1. Introduction

Fluorescent materials have been widely used in bio-sensing and imaging due to their excellent sensitivity and versatility. Many fluorescent imaging techniques have been developing in the past two decades including confocal microscopy, total internal reflection fluorescence (TIRF) microscopy and two-photon excitation microscopy that can confine light into a small volume to realize high signal-to-noise-ratio (SNR). Greater signal enhancement and higher collection efficiencies have been pursued through a combination of optical and material approaches as well as highly sensitive detector designs for single molecule imaging or low molecular concentration detection. Along with these, two enhancement mechanisms have become well established in recent years including surface plasmon resonance (SPR) [1] and photonic crystal resonance (PCR) [2]. Many elaborate designs [3–8] have been created to sculpture the substrate into an enhancement device within which localized excitation field can be further increased. For SPR enhancement, structured metallic thin films [3–5] and nano-particles [9] are used to generate surface plasmon modes with highly enhanced near field intensities. However, photo-stability is a main concern associated with metallic surfaces because photo quenching greatly reduces the lifetime and absorbs the energy through non-radiative processes. Usually a spacing layer (~10 nm) is needed to reduce the quenching effect, which also reduces the obtainable signal enhancement factor. Photonic crystal structures comprised of different dielectric or semiconductor materials have the advantage of low energy loss. However, the presence of an optical interface will affect the angular radiation distribution, which usually leads to lower collection efficiencies [10,11]. An objective lens with a high numerical aperture (NA) is typically used in fluorescence imaging to maximize the signal collection. Techniques such as solid immersion lens [12], numerical aperture increasing lens [13] and paraboloid collector [14] have been devised to maximize the collection of the fluorescence signal in the object space. Due to a large portion of the fluorescence emission is coupled into the substrate that will never reach the detector, the collection efficiency is typically much lower than 50% even with these improved objective lenses. In this letter, a simple one-dimensional photonic band gap (1D PBG) multilayer structure is designed and fabricated to significantly enhance the fluorescence signal through the combination of a PCR enhancement and collection efficiency improvement with omnidirectional reflection.

To avoid photo-quenching in metal-based enhancer, Cunningham et al used 1D gratings and 2D photonic crystal surfaces as alternatives to SPR enhancer to effectively enhance the fluorescence emission by increasing the excitation field strength [7,15]. A simple 1D planar photonic crystal structure with alternating high and low refractive index thin films has been demonstrated by Ye et al [16] and Descrovi et al [17] for fluorescence enhancement, a concept proposed by Thomas et al [18] and Haus et al [19]. In a typical reflective fluorescence microscope illustrated in Fig. 1(a) , the molecular analyte is excited from the top at a shorter wavelength. The emission, which usually occurs at a longer wavelength, can be captured by the same objective lens to form an image. However, a significant amount of emitted signal is either lost in the substrate due to high index bending effect or scattered directly into the ambience, leading to low collection efficiency and limiting the obtainable fluorescence signal enhancement.

 

Fig. 1 (a) Illustration of a reflective fluorescence microscope; (b) Proposed fluorescence enhancer setup with multilayer 1D PBG coatings on the surface of the glass substrate.

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In this paper, we propose and experimentally demonstrate a novel fluorescence enhancer that is compatible with TIRF microscopy. As illustrated in Fig. 1(b), the excitation light coming from the bottom of the substrate at the resonant angle will be enhanced within the 1D PBG multilayer thin films forming a PCR due to field localization, leading to higher fluorescence emission. At the same time, the 1D PBG multilayer thin films structure is designed as an omnidirectional reflector for the red-shifted fluorescence signal. Omnidirectional reflection will direct most of the emitted fluorescence signal into the objective lens, giving rise to high signal collection efficiency. With these two factors of improvement, the fluorescence signal can be greatly increased for single molecule imaging or low molecular concentration detection.

2. Theory and calculation

The concept of PBG originates from late 80s [20] with the simple idea of using dielectric materials to artificially create a stop band similar to the electronic bands in semiconductor crystals. Light wave with frequency that falls into this band gap will be reflected back regardless of incident angle and polarization. This “perfect mirror” can be realized by periodic thin films with alternating high and low index materials with infinite number of periods.

