The location of electronic energy levels in molecules and the probability and polarization of radiative transitions between ground and excited state can be accurately predicted by quantum mechanical theory [1]. For most molecules, no excited singlet states (S₂, S₃…) have been observed to emit light upon excitation, the same being true for triplet states (T₂, T₃…). This is summarized in Kasha’s rule: The emitting electronic level of a given multiplicity is the lowest excited level of that multiplicity [2].

As the first authentic example of a molecule which violates Kasha’s rule, azulene has been the subject of numerous spectroscopic and photophysical investigations [3–6]. Beer and Longuet-Higgins discovered the second excited level emits light upon excitation, producing the anomalous (S₂ to S₀) fluorescence of azulene in 1955 [6], whereas that normal S₁ to S₀ fluorescence and T₁ to T₀ phosphorescence were much too weak to be observed. The absence of S₁ to S₀ fluorescence suggests that S₁ to S₀ internal conversion is a very rapid progress in Azulene, this supposition being confirmed by the lifetime of the S₁ state being determined as 1.9 psec [7]. Compared with the analogous spacing of the isomer Naphthalene, Azulene has much wider spacing between its zero –point levels of the first two excited singlet states (S₁, S₂) [8]. The anomalous fluorescence of Azulene subsequently occurs because internal conversion fails to take place between the S₂ and S₁ level, on account of there wide energy separation.

Azulene, with 7-membered rings, is an allelochemical. It can penetrate into the cell, interact with cellular components such as DNA-containing organelles or tubulin of the cytoplasm, and selectively fluoresce upon interaction with different cellular compartments [9]. This activity makes it as valuable as other indicator fluorescent dyes. However, the detection is limited by Azulene‘s weak S₂ fluorescence. In this regard, the plasmonic enhancement of fluorescence of Azulene is very important, and may facilitate the studying of allelopathic mechanisms at the cellular level.

In recent years, our laboratories have both studied and demonstrated many applications of metal-enhanced fluorescence (MEF) [10–12], metal enhanced phosphorescence [13], metal-enhanced chemiluminescence [14] and Surface Plasmon Coupled Fluorescence for directional emission [15]. These have included the increased detectability and photostability of fluorophores [16] and chemiluminescent species [14], improved DNA detection [17], the release of self-quenched fluorescence of over labeled proteins [18], enhanced wavelength-ratiometric sensing [19], and the application of metallic surfaces to amplified (ultra fast and sensitive) assay detection [20]. Our laboratorys’ current interpretation of MEF has been explained by a model whereby non-radiative energy transfer occurs from excited distal fluorophores, to the surface plasmon electrons in non-continuous films (Fig. 1 top), in essence a fluorophore induced mirror dipole in the metal. The surface plasmons in turn, radiate the emission of the coupling fluorophores [10–12]. This explanation has been facilitated by our recent observation of surface plasmon coupled emission (SPCE) [21], whereby fluorophores distal to a continuous metallic film can directionally radiate fluorophore emission at a unique angle from the back of the film. However, all previous MEF and MEP studies were exclusively focused on S₁ and T₁ electronic states respectively and their emission.

Figure 1 Graphical representation of our laboratory’s current interpretation of Metal-Enhanced Fluorescence (MEF) (Top), and Metal-Enhanced S2 emission (Bottom). IC-Internal Conversion, VR-Vibrational energy relaxation. Ag-Silver nanoparticle (SiFs), MEF-Metal-Enhanced (more ...)

In this letter we report our observations on the photophysical characteristics of Azulene, which has well-known S₂ emission, in close proximity to SiFs. We have observed both the enhanced absorption and S₂ emission of Azulene from SiFs (which is ≈ 2-fold brighter) as compared to a glass substrate, a control sample at room temperature (RT) and at 77 K. Furthermore, a shorter Azulene fluorescence lifetime was also observed, consistent with our previous observations of Metal-enhanced Fluorescence [10–21]. These observations are helpful in our understanding of not only the excitation/relaxation of azulene, but in our laboratories continued efforts to develop a unified theory to explain plasmon-lumophore (and fluorophore) interactions.

300 μL of Azulene (1.0 × 10⁻³ M) in EPA solution (Diethyl - Ether: Isopentane: Ethanol = 5:5:2 (v/v)) was dropped in a sandwich format between the glass slides and the silver island films, respectively. The Fig. 2 insert shows the experimental sample geometry. The glass/SiFs surfaces were placed in liquid nitrogen for 2 mins and used for low temperature (77K) measurements. Absorbance spectra were taken using a Varian Cary 50 UV-Vis. Spectrophotometer. Fluorescence measurements were undertaken using a Cary Eclipse fluorescence spectrophotometer.

