Spatially Inhomogeneous Enhancement of Fluorescence by a

Anatoli Ianoul* and Andrew Bergeron. Department of Chemistry, Carleton UniVersity, 1125 Colonel By DriVe, Ottawa ON Canada. ReceiVed June 30, 2006...
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Langmuir 2006, 22, 10217-10222

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Spatially Inhomogeneous Enhancement of Fluorescence by a Monolayer of Silver Nanoparticles Anatoli Ianoul* and Andrew Bergeron Department of Chemistry, Carleton UniVersity, 1125 Colonel By DriVe, Ottawa ON Canada ReceiVed June 30, 2006. In Final Form: September 7, 2006 Near-field scanning optical microscopy (NSOM) was applied to study the effect of a two-dimensional array of silver nanoparticles on the spatial distribution and magnitude of fluorescence signal enhancement for a monolayer of Rhodamine 6G (Rh6G). Twenty polyelectrolyte monolayers were deposited between the nanoparticles and the dye by a layerby-layer deposition technique resulting in a 15-20 nm separation cushion, necessary to minimize the fluorescence signal quenching. The fluorescence signal in NSOM images was found to be distributed inhomogeneously as small (100-200 nm in diameter) fluorescent clusters with typically 5-30 times higher fluorescence intensities than a sample without nanoparticles. The position and relative intensity of the clusters was found to be dependent on the excitation wavelength, suggesting that the enhancement originates from the nanoparticle surface plasmon resonance.

Introduction Fluorescence intensity can be greatly enhanced near the surface of metal nanostructures.1-5 This phenomenon has been extensively studied and is of particular interest in such areas as the development of biosensors with improved detection limits.1 For a fluorophore located near the metal surface, three mechanisms should be considered: (1) fluorescence quenching due to the nonradiative energy transfer from fluorophore to the metal; (2) increase of the intrinsic radiative rate of the fluorophore; and (3) local enhancement of the strength of incident light at the metal surface.1-5 Depending on the separation between the fluorophore and metal surface, these three mechanisms play a more or less dominant role. When the separation is between 5 and 20 nm, the fluorescence signal is likely to be enhanced.1-5 Numerous studies on the distance dependence of metal-induced fluorescence enhancement and quenching have been performed.1-11 Different types of fluorescence-enhancing surfaces have been proposed and used: fractal silver surfaces,2a silver nanorods,2b triangular silver nanoplates,2c colloidal metal films,7 single gold nanoparticles,3 and thick silver films.5 Enhancement factors as high as 90 have been reported.6 Although a wealth of information has been gathered on the effect of two-dimensional (2D) metal nanostructures on the * To whom correspondence should be addressed. Phone: (613) 520-2600 × 6043. Fax: (613) 520-3749. E-mail: [email protected]. (1) (a) Lakowicz, J. R. Anal. Biochem. 2001, 298, 1-24. (b) Geddes, C. D.; Lakowicz, J. R. J. Fluoresc. 2002, 12, 121-129. (c) Lakowicz, J. R. Anal. Biochem. 2005, 337, 171-194. (2) (a) Parfenov, A.; Gryczynski, I.; Malicka, J.; Geddes, C.; Lakowicz, J. R. J. Phys. Chem. B 2003, 107, 8829-8833. (b) Aslan, K.; Leonenko, Z.; Lakowicz, J. R.; Geddes, C. D. J. Phys. Chem. B 2005, 109, 3157-3162. (c) Aslan, K.; Lakowicz, J. R.; Geddes, C. D. J. Phys. Chem. B 2005, 109, 6247-6251. (3) Anger, P.; Bharadwaj, P.; Novotny, L. Phys. ReV. Lett. 2006, 96, 113002. (4) Rigneault, H.; Capoulade, J.; Dintinger, J.; Wenger, J.; Bonod, N.; Popov, E.; Ebbesen, T. W.; Lenne, P.-F. Phys. ReV. Lett. 2005, 95, 117401. (5) Kawasaki, M.; Mine, S. J. Phys. Chem. B 2005, 109, 17254-17261. (6) Lee, I. S.; Suzuki, H.; Ito, K.; Yasuda, Y. J. Phys. Chem. B 2004, 108, 19368-19372. (7) Sokolov, K.; Chumanov, G.; Cotton, T. M. Anal. Chem. 1998, 70, 38983905. (8) Campion, A.; Gallo, A. R.; Harris, C. B.; Robota, H. J.; Whitmore, P. M. Chem. Phys. Lett. 1980, 73, 447-450. (9) Kittredge, K. W.; Fox, M. A.; Whitesell, J. K. J. Phys. Chem. B 2001, 105, 10594-10599. (10) Dulkeith, E.; Ringler, M.; Klar, T. A.; Feldman, J.; Munoz, A.; Parak, W. J. Nano Lett. 2005, 5, 585-589. (11) Schneider, G.; Decher, G. Nano Lett. 2006, 6, 530-536.

