Steve Blair

Jérôme Wenger

Enhancing Fluorescence with Sub-Wavelength Metallic Apertures

INTRODUCTION

The ability to reliably produce nanometric structures with a resolution down to a few nanometers opens the way for promising photonics applications [START_REF] Barnes | Surface plasmon subwavelength optics[END_REF][START_REF] Ozbay | Plasmonics: Merging Photonics and Electronics at Nanoscale Dimensions[END_REF]. With these nanodevices, light can be locally confined, inducing large absorption and diffusion cross sections and enhancing the local electromagnetic fields. Such effects hold strong relevance for molecular detection methods based on photonic emission (fluorescence and Raman scattering) that have recently gained a wide popularity in various fields such as biology, medicine, chemistry and materials sciences. The sensitivity of most applications relies directly on the intrinsic molecular brightness. Therefore, increasing the optical emission of molecules using metallic nanostructured substrates has become a key issue [START_REF] Lakowicz | Radiative decay engineering 5: metal-enhanced fluorescence and plasmon emission[END_REF][START_REF] Aslan | Metal-enhanced fluorescence: an emerging tool in biotechnology[END_REF][START_REF] Lakowicz | Plasmonics in biology and plasmon-controlled fluorescence[END_REF].

The fluorescence emission of a single molecule can be greatly enhanced by properly tailoring its photonic environment, opening new opportunities for molecular detection. Since the founding works of Purcell [START_REF] Purcell | Spontaneous emission probabilities at radio frequencies[END_REF], Drexhage [START_REF] Drexhage | Interaction of light with monomolecular dye layers[END_REF] and Kleppner (8), it is well recognized that the spontaneous de-excitation of a quantum emitter can be controlled by its environment, leading to modifications of the total de-excitation rate and spatial emission distribution. Following Fermi's golden rule, the spontaneous deexcitation rate is proportional to the local density of states (LDOS) [START_REF] Barnes | Fluorescence near interfaces: the role of photonic mode density[END_REF][START_REF] Lakowicz | Radiative decay engineering 5: metal-enhanced fluorescence and plasmon emission[END_REF], which can be altered by a wide range of structures, such as planar interfaces [START_REF] Barnes | Fluorescence near interfaces: the role of photonic mode density[END_REF], nanoparticles [START_REF] Anger | Enhancement and quenching of single molecule fluorescence[END_REF][START_REF] Kuhn | Enhancement of single-molecule fluorescence using a gold nanoparticle as an optical nanoantenna[END_REF], or nanoantennas [START_REF] Farahani | A single quantum dot coupled to a scanning optical antenna: A tunable superemitter[END_REF][START_REF] Muskens | Strong enhancement of the radiative decay rate of emitters by single plasmonic nanoantennas[END_REF].

Up to now, a large part of the international scientific attention was devoted to the study of metallic nanoparticles (either single particles or in a colloid ensemble) [START_REF] Lakowicz | Radiative decay engineering 5: metal-enhanced fluorescence and plasmon emission[END_REF][START_REF] Lakowicz | Radiative decay engineering: the role of photonic mode density in biotechnology[END_REF]. Milling nanometric apertures in a metallic film is an intuitive way to manufacture new nanophotonics devices that are robust and highly reproducible. Although this concept appears very simple, such apertures exhibit attractive physical properties, such as localization of excitation light, strong isolation from emission produced by unbound species, and an increase in apparent absorption and emission yield. The simplicity of the structures and their ease of use should further expand their application towards the real-time detection and identification of a small number of molecules.

The purpose of this chapter is to overview some of the key results in fluorescence emission from sub-wavelength metal apertures, both single apertures and in array arrangements. While a comprehensive theory is currently lacking, sufficient understanding can be obtained from computational models to qualitatively describe experimental results.

BACKGROUND

The publication of the unexpected optical properties of arrays of subwavelength metal apertures by Ebbesen and co-workers in 1998 [START_REF] Ebbesen | Extraordinary optical transmission through subwavelength hole arrays[END_REF] sparked tremendous interest in the light transmission properties of ordered and disordered arrangements of metal apertures. When normalized to the transmission of an isolated aperture [START_REF] Lezec | Diffracted evanescent wave model for enhanced and suppressed optical transmission through subwavelength hole arrays[END_REF], transmission enhancements of about 3 were obtained, where very little transmission would be expected from an isolated aperture (the original series of publications normalized total transmission to the Bethe-Bouwkamp theory [START_REF] Bethe | Theory of diffraction by small holes[END_REF][START_REF] Bouwkamp | On Bethe's theory of diffraction by small holes[END_REF] for transmission through a single aperture T  (d/λ) 4 , where d is the diameter, which produced enhancement factors greater than 10 3 ). This enhancement was attributed to coupling into surface plasmon polariton (SPP) modes of the metal film, mediated by the periodic arrangement of apertures.

Significant attention has also been directed towards the properties of an individual circular [START_REF] Wannemacher | Plasmon-supported transmission of light through nanometric holes in metallic thin films[END_REF][START_REF] Levene | Zero-mode waveguides for single-molecule analysis at high concentrations[END_REF][START_REF] Degiron | Optical transmission properties of a single subwavelength aperture in a real metal[END_REF][START_REF] Yin | Surface plasmons at single nanoholes in Au films[END_REF][START_REF] Popov | Surface plasmon excitation on a single subwavelength hole in a metal sheet[END_REF][START_REF] Catrysse | Propagating modes in subwavelength cylindrical holes[END_REF] or rectangular [START_REF] Brolo | Increased cut-off wavelength for a subwavelength hole in a real metal[END_REF][START_REF] Pardo | Waveguiding in nanoscale metallic apertures[END_REF] aperture. Metal apertures support propagating modes with extended cutoff wavelength [START_REF] Brolo | Increased cut-off wavelength for a subwavelength hole in a real metal[END_REF][START_REF] Shin | Effect of the plasmonic dispersion relation on the transmission properties of subwavelength cylindrical holes[END_REF][START_REF] Pardo | Waveguiding in nanoscale metallic apertures[END_REF], along with a surface plasmon mode propagating down the walls that has no cutoff [START_REF] Shin | Effect of the plasmonic dispersion relation on the transmission properties of subwavelength cylindrical holes[END_REF]. The situation for a finite-height aperture is somewhat more complex in that localized plasmon (LSP) modes exist at the entrance and exit [START_REF] Degiron | Optical transmission properties of a single subwavelength aperture in a real metal[END_REF][START_REF] Gray | Surface plasmon generation and light transmission through isolated nanoholes and arrays of nanoholes in thin metal films[END_REF][START_REF] Shuford | Electric field enhancement and light transmission in cylindrical nanoholes[END_REF]; the effects of LSP modes are more pronounced for noble metals such as silver and gold [START_REF] Gérard | Nanoaperture-enhanced fluorescence: Towards higher detection rates with plasmonic metals[END_REF]. These modes are analogous to the LSP modes of metallic nanoparticles via Babinet's principle [START_REF] Prikulis | Optical spetroscopy of nanometric holes in thin gold films[END_REF] and can couple to the propagating modes of the nanocavity, resulting in surprisingly high transmission [START_REF] Heng | Characterization of light collection through a subwavelength aperture from a point source[END_REF][START_REF] Shuford | Electric field enhancement and light transmission in cylindrical nanoholes[END_REF].

A paper in 1986 suggested that fluorescence enhancement and reduced observation volumes could be obtained from random arrangements of small metal apertures [START_REF] Fischer | Submicrometer aperture in a thin metal film as a probe of its microenvironment through enhanced light scattering and fluorescence[END_REF]. In 2003, single molecule detection in an epifluorescence arrangement from a single sub-wavelength aperture was demonstrated in a high-concentration environment [START_REF] Levene | Zero-mode waveguides for single-molecule analysis at high concentrations[END_REF] (in which no fluorescence enhancement was reported), followed by a report of enhancement from a fluorescing monolayer self-assembled within an ordered array of apertures [START_REF] Liu | Fluorescence enhancement from an array of subwavelength metal apertures[END_REF] under trans-illumination. Subsequently, there have been reports of enhanced Raman scattering [START_REF] Brolo | Nanohole-enhanced Raman scattering[END_REF][START_REF] Wenger | Raman scattering and fluorescence emission in a single nanoaperture: optimizing the local intensity enhancement[END_REF], real-time monitoring of molecular capture [START_REF] Liu | Biosensing based upon molecular confinement in metallic nanocavity arrays[END_REF], electroluminescence of an organic LED [START_REF] Liu | Efficiency enhancement of an organic light-emitting diode with a cathode forming two-dimensional periodic hole array[END_REF], and fluorescence from a polymer layer [START_REF] Brolo | Enhanced fluorescence from arrays of nanoholes in a gold film[END_REF][START_REF] Kim | Laser-induced fluorescence within subwavelength metallic arrays of nanoholes indicating minimal dependence on hole periodicity[END_REF]. Computational modeling of dipole emission [START_REF] Liu | Enhanced fluorescence transduction properties of metallic nanocavity arrays[END_REF] provided a partial explanation for the enhancement that can be attributed to a single aperture. The fact that significant enhancement in excitation also occurs within a single nanoaperture was first demonstrated in a series of papers on single-molecule epifluorescence in round [START_REF] Rigneault | Enhancement of single-molecule fluorescence detection in subwavelength apertures[END_REF][START_REF] Wenger | Raman scattering and fluorescence emission in a single nanoaperture: optimizing the local intensity enhancement[END_REF] and rectangular [START_REF] Wenger | Single molecule fluorescence in rectangular nano-apertures[END_REF] apertures. Excitation enhancement is further supported by computational models [START_REF] Popov | Field enhancement in single subwavelength apertures[END_REF][START_REF] Yuan | Field enhancement of nano-sized metal aperture with and without surrounding corrugations through resonant surface plasmons[END_REF][START_REF] Shuford | Electric field enhancement and light transmission in cylindrical nanoholes[END_REF][START_REF] Mahdavi | Modeling fluorescence enhancement from metallic nanocavities[END_REF]. Additional studies demonstrated single molecule detection under transillumination [START_REF] Leutenegger | Confining the sampling volume for Fluorescence Correlation Spectroscopy using a sub-wavelength sized aperture[END_REF], with comparatively lower enhancement levels, and in C-shaped apertures [START_REF] Fore | Pulsed-Interleaved Excitation FRET Measurements on Single Duplex DNA Molecules Inside C-Shaped Nanoapertures[END_REF].

