 ± 3 000 M−1 cm−1) and RF (εRF (λmax) = 51 000 ± 2 800 M−1 cm−1).22 These results provide a spectroscopic confirmation that RZ is reduced to RF at this potential. In the same experiment, the potential was subsequently stepped further to −0.65 V vs Ag|AgCl for 11 min and then to −0.30 V vs Ag|AgCl for 7 min (Figure S-2). At −0.65 V vs Ag|AgCl, the spectrum, with a maximum at λmax = 570 nm, decreased continuously in a manner analogous to what was observed above for the reduction of RF to DH. At −0.30 V vs Ag|AgCl, the absorbance increased in the whole wavelength range, the maximum being still located at λmax = 570 nm. The latter spectrum was reminiscent of RF, with no significant contribution from RZ at 600 nm. This demonstrates that RZ cannot be formed by the electrochemical oxidation of RF, in full agreement with the conclusions drawn above from the voltammetric experiments. In Situ Fluorescence Spectroscopy. Using the same spectroelectrochemical cell and the same methodology as for absorption spectroscopy, in situ fluorescence spectroscopy measurements were also performed. Figure 3a presents a series of fluorescence emission spectra recorded from a solution of RF at an applied potential of −0.65 V vs Ag|AgCl. At open circuit potential, the RF spectrum has an intense fluorescence peak culminating at 592 nm. When the cell is switched on, the fluorescence spectrum continuously decreases, because RF is being transformed to the nonabsorbing, nonfluorescent DH. In the case of RZ, Figure 3b shows that the maximum fluorescence intensity is located at 638 nm. Upon reduction of RZ at −0.45 V vs Ag|AgCl, a pronounced fluorescence peak emerges and

between the cathodic peak current and the square root of the scan rate (see inset in Figure 1a) indicates that the reduction is diffusion-controlled. From the slope of the curve, and using the Randles-Ševčiḱ equation, the diffusion coefficient of RF is estimated at DRF = 1.9 × 10−6 cm2 s−1. The CV of RZ (Figure 1b) exhibits the same pair of peaks, preceded at less negative potentials by a first cathodic peak with a maximum at −0.40 V vs Ag|AgCl. This peak is also diffusioncontrolled (see inset in Figure 1b) and can be attributed to the 2 e− reduction of RZ to RF according to the reaction depicted in Scheme 1.28−31 This reaction involves a coupling between an electron transfer step and a chemical step (the dissociation of the N−O bond). The absence of an anodic response in the voltammogram during the backward sweep indicates that the reaction is not chemically reversible. The diffusion coefficient of RZ can be roughly estimated from the slope of the ip versus υ1/2 plot using the equation corresponding to an ErevCirr scheme,32 leading to a value of DRZ = 2.3 × 10−6 cm2 s−1. This value is slightly higher than those reported by Khazalpour and Nematollahi,31 ranging between 7.98 × 10−7 cm2 s−1 and 1.39 × 10−6 cm2 s−1 in aqueous solutions of lower pH than in the present study (7.3 against 10.0). The voltammetric investigations establish that RZ is irreversibly reduced to RF, which can be further reversibly reduced to DH at more negative potentials. Spectroelectrochemical Characterizations. In Situ Absorption Spectroscopy. RZ, RF, and DH exhibit very distinct spectral properties, so in situ spectroelectrochemistry is ideally suited to confirm the electrochemical interconversion between these various species. Absorption spectra were recorded in the visible range under electrochemical control, using a specially designed commercial spectroelectrochemical cell. Figure 2a presents a set of absorption spectra collected from a RF solution. A first spectrum was recorded at open circuit potential (i.e., without electrochemical control); this spectrum is labeled t = 0 s. A potential of −0.65 V vs Ag|AgCl

Figure 2. Time evolution of the in situ absorption spectra recorded in a 0.1 mM resorufin solution (a) and a 0.1 mM resazurin solution (b). The electrode potential was held at −0.65 V vs Ag|AgCl in part a and −0.45 V vs Ag|AgCl in part b. D


