Article pubs.acs.org/Langmuir
Single-Molecule, Single-Particle Approaches for Exploring the Structure and Kinetics of Nanocatalysts Takashi Tachikawa and Tetsuro Majima* The Institute of Scientific and Industrial Research (SANKEN), Osaka University, Mihogaoka 8-1, Ibaraki, Osaka 567-0047, Japan ABSTRACT: In this Article, we focus on the in situ observation of photochemical reactions on individual nanoobjects of solid catalysts using single-molecule, single-particle fluorescence spectroscopy. The use of high-resolution imaging techniques with suitable fluorogenic probes enables us to determine the location of the catalytically active sites that are related to the structural heterogeneities on the surface of the solid catalyst and the temporal fluctuation of photochemical reactivity. Furthermore, we present the real-time observation of metastable gold nanoclusters in polymer matrices at the singlecluster level. This Article encourages readers to explore the nanoworld in terms of practical applications in many fields such as fundamental physics and chemistry.
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molecules, polymer segments, fluorescent proteins, and luminescent quantum dots (QDs).15−21 To facilitate the observation of very weak emissions from a single fluorophore, many detection methods such as total internal reflection fluorescence microscopy (TIRFM) and confocal fluorescence microscopy have been developed.16 TIRFM is advantageous in visualizing the fluorescence from single molecules immobilized or located at a glass/solution interface with very low background noise using an evanescent field and thus has been applied to the investigation of the structural dynamics of dye-labeled biomolecules such as DNA and proteins. Confocal microscopy is widely used for the detection of a single molecule immobilized on a glass surface or in a polymer matrix, as well as for the detection of a molecule freely diffusing in solution; this technique is often referred to as fluorescence correlation spectroscopy (FCS).22−25 In addition, by measuring the delay between the arrival of the excitation pulse and the detection of the emitted photon with a time-correlated single-photon counting (TCSPC) system, it is possible to reconstruct a fluorescence decay curve for a single dye molecule in the excited state. These single-molecule detection techniques have the following advantages, which make them superior to the conventional ensemble-averaged ones that rely on a bulk sample: extremely high sensitivity, the ability to observe properties that cannot be observed by ensemble measurements (e.g., a subpopulation existing in the sample or temporal fluctuations of photochemical reactivity), and the elimination of the need for synchronization. Therefore, the single-molecule/
oble metal nanoparticles (or clusters) and their composites with organic and inorganic materials have potential applications such as for developing biosensors, luminescent probes, catalysts, photovoltaic cells, and photonic (plasmonic) devices.1−6 The reduction of size and dimensionality of the materials results in a drastic change in their electronic properties because the spatial length scale of the electronic motion is reduced with decreasing size.7 If the particle sizes are smaller than a few nanometers, the classical model for electromagnetic field enhancement, which is a major mechanism for surface-enhanced Raman spectroscopy (SERS),8 is modified by quantum size effects.9 The size of nanoparticles is also pivotal for their catalytic properties as a consequence of the size dependences of the adsorption free energies of the substrates and the activation barriers associated with rate-limiting elementary steps such as oxygen dissociation on the surface.10−12 In addition to the particle size effects, optical properties of metal nanoparticles are highly dependent upon their shape.13,14 Therefore, in order to design and develop an efficient system for the above-mentioned applications, it is necessary to understand the relationship between the structure and function of these nanoparticles at the single-particle level. Furthermore, chemical reactions occurring at the surface of an isolated solid catalyst have been found to be intrinsically heterogeneous and closely related to many factors such as the structural dispersion of the nanoparticles, the spatial distribution of reactive sites, surface restructuring dynamics, the conformation of adsorbates, and the electronic interactions between adjacent components. With the advent of advanced microspectroscopy techniques, it will be possible to resolve these types of issues. Single-molecule fluorescence spectroscopy is emerging as a promising tool for studying the photophysical and photochemical processes of all types of molecular systems such as dye © 2012 American Chemical Society
Special Issue: Colloidal Nanoplasmonics Received: January 12, 2012 Revised: February 10, 2012 Published: February 10, 2012 8933
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single-particle fluorescence spectroscopy has been applied to explore the inherent features of heterogeneous reactions on various solid catalysts such as layered double hydroxide (LDH),26,27 gold nanoparticles,28,29 carbon nanotubes,30−32 titanium dioxide (TiO2),33−41 luminescent QDs (e.g., CdSe/ ZnS and CdTe),42−45 and synthetic zeolites (ETS-10, TiMCM-41, ZSM-22, and ZSM-5).46−48 These studies can be classified into three types of experiments as follows: “singleparticle−many-molecules”,41,46 “single-particle−single-molecule”,26−32,34−36,47,48 and “particle-film (assembly)−singlemolecule”33,37−40,42 experiments (Figure 1). In addition, “single
Figure 2. Illustration of the experimental setup for the single-molecule fluorescence spectroscopy based on TIRFM.
