Fluorophore Coupling to Internal Modes of Bragg Gratings

Abstract

Multilayer structures with two dielectrics having different optical constants and no structural features in the x−y plane can display photonic band gaps (PBGs) and are called one-dimensional photonic crystals (1DPCs). If the top layer thickness is carefully selected, the electromagnetic energy can be trapped at the top surface. These highly enhanced fields are called Bloch surface waves (BSWs). The BSW resonance angles are sensitive to the dielectric constant above the top dielectric layer. As a result, BSW structures have been used for surface plasmon resonance-like measurements without the use of a metal film. However, the emphasis on surface-localized BSWs has resulted in limited interest in fluorophore interactions with other optical modes of 1DPCs or Bragg gratings without the different thickness top layer. Herein, three different fluorescent probes were used to cover the short, center, and long wavelengths of the PBG. We demonstrate efficient coupling of fluorophores to both the BSW and internal modes (IMs) of a 1DPC. Coupling to the IM is expected to be low because of the micron-scale distances between the fluorophores and IM, which exists inside the Bragg gratings. At different wavelengths or observation angles, the IM-coupled emission (IMCE) can occur with the first three modes of the multilayer. This coupling is not dependent on a BSW mode. IMCE was also observed for a monolayer of fluorophore-labeled protein. IMCE enables sensitive detection of surface-bound fluorophores. Applications are anticipated in high sensitivity detection and super-resolution imaging.

Graphical Abstract

INTRODUCTION

Detection of fluorophores on glass surfaces and slides has grown steadily during the past 3 decades. For example, the introduction of biomolecule-specific fluorophores stimulated the use of fluorescence microscopy for cell imaging.¹ This trend was further enhanced by confocal detection and/or multiphoton excitation, which decreased out-of-focus emission and increased contrast.² There has also been a rapid growth of fluorescence detection of surface-bound fluorophores for analytical applications such as immunoassays,³ proteomics,⁴ and DNA sequencing.⁵ All these applications use the free-space emission of biomolecules labeled with specific fluorophores. To avoid laborious labeling procedures and possible perturbations of the biomolecules by the fluorophores, there is high interest in label-free methods. The most widely used label-free method is surface plasmon resonance (SPR), which uses glass slides with a thin layer of gold, about 40 nm thick, that displays intense absorption at a specific angle above the critical angle.⁶ The SPRs display a small angular shift which depends on the mass of a biomolecule binding to the surface and small changes in refractive index at the gold–water interface. Because these angular shifts are small, less than 1°, several methods have been described, which increase the sensitivity of the SPR, such as phase-sensitive detection,⁷ substrates with gratings,⁸ waveguides,⁹ or metallic particles.¹⁰

During the past decade, there has been rapid development of plasmonic substrates which use enhanced fluorescence to provide selective detection of surface-bound fluorophores. One of the earliest methods is surface plasmon-coupled emission (SPCE) wherein excited-state fluorophores near the metal surface create plasmons, which are detected at the plasmon resonance angle for the emission wavelength.^11,12 SPCE has been extended to include metal films coated with a single dielectric layer to create metal–dielectric waveguides (MDWs).¹³ For SPCE and MDW structures, the incident light is trapped at the top dielectric–air or water interface, resulting in an enhanced excitation field. The emission at each wavelength is coupled through the substrate at a single angle from the normal z-axis, which provides selective detection of surface-bound fluorophores. Metallic surfaces have the disadvantage of quenching emission from fluorophores within 10 nm of the metal surface.^14,15 This quenching effect has been avoided by the use of all-dielectric Bloch surface wave (BSW) structures with photonic band gap (PBG), which trap light at the surface because of the high incident wavevector of the surface-bound electromagnetic waves.²¹ The trapped state is known as a BSW,¹⁶ which should not be confused with Bloch waves which exist within any periodic structure.¹⁷ In the present report, we refer to Bloch waves as internal modes (IMs) of the structure. We consider only one-dimensional photonic crystals (1DPC) which have multiple planar films with different refractive indices. If the top dielectric layer has a different and suitable thickness, the incident light can be trapped at the top layer.^18,19 1DPC structures contain only transparent optical materials and have low optical losses. As a result, the BSW resonances show a very narrow angular distribution and a corresponding strong field enhancement at the surface.^20,21 The BSW resonance angle is sensitive to the dielectric constant above the structure which permits label-free SPR-like detection of surface binding.²² Similar to surface plasmons, the BSWs have an evanescent field which penetrates a short distance into the sample. This evanescent field has been used for enhanced excitation of surface-bound fluorophores. The excited-state fluorophores can couple with the BSW field and radiate through the substrate over a narrow range of wavelengths and angles.^23,24 This phenomenon is called BSW-coupled emission (BWCE).

