Anal. Chem. 1998, 70, 2646-2650
Near-Field Surface-Enhanced Raman Imaging of Dye-Labeled DNA with 100-nm Resolution Volker Deckert, Dieter Zeisel, and Renato Zenobi*
Laboratorium fu¨ r Organische Chemie, ETH Zu¨ rich, Universita¨ tsstrasse 16, CH-8092 Zu¨ rich, Switzerland Tuan Vo-Dinh
Oak Ridge National Laboratory, Advanced Monitoring Development Group, Oak Ridge, Tennessee, 37831-6101
Raman chemical imaging on a scale of 100 nm is demonstrated for the first time. This is made possible by the combination of scanning near-field optical microscopy (SNOM or NSOM) and surface-enhanced Raman scattering (SERS), using brilliant cresyl blue (BCB)labeled DNA as a sample. SERS substrates were produced by evaporating silver layers on Teflon nanospheres. The near-field SERS spectra were measured with an exposure time of 60 s and yielded good signal-to-noise ratios (25:1). The distinction between reflected light from the excitation laser and Raman scattered light allows the local sample reflectivity to be separated from the signal of the adsorbed DNA molecules. This is of general importance to correct for topographic coupling that often occurs in near-field optical imaging. The presented data show a lateral dependence of the Raman signals that points to special surface sites with particularly high SERS enhancement. Raman microscopy has proved to be a powerful tool for highresolution imaging1 of different chemical compounds. The use of diffractive optics, however, limits this method to a lateral resolution to ∼λ/2, given by the Rayleigh criterion. To overcome this limitation, we are combining near-field optical imaging2,3 and Raman spectroscopy. Instead of lenses, a near-field optical fiber tip, which acts as a point light source with subwavelength dimensions, is used for illumination. As long as the distance between aperture and sample is much smaller than the wavelength of the light, the size of the illuminated spot is determined by the tip aperture dimensions. So far, the combination of high lateral resolution in the optical near-field and spectroscopic information has been mostly restricted to fluorescence and luminescence experiments.4-10 Only a few publications dealt with vibrational spectroscopy in the optical near-field. In one example, infrared
spectra were obtained using a photon scanning tunneling microscope (PSTM) configuration and a free-electron laser as a tunable infrared light source.11 Near-field Raman investigations are difficult because of the exceedingly low Raman cross sections and the limitation to low laser power due to small transmission coefficients of the near-field apertures. Consequently, in the few reported near-field Raman experiments, strong Raman scatterers12-14 were measured. Another possibility to increase the Raman scattering cross section is to utilize surface enhancement effects.15,16 Surface-enhanced Raman scattering (SERS)17-19 provides a good way to overcome the above-mentioned sensitivity problems of Raman spectroscopy. Recently, even single-molecule detection with SERS has been claimed.20,21 Scanning near-field Raman spectroscopy provides the unique opportunity of measuring simultaneously the shear-force topography22 of the SERS substrates and Raman scattering from the sample with a lateral resolution on the order of the roughness of the SERS substrate. For the first time, SERS imaging on a 100-nm scale is demonstrated here. We recorded near-field Raman spectra of dye-
* To whom correspondence should be addressed. E-mail: zenobi@ org.chem.ethz.ch. (1) See, for instance: Raman microscopy: developments and applications; Turrell, G., Corset, J., Eds; Academic Press: London, 1996. (2) Heinzelmann, H.; Pohl, D. W. Appl. Phys. A. 1994, 59, 89-101. (3) Betzig, E.; Trautman, J. K. Science 1992, 257, 189-195. (4) Betzig, E.; Chichester, R. J. Science 1993, 262, 1422-1425. (5) Higgins, D. A.; Bout, D. A. V.; Kerimo, J., Barbara, P. F. J. Phys. Chem. 1996, 100, 13794-13803. (6) Van Hulst, N.; Moers, M. H. P. IEEE Eng. Med. Biol. 1996, 51-58.
(7) Dunn, R. C.; Holtom, G. R.; Mets, L.; Xie, X. S. J. Phys. Chem. 1994, 98, 3094-3098. (8) Enderle, Th.; Ha, T.; Ogletree, D. F.; Chemla, D. S.; Magowan, C.; Weiss, S. Proc. Natl. Acad. Sci. U.S.A. 1997, 94, 520-525. (9) Meixner, A. J.; Zeisel, D.; Bopp, M. A.; Tarrach, G. Opt. Eng. 1995, 34, 2324-2332. (10) Ambrose, W. P., Goodwin, P. M.; Martin, J. C.; Keller, R. A. Science 1994, 265, 364-367. (11) Piednoir, A.; Licoppe, C.; Creuzet, F. Opt. Commun. 1996, 129, 414-422. (12) Smith, D. A.; Webster, S.; Ayad, M.; Evans, S. D.; Fogherty, D.; Batchelder, D. Ultramicroscopy 1995, 61, 247-252. (13) Jahncke, C. L.; Paesler, M. A.; Hallen, H. D. Appl. Phys. Lett. 1995, 67, 2483-2485. (14) Grausem, J.; Humbert, B.; Burneau, A.; Oswalt, J. Appl. Phys. Lett. 1997, 70, 1671-1673. (15) Zeisel, D.; Dutoit, B.; Deckert, V.; Roth, T.; Zenobi, R. Anal. Chem. 1997, 69, 749-754. (16) Emory, S. R.; Nie, S. Anal. Chem. 1997, 69, 2631-2635. (17) Fleischmann, M.; Hendra, P. J.; McQuillan, A. J. Chem. Phys. Lett. 1974, 26, 183. Jeanmaire, D. L.; Van Duyne, R. P. J. Electroanal. Chem. 1977, 84, 1-20. Albrecht, M. G.; Creighton, J. A. J. Am. Chem. Soc. 1977, 99, 5215-5217. (18) Otto, A.; Mrozek, I.; Grabhorn, H.; Akeman, W. J. Phys. Condens. Matter 1992, 4, 1143-1212. Wokaun, A. Mol. Phys. 1985, 56, 1-33. (19) Moskovits, M. Rev. Mod. Phys. 1985, 57, 783-826. (20) Kneipp, K.; Wang, Y.; Kneipp, H.; Perelman, L. T.; Itzkan, I.; Dasari, R. R.; Feld, M. S. Phys. Rev. Lett. 1997, 78, 1667-1670. (21) Nie, S.; Emory, S. R. Science 1997, 275, 1102-1106. (22) Betzig, E.; Finn, P. L.; Weiner, J. S. Appl. Phys. Lett. 1992, 60, 2484-2486.
2646 Analytical Chemistry, Vol. 70, No. 13, July 1, 1998
S0003-2700(97)01304-8 CCC: $15.00
© 1998 American Chemical Society Published on Web 05/15/1998
labeled DNA adsorbed on SERS-active substrates and investigated their correlation with substrate topography on a nanometer scale. A brilliant cresyl blue (BCB)-labeled DNA sequence was selected as the analyte for SERS detection due to the importance of DNA in medical applications.23 Combining the high spatial resolution of the SNOM with optical spectroscopy allows a distinction between reflected and Raman scattered light, for separating the local sample reflectivity from the signal of the adsorbed DNA molecules. This is of general importance to correct for topographic coupling that often occurs in near-field optical imaging.24-26 EXPERIMENTAL SECTION Setup. The basic near-field Raman setup has been previously described.15 Several modifications were made to allow slow-scan imaging. For synchronization of the near-field microscope scan and the CCD readout, each instrument was programmed to trigger its counterpart via an RS-232 serial line. This ensures a precise correlation between lateral position and optical spectrum. For this we used a special lithography software (TopoLith 1.1, Paul Bucher Co., Basel, Switzerland) and adapted the source code of the scanning software and the CCD data acquisition program (MAPS 2.0, Photometrics Ltd.) for our purpose. During the whole measurement, the tip was kept in shear-force feedback. The tip was set at the desired lateral position until the CCD camera finished data collection. The tip was then moved to the next position. The light was collected in reflection using a microscope objective (NA ) 0.65) and focused onto a 100-µm glass fiber that acted as a confocal element. The lateral resolution with this setup was 80-100 nm. Laser power at the sample was 1 µW. All Raman spectra were measured with the 488-nm line of the Ar+ laser. Using a 50-µm slit in the spectrometer gives us a spectral resolution of
