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Direct Observations of Graphene Dispersed in Solution by Twilight Fluorescence Microscopy Yutaka Matsuno, Yu-Uya Sato, Hikaru Sato, and Masahito Sano J. Phys. Chem. Lett., Just Accepted Manuscript • Publication Date (Web): 16 May 2017 Downloaded from http://pubs.acs.org on May 18, 2017

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The Journal of Physical Chemistry Letters

Direct Observations of Graphene Dispersed in Solution by Twilight Fluorescence Microscopy

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Yutaka Matsuno , Yu-uya Sato , Hikaru Sato and Masahito Sano* ǂ

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Department of Organic Materials Science, Yamagata University 4-6-13 Jyonan, Yonezawa, Yamagata 992-8510 Japan

ACS Paragon Plus Environment

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ABSTRACT. Graphene and graphene oxide (GO) in solution were directly observed by a newly developed twilight fluorescence (TwiF) microscopy. A nanocarbon dispersion was mixed with a highly concentrated fluorescent dye solution and placed in a cell with a viewing glass at the bottom. TwiF microscopy images the nanocarbon material floating within a few hundred µm of the glass surface by utilizing two optical processes to provide a faintly illuminating backlight and visualizes GO as either a dark image by absorption and energy transfer processes or a bright image by alternation of fluorophore chemistry and autofluorescence. Individual graphene and GO sheets ranging from sub-micron to sub-millimeter widths were clearly imaged at different wavelengths, which were selectable based on the dye used. Graphene could be differentiated from GO coexisting in the same solution. Partial transparency revealed layering and network structures. Motions in tumbling flow were recognized in real time. An effect of changing the solvent and the process of adhesion on the glass surface were followed in situ.

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Graphene is an atomically thin but macroscopically wide carbon sheet1 with excellent electrical, thermal, and mechanical properties.2 This film-like morphology allows placement of graphene sheets on solid substrates for fundamental studies and various applications. However, mechanical strain and chemical interactions with the substrate surface complicate studies of the intrinsic properties and lead to degradation of the electric performance of these materials.3 One method to minimize these unwanted effects is to bring graphene in a liquid.4 Graphene oxide (GO) has been under intensive investigation for use in practical applications.5 The abundant oxygen containing functional groups make GO readily dispersible in various solvents. GO in the form of a dispersion is frequently used in mixtures with other materials, in chemical modifications for further processing, and in device fabrication before reduction to graphene-like sheets.6 To understand and control graphene chemistry, it is of great importance to visualize and characterize individual graphene or GO sheet in liquid environments. The most popular microscopy techniques applied to date to visualize these nanocarbons are scanning electron microscopy and atomic force microscopy (AFM),7 as well as Raman mapping.8 An ordinary bright-field microscope can be used to observe graphene on a solid surface via an interference effect but restricts the solid to optically matched SiO2/Si in air.1,9 Fluorescence quenching microscopy10,11 applies the intrinsic properties of the nanocarbons to quench the fluorescence from a dye overlayer under an epifluorescence microscope. Nonlinear photoluminescence microscopy12 and four-wave-mixing microscopy13 utilize a femtosecond laser. Polarizing microscopy is used to image graphene in liquid crystals.14 Most of these microscopies require the nanocarbons to be fixed on a solid support and are not able to visualize each nanocarbon sheet floating in simple solutions.


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To make a 97.7% transparent15, atomically thin graphene sheet visible in liquid environments, we reasoned that utilizing highly localized illumination, stronger contrast mechanisms than absorption, and orientations that face the macroscopically large surface toward an objective lens would be necessary. The fluorescence emitted by dye molecules dissolved in solution near a solid surface simultaneously satisfies these conditions because the intensity can be spatially controlled at nanometer scales and modified by interactions with nanocarbons. Naturally occurring convective flow may orient a flat object parallel to the solid surface. We developed twilight fluorescence (TwiF) microscopy based on a commercially available total internal reflection fluorescence (TIRF) microscope in the objective lens configuration. A nanocarbon dispersion is mixed with a fluorescent dye solution and placed in a cell with a viewing glass at the bottom. Typical biological applications of TIRF microscopy involved the observation of bright fluorescent objects in a dark background,16 necessitating a low dye concentration in the sample solution. A unique feature of TwiF microscopy is the use of a highly concentrated dye solution in order to confine the fluorescence illumination within a hundred µm from the glass interface. The resulting illumination is dim enough so that the same nanocarbon to appear either dark or bright when the nanocarbon causes a small change in the fluorescence intensity or spectrum of the dye molecules in its vicinity. In order to differentiate these imaging modes, the dark (bright) mode corresponds to when a target object appears dark (bright). In the TIRF microscope with the objective lens configuration, the excitation beam is incident from the glass side to the solution phase at the TIR angle (Figure 1a). The induced evanescent field excites only those dye molecules within a distance on the order of the excitation wavelength from the glass surface. Additionally, the same excitation beam, consisting of light from a mercury lamp that is passed through a slit to configure the beam suitable for the objective of the


