Supporting Information for Matching nanoantenna field confinement to FRET distances enhances Förster energy transfer rates

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1 Supporting Information for Matching nanoantenna field confinement to FRET distances enhances Förster energy transfer rates Petru Ghenuche, Mathieu Mivelle, Juan de Torres, Satish Babu Moparthi, Hervé Rigneault, Niek F. Van Hulst,3, María F. García-Parajó,3, and Jérôme Wenger CNRS, Aix-Marseille Université, Ecole Centrale Marseille, Institut Fresnel, Campus de St Jérôme, 3397 Marseille, France ICFO-Institut de Ciencies Fotoniques, Mediterranean Technology Park, 886 Castelldefels, Spain 3 ICREA-Institució Catalana de Recerca i Estudis Avançats, Barcelona, 8, Spain jerome.wenger@fresnel.fr This document contains the following supporting information:. Vertical section of experimental configuration. Scanning electron microscopy images 3. Dark-field spectroscopy 4. FCS analysis procedure 5. Acceptor fluorescence in the box aperture 6. Comparison of fluorescence decay traces 7. Dipole intensity distributions 8. Fluorescence time traces for burst analysis 9. Reference FRET histogram for isolated donor (no energy transfer). FRET rates and comparison between the two measurement methods. Quantification of radiative and non-radiative decay rates. Excitation power dependence

2 Vertical section of experimental configuration a b l 55nm Al Al Al Al Glass 8 nm 5 Intensity enhancement Water Figure S: (a) Sketch of the experimental configuration showing donor-acceptor fluorescent pairs on DNA double strands diffusing in the nanogap antenna. (b) Vertical cross-section of the excitation intensity enhancement at 55 nm with polarization parallel to the nanogap. Scanning electron microscopy images 79.7 nm 79.6 nm 78. nm 79.8 nm 44.5 nm 49. nm 49. nm 5. nm.4 nm 53.8 nm.7 nm Figure S: Scanning electron microscope images of two representative antennas fabricated by focused ion beam milling in a 5 nm thick aluminum film deposited on glass coverslip.

3 3 Dark-field spectroscopy reveal the antenna contribution Scattered intensity (a.u.) Wavelength (nm) Figure S3: Dark-field scattering spectra of the aluminum gap antenna (red curves) and the box aperture (blue curves) for linear illumination polarizations parallel (solid lines) and perpendicular (dashed lines) to the box and antenna main axis. The presence of the dimer antenna inside the box aperture induces a significant red-shift, which is only visible for the polarization parallel to the dimer axis. 3

4 4 FCS analysis procedure The analysis of the FCS data considers two species with different fluorescence brightness: N molecules in the dimer hot spot volume with brightness Q, and N background molecules with brightness Q diffusing away from the hot spot []. The fluorescence intensity correlation function can be written [] G(τ) = F (t).f (t + τ) F (t) = + N Q G d (τ) + N Q G d(τ) (N Q + N Q ) () where G d (τ) and G d(τ) are the normalized functional forms of the correlation function for each species taken individually based on a three dimensional Brownian diffusion model: G di (τ) = ( + τ/τ d,i ) + s i τ/τ d,i () τ d,i stands for the mean residence time (set by translational diffusion) and s i the ratio of transversal to axial dimensions of the analysis volume (i = or ). Since Atto55 and Atto647N dyes undergo negligible photoblinking related to transitions to a triplet state or photoisomerization at µw excitation power (as compared to cyanine derivatives such as Cy5 or Alexa Fluor 647) [3], our model does not consider photoblinking and focus on translational diffusion which is largely sufficient to model the experimental traces. An essential feature of FCS is that the different fluorescent species contribute to the amplitude of G(τ) in proportion to the square of their relative fluorescence brightness, as shown in Eq. (). Fitting the FCS correlation traces quantifies the relative amplitudes ρ i of the contribution from each species without the need for any extra information: G(τ) = + ρ G d(τ) + ρ G d (τ) (3) By comparing Eqs.() and (3), the definitions of ρ i are straightforward: ρ = ρ = N Q (N Q + N Q ) (4) N Q (N Q + N Q ) (5) To quantify the number of molecules N in the antenna hot spot and their brightness Q, we introduce the known value of the average total fluorescence intensity F = N Q + N Q and take advantage of the relation: ρ Q + ρ = (6) Q F The combination of Eqs. (4) and (6) yields the expression for the desired parameters: Q = ρ F ρ Q (7) 4

5 N = ( ρ F Q ) ρ (8) These expressions enable computing N and Q based on the measurement of F and the FCS fitting quantifying ρ and ρ. The only supplementary parameter is the brightness per molecule Q away from the hot spot, which we fix according to the brightness per molecule found with FCS for the antenna with perpendicular orientation (in this case the contribution from the hot spot cancels out). Table S summarizes the results for the FCS fit. Acceptor Acceptor Acceptor Donor Isolated FRET.nm FRET 6.8nm Isolated F (khz) ρ ρ τ (µs) τ (µs) Q (khz) Q (khz) N N Fluorescence Enhancement Detection Volume (zl) 3 5 Table S: Fitting parameter results for the FCS curves displayed in Fig. c. The experimental conditions are identical between cases: concentration µm, excitation power µw with linear polarization aligned along the dimer axis. The fluorescence enhancement is defined respective to the reference confocal brightness per molecule of. khz for the isolated Atto647N (.95 khz for the isolated donor Atto55). 5

6 5 Acceptor fluorescence in the box aperture a Box aperture Acceptor fluorescence µm b c 3 nm nm Acceptor kcounts / ms 4 FRET 6.8nm FRET.nm Acceptor only 4 Time (s) 6 Correlation G-.3.. FRET 6.8nm FRET.nm Acceptor only... Lag time (ms) Figure S4: (a) Scanning electron microscope image of a box