Single layer graphene for estimation of axial spatial resolution in

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Single layer graphene for estimation of axial spatial resolution in confocal Raman microscopy depth profiling Carol Korzeniewski, Jay P. Kitt, Saheed Bukola, Stephen E. Creager, Shelley D. Minteer, and Joel M. Harris Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b04390 • Publication Date (Web): 04 Dec 2018 Downloaded from http://pubs.acs.org on December 10, 2018

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Analytical Chemistry

Single Layer Graphene for Estimation of Axial Spatial Resolution in Confocal Raman Microscopy Depth Profiling Carol Korzeniewski,*a,b Jay P. Kitt,b Saheed Bukola,c Stephen E. Creager,c Shelley D. Minteer,b Joel M. Harrisb Abstract Single layer graphene (SLG), with its angstrom-scale thickness and strong Raman scattering cross section, was adapted for measurement of the axial (Z-direction) probe beam profile in confocal Raman microscopy depth-profiling experiments. SLG adsorbed to a glass microscope coverslip (SLG/SiO2) served as a platform for estimation of axial spatial resolution. Profiles were measured by stepping the confocal probe volume through the SLG/SiO2 interface while measuring Raman scattering from the sample. Using a high numerical aperture (1.4 NA) oil immersion objective, axial profiles were derived from the graphene 2D vibrational mode and fit to a Lorentzian instrument response function (IRF). Subsequently, the Z-direction spatial resolution in depth-profiling studies of polymer interfaces was estimated through convolution of the Lorentzian IRF with a step function representing the ideal junction separating the phases of interest. In the study of a bipolar polymer membrane, confocal Raman depth profiles of the AEM/CEM (anion exchange membrane / cation exchange membrane) interface show the transition region is broader than the limiting response and are consistent with roughness at the boundary on the order of a few micrometers. Using ClO4- as a Raman active mobile ion probe, application of self-modeling curve resolution (SMCR) to spectral datasets within a profile showed ClO4- ions track the spatial distribution of the AEM phase. Finally, in measurements on a liquid-solid interface formed between 1-octanol and a polydimethylsiloxane (PDMS) membrane, the IRF derived from fitting the experimental profile was slightly narrower than those obtained from profiling SLG, indicating the potential to use polymer-liquid interfaces formed from widely available materials and reagents for estimation of axial spatial resolution in confocal Raman depth-profiling. * Corresponding Authors: [email protected]; [email protected] aDepartment

of Chemistry and Biochemistry, Texas Tech University, Lubbock, TX 79409

bDepartment

of Chemistry, University of Utah, Salt Lake City, UT 84112

cDepartment

of Chemistry, Clemson University, Clemson, SC 29634

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Introduction Confocal Raman microscopes enable analyses to be restricted to ultra-small sample volumes, on the order of a few femtoliters (fL), by combining tight (diffraction limited) focusing of the excitation beam with an aperture in the image plane that restricts light originating from outside the excitation volume from reaching the detector (Figure S1).1-4 While often applied to spatially resolve chemical composition within materials that possess some type of heterogeneity, in recent years confocal Raman microscopy has advanced as a rapid and sensitive platform for performing chemical assays in ultra-small volumes based on detection of molecules extracted into individual, micron-scale porous particles.5-12 Additionally, when combined with optical trapping, confocal Raman microscopy has enabled changes within an individual particle to be followed in response to stimuli, such as temperature change,13,14 analyte partitioning10 and receptor mediated interactions.15 The potential to adapt confocal Raman microscopy for characterization of buried interfaces within materials has been of longstanding interest.1-3,16-21 However, depth-profiling applications have been limited, because phenomena such as refraction, spherical aberration and stray scattering through the probe volume can degrade spatial resolution.1,2 Over the years, it has been shown that the first two of these effects can be greatly mitigated through the use of high numerical aperture, oil-immersion microscope objectives.1-3,18 Building on the advantages of oil-immersion optics, the reported work demonstrates a simple approach for confocal probe volume characterization that enhances the utility of confocal Raman microscopy for materials depth-profiling. Robust interfaces, which are easy to prepare and adapt for measurement of the axial (Z-direction) confocal probe volume profile, are described. In one example, single layer graphene (SLG) adsorbed to a glass microscope coverslip (SLG/SiO2) is