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images in Figure S-3). Upon complete immersion, it corresponds to an electrode of cylindrical geometry, the base of the cylinder facing the microscope objective. Such a geometry can be easily delimited and visualized in 3D by CLSM, as will be shown hereafter. The voltammetric and spectroelectrochemical characterizations reported above have shown that RZ, poorly fluorescent, can be reduced to the highly fluorescent RF at potentials around −0.45 V vs Ag|AgCl. At more negative potentials, RF is further reduced to the nonfluorescent DH. Therefore, we focused our EC-CLSM investigation on the formation of RF by the electroreduction of RZ at −0.45 V vs Ag|AgCl. Figure 4 gathers a set of

Figure 3. Time evolution of the in situ fluorescence emission spectra recorded in a 0.1 mM resorufin solution (a) and a 0.1 mM resazurin solution (b). The electrode potential was held at −0.65 V vs Ag|AgCl in part a and −0.45 V vs Ag|AgCl in part b. Excitation wavelength λex = 543 nm.

increases around 585−592 nm, which is consistent with the formation of the highly fluorescent RF dye. These spectroelectrochemical experiments provide a chemical confirmation of the reaction scheme depicted in Scheme 1, i.e., that the product of the electrochemical reduction of RZ is, indeed, RF, which is further electrochemically reducible to DH. A proper choice of the applied potential enables us to control finely the interconversion between these species at electrode surfaces. In Situ Fluorescence Microscopy. The three fluorogenic molecules clearly exhibit distinct emission spectra; therefore, these are perfect candidates for the coupling between electrochemistry and fluorescence microscopy. The idea is to take advantage of the electrochemically induced fluorescence modulation for imaging purposes. The experimental setup employed to perform in situ fluorescence microscopy experiments consists of a three electrode spectroelectrochemical cell placed on top of an inverted fluorescence CLSM. In our configuration, the excitation beam passes through the objective, then through a very thin (∼0.25 mm) optical window, through a layer of electrolyte (typically several millimeters), and is focused on the working electrode (focal plane). In other words, the electrode surface is facing the objective for a direct observation of fluorescent processes occurring locally. The present setup is very convenient and adapted to a large choice of electrode materials or microscope objectives. Indeed, this spectroelectrochemical cell is similar to conventional three electrode electrochemical cells, and any electrode can be inserted to the cell, regardless of its size (micro- or macroelectrode), provided that the electrode surface is properly oriented to be imaged. In addition, the objective can be easily changed, depending on the requirements in terms of working distance, numerical aperture, or magnification. The RZ−RF system was investigated using the EC-CLSM setup. The working electrode was a 315 μm diameter goldcoated optical fiber bundle (see scanning electron microscopy

Figure 4. Time series of fluorescence CLSM images recorded in situ in a 0.1 mM resazurin solution. The acquisition was started at an open circuit potential, and then the cell was switched on at a potential of −0.45 V vs Ag|AgCl at t ≈ 8 s. The number in the upper left corner of the frames gives the time (in seconds) at which each image collection was started. The white circle indicates the exact position of the cylindrical electrode. In the last frame, the white squares delimit the two regions of interest discussed in the text.

fluorescence images acquired during a potential step experiment, in the presence of 0.1 mM RZ in solution. The potential was initially held at 0 V vs Ag|AgCl, where RZ is stable, and then stepped to −0.45 V vs Ag|AgCl after ∼8 s. Immediately after the potential step, the fluorescence intensity increases markedly over the entire electrode area, confirming the electrochemical generation of a highly fluorescent species at the electrode. A closer examination of the images shows that after a few seconds, the fluorescent area is slightly larger than the electrode area. This is consistent with the buildup of a concentration profile of RF by diffusion around the electrode. A slight “plume” effect oriented toward the lower left corner of the images is also noticeable. We ascribe this phenomenon to the transport of RF toward the counter-electrode. Indeed, repeating the experiment after changing the position of the counter-electrode in the cell resulted in modifying the direction of the plume accordingly (Figure S-4). The time evolution of the fluorescence intensity was monitored in two specific regions of interest (ROI): one E