filters to remove the undesired scattered light, and then imaged using an electron-multiplying charge-coupled device (EMCCD) camera. To measure the single-molecule fluorescence spectra, only the emission that passed through a slit is brought into a spectrograph and imaged by an EM-CCD or intensified CCD camera. The spectrum is typically integrated for a few seconds to obtain enough signal-to-noise ratio and analyzed by a personal computer (PC). The details of the experimental setup, instrumentation, standard protocols, and analytical procedures for a variety of single-molecule fluorescence measurements are described in the literature.56 There is significant current interest in the development of molecular probes for the characterization of reaction processes in a nanoscale environment.57−59 Synthetic fluorogenic probes have been utilized to detect biologically relevant species and specific gene products in vitro and in vivo due to their simplicity and sensitivity.60,61 Another interesting application of such probes is the exploration of chemical reactions occurring on the heterogeneous catalysts at high spatial and temporal resolution by the rapidly developing technology of singlemolecule fluorescence spectroscopy. In 2006, Roeffaers et al. studied the spatial distribution of catalytic activity on a layered double hydroxide using a widefield microscope and a suitable fluorogenic probe (5carboxyfluorescein diacetate, Figure 3A).26 They discovered that transesterification occurs primarily on the {0001} planes of the crystal, whereas ester hydrolysis proceeds on the lateral {1010} facets of the crystal. Naito et al. described the methodology for evaluating the photocatalytic activity of individual TiO2 nanotubes, which have a porous structure containing a straight macropore (pore size: 100−150 nm) and mesopores between the anatase nanoparticles (pore size: 5−10 nm), by the single-molecule counting of •OH using a specific fluorescent probe, 3′-(paminophenyl) fluorescein (APF) (Figure 3B).33 The singlemolecule observation of fluorescein products generated by the
Figure 1. Objects of the single-molecule−single-particle experiments.
site−single molecule” experiments have been carried out in order to investigate the chemical reactivity of immobilized probe molecules or functionalized oxide supports at the surfacesolution (air) interface.49−51 In this Article, we focus on the single-molecule, singleparticle observation of chemical reactions on metal nanoparticles (clusters) and their composites by using advanced fluorescence microscopic techniques. The feasibility of studying heterogeneous reactions at the single-molecule, single-particle level permits us to devise completely new experimental schemes. For instance, one can readily distinguish between reactive and inert molecules (particles) without ensemble averaging. Interested readers are referred to recent comprehensive reviews, which include more in-depth discussion of these topics.52−55 A typical experimental setup for the single-molecule fluorescence spectroscopy based on TIRFM is shown in Figure 2. A circular-polarized light emitted from a continuous wave (CW) laser (e.g., diode lasers) passing through an objective lens (high NA value is recommended) after reflection by a dichroic mirror was totally reflected at the cover glass-solution interface. This results in the generation of an evanescent field (within the penetration depth d ∼ 200 nm), which makes it possible to detect a single fluorescence dye molecule. The position of catalyst nanoparticles immobilized on the cover glass can be determined from the optical transmission or atomic force microscope (AFM) images. In our studies, the 365 nm light, which was emitted by a light emitting diode (LED), was passed through the objective to excite the photocatalyst nanoparticle. The fluorescence emission from the fluorescent products generated on a single nanoparticle is collected using the same objective, passed through long-pass and bandpass 8934
