We demonstrate that individual carbon nanotubes (CNTs) can be visualized with fluorescence microscopy through noncovalent labeling with conventional fluorophores. Reversal of contrast in fluorescence imaging of the CNTs was observed when performing labeling procedure in a nonpolar solvent. Our results are consistent with a CNT-fluorophore affinity mediated by hydrophobic interaction. The reverse-contrast images also provide clear indication of nanotube location. © 2003 American Institute of Physics

Cheney, Richard E.

Falvo, Michael R.

Prakash, Rohit

Superfine, Richard

Washburn, S.

Carolina Digital Repository

APPLIED PHYSICS LETTERS VOLUME 83, NUMBER 6 11 AUGUST 2003Visualization of individual carbon nanotubes with fluorescence microscopyusing conventional fluorophoresRohit PrakashDepartment of Physics and Astronomy, University of North Carolina, Chapel Hill, North Carolina 27599S. Washburn and Richard SuperfineDepartment of Physics and Astronomy, Curriculum for Applied and Materials Science,Department of Computer Science, University of North Carolina, Chapel Hill, North Carolina 27599Richard E. CheneyDepartment of Cell and Molecular Physiology, University of North Carolina,Chapel Hill, North Carolina 27599Michael R. Falvoa)Curriculum for Applied and Materials Science, University of North Carolina,Chapel Hill, North Carolina 27599~Received 25 April 2003; accepted 9 June 2003!We demonstrate that individual carbon nanotubes~CNTs! can be visualized with fluorescencemicroscopy through noncovalent labeling with conventional fluorophores. Reversal of contrast influorescence imaging of the CNTs was observed when performing labeling procedure in a nonpolarsolvent. Our results are consistent with a CNT-fluorophore affinity mediated by hydrophobicinteraction. The reverse-contrast images also provide clear indication of nanotube location. ©2003American Institute of Physics.@DOI: 10.1063/1.1599042#oalinpessc-ronedepyoeoows-lealuleoofaheNTInthr 5tingadrafer-.sed0There have been several reports on efforts to attach mecules and proteins to carbon nanotubes~CNTs! throughcovalent1–4 as well as noncovalent interactions.5–8 In someof these, the goal of specific immobilization of biologicmolecules on CNTs for biological and chemical sensapplications7,9 was explored. Other work has focused on otical properties of molecules adsorbed onto CNTs. In thstudies, optical behavior of adsorbed molecules was uboth as a mode of probing molecule/CNT interactions2 awell as a method of labeling the CNTs for fluorescenmicroscopy.5 Unlike covalent functionalization, which requires damage to the atomic lattice and the electronic perties of the CNT, labeling through noncovalent attachmeallows for the entire length of the CNTs to be visualizwithout perturbing thesp2 bonded graphene sidewall. Likmany biological structures studied with optical microscoCNTs are too small to observe consistently without the aidfluorescent tags. Larger CNTs can be imaged with phasdifferential interference contrast imaging with the helpvideo enhancement, but reliable fluorescence labeling allfor much smaller CNTs to be visualized with optical microcopy. We present here a preliminary study of the noncovabinding of several common fluorescent dyes to multiwCNTs in both polar and nonpolar solutions. These rescomplement recent reports of attachment of a fluorescpolymer to single-wall CNTs and subsequent imagingsingle-wall CNT bundles in a polar solvent~1,2dicholorethane!.5 This is a report of fluorescence imagingindividual CNTs in biologically relevantenvironments.We labeled the CNTs using two procedures. Inmethod 1,CNTs were deposited from suspension onto glass slidesa!Electronic mail: falvo@physics.unc.edu1210003-6951/2003/83(6)/1219/3/$20.00Downloaded 08 Aug 2003 to 152.2.131.62. Redistribution subject to AIl-g-eedep-ts,forfsntltsntfndthe solvent removed by evaporation upon spin coating. Tflourophore solution was prepared by adding 1.5ml of thefluorophore solution~5 mg/ml in 100% methanol! to 99ml ofdiH20. This solution was then passed over the adsorbed Cin a simple flow cell mounted in an optical microscope.method 2, 250ml of the CNT suspension was combined withe fluorophore/water solution in a test tube and mixed fos prior to spin coating onto a glass slide for imaging~spun350 ml in seven 50ml aliquots onto glass slide!. For bothmethods, the CNT suspensions were prepared by sonica;1 mg of arc prepared CNT soot