FLUORESCENCE ENHANCEMENT BY A SINGLE APERTURE

Nanometric apertures milled in an opaque metallic film allow for a simple and elegant way of generating a reduced analysis volume. As illustrated on Figure 17.1, the nanostructure itself acts as a pinhole filter directly located at the object plane of a standard epi-illumination microscope, similar to the original idea proposed by E. H. Synge in 1928 (49). When the aperture diameter is reduced sufficiently below the cut-off diameter of the fundamental excitation mode that may propagate through the (waveguiding) hole, the light inside the aperture is primarily confined to a rapidly decaying evanescent mode, with a decay length of a few tens of nanometers (such devices have thus been named zero-mode waveguides [START_REF] Levene | Zero-mode waveguides for single-molecule analysis at high concentrations[END_REF]). Beyond the optical confinement to isolate a single molecule in a highly concentrated (micromolar) solution, nanometric apertures can significantly enhance the fluorescence emission of a single molecule by properly tailoring its photonic environment. A nanometric aperture can affect the fluorescence emission in three ways: (i) by locally enhancing the excitation intensity, (ii) by increasing the emitter's radiative rate and quantum efficiency, and (iii) by modifying its radiation pattern, towards a higher emission directionality to the detectors. Determining the specific influence of these processes is a crucial issue to characterize nanodevices for enhanced fluorescence, and has been a topic of great interest for the last decade [START_REF] Barnes | Fluorescence near interfaces: the role of photonic mode density[END_REF][START_REF] Fort | Surface enhanced fluorescence[END_REF]. The inherent challenge in this task originates from the fact that the detected signal results from a product of excitation and emission processes. Excitation depends on the interaction between the driving field and the nanostructure, while at moderate optical intensity, the emission efficiency is set by the balance of radiative and non-radiative decays and the modification of the radiation pattern.

To our knowledge, the first implementation of nanoaperture-enhanced fluorescence dates back to 1986 [START_REF] Fischer | Submicrometer aperture in a thin metal film as a probe of its microenvironment through enhanced light scattering and fluorescence[END_REF], where apertures of randomly-distributed diameters between 180 and 480 nm in thin films of silver or gold supported by glass slides were used as probes to detect the properties of an adjacent liquid containing fluorescent molecules. Even in the absence of efficient detectors and laser sources, sensitive measurements of refractive-index differences of the order of 10 -4 were demonstrated, and a global enhancement of the fluorescence brightness was reported. Yet, the lack of efficient detectors and laser excitation together with a rather involved experimental configuration and distribution of aperture sizes complicated the analysis of the measured data.

More recently, a single sub-wavelength nanoaperture was demonstrated to significantly enhance the fluorescence rate emitted by a single fluorescent molecule. Using single rhodamine 6G molecules in isolated 150nm diameter apertures milled in an aluminum film, a 6.5 fold enhancement of the fluorescence rate per molecule was reported as compared to free solution [START_REF] Rigneault | Enhancement of single-molecule fluorescence detection in subwavelength apertures[END_REF]. This phenomenon was further investigated with a combination of FCS and fluorescence lifetime measurements to resolve the photokinetic rates of rhodamine 6G molecules in a water-glycerol mixture [START_REF] Wenger | Radiative and nonradiative photokinetics alteration inside a single metallic nanometric aperture[END_REF]. The fluorescence lifetime appeared to be dramatically reduced inside the aperture, indicating that the molecular energy levels' branching ratios were strongly affected. For a properly tailored nanoaperture diameter of 150 nm, the local density of states alteration brought by the aperture allowed for a higher radiative rate without decreasing the fluorophore quantum efficiency (fluorescence quenching). The combination of this effect together with an increase in the local excitation intensity led to the overall fluorescence enhancement. 17.3 1. Background F in a nanoaperture is defined as the ratio of the detected fluorescence count rate per molecule in the aperture CRMaper and in open solution CRMsol for the same (fixed) excitation power, that is F = CRMaper / CRMsol. To a good approximation, most of the fluorescent molecules can be treated as a system of three energy levels in steady state (singlet ground state S0, first excited singlet state S1 and dark non-fluorescing state D). Under steady-state conditions, the fluorescence rate per molecule CRM is given by ( 52)

 CRM     Ie 1 Ie / Is ( 1
)
where  is the light collection efficiency,  = krad / ktot is the quantum yield, and Is = ktot /  * 1/(1+kisc / kd) is the saturation intensity.  Ie stands for the excitation rate, where  denotes the excitation cross-section and Ie the excitation intensity. krad and knrad are the rate constants for radiative emission and non-radiative deexcitation from S1 to the ground state S0. kisc and kd are the rate constants for inter-system crossing to the dark state D and relaxation to the ground state S0 respectively. The total deexcitation rate from the excited singlet state S1 is noted ktot, and is the inverse of the excited state lifetime.

In the low excitation regime Ie << Is, Eq. ( 1) reduces to

CRMlow =  Ie , (2)
which indicates that the fluorescence rate per molecule is proportional to the fluorescence collection efficiency, the dye quantum yield and the local excitation intensity. The fluorescence enhancement F can therefore be expressed as

  F  CRMaper CRMsol      I e    krad  ktot  I e (3)
where , , Ie are the enhancements in the collection efficiency, quantum yield and excitation rate respectively (the free solution serves as a reference for enhancements calibration). krad and ktot stand for the enhancements in radiative and total decay rates. This expression highlights the complex balance between the collection, excitation and emission efficiencies needed to obtain a significant total fluorescence enhancement.

2. Experimental studies on fluorescence from nanoapertures

Two distinct sets of experimental studies have been performed, both providing information on the enhancement of fluorescence from metal nanoapertures. The first set of experiments has been performed using self-assembled monolayers of fluorescing molecules while the second set of experiments has been performed on single molecules diffusing from solution. Despite the very different measurement methodologies, both sets of experiments provide corroborating data on enhanced fluorescence in nanoapertures.

1. Studies with self-assembled monolayers

Disordered arrangements of nanoapertures can be used to approximate the properties of an individual aperture while allowing measurements to be made across a large area. When the reciprocal space of the arrangement lacks the strong isolated peaks of a periodic lattice, SPP excitation on the surface is not coherently reinforced, thereby reducing inter-aperture interactions, and allowing the interpretation of the results as an ensemble across multiple, independent, apertures.

Similar to the early work by Fischer [START_REF] Fischer | Submicrometer aperture in a thin metal film as a probe of its microenvironment through enhanced light scattering and fluorescence[END_REF], fluorescence enhancement has been studied in disordered arrangements of apertures, but with the important difference of having well-defined sizes [START_REF] Liu | Biosensing based upon molecular confinement in metallic nanocavity arrays[END_REF][START_REF] Liu | Enhanced fluorescence transduction properties of metallic nanocavity arrays[END_REF][START_REF] Mahdavi | Modeling fluorescence enhancement from metallic nanocavities[END_REF]. In these experiments, apertures of ~200nm diameter and average spacing of 1m were defined in PMMA using electron beam lithography (EBL) and dry etched into a 70nm layer of gold on a quartz substrate. The nanostructured metal films were coated on the top surfaces with a fluorescing monolayer, which consisted of avidin labeled with Cy-5 dye. Cy-5 has peak absorption near 649nm and peak emission near 670nm. A solution of 1M avidin in phosphate buffered saline (PBS, pH 7.5, containing 0.02% sodlium azide as a preservative) was labeled with Cy-5, with a ratio of Cy-5 to avidin of 0.95 [START_REF] Liu | Biosensing based upon molecular confinement in metallic nanocavity arrays[END_REF]. This solution was then diluted to the desired Cy-5 concentration (10nM for 1% dye concentration) in 1M pure avidin in PBS. The diluted solution was then used to coat the sample surface, and left for 3 hours at room temperature to allow formation of the labeled monolayer. Then, the unadsorbed species were removed by washing the samples in TE buffer, which contained 10mM Tris buffer (pH7.4) and 1 mM EDTA. Finally, the samples were dried in vacuum for 1 hour and immediately used. Given the size of an individual avidin molecule, the average distance between the fluorophore and the surface was estimated at about 5nm.

Figure 17.2 shows the experimental setup for measurement of light transmission and Cy-5 fluorescence. Detection was performed from the backside to reduce collection of fluorescence emission from the top surface. Incident light from a narrow-line tunable diode laser set to 653nm was p-polarized and attenuated by an OD 3 filter to reduce photobleaching effects during long measurements. The light was spectrally filtered by a 1.5nm width band-pass filter to suppress broad-band spontaneous emission, and passed through a chopper before being focused on the sample. A photomultiplier tube (PMT) was mounted on an adjustable-length arm attached to a computer controlled rotation stage, while the sample was mounted at the center of rotation of this stage on a manual rotation stage. With this configuration, the laser incidence angle and the transmission detection angle could be adjusted either collectively or independently. Transmission of incident laser light was measured in the zeroth-order by a photodetector. For fluorescence transmission measurements, the detector was removed and the fluorescence signal imaged through a 670nm bandpass filter onto the PMT. The PMT was connected to a lock-in amplifier, the output of which was read by a computer.

Figure 17.2:

Experimental setup for measuring fluorescence from nanoaperture arrays. Narrow-line incident light (635nm) illuminates the sample from the top side, while detection occurs through the back side, using a standard fluorescence emission filter. The sample and detector can be independently rotated.