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TIRF microscope, contains a small portion of the rays that are not parallel to the optical axis. These off-axis rays do not satisfy the TIR condition and are transmitted to the solution phase as a refracted beam. At high dye concentrations, the refracted light diminishes within several to a few hundred µm due to absorption by the dye molecules themselves. Consequently, the apparent fluorescence viewed from outside is diminished, a phenomenon known as the inner filter effect (IFE).17 The fluorescent light emitted by dye molecules that are excited by both the evanescent field and IFE beams is used for TwiF illumination. This description suggests a close similarity of TwiF microscopy to quasi-TIRF microscopy. The use of an incoherent light source has an advantage of simple operations over a laser quasi-TIRF microscope where a precise adjustment of the incident angle is required. In TwiF microscopy, the illumination intensity is maximal at the glass surface and then decays nearly exponentially within approximately 0.2-100 µm, resembling the twilight sky near the horizon. In this study, GO sheets possessing 2-200 µm widths and dry thicknesses of 1-3 nm, as measured by AFM, were used (Supporting Information, Figure S1). With a 100x objective lens, we should be able to observe a sheet as small as 500 nm wide. Although this observation might be possible for sheets adhered to the glass surface, floating sheets less than approximately 2 µm wide undergo rapid Brownian motions and appear blurry in the present non-viscous solutions. Thus, small fragments were removed, and the concentration of GO was kept low to avoid the IFE due to GO. A method to disperse large sheets of graphene in solution has not been developed. We limited this study to mechanically exfoliated graphene sheets adhered on a solid surface. All GO sheets that have been observed by TwiF microscopy oriented their large surface nearly parallel to the glass surface. Some sheets adhered to the surface, while others slowly floated and drifted (Video 1). When the solution was agitated, the sheets temporarily moved chaotically but


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settled down to a slow drift with a parallel orientation within a short period. On the other hand, crumpled GO sheets (aggregates) moved so fast that they appeared blurry at the usual imaging rate. The Reynold number of a fastest flow that can be imaged with the present configuration is estimated to be an order of 10-3-10-2. These observations indicate that naturally occurring laminar flow orients the flat sheets with their large surface parallel to the glass surface. All images presented in this paper should be understood as a captured frame of these sheets. When a nanocarbon material reduces the fluorescence intensity of nearby dye molecules compared with that of more distant molecules, the nanocarbon appears as a dark image. GO sheets were dispersed in water with 0.1 mM rhodamine-b (Rh-b), the concentration above which the IFE appears in a bulk solution (Figure 1b). Then, the identical location of the sample solution was observed using different microscope setups, which were exchangeable by selecting an excitation beam with a mirror without disturbing the sample solution. Absorption of the light by a single layer of graphene was undetectable, and only thick multilayered flakes were barely visible by a bright-field setup (Figure 2a). Fluorescence quenching offers other contrast mechanisms. Rh-b molecules are known to adsorb on GO surfaces and can be quenched by Dexter energy transfer.18-20 More importantly, distant molecules away from graphene may be quenched by Förster resonance energy transfer (FRET) through Coulombic interactions.21 The dependence of FRET on the molecule-graphene distance r is r-4, compared with r-6 for moleculemolecule, suggesting that dye molecules located over 20 nm from the graphene surface can be quenched.22,23 With the added contrast of FRET, an epifluorescence quenching image (Figure 2b) shows more sheets than a bright-field image (Figure 2a). However, the fluorescence background is too bright to image thinner sheets. Actually, the epifluorescence image is sufficiently bright to be observed directly by the naked eye, whereas the TwiF image must be viewed by a high