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Analytical Chemistry located far away from the electrode (ROI 1) and the other located within the electrode area (ROI 2). Both ROIs had the same dimensions, 50 × 50 pixels, giving an overall amount of 2500 pixels to perform the analysis. The mean fluorescence intensity of these ROIs was measured for each image and is plotted in Figure 5 as a function of the elapsed time. It can be

Figure 5. (a) Potential step applied for the in situ fluorescence CLSM images displayed in Figure 4. (b) Time evolution of the corresponding mean fluorescence intensities recorded in ROI 1 and ROI 2 displayed in the bottom right frame of Figure 4.

seen that the fluorescence recorded in ROI 1 remains constant and relatively low regardless of the value of the applied potential. At the electrode surface (i.e., ROI 2), the fluorescence intensity increases markedly immediately upon switching the potential to −0.45 V vs Ag|AgCl, then levels off after 7−10 s to a roughly constant value. This indicates that the average concentration of RF in the confocal volume does not change significantly at long times, in agreement with the formation of a steady-state concentration-distance profile of RF at the ∼315 μm diameter electrode. A key feature of confocal microscopy is the possibility to monitor the spatial distribution of fluorescence not only in the plane of the electrode surface (“x−y plane”) but also along the direction normal to the surface (“z-axis”). Figure 6 presents the results of such an experiment, performed under potential control. The potential was set at −0.45 V vs Ag|AgCl, conditions in which RF is produced at the electrode surface. A sufficient time to reach a (quasi) steady-state of RF concentration was elapsed before starting the images acquisition. The microscope objective was displaced along the z-direction by 5 μm increments, and confocal fluorescence images were recorded at each position. The corresponding series of images allows one to reconstruct either a 2dimensional or 3-dimensional z-stack, providing thus a direct mapping of the fluorogenic RF diffusion layer. Figure 6a displays a 2D image in the x−y plane, recorded at a position z located deep inside the fiber. This image corresponds to the “bottom” slice of the z-stack. The heart of the optical fiber bundle electrode appears as a dark disk because no fluorescence is collected from its interior. A significant fluorescence is noticeable at the gold-coated fiber surface, where RF is electrochemically generated, and its intensity decreases away from the disk edge, in agreement with the radial diffusion of RF toward the solution. Figure 6b is a map of the fluorescence intensity along the z-direction, reconstructed from the stack. It corresponds to the plane parallel to the z-axis, cutting the x-y plane along the top left−bottom right diagonal of Figure 6a,

Figure 6. Mapping of the diffusion layer of RF generated by electrochemical reduction of resazurin at −0.45 V vs Ag|AgCl. (a) In situ CLSM image recorded at a distance z far away from the electrode surface in the direction opposite to the objective. The white circle delimits the area that was used to plot the intensity profile in panel c. (b) Fluorescence intensity map along the z-direction, reconstructed from the z-stack analysis. The map corresponds to a cross-section taken along the top left−bottom right diagonal of the x−y plane displayed in panel a. (c) Comparison between the mean fluorescence intensity profile along the z-direction, averaged in the ROIs displayed in panel a, and the theoretical concentration profile of a species electrogenerated at the electrode surface. The upper limit of the z-axis is fixed at the value of the cylindrical electrode radius, r0. The dotted line is a guide to the eyes to estimate the thickness of the diffusion layer.

and observed from the top right corner. The dark area at the bottom center part of Figure 6b is again the core of the working electrode. The fluorescence intensity profile is roughly hemispherical on top of the electrode, then follows more or less the shape of the fiber on its sides. This is in good qualitative agreement with the concentration profile expected at steady state for the radial diffusion a species generated at the surface of a cylindrical electrode.33 It is thus likely that the fluorescence intensity is a good measure of the relative RF concentration and can be used quantitatively, as was found by Rudd et al.9 on their EC-CLSM measurements of pH gradients using fluorescein. In Figure 6c, the fluorescence intensity profile along the z-axis is confronted to a theoretical steady-state concentration profile. The experimental data points represent the mean fluorescence intensity recorded in a circular ROI of 30 pixels in diameter, centered on the electrode (ROI displayed in Figure 6a), for each slice of the z-stack starting from the edge of the electrode, toward the bulk of the solution. The mean fluorescence F