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Figure 3. (A) Transesterification of 5-carboxyfluorescein diacetate with 1-butanol. Upon catalytic conversion, a highly fluorescent fluorescein molecule is formed. Reprinted with permission from ref 26. Copyright 2006 Nature Publishing Group. (B) Detection of •OH radicals with 3′-(paminophenyl) fluorescein (APF) during TiO2 photocatalytic reactions. Reprinted with permission from ref 33. Copyright 2009 American Chemical Society. (C) Production of highly fluorescent resorufin from nonfluorescent resazurin by reduction reactions. Reprinted with permission from ref 29. Copyright 2010 American Chemical Society. (D) Production of highly fluorescent HN-BODIPY from nonfluorescent DN-BODIPY by reduction reactions. Reprinted with permission from ref 36. Copyright 2011 American Chemical Society. (E) Acid-catalyzed condensation of furfuryl alcohol producing the conjugated products that show a pronounced visible light absorption. Reprinted with permission from ref 47. Copyright 2009 WILEYVCH Verlag GmbH & Co. KGaA, Weinheim. (F) Phenylbutadienyl-substituted BODIPY designed for epoxidation reaction on a titanosilicate TiMCM-41. The red fluorescent BODIPY probe is epoxidized with tert-butylhydroperoxide on the Ti sites in the mesopores of a Ti-MCM-41 particle. Upon catalytic conversion, yellow emitting BODIPY products are formed. Reprinted with permission from ref 48. Copyright 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
(ET) is a fundamental process in physics, chemistry, and biology, playing a crucial role in natural and artificial energy conversion systems.62 Chen and co-workers detected the single-molecule turnover of nonfluorescent resazurin to its fluorescent product resorufin (Figure 3C) by the addition of NH2OH on a gold nanoparticle.28,29 Based on the stochastic analysis of off-on fluorescence bursts, it was found that the particle size strongly influences the surface-restructuring-
photocatalytic reaction revealed the importance of the transport behavior of reagents through the porous structures on the photocatalytic activity and the existence of the spatial heterogeneity of reactive sites even in an isolated TiO2 nanotube. Recently, two groups reported in situ observation of singlemolecule catalytic events on a catalyst nanoparticle using redox responsive fluorogenic probes. Interfacial electron transfer 8935
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Figure 4. Optical transmission (A) of a single 8 nm Au/TiO2 particle immobilized on a cover glass and fluorescence images (B) of the same particle in Ar saturated DN-BODIPY solution (2.0 μM in methanol) under 488 nm laser and UV irradiation. The fluorescence images display a series of successive images exhibiting on and off events. The scale bars are 500 nm. (C, F) Fluorescence intensity trajectories obtained for individual TiO2 (C) and 14 nm Au/TiO2 (F) particles in Ar-saturated DN-BODIPY solution under 488 nm laser and UV irradiation. (D, G) The locations of fluorescence bursts determined using centroid analysis of each fluorescent spot obtained for individual TiO2 and 14 nm Au/TiO2 particles. The integration time per frame was 50 ms. See the corresponding colors in panels (C) and (F) for panels (D) and (G), respectively. (E, H) The spatial distribution of reactive sites was determined by fitting a two-dimensional Gaussian function to the fluorescence spot distributions collected from identical individual TiO2 and 14 nm Au/TiO2 particles. The red circles in panel (H) indicate the highly active reaction centers. The particle size of TiO2 is about 150 nm. Reproduced from ref 35 by permission of The Royal Society of Chemistry.
workers (Figure 3E and F). 47,48 Their high-resolution reconstruction method is based on catalytic conversion of fluorogenic substrates and is referred to as NASCA (Nanometer Accuracy by Stochastic Chemical reActions) microscopy. Charge transport across the heterogeneous interface of metal−semiconductor and semiconductor−semiconductor nanocomposites has attracted a great deal of research interest, because this process largely governs the performance of photovoltaic devices, batteries, fuel cells, and (photo)catalysts used for water splitting and environmental purification.4,63 Wang et al. investigated the photocatalytic redox reaction of a fluorogenic probe, DN-BODIPY, over isolated TiO2 and Au nanoparticle-loaded TiO2 (Au/TiO2) nanoparticles.35 A series of Au/TiO2 particles (5, 8, and 14 nm Au/TiO2) were prepared by the deposition-precipitation method using HAuCl4 and anatase TiO2 (100−200 nm size) as the raw materials. Photoinduced reactions, which are based on the semiconductor properties of metal oxides, are basically initiated by the band gap excitation (in the case of anatase TiO2, UV light irradiation (λ < 390 nm) corresponding to its band gap of 3.2 eV) to generate the valence band (VB) holes (h+) and conduction band (CB) electrons (e−).64 These charge carriers are immediately captured at various trap sites in the bulk material or on the surface to oxidize or reduce the adsorbates. The incorporation of noble metals with a large work function (e.g., Au and Ag) onto metal oxide supports considerably