in 20 ml of methanol.Two optical microscopes were used for this work:Zeiss Axiovert 100TV with Hamamatsu Orca-II coolecharge coupled device~CCD! camera~Figs. 2 and 3!, and aNikon inverted Diaphot 200 with Spot 2 cooled-CCD came~Fig. 4!. Samples were imaged using phase contrast, difential interference contrast~DIC! and fluorescence imagingA tetramethyl rhodamine isothiocyanate filter cube was ufor all fluorescence imaging,~excitation filter 530–560 nm,dichroic mirror wavelength 570 nm, barrier filter 590–65nm!. The following dyes were used in this study:~1! DiIC 16(3) 1,18-dihexadecyl-3,3,38,38- tetramethylin-docarbocyanine perchlorate~molecular probes D-384!;~2! DiOC6(3) 3,38-dihexyloxacarbocyanine iodide~molecu-lar probes D-273!;~3! DiOC5(3) 3,38-dipentyloxacarbocyanine iodide~mo-lecular probes D-272!;~4! DiOC2(3) 3,38-diethyloxacarbocyanine iodide~molecu-lar probes D-14730!;~5! BODIPY® FL C 16 4,4-difluoro-5, 7-dimethyl-4-bora-3a, 4a-diaza-s-indacene-3-hexadecanoic acid~molecularprobes D-3821!; and9 © 2003 American Institute of PhysicsP license or copyright, see http://ojps.aip.org/aplo/aplcr.jspucieiefug.ThsorsegrcatTmnga.eheohep-elonnsTendeureningbonng1220 Appl. Phys. Lett., Vol. 83, No. 6, 11 August 2003 Prakash et al.~6! Alexa Fluor® 488 hydrazide, sodium salt~molecularprobes A-10436!.Of the six fluorophores tested in this study we saw scessful labeling of CNTs with only the DiIC16 and theDiOC6. We saw very limited success with DiOC5 and nolabeling at all with DiOC2, BODIPY or hydrazide. Here wedefine successful labeling as that which produced sufficimaging contrast relative to background to locate CNTsfluorescence images~where the CNT is bright relative to thbackground!. The molecular structure of the two successand one of the unsuccessful fluorophores are shown in FiDiOC5 and DiOC2 have identical structure to DiOC6 asidefrom the number of methylene groups in the sidechains.hyrodzide fluorophore is similar to BODIPY in that it hamostly polar moieties extending from the heterocyclic ption of the molecule.In Fig. 2, CNTs are clearly visible in both the phacontrast image~top! and labeled with DiOC6 in the fluores-cence image~bottom!. The dye labeling was performed usinmethod 1 as described earlier. In some trials, atomic fomicroscopy~AFM! was performed on samples to verify thCNTs observed in the optical images were individual CN~Fig. 5!. We found CNTs with diameters as small as 14 ncan be visualized with phase contrast and DIC imagiThough we show no DIC images, we observed no systemadvantage of phase contrast versus DIC in imaging CNTsaddition to the CNTs, carbonaceous debris was preswhich is routinely seen accompanying CNT material. Tdye readily labeled this material also, as is expected duits graphitic composition. Figure 3 shows another caseCNTs labeled with dye~method 2!. In this case the dye isDiIC16. We note that since the CNTs are well below tdiffraction limited resolution of the optical system, their aparent widths will be equal to the resolution limit in both thDIC and fluorescence images.10 In the case of our opticasystem, this limit will be roughly 300–600 nm dependingthe numerical aperture values for the objective and condelenses.The noncovalent attachment of the dye to the CNprobably occurs through hydrophobic interaction. Graph~the sp2 bonded graphite plane! is hydrophobic. CNTs areessentially graphene sheets wrapped into seamless cylinFIG. 1. Fluorophore structures.Downloaded 08 Aug 2003 to 152.2.131.62. Redistribution subject to AI-ntnl1.e-e.ticInnt,etofersers.The dyes used in this study lack the polyaromatic structthat would best facilitate binding to the CNTs throughpstacking as has been suggested for molecules contaipyrene moieties.7 The success of the DiIC16 and the DiOC6in contrast to the poor results of the DiOC5 and DiOC2 sug-gest that the binding was mediated through the hydrocarchains. The DiIC16 and DiOC6 having longer hydrophobichydrocarbon chains, would bind more stably if the bindiFIG. 