Figure 2 Absorption spectra of Azulene sandwiched between 2 silvered (SiFs) and unsilvered slides respectively. EPA, Diethyl - Ether: Isopentane: Ethanol = 5:5:2 (v/v), SiFs- Silver Island Films.

Fluorescence lifetimes were measured using the time-correlated single photon counting technique, using a PicoQuant modular fluorescence lifetime spectrometer (Fluo Time 100) with a PicoQuant LDH-P-C-400 laser as the light source. The intensity decays (Figure 4) were subsequently analyzed in terms of the multi-exponential model:

Figure 4 Fluorescence Intensity decays of Azulene from between silvered (SiFs) and unsilvered glass slides at room temperature (RT). Data from least square reconvolution analysis is shown in Table 1.

(1)

Where α_i are the amplitudes and τ_i are the decay times, . The fractional contribution of each component to the steady state intensity is given by:

(2)

The mean lifetime of the excited state is given by

(3)

and the amplitude-weighted lifetime is given by

(4)

The values of α_i and τ_i were determined by nonlinear least squares impulse reconvolution, with a goodness-of-fit X² criterion.

Fig. 2 shows the absorption spectra of Azulene from between the SiFs and from glass. SiFs and glass without Azulene were used as reference backgrounds for the Azulene absorption measurements, respectively. From Fig. 2, it can be seen that the Azulene has a larger absorbance on the SiFs as compared to glass. The enhanced absorption of dye molecules near to metallic surfaces was first reported by Glass et. al. in 1980 and later confirmed by other groups [23–25]. When a lumophore is placed near metal, there is a very strong net absorption effect, a result of the coupling of the molecular dipoles with the localized electromagnetic field of the metallic particle’s surface plasmon resonance [12]. In essence, conducting metallic particles can modify the free space absorption condition in ways that increase the photonic mode density and incident electric field felt by a lumophore [10–12]. These enhanced absorption phenomenon can lead to surface enhanced luminescence phenomena, such as Metal-Enhanced Fluorescence (MEF) and Metal-Enhanced Phosphorescence (MEP) [13], phenomena described by our groups [10–13].

Fig. 3-top shows the S₂ fluorescence emission spectra of Azulene from SiFs and from glass at RT, consistent with previous reports [1–3] The enhanced fluorescence intensity was ≈ 1.5-fold brighter from the silver, (i.e. ratio of emission from SiFs/glass). It should be noted that the true metal-enhanced fluorescence enhancement factor is ≈ 37-fold. This is because the MEF phenomenon is distance dependent [10–12], where with a sample thickness of ~1 micron and an enhanced interaction region < 20 nm [10–12], then only 4 % of the sample is within the MEF enhancement region.

Figure 3 Fluorescence emission spectra, λex = 338 nm, of Azulene sandwiched between 2 silvered (SiFs) and unsilvered slides at room temperature (RT), (Top), and the emission spectra at 77 K, (Bottom). All spectra are the mean of 3 separate and independent (more ...)

In addition to the metal-enhanced S₂ fluorescence at RT, we have observed that temperature can also influence the magnitude of the MEF phenomenon, hitherto, an unreported observation. It can be expected that if all other factors remain constant, the fluorescence intensity will increase with decreasing temperature [26]. Figure 3 Bottom shows the fluorescence emission spectra of Azulene from between both SiFs and from glass at 77K. It can be seen that the enhanced fluorescence intensity was >2-fold brighter from silver as compared to glass. Table 1 summarizes the fluorescence spectral data for Azulene at RT and at 77K sandwiched between both glass and silver slides. The enhancement ratio for Azulene increases from 1.3 (at RT) to 2.0 (at 77K). At first, one may consider this increase in ratio to be due to a property of the Azulene itself at low temperature. However, this ratio is calculated by the division of the emission intensity of Azulene from SiFs by the emission intensity from glass, also measured at 77 K. This suggests that the further increase in MEF at low temperature (i.e. increase in ratio) is indeed a property of the plasmons themselves and the coupling of the Azulene to silver surface plasmons at low temperature. Given that the absorption spectrum of the SiFs remains unchanged at low temperature (data not shown), then we speculate that the further enhanced MEF is due to a reduced damping of the coupled S₂ emission. Further low temperature studies are underway in this regard.