In practice, finite number of periods has to be used. Figure 2(a) illustrates a 3-period 1D PBG structure on glass substrate. For an infinite period structure, electro-magnetic waves traveling through it can be described by the Bloch wave solution in the form of [18]

 

Fig. 2 (a) Illustration of the electro-magnetic waves with different polarizations incident on a 3-period 1D PBG structure; n1, n2 and h1, h2 are the corresponding refractive indices and film thicknesses for the repeating layers; (b) Projected band diagram of infinite period 1D PBG with n1 = 3.23, n2 = 1.45, n0 = 1.0 and h1/h2 = n2/n1; a = h1 + h2 is the period of the bi-layer thin films. The green zone is propagating band and the blue zone is stop band. The minus sign for lateral wave-vector is for TM wave. The two red lines are corresponding to the light lines $\omega =c{k}_y/n_0$ for TE and TM wave respectively.

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The wavelength range corresponding to the band gap is 569~713 nm. For finite period of such 1D PBG structures, more periods provide better omnidirectional reflection and sharper band-edges. The reflectance of a 3-period design for both TE and TM wave incident from different angles can be calculated using transfer matrix method [22]. Figures 3(a) and 3(b) show the reflectance as a function of wavelength, from which we can see that the band gap for TM wave is narrower than TE wave. This agrees well with Fig. 2(b) as the point C is much lower than point B. The reflectance for the emission at 633 nm remains above 91% indicating omnidirectional reflection for both polarizations at all incident angles. This effect enables the 1D PBG structure to reflect most of the fluorescence emission towards detection and prevents the coupling into the substrate, leading to higher collection efficiency.

 

Fig. 3 Calculated reflectance of a 3-period 1D PBG design for (a) TE & (b) TM waves at 0°, 20°, 40°, 60°, 80° and 89° incident angles as a function of wavelength; Collection efficiency as a function of distance between the dipole molecules and the top surface of 1D PBG structure for (c) TE excitation with 3-period 1D PBG structure; (d) Bare glass substrate.

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The surface bounded molecule can be modeled as a point dipole with the radiation distribution described in [14]. The collection efficiency can be calculated as the ratio of integrated flux intensity$S$ of electro-magnetic wave in a solid angle element_{$d{\Omega }^2$}.

As described earlier, the excitation light coming from the bottom of the substrate can be enhanced within the multilayer structure at the resonant angle higher than the total internal reflection angle. As each interface between the thin films transmits and reflects light, localized electric field can be built up by interference of forward and backward waves. A strong evanescent field will be generated on the top surface to excite the fluorescent molecules. By scanning all incident angles, a peak enhancement of 9.2 folds for the electric field of the TE excitation (λ_ex = 532 nm) is found at the incident angle of 44.07° with FWHM = 0.22° for the 3 period design shown in Fig. 4(a) . Theoretically, this peak can be further increased if more periods are added to the structure. At the same time, the angular width will become much narrower, which will require stricter beam alignment. The refractive index we used here for GaP at 532 nm (n₁ = 3.35-0.039i) was measured with a spectroscopic ellipsometer. The imaginary part – the extinction coefficient, although very small, will also affect the magnitude of the enhancement. Techniques for improving the extinction coefficient in GaP have been reported in our previous work [21]. Figure 4(c) illustrates the electric field distribution for TE wave throughout the device with the refractive index profile plotted as a function of the propagation distance. At z<0, it shows the interference pattern within the substrate. From 0 micron to around 0.5 micron, the field builds up gradually within the 3 periods of the thin film structure and finally decays as an evanescent wave penetrating into the top surface for about 1 micron.

 

Fig. 4 Electric field of the excitation light on top surface of the 3-period 1D PBG structure for both (a) TE and (b) TM waves as a function of the incident angle within the substrate; Electric field of the excitation light as a function of propagating distance for (c) TE and (d) TM waves.

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For this particular design, the TM excitation does not offer the enhancement effect shown in Figs. 4(b) and 4(d). However, if an extra layer of 55 nm GaP thin film is added on top of the 3-period thin film structure, which then becomes 3.5-period structure, TM wave PCR can also be found as shown in Fig. 5 . In this case, the transmitted field has a peak enhancement of 8.9 folds at the incident angle of 45.07° with a FWHM = 1.27°, which is much larger than the angular width of TE enhancement. The wide resonant angle benefits the optical alignment especially for typical TIRF system that employs an objective lens to focus a cone of light onto the molecules.

 

Fig. 5 Electric field of the excitation light on top surface of the 3.5-period (an extra layer of 55 nm GaP) 1D PBG structure for both TE (top) and TM (bottom) waves as a function of the incident angle within the substrate.