Figure 17.3 shows the transmission and fluorescence enhancement from a disordered nanoaperture array versus incidence angle. Both quantities are nearly constant with angle, indicating minimal influence from SPP coupling as expected. In these measurements, a reference fluorescence level was obtained from a bare substrate that underwent the same chemical modifications as the nanoaperture samples. Since the illumination area and collection optics were the same, a direct ratio of fluorescence signal could be used to determine the enhancement factor. This ratio must be corrected for the fill fraction of the nanoapertures, since with backside detection, fluorescence from the top surface was negligible (as determined by measuring fluorescence from the backside of a uniform gold film with the same surface treatment), and fluorescence emission originated from within the apertures. Since the avidin monolayer covered the entire interior surface area of the apertures, the fill fraction was 7.5% (rather than 3.1% based upon the nanoaperture diameter), leading to a fluorescence enhancement factor of 7, averaged across all angles of incidence. thick gold film, with average spacing of 1m. In these measurements, the sample angle was varied with respect to the incident light, while detection occurred in a direction collinear with the incident light. Fluorescence is normalized to a quartz substrate with the same monolayer and corrected for fill fraction.

Performing the same measurements on samples where the gold surfaces had been passivated with mPEG-thiol (see section 17.4.1), leaving the subsequently selfassembled monolayers only at the bottom of the nanocavities, average fluorescence enhancement (across multiple samples) of 6.90.7 for Cy-5 has been obtained [START_REF] Mahdavi | Modeling fluorescence enhancement from metallic nanocavities[END_REF]. In this case, the fill fraction for fluorescence emission was 3.1%. What the comparison of these measures shows is that the enhancements with and without coverage of the aperture sidewalls are about the same, a conclusion that is supported by simulation results [START_REF] Mahdavi | Modeling fluorescence enhancement from metallic nanocavities[END_REF].

2. Single molecule studies

In order to understand the physical phenomena behind nanoapertureenhanced fluorescence, investigating an isolated nanoaperture is of major interest, which eliminates any effects of aperture coupling. Further, data from a single molecule may reveal information hidden by ensemble measurements, such as variances in kinetic rates, memory effects, or transient states [START_REF]Single-Molecule Detection in Solution -Methods and Applications[END_REF].

In order to get an accurate estimation of the CRM and fluorescence enhancement factor of a single nanoaperture, it is of crucial importance to simultaneously quantify the total fluorescence intensity and the number of emitters from the aperture. Fluorescence Correlation Spectroscopy (FCS) is a robust method capable of both [START_REF] Rigler | Fluorescence correlation spectroscopy, Theory and Applications[END_REF][START_REF]Single-Molecule Detection in Solution -Methods and Applications[END_REF]. FCS can in principle provide information about any molecular dynamic process on the nanosecond and longer time scale that induces a change in fluorescence intensity. For instance, fluctuations occur when molecules Experiments have also been conducted on rectangular sub-wavelength apertures milled in aluminum films [START_REF] Wenger | Single molecule fluorescence in rectangular nano-apertures[END_REF]. This shape allows the activation of two distinct modes by changing the angle between the linearly-polarized incoming electric field and the longitudinal axis of the aperture. Since the waveguide's cut-off is different for each axis, the penetration depth of the excitation light within the aperture can thus be significantly tuned, offering new possibilities to dynamically tailor the observation volume in FCS. Whereas for both polarization directions the molecular environment remained almost the same (as shown by the same fluorescence lifetime), the fluorescence rate enhancement varied strongly with the polarization direction: a high transmission of the excitation field did not yield any fluorescence enhancement whereas an evanescent coupling of the pump field induced a significant increase in the detected count rate per molecule. These results confirmed that the fluorescence enhancement found for rhodamine 6G was mainly related to the excitation near field intensity within the sub-wavelength aperture. Rhodamine 6G (Rh6G) dyes were excited at 488 nm, and their fluorescence was collected in the 510-560 nm range. Alexa Fluor 647 (A647) dyes were excited at 633 nm, and their fluorescence was collected in the 650-690 nm range. These curves show significant differences between apertures milled in gold or in aluminum, and highlight the need for a layer exhibiting a strong metallic character at the excitation wavelength to obtain enhanced fluorescence.

All the above-mentioned experiments have been conducted with a high quantum yield dye and aluminum films, which exhibits a reduced plasmonic response in the visible range. A metal supporting strong surface plasmon resonances in the visible range, such as gold, allows for even larger fluorescence enhancement factors. Two recent studies have focused on the case of Alexa 647 dye (quantum yield in water solution 30%) and nanoapertures milled in gold films [START_REF] Gérard | Nanoaperture-enhanced fluorescence: Towards higher detection rates with plasmonic metals[END_REF][START_REF] Wenger | Emission and excitation contributions to enhanced single molecule fluorescence by gold nanometric apertures[END_REF]. Nanometric apertures milled in gold exhibit significantly higher (50%) fluorescence enhancement factors than apertures in aluminium, with a maximum enhancement of 12 for a 120 nm diameter aperture in gold, as shown in Figure 17.5. This effect is related to a larger enhancement of the excitation intensity and radiative rate. Due to the intrinsic permittivity of gold and its larger skin depth than aluminum, the maximum enhancement for gold also occurs at a smaller aperture diameter, which is beneficial for single-molecule detection at high concentrations. Comparison with numerical simulations reveals that the enhancement factor is maximum when the group velocity of the guided mode inside the aperture is close to zero, that is, when the photonic density of modes is maximum. This diameter is significantly shifted (up to 50 nm) as compared to the intuitive position of the cut-off of the fundamental guided mode due in part to the skin depth of the metal. Such a significant shift has to be considered carefully while designing nanoapertures for high-efficiency single-molecule analysis. In a second contribution [START_REF] Wenger | Emission and excitation contributions to enhanced single molecule fluorescence by gold nanometric apertures[END_REF], the respective roles of excitation and emission in the enhanced fluorescence have been determined for a broad range of nanoapertures with diameters from 80 to 310 nm. For this purpose, a specific fluorescence characterization procedure combining FCS with fluorescence lifetime measurements has been developed, with results presented in Figure 17.7. This data allows discrimination among different physical effects and relation of photokinetic enhancements to the local photonic density of states; for example, the increase in radiative rate at saturation excitation power, as shown in Figure 17.8. For aperture diameters above 175 nm (at 633 nm excitation with gold films), the photokinetic rates increase as the aperture diameter is decreased. This region corresponds to a propagative excitation field. As the aperture diameter is decreased, more electromagnetic confinement is obtained at the aperture entrance, which translates into an increase in the emission enhancement along with the excitation enhancement. On the other hand, for aperture diameters below 100 nm, a decrease in the fluorescence enhancement is observed as the aperture diameter is reduced. Diameters below 100 nm lead to strongly evanescent fields inside the aperture and large propagative losses, which contributes to the reduction in emission and excitation enhancement. Lastly, diameters between 100 and 175 nm appear as a trade-off between the two extreme cases. This region is close to the position where the group velocity is minimum, leading to a maximum of the photonic density of states. Moreover, the metal-dielectric interface set by the aperture may allow fluorescence energy transferred to a localized surface plasmon to be coupled out into the radiated field at the aperture rim, contributing to the emission. Combining FCS with fluorescence lifetime measurements turns out to be very efficient in resolving the contributions of excitation and emission to the overall fluorescence process. This procedure can be straightforwardly extended to other types of plasmonic nanostructures, as long as the observation volume is well defined. The limit of this method is that all the presented results account for spatial averaging over all the possible molecular orientations and positions inside the analyzed volume. There is no sensitivity to individual molecular trajectories or dipole orientations, but one ends up directly with global figures to characterize the emitted fluorescence. Besides, distinguishing between the contributions of the radiative rate and the collection efficiency remains a challenge, mainly because of the intrinsic difficulty to reliably measure collection efficiency. Lastly, the fluorescence enhancement factors are spectrally averaged within the fluorescence bandpass detection window. However, further investigations can provide some additional knowledge on these last two points, as we will discuss hereafter. To address the question of spectral averaging within the fluorescence bandpass filters, fluorescence spectra were taken from a single gold nanoaperture (30) using a spectrograph with nitrogen-cooled CCD detector. The parallel use of FCS quantifies the number of detected molecules per experimental run, which allows the a posteriori normalization of the raw fluorescence spectrum, and the calculation of the fluorescence spectrum per single molecule. Experiments performed on Alexa 647 molecules excited at 633 nm revealed that the detected count rate per molecule was significantly enhanced over the whole emission spectral range, with a maximum enhancement for 120 nm diameter apertures, as observed in FCS. The shapes of the spectra do not change noticeably between the different apertures and the free solution. The same behavior has been observed for apertures in aluminium, and for Alexa 680 dyes. In the case of single nanoapertures, we point out that white light transmission measurements through a single aperture of diameter 120-300 nm yielded transmission curves almost flat over the 650-710 nm spectral range covered by the dye emission. This range, therefore, seems too narrow to monitor any clear spectral link between white light transmission and fluorescence.

3. Simulation results

Rigorous electromagnetic computations of light diffraction in a cylindrical geometry confirmed the excitation intensity enhancement observed experimentally [START_REF] Popov | Field enhancement in single subwavelength apertures[END_REF]. At low excitation powers (well below saturation), the fluorescence enhancement was linked to the appearance of the fundamental mode propagating inside the aperture. The cutoff condition led to modes with a low group velocity, and to an increased local density of states allowing a higher excitation intensity as compared to a diffraction-limited beam. These results are verified by the computational studies presented in this section.