fluorescence signal enhancement and quenching, experimental data on the lateral distribution of these effects are quite limited. These data would allow finding a relationship between the enhancing properties and the topography of the nanostructures and thus facilitate the development of sensor platforms with high enhancement factors. Therefore, in this work, we employed near-field scanning optical microscopy (NSOM) to study the spatially resolved optical properties of a monolayer of silver nanoparticles and their effect on the fluorescence signal of Rhodamine 6G (Rh6G) dye. We used 2D arrays of metal nanoparticles prepared by a layer-bylayer deposition technique and studied effect of this substrate on the fluorescence of a dye monolayer. The dye was deposited on a controlled separation from the top of a nanoparticle monolayer to provide favorable conditions for the resulting fluorescence enhancement. The results of this work indicate that up to a 30fold fluorescence enhancement can be achieved in small clusters with average lateral dimensions between 100 and 150 nm, depending on the excitation wavelength. Experimental Section Chemicals. Poly(styrenesulfonic acid sodium salt) (PSS; MW 15200; Fluka), poly(diallydimethylammonium chloride) (PDADMAC; 35% ww, MW∼17000), Rh6G, ammonia 30%, ACS grade sodium chloride (Sigma-Aldrich), and 30% hydrogen peroxide (Caledon) were used as purchased. Preparation of Silver Nanoparticles. AgNO3 (90 mg) was dissolved in 500 mL of Milli-Q water in an Erlenmeyer flask. The solution was brought to a vigorous boil with stirring. A 1% solution of sodium citrate (10 mL) was added dropwise to the boiling AgNO3 solution. The mixture was boiled for an hour and then cooled to room temperature. The silver sols exhibited a grayish-green color. Polyelectrolyte (PE) Solution Preparation. The PE solutions were prepared at 1 mg/mL in 1mM NaCl using 18 MΩ Milli-Q water as a solvent. Preparation of Quartz Substrate. Chemglass quartz cover slips were prepared by washing them in a 70 °C bath of 5:1:1 by volume H2O/H2O2/NH3. The slides were then immersed in PDADMAC solution for 15 min to create a positively charged surface. Preparation of Silver Colloid Monolayers. The glass slides prepared in the previous step were covered with Ag nanoparticles by dipping them in a Ag colloidal solution for different periods of time.

10.1021/la061894p CCC: $33.50 © 2006 American Chemical Society Published on Web 10/18/2006