coupled catalytic dynamics. They also applied the same probe system to explore the kinetics of electrocatalysis on individual carbon nanotubes.30 Tachikawa and co-workers reported a new class of highly sensitive fluorogenic probes, DN-BODIPY (8-(3,4-dinitrophenyl)-1,3,5,7-tetramethyl-4,4-difluoro-4-bora-3a,4adiaza-s-indacene), which can be used to identify photocatalytically active redox sites over individual TiO2 particles (Figure 3D).34−36 Prior to reduction of DN-BODIPY, the two nitro (NO2) groups of the probe significantly decrease the lowest unoccupied molecular orbital (LUMO) energy level of the benzene moiety introduced at the meso-position of the BODIPY core. This conjugation completely quenches the fluorescence by an intramolecular ET from the excited fluorophore to the dinitrobenzene moiety. The reduction of one of the NO2 groups to a hydroxylamino (NHOH) moiety results in an increase in the electron density. This effect is almost neutralized by the remaining NO2 group, thereby dramatically suppressing the intramolecular ET process. By accepting electrons from the photoexcited catalysts, nonfluorescent DN-BODIPY (Φfl < 10−4 in methanol) can be reduced to form a highly fluorescent product, that is, 4hydroxyamino-3-nitrophenyl-BODIPY (HN-BODIPY, Φfl = 0.50 in methanol). Different strategies for resolving single catalytic turnovers on an isolated catalyst have been developed by Roeffaers and co8936
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Figure 5. (A) Schematic of kinetic mechanism for photocatalytic reduction over Au/TiO2. (B) Typical fluorescence intensity trajectory observed for a single 8 nm Au/TiO2 particle in an Ar-saturated DN-BODIPY solution under 488 nm laser and UV light irradiation. (B) Off and (C) on time distributions constructed for many single 8 nm Au/TiO2 particles. These distributions are fitted with a single-exponential decay function to obtain the average values of τoff and τon. Insets show the DN-BODIPY concentration dependence of ⟨τoff⟩ (C) and ⟨τon⟩ (D) obtained for TiO2 (black) and 8 nm Au/TiO2 (red). The solid lines in the inset of panel (C) were obtained from eq 1. The solid lines in the inset of panel (D) are guides for the eye. Reproduced from ref 35 by permission of The Royal Society of Chemistry.
Figure 6. (A) Fluctuation of R over a single 5 nm Au/TiO2 particle. (B) Distribution of ⟨R⟩ and its RSD over 10 different individual particles for TiO2 (black), 5 nm Au/TiO2 (blue), 8 nm Au/TiO2 (green), and 14 nm Au/TiO2 (red). Reproduced from ref 35 by permission of The Royal Society of Chemistry.
Figure 4C and F shows the trajectories of the fluorescence intensity obtained for individual TiO2 and 14 nm Au/TiO2 particles in Ar-saturated DN-BODIPY solution under a 488 nm laser and UV irradiation, respectively. Interestingly, when compared to those for a single TiO2 particle (over 10 centers, panels D and E), a single 14 nm Au/TiO2 particle had a limited number of reactive centers (2−3 centers, panels G and H), which were equal to the number of Au nanoparticles loaded onto one TiO2 particle. These results suggest that the reduction of DN-BODIPY over Au/TiO2 occurs more easily on the surface of Au nanoparticles than on a bare TiO2 surface. Each decrease in intensity marks the disappearance of the fluorescent product from the surface of the nanoparticle (Figure 5A and B).35 Note that photobleaching or blinking of individual HN-BODIPY molecules occurs on much longer time
enhances the charge separation efficiency via ET from the CB of semiconductor metal oxides to metals on the surface.65,66 Figure 4A and B shows the optical transmission image under white light illumination and the fluorescence image captured for a single 8 nm Au/TiO2 particle in Ar-saturated methanol containing DN-BODIPY under a 488 nm laser and UV irradiation, respectively. Individual particles showed a number of fluorescence bursts that had signals higher than the background (Figure 4C and F). Such a sudden increase in intensity corresponds to the generation of the fluorescent product (i.e., HN-BODIPY) (also see Figure 5A). The locations of the fluorescence bursts were determined with an accuracy of about 20−30 nm by employing a two-dimensional Gaussian function to the distribution of the fluorescent spots (see the red dots in the transmission image of Figure 4A). 8937
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Figure 7. (A) Fluorescence image under excitation of a 405 nm laser for 60 s showing the photofabricated Aun. Inset shows the typical fluorescence trajectories observed for single Aun. (B) Time-dependent change in the number of fluorescent species. (C) Single-cluster fluorescence spectra. (D) Histogram of single-cluster emission peak. Emission maxima for typical single Aun are ca. 2.2 and >2.4 eV. Reprinted with permission from ref 86. Copyright 2009 American Chemical Society.
