1. Introduction

In an effort to develop more efficient fluorescence-based immunoassays, metallic surfaces or particles have been explored as substrates for enhancing molecular fluorescence [1–4]. By exciting surface plasmons (SPs) from the metallic surfaces or nanoparticles, local electromagnetic (EM) field around the fluorophores is enhanced, resulting in increased efficiency of fluorescence excitation [4]. SPs are the oscillations of the free electrons located on the surface of a metal film, and typically are excited by incident light through prism-coupled total internal reflection (TIR) or grating-coupled diffraction methods [5]. The prism-coupled excitation scheme can also be replaced by a highly oblique, collimated beam produced by focusing a light beam at the outer region of the back focal plane (BFP) of a high numerical aperture (NA) objective [6]. When the wave vector of the incident evanescent, transverse magnetic (TM) light matches that of the SPs, the phenomenon of surface plasmon resonance (SPR) occurs, and the local EM field associated with SPR becomes greatly enhanced [5]. As a result, fluorescence intensity from chromophores located on the metal surface can become greatly enhanced via the SP excitation mechanism.

Due to their complex dielectric constants with a large negative real part, gold (Au) and silver (Ag) films are ideal candidates for SP excitation in the visible and near infrared (NIR) range. Theoretical simulations also indicate that Ag film provides superior electric field enhancement at the NIR region which can result in the enhancement of two-photon excited fluorescence (TPEF). When the thickness of the Ag film is about 50 nm, the SP excitation based on the objective-based TIR method can be optimized to greatly enhance the local electric field. However, collection of the excited fluorescence by the objective is significantly reduced due to the presence of the 50 nm Ag film resulting in a decrease of the fluorescence coupled emission efficiency. Therefore, to improve SP-enhanced TPEF using the objective-coupled TIR method, a thinner Ag film capable of local electric field enhancement and improved fluorescence detection efficiency should be adopted [7]. Moreover, simulation shows that a decrease in the Ag film thickness would result in a decreased SPR angle. As a result, the requirement of achieving high incident angle through a microscope objective can be relaxed.

In addition to thickness of the Ag film, its surface roughness is also an important parameter in the optimizing SP effects. In particular, the surface roughness of an Ag film deposited on a dielectric substrate by the use of techniques such as sputtering, e-beam evaporation, and chemical vapor deposition, is a few nanometers in root-mean-square (RMS) [8–11]. In recent studies, an ultrasmooth Ag film was achieved by e-beam evaporation of the Ag source on Si(100) substrates by the use of a germanium (Ge) nucleation layer [12]. Through this technique, a surface roughness below 1.0 nm (RMS) can be achieved. A similar by e-beam evaporation technique deposits the Ag film on a nickel (Ni) seed layer located on a silicon or quartz substrate [13]. In this manner, the surface roughness can be reduced from > 4 nm for a pure Ag film to around 1.3 nm RMS for the Ag/Ni film. Therefore, a smooth Ag/Ni film located on the quartz substrate can be used to optimize the SP phenomenon for plasmonic and metamaterials applications [13]. Specifically, artifacts associated with rough surfaces (strong scatterings, broadened SPR curves, and decreased SPR sensitivities) can be reduced [5,13,14].

The emission efficiency of the fluorophores not only is affected by the local SP-enhanced electric field, but is also influenced by quenching of the metal film. The fluorescence quantum yield and emission coupling yield combined with the local electric field enhancement dominate the overall fluorescence emission efficiency. By appropriately modifying the distance of a metal film on a substrate, the SPs can contribute to an increase in the fluorescence quantum yield, a reduction in the fluorescence lifetime for improved photostability, and a specific orientation in the typically isotropic emission [15,16]. These effects are not due to the reflection of the emitted fluorescence, but are the result of the fluorophore dipole interacting with free electrons in the metal. Our previous study has theoretically and experimentally demonstrated that the TPEF can be enhanced and quenched by depositing silver and dielectric films with different thicknesses [7]. Furthermore, another of our studies has shown that the SP-enhanced TPEF evanescence wave microscopy can increase the brightness and acquisition frame rate in imaging live cell membrane [17]. Moreover, the surface plasmon-coupled emission (SPCE) phenomenon [18–23] should be investigated for improving TPEF imaging when objective-based evanescence wave microscopy is used.