2. Phase contrast image~top! and fluorescence image~bottom! ofCNTs labeled with DiOC6 . Scale bars are 10mm in length.FIG. 3. Phase contrast~ op! and fluorescence image~bottom! of CNTs la-beled with DiIC16 . Scale bars are 5mm in length.P license or copyright, see http://ojps.aip.org/aplo/aplcr.jsptoeenraghaerethtel iufssinnhthtofoas-merentlyess-sun-..m.oc..a,tt.oc.C.geref5thethetion1221Appl. Phys. Lett., Vol. 83, No. 6, 11 August 2003 Prakash et al.happens through this part of the molecule~s e Fig. 1!.To better elucidate the nature of the binding of the dyethe CNTs, we repeated the experiments using a nonpsolvent~nonaqueous!. The binding affinity of the dye to theCNTs in the nonpolar solvent should be significantly lowthan in the aqueous case~assuming hydrophobic binding!because of the increased solubility of the dye in the solvOur results are consistent with this hypothesis. Figureshows a fluorescence image of CNTs mixed with DiIC16~method 1! using 1,2-dichloroethane~DCE! in the prep in-stead of water. The fluorescence image shows a strikingversal of contrast when compared to the fluorescence imof Figs. 2 and 3. We interpret this image as indicating tthe binding affinity of the fluorophore for the CNTs is lowthan to the underlying glass substrate in the nonpolar solvWe see a background of fluorescence from dye lying onsubstrate and low fluorescence signal~implying less dye!coming from the CNTs.Note that in these images, the intensities were adjusfor maximum contrast. The absolute fluorescence signatensities indicate that the coverage of fluorophore on thederlying substrate was actually less for the DCE case thanthe aqueous case, though the contrast and the brightnethe images imply otherwise. This suggests that the bindaffinity of the fluorophore decreased for both the CNTs athe glass substrate when going from water to DCE. Tdecrease in affinity, however, was much more severe inCNT case and we observed a reversal of contrast. Oet al. mention a similar solvent related contrast effectsingle-walled CNT bundles in their report.5 As for the ab-FIG. 4. Reversal of contrast in nonpolar solvent. Fluorescence imagCNTs labeled with DiIC16 . For this experiment, the CNTs and dye wemixed in a nonpolar solvent~DCE!. Scale bar is 10mm in length.Downloaded 08 Aug 2003 to 152.2.131.62. Redistribution subject to AIolarrt.4e-estnt.edn-n-orofgdisebersence of quenching through energy transfer to the CNTshas been reported,1–3 we briefly note that the AFM measurements were made of CNT before and after labeling for sotrials to determine the thickness of the adsorbed dye~se Fig.5!. These data indicate a significant buildup of fluoropho( 10 nm) on the CNTs. The energy transfer was apparesufficiently attenuated at these distances to allow for succful fluorescence imaging.5The authors thank Tim O’Brien for helpful discussionon fluorescence microscopy and the National Science Fodation for supporting this work.1V. Georgakilas, K. Kordatos, M. Prato, D. M. Guldi, M. Holzinger, and AHirsch, J. Am. Chem. Soc.124, 760 ~2002!.2L. W. Qu, R. B. Martin, W. J. Huang, K. F. Fu, D. Zweifel, Y. Lin, Y. PSun, C. E. Bunker, B. A. Harruff, J. R. Gord, and L. F. Allard, J. ChePhys.117, 8089~2002!.3J. E. Riggs, Z. X. Guo, D. L. Carroll, and Y. P. Sun, J. Am. Chem. S122, 5879~2000!.4W. J. Huang, S. Taylor, K. F. Fu, Y. Lin, D. H. Zhang, T. W. Hanks, A. MRao, and Y. P. Sun, Nano Lett.2, 311 ~2002!.5K. Otobe, H. Nakao, H. Hayashi, F. Nihey, M. Yudasaka, and S. IijimNano Lett.2, 1157~2002!.6M. Shim, N. W. S. Kam, R. J. Chen, Y. M. Li, and H. J. Dai, Nano Le2, 285 ~2002!.7R. J. Chen, Y. G. Zhan, D. W. Wang, and H. J. Dai, J. Am. Chem. S123, 3838~2001!.8F. Balavoine, P. Schultz, C. Richard, V. Mallouh, T. W. Ebbesen, andMioskowski, Angew. Chem., Int. Ed.38, 1912~1999!.9M. Shim, A. Javey, N. W. S. Kam, and H. J. Dai, J. Am. Chem. Soc.123,11512~2001!.10D. B. Murphy,Fundamentals of Light Microscopy and Electronic Imagin~Wiley, New York, 2001!.ofFIG. 5. AFM image of a CNT~left! before the addition of DiOC6 . Fluo-rescence image~right! after the DiOC6 was added. Inset: AFM image oCNT after addition of DiOC6 . The CNT indicated by the arrow was 18 nmin height before the addition of DiOC6 , and 32 nm after. The scale bar ismm in length. Note that the smaller CNT below the one indicated byarrow is not labeled by the fluorophore. It is not clear to us why this iscase but we speculate it has to do with its small size. Further investigawill be needed to answer this question.P license or copyright, see http://ojps.aip.org/aplo/aplcr.jsp

Visualization of individual carbon nanotubes with fluorescence microscopy using conventional fluorophores

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Visualization of individual carbon nanotubes with fluorescence microscopy using conventional fluorophores

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