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sensitivity camera. As Figure 2c shows, the TwiF illumination is dim enough to reveal thinner sheets with better contrast, yet strong enough to make the GO sheets appear partially transparent. This partial transparency allows analysis of the layering and network structures. Similarly, graphene sheets adhered on an ordinary glass surface in solution can be imaged with better contrast than by interference imaging (Figure 2d). A requirement for efficient FRET is a large spectral overlap of the dye’s fluorescence and the object’s absorption21 (Figure 3a). By choosing a suitable dye, only the objects that absorb the emission wavelengths are selectively imaged. Since GO absorbs over a wide wavelength range, it can be imaged by any visible light (Figure 3b-d). GO sheets in Rh-b and bisbenzimide Hoechst 33342 (H33342) tend to adhere on the glass surface and are imaged sharply. GO sheets in fluorescein (FL) float in the solution and small protrusions or rough parts on the sheet edge execute Brownian motions. Consequently, the floating GO sheets appear smooth and blurry. A slight increase in the dye fluorescence on GO or a decrease in the solution fluorescence gives a bright GO image. Aprotic polar solvents, such as N-methyl-2-pyrrolidone (NMP) and N,N-dimethylformamide, are often used as dispersing solvents for graphene24 and carbon nanotubes25. GO sheets in NMP with rhodamine-6G (Rh-6G) appeared brighter than the surrounding media (Figure 4a). Xanthene dyes are known to form non-fluorescent H-aggregates as well as fluorescent J-aggregates.26-29 In NMP solution, increasing the concentration above that of the IFE shifted the equilibrium toward J-aggregates (Supporting Information, Figure S2). Thus, the locally increased concentration of Rh-6G on the GO surface due to adsorption had enhanced the fluorescence. It is also possible that the atomically rough GO surface or various functional groups on the GO had shifted the equilibrium toward J-aggregates. Because the absorption of GO in the long wavelength region was not too large (Figure 3a), the fluorescent


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light escaping from FRET was sufficient to give a bright contrast. In fact, atomically flat, chemically inert graphene produced dark images in NMP with xanthene dyes. These results were immediately applied to differentiate dark graphene from bright GO coexisting in solution (Figure 4b). There is a way to obtain a bright image of GO without addition of a dye. GO is known to autofluoresce.30-40 Although the autofluorescence of GO sheets wider than a few µm was very weak by the visible excitation, the sheets could be made observable with UV excitation (Figure 4c). We implemented an optical fiber in the microscope to guide a small section of the fluorescence light to a spectrometer so that the fluorescence spectrum from a single GO sheet could be measured (Figure 4d). Both intensity and TwiF spectra of autofluorescence were found to vary between each as-synthesized GO sample, and sheet to sheet within a sample, and even depended on the position within a given sheet and on the layering. It was, however, still useful to image GO with autofluorescence, as it was free of the dye effects. For instance, we noticed that a GO sheet appeared with one of two distinct features exclusively in TwiF microscopy images: a uniformly flat plate or wedged polygon (Figure 5a). The coexistence of these features in the same solution was recognized in both dark and bright modes using different dyes as well as by autofluorescence without dye (Figure 4c). Thus, both features reflect the structure of GO in solution. Since the wedged polygon image has rarely been reported in previous studies on dry GO sheets on a solid substrate, this feature may be stable only in liquid environments. The open-air architecture of the sample cell allows various solution manipulations. For instance, sonication is often used to exfoliate multilayered GO flakes to form thinner sheets, with the risk of tearing GO into small pieces. A user can optimize the sonication procedure to obtain the desired size and layering properties by monitoring the changes (Figure 5b). We can study the