In addition to the importance of surface thickness, roughness, and spacer, the stability of the silver film to resist water damage is also crucial for practical and dynamical imaging. Therefore, in this study, we attempt to investigate the optimal condition for SPCE induced TPEF imaging under aqueous solution by fabricating an Ag film with an appropriate thickness, a low surface roughness, and a dielectric protection spacer. Finally, TIR TPEF images taken using the objective-coupled evanescence wave microscopy through the enhancement of the SPCE are demonstrated.

2. Experimental setup

2.1. Objective-coupled evanescence wave microscope

Shown in Fig. 1 is the schematic diagram of the oil immersion objective-based evanescence wave microscope. A 700-1000 nm femtosecond titanium-sapphire (ti-sa) laser (Tsunami, Newport) with a pulse width of 100 fs and repetition rate of 80 MHz was used as the excitation source. After passing through a half-wave plate and a linear polarizer for polarization and power control, the laser source is reflected by mirrors and guided into the microscope by a focusing lens. A mirror and a lens were fixed on a linear translation stage in order to adjust the position of the focal point on the back focal plane (BFP) of the high NA oil-immersion objective (60 × , NA = 1.49, Nikon). After passage through the objective, a quasi-collimating laser beam was formed. The incident angle of the quasi-collimating beam was manipulated by adjusting the focal position on the BFP via movement of the linear translation stage, which allows for adjustment of the incident angle up to 79.5°. The fluorescence was collected by the same objective and passed through a dichroic mirror, a shortpass filter (SPF, λ < 680 nm, Semrock), a bandpass filter (BPF, λ = 500-535 nm, Semrock), and an imaging lens prior to image acquisition by a high-speed frame rate EMCCD camera (iXon DV885, Andor).

 

Fig. 1 Schematic diagram of the optical setup of the TPEF evanescence wave microscopy based on the objective-coupled TIR method. Red and green lines indicate the respective optical paths of the excitation source and fluorescence collection.

Download Full Size | PDF

2.2. Thin film deposition

A BK7 glass cover slip was used as the substrate and was first cleaned by isopropyl alcohol, acetone, and ethanol in sequence. Next, the cover slip was cleaned in Piranha solution (H₂SO₄:H₂O₂ (3:1)), and then dried with N₂ gas before the sputtering process. Radio-frequency (RF) magnetron sputtering was used to deposit various thin films on the BK7 cover slip. The working gas was argon with pressure was set at the 0.2 mtorr for sputtering. The deposition rate can be adjusted by controlling the sputtering power for various materials. In our experiments, the sputtering powers of Ge layer, Ag film, and silicon dioxide (SiO₂) layer were 20 W, 25 W, and 200 W, respectively. The deposition rates of the Ge layer, Ag film, and SiO₂ layer were 0.0075 nm/s, 0.057 nm/s, and 0.056 nm/s, respectively.

3. Results and discussions

3.1. Silver film versus gold film with appropriate thickness

Both Au and Ag are suitable as the metal film for SP excitation in the visible light and NIR regions. Au is a preferred choice for bio-applications due to its inert nature. However, a superior enhancement of local electric field can be achieved by exciting the SPs using an Ag film at the excitation wavelength of 780 nm. At 780 nm, the refractive indices of the water solution, Ag film, and BK7 cover slip (H₂O/Ag/BK7) are 1.3290, 0.1432 + j5.1304, and 1.5188, respectively [24]. By the use of these parameters in the Fresnel equations, the electric field enhancement of the Ag film at the SPR angle as a function of thickness can be obtained and the results are shown in Fig. 2(a) . The thickness of the Ag film with the maximum electric field enhancement is about 50 nm. The maximum enhancement is about 60-fold for the vertical electric field (${\left|E_z\right|}^2$), but is only about 4-fold for the horizontal electric field (${\left|E_x\right|}^2$).

 

Fig. 2 (a) Electric field enhancement and (b) fluorescence coupled emission as function of Ag film thickness. (c) Electric field enhancement and (d) fluorescence coupled emission as function of the Au film thickness.

