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Long-Range Single-Molecule Förster Resonance Energy Transfer between Alexa Dyes in Zero-Mode Waveguides

Zero-mode waveguide (ZMW) nano-apertures milled in metal films were proposed to improve the Förster resonance energy transfer (FRET) efficiency and enable single-molecule FRET detection beyond the 10 nm barrier, overcoming the restrictions of diffraction-limited detection in a homogeneous medium. However, the earlier ZMW demonstrations were limited to the Atto 550–Atto 647N fluorophore pair, asking the question whether the FRET enhancement observation was an artifact related to this specific set of fluorescent dyes. Here, we use Alexa Fluor 546 and Alexa Fluor 647 to investigate single-molecule FRET at large donor–acceptor separations exceeding 10 nm inside ZMWs. These Alexa fluorescent dyes feature a markedly different chemical structure, surface charge, and hydrophobicity as compared to their Atto counterparts. Our single molecule data on Alexa 546–Alexa 647 demonstrate enhanced FRET efficiencies at large separations exceeding 10 nm, extending the spatial range available for FRET and confirming the earlier conclusions. By showing that the FRET enhancement inside a ZMW does not depend on the set of fluorescent dyes, this report is an important step to establish the relevance of ZMWs to extend the sensitivity and detection range of FRET, while preserving its ability to work on regular fluorescent dye pairs.

Single molecule Förster resonance energy transfer (smFRET) is a highly sensitive approach to investigate intra and inter-molecular distances on the nanometer scale, 1 revealing dynamic information about biomolecular structures and interactions. 2,3 However, the energy transfer efficiency quickly vanishes when the donor-acceptor separation grows, making smFRET measurements highly challenging at distances above 10 nm. 4,5 Extending smFRET to large biomolecular constructs requires the use of elaborated donor-acceptor constructs, and several strategies have been investigated using lanthanides, 6-8 quantum dots, 9,10 multi-color cascaded systems, 11,12 gold nanoparticles quenchers, [13][14][15] metal-induced energy transfer, 16,17 or multiple fluorophores. [18][19][20] Because they do not rely on the organic fluorescent dyes pairs conventionally used in smFRET like Cy3-Cy5 for instance, these advanced approaches further complicate the sample preparation and data analysis. For many applications it would be desirable to extend the smFRET range using regular fluorophore pairs.
Since the works by Purcell and Drexhage,21,22 it is established that the fluorescence emission decay rate is not determined only by the molecular structure, but also depends on the photonic environment surrounding the molecule. The presence of a mirror (or a more elaborated optical component) can affect the fluorescence decay kinetics and the fluorescence lifetime. In a conceptually similar fashion, the dipole-dipole interaction leading to FRET can also be influenced by the photonic environment in some cases. [23][24][25][26] This opens a broad field of research using mirrors, [27][28][29][30][31] microcavities, 23,26,32,33 nanoapertures, [34][35][36][37][38][39][40] nanoparticles, [41][42][43][44][45][46][47][48] nanogap antennas, [49][50][51][52][53] or hyperbolic metamaterials. 54,55 Tuning FRET with nanophotonics can potentially overcome the 10 nm barrier in diffraction-limited confocal microscopes while still using conventional fluorophore pairs. However, reaching an enhancement of the FRET efficiency requires a delicate balance between the FRET rate and the other donor radiative and non-radiative processes, 47,48,51,55 while in many cases the FRET efficiency can end up being quenched by the nanophotonic element. 27,33,48,50,51 We have recently shown that nanoapertures milled in an opaque aluminum film (so called zero-mode waveguides ZMWs 56,57 ) can improve the FRET efficiency and enable smFRET detection beyond the 10 nm barrier. 58 ZMWs are promising devices to perform smFRET on large biomolecular constructs with conventional dyes. However, the demonstration was so far limited to Atto 550 -Atto 647N FRET pairs. 58 Both of these fluorescent molecules bear a positive charge after DNA labelling, and have been found to be quite hydrophobic. 59,60 They bear also a higher affinity for glass or metal surfaces, which was observed for Atto 550 and Atto 647N dyes as compared to their cyanine or Alexa Fluor counterparts. [59][60][61][62] Although care was taken in our previous work to properly passivate the ZMW surface, 58,62 we cannot fully exclude that the observed enhanced FRET could be related to this specific choice of FRET pair from Atto dyes.