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Analytical Chemistry intensity sharply decreases, almost linearly, revealing the RF concentration profile. In order to compare this to a theoretical concentration profile, the shape of the electrode was first approximated to an inlaid disc, for which an analytical solution to the diffusion equations, at steady state, is available.34,35 In such a case, and for a species generated at the electrode surface (z = 0), the concentration along the z-axis is given by the expression:34−36 ⎛r ⎞ c 2 = a tan⎜ 0 ⎟ ⎝z⎠ π cz = 0

where r0 is the radius of the disc. This equation was used to plot the solid line in Figure 6c. It predicts that the concentration falls by a factor of 2 at a distance r0 away from the electrode and that the thickness of the diffusion layer is equal to πr0/2.36 Clearly, the experimental concentration (fluorescence) profile is much sharper than the theoretical one. Although the chosen theoretical framework does not reflect the exact shape of the electrode, such an approximation is not at the origin of the observed discrepancy between the experimental and theoretical data. Indeed, Dickinson et al.33 have used numerical simulations to calculate the concentration profiles at fully conducting cylinder electrodes, which better reflect the shape of our fiber bundles electrode. From their results, it is inferred that at steady state, the profile along the z-axis decreases less sharply than that for an inlaid disk-shaped electrode, in contrast with our present observation of a steeper decrease. The occurrence of a diffusion length smaller than predicted very likely originates from the effect of natural (or “spontaneous”) convection. Amatore and colleagues36−38 have demonstrated that at long electrolysis times, natural convection plays a significant role in the establishment of the steady state and has for consequence to decrease the diffusion layer thickness. Most particularly, it was shown that the effect of natural convection becomes increasingly important as the size of the electrode increases. While Rudd et al.9 noticed that the effect of convection was rather small at their 25 μm diameter electrode, the much larger size of our electrode (315 μm diameter) should exert a more significant influence of the spontaneous convection, which very likely explains the observation of a diffusion layer thinner than expected. The almost linear decrease of the concentration (fluorescence) is a further indication of occurrence of spontaneous convection and the establishment of the Nernst diffusion layer.37 Even though it is implicit in the above experiments that the enhancement of the fluorescence at −0.45 V vs Ag|AgCl stems from the electrochemical production of RF, this can be directly demonstrated by performing a wavelength scan with the ECCLSM setup. The confocal microscope employed in this study is equipped with a wavelength selector in front of the PMT, which allows us to select the emission bandwidth of the collected fluorescence. During a wavelength scan experiment, a series of images were recorded with the emission bandwidth set to 10 nm and centered at a given wavelength, this latter being increased by 5 nm steps between each image. Figure 7 presents the results of such wavelength scan experiments, conducted at an applied potential of 0 V (top row, panels A and B) or −0.45 V (bottom row, panels C and D) vs Ag|AgCl. At 0 V, RZ remains stable and is not reduced at the electrode surface. The image depicted in Figure 7a was recorded at this potential and at a wavelength of 640 (±5) nm, which is close to the maximum of the fluorescence emission spectrum of RZ (see

Figure 7. Wavelength resolved in situ CLSM imaging in a 0.1 mM resazurin solution. (a) Image recorded at 640 (±5) nm with an applied potential of 0 V vs Ag|AgCl, at which resazurin is stable. (b) CLSM fluorescence emission spectra reconstructed from a series of images taken at different wavelength bandwidths. The displayed spectra correspond to ROI 1 and ROI 2 of panel a. (c) Image recorded at 580 (±5) nm with an applied potential of −0.45 V vs Ag|AgCl, at which resazurin is electrochemically converted to resorufin. (d) Corresponding CLSM fluorescence spectra reconstructed from ROI 1 and ROI 2 of panel c.