Download Full Size | PDF

The radiation pattern of the fluorescent dye is similar to dipole radiation. As the fluorescence penetrates the silver layer, the radiation pattern transmitted through the layer is given by [7,25,26]

Figures 2(c) and 2(d) show the electric field enhancement and the fluorescence coupled emission for the Au film. The refractive index of the Au film is n ₁ = 0.1754 + j4.9123 at 780 nm and n ₁ = 0.6318 + j2.0984 at the center emission wavelength of 515 nm. The trend of electric field enhancement for the Au film is similar to that of the Ag film. At the film thickness of 50 nm, the maximum enhancement is about 45-fold for the vertical electric field and 4-fold for the horizontal electric field. However, the fluorescence coupled emission power for the VED dramatically drops when the thickness is increased above to 15 nm. For the HED, the fluorescence coupled emission is also absorbed by the Au film. Based on considerations of the electric field enhancement and fluorescence coupled emission efficiency, the Ag film is superior to the Au film as a substrate for TPEF. Moreover, the appropriate thickness of the Ag film should be between 32 nm and 50 nm, as found in Figs. 2(a) and 2(b). However, it is difficult to find an appropriate thickness of the Au film with an acceptable enhancement and coupling, as observed in Figs. 2(c) and 2(d). The TPEF intensity collected by the high NA oil-immersion objective is proportional to the square of the intensity enhancement and the fluorescence collection efficiency. A thinner Ag film can provide superior fluorescence collection efficiency. However, the enhancement of the local electric field intensity is lower than that of an Ag film with a 50 nm thickness. Therefore, the thickness of the Ag film was chosen to be 40 nm for SPCE TPEF imaging.

3.2. Silver film with optimal evenness on cover slip

The surface evenness of an Ag film deposited directly on a glass substrate is usually a few nanometers (RMS). In an attempt to improve upon the smoothness of the Ag film, a seed layer of Ge was used in depositing a 40 nm Ag film on the cover slip (0.17 mm). The surface morphology of the sample was measured using the tapping mode of a NT-MDT supplier atomic force microscope (AFM). The scan size and scan rate were 1 μm x 1 μm (512 x 512 pixels) and 1 Hz per line, respectively. Following typical cleaning procedures, the average RMS surface roughness of the cover slip was determined to be less than 0.3 nm within a randomly selected region of 1 μm². Figures 3(a) and 3(b) show the respective AFM images of the Ag film without and with the Ge seed layer on the cover slips. As a reference, the surface morphology of the Ge layer on the cover slip is shown in Fig. 3(c). In this case, a surface roughness of about 0.4 nm was found. In addition, histograms of the two-dimensional (2D) surface-height values of Figs. 3(a)-3(c) are respectively shown in Figs. 3(d)-3(f). The average RMS surface roughness of a pure Ag film is ~4.7 nm, with a peak-to-valley height of ~27.0 nm. In comparison, the average RMS surface roughness and the peak-to-valley height of the Ag/Ge film improved to ~1.0 nm and ~8.0 nm, respectively. Therefore, the Ag/Ge film deposited on the cover slip by direct RF sputtering exhibits a smaller RMS surface roughness and a narrower peak-to-valley surface topological height distribution as compared to the case when the Ag film was directly deposited onto the cover slip. SPs on rough surfaces lead to phenomena such as broadened SPR curves and decreased SPR sensitivities. The influence of surface roughness can be evaluated via a variation of metal permittivity [5,13,14,27]. This will add a small deviation Δk _sp from the SP wave vector on smooth metal surface. The Δk _sp is the increase in damping resulting from surface roughness. If the surface roughness of the silver film is too large, local hot spots from the uneven surface could degrade image quality. The improvement in surface smoothness can result in improved uniformity in SPCE induced TPEF images.

 

Fig. 3 AFM topography analysis of: (a) 40 nm Ag film, (b) 40 nm Ag film with a 2 nm Ge seed layer, and (c) 2 nm Ge on cover glasses. (d)-(f) are the corresponding histograms of the 2D surface-height values of (a)-(c).