SPR, SPCE, and BWCE all depend on evanescent fields which have short penetration lengths into the sample. As a result of the need for evanescent wave–sample interactions, research has focused on fluorophore coupling to the surface modes.^13,25 However, 1DPCs also display IMs wherein the optical field is localized within the multilayer structure. Because the IMs have shorter evanescent tails penetrating into the sample, they have been ignored for detection of surface-bound fluorophores. Stated otherwise, fluorophores coupling to IMs is generally thought to be weak because the fluorophores are hundreds of nanometers or several microns away from the locations where most of the IM energy is confined. Fluorophore coupling to IMs has been detected in previous angle-dependent measurements of the coupled emission.²¹ IM-coupled emission (IMCE) has also been observed using back focal plane imaging of reflected light or coupled emission.^26,27 However, because of the emphasis on the surface mode coupling, the emission coupling to IMs has rarely been examined and usefulness of IMCE has not yet been recognized.

In the present report, we characterize the properties of IMCE. The IMCE intensities are comparable to BSW-coupled emission intensities but occur at different angles and wavelengths through the substrate. IMCE does not require the presence of a BSW and can be used with any Bragg grating (BG). We show that the IMCE intensity depends on the reflectivity spectrum of the BG. The IMs are almost completely inside the 1DPC or BG, and the coupling angles are almost independent of the refractive index above the structure, which makes IMCE convenient for detection of surface-bound fluorophores in aqueous solution without concern for a specific top layer thickness or change in the coupling angle. The BG and 1DPC multilayer structures have no nanoscale features in the x−y plane, can be fabricated by vapor deposition,²⁸ and are therefore available for large area samples at low cost. They can be made with any dielectric and designed for use over a wide range of wavelengths from the UV to the NIR. 1DPCs can also be fabricated by spin coating of polymers²⁹ inorganic sol gels,³⁰ or with a small contrast ratio between the two dielectric materials.³¹ These properties of 1DPCs will enable the use of IMCE in a wide range of bioaffinity assays, spotted array applications, and potentially point-of-care devices.

MATERIALS AND METHODS

The fluorescent probes rhodamine 6G (Rh6G) and Nile blue (NB) were obtained from Invitrogen, Inc. [Ru(bpy)₂dppz]-(PF₆), where bpy is 2,2′-bipyridine, dppz is dipyridophenazine and PF₆ is hexafluorophosphate, was obtained from Sigma-Aldrich and hereafter called Ru(dppz). All probes were used without further purification. The molecular structure and emission spectra of these probes in poly(vinyl alcohol) (PVA) are shown in Figure 1. These probes were selected to overlap with or be close to the PBG of the 1DPC. Each probe was dissolved in 1% PVA in water and spin-coated on glass slides on the 1DPC using conditions described previously to obtain a 45 nm thick layer of PVA.²¹ Rhodamine B-labeled streptavidin (RhB-SA) was obtained from Invitrogen, Inc. A monolayer of RhB-SA, with a thickness near 4 nm, was obtained by covering the 1DPC with a 1 mg/mL solution in pH 7.2 phosphate buffer for 30 min at 4 °C, followed by rinsing with the same buffer without SA.

Figure 1.

Chemical structures of the probes and emission spectra in PVA on glass used in the present report

The 1DPC was made by plasma-enhanced chemical vapor deposition of silica (SiO₂) and silicon nitride (Si₃N₄) on standard microscope slides. This structure consisted of alternating layers of the low (L) and high (H) refractive index (n) dielectrics (Figure 2B). The top layer of the lowrefractive index SiO₂ was a different thickness needed to support a BSW at wavelengths near 580 nm, but this thickness does not support BSW at wavelengths above 650 nm.²¹ Simulated reflectance was obtained using TFCalc.³² Simulations of dipole emission or incident fields were calculated using software from Lumerical, Inc.³³ and confirmed by a code based on the algorithm used in ref 34. The low (L) refractive index of SiO₂ was used with a null imaginary part. The high (H) refractive index of Si₃N₄ ranged from n_H = 2.196 + i0.033 at 550 nm to n_H = 2.144 + i0.016 at 633 nm.²¹ The imaginary component resulted in a finite angular width, which facilitated some of the simulations.