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interactions of graphene with its surroundings. In dispersing solvents, graphene-solvent interactions are favorable, and a graphene sheet spreads flat to maximize its contact area, behaving as if it were under a repulsive osmotic pressure.41 Contrarily, graphene-graphene interactions become dominant in non-dispersing solvents, and graphene surfaces attract each other. When non-dispersing ethanol was added to dispersing water, a floating GO sheet was crumpled immediately and moved out of the viewing area fast. However, a flat GO sheet that had adhered on a glass surface remained stationary. Because some parts of the sheet were strongly adhered to the surface, the intra-sheet graphene-graphene attraction teared the sheet into many pieces with complementary edges over the course of crumpling (Figure 5c). In addition, the adhesion process can be followed in real time. A large GO sheet did not attach its entire surface at once but rather part by part, as if it consisted of smaller sheets jointed by hinges (video 2). Such a space-filling adsorption process recovers a uniformly flat morphology of the GO sheet. Making the glass surface hydrophilic using an oxygen plasma treatment and pressuring the solution toward the glass surface facilitated the preferential attachment of large sheets over 100 µm wide (Figure 5d). We believe that only large sheets, which have high affinity for the hydrophilic surface and are oriented parallel to the surface, can remain on the glass surface, whereas small sheets are swept away under turbulent flow. The exponentially decaying illumination of TwiF microscopy enhances the contrast in the depth direction, clearly revealing three-dimensional movements. Motions such as tumbling flow (video 3) were easily recognized. An atomically thin GO sheet could flip over its face without being curled or folded. This observation indicates a small hydrodynamic resistivity against flipping, suggesting that the sheet is highly porous or is coated by a thick layer of the dye.


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We have demonstrated that the newly developed TwiF microscopy/spectroscopy allows us to study physical chemistry of graphene and GO at a single sheet level in solution. Its illumination is realized by high concentrations of dye (as opposed to diluting the dye solution to reduce the intensity) and a stray light from the excitation beam (as opposed to removing stray light for clearer imaging). The contrast depends on the interactions of an object with dye molecules, as demonstrated here with energy transfers or local modulations of fluorophores. This mechanism suggests that various types of materials other than nanocarbons can be directly observed. If the fluorescent responses of the dye are known beforehand, TwiF microscopy/spectroscopy may be used to characterize the local properties of the target in liquid environments.

EXPERIMENTAL METHODS Twilight fluorescence microscopy. TwiF microscopy was based on a TIRF microscope assembly on an inverted microscope (Eclipse Ti-U, Nikon Instruments) with a 100x or 60x objective lens (both with an NA of 1.49, 60x lens for NMP) and a mercury lamp as the excitation source. A highly sensitive DS-Qi2 monochromatic camera was used, and coloring was performed by a computer to reproduce the color observed through an eyepiece. The excitation (λexc) and emission (λem) wavelengths used (in units of nm) were 525 < λexc < 545 and 580 < λem < 635 for rhodamine-b (Rh-b) and rhodamine-6G (Rh-6G), 450 < λexc < 490 and 520 < λem for fluorescein (FL), and 330 < λexc < 380 and 420 < λem for bisbenzimide Hoechst 33342 (H33342). A drop of the sample solution is mixed with a dye solution in the identical solvent. Then, the mixture is placed on the TIRF glass plate. Minor refocusing may be necessary before the TwiF microscopy observations. To determine the optimum dye concentration, several stock solutions are prepared with the dye concentration ranging from 0.01-10 mM Rh-b in water. The simple


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and quick operation of the TwiF microscope allows us to directly determine the optimal concentration in a short period of time. The concentration used in this report was 0.1 mM for all dyes, unless otherwise specified. Twilight fluorescence spectroscopy. The fluorescence light going into the camera is redirected to a spectrometer (Shamrock 63 Spectrograph with an iVac 324 FI CCD, Andor) by an optical fiber. For rhodamine in NMP, a 575 nm < λem filter was used. Sample preparation. A graphene sheet on a glass plate was prepared by mechanically exfoliating HOPG using adhesive tape. GO flakes were prepared by Hummers method42 and exfoliated by sonication for an appropriate duration determined from TwiF microscopy observation. The GO solution was filtered to remove small fragments less than 1 µm wide. Solution

spectroscopy.

Absorption

spectra

were

recorded

on

a

Jasco

V-570

spectrophotometer using calibrated 0.1, 0.5 and 1.0 cm square cuvettes. Fluorescence spectra were measured on a Hitachi F-2500 fluorescence spectrophotometer using a 1.0 cm square cuvette at 90º detection.