Figure 6B, most of the bare TiO2 particles exhibited lower activity (⟨R⟩, 0.23−0.60 s−1) and greater temporal variation (RSD, 30%−51%) in the photoreduction of DN-BODIPY than Au/TiO2 particles. This result implies that Au nanoparticles can preferentially trap electrons from TiO2 and then deliver them to DN-BODIPY. In addition, bare TiO2 particles possess a much broader distribution in activity, which is always masked in ensemble-averaged measurements. This explains the fact that the surface of TiO2 has a number of electron trapping sites with different energies and structures, which results in an extremely broad distribution in the reaction rates.68,69 The enhancement of photochemical activity by coupling TiO2 photocatalysts and metal nanostructures under visible light irradiation has attracted a great deal of interest.70 The following mechanisms have been proposed: (i) photoinduced heating of metal nanostructures,71,72 (ii) electron transfer from metal to TiO2 mediated by surface plasmons on illuminated metal nanoparticles,73,74 and (iii) plasmon-mediated energy transfer from metal nanoparticles to TiO2.75,76 Both (ii) and (iii) would increase the concentration of e−/h+ pairs in the composite. We hope that single-molecule, single-particle analysis provides supportive and straightforward evidence for visible-light-enhanced (photo)catalytic reactions. Photoluminescence is a particularly useful tool for the identification of crystal structures, particle sizes, and defect states, because the positions of emission peaks are strongly dependent on these structural characteristics of nanoparticles or clusters.77−79 Subsequent changes of emission intensity and spectrum also give us information about the underlying reaction chemistry. Noble-metal clusters, that is, those composed of several tens of atoms, have attracted considerable attention for a variety of reasons ranging from a fundamental scientific interest in nanoscopic materials to technological applications.79−82 A
scales under similar laser intensities, which suggests that the observed sudden decreases in intensity are attributed to the dissociation of adsorbed HN-BODIPY. The statistical properties of the fluorescence bursts provide valuable information regarding the kinetic mechanism of photocatalysis over individual Au/TiO2 particles. In order to obtain the adsorption constant for substrate, the dependence of the product formation rate, the reciprocal of the average value of τoff (⟨τoff⟩−1) on the substrate concentration was analyzed by the Langmuir−Hinshelwood equation (Figure 5C):28,67 ⟨τoff ⟩−1 =
γeff Kad[S] 1 + Kad[S]
(1)
where Kad is the equilibrium adsorption constant for substrate (Kad = kad[S]/k−ad) and γeff represents the reactivity of all catalytic sites (γeff = kns, where k is the rate constant for one catalytic site and ns is the total number of substrate binding sites on one particle). The Au/TiO2 systems showed larger Kad values (0.8−1.1 μM−1) than that of the TiO2 system (0.5 μM−1), which is of the same order of magnitude as those for nitrobenzenes. In contrast, the DN-BODIPY concentration has no significant effect on ⟨τon⟩−1, suggesting that the disappearance of HN-BODIPY from the surface of the nanoparticles does not involve the substrate-assisted steps (Figure 5D).28 Figure 6A shows the time profile for the turnover rate (R) of the on−off cycle every 10 s over one Au/TiO2 particle during 10 min of UV irradiation. It is clear that the R values over the same particle vary temporally, which is indicative of the fluctuation in the photocatalytic activity of one particle at different times. To quantify the fluctuation in activity, the mean value of the turnover rate (⟨R⟩), and its relative standard deviation (RSD) over each particle were calculated. Based on the statistical analysis of 10 different particles, as shown in 8938
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Figure 8. Fluorescence images of Aun (A, C) and Aum (B, D) under the excitation of a CW laser in the Ar- and O2-saturated atmospheres. Aun and Aum were excited at 405 nm 532 nm, respectively. The scale bars are 5 μm. Proposed energy-level diagrams ad reaction pathways of Aun (E) and Aum (F). Fl and Phos denote fluorescence and phosphorescence, respectively. ET, BET, and ISC denote electron transfer, back electron transfer, and intersystem crossing, respectively. Reprinted with permission from ref 96. Copyright 2009 American Chemical Society.