Table 1 Fluorescence spectral data for Azulene at RT and 77 K sandwiched between both glass and silvered slides (SiFs). The enhancement factor was calculated as the “peak” emission intensity ratio SiFs/Glass. Integrated area ratio is the ratio (more ...)

The full width half maximum (FWHM) for Azulene at RT and at 77K are also shown in Table 1. FWHM for the fluorescence emission spectra of Azulene on SiFs at 77K was ≈ 9 nm which is narrower, as expected, as compared to that measured at room temperature (≈ 18 nm). Also, the emission wavelength maximum at 77K is identical to that of the spectra measured at RT. Most organic fluorophores also emit phosphorescence when in a rigid medium [6]. It is however, not surprising that phosphorescence was not observed for Azulene, because assuming a fluorescence-phosphorescence frequency difference of 2,000–10,000 cm^{− 1}, the phosphorescence T₁ energy level of Azulene is higher than the lowest excited S₁ singlet level at 14,000 cm^{− 1} [27]. The radiationless transition to S₀ couples with the highly forbidden triplet-singlet transition which gives rise to classical phosphorescence [4].

In addition, we studied the intensity decays of Azulene in the presence of SiFs. These decays were used to calculate the respective lifetimes shown in Table 2, using non-linear least squares regression analysis. From Figure 4 and Table 2, we can see both a reduced mean lifetime (τ _mean) and amplitude-weighted lifetime (<τ>) for fluorophores near-to silver (0.09 ns) as compared to the glass control sample (0.34 ns). In fact, shorter lifetimes for fluorophores in close proximity to silver nanostructures, coupled with enhanced emission intensities in the same system, is indicative of the MEF S₁ phenomenon, and has been reported and explained by our group many times previously [10–13]. In fluorescence, the spectral observables are governed by the magnitude of Γ, the radiative decay rate, relative to the sum of the non-radiative decay rates, k_nr, such as internal conversion and quenching. In the absence of metallic particles or surfaces, then the quantum yield, Q₀ and fluorescence lifetime τ₀ are given respectively by:

Table 2 Fluorescence intensity decay analysis of Azulene at room temperature. αi are the amplitudes and τi are the decay times. <τ> the amplitude-weighted and mean is the mean lifetime. SiFs-Silver Island (more ...)

(5)

(6)

In all of our applications of S₁ MEF to date, we have found that the enhanced fluorescence signals (Quantum yields – Q_m) of fluorophores in close proximity (< 10 nm) to metallic nanostructures could be well described by the following equations [10–12, 16]:

(7)

where Γ is the unmodified radiative decay rate, Γ_m is the metal-modified system radiative decay rate and k_nr are the non-radiative rates, Figure 1-top. Similarly, the metal-modified lifetime, τ_m, of a fluorophore is decreased by an increased radiative decay rate:

(8)

From equations 7 and 8, we can see that as the value of the system Γ_m increases, the quantum yield Q_m increases, while the lifetime, τ_m, decreases.

As described in the introduction, S₁ to S₀ has a narrow energy gap for Azulene and the S₁ to S₀ internal conversion rate is a very rapid process. In this work, we saw no evidence that the S₁ state of Azulene can couple or indeed be enhanced by surface plasmons. For the S₂ state, enhanced S₂ emission is currently thought to occur due to the efficient non-radiative transfer to surface plasmons which, in turn, efficiently radiate, similarly to what is observed for many other fluorophores’ S₁ emission [10–13]. As shown in figure 1 bottom, a highly efficient S₂ emission coupling to surface plasmons, where the plasmons efficiently and quickly radiate the coupled emission, results in a reduced fluorophore lifetime and an enhanced observed S₂ emission intensity. These findings are consistent with equations 7 and 8, which have been used successfully to describe Metal-Enhanced S₁ emission. To the best of our knowledge, this is the first observation of enhanced S₂ emission.

In this paper we report the first observation of metal-enhanced S₂ fluorescence. Azulene in close proximity to SiFs can undergo enhanced S₂ fluorescence, a 2-fold increase was observed as compared to an identical control sample containing no silver at 77K. In addition, the amplitude-weighted lifetime was reduced ≈ 4 fold near to silver. This finding suggests that photon induced electronically excited states at RT, and also at low temperature, can both induce and couple to surface plasmons, facilitating enhanced S₂ fluorescence emission.

This work was supported by the NIH, NCRR RR008119. The authors would also like to thank UMBI for salary support.