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used to determine the spectrometer instrument response function (IRF). Subsequently, this IRF is applied to estimate the limiting Z-direction spatial resolution in studies of polymer and mobile ion composition at the buried junction separating the anion- and cation-exchange phases of a bipolar membrane.22-26 In another example, the limiting IRF is derived from profiles of an interface formed by a several micron layer of bulk liquid in contact with a smooth polymer membrane. The latter has potential for more general application due to component availability and the possibility to select from among a variety of materials and reagents. In the study of buried polymer interfaces, nanometer-scale spatial resolution in depth profiling materials under ambient, and even in situ electrochemical conditions, is possible through the use of neutron reflectometry, albeit with constraints on scanning depth (< 500 nm) and interface uniformity.27-30 Although the Z-direction spatial resolution is lower, with a working depth of 200 m or greater, and ability to sample within a variety of chemical environments, confocal Raman microscopy is more easily adapted for direct spatial mapping materials of practical interest, such as the bipolar membranes investigated.1-3,18,21 The reported strategies for estimation of limiting resolution in sub-surface profiling with confocal Raman microscopy improve quantitative understanding and advance the technique as an aid to scientific discovery. Experimental Samples and Reagents. Single-layer graphene (SLG) was transferred onto a glass cover slip using the trivial transfer technique.31 In brief, the trivial transfer graphene (TTG) sample consisting of SLG on a poly-(methyl methacrylate) (PMMA) film adherent to a polymer support was purchased from ACS Material (Pasadena CA, USA). The TTG was released onto a deionized water surface to remove the graphene with PMMA from the polymer support. The graphene with PMMA was then picked up from the water surface with the cover slip. SLG with

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PMMA on the cover slip was allowed to dry at ambient temperature. Then, the PMMA was removed by immersing the cover slip into acetone that had been preheated to 50 C. The PMMA was rinsed off by the acetone leaving behind only the SLG on the glass cover slip. The transfer technique of SLG onto Nafion membrane was similar to our prior work.32 Briefly, chemical vapor deposition graphene on copper foil (ACS Material) of size 2.0 cm x 2.0 cm was cut from a larger piece and placed on top of a Nafion-211 (Fuel Cell Store, College Station, TX, USA) membrane disk (19.1 mm diameter) that was supported by polytetrafluoroethylene-reinforced fiberglass.32 This assembly was hot pressed together in a Carver hot press at 140 C, 600 lbf, for 2 min. Thereafter, the underlying Cu was removed in an etching solution of 0.3 M (NH4)2S2O8 in water, leaving behind only the SLG on the Nafion membrane. Just prior to spectral measurements, to ensure samples were free of contaminants and the membrane was fully ion exchanged, samples were very briefly (a few minutes) immersed in warm (60 C) 0.5 M NaCl containing 0.3 % H2O2 and subsequently rinsed in deionized water. Surface water was removed by patting lightly with a Kimwipe before placing the membrane onto the optical stage for analysis. Bipolar membranes (Fumasep FMB) were from FumaTech (Bietigheim-Bissingen, Germany) and provided by the Lewis group (Caltech). Samples were stored in 1 M NaCl, and just prior to spectral measurements, were treated as described above for graphene-modified Nafion membranes, except the immersion periods were typically longer (15 min). Samples exchanged 0.1 M NaClO4 solution were rinsed and subsequently stored in ultrapure water for 48 hrs prior to spectral measurements to reduce the likelihood of accumulating ClO4- in the cation exchange phase through Donnan exclusion failure. Polydimethylsiloxane (PDMS) membrane was cut from