Here, we build on the methodology previously developed for smFRET inside a ZMW, 58 and explore the enhancement of smFRET efficiency for the Alexa Fluor 546 -Alexa Fluor 647 donor-acceptor pair. This FRET pair, although spectrally quite similar to the Atto 550 -Atto 647N pair, has a markedly different chemical structure and behavior (Fig. 1a). Both Alexa 546 and Alexa 647 feature a negative charge once labeled to DNA, while the Atto 550 and 647N dyes have a positive charge. It was observed that these Alexa dyes are more hydrophilic than their Atto counterparts, 59,60 and that the Atto 550 and 647N could induce surface adhesion of the DNA molecules while the Alexa 546 and 647 did not. 62 To assess the relevance of ZMWs for smFRET enhancement, it is thus necessary to quantify their performance for a clearly different set of dyes than the Atto 550 -Atto 647N pair used so far. 35,36,50,58 Our new measurements for Alexa 546 -Alexa 647 smFRET inside aluminum ZMWs demonstrate enhanced smFRET efficiencies at separations exceeding 10 nm, confirming the earlier conclusions drawn with the Atto dyes. The detailed characterization reported here is an important step to establish the relevance and validity of ZMWs to extend the FRET detection range. As additional advantage, all the smFRET measurements in the ZMWs are performed at 100 nM concentration, which is a thousand-fold more concentrated than the conditions typically used for confocal detection. This brings smFRET analysis closer to physiological concentrations. 63,64  images of the pattern with ZMWs and a single ZMW of 110 nm diameter milled in an aluminum film.

Results and Discussion
The FRET sample consists of double stranded DNA molecules of 51 base pair length, labeled with a single Alexa 546 donor and a single Alexa 647 acceptor. The donor-acceptor distance is fixed to 30 or Our experiments monitor the FRET events stemming from individual molecules diffusing across the detection volume (Fig. 1b). To clearly quantify the FRET efficiency and avoid the issues related to incomplete fluorophore labelling, we implement pulsed interleaved excitation (PIE) using two alternating laser excitations to excite the donor and the acceptor dyes in a sequential manner. 65 The main difference as compared to a conventional diffraction-limited microscope is the use of zeromode waveguide (ZMW) nanoapertures to confine the light into attoliter volumes. 56,57 The ZMWs used here are milled in a 100 nm thick aluminum film with a diameter of 110 nm (Fig. 1c,d). While the use of Atto 550 -Atto 647N dyes requires the ZMW to be passivated with a silane-modified polyethylene glycol in order to avoid surface adsorption of the DNA molecules, 62 for Alexa 546 -Alexa 647 we find that similar results can be obtained with and without the surface passivation step. This additional advantage of the Alexa FRET pair further simplifies the experiment preparation. Brighter detection events are directly obtained with the ZMW (Fig. 2d-f) as compared to the confocal reference ( Fig. 2 a-c), which illustrates one specific advantage of the fluorescence enhancement occurring inside ZMWs as an improvement for the net detected fluorescence brightness. 67 We analyze the total fluorescence time trace using fluorescence correlation spectroscopy (FCS) to compute the temporal autocorrelation and estimate the average number of emitters and their average brightness.
Following our previous studies, 58,67 this quantifies the fluorescence brightness enhancement factor for isolated donor and acceptor molecules. For Alexa 546, we find a gain of 11.4 ± 0.9 in a 110 nm ZMW, while for Alexa 647, the enhancement is 15.9 ± 1.2. The fact that a higher enhancement is observed for the red dye is mostly related to the lower quantum yield of the dye (33% for Alexa 647 and 79% for Alexa 546), as low quantum yield emitters lead to the observation of higher enhancement factors. 68 After the PIE post-selection, we compute the FRET efficiency EFRET for each burst, taking into account the donor crosstalk, the acceptor direct excitation and the different quantum yields and detection efficiencies between the dyes. The influence of the ZMW is fully taken into account by calibrating the correction parameters for each ZMW independently. The aluminum ZMWs used here are optically weakly resonant components, which do not noticeably modify the fluorescence spectrum of the dyes.