of atoms) correspond to the clusters emitting at 2.2 and >2.4 eV, respectively. Even though the photochemical reactivity of quantized noble metal clusters is a subject of great interest, there have been no reports on the reactivity of excited clusters because of their instability without protective ligands. Sakamoto et al. solved this problem by embedding bare Au clusters in a polymer matrix and successfully investigated the photochemical reactions between ligand-free clusters and oxygen molecules.96 Figure 8A−D displays the fluorescence images of individual Aun and Aum (m > n) clusters observed by employing the appropriate combination of sample and light source. The Aum sample was prepared by spin coating an acetone solution of aged PVAc film containing Aum. The use of a 532 nm CW laser enabled us to detect the lager Au clusters formed in the sample. The fluorescence spectral measurements revealed that the observed spectrum of the Aum sample was composed of at least two peaks at 1.97 and 2.04 eV, which correspond to Au21 and Au19, respectively. The images highlight a significant difference in the photochemical reactivity of Aun (n < 12 or 17) and Aum (m = 19 or 21) toward molecular oxygen, which is one of the most ubiquitous quenchers of the excited states. As shown in Figure 8C, oxygen molecules efficiently quenched the fluorescence of Aun (n < 12 or n = 17). It should be noted that approximately 50% of the decrease was recovered by removing oxygen, inferring that the quenching mechanism does not involve strong interactions such as chemisorption. Meanwhile, the fluorescence intensity of Aum (m = 19 or 21) increased with increasing oxygen concentration (Figure 8D). From these experimental results, two different quenching processes were proposed (Figure 8E and F). The driving force for photoinduced ET is expressed by the oxidation/reduction potentials of donor/acceptor molecules and the energy gap between the ground and excited states (ΔEET). The smaller ΔEET of Aum than that of Aun could explain the difference in reactivity to electron-accepting oxygen. The ground state oxygen (3O2) is also an efficient triplet quencher. The oxygen-induced enhancement of the fluorescence intensity observed for Aum probably originated from the depopulation of the dark states by 3O2.97,98 Because singlet oxygen (1O2) was
number of strategies have been proposed to fabricate noblemetal clusters by employing biomolecules,83−85 polymers,86−88 and zeolites89 as templates or supports. Metal clusters with sizes comparable to the Fermi wavelength exhibit characteristics that are different from those of both bulk materials and large nanoparticles. In such a size regime, metal clusters exhibit molecule-like transitions owing to the discretion of the density of states and feature distinct fluorescence bands in the UV to near-infrared region.79,90−94 Here, we demonstrate the methodology for studying the photoreactivity of Au clusters at the single-cluster level. Au clusters formed and grew during a 405 nm CW laser irradiation to a poly(vinyl acetate) (PVAc) film containing the radical precursor 2-hydroxy-4′-(2-hydroxyethoxy)-2-methylpropiophenone and the metal precursor HAuCl 4.86 Upon photoexcitation, the radical precursor yielded radicals, which work as reducing agents for the Au ion to generate a Au atom. Figure 7A shows the fluorescence image captured during the 405 nm laser excitation of the film in an Ar atmosphere.86 Only a very few luminescent spots were observed in the initial several tens of seconds, whereas the number of fluorescent species increased after successive laser excitation (Figure 7B). The individual Au clusters underwent repeated cycles of emission (so-called “photoblinking”95), suggesting that the observed spots were single fluorescent clusters. As shown in Figure 7C and D, the fluorescence spectra and histogram of peak energies clearly indicate two main species with peaks at 2.2 and >2.4 eV. No clear spectral shift was observed during the duration of the experiment, indicating that these clusters were stable without further growth. The fluorescence spectra of Aun, which is characteristic to the number of atoms (n), motivated us to identify a Aun sample with a clear resolution of the number of atoms during the growth process. According to the literature, the emission energy of Au clusters (Eem) can be expressed by the jellium model:79 Eem = EFermi n1/3
(2)
where EFermi is the Fermi energy of bulk Au. From this relationship, it was suggested that Au9−20 clusters emit in the region from 2.1 to 2.6 eV and Au17 and Aun