A generalized computational geometry for a single aperture is illustrated in Figure 17.9; the size of the computational space is 1.0×1.0×1.1μm, with scattering boundary conditions on all six faces. Electromagnetic computations were performed using COMSOL Multiphysics version 3.4. A quartz substrate is assumed, on top of which a 100nm or 150nm thick layer of gold or aluminum is placed; the upper region is air or water. Dielectric properties of gold and aluminum are incorporated via the complex dielectric constant, as measured by spectroscopic ellipsometry from 300 to 1,600nm. A single aperture is placed in the metal layer. As an illustration of the fluorescence excitation and emission enhancement mechanisms within an aperture, Figure 17.10 plots the intensity distribution (sum of the intensities of all three field components) in the xz cross-section of a gold nanoaperture of 125nm diameter in water and 100nm thickness upon excitation by an x-polarized plane wave at 635nm wavelength originating from the substrate side (left half) and the enhancement of emission from a dipole (at 670nm wavelength, averaged across all three dipole orientations) located within the nanoaperture, as measured at a plane just below the metal/substrate interface (right half). In the excitation map, there are two contributions. The first is a component of relatively uniform lateral intensity, but exponential decay through the depth of the aperture; this is the fundamental aperture mode in cutoff. The second component is the localized surface plasmon (LSP) resonances, which are most prominent at the terminal regions of the aperture (29) and along the direction of the incident polarization. These resonances are analogous to LSP resonance modes of metal nanoparticles, and are most pronounced at the incidence side. They also bear strong connection with the electromagnetic concentration at the apex of a metallic tip. Similar contributions are present for dipole emission. Emission to the far field is enhanced the most when the dipole is located near the measurement plane, and especially, near the aperture rims where LSP resonances occur. As the dipole moves away from the substrate, the emission decays exponentially. The excitation and emission enhancement characteristics of nanoapertures will next be discussed in more detail.

1. Excitation enhancement

In the calculations of this section, a plane wave (at a specific wavelength) is launched 500nm from below the aperture. Average excitation enhancement is calculated by integrating the total intensity within a defined volume of an aperture (a 10nm thick cross-section slice, as illustrated in Figure 17.9) and dividing by the integrated intensity within the same volume, but in the absence of the metal layer. Figure 17.11 plots the averaged intensity within these cross-section volumes for aluminum and gold nanoapertures of 150nm thickness and varying diameters. In these simulations, the excitation light (at 488, 532, or 635nm) originates from the substrate side, and the upper region is air. Even though the plasmonic resonance dominates the cross-section intensity map, as shown in Figure 17.10, it only occupies a small region of the aperture, and does not significantly affect the enhancement averaged within the 10nm thick volumes. This is shown in xy cross-section for a 125nm diameter gold nanoaperture in Figure 17.12, where the cross-section is taken 5nm above the substrate.

Numerous observations, consistent with experimental results, can be made from the calculations. For example, as the wavelength increases, in general, the aperture diameter providing the maximum enhancement increases. Further, the intensity decreases exponentially with distance away from the substrate; for the larger ratios of diameter to wavelength, the decay constant is clearly reduced, indicating non-cutoff conditions associated with a dramatic reduction in intensity enhancement.

Aluminum apertures provide enhancement over the visible spectrum, whereas gold drops dramatically in the green and blue (compare to Figure 17.5). Nevertheless, in the red, gold provides nearly twice the enhancement of aluminum, as expected [START_REF] Gérard | Nanoaperture-enhanced fluorescence: Towards higher detection rates with plasmonic metals[END_REF]. For aluminum apertures, the optimal aperture diameter for 488nm excitation is in the 100-150nm range, and for 635nm illumination, the optimal aperture diameter is near 175nm; these results are consistent with Figure 17.5. It is interesting that for small aluminum apertures (75nm diameter) and red excitation, no enhancement is expected. This is consistent with the original Cornell results [START_REF] Levene | Zero-mode waveguides for single-molecule analysis at high concentrations[END_REF]. Similarly, for the gold aperture calculations, the enhancement is relatively flat with diameter for 488nm excitation, as expected from Figure 17.5, and less than a factor of 2, while a slight peak in enhancement is obtained for smaller aperture diameters under 532nm illumination. Under 635nm excitation, the optimal aperture diameter is near 125nm, also expected from the experimental results. For high quantum efficiency dyes, such as Rh6G, excitation enhancement will be the dominant mechanism of increased fluorescence count rate in the nonsaturating regime according to Eq. 3. The calculated results of Figure 17.11 have good quantitative agreement with measurements on enhanced fluorescence from Rh6G under 488nm illumination. Lower quantum efficiency dyes, such as Cy-5/Alexa647 for example, can also experience significant increase in quantum yield.

2. Emission

The emission properties of a fluorophore in a single nanoaperture have been theoretically studied [START_REF] Liu | Enhanced fluorescence transduction properties of metallic nanocavity arrays[END_REF][START_REF] Mahdavi | Modeling fluorescence enhancement from metallic nanocavities[END_REF]. These calculations do not depend on the excitation mechanism, and are therefore valid for chemiluminescence, electroluminescence, and photoluminescence, making the conclusions relevant to a number of biotechnology applications.

Dipole emission is a rather more complicated process to simulate than excitation, so some simplifications will be made here. The calculations presented here follow that of reference [START_REF] Mahdavi | Modeling fluorescence enhancement from metallic nanocavities[END_REF], in which the increase in radiative output of a dipole lying within a nanoaperture, as measured in a plane just below the interface with the substrate, is determined. The enhancement is calculated by taking the ratio of the power flow through this plane divided by the power flow through this same plane, but in the absence of the metal, for all three dipole orientations. This calculated enhancement corresponds approximately to the measured fluorescence enhancement in the saturation regime under strong excitation due to modification of the radiative rate and directionality -krad. The more general case of estimating total fluorescence enhancement in the non-saturating regime, where the quantum efficiency of a given fluorophore needs to be determined, is deferred to a later publication.

Figure 17.13 plots the emission enhancement averaged over the 15 10nm thick volumes of 150nm tall apertures. Within each volume, dipoles are placed uniformly along the radius in 10nm increments; smaller increments are used near the edge. The averaged enhancement within each volume is a weighted average of the enhancement from each of these dipole locations, weighted by r 2 , where r is the distance from the aperture axis. There are some similarities between these results and the excitation enhancement results. In particular, the enhancement for gold is significantly greater than for aluminum, even at 570nm. Further, the diameter for maximum enhancement increases with the emission wavelength, with the diameters roughly corresponding to the optimal diameters in the excitation cases at the same wavelengths. The LSP modes also play a role in the emission process. This is evidenced in the plots by the peaks in emission enhancement in V1 and V15, leading to nonexponential decay throughout the aperture. The effect at V1 is strongest for the larger aperture diameters because of the reduced attenuation in propagation through the apertures. The LSP resonances are more pronounced in emission than in excitation. The difference is that any dipole positioned near the rims of the aperture interacts with the LSP resonance. In other words, the LSP enhancement occurs at any location near the rims, and therefore provides a greater contribution to the weighted-average enhancement.

Comparing to Figure 17.8, good agreement is obtained with the calculations. Calculations predict that the greatest change in radiative rate for a red dye (such as A647) occurs for a gold nanoaperture of 125-150nm diameter. Looking at the enhancement values calculated for V1, enhancements in the range 6-7 are anticipated, which is also agrees well with the experimental data.

3. Radiation pattern

An open question is to what extent a nanoaperture modifies the radiation pattern of emitters placed inside it. Any beaming effect towards the detectors would increase the molecular detection efficiency and, thus, the enhancement factors. According to recent calculations [START_REF] Popov | Single-scattering theory of light diffraction by a circular subwavelength aperture in a finitely conducting screen[END_REF], the far-field directivity of the beam transmitted through a single sub-wavelength aperture is highly dependent on the hole size and on the permittivity of the material surrounding the aperture. From these calculations, the directivity of the beam tends to increase when the modulus of the permittivity decreases and when the aperture size increases. For instance, gold has a lower permittivity than aluminum, and should therefore provide a better directivity. Yet, to yield similar fluorescence enhancements, apertures in gold have sizes much lower than with aluminum. Altogether, the competition between these two effects leads to a similar far-field directivity for both metals. Experimentally, no beaming effect could be detected within the collection half-angle of 64° with neither gold nor aluminum from single aperture studies [START_REF] Gérard | Nanoaperture-enhanced fluorescence: Towards higher detection rates with plasmonic metals[END_REF]. Further, experimental results of the next section, where far-field radiation patterns from aperture arrays are shown, do not provide conclusive evidence of beaming effects either. Therefore, in the absence of detailed computational or experimental study of dipole radiation patterns from nanoapertures, any effect of directionality on measured fluorescence enhancement cannot be quantified at this time. However, it should be noted that the calculations of the previous section intrinsically incorporated directionality effects, under the assumption that detection is performed across a plane.

APERTURE ARRAYS AND STRUCTURED APERTURES

The extraordinary optical transmission (EOT) phenomenon discussed in Section 2 is generally attributed to coupling into surface plasmon polariton (SPP) modes of the metal film, mediated by the periodic arrangement of nanoapertures. The explanation of the extraordinary transmission through an array of nanoapertures can be built upon the properties of individual nanoapertures -the entire system can be viewed as an array of coupled resonators, where the coupling in general occurs through surface waves, which, under the proper phase matching conditions, are in fact the SPP waves.

The periodic arrangement adds two additional fluorescence enhancement mechanisms -increased excitation intensity due to SPP excitation [START_REF] Liu | Fluorescence enhancement from an array of subwavelength metal apertures[END_REF] and modification of the radiation emission pattern. The latter effect has been referred to as ``beaming'' [START_REF] Lezec | Beaming light from a subwavelength aperture[END_REF], or more appropriately for fluorescence, surface plasmon coupled emission (SPCE) [START_REF] Lakowicz | Radiative decay engineering 3: Surface plasmon-coupled directional emission[END_REF], and results from the coupling of multiple nanocavities via the interstitial surface regions.

In the 2-D periodic metallic structure, coupling between radiation modes and surface plasmons is described by

 k t  nK ˆ x  mK ˆ y  k sp ( 4
)
where n and m are integers. Because we use a square lattice, the coupling angles along the x and y axes are identical. The dispersion relation for a surface plasmon polariton (SPP) at a planar interface between a metal and a dielectric is given by

 k sp   c  m  d  m   d ( 5
)
where m and d are the dielectric constants of the metal and dielectric material, respectively. Note that, due to the periodicity of an aperture array, the dispersion relation of equation 5 is modified by the details of the aperture size, shape, and spacing (58, 59, 60).