Figure 2.

(A) Optical geometry for emission measurements with a 1DPC using RK excitation. The incident wavelength is 470 nm for Rh6G and Ru(dppz) and 635 nm is for NB. (B) Dimensions and optical properties of the 1DPC. The far-field optical properties for 580, 600, and 675 nm incident wavelengths are shown in Figures 4, S1, and S2, respectively.

The experimental geometry is moderately complex, and to avoid ambiguity is shown in detail (Figure 2A). The 1DPC is mounted on a hemicylindrical prism and optically coupled with index matching fluid (glycerol) to avoid total internal reflection (TIR) from the glass slide.³⁵ The prism allows light above the glass–air critical angle to enter and create an evanescent field at the top SiO₂−PVA layer. The prism also allows the coupled emission above the critical angle to be measured in the farfield. The z-axis is the laboratory vertical axis and is along the long axis of the prism. The incident light for excitation can be through the prism, known as the Kretschmann (KR) configuration, or directly from the air side of the sample known as the reverse Kretschmann (RK) configuration. The terms KR and RK usually refer to the incident light, but we use these same terms to describe the emission. In this report, the excitation was always in the RK configuration from the air side at 180°. Similar results were obtained for KR excitation (not shown). Emission is measured on the prism side (KR) from 0 to 80° and from the air side (RK) from 95 to 140°. The RK emission is also described as free-space emission. The prism has a diameter of 1 in. and the end of the observation fiber, with a diameter of 1.0 mm, is placed 2 cm away from the prism surface or about 2.5 cm from the illuminated spot. Most of the measurements are S-polarized, indicating that the excitation and emission polarizer are aligned with the vertical z-axis and parallel to the surface of the 1DPC. For P-polarized emission measurements, the excitation polarizer remains aligned with the z-axis, but the emission polarizer is tilted perpendicular to the z-axis. In some cases, the incident light was along the y-axis, but the S-coupled emission was nearly the same as with incident light polarized along the z-axis. A long-pass filter is used to remove reflected or scattered incident light. The emission passing through the fiber is measured with either an Ocean Optic SD2000 spectrofluorometer or a Pico Quant MicroTime 300 spectrofluorometer.

RESULTS AND DISCUSSION

Simulated Optical Properties of the 1DPC.

In previous publications, we reported coupled emission from photonic and plasmonic structures. For thin metallic films, the coupled emission occurred at the SPR angle,^11,12 and for dielectric photonic structures, the coupled emission occurred at the wavelength and angle of the BSW resonance.²¹ Coupled emissions with the photonic structure at angles larger than the BSW angles were noted but not discussed.²¹ The fluorophores in Figure 1 were selected because the emission spectra of Rh6G, NB, and Ru(dppz) overlap with the resonances of the 1DPC. The simulated reflectance spectra show strong narrow S-polarized dips in reflectivity, which depend on the incident wavelength (Figure 3). The sharp resonances at the shorter wavelengths are due to the S-polarized BSW modes. Wider but less pronounced dips in the reflectivity are seen at longer wavelengths, which are shown below to be due to IMs of the 1DPC. By design, the present 1DPC does not display a BSW mode above 650 nm to allow us to determine if the BSW mode contributed to IMCE.

Figure 3.

(A) Wavelength and (B) angle-dependent reflectivity spectra of the 1DPC. The numbers refer to the incident angle (A) or incident wavelength (B).

The optical modes of the 1DPC can be observed by the angle-dependent reflectivity at a single wavelength (Figure 3B). The BSW modes are seen as sharp resonances near 45°, and the IM appears at larger observation angles. These results show BSW modes for 555 and 600 nm, but BSW modes are essentially absent at 675 nm. The IMs are still present for 675 nm and would be present without the thicker top layer of SiO₂ (not shown). The IMs are very robust and exist even if the spaces above the 1DPC is all water (n = 1.33). In contrast, the BSW mode is not present when the 1DPC in Figure 2B is covered with water. A different 1DPC structure is needed to support a BSW mode in water.