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Bisco HT-6240 sheet (0.31”, Rogers Corp., Carol Stream, IL, USA). 1-Octanol was from SigmaAldrich (St. Louis, MO USA). Chemical reagents were reagent grade or better and used as received. Aqueous solutions were prepared from 18 MΩ-cm water delivered from a Milli-Q water purification system. Raman Microscopy. The confocal Raman microscope has been described in detail.3,33 Briefly, Raman scattering was excited by a Kr+ laser operating at 647.1 nm (Coherent, Santa Clara, CA, USA) and 3 mW (graphene/substrate samples) or 30 mW (bipolar membrane) power. Following expansion using a 4 beam expander (50-25-4X-647, Special Optics Inc., Wharton, NJ), the laser beam was directed into the rear entrance port of a Nikon TE 200 inverted microscope (Nikon TE-200, El Segundo, CA, USA) and reflected off of a dichroic mirror (Semrock, Lake Forest, IL, USA), slightly overfilling the back aperture of a 100, 1.4 NA oil immersion objective (Nikon Plan Apo, El Segundo, CA, USA). The focused spot from the objective was directed through immersion oil contacting the underside of a microscope coverslip (No. 1.5 thickness, BK-7 glass). Membrane samples were positioned on top of the coverslip and held in place with a covering quartz microscope slide. The microscope objective collected scattered light from the sample, which was passed back through the dichroic mirror and a final high pass filter (Semrock, Lake Forest, IL, USA) before being focused onto the entrance slit of a grating monochromator (Bruker 500-IS, Preston, Victoria, Australia). The diffraction grating, 300 lines/mm blazed at 750 nm, and 50 m entrance slit provided a spectral resolution of 3 cm-1. Raman scattered light was detected using a charge-coupled device (CCD) camera (Andor, iDus DU401A, South Windsor, CT, USA). The confocal aperture was defined using the entrance slit of the monochromator in the horizontal dimension and by binning three rows of pixels on the CCD camera (78 m) in the

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vertical dimension (Figure S1).17,18 The vertical dimension was optimized for each sample. To account for any sensitivity of the confocal aperture position to the refractive index properties of the sample, the CCD rows were selected while the interface of interest was positioned within the excitation beam focal region (Figure S2). The collection aperture, together with the tight excitation beam focus (0.6 μm diameter)3, defines an ideal, diffraction-limited confocal probe volume with 90% detection efficiency within a depth along the Z-direction of ± 1.2 μm;3 note, this depth was incorrectly stated as a factor of two smaller in a recent related paper.34 Raman spectra of samples containing SLG were recorded by averaging 20 spectra collected with exposure periods of 5 s. For bipolar membrane samples, typically 20 spectra collected during 10 s exposure periods were averaged. Data were processed using custom scripts executed in Matlab (version 8.6; MathWorks, Natick, MA, USA). After subtraction of a dark current offset, sample spectra were corrected for instrument response35 by ratioing to a white-light reference spectrum.7 Baselines were corrected using a rolling-circle36 high pass filter. Lorentzian peak fitting was performed using OriginPro (version 8.0, OriginLab Corp., Northampton, MA, USA). Convolution calculations and spatial profile fitting were carried out using Microsoft Excel (Redmond, WA, USA). Matlab (version R2015a) supported self-modeling curve resolution (SMCR)37,38 analysis of Raman spectra collected as a function of depth into a sample, as described previously39,40 for the examination of Raman datasets. Results and Discussion Single-layer graphene for depth profiling confocal depth resolution. The strategy for adapting SLG to map the axial dimension of the confocal probe volume in Raman depthprofiling experiments is shown in Figure 1. The spectra in Figure 1A were recorded as the probe was stepped vertically through a graphene layer adherent to a microscope coverslip. Starting just

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Figure 1. (A) Confocal Raman spectra of SLG following deposition on a microscope coverslip, as a function of the probe Z-spatial position. The Z = 0 position is 12 m below the coverslip. The region of the graphene 2D band is shown. (B) The graphene 2D peak intensities in (A) plotted as a function of Z-spatial position. The solid line (red) is the least-squares fit to a Lorentzian function with FWHM = 1.7 m.

below the coverslip, at the position labeled Z = 0, the change in graphene 2D peak scattering was tracked as the beam focus was translated upward. In the spectra shown, the scattering intensity grows as the region of tight excitation beam focus comes into alignment with the graphene plane, near Z = 12 μm, and decreases as the focus translates further through the layer. For reference, a full SLG spectrum recorded with the probe near the point of optimal alignment is included in Figure S3 alongside a spectrum of basal plane HOPG. For both, the 2D and G band positions and shapes are consistent with earlier reported Raman spectra of the materials excited in the red at frequencies near 647 nm.32,41,42 Since SLG has atomic-scale thickness, orders of magnitude smaller than the confocal Raman probe volume Z-dimension, the scattering collected while translating the probe volume through the SLG/SiO2 interface with Z normal to the graphene plane traces the probe volume detection efficiency profile (Figure S4).2-4,18 The profile derived from the graphene 2D peak intensities in Figure 1A is plotted in Figure 1B and follows the Lorentzian response expected for the microscope.3 The full width at half maximum (FWHM) is a measure of the probe volume depth