1. Experiments

In these experiments, periodic 2-D arrays of nanoapertures were produced in gold films using e-beam lithography and dry etching, as shown in the inset image of Figure 17.2. The underlying substrate for these structures is quartz. The films were coated on the top surfaces with a fluorescing monolayer, consisting of avidin labeled with Cy-5 dye, as before. The experimental setup of Figure 17.2 was used, which allowed measurements of fluorescence versus incidence angle and the radiation emission pattern.

As has already been established for fluorescence emission from individual nanoapertures, it is advantageous to perform detection from the backside of the substrate in order to minimize the contribution of background fluorescence. For experimental simplicity, all measurements were made in the trans-illumination geometry.

1. Effects of EOT

The EOT phenomenon in arrays of nanoapertures is the result of light coupling through the apertures. That being the case, it can reasonably be expected that increased light intensity within the apertures results. The first evidence that light transmitted directly through the apertures was obtained from near-field scanning optical microscopy across the backside of a sample [START_REF] Thio | Plasmon-enhanced transmission through hole arrays in Cr films[END_REF], which clearly indicated localization of light at the apertures. Evidence of intensity enhancement within the apertures was provided in 2003 (34) using fluorophores as local intensity probes. In these measurements, aperture diameters of 100nm and 150nm were used, with spacings of 1m and 750nm in 70nm thick gold films. In all cases, fluorescence intensity reached its maximum value under the conditions of EOT. Furthermore, even under non-EOT conditions, enhanced fluorescence was obtained, presumably owing to the enhancement effects of the individual apertures.

Figure 17.14 plots transmission and fluorescence emission versus incidence angle for an array of ~200nm diameter apertures with 1m spacing in 70nm thick gold (fluorescence from a disordered array, Figure 17.3, is also plotted for comparison) [START_REF] Liu | Biosensing based upon molecular confinement in metallic nanocavity arrays[END_REF].

The fluorescence curves have been normalized against fluorescence from a bare glass substrate and corrected for the fill fraction of the apertures, which is 7.5% (including the bottom of the aperture and the sidewalls). It is clear that the fluorescence curve follows transmission. The first transmission peak occurs at an incidence angle of 25°, corresponding to the excitation of the (1,0) surface-plasmon mode at the metal-air interface; the location of this peak can be shifted (for a given wavelength) by changing the aperture spacing. The fluorescence also peaks at this angle, suggesting a corresponding increase in excitation intensity. On either side of this peak is evidence of suppression in the excitation light, where the fluorescence levels drop below that of a disordered array (which approximately represents the fluorescence enhancement from an individual aperture). thick gold film, with spacing of 1m. In these measurements, the sample angle was varied with respect to the incident light, while detection occurred from the backside in a direction collinear with the incident light. Fluorescence from a disordered array is also plotted for reference. Fluorescence is normalized to a quartz slide with the same monolayer, and corrected for fill fraction.

In these experiments presented so far, and in related experiments by others [START_REF] Brolo | Enhanced fluorescence from arrays of nanoholes in a gold film[END_REF][START_REF] Brolo | Surface plasmon-quantum dot coupling from arrays of nanoholes[END_REF][START_REF] Kim | Laser-induced fluorescence within subwavelength metallic arrays of nanoholes indicating minimal dependence on hole periodicity[END_REF], all of the samples had fluorophores coating the entire surface, not just within the nanoapertures. The question then arises as to what components of fluorescence emission are detected through the backside.

2 SPCE

It is well established that fluorophores located near a metal surface can directly transfer energy from their excited state to surface plasmon modes [START_REF] Weber | Energy transfer from an excited dye molecule to the surface plasmons of an adjacent metal[END_REF]. When the metal has a surface texture, these surface plasmon modes can directionally scatter to the far field [START_REF] Kitson | Surface plasmon energy gaps and photoluminescence[END_REF]. This effect has been most widely studied in reflection, but it is known that it can occur in transmission as well [START_REF] Gruhlke | Surface-plasmon cross coupling in molecular fluorescence near a corrugated thin metal film[END_REF]. SPCE in transmission has been studied in arrays of sub-wavelength apertures such that the magnitude of this component can be compared to the direct emission from the apertures [START_REF] Liu | Fluorescence transmission through 1-D and 2-D periodic metal films[END_REF]. The structures used in the experimental results reported here were 2-D periodic arrays of apertures in 70nm thick gold film, with aperture spacings of 1m and aperture diameters of 200nm [START_REF] Liu | Biosensing based upon molecular confinement in metallic nanocavity arrays[END_REF].

The measurement of fluorescence emission from the 2-D patterns is shown in Figure 17.15. In this measurement, the sample is fixed at normal incidence to the excitation light and the detector is scanned. Because of the symmetry of diffraction from the 2-D structure, the fluorescence emission pattern was only measured for angles along the x axis. The emission pattern exhibits a broad background with isolated peaks at angles corresponding to the calculated scattering angles using equation ( 4). When measured through an s-oriented analyzer, the emission pattern consists only of the broad background, as the p-polarized SPCE is blocked [START_REF] Liu | Fluorescence transmission through 1-D and 2-D periodic metal films[END_REF]. The broad background pattern in the measurements is therefore due to direct fluorescence emission from the apertures (in addition to a much weaker contribution due to the direct transmission of fluorescence through the metal), which is the dominant transmission mechanism. Emission from the apertures is incoherent in that there is no correlation from one aperture to another; the collective emission pattern is therefore representative of the emission from a single aperture under far-field measurement. For reference, the fluorescence emission pattern from a glass substrate is also plotted in Figure 17.15. This pattern is similar to that obtained from the nanoapertures. One might be tempted to conclude that the emission pattern is slightly more directional from the glass surface than from the nanoapertures. However, more thorough experimental study should be performed before drawing any such conclusions.

3. Eliminating SPCE

Even though the SPCE component is weak upon backside detection, it can be reduced by using backside illumination and/or controlling the fluorescence collection angle [START_REF] Liu | Biosensing based upon molecular confinement in metallic nanocavity arrays[END_REF]. For biotechnology applications, it is desirable to prevent molecular capture on the surface since transduction of these molecules would be less efficient than the transduction of molecules bound within the nanoapertures. This can be accomplished by chemical passivation of the top surface, which minimizes subsequent binding [START_REF] Liu | Biosensing based upon molecular confinement in metallic nanocavity arrays[END_REF]. An illustration of passivated versus non-passivated structures is shown in Figure 17.16. In the passivation procedure for gold (note that a passivation procedure for aluminum has recently been demonstrated ( 67)), mPEG-thiol in powdered form is dissolved in ethanol to a concentration of 1μM. The solution is applied to the samples for 24h in a nitrogen glovebox at room temperature and atmospheric pressure, then rinsed in ethanol and dried under a nitrogen gun. The labeled avidin solution is then applied as before. After this procedure, a reference gold surface did not produce any measurable fluorescence, while a reference quartz surface produced a fluorescence signal roughly 85% of the level of a coated quartz surface without passivation (which is probably the result of a reduction in bound surface concentration).

The unpolarized fluorescence emission pattern from the passivated nanoaperture array is shown in Figure 17.17. The absence of the distinct fluorescence peaks (as compared to Figure 17.15) demonstrates that the SPCE component is eliminated through prevention of immobilization of Cy-5/avidin on the top surface.

Again, the emission pattern from a glass substrate is also shown for reference. Figure 17.18 shows the fluorescence output as a function of incidence angle for the passivated sample. At the surface-plasmon incidence angle, the total fluorescence enhancement compared to the reference is 12 (normalized to the 3.1% fill-fraction of the bottom surface of the nanoapertures), which is comparable to the enhancement obtained under full interior surface coverage (Figure 17.14). Therefore, the fluorescence enhancement (per unit area) is comparable for fluorophores on the bottom as for fluorophores on the sidewalls with backside detection, as before with individual apertures. As a conclusion to the experimental results, a more complete study of fluorescence enhancement from nanoaperture arrays was recently reported [START_REF] Mahdavi | Modeling fluorescence enhancement from metallic nanocavities[END_REF], where measurements were made across multiple samples. From these results, an enhancement factor of 12±1 was obtained using the same sample geometries described above.

2. Simulations

Considerable effort has been devoted to modeling the optical transmission through arrays of nanoapertures in order to maximize transmission; this work will not be further reviewed here. What is of interest is how the array modifies the enhancement mechanisms obtained from a single aperture, a topic which has received comparably little attention [START_REF] Salomon | Distribution of optical transmission of periodic subwavelength holes in a metal film[END_REF]. As a result, only a brief overview of array effects will be presented here, in anticipation that more comprehensive studies will be performed in the future.

We presently make the assumption that the emission of a molecule within an aperture is not significantly affected by the presence of neighboring apertures in an array. Two conditions must hold in order for this assumption to be valid: 1) the apertures are sufficiently separated such that the dipole-like LSP modes, into which molecular emission can be coupled, do not overlap, and 2) coupling of molecular emission from the apertures into surface waves ( 19) is weak; clearly, both effects will have an exponential-like decay with aperture separation. There is some experimental evidence that supports these assumptions. For example, if efficient aperture coupling occurred in emission (where the top surface is passivated and molecules are localized within the apertures), then distinct scattering peaks would be observed in Figure 17.18 at angles corresponding to the dispersion relation of SPP waves at the metalsubstrate interface. No such peaks are apparent. It is possible that the aperture spacing in these experiments was too large for efficient coupling to occur. Further, a recent study suggested the fluorescence emission from an aperture array is dominated by the aperture [START_REF] Kim | Laser-induced fluorescence within subwavelength metallic arrays of nanoholes indicating minimal dependence on hole periodicity[END_REF]. Nevertheless, more investigation needs to be performed.