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Figure 1. AFM topography images of a monolayer of silver nanoparticles deposited by a layer-by-layer method for different times of incubation in a nanoparticle solution. The nanoparticle surface density increases with incubation time. Multilayer Deposition. The PE PSS/PDADMAC layers were deposited by successive dipping of the substrate slide (with or without a monolayer of silver nanoparticles) into the solutions containing the PE for 15 min, after which the substrate was rinsed in 18 MΩ Milli-Q water, and then dipped into the oppositely charged PE for 15 min. This process was repeated until the desired number of layers was achieved. For NSOM, imaging samples with 20 layers of PE pairs were used. Deposition of Rh6G. Monolayers of Rh6G were prepared using the method described by Wang.12 A dye (10-3 M Rh6G in HCl, pH 2.5) was deposited on the substrate by immersing the slide (with or without the nanoparticle layer) in the dye solution for 20 min. After immersion, the slide was rinsed thoroughly with deionized water and dried under a stream of nitrogen. Thickness Determination. The successive build-up of the layers was monitored using a CARY 3 UV-vis spectrophotometer, to ensure that the absorbance at 227 nm, and the subsequent concentration, varied linearly with the number of layers. The thickness of the samples was determined with atomic force microscopy (AFM). Raman Spectroscopy. Surface-enhanced Raman measurements were performed using a single grating monochromator (Jobin Yvon, focal length 640 mm) equipped with a liquid nitrogen-cooled CCD camera (Princeton Instruments) and a notch filter to remove the excitation wavelength. An air-cooled argon ion laser (Melles Griot) operating at 488 nm and 20 mW was used for excitation. The spectra resolution of the instrument was ∼4 cm-1. The accumulation time was 60 s. Imaging. AFM measurements were carried out on a multimode Nanoscope III atomic force microscope (Digital Instruments, Santa Barbara, CA) in the repulsive mode in air. A J scanner (maximal scan area of 120 µm2) and 200-µm-long soft cantilevers with integrated pyramidal silicon nitride tips (spring constant of 60 mN/ m) were used for all measurements. The imaging force was approximately 2-4 nN, and the scan rate was typically 0.5 Hz. Details of the NSOM instrument and procedure are presented elsewhere.13-15 Briefly, bent NSOM probes were prepared from high GeO2-doped fibers with a core diameter of 3 µm using a twostep chemical etching method followed by aluminum deposition and focused ion beam milling to produce a flat circular aperture. Probes with aperture diameters of ∼50 nm were used in the present work (estimated from scanning electron microscopy images). NSOM experiments were carried out on a combined AFM/NSOM microscope based on a Digital Instruments Bioscope mounted on an inverted fluorescence microscope (Zeiss Axiovert 100). A separate x-y piezo scanner (Polytec PI, Auburn, MA; 50 µm lateral scan (12) Wang, Y.; Hu, C. Thin Solid Films 2005, 476, 84-91. (13) Ianoul, A.; Strekal, N.; Maskevich, S. J. Nanosci. Nanotechnol. 2006, 1, 61-65. (14) Burgos, P.; Lu, Z.; Ianoul, A.; Hnatovsky, C.; Viriot, M. L.; Johnston, L. J.; Taylor, R. S. J. Microsc. 2003, 211, 37-47. (15) Ianoul, A.; Grant, D.; Rouleau, Y.; Bani, M.; Johnston, L. J.; Pezacki, J. P. Nat. Chem. Biol. 2005, 1, 196-202.

range) was used for sample scanning. A continuous-wave mixedgas ion laser (Coherent, Innova 70 Spectrum) was used for excitation purposes (488 or 568.5 nm, 20mW, linear polarization). The NSOM signal was collected with a 63× (0.75 NA) objective, with appropriate band-pass (Omega Optics) and notch filters (Kaiser Optical Systems, Ann Arbor, MI) to remove residual excitation and red alignment laser light, and was detected using an avalanche photodiode detector (Perkin-Elmer Optoelectronics, SPCM-AQR-15, Vaudreuil, Canada). Images were recorded in tapping mode at a scan rate of 0.25 Hz and a resolution of 512 × 512 pixels. Images were processed using standard Nanoscope software as well as Image J software (NIH). Scatter plots were obtained using the “Colocalization Finder” plug-in of the Image J software. Prior to building a scatter plot, images were aligned using the corresponding topography images. Cluster size analysis was performed using the original nonprocessed NSOM images with custom-made software. The software allows the determination of the number of clusters, their location in the image, as well as their height and half-width.

Results and Discussion Monolayers of Ag Nanoparticles. Monolayers of silver nanoparticles were prepared by a layer-by-layer deposition method. The method is based on the consecutive deposition of positively and negatively charged layers of polymer on a substrate (Figure 1, scheme at the top). Because silver nanoparticles are charged negatively in solution, the same approach can be used for the fabrication of a monolayer of Ag nanoparticles on the top of a positively charged PE layer.16 In Figure 1, AFM images of films prepared by immersing positively charged substrates in a solution of silver nanoparticles for 15 min, 120 min, and 24 h are presented. With longer immersion times, the surface density of the nanoparticles increases from ∼1.5 particles per square micrometer after 15 min of incubation to ∼4 particles after 120 min (Figure 1). Finally, after 24 h, the substrate is almost completely covered with a monolayer of Ag nanoparticles. The size distribution of the nanoparticles used is quite broad, as can be seen from the figures, with the average nanoparticle diameter being ∼70 nm. Occasionally, nanorods are observed. The increase in the surface density of the nanoparticles can also be monitored with the UV-vis absorption spectra of the slides. As the time of deposition increases, the intensity of the band around 370 nm corresponding to the plasmon resonance of Ag nanoparticles increases (Figure 2). Simultaneously, a broad band near ∼700 nm appears as a result of interparticle coupling. (16) Hu, X.; Cheng, W.; Wang, T.; Wang, Y.; Wang, E.; Dong, S. J. Phys. Chem. B 2005, 109, 19385-19389.