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along the Z-direction encompassing the region of 50% detection efficiency (Figure S4).1 Aberrations from refractive index mismatches between the objective and sample43 (the objective is designed for imaging from oil immersion fluid into water), together with the limitations of image transfer and filling the numerical aperture of the objective broaden the probe volume relative to an ideal, diffraction-limited response. The measured profile in Figure 1B that was achieved is consistent with the 2 μm FWHM observed earlier,4,18,44 particularly in related measurements by Froud and co-workers18 using a comparable oil-immersion objective to collect scattering from a thin silicon layer and by de Grauw, et al.4 using a water-immersion objective to profile a thin polystyrene film. These results are also consistent with the depth profile found earlier in studies of this instrument that employed polymer beads immersed in water. In this earlier work, the small size of the particles (smaller than the excitation spot size) and the refractive index of water surrounding the Figure 2. Confocal Raman depth profiles recorded from a membrane composed of SLG immobilized to the surface of Nafion 211.32 The membrane was oriented with the SLG side toward the microscope coverslip. The Z = 0 position is 6 m below the coverslip. The solid red line is the least-squares fit of the graphene 2D band peak frequencies (black symbols) to a Lorentzian function with FWHM = 1.9 m. The blue line is the convolution of this Lorentzian response function with a step function representing the ideal graphene/Nafion interface. The blue symbols are the peak intensities for the Nafion band at 730 cm-1 associated with vibrations centered on the TFE backbone. The green dashed line is the convolution result obtained with FWHM = 1.7 m.

beads, which matches the objective design, generated slightly narrower depth profiles.3 The measured profile from an atomicallythin layer estimates the IRF for the confocal microscope and spectrometer and is valuable for assessing limiting resolution in depthprofiling applications.1,2,18,20 As an example, Figure 2 shows the correspondence between

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the experimental confocal Raman depth profile of polymer near a SLG/polymer interface and the limiting profile for the polymer calculated using the IRF derived from graphene 2D vibration scattering intensities. The black symbols in the plot trace the response from the SLG layer, which is Lorentzian with FWHM of 1.9 μm, slightly greater than profiles recorded from the more rigid SLG/SiO2 interfaces studied (Figure 1). The blue symbols trace the bulk membrane phase based on intensity in the strong vibration at 730 cm-1 centered on ionomer backbone tetrafluoroethylene (TFE) groups (Figure S5).45-47 The solid blue line that follows the TFE marker experimental points is the result of calculating the convolution of the SLG Lorentzian line shape in the plot with a step function representing the ideal SLG/polymer interface. The dashed line that closely overlays the solid blue line, but has a slightly sharper rise, is the convolution result obtained by substituting the Figure 1B IRF. The 0.2 μm difference in FWHM for the two graphene profiles has a negligible effect on the derived polymer profile in comparison to the variation in spectral peak intensities. We anticipate the slightly narrower IRF measured in experiments with the SLG/SiO2 platform reflects differences in substrate rigidity and response to SLG heat dissipation under laser irradiation due to its very strong absorption cross section at the excitation frequencies used. Indeed, to prevent damage, a low laser power (3 mW) was required to depth profile samples containing SLG.32,42 The convolution results in Figure 2 show good consistency with the experimental scattering intensities and indicate the graphene profiles closely approximate the spectrometer IRF, which can be used to detect real distributions in composition along the Zdimension (see below). Application to two immiscible contacting bulk phases. The convolution relationship applied to obtain the polymer profile in Figure 2 was subsequently adapted to extract the limiting IRF from a profile of two contacting bulk phases, as demonstrated in Figure 3 for the liquid-solid

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interface formed between 1-octanol and a PDMS membrane. Since the refractive index values (n) for the components are very close (n  0.03), aberrations associated with refractive index change at the boundary are expected to be small.1 The symbols in the plot trace the Raman scattering intensities for spectral bands that are well resolved and unique to each phase (Figure S6). Again, approximating the interface as a step function, its convolution with the spectrometer IRF reflects the experimentally observed profile. In Figure 3, the solid line was derived from an iterative fit to the convolution function using the FWHM variable in the Lorentzian IRF as an adjustable parameter. In the optimized profile, the FWHM is close to the values obtained from measurements on SLG/SiO2 interfaces. This result indicates the simple 1-octanol/PDMS system is well-suited as a platform for estimation of the probe volume detection efficiency profile in confocal Raman microscopy depth-profiling experiments. Although the 1-octanol and PDMS components were chosen for their availability, complementary spectral features and closely matched refractive index, a variety of other