ASSOCIATED CONTENT Supporting Information. Supporting Information is available free of charge via the Internet at http://pubs.acs.org and includes the characterization of GO, absorption and fluorescence spectra of Rh-6G in NMP. Web-Enhanced Object. A WEO is available in the HTML version of the paper. Video 1: drifting GO sheets in NMP. Videos 2: adhesion of GO on glass in water with Rh-b. A GO solution was added to a pure Rh-b solution on the glass surface. Video 3: tumbling motion of GO in NMP after agitating the solution.


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AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] ORCID Masahito Sano: 0000-0002-9050-5269 Notes The authors declare no competing financial interests.

ACKNOWLEDGEMENTS A part of this work is based on results obtained from a project commissioned by the New Energy and Industrial Technology Development Organization (NEDO).

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M.; Chen, C-W. Tunable Photoluminescence from Graphene Oxide. Angew. Chem. Int. Ed. 2012, 51, 6662-6666. (35) Kochmann, S., Hirsch, T.; Wolfbeis, O. S. The pH Dependence of The Total Fluorescence of Graphene Oxide. J. Fluoresc. 2012, 22, 849-855. (36) Shang, J.; Ma, L.; Li, J.; Ai, W.; Yu, T.; Gurzadyan, G. G. The Origin of Fluorescence from Graphene Oxide. Sci. Rep. 2012, 2, 272. (37) Li, M.; Cushing, S. K.; Zhou, X.; Guo, S.; Wu, N. Fingerprinting Photoluminescence of Functional Groups in Graphene Oxide. J. Mater. Chem. 2012, 22, 23374-23379. (38) Thomas, H. R.; Vallés, C.; Young, R. J.; Kinloch, I. A.; Wilson, N. R.; Rourke, P. Identifying The Fluorescence of Graphene Oxide. J. Mater. Chem. C 2013, 1, 338-342. (39) Cushing, S. K.; Li, M.; Huang, F.; Wu, N. Origin of Strong Excitation Wavelength Dependent Fluorescence of Graphene Oxide. ACS Nano 2014, 8, 1002-1013. (40) Du, D.; Song, H.; Nie, Y.; Sun, X.; Chen, L.; Ouyang, J. Photoluminescence of Graphene Oxide in Visible Range Arising from Excimer Formation. J. Phys. Chem. C 2015, 119, 20085-20090. (41) Flory, P. J. Principles of Polymer Chemistry; Cornell University Press: Ithaca, New York; 1953. (42) Hummers, W. S. Jr.; Offeman, R. E. Preparation of Graphitic Oxide. J. Am. Chem. Soc. 1958, 80, 1339.


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Figure 1. TwiF microscopy and inner filter effect. (a) Schematic showing the mechanisms. (b) Concentration dependence of the fluorescence intensity of Rh-b in water, measured by a spectrophotometer using a 1.0 cm cuvette. The region of decreasing intensity above approximately 0.01 mM corresponds to the inner filter effect.


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Figure 2. TwiF microscopy and comparison with other microscopies. Images of GO in water with Rh-b taken by (a) bright-field, (b) epifluorescence, and (c) TwiF microscopy configurations at the same location. (d) TwiF microscopy image of graphene in water with Rhb.


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Figure 3. Dark mode in water. (a) Absorption spectrum of GO and fluorescence spectra of the dyes. TwiF microscopy dark images of GO with (b) Rh-b, (c) FL, and (d) 0.05 mM H33342.


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Figure 4. Bright mode in NMP. (a) GO in a 0.5 mM Rh-6G solution. (b) TwiF microscopy image of GO and graphene sheets in an Rh-b solution. The left image focuses on the graphene sheets attached to the glass surface, and the right image focuses on the GO sheets floating in the solution. (c) Autofluorescence image and (d) its spectrum of a GO sheet without dye (330 < λexc < 380 nm).


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Figure 5. Applications of TwiF microscopy. (a) TwiF microscopy images of GO in NMP with Rh-b, featuring a wedged polygon (marked A) and uniformly flat plate (B). (b) TwiF microscopy images of an aqueous GO solution with Rh-b after various sonication durations. (c) TwiF microscopy image of GO sheets adsorbed on glass in water with Rh-b before (left) and after (right) addition of ethanol. (d) TwiF microscopy image of large GO sheets in water with Rh-b.


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Direct Observations of Graphene Dispersed in ... - ACS Publications

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