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3. Fabrication and experiment

The 3-period 1D PBG structure was fabricated layer by layer using RF magnetron sputtering (100 W, 4 mTorr) method on a fused silica substrate. Since the sputtered GaP thin films usually have large extinction coefficients due to stoichiometric imbalance and structural misalignment, a post-deposition high temperature (700 °C, 10 minutes) anneal was conducted after depositing each layer of GaP. This process improves the recrystallization and further reduces the energy loss for the excitation light. A stylus profiler was used to confirm the thickness of each layer. The transmission spectrum at normal incidence was monitored on an optical thin film analyzer right after each deposition to match our calculation results based on the ellipsometry data. In this way, the fabrication error can be minimized to ensure the smallest drifting of the PCR. The transmission spectrum of the 3-period sample is shown in Fig. 6(a) . The measured spectrum agrees very well with the predicted values.

 

Fig. 6 (a) Transmission spectrum of the fabricated 3-period 1D PBG sample compared with calculated spectrum based on ellipsometry measurements; (b) Result of omni-directional reflection test using a HeNe laser incident on the top surface of the 1D PBG sample.

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The omnidirectional reflection effect is tested using a HeNe laser as shown in Fig. 6(b). With the incident angle changing from 5° to 75° with respect to the top surface of the 1D PBG structure, the reflectance remains around 80%. This number is lower than the calculated 90%, which could be attributed to the absorption and scattering losses in the films.

The Qdots (R) 625 ITK carboxyl quantum dots from Invitrogen were used as the fluorescent material due to its excellent stability and lifetime. The peak emission wavelength (λ_em = 625 nm) is slightly smaller than HeNe laser but still lies in the band gap. The quantum dots were diluted in isopropyl alcohol with volume ratio 1:10 and 20 µl is then spin-coated onto the PBG surface at 1000 rpm for 20 seconds. After room temperature drying, the quantum dots will settle down and become surface-bounded. A high spin speed will bring the quantum dots closer to the surface where evanescent field can penetrate. However, if the spin is too fast a large portion of dots will be lost, which requires stronger excitation light to pump. At lower speed such as 100 rpm, the quantum dots will accumulate and become clusters that lift some molecules off the surface. The off-surface molecules will then cause background noise due to scattering excitation and they most likely gives lower emission due to the limited depth of evanescent field.

The setup in Fig. 7 is used to test the field enhancement effect of the 1D PBG structure. The polarization direction of the excitation light can be controlled using a half-wave plate after mirror 1. The prism is fixed with the top surface at the center of the rotation stage. The PBG sample is placed on the prism using index-matching oil. By rotating the stage, the incident angle of the excitation light can be increased slowly to find the PCR angle. The fluorescence signal was then captured by an objective lens to form an image on the CCD camera. For the 3 period 1D PBG sample, a strong enhancement image was found at the incident angle of 45.03° with TE excitation light. This angle is very close to the predicted 44.07° if rotation stage alignment error is taken into consideration. An average enhancement of 50 folds was achieved compared with a regular glass substrate at the same incident angle and excitation power shown in Figs. 8(a) and 8(b) (see Media 1). No enhancement effect was found by switching the excitation light to TM polarization. However, if the 3.5-period 1D PBG sample is used, a brighter enhanced fluorescent image was captured with an average enhancement of 69 folds compared with bare glass substrate shown in Figs. 8(c) and 8(d) for TM polarized excitation. The resonance angle is 44.67° which is also very close to the predicted 45.07°. By switching to TE polarization for the excitation, no enhancement effect was found.

 

Fig. 7 Experimental setup of fluorescence enhancement test of 1D PBG structure.

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Fig. 8 Fluorescence images capture by the CCD camera for (a) 1D PBG sample with TE enhancement; (b) bare glass substrate with TE excitation at the same incident angle; (c) 1D PBG sample with TM enhancement; (d) bare glass substrate with TM excitation at the same incident angle (Media 1).

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4. Conclusion

In conclusion, a 1D PBG multilayer structure is designed to achieve fluorescence enhancement through combined PCR and omnidirectional reflection. Both TE and TM polarized excitation light can be enhanced at the resonant angle by switching the sequence of the bi-layer period. This represents an advantage over SPR enhancement, which only works for TM polarized excitation. An average enhancement of 50 to 69 folds has been experimentally confirmed with GaP and SiO₂ as the high and low index materials. The omnidirectional reflection effect contributes to higher collection efficiency by prohibition of Fresnel transmission of the fluorescence emission into the substrate. Such a simple structure has other advantages of avoiding complex surface sculpturing and better photo-stability. The fluorescence enhancer is compatible with the traditional TIRF microscopy and may find broad applications in bio-sensing and imaging.