We therefore assume that the major effect of the array is in increased collection of light within the apertures. In the simulation results of this section, we only consider briefly the excitation component to enhanced fluorescence emission. As already established by experiments, the light intensity within periodic arrangements of nanoapertures increases in association with EOT and is a result of aperture coupling effects via surface waves under external excitation. This coupling therefore modulates the response obtained for an isolated aperture. Simulations were performed in the same manner as described before, except for the fact that periodic boundary conditions were used along the sides of the simulation space boundary.

Figure 17.19 plots the intensity enhancement averaged over the lower 10nm volume of the nanoaperture (i.e. V1) for the cases of a single aperture and an array, with illumination from the substrate side. For these calculations, 150nm apertures in a 100nm thick gold layer were considered. For the array, the aperture spacings are 400nm, 500nm and 600nm. It is clear from the calculation that the array produces strong modulation of the intensity enhancement of a single aperture, where significantly greater enhancement can be obtained. Further, the locations of the enhancement peaks can be shifted based upon the aperture spacing, with greater enhancements occurring in the red part of the spectrum (where the enhancement line shapes resemble Fano-type resonances). In the blue/green part of the spectrum, the enhancement drops dramatically owing to the dielectric properties of gold. To our knowledge, detailed optimizations of excitation enhancement with aperture arrays have not yet been performed, so this is an area of further study.

3. Structured apertures

In addition to nanoaperture arrays, structured apertures could provide higher fluorescence rate enhancement than single apertures, while still enabling efficient signal-to-background discrimination. Examples include the bull's eye structures [START_REF] Lezec | Beaming light from a subwavelength aperture[END_REF][START_REF] Popov | Surface plasmon excitation on a single subwavelength hole in a metal sheet[END_REF], nanopockets [START_REF] Liu | Fluorescence enhancement of quantum dots enclosed in Au nanopockets with subwavelength aperture[END_REF] or combined slit and aperture [START_REF] Liu | Nanoillumination based on selffocus and field enhancement inside a subwavelength metallic structure[END_REF].

As already mentioned for aperture arrays, the periodicity of the structure can lead to significant local field enhancement by resonantly exciting surface plasmons. To translate this resonance effect to an isolated aperture, the metal surface surrounding the aperture can be structured in a periodic manner in order to efficiently excite the SPP. Most designs use concentric grooves around a central nanoaperture, which is called "bull's eye aperture" [START_REF] Lezec | Beaming light from a subwavelength aperture[END_REF][START_REF] Garcia-Vidal | Focusing light with a single subwavelength aperture flanked by surface corrugations[END_REF][START_REF] Martin-Moreno | Theory of highly directional emission from a single subwavelength aperture surrounded by surface corrugations[END_REF].

When the excitation light hits the grooves at resonance, extra focusing is obtained, which gives rise to an enhanced electromagnetic field [START_REF] Yuan | Field enhancement of nano-sized metal aperture with and without surrounding corrugations through resonant surface plasmons[END_REF][START_REF] Shuford | Electric field enhancement and light transmission in cylindrical nanoholes[END_REF]. This phenomenon has been first demonstrated for enhanced optical transmission trough a single (structured) nanoaperture [START_REF] Lezec | Beaming light from a subwavelength aperture[END_REF]. More recently, enhanced second harmonic generation [START_REF] Nahata | Enhanced nonlinear optical conversion from a periodically nanostructured metal film[END_REF] was obtained and ultra-compact silicon detectors have been demonstrated [START_REF] Ishi | Si nanophotodiode with a surface plasmon antenna[END_REF]. Very elegantly, the bull's eye antenna structure compensates for the reduced active nanoaperture area. This opens promising opportunities for advanced optoelectronics devices assisted by plasmonics.

When the output surface surrounding the aperture is corrugated, a narrow beam can be generated, with a divergence of a few degrees [START_REF] Lezec | Beaming light from a subwavelength aperture[END_REF]. The backside grooves are used to scatter the surface waves into freely propagating light, which contributes to the total detected intensity. In addition, interference between the scattered light and the light emerging from the hole generates a low-divergence beam, which can be more efficiently collected. However, this process has not been studied in the context of dipole emission from a structured aperture.

From the point of view of nanoaperture-enhanced fluorescence, molecules are only sensitive to the local excitation intensity. Therefore, the effective excitation intensity is the critical parameter that has to be considered for practical applications. With the bull's eye aperture surrounded by concentric grooves, both the surface structure and the incident wave have to cover a diameter of about 4 times the wavelength. This dramatically limits the incoming intensity (i.e. constant power distributed across a larger surface area). Therefore, even with large enhancements, the excitation intensity in the central aperture may not be significantly higher that the intensity reached with a tightly focused laser beam without the nanostructure.

In the case of a bull's eye structure, the relative electromagnetic enhancement is about 100 (computed for an incoming plane wave and consistent with experiment (72)), but the structure radius and thus the laser beam focus spot are about 4 times the diffraction limit (minimum focus size). If we compare the relative intensity enhancement to the case of a diffraction-limited spot, the intensity with the bull's eye is increased by 100x(1/4)² = 6.25, which is about the enhancement with a single (bare) aperture with a tightly focused beam. Of course, this argument has to be supplemented with the influence the emission rate and the directivity of the fluorescence beam, which contribute to the overall enhanced fluorescence. However, the situation is better with arrays of structures, where uniform illumination would be used.

Recently, the use of a single nanoaperture surrounded by a single channel groove was suggested [START_REF] Bonod | Field enhancement in a circular aperture surrounded by a single channel groove[END_REF]. Such a single channel is much simpler to fabricate than the largerarea bull's eye, and is shown in Figure 17.20. Numerical analysis of diffraction shows the possibility of a 50-fold increase of the electric field intensity inside the central aperture, when compared to the incident field. The simultaneous excitation of cavity modes in the central aperture and in the surrounding coaxial channel can lead to almost 50-fold increase in the electric field intensity in the central aperture, compared to the intensity of the incident field. The coupling between the two cavity modes in the central aperture and in the surrounding coaxial channel is made through the surface plasmon wave that propagates along the metal-cladding interface. This coupling is the strongest when the field of the channel cavity mode matches the field of the plasmon surface wave, i.e, when the channel modes are below their cut-off.

APPLICATIONS

This review of nanoaperture enhanced fluorescence reveals some unique properties of nanoapertures useful for biotechnology applications. The primary advantage is in a highly localized observation volume, resulting in the reduction of background signal from sources lying outside the apertures. Related to this is a welldefined analysis volume, where localization of chemical reactions can be obtained.

1. Enhanced single molecule analysis in solution

A central issue in single-molecule analysis is to discriminate the fluctuating signal to the background noise, which mainly originates from laser-induced Rayleigh and Raman scattering, and from the electronic noise of the photon-counting detectors. To increase the signal-to-background ratio, a first strategy is to maximize the collected signal by using a high numerical aperture (NA) objective and naturally bright fluorophores. A second strategy is to reduce the background by rejecting the scattered light with high-quality optical filters (spectral filtering) and by restricting the detection volume (spatial filtering). Therefore, the common approach uses a confocal microscope with a high NA objective, which provides detection volumes of about 0.5 fL (= 0.5 µm 3 ). This limits the concentration for single-molecule analysis to a few nM. For artificial environments, this low concentration may not be a problem; however, it may be a crucial limitation for biological and biochemical studies which typically involves concentrations in the M to the mM range [START_REF] Levene | Zero-mode waveguides for single-molecule analysis at high concentrations[END_REF]. Many enzymatic reactions are also naturally effective at ligand concentrations in this range. Arbitrarily reducing the ligand concentration may lead to chemical pathway alteration and artifacts. Performing FCS at biologically more relevant concentrations requires the development of analysis volumes with nanometric dimensions, yielding a volume reduction by at least three orders of magnitude as compared to standard confocal microscopy.

To further improve the FCS technique and go beyond confocal microscopy, the use of micro-and nanostructures is of major interest. By tailoring the molecules environment, photonic structures can enhance the fluorescence emission and collection efficiencies. This simultaneously increases the molecular brightness and reduces the detection volume, leading to a lower amount of background scattered light, and to a higher concentration available for single-molecule studies, as shown in Figure 17.6. Several methods allowing a simultaneous decrease of the detection volume and increase of the fluorescence signal have been proposed so far, and are reviewed in [START_REF] Blom | Fluorescence fluctuation spectroscopy in reduced detection volumes[END_REF].

In spite of their conceptual simplicity, nanoapertures possess appealing properties to increase the effectiveness of fluorescence-based single-molecule detection, such as localization of excitation light, strong isolation from emission produced by unbound species, and an increase in apparent absorption and emission yield [START_REF] Craighead | Future lab-on-a-chip technologies for interrogating individual molecules[END_REF][START_REF] Mannion | Nanofluidic structures for single biomolecule fluorescent detection[END_REF][START_REF] Genet | Light in tiny holes[END_REF]. The significant fluorescence increase obtained for small-radius apertures appears especially interesting because it allows for the possibility to significantly reduce the observation volume while still detecting a sufficient signal. This yields an efficient signal-to-background discrimination, even with attoliter volumes and single molecule resolution. Using nanoapertures, a large range of biologic processes have been efficiently monitored with single molecule resolution at micromolar concentrations [START_REF] Levene | Zero-mode waveguides for single-molecule analysis at high concentrations[END_REF][START_REF] Samiee | Lambda repressor oligomerization kinetics at high concentrations using fluorescence correlation spectroscopy in zero-mode waveguides[END_REF][START_REF] Leutenegger | Confining the sampling volume for Fluorescence Correlation Spectroscopy using a sub-wavelength sized aperture[END_REF][START_REF] Wenger | Dual-color fluorescence crosscorrelation spectroscopy in a single nanoaperture : towards rapid multicomponent screening at high concentrations[END_REF].

Moreover, the studies on nanoaperture-enhanced fluorescence point out that for a properly tailored aperture, count rates per molecule greater than a few hundred thousands photons per second were readily obtained, whereas for a single molecule in open solution, fluorescence saturation prevents the count rate from exceeding a few tens of kilocounts per second. This allows for fast and reliable screening for single molecules.