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Figure 2. UV-vis absorption spectra for a monolayer of silver nanoparticles for different incubation times.

Figure 3. Dependence of the maximum absorption intensity measured at 370 nm on the incubation time.

The intensity of the absorption band of the nanoparticle monolayer at 370 nm initially increases linearly with time (Figure 3). After around 5 h of incubation, deviation from the linearity is observed, and after around 24 h of incubation, saturation occurs. Longer incubation times result in even higher absorption. This is, however, accompanied by an increase in the number of large nanoparticle aggregates on the substrate surface. Therefore, in this work, we used nanoparticle monolayers prepared by immersing the substrates in the nanoparticle solution for 24 h. Complete and Uniform Deposition of Rh6G on a PE Multilayer without Ag Nanoparticles. In this work, a layerby-layer deposition technique was used to prepare a monolayer of Rh6G. Rh6G molecules carry positive charge in solution, and therefore a monolayer of Rh6G can be deposited on a negative PE layer.12 This positive charge should also prevent the dye from penetrating the multilayer. To test whether the Rh6G deposition on the surface is complete and uniform, we prepared a sample containing 20 alternating PSS/PDADMAC layers and a monolayer of Rh6G dye deposited on the top (Figure 4, scheme). We used NSOM to probe the lateral distribution of Rh6G. In Figure 4, topography (A) and NSOM fluorescence (B) images of the sample are presented. Corresponding cross-sections are presented as well (Figure 4A′,B′). A part of the sample was physically removed (scratched, as can be seen at the top of both images and in the cross-sections) to provide a zero (no fluorescent sample) NSOM signal level for accurate intensity measurements. During the measurements, laser light was blocked for a short period of time (Figure 4B, dark horizontal line at the top and Figure 4B′) to obtain a zero instrument level. From the topography image (Figure 4A), we can see that the multilayer film prepared by this technique is relatively uniform (part of the sample below the scratch), with occasional small clusters on the surface. In the corresponding NSOM fluorescence image (Figure 4B, measured in a 580-600 nm spectral range), we observe a complete and uniform distribution of the Rh6G fluorescence signal. Near the scratch (top part of Figure 4A) one observes some large aggregates that were formed probably during the scratch formation. The fluorescence signal intensity of these aggregates (Figure 4B) is much higher than that of the rest of

Figure 4. NSOM topography (A) and fluorescence (B) images and corresponding cross-sections (A′ and B′) of a sample containing 20 PSS/PDADMAC layers and a monolayer of Rh6G deposited on the top using a layer-by-layer technique. The bottom panel shows corresponding cross-sections. A part of the sample corresponding to the top area of the images was scratched. During imaging, the laser beam was blocked for a short period of time to determine the NSOM zero intensity level (indicated as “laser-off” in the crosssection).

the sample. This indicates that these aggregates are formed from the removed polymer film containing Rh6G. Using the scratched area of the sample as a reference, we estimated the thickness of the film as well as the fluorescence signal intensity. From the topography cross-section (Figure 4A′), we can see that the film is 20-30 nm thick. In the corresponding NSOM fluorescence cross-section (Figure 4B′), we observe a decrease in the fluorescence signal intensity by about 20 kHz (counts per second) coinciding with the position of the scratch. The NSOM signal corresponding to the bare substrate (scratch) can be used as the internal intensity standard. This allows quantitative comparison of NSOM images for different samples, assuming the NSOM signal of the bare substrate for all samples is the same. From Figure 4B′ we find the ratio of the average fluorescence signal to the bare substrate signal to be around 1. Surface-Enhanced Raman Spectra of Rh6G on a Film of Ag Nanoparticles. As a next step, samples containing both Ag nanoparticles and Rh6G dye were prepared according to the scheme shown in the Figure 5. These samples contain a monolayer of nanoparticles followed by a variable number of PE layers with a monolayer of Rh6G as a top layer. Substrates prepared from Ag nanoparticles by such an approach have been used as substrates in surface-enhanced Raman scattering (SERS) spectroscopy.17 Raman spectra for the samples are presented in Figure 5. The Raman spectrum for the Rh6G monolayer deposited on PE film without nanoparticles is very weak and cannot be resolved under the experimental conditions used (Figure 5A). However, one can observe a weak background due to the Rh6G fluorescence signal. The addition of nanoparticles to the sample leads to a dramatic increase in the Raman signal (Figure 5B-D). The bands (17) Goulet, P. J. G.; dos Santos, D. S., Jr.; Alvarez-Puebla, R. A.; Oliveira, O. N., Jr.; Aroca, R. F. Langmuir 2005, 21, 5576-5581.