Figure 3. Depth profiles derived from confocal Raman spectra of the solid-liquid interface region formed between a PDMS membrane contacting a 1-octanol thin film. The Z = 0 position is 14 m below the coverslip. The symbols track the change in peak intensities with probe beam Z-spatial position for the PDMS (black, 490 cm-1 band) and 1-octanol (blue, 1064 cm-1 band) phases. The red line shows the convolution of a Lorentzian response function (FWHM = 1.6 m) with a step function representing the ideal, atomically smooth 1-octanol / PDMS interface.

suitable materials and reagents exists that can be similarly adapted. Characterizing the interface compositions of a bipolar membrane. In subsequent studies, the IRF derived from profiling the SLG/SiO2 interface was applied to estimate limiting spatial

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resolution in the analysis of the region separating anion- and cation-exchange phases within a bipolar membrane. Bipolar membranes typically contain anion and cation exchange materials in two separate but adjacent layers, sometimes with a much narrower (nanoscale) layer of a watersplitting catalyst in between.22,23 The membranes enable polymer electrolyte fuel cells and electrolyzers to operate near the pH optimum of the catalytic reactions, providing a route to improved energy conversion efficiency and substantial cost reductions.22-26 Realizing these gains, however, depends upon achieving an ideal spatial distribution of components within the interfacial region.22 Measurement techniques capable of accessing the buried interface and probing variations in structure and composition across the layers would greatly assist the development of bipolar membranes for improved efficiency in energy conversion applications. Figure 4 demonstrates the spatial resolution attained in confocal Raman depth profiling near the anion-exchange membrane / cation-exchange membrane (AEM/CEM) junction of a bipolar membrane. The black symbols trace the experimental profile for the CEM component based Figure 4. Depth profile derived from confocal Raman spectra of the AEM-CEM interface region of a Fumasep FBM bipolar membrane (see Figure 5). The peak intensity of the unique CEM-phase marker at 1128 cm-1 is plotted as a function of the measurement position (black symbols). The solid lines show the result of calculating the convolution of a Lorentzian response function, with FWHM = 1.7 m (red) or 3.5 m (black), and a step function representing the ideal, atomically sharp AEM-CEM interface. A photo taken through the microscope eyepiece is included as an inset and shows the region of beam focus (“X”) and segment of PEEK mesh support.

on a feature unique to the phase at 1128 cm-1 (Figure 5). The black line shows the profile is well fit by a Lorentzian IRF that has a FWHM just greater than two times the limiting IRF derived in measurements on SLG. The broadening across the AEM-CEM gap suggested by the profiles is consistent with the membrane processing

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conditions. During processing, the AEM and CEM surfaces forming the boundary are intentionally roughened to increase the contact area, and polyelectrolyte coatings and a water splitting catalyst are applied.48 These modifications contribute a few microns separation between the bulk AEM and CEM phases. The chemical modifying layers are too narrow to be resolved individually in the depth profiling experiment, but the broadening in the CEM phase profile relative to the limiting response reflects the presence of these layers. Figure 5 shows selected spectra from the dataset associated to the black symbols in Figure 4. The band at 1128 cm-1 gives the clearest evidence for appearance of the CEM phase. Other prominent bands in the spectra appear above 1250 cm-1 and display only weak sensitivity to the probe beam axial position, indicating the chemical functionality associated with the vibrational modes is common to both the AEM and CEM layers. The inset photo in Figure 4 was taken through the microscope objective while the objective focused the probe within a bipolar membrane on the stage. The photo shows the outline of the PEEK (polyether ether ketone) polymer mesh supporting the bipolar membrane and probe beam location within the plane (x,y) normal to the Zdirection. In recording the Figure 5 spectra, the probe beam was positioned toward the center of one of the square-shaped areas of the PEEK mesh. As expected, spectral features that distinguish PEEK from other membrane components, particularly PEEK bands near 1144 cm-1 and 1645 cm-1 (Figure 6) are not evident in Figure 5. However, as shown below, PEEK features

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Figure 5. Confocal Raman spectra selected from the set associated with the Figure 4 profile. The membrane was ion exchanged in 0.5 M NaCl and rinsed briefly in deionized water just prior to measurements. At Z = 0, the confocal probe volume is positioned within the AEM phase and 15 m below the AEMCEM interface.