The optical modes (BSW or IM) associated with the dips in reflectivity (Figure 3) were assigned by simulations of the field intensity (|E|²) distributions in the 1DPC for illumination at the resonance wavelengths and angles. At 580 nm with S-polarized incident light at 44.1°, the intensity is strongly localized at the surface of the 1DPC and enhanced 1400-fold (Figure 4A), which is characteristic of a BSW mode.^36,37 The optical field is localized by two effects. Light cannot escape from the top because of TIR. Light propagation through the bottom is restricted by the PBG and can only escape by tunneling. The electric field distribution with 580 nm incident light changes for different angles of illumination. The first IM (IM1) for illumination at 57.8° is inside the 1DPC and has a maximum intensity in the center of the 1DPC with a symmetrical intensity distribution (Figure 4B). The second and third IMs (IM2 and IM3) for illumination at 68.2 and 85°, respectively, have more complicated intensity distribution (Figure 4C,D). Similar results were obtained at 600 nm where the BSW field was enhanced nearly 900-fold (Figure S1). At 675 nm, the 1DPC showed similar shaped internal IM1 and IM2 modes (Figure S2). At 675 nm, the BSW mode is shifted close to the critical angle and there was only a fourfold field enhancement of the BSW mode at 675 nm at the 1DPC surface, which is comparable to TIR.³⁵ This is in contrast to the apparent BSW mode at shorter wavelengths where BSW field intensities are greatly enhanced at 580 and 600 nm. The difference between a TIR and BSW mode can be seen in Figure S2A where the TIR starts abruptly at the PVA–air interface and the BSW mode is localized in both the PVA and air space. The IM field intensities at the surface for 675 nm are comparable to or lower than those found for TIR. BSW-coupled emission at 675 nm is not expected because the BSW mode is no longer significant (Figure S2). For all three wavelengths, the maximum intensity location of IM1 is about 1000 nm from the surface and not within the near-field of the excited-state fluorophores. The most intense region of the IM2 mode is about 500 nm from the surface. Fluorophore coupling is expected to be very weak at these long distances. Coupling between two fluorophores for fluorescence resonance energy transfer (FRET) occurs at distances no greater than 8 nm³⁸ and FRET between fluorophores and a continuous layer of gold may occur to about 16 nm.^14,39 Coupled emission via the IMs is expected to be weak at any wavelength or angles because they have very small amplitudes at the top surface where the fluorophores are localized and are too distant from the IMs for FRET-type near-field interactions.

Figure 4.

Field intensity (|E|²) distribution for 580 nm incident light. (A) BSW resonance at 44.1°, (B) IM1 at 57.8°, (C) IM2 at 68.2°, and (D) IM3 at 85.0°. Both S- and P-polarizations are shown.

Coupled Emission Spectra for Rh6G.

The coupled emission of Rh6G is highly modified when placed on the 1DPC (Figure 5). On a plain glass slide, the emission spectrum (dashed lines in Figure 5) is not significantly dependent on the observation angle. On the 1DPC, the apparent emission spectra become very narrow and confined to a small range of wavelengths which do not appear to follow the free-space emission spectra. The coupled emission shifts to shorter wavelengths as the observation angle is increased in the BSW range, 41 to 47° (Figure 5A). At larger angles from 50 to 70°, the emission maxima are coupled to the first IM and also shift to shorter emission maxima at larger observation angles (Figure 5B). The coupled emission maxima are strongly dependent upon the observation angle and confined to a small range of angles (Figure 6A). On the other hand, P-polarized emission was insensitive to the observation angle (Figure S3).

Figure 5.

Rh6G, S-polarized emission in the BSW range (A) and in the IM1 range (B). Dashed lines show the intensity-normalized Rh6G emission spectrum from glass, which is insensitive to the observation angle. (C) Normalized Rh6G, S-polarized emission in the BSW range and in the ranges for IM1 and IM2. Some selected angles are also listed.

Figure 6.

(A)Rh6G and (B) Ru(bpy)₂(dppz)(PF₆)₂ angular emission intensity distribution from the 1DPC. RK illumination using 470 nm at 180°.

When we first observed these spectral shifts, we thought the emission spectra were red-shifted at larger angles. A similar effect was observed for a number of different fluorophores. We later realized that the BSW-coupled modes occurred from 41 to 47° and that new modes appeared at larger angles for 50 to 70°, which we now assign to IMCE. We were hesitant to reach this conclusion because the peak intensity envelope of the first IM1-coupled emission was only about 10-fold less than the BSW-coupled emission. These relative intensities are very different from the 1000-fold enhanced intensities of the BSW modes and the fourfold enhancements of the IM1 mode at the surface with light incident at the resonance value for each wavelength. However, the difference in emission intensities could not be due to an amplified excitation field because the experiments were performed with RK excitation at 180° and emission S-polarized along the z-axis (Figure 2A). Because of the RK mode of excitation, the relative intensities reflect the coupling efficiency to the BSW or IM1 modes. The connection of the observed maxima with each mode can be clarified by plotting the normalized intensities (Figure 5C). The relatively high intensities of the IMCE (Figure 5B) suggest that it could be useful for surface-selective detection.