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Figure 5. SERS spectra for a sample containing a monolayer of Ag nanoparticles covered with variable number of PSS/PDADMAC PE layers and a layer of Rh6G on the top: (A) 20 PSS/PDADMAC layers and a monolayer of Rh6G without Ag nanoparticles; (B) a monolayer of Ag nanoparticles, 1 PSS/PDADMAC layer, and a monolayer of Rh6G; (C) Ag nanoparticles, 8 PSS/PDADMAC layers, and Rh6G; (D) Ag nanoparticles, 20 PSS/PDADMAC layers, and Rh6G.

observed at 1364, 1513, 1581, and 1660 cm-1 are characteristic of the Rh6G SERS spectrum. With an increasing number of PE layers (and therefore separation) between the nanoparticles and the dye, the SERS signal becomes weaker, whereas the intensity of the fluorescence background increases by a factor of 3. This is in agreement with previous observations and is related to the distance dependence of the Raman signal enhancement and also to the distance dependence of the fluorescence signal enhancement combined with fluorescence quenching by a metal surface.1 Thus, the intensity of the fluorescence signal from the dye is the highest (i.e., the enhancement of the signal is the strongest) for 20 PSS/ PDADMAC layers. To determine the spatial distribution of this enhancement, we applied NSOM. Spatially Inhomogeneous Light Transmission by a Monolayer of Ag Nanoparticles. NSOM enables imaging the optical properties of the samples with resolution defined by the NSOM probe aperture. In this study, we used NSOM probes with apertures of ∼50 nm, which provide sufficient resolution to probe the optical properties of the monolayers of Ag nanoparticles with an average size of ∼70 nm. The images in Figure 6 represent the topography (left) and the NSOM transmission signal intensity (right) for a nanoparticle layer prepared at relatively low particle surface density to obtain clear correlation between the position of the nanoparticle and the corresponding optical signal. The average height of the nanoparticles was found to be ∼60 nm (Figure 6, left), consistent with the average nanoparticle size. At 568 nm, the optical image shows dark areas corresponding to the decreased intensity of the light transmitted by the sample (Figure 6, right). The positions of these areas coincide with the position of the nanoparticles in the topography image, suggesting that the nanoparticles transmit light less effectively than the substrate alone. The reduced light transmission is most probably due to either light scattering by the nanoparticles or light absorption by the surface plasmons, since, in the UV-vis spectra of the films, we observe some absorption of light at 568 nm (Figure 2). Spatially Nonhomogeneous Fluorescence of Rh6G on a Monolayer of Ag Nanoparticles. Finally, we applied NSOM to probe the spatial distribution of the Rh6G fluorescence signal on the surface of Ag nanoparticles. The images in Figure 7

Ianoul and Bergeron

Figure 6. NSOM topography (left) and transmission (right) images for a layer of Ag nanoparticles at low surface density. The transmission was measured at 568 nm.

Figure 7. NSOM topography (A), transmission (B), and fluorescence (C) images for a monolayer of Ag nanoparticles covered with 20 PSS/PDADMAC layers and a layer of Rh6G on the top. The crosssection (D) allows measuring the NSOM fluorescence signal intensity. The scatter plot (E) was obtained by plotting the intensity of each pixel in the transmission image (B) as one coordinate and the intensity of the fluorescence image (C) of the same pixel as another coordinate. The plot shows a negative correlation, suggesting colocalization of areas of low transmission signal with areas of high fluorescence signal. The excitation wavelength is 568 nm.

correspond to the topography (A), the NSOM transmission at 568 nm (B), and the NSOM fluorescence measured in the 580600 nm spectral range (C) of a sample consisting of a monolayer of Ag nanoparticles coated with 20 PE layers and a monolayer of Rh6G. The particle surface density for this sample is higher than the that of the sample in Figure 6, as can be seen from the topography image (Figure 7A). The bottom part of the sample was removed (scratched) to obtain the topography and optical intensity reference levels. A laser beam was temporarily blocked during the scan to obtain a zero intensity level (dark stripe at the bottom of the NSOM transmission image B). The choice of Rh6G is justified by the relatively high quantum yield for this dye and the significant overlap of the excitation spectrum with the absorption spectrum of the nanoparticle monolayer to probe the substrate effect on the incident light.