Download Full Size | PDF

3.3. Dielectric spacer for protection from water

For bio-imaging applications such as the visualization of live cell membrane, the observation period could range from a few hours to over a day [28]. Therefore, the Ag film needs to be robust in solution under long-term and high-flux illumination. We found that after 15 seconds, the Ag film in aqueous environment was seriously damaged when the power of the 780 nm, femtosecond laser source was increased to 30 mW (illumination spot: 20 μm in diameter). This effect may be due to the interaction between the Ag film and the aqueous solution [29]. Figure 4(a) shows the damage of the Ag/Ge film in the aqueous solution by white-light imaging. The propagation of the incident laser beam is from the right to the left, and the propagation of the SP wave is parallel to that of the incident beam. Figure 4(b) shows the two-photon luminescence (TPL) image of the damaged Ag/Ge film. The TPL on the right side of the image is brighter than that at the left side. The asymmetric TPL pattern should be related to the propagation of the SP wave. Similar damage to the Ag/Ge film by the ti-sa laser operating at the continuous wave (CW) mode was also observed (at the same wavelength and power). The white light image of the damage specimen is shown in Fig. 4(c). However, the damage phenomenon in the Ag/Ge film disappears when the aqueous solution was mixed with fluorescent dyes at a high concentration. Figure 4(d) displays a Gaussian SPCE TPEF image when 1 mM Rhodamine 6G (R6G) solution placed on top of the Ag/Ge film is excited by the 780 nm femtosecond laser at 30 mW for several minutes. The TPEF image of a Gaussian beam profile shape demonstrates that the Ag/Ge film remains in good quality. When the R6G concentration is reduced to 0.1 mM, the Ag/Ge film can resist the irradiance longer. However, the Ag/Ge film was eventually destroyed. The experimental results imply that the Ag-water interaction is lessened when the aqueous solution is supplemented with highly concentrated fluorescent dyes. The fluorescent dyes can reduce the damage of the Ag film. However, their fluorescence may contribute to background signal in SPEC TPEF imaging applications.

 

Fig. 4 (a) White-light and (b) TPL images of the damaged Ag/Ge film in H₂O after 15-second femtosecond laser irradiance (780 nm at 30 mW). (c) White-light image of Ag/Ge film in H₂O after 15-second CW laser irradiance showing the damage to the film. (d) Gaussian SPCE TPEF image with 1 mM R6G solution placed on top of the Ag/Ge film.

Download Full Size | PDF

In order to prevent the damage to the Ag film in aqueous solution, a dielectric spacer can be employed and deposited on the top of the Ag film. In addition to preventing the Ag-water interaction, the dielectric spacer can also reduce the fluorescence quenching effect by the Ag film and hence increase the quantum yield of the fluorescent dyes [7]. In order to simulate the fluorescence quantum yield in a five-layer structure such as 4/3/2/1/0 (H₂O/SiO₂/Ag/Ge/BK7), each fluorescent molecule may be regarded as an individual electric dipole. The vibrated characteristic of the dipole moment is modulated by its reflected electric field. The power dissipation is relation to the damping rate of the vibrated dipole and is also the function of the orientation of the dipole. To consider the orientation of the dipole, the two types of the power dissipation can be denoted as the VED and the HED for the five-layer structure, which are perpendicular and parallel to the interface, respectively. They can be expressed as [7,30,31]

Because the power dissipation is a function for the orientation of the dipole, the modified quantum yield can be separated into Q_VED and Q_HED. Therefore, the dissipated power spectrum of the fluorescent dye can be calculated to evaluate the modified quantum yield from the theoretical models. The refractive indices of the aqueous solution, dielectric spacer, Ag film, Ge layer, and BK7 cover slip (H₂O/SiO₂/Ag/Ge/BK7 as) are 1.3360, 1.4615, 0.1300 + j3.055, 4.5867 + j2.4516, and 1.5205 at the emission wavelength of 515 nm, respectively. The corresponding thicknesses of the Ge layer and Ag film are 2.0 nm and 40.0 nm. Figure 5 shows the modified quantum yield as a function of thickness of the dielectric spacer. The modified quantum yield of the VED is smaller than that of the HED because the emission intensity of the fluorescent dyes is quenched by the SPs for the VED. Without a spacer, the quenching significantly diminishes the quantum yield in the VED. The fluorescence emission intensity is determined by the enhancement of electric field intensity, fluorescence coupled emission efficiency, and fluorescence quantum yield. Therefore, the dielectric spacer thickness was designed to be 20 nm to achieve a maximum SPCE TPEF enhancement. Furthermore, the Ag film in aqueous solution, when protected by a 20 nm SiO₂ spacer, sustains no damage by the femtosecond laser with a 780 nm wavelength at 30 mW, even for an exposure up to one hour. Uniform white-light images of the SiO₂/Ag/Ge film in H₂O after long-term exposure by the femtosecond laser at 780 nm and 30 mW can be observed. The observation provides evidence of the protective function that the dielectric spacer serves. Therefore, the optimal condition of the Ag film deposited on the cover slip in aqueous solution for the SPCE TPEF imaging is achieved by adopting an Ag film with the appropriate thickness of 40 nm, a surface roughness below 1.0 nm via the Ge seed layer, and a 20 nm SiO₂ protection spacer.