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can be observed when profiling deeper into the membrane,1,2,20 especially with the probe position within the in the x,y plane closer to the mesh (Figure S7). To improve spatial resolution in confocal Raman microscopy depth-profiling, multivariate analysis techniques provide potential opportunities.21 In the reported work, SMCR37,38 was adapted to more closely scrutinize bipolar membrane spectra. SMCR is a multivariate curve resolution technique that uses eigenvector decomposition of the spectral data to resolve correlated changes in the spectra as a function of the experimental parameter varied (depth, in this case).37,38,49 The results of SMCR analysis are a set of component spectra which describe the chemical composition of the sample, and a set of contribution vectors which describe how the chemical composition changes as a function of the experimental parameter. Figure 6 displays the pure components derived from application of SMCR to a dataset (Figure S7) recorded as a function of probe depth within a bipolar membrane. Each SMCR component is plotted along with an experimental spectrum of the phase to which it is assigned. For the CEM phase, the experimental spectrum and SMCR-derived spectral vector match well. The agreement between spectra associated with the AEM phase is similar. For these experiments, the membrane was exchanged in an aqueous 0.1 M NaClO4 solution Figure 6. Spectral vectors derived from SMCR analysis of confocal Raman spectra (Figure S7) recorded while profiling across the AEM-CEM bipolar membrane interface (black) compared to experimental spectra of the pure component phases (blue, red, cyan, as indicated).

with the aim of incorporating a marker (ClO4- in this case) capable of providing insights into the spatial distribution of charge compensating mobile ions near the AEM/CEM interface. Therefore, in addition

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to bands from the anion exchange polymer, the AEM phase spectral vector includes the ClO4- ion vibrational feature at 927 cm-1. It is notable that the ClO4- feature appears in only one of the orthogonal spectral vectors identified through SMCR analysis, as it indicates the membrane effectively excludes ClO4- ions from the CEM phase. The third component detected in the SMCR analysis is traceable to the PEEK mesh membrane support. The features likely arise from Raman scattered photons that originate in illuminated areas of the membrane outside the excitation beam focus, but which scatter back through the confocal probe volume.1,2,20 Without application of SMCR, there is potential for the PEEK feature at 1144 cm-1 to overlap the CEM phase marker at 1128 cm-1 as the beam is translated more deeply into the CEM phase. SMCR analysis provides discrimination against this PEEK interference in developing profiles of the CEM phase. The concentration vectors for the AEM and CEM components identified through SMCR analysis of spectra from the bipolar membrane depth profile are plotted in Figure 7. In contrast to Figure 4, the use of

Figure 7. Concentration coefficients derived from SMCR analysis of the Fig. S7 dataset for the components indicated (ClO4- exchanged AEM phase (black), CEM phase (red) and PEEK mesh support (cyan)). The solid lines show the result of calculating the convolution of a Lorentzian response function, with FWHM = 1.7 m (red) or 4.5 m (black), and a step function representing the ideal, atomically sharp AEM-CEM interface.

SMCR to identify responses associated with pure components enables a profile for the AEM phase to be constructed. The lack of spectral features unique to the AEM polymer in the bipolar membrane limited the ability to include a profile for the phase in Figure 4. The solid lines in the Figure 7 plot show the result of calculating the convolution of a step function, serving as a model for the ideal AEM/CEM interface, and IRFs with different FWHM values. For the data segments