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Figure 8. NSOM topography (A), transmission (B), and fluorescence (C) images for the same sample shown in Figure 7. The scatter plot (D) shows a negative correlation, suggesting colocalization of areas of low transmission signal with areas of high fluorescence signal. The excitation wavelength is 488 nm.

Overlap between the emission spectrum of Rh6G and the monolayer absorption spectrum is reduced, thus enabling the detection of fluorescence in transmission mode. Because of the poor topography resolution of the NSOM setup (400-500 nm), we could only observe an average topography image (Figure 7A), with the characteristic size of the features being close to the size of the NSOM probe. Nevertheless, the uniform distribution of the nanoparticles as well as the characteristic thickness of the film confirms that the nanoparticles formed a uniform and continuous monolayer. In the corresponding NSOM transmission image (Figure 7B), we observe nanometer-scale inhomogeneities similar to the Figure 6B image. However, because of the higher nanoparticle surface density, the boundary between the dark and bright areas in Figure 7B is not defined as well as it is in Figure 6B. Several papers have reported on the near-field spectroscopy of a single nanoparticle and observed enhanced or reduced transmission of light by the nanoparticle.18-24 The enhanced transmission was explained as excitation of the surface plasmon resonances by the evanescence wave of the NSOM tip and consequent radiation of the propagating modes in the far field.18,20-24 The magnitude of enhancement was found to depend on the wavelength and polarization of light, the NSOM probenanoparticle separation, as well as the size and material of the nanoparticles. The reduction of transmission, in contrast to the enhancement, was observed when the excitation wavelength was not in resonance with the nanoparticle plasmon band.21,24 This is similar to experiments in this work, since the excitation wavelength of 568 nm is not in resonance with the main plasmon absorption band near 370 nm. When clusters of nanoparticles are considered,20 several individual surface plasmon resonances are observed with highly location-dependent spectroscopic properties. Therefore, we believe that the decreased transmission of light at 568 nm indicates the location in the film where localized surface plasmon resonances are likely to be excited at this wavelength. Also, because of the more complex nature of plasmon excitations in this dense monolayer, no clear correspondence between the position of the nanoparticle and the transmission light intensity can be made.20 (18) Klar, T.; Perner, M.; Grosse, S.; von Plessen, G.; Spirkl, W.; Feldman, J. Phys. ReV. Lett. 1998, 19, 4249-4252. (19) David, T.; Chicanne, C.; Richard, N.; Krenn, J. R.; Scheurer, F.; Ounadjela, K.; Hehn, N.; Lacroute, Y.; Goudonnet, J. P. ReV. Sci. Instrum. 1999, 70, 45874594. (20) Markel, V. A.; Shalaev, V. M.; Zhang, P.; Huynh, W.; Tay, L.; Haslett, T. L.; Moskovits, M. Phys. ReV. B 1999, 59, 10903-10909. (21) Benrezzak, S.; Adam, P. M.; Bijeon, J. L.; Royer, P. Surf. Sci. 2001, 491, 195-207. (22) Kim, J.; Kim, J.; Song, K. I. B.; Lee, S. Q.; Kim, E. U. N. K.; Choi, S. E. U. L.; Lee, Y.; Park, K. H. O. J. Microsc. 2002, 209, 236-240. (23) Bakker, R. M.; Drachev, V. P.; Yuan, H.-K.; Shalaev, V. M. Opt. Express 2004, 12, 3701-3706. (24) Imura, K.; Nagahara, T.; Okamoto, H. Chem. Phys. Lett. 2004, 400, 500505.