We point out that for experiments conducted on ultra-small structures (diameters typically below 70 nm), the signal to noise ratio comes close to one, as a consequence of different effects. First, the fluorescence emission is strongly affected by the metal in the close vicinity of the structure, leading to a decrease in fluorescence enhancement [START_REF] Rigneault | Enhancement of single-molecule fluorescence detection in subwavelength apertures[END_REF][START_REF] Mahdavi | Modeling fluorescence enhancement from metallic nanocavities[END_REF]. Second, the background noise becomes then significant, due to the metal auto-fluorescence and the large pool of dyes above the aperture that induce some fluorescence leakage back to the detector. It should be noted that the weakly-fluctuating nature of the background still enables a discrimination against the signal, even if it tends to decrease the amplitude of the FCS correlation curve. Therefore, detecting the presence of a millisecond-scale enzymatic reaction event or a microsecond-scale diffusion event is always possible even for a low signal-to-noise ratio, which is a general property of FCS.

2. Real time single molecule DNA sequencing

Performing high-throughput, high-accuracy DNA sequencing at low costs has become a major issue, largely attracted by the growing potential of quantitative genomics. For this cutting edge application, nanometric apertures offer specific advantages as they enable the observation of single fluorophores against a dense background by maintaining a high signal-to-noise ratio. Moreover, nanoapertures can be operated in massive parallelism, allowing for high-speed DNA analysis.

This concept has been developed since 2004 by Pacific Biosciences, a private company formed by researchers at Cornell University. Each nanoaperture forms a nano-observation chamber for watching DNA polymerase as it performs sequencing by synthesis (Figure 17.21). Within each chamber, a single DNA polymerase molecule is attached to the bottom surface so that it persists in the detection volume. Fluorescently labeled nucleotides diffuse into the reaction solution at high concentrations to promote enzyme speed and accuracy. When the DNA polymerase encounters the nucleotide complementary to the next base in the template, it is incorporated into the duplicated DNA chain. During the incorporation process, the enzyme holds the nucleotide in the detection volume for a few tens of milliseconds, much longer than the average residence time of the diffusing nucleotides. This causes a millisecond fluorescence flash to occur, which is detected by the collection system. As each nucleotide (A, T, G, C) bears a different color marker, the spectral analysis of the fluorescence flash allows to discriminate the incorporated nucleotide, which constructs the DNA sequence step by steps. Then, as part of the natural incorporation cycle, the polymerase cleaves the bond holding the fluorophore in place and the dye diffuses out of the detection volume. This ensures that the duplicated DNA strand is purely natural, i.e. it doesn't bear any fluorescing material. Following incorporation, the signal quickly returns to the baseline. Then, the polymerase advances to the next base and the process continues to repeat, adding nucleotides one by one. DNA sequencing is observed as it occurs in real-time across thousands of nanoapertures that can be operated simultaneously. Acting uninterrupted, the DNA polymerase keeps on incorporating bases at a speed of tens per second. Researchers at Pacific Biosciences demonstrated that DNA strands thousands of nucleotides in length could be accurately sequenced.

This sequencing has a wide range of applications, yet many technological issues have to be addressed. These mostly deal with polymerase enzymatic activity and large scale nanoaperture detection. Selective aluminum passivation for targeted immobilization of single DNA polymerase molecules in nanoapertures is described in [START_REF] Korlach | Selective aluminum passivation for targeted immobilization of single DNA polymerase molecules in zero-mode waveguide nanostructures[END_REF]. Improved fabrication techniques of nanoapertures on a large scale wafer are described in [START_REF] Foquet | Improved fabrication of zeromode waveguides for single-molecule detection[END_REF].

3. Sub-diffraction diffusion analysis within lipidic membranes

Revealing the dynamic organization of the cell plasma membrane at the submicron level is a challenging task. On one hand, electron microscopy resolves nanometric features, and yet it cannot be easily applied to live cells under physiological conditions. On the other hand, standard optical microscopy unravels details of live cell membranes, but is limited to a resolution of about 200 nm. Therefore, new methods providing dynamic information on lipids and proteins in live cell membranes with nanometric resolution are required.

The first proof-of-principle towards the use of nanometric apertures to probe the plasma membrane of a live cell on a sub-diffraction area was reported in 2005 [START_REF] Edel | High spatial resolution observation of single molecule dynamics in living cell membranes[END_REF]. A single aperture acts as a pinhole directly located under the cell membrane and restricts the observation area below the diffraction limit. Recently, cell membrane invagination within the nanostructure was shown to be highly dependent upon actin filaments but not on microtubules [START_REF] Moran-Mirabal | Cell investigation of nanostructures: zero-mode waveguides for plasma membrane studies with single molecule resolution[END_REF]. The same concept also works for lipid bilayers, which are especially relevant to study systems involving ligand-receptor interactions [START_REF] Samiee | Zero Mode Waveguides for Single-Molecule Spectroscopy on Lipid Membranes[END_REF]. Moreover, much information on the molecular diffusion is obtained by performing measurements with increasing nanoaperture diameters [START_REF] Wenger | Singlefluorophore diffusion in a lipid membrane over a subwavelength aperture[END_REF]. Since the diffusion of molecules in the cell plasma membrane is highly sensitive to heterogeneities, monitoring the lateral diffusion of lipids and membrane proteins is highly relevant in order to reveal the structure and the role of the membrane. Experiments reported in [START_REF] Wenger | Diffusion analysis within single nanometric apertures reveals the ultrafine cell membrane organization[END_REF] showed that the aperture limited the observed membrane area, but did not alter the diffusion process. Extensive FCS analysis on fluorescent-labelled ganglioside in cell membranes revealed membrane structures of 30 nm radius, well below the optical resolution of confocal microscopes.

Enhanced fluorescence with subwavelength apertures

Blair et al.

Nanoapertures thus appear as valuable tools to monitor the diffusion of molecular components in the plasma membrane at high spatial and temporal resolutions, with the cells being kept under physiological conditions and with ultralow amounts of incident laser light. Combined with FCS, this yields a technique having both high spatial and temporal resolution together with a direct statistical analysis.

4. Biosensing applications of sub-wavelength apertures

Detecting molecules in real-time with high sensitivity and molecular specificity is of great interest in many fields of bioscience. Intensive world-wide research on new biosensing techniques is motivated by numerous applications in clinical diagnostics, genetic screenings, proteomics, and single-molecule detection. In this context, the combination of molecules and subwavelength apertures is a promising area of application. The electromagnetic field enhancement, the sensitivity to the dielectric medium in contact with the surface and the simplicity of integrating the arrays have motivated efforts to use them to detect molecules and enhance spectroscopic contrasts [START_REF] Liu | Enhanced fluorescence transduction properties of metallic nanocavity arrays[END_REF].

A first application of the fluorescence enhancement was devoted to the detection of DNA binding events [START_REF] Liu | Biosensing based upon molecular confinement in metallic nanocavity arrays[END_REF]. To perform this affinity sensing, nanoapertures in gold were spotted with probe molecules specific to a target of interest. In order to immobilize probe molecules directly on the bottom (quartz) surface of the apertures, exposed gold surfaces were first passivated with mPEGthiol, leaving only the exposed quartz surface available for avidin monolayer formation. Biotinylated oligonucleotide probes of 20-base length were then immobilized. During hybridization, 20mer target oligos accumulate within the nanoapertures due to specific recognition and binding to the probes. Bound targets produced enhanced fluorescence in transmission, while (non-enhanced) fluorescence emitted from unbound molecules in solution lying outside did not couple efficiently through the structure. Performing detection through the backside of the substrate therefore yields high signal-to-background rejection, enabling real-time detection, as demonstrated in these proof-of-principle experiments [START_REF] Liu | Biosensing based upon molecular confinement in metallic nanocavity arrays[END_REF].

Nanoaperture arrays are particularly relevant for integration with high-throughout screening (HTS) methodologies, for applications in drug screening or evolutionary biotechnologies [START_REF] Koltermann | Rapid assay processing by integration of dual-color fluorescence cross-correlation spectroscopy: high throughput screening for enzyme activity[END_REF]. Combined with FCS or coincidence analysis [START_REF] Winkler | Confocal fluorescence coincidence analysis: an approach to ultra high throughput screening[END_REF], nanostructures appear very well suited to design miniaturized HTS fluorescence sensors [START_REF] Wenger | Dual-color fluorescence crosscorrelation spectroscopy in a single nanoaperture : towards rapid multicomponent screening at high concentrations[END_REF].

CONCLUSIONS AND FUTURE DIRECTIONS

In summary, we have tried to overview the state of the field of nanoaperture enhanced fluorescence. While much is known currently about the photophysics of an isolated aperture, it is clear that much more work needs to be performed in order to understand, and maximize, fluorescence enhancement effects from arrangements of apertures and structured apertures. In particular, the relative contributions of radiative and non-radiative processes have only been studied in a couple of experiments, and limited computational work has been performed. In addition, the role of emission directionality is not clear at present and requires further study.

In addition to the directions for further study mentioned above, there are quite a few additional avenues of exploration. For example, the blue/UV range has not been explored. EOT in the blue has been demonstrated with aluminum aperture arrays [START_REF] Ekinci | Extraordinary optical transmission in the ultraviolet region through aluminum hole arrays[END_REF]; one might reasonably expect fluorescence enhancement to also occur in this regime (in particular, Figure 17.11 shows significant excitation enhancement in the blue/green). In addition, the infrared region is promising since surface plasmon dissipation losses are significantly reduced as compared to the visible range, and very large enhancement would be expected.

Despite some of the limitations of the current state of knowledge, nanoapertures are enabling significant research and even commercial applications, largely due to their unique properties of simultaneous enhancement, background isolation properties, and molecular localization properties.