Therefore, as we detail in the Methods section, most correction parameters (for crosstalk and direct excitation) are unchanged in the ZMW as compared to the confocal case.  estimate for the average FRET efficiency in the confocal case for the 40 base pairs separation. However, this approach is limited by the uncertainties on both R and R0 and the assumption of perfectly random orientation for both dyes, which may not be fully verified by our real sample. Thanks to the optical confinement occurring in the ZMW, the FRET efficiency is improved up to 8.5 % ± 0.2 % inside the 110 nm ZMW. Moreover, the full statistical distributions are clearly different, and smFRET is better detected in the ZMW case thanks to higher average FRET efficiencies and narrower distributions. In the confocal case, the standard deviation of the Gaussian distribution (Fig. 3a,c) is 12%, while it is reduced inside the ZMW to 8% thanks to the higher fluorescence brightness.
We also investigate shorter separations of 30 base pairs (D-A distance ~ 10.2 nm). As the acceptor is brought closer to the donor, the average FRET efficiency is increased to 7.8 % ± 0.3 % in the confocal setup ( Fig. 3c) and is further enhanced to 11.5 % ± 0.2 % in the ZMW (Fig. 3d). Computing the gains in the average FRET efficiencies brought by the ZMW, we find a gain of 2.2× (± 0.2) for the 40 bp separation and 1.5x (± 0.1) for the 30 bp case. First, these values demonstrate that the ZMW can indeed improve the net detected FRET efficiency for Alexa FRET pairs, which is especially relevant at large D-A separations exceeding 10 nm where confocal microscopes face their limit of detection. It was observed previously with nanoapertures, 35 nanoantennas, 50 and planar microcavities 26 that the enhancement factors for the FRET rate were higher for the samples corresponding to the larger D-A separations. In other words, the influence of the nanophotonic structure is more pronounced when the D-A separation is larger. We retrieve this feature here. The main reason behind this is that the nanophotonic structure influence on the FRET rate is quite weak as compared to the FRET rate between two closely separated fluorescent dyes in a homogeneous environment. Therefore, one has to go to D-A distances greater than 10 nm so that the ZMW relative influence becomes more prominent. 58   While the various results demonstrate the FRET enhancement inside a ZMW of 110 nm diameter, we now investigate the influence of the ZMW diameter on the energy transfer between Alexa dyes. The same procedure as for Fig. 3 is applied for ZMW diameters ranging from 80 to 150 nm (Fig. 5a). A gradual shift of the mean FRET efficiency is observed on the distributions, which is summarized as a function of the ZMW diameter in Fig. 5b. Here the 110 nm diameter used in Fig. 3 is near the optimum.
Using a large diameter, the fluorescence enhancement decreases and the FRET results tend to retrieve the confocal values. Using a lower diameter, the quenching losses due to direct energy transfer to the metal increase, which compete with FRET to the acceptor dye and reduce the observed FRET efficiency.

Conclusions
Our data demonstrate here that the phenomenon of smFRET enhancement inside a ZMW is quite general and does not depend on the type of fluorescent dyes used. Performing smFRET measurements on Alexa 546 -Alexa 647 pairs, which feature a markedly different chemical structure, surface charge and hydrophobicity as compared to their Atto 550 -Atto 647N counterparts, we retrieve the same conclusions about the quantitative performance of ZMWs to enhance smFRET. Notably, we could achieve over a two-fold enhancement of the net detected FRET efficiency for dyes separated by more than 10 nm. This significantly improves the sensitivity and detection range of smFRET, while preserving the ability to work on conventional fluorophore pairs. The only difference as compared to a classical Therefore, preliminary calibrations should be performed in order to enable relevant distance measurements using FRET in ZMWs (and this work contributes to it), but conceptually there is no reason why quantitative distance measurements would not be possible using FRET inside ZMWs.