Coupled Emission of NB.

We were surprised by the relatively high intensity of the IM1-coupled emission from Rh6G and questioned if the absence of a BSW mode facilitated coupling to the IM1 intensity. To clarify this question, we selected the probe NB. NB has longer emission wavelength over 650 nm (Figure 7), where the BSW mode is no longer significant (Figure S2). The 660 nm emission maximum of NB is much longer than the emission maxima of Rh6G at 550 nm. BWCE was not observed for NB on the 1DPC, presumably because the BSW mode is weak or nonexistent at this wavelength. The emission is still strongly directional (Figure 7C), but the combination of observation angle and wavelength is consistent with coupling to the IM1 and IM2 modes (shown below). As before, the P-polarized emission spectra were mostly the same at all observation angles (Figure S4). Accordingly, at 660 nm, the 1DPC in Figure 2B is no longer sustaining a BSW mode and present results suggest that IMCE can be observed with any BG without a BSW mode.

Figure 7.

S-polarized NB emission in the IM1 range (A) and in the IM2 range (B). Dashed lines show the intensity-normalized NB emission spectrum from glass, which is insensitive to the observation angle. Note that the wavelength axis is different from that used in Figure 5. (C) NB emission intensity distribution from 1DPC. RK illumination polarized along the Y-axis using 635 nm at 180°.

Coupled Emission with Ru(dppz).

Coupling of NB with the IM1 and IM2 modes at wavelengths larger than the BSW suggested that the BSW modes are not necessary for coupling to the IMs. The absence of a BSW pathway may increase coupling to the IMs. To further demonstrate coupled emission to the IMs, we examined a metal–ligand complex Ru(dppz) with a wide emission spectrum ranging from 540 to 800 nm (Figure 8, dashed lines). Because of the width of the Ru(dppz) emission, the angle-dependent spectra were expected to contain contributions of coupled emission from more than one mode. We reasoned that detection of both BWCE and IMCE from a single fluorophore would reveal the relative efficiency of BSW and IM coupling. At observation angles of 41 to 46°, coupled emission is observed from both the BSW and IM1 modes, but in different emission wavelength ranges. This result is consistent with the NB results which showed IM1-coupled emission near 675 nm (Figure 7). Once again the P-polarized emission spectra did not show consistent angle-dependent shifts (Figure S5). The IM1 intensity is about fivefold less than the BSW intensity (Figure 8A). These results confirm that IMCE provides high coupled emission intensities with RK incident light, without the high surface field enhancements with KR incident light. A similar argument can be made by comparison of the IM1 versus IM2 relative intensities (Figure 8B). The connections between the wavelength-dependent emission maximum and the optical modes can be seen in the normalized intensities (Figure 8C), where some emission is even seen with the IM3 mode.

Figure 8.

BSW-, IM1-, and IM2-coupled emission from Ru(bpy)₂(dppz)(PF₆)₂ on 1DPC. Dashed lines show the intensity-normalized ruthenium complex emission spectrum from glass λ_ex = 470 nm. (C) Corresponding normalized emission in the BSW, IM1, IM2, and IM3 ranges. Some selected angles are labeled.

Free-Space Emission Intensities.

The coupled emission of Ru(dppz) is confined to a very narrow range of angles, and contributions from the BSW, IM1, and IM2 modes can be resolved by the smaller peaks at higher angles (Figure 6B). If the BSW mode was not present, the smaller peaks would be dominant. We noticed that the free-space emission with Ru(dppz) was very low, as was the free-space intensity of Rh6G (Figure 6). In contrast, the free-space emission of NB was high (Figure 7C). These results are consistent with the observed coupling of Rh6G to the BSW mode and Ru(dppz) coupling with the BSW, IM1 and IM2 modes, resulting in their low free-space intensities. In contrast, NB could not couple with a BSW mode and could only couple to the IM1 and IM2 modes. As a result, the free-space emission from NB is higher than the other two fluorophores.

Dispersion Diagrams and Mode Coupling.

The dispersion diagram for the 1DPC in Figure 9 shows the angle and wavelength-dependent properties of the structure. Superimposed on this diagram are the angle-dependent maxima for the three fluorophores studied. The agreement between the simulations and experimental data is not perfect, which is probably the result of the actual 1DPC having slightly different dimensions and/or optical properties than the simulated 1DPC. However, it is clear that the apparent emission maxima of the three different fluorophores on the 1DPC are correlated with the listed modes. The IM1 modes can be observed at the same angles as the BSW mode. Because its long emission spectrum, NB only couples to IM1 and IM2, whereas Rh6G couples to BSW, IM1, and IM2 and due to its wide emission spectrum Ru(dppz) couples to four modes, BSW and IM1, IM2 and IM3. The striking feature of Figure 9 is that the angle-dependent emission maxima are independent of fluorophore and a characteristic of the optical modes of the 1DPC. More specifically, the same emission maximum is observed for any fluorophore on the 1DPC or Bragg structure when measured at the same observation angle. Another important result is that fluorophores can couple with any optical mode in the 1DPC if there is spectral and angular overlap. This property is similar to FRET, which occurs with any donor–acceptor pair and depends only on spectral overlap.³⁸

Figure 9.

Dispersion diagram for the 1DPC. The figure shows the reflectivity for a range of wavelengths and incidence angles. The dots represent the angle-dependent emission maxima and respective angles.

Sensitivity of IMCE.

To demonstrate the use of IMCE to measure low intensity fluorescence, we examined the 1DPC coated with a single monolayer of RhB-SA. The effective probe concentration for the monolayer is many-fold lower than the previous measurements with labeled PVA layers. The BSW-coupled emission was found to be at least 10-fold more intense than the free-space emission measured in the RK-direction. The IMCE intensity is about two-fold larger than the free-space intensity (Figures 10 and S6). Thus, IMCE is not as sensitive as BWCE, but the intensity is comparable to or larger than the free-space emission. The emission spectra of RhB-SA displayed the same dependence on observation angle for the BSW and IMs (Figure 10). The angle-dependent emission maxima follow the dispersion diagram shown in Figure 9 (data not shown). These results suggest that IMCE can be used for a wide range of surface-bound bioassays.

Figure 10.

S-polarized emission from a monolayer of RhB-SA on 1DPC in the BSW range and in the IM1 range. Normalized spectra are shown in the right panel. Only few selected spectra are labeled for clarity.

The emphasis of this paper is to demonstrate that coupled emission can be observed with a BG, independent of the presence of a BSW surface mode. The use of a BG is in contrast to the use of BSW structure for sensing, as seen by their use as a substitute for SPR^40,41 and fluorescence detection of surface-bound fluorophores.^42,43 A common feature of these BSW applications is on the use of localized surface waves and interactions with the sample. As discussed above, BSW are not present on all multilayer structures and a change in the sample solution can eliminate the BSW resonance in the sample. The present paper attempts to overcome this narrow vision of sensing with multilayer BSW structures and to expand their use by coupling to IMs using Bragg structures. These IMs are very robust and weakly, if affected at all, by the sample dielectric constant. Our results show that excited-state fluorophores can couple with the IMs over micron distances and display strongly directional emission. Importantly, the intensities of the IM-coupled emission can be comparable to those of BSW-coupled emission.

CONCLUSIONS

This paper agrees with the results from an earlier report,²³ which reported strong modification of the emission spectra of a rhodamine dye on a BSW structure. The rhodamine spectrum change is from a typical single wide emission on glass to two narrow emission spectra on a BSW structure. While not certain, it seems likely that the spectral redistribution reported was separate coupling to a BSW and an IM.²³ This not a criticism of the authors but merely an alternative explanation for their results.

Another potential use of IMCE is for detection of supercritical angle fluorescence (SCF), which occurs at angles above the critical angle.⁴⁴ This emission can be collected using parabolic reflective detectors.⁴⁵ SCF has already been used without a BSW structure or BG for high sensitivity immunoassays without wash steps⁴⁶ and detection of single nucleotide polymorphisms without a microscope objective.⁴⁷ In the case of fluorescence microscopy, SCF contains high wavevector components, which increases spatial resolution to provide subdiffraction-limited spatial resolution.^48–50 The SCF results were obtained without BG. The use of BG substrates may provide a method to suppress the lower wavevector components, which decrease spatial resolution.

In summary, a simple multilayer BG structure can couple the emission of near-surface fluorophores into IMs at different angles and wavelengths can be easily fabricated without the delicate structural design to obtain a BSW resonance. These structures are also widely available from optic companies. IMCE can have an impact on the development of new clinical sensing instruments and point-of-care devices.