Figure 17 . 1 :

 171 Figure 17.1: (a) Arrangement for single nanoaperture enhanced fluorescence. A fluorescent molecule located at the bottom of the aperture emits significantly more photons as the same molecule put in the diffraction limited spot of a high-numerical aperture microscope objective. (b) SEM image of 150, 220, 360 nm diameter apertures milled by focused ion beam on a 200 nm thick aluminum film deposited over a standard glass coverslip, and close-up view of a 150 nm aperture [image courtesy of J. Dintinger].

Figure 17 . 3 :

 173 Figure 17.3: Transmission of excitation light and fluorescence emission from Cy-5 from a disordered arrangement of 200nm diameter nanoapertures in 70nm thick gold film, with average spacing of 1m. In these measurements, the sample angle was varied with respect to the incident light, while detection occurred in a direction collinear with the incident light. Fluorescence is normalized to a quartz substrate with the same monolayer and corrected for fill fraction.

  diffuse in and out of an observation volume, or when reaction kinetics or conformational changes induce a change in the fluorescence brightness. Statistical analysis of the fluctuations is generally achieved by computing the fluorescence intensity correlation function, that is, the temporal fluctuations of the fluorescence intensity are recorded, and the autocorrelation of this signal is computed. Numerical analysis of the FCS data then provides the average number of detected molecules which is used to compute the fluorescence count rate per molecule CRM.The FCS setup for working with single nanoapertures is shown in Figure17.4. Initial measurements were performed with round aperture in 300nm thick aluminum films, with aperture diameters ranging from 110 to 420nm[START_REF] Rigneault | Enhancement of single-molecule fluorescence detection in subwavelength apertures[END_REF]. These apertures were prepared by focused ion beam milling. Total fluorescence enhancement factors of up to nearly 7 were obtained for Rh6G dye, excited at 488nm, as reproduced in Figure17.5b. Following the first demonstration by Cornell University[START_REF] Levene | Zero-mode waveguides for single-molecule analysis at high concentrations[END_REF], it was shown that nanoapertures could result in significant reduction in observation volumes, leading to the ability to perform single molecule measurements a high solution concentrations, as shown in Figure17.6.

Figure 17 . 4 :

 174 Figure 17.4: Principle of fluorescence correlation spectroscopy: the fluorescence intensity temporal fluctuations originating from a well-defined volume are recorded and correlated to estimate the average number of molecules observed and the characteristic fluctuation time. This data is used to compute the average detected fluorescence rate per molecule in the observation volume.

Figure 17 . 5 :

 175 Figure 17.5: Experimental single molecule fluorescence enhancement versus aperture diameter, for apertures milled in gold (a) and aluminum (b) films.Rhodamine 6G (Rh6G) dyes were excited at 488 nm, and their fluorescence was collected in the 510-560 nm range. Alexa Fluor 647 (A647) dyes were excited at 633 nm, and their fluorescence was collected in the 650-690 nm range. These curves show significant differences between apertures milled in gold or in aluminum, and highlight the need for a layer exhibiting a strong metallic character at the excitation wavelength to obtain enhanced fluorescence.

Figure 17 . 6 :

 176 Figure 17.6: Observation volume measured by FCS for nanoapertures milled in aluminum (rhodamine 6G dyes, 488 nm excitation) and the corresponding concentration to ensure an average number of one molecule in the observation volume (right scale).

Figure 17 . 7 :

 177 Figure 17.7: (a) Fluorescence rates per molecule versus excitation power in free solution and in single gold nanoapertures of 80, 120 and 275 nm diameter. Circles are experimental data, lines are numerical fits according to three energy states model. Nanoapertures not only allow for large fluorescence enhancements at low excitation powers, but also provide count rates per molecule largely above a few hundred thousands events per second. (b) Corresponding normalized fluorescence decay traces measured in free solution and in single nanoapertures, showing a clear lifetime reduction as the aperture diameter is decreased.Combining FCS with fluorescence lifetime measurements turns out to be very efficient in resolving the contributions of excitation and emission to the overall fluorescence process. This procedure can be straightforwardly extended to other types of plasmonic nanostructures, as long as the observation volume is well defined. The limit of this method is that all the presented results account for spatial averaging over all the possible molecular orientations and positions inside the analyzed volume. There is no sensitivity to individual molecular trajectories or dipole orientations, but one ends up directly with global figures to characterize the emitted fluorescence. Besides, distinguishing between the contributions of the radiative rate and the

Figure 17 . 8 :

 178 Figure 17.8: Fluorescence enhancement at saturation of the absorption / emission cycle, for A647 molecules in single nanoapertures milled in gold. This factor represents the product of the gain in collection efficiency  by the enhancement in radiative rate krad. This figure highlights the contribution of the enhanced emission independently of the altered excitation intensity.

Figure 17 . 9 :

 179 Figure 17.9: Illustration of general computational geometry for a single nanoaperture.

Figure 17 . 10 :

 1710 Figure 17.10: Image of calculated excitation (left half) and radiative emission (right half) of a molecule within a single nanoaperture. Illumination at 635nm wavelength is from below, where the left half image indicates the intensity distribution within the aperture. Emission on the right half is plotted as a function of dipole position, where the color map indicates the radiative enhancement at 670nm wavelength as detected on a plane just below the aperture/substrate interface. The white space around the emission map results from the lack of data points for dipole positions at the boundaries (a finite dipole size, 1nm, was assumed).

Figure 17 . 11 :

 1711 Figure 17.11: Calculated excitation enhancement at three different wavelengths for nanoaperture diameters ranging from 75nm to 250nm in both aluminum (left) and gold (right) films of 150nm thickness. The intensity enhancement is averaged within 10nm thick volumes within the apertures, with V1 representing the volume adjacent to the substrate. Plane wave excitation occurs from the substrate side. The upper region is air.

Figure 17 . 12 :

 1712 Figure 17.12 : Calculated intensity map in the xy plane of a 125nm diameter gold nanoaperture at a position 5nm above the substrate. Excitation is at 635nm wavelength from the substrate.

Figure 17 . 13 :

 1713 Figure 17.13: Calculated radiative emission enhancement at two different wavelengths for a dipole placed with nanoapertures of diameters ranging from 75nm to 200nm in both aluminum (left) and gold (right) films of 150nm thickness. The intensity enhancement is averaged for dipoles located within 10nm thick volumes within the apertures, with V1 representing the volume adjacent to the substrate. Emitted power is measured through a plane on the substrate side. The upper region is air.

Figure 17 . 14 :

 1714 Figure 17.14: Transmission of excitation light and fluorescence emission from Cy-5 from a periodic arrangement of 200nm diameter nanoapertures in 70nmthick gold film, with spacing of 1m. In these measurements, the sample angle was varied with respect to the incident light, while detection occurred from the backside in a direction collinear with the incident light. Fluorescence from a disordered array is also plotted for reference. Fluorescence is normalized to a quartz slide with the same monolayer, and corrected for fill fraction.

Figure 17 . 15 :

 1715 Figure 17.15: Unpolarized angular emission profile from a Cy-5/avidin fluorescing monolayer covering the entire surface of a nanoaperture array of 200nm diameter in 1m spacing in 70nm thick gold. In this measurement, the sample was fixed at normal incidence and the detector on the back side was scanned (zero degrees is normal to the sample). Angular fluorescence emission from a glass substrate is shown for reference.

Figure 17 . 16 :

 1716 Figure 17.16: Illustration of non-passivated (left) and passivated (right) nanoaperture samples. For the non-passivated samples, a fluorescing monolayer covers the entire exposed surface, while only the bottom surfaces of the nanoapertures are covered in the passivated samples.

Figure 17 . 17 :

 1717 Figure 17.17: Unpolarized angular emission profile from a Cy-5/avidin fluorescing monolayer covering the exposed glass surface regions of a passivated nanoaperture array of 200nm diameter in 1m spacing in 70nm thick gold. In this measurement, the sample was fixed at normal incidence and the detector on the back side was scanned (zero degrees is normal to the sample). Angular fluorescence emission from a glass substrate is shown for reference.

Figure 17 . 18 :

 1718 Figure 17.18: Fluorescence emission from a Cy-5/avidin fluorescing monolayer covering the exposed glass surface regions of a passivated nanoaperture array of 200nm diameter in 1m spacing in 70nm thick gold. In this measurement, the sample was rotated with respect to normal incidence. The average fluorescence level from a glass substrate is indicated for reference.

Figure 17 . 19 :

 1719 Figure 17.19: Calculated excitation intensity enhancement as a function of wavelength at the bottom 10nm slice (V1) of a single 150nm diameter nanoaperture in 100nm thick gold compared to an array of nanoapertures of spacings 400nm, 500nm and 600nm. Excitation occurs from the substrate side and the upper region is air.

Figure 17 . 20 :

 1720 Figure 17.20: (a) Schematic representation of a metallic film with a circular aperture and a circular channel around it. The excitation beam impinges of the corrugated surface. (b) Field enhancement averaged at the entrance plane of the aperture as a function of the channel depth. The aperture is milled in aluminum, with a diameter of 150 nm (488 nm excitation wavelength). The groove has 400nm and 560nm inner and outer diameters respectively. [Image courtesy of N. Bonod and E. Popov].

Figure 17 . 21 :

 1721 Figure 17.21: Single nanometric aperture for real time single molecule DNA sequencing. A DNA polymerase molecule attached to the bottom surface is used to successively incorporate fluorescent nucleotides complementary to the DNA strand, causing fluorescence bursts for each incorporation process (see text for details). Image copyright of Pacific Biosciences Inc., reprinted with permission.

17.7 ACKNOWLEDGEMENTS S. B. acknowledges Y. Liu for early experimental and computation work, and F. Mahdavi and M. Diwekar for new contributions to this chapter. Financial support was provided by the National Institutes of Health, the National Science Foundation, and the State of Utah Centers of Excellence Program. J. W. acknowledges H. Rigneault, E. Popov, T.W. Ebbesen, N. Bonod, D. Gerard and financial support from the Agence Nationale de la Recherche ANR and Centre National de la Recherche Scientifique CNRS.