Figure 3). It can be seen that the fluorescence intensity is quite homogeneous throughout the image, though the shape of the electrode can be guessed in the form of a disk of slightly lower fluorescence intensity. The influence of the wavelength is obtained by analyzing the variation of fluorescence intensity in two regions of interest, ROI 1 located far away from the electrode, and ROI 2 located within the area of the electrode surface. The resulting plots of the mean fluorescence intensity versus wavelength are shown in Figure 7b. The two data sets corresponding to ROI 1 and ROI 2 are almost superimposed in the whole wavelength range, and the obtained curve is reminiscent of the emission spectrum of RZ. These observations are, as expected, consistent with the spectroelectrochemical behavior of RZ, but it is noteworthy that the wavelength scan analysis provides a direct molecular (i.e., spectroscopic) information regarding the chemical nature of the fluorescent species producing the recorded images (microscopic imaging). Figure 7c,d shows the results of a similar experiment performed at −0.45 V vs Ag|AgCl. The image displayed in Figure 7c was acquired at a wavelength of 580 (±5) nm, i.e., close to the maximum of the fluorescence emission spectrum of RF. In contrast to Figure 7a, the fluorescence intensity is not homogeneous at all, being clearly much higher in the area of the electrode (with, as observed in Figure 4, a “plume” pointing toward the counter-electrode). This is in agreement with the production of RF at the electrode at the applied potential of −0.45 V vs Ag|AgCl. Figure 7d shows the evolution of the mean fluorescence intensity with the emission wavelength in two ROIs, located at the same positions as in the previous experiment. Far away from the electrode, in ROI 1, the curve is once again quite similar to the emission spectrum of RZ. The G



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mean fluorescence intensity is significantly larger in ROI 2 than in ROI 1, and this in the whole spectral domain. Particularly noticeable is the emergence of a pronounced peak in the wavelength range 560−610 nm. The appearance of this peak gives a direct chemical evidence for the formation of RF, whose fluorescence emission spectrum displays a peak exactly in this range (see Figure 3).

ACKNOWLEDGMENTS This work has been supported by the Centre National de la Recherche Scientifique (CNRS). The support from WallonieBruxelles International and the Fonds de la Recherche Scientifique, the Ministry of Foreign Affairs, and the Ministry of Higher Education and Research is gratefully acknowledged in the frame of the Hubert Curien Partnerships (Program Tournesol No. 29120QE). The cell used for the in situ microscopy experiments was fabricated by Brian Ditchburn, glassblower at the University of British Columbia (UBC, Canada) and kindly offered by Professor Dan Bizzotto, UBC. The authors thank Patrick Garrigue for his skillful assistance in the preparation of the gold-coated fiber bundle electrode.

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CONCLUSIONS To summarize, a general approach coupling electrochemistry and in situ confocal fluorescence microscopy is illustrated. RZ and RF phenoxazines were selected as model electroactive fluorogenic dyes. The different redox state of these dyes exhibit very distinct optical properties which markedly affect their corresponding absorption and emission behavior. The reversible reduction of RF to DH is responsible for a fluorescence extinction whereas the irreversible reduction of the poorly emitting RZ to RF is accompanied by an emission enhancement due to the large increase in quantum yield. This electroinduced conversion is first studied in details by a series of in situ spectroelectrochemical experiments. Then, the direct generation of these species at the vicinity of an electrode surface is monitored under a CLSM. The electrochemical reactivity is evidenced by local fluorescence modulation which is not only recorded in the x−y plane of the electrode but also alongside the z axis. The latter experiment which is a so-called z-stacking allows one to build up and image the diffusion layer surrounding the electrode. Thanks to this original approach and particularly the space resolution of the microscope, surface heterogeneity of the electrode surface could be potentially studied. Indeed, the proof-of-principle experiments reported here with a 315 μm in diameter electrode could be potentially scaled down to smaller microelectrodes by using highresolution/magnification objectives or extended to the study of the local reactivity at modified and heterogeneous electrodes. In the future, such an EC-CLSM coupling will be applied to perform various analytical tasks such as in situ monitoring of the evolution of a biorelevant analytes concentration.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.6b00477. Additional data regarding in situ absorption spectroscopy (Figures S-1 and S-2), SEM image of a gold-coated optical fiber bundle (Figure S-3), and the influence of the position of the counter-electrode in the EC-CLSM setup (Figure S-4) (PDF)

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Corresponding Author

*Phone: +32 2 650 35 80. E-mail: [email protected]. Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest. H


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Coupling Electrochemistry with Fluorescence Confocal Microscopy To

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