 

Fig. 5 Quantum yield of the fluorescent dyes on the Ag film as a function of the dielectric spacer thickness.

Download Full Size | PDF

The greater the fluorescence intensity and the higher signal-to-noise ratio, the shorter the required exposure time, and hence a faster frame rate can be achieved through the SP enhancement technique. Figure 6(a) shows a Gaussian SPCE TPEF image of 1.0 mM R6G solution on the SiO₂/Ag/Ge/BK7 being excited by the femtosecond laser at the SPR angle. By inserting an extra lens before the imaging lens at an appropriate position, the image at the BFP of the high NA objective can be observed. Figure 6(b) shows the SPCE TPEF image of the R6G at the BFP of the objective. An SPCE circle was observed because the fluorescence is mostly from the SPCE. Meanwhile, the colorful multiple circles were detected by a color CCD camera. Based on the SPCE, the fluorescence emission is highly directional and therefore can be effectively collected. The emission spectrum of the R6G solution is from 520 nm to 650 nm, with an emission peak at 566 nm. The colorful multiple circles reveal that the TPEF signal of the R6G is simultaneously emitted with a longer emission wavelength (i.e. smaller wave vector) in the inner circle (i.e. smaller SPCE angle) and shorter emission wavelength (i.e. larger wave vector) in the outer circle (i.e. larger SPCE angle) via the SPCE. The intensity of the SPCE circle is uniform and highly symmetrical because the refractive index of the R6G solution is uniform in the laser illuminated area. Some spots inside the circles and near the bottom of the circles can be found. This is due to damage by the femtosecond laser in previous experiments where more than 50 mW was inaccurately focused on the lens material near the BFP of the high NA objective. Therefore, power adjustments of a femtosecond laser as the light source for an objective-based evanescence wave microscope needs to be carefully adjusted.

 

Fig. 6 SPCE TPEF images of R6G at (a) the Ag surface and (b) the BFP of the objective.

Download Full Size | PDF

Figure 7(a) shows the SPCE TPEF image of a fluorescent bead pattern on the SiO₂/Ag/Ge cover slip. The fluorescent beads with a diameter of 1.0 μm are carboxylate-modified yellow-green polystyrene beads (F8888, Molecular Probes). The emission spectrum of the fluorescent bead is from 500 to 600 nm, with an emission peak at 515 nm. The pattern resulted from some beads in aqueous solution falling on the cover slip. The emission intensities of the patterned fluorescent beads were non-uniform and this effect is most likely due to different electric field enhancements and fluorescence coupling efficiency resulting from the different distances of the fluorescent beads from the SiO₂ film surface. According to theoretical analysis, the local electric field decays exponentially with the distance from the SiO₂ surface. Figure 7(b) shows the SPCE TPEF image of the fluorescent bead pattern at the BFP of the objective. Figure 7(b) also shows an imperfectly circle. The intensity of the SPCE circle is influenced by the shape of the fluorescent structure, the illuminated position, and the polarization of the incident laser spot [21,22,32]. The equivalent refractive indices resulted from the different distances of the polystyrene fluorescent beads from the SiO₂ surface and are slightly varied, and hence induce different SPCE angles. In our experiment, the illuminated area of the quasi-collimating laser beam is filled with the fluorescent beads at different distances. As a result, the SPCE induced TPEF image in the BFP from can be complex.

 

Fig. 7 SPCE TPEF images of a fluorescent bead pattern at (a) the Ag surface and (b) the BFP of the objective.

Download Full Size | PDF

4. Conclusions

This study has developed an optimizing configuration of an Ag film on a cover slip for SPCE TPEF imaging under aqueous solution. The thickness of the Ag film is designed to be 40 nm and can provide high local electric field enhancement and fluorescence coupled emission efficiency via the SP excitation induced fluorescence excitation. The deposited Ag film with a 2.0 nm Ge seed layer, fabricated by utilizing the RF sputtering process, exhibits a surface roughness below 1.0 nm for improved SPR performance and image quality. Moreover, the Ag film with a 20 nm SiO₂ spacer not only avoids damage caused by the Ag-water interaction under high laser power irradiance, but also reduces the fluorescence quenching effect by the Ag film. Based on the SiO₂/Ag/Ge configuration, effective TIR TPEF imaging can be achieved through SPCE.