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compared, the measured AEM profiles are broader than the limiting profile calculated using the SLG/SiO2 derived IRF (FWHM = 1.7 m). The data follow the convolution result obtained when the IRF FWHM is 4.5 m, slightly larger, but comparable to the responses in Figure 4 (FWHM = 3.5 m). The rising portion of the CEM phase profile is similarly broadened. As discussed above, the roughening and catalyst layer insertion within the AEM/CEM gap can explain the lack of sharpness in the composition transition in Figure 7. For depths greater than the 20 m position in Figure 7, significant asymmetry develops in the responses. The asymmetry does not appear to be linked to the presence of Raman active mobile ions in the AEM phase, since the irregularities also appear in concentration vectors obtained from depth profiles on bipolar membrane exchanged in aqueous NaCl solution. The unanticipated asymmetry may be related to back reflection of light from the roughened, catalyst modified AEM/CEM junction. Reflection of excitation or Raman scattered photons back toward the detector is expected to enhance signals from the AEM, while the reduced light transmission across the AEM/CEM junction will diminish signals from the CEM phase. Effects of light back reflection can also explain the persistence of the concentration vectors for the AEM phase, including exchanged ClO4- ions, as the probe beam is advanced within the CEM. Similar asymmetry was not observed in profiles of the 1-octanol/PDMS system (Figure 3). To attain deeper understanding, further study is planned using model AEM/CEM interfaces that have controlled composition and structure. Conclusions SLG transferred to a glass microscope coverslip is a sensitive platform for measurement of the axial probe volume detection efficiency profile of a confocal Raman microscope. This beam profile, after convolution with a model of the interface under study, enables estimation of the 15



expected limiting Z-direction spatial resolution for the experiment. In confocal Raman microscopy experiments performed with a high NA oil-immersion objective, the beam profile derived from measurements on SLG/SiO2 allowed chemical composition variation to be distinguished from instrument limited broadening in depth profiling through polymer interfaces. The profiles recorded did not suffer light collection efficiency losses across the range of depths probed (< 100 m), as anticipated for the immersion optics.1,18,46 In the study of bipolar membranes, depth profiles through the AEM/CEM junction were broader than the limiting instrument response and indicate the pure AEM and CEM phases have a separation of a few micrometers. Although not initially expected, the results are consistent with known properties of the bipolar membranes examined.48 Since the AEM/CEM interface within a bipolar membrane is key to efficiency in energy conversion applications,22,24-26 the ability to discern differences in chemical composition through the region provides valuable information for guiding the design of next generation membranes. To enhance spatial resolution, strategies for filtering out of focus light in the pupil plane1,50 are under investigation. Since 90 % detection efficiency is represented by 6  FWHM for a Lorentzian axial beam profile, discrimination of pure phases in Z-profiling is extremely challenging.1 The approach presented provides simple and inexpensive means for determination of the limiting spectrometer IRF, enabling close estimates of the spatial resolution attainable in measurements on the system of interest.1,2,18,20 In relation to the latter, the system must be adaptable to the microscope stage and constraints of the objective working distance (< 200 m). Furthermore, although the ability to resolve adjacent features in a depth profile is limited by the IRF, factors such as variations in scattering cross section for the different materials1 and

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sensitivity of light collection efficiency to probe depth (< 100 m for immersion objectives1,18,46) can add further constraints. The SLG/SiO2 samples used in the reported work are simple to prepare and have good durability, providing reproducible SLG Raman spectra over many months as long as laser powers are kept to  3 mW. Similar to SLG/SiO2, a profile of the interface separating a smooth polymer membrane surface in contact with a liquid of closely matched refractive index (e.g., 1octanol/PDMS) also enabled estimation of the limiting spectrometer IRF. The reported strategies assist interpretation of spectra derived from confocal Raman microscopy depth-profiling and will find broad application as systems of interest (i.e., buried interfaces,32 materials under electrochemical potential control34,51,52) grow in complexity. Associated Content Supporting Information The supporting information is available free of charge on the ACS Publications website. Formation of a confocal aperture; confocal aperture alignment; SLG full Raman spectrum; confocal Raman probe volume axial detection efficiency profile; SLG-Nafion confocal Raman depth profile spectral dataset; 1-octanol/PDMS confocal Raman depth profile spectral dataset; bipolar membrane confocal Raman depth profile spectral dataset Acknowledgements The authors gratefully acknowledge support from the Office of Science, U.S. Department of Energy, through grants DE-FG03-93ER14333 (Utah) and DE-SC0018151 (Clemson) and the U.S. ARO MURI program through grant W911NF-14-1-0263 (Utah). We thank Nate Lewis and

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For table of contents only.

Confocal Raman microscopy Z-direction probe beam calibration

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Single layer graphene for estimation of axial spatial resolution in

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