Unlike the Rh6G deposited on bare glass (Figure 4B), the NSOM fluorescence image measured in a 580-600 nm spectral range for the Rh6G monolayer deposited on a monolayer of Ag nanoparticles is very speckled (Figure 7C). A number of small bright spots can be observed with an estimated surface density of around six clusters per square micrometer. From the crosssection of the fluorescence image (Figure 7D), we estimated that the intensity of individual clusters is between 0.1 and 0.6 MHz. The transmission by the bare glass substrate used as an internal intensity standard was measured to be around 20 kHz. Therefore, we obtain a fluorescence intensity 5-30 times higher than that for the substrate without Ag nanoparticles (Figure 4B′). The scatter plot presented in Figure 7E was obtained by plotting the intensity of each pixel in the NSOM transmission image (Figure 7B) as one coordinate and the intensity of the same pixel in the NSOM fluorescence image (Figure 7C) as another coordinate. If there is a correlation between the two images, it should appear as a linear plot. A positive slope of the plot indicates a positive correlation, and a negative slope indicates a negative correlation. There is a clear negative correlation between the two intensities (Figure 7E), indicating that the minimum intensity in the transmission image corresponds to the maximum intensity in the fluorescence image. Therefore, the position, density, and intensity of the clusters in the fluorescence image (Figure 7C) are related to and are most probably defined by the optical properties of Ag nanoparticles. To confirm that the obtained correlation is not an artifact resulting from heterogeneous deposition of the dye on the surface, the same experiment was performed with a different excitation wavelength. The images in Figure 8 show the topography (A), the NSOM transmission (B) and the NSOM fluorescence images (C, detected in a 580-600 nm spectral range) for the same sample shown in Figure 7, with the area of the sample being measured using 488 nm excitation. The position and relative intensity of the dark areas in the NSOM transmission images obtained at 488 nm (Figure 8B) and 568 nm (Figure 7B) are quite different. This points out the location-dependent spectroscopic properties of the monolayer of Ag nanoparticles. The NSOM fluorescence image at 488 nm (Figure 8C) is similar to the image obtained at 568 nm (Figure 7C) in that it also contains small clusters. However, the position of these clusters is not exactly the same, and some new clusters appeared. The corresponding scatter plot (Figure 8D) for transmission and fluorescence images at 488 nm also shows a strong negative correlation, indicating that the minimum intensity in the transmission image corresponds to the maximum intensity in the fluorescence image. Therefore, the correlation between the local optical properties of metal nanoparticles (NSOM transmission), which are due to the excitation of surface plasmon

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resonances, and the corresponding lateral distribution of the dye fluorescence signal is present at different excitation wavelengths used. Enhancement of the fluorescence signal can be a result of two phenomena: increase in the radiative decay rate and increase in the local field intensity at specific spots on the surface.1 Although our experimental setup does not allow us to perform fluorescence lifetime measurements, we can conclude that fluorescence enhancement due to an increase in the radiative decay rate is unlikely, since the quantum yield of Rh6G is very high.1 In this work, we find that the enhancement of the fluorescence correlates with the transmission signal of the Ag nanoparticles. Since optical heterogeneities in the transmission image spatially coincide with localized surface plasmon resonances on the surface of Ag nanoparticle monolayers, it is reasonable to suggest that the enhancement of the fluorescence observed in this work is primarily due to the local enhancement of the field. If this is the case, the obtained spatial distribution of the fluorescence signal gives us an idea of the number and relative enhancement factors for specific spots on the surface of the monolayer of Ag nanoparticles. It is interesting that the average cluster size for fluorescence images obtained at 488 nm excitation is smaller than that for 568 nm [110 nm compared to 140 nm (Figure 9)], suggesting that the size of these spots increases with increasing wavelength. This is reasonable, since localized surface plasmons at 568 nm are expected to be excited in larger nanoparticles than for 488 nm. Finally, in this work we imaged the inhomogeneous enhancement of the fluorescence for a monolayer of Rh6G deposited on the surface of silver nanoparticles. We found that the size and density of the heterogeneities depend on the excitation wave-

Ianoul and Bergeron

Figure 9. Cluster size distribution for NSOM fluorescence images measured using 488 nm (white, Figure 8C) and 568 nm (black, Figure 7C) excitation wavelengths.

length. Enhancement of the fluorescence by a factor of 5-30 due to the enhancement of local field near the surface of metal nanoparticles was observed. It is of further interest to investigate the spatial distribution of the Raman signal enhancement on a monolayer of silver nanoparticles, and this work is currently underway. Acknowledgment. We would like to thank Linda J. Johnston for access to the NSOM instrument, Z. Lu for NSOM tips fabrication, and Harrison Westwick for help with the preparation of the manuscript. Financial support was provided by NSERC, CFI, and Carleton University. LA061894P