Zero-mode waveguide fabrication.
A 100 nm thick layer of aluminum is deposited on a clean glass coverslip by electron-beam evaporation (Bühler Syrus Pro 710). 58 The deposition rate is 10nm/s at a chamber pressure of 5. 10 -7 mbar. 73  FRET efficiency measurements. The procedure to compute the FRET efficiency histograms follows the standard approach in smFRET. 1,74 First, we select the single molecule detection events and separate them from the background noise, applying a threshold criterion so that the sum of the signals in donor and acceptor channels exceeds 25 counts per ms for the ZMW (12 counts per ms for the confocal case).
A second threshold is used to check the presence of the red dye upon the excitation by the red laser.
We choose the value at 12 counts per ms in acceptor channel with red excitation (3 counts per ms for the confocal case due to the lower fluorescence brightness). We ensure that these levels have a negligible influence on the measured average FRET efficiencies.
The FRET efficiency is then calculated as 1,58 The direct excitation δ corresponds to the fraction of the acceptor fluorescence due to direct excitation by the green laser as compared to acceptor emission signal upon the red laser. This parameter is measured when a DNA sample containing only the acceptor dye is excited: δ = . For the confocal reference, we find δ=0.11, which also does not change for ZMWs diameters except for the 80 nm ZMW where we have δ=0.08.
The γ correction factor takes into account the differences in the fluorescence quantum yields (φd, φa) and the detection efficiencies (κd, κa): = . For the Alexa 546-Alexa647 FRET pair in our confocal setup, we compute γconf=0.43 ± 0.02 from the knowledge of the fluorescence spectrum and quantum yield of each dye. The photodiode response is also accounted for in the calculation. Alternatively, the γ correction factor can also be estimated from the measured stoichiometry S, 1 Fig. 3 and 5) shows that the gain in the acceptor emission is enough to compensate for the increased γ factor. For the donor only TCSPC data, we use 10 times higher concentrations (average number of detected molecules about 1) to achieve a better signal to noise ratio. As a consequence of the higher background contribution in the FRET cases, a peak at t = 0 is seen for the FRET TCSPC data.
This peak corresponds to a sum of laser light scattering, metal photoluminescence and Raman scattering. This contribution is interpolated with a fixed 20 ps component (shorter than the 38 ps IRF resolution) to achieve a complete fitting of the TCSPC decay, but this contribution is then discarded for the lifetime analysis as it only corresponds to noise. The background and scattering contributions are recorded by performing a separate experiment in the same conditions using only the buffer in the absence of DNA sample. For the TCSPC analysis, we ensure that more than 92% of the total detected photons are considered for the fits. The data for the confocal reference (FRET and donor only) are fitted with a single exponential decay (excluding the fixed 20 ps contribution from laser scattering). For the ZMWs, a biexponential function with fast and slow components provides a better fit with flat residuals. As observed previously for Atto dyes, 58 Table 1. The tail seen for long delay times higher than 5 ns is only due to the background level, there is no supplementary long lifetime. In the ZMW case, the intensity-averaged lifetimes are used to compute the average FRET efficiency. We find empirically that these values provide a better match with the separate burst intensity analysis (Fig. 3) than the amplitude-averaged data. However, our claim of enhanced FRET efficiency in the ZMW is maintained for all the approaches (intensityaveraged, amplitude-averaged or direct comparison between long lifetime components). Table 1. Results obtained from the numerical fit to the TSCPC histograms shown in Fig. 4a. In the case of a biexponential fit, τ1 and τ2 are the individual lifetimes of each component and α1 and α2 are their respective normalized amplitudes. τamp = (α1 τ1 + α2 τ2)/(α1 + α2) is the amplitude-averaged lifetime, while τint = (α1 τ1 2 + α2 τ2 2 )/( α1 τ1 + α2 τ2) denotes the intensity-averaged lifetime. The 20 ps scattering peak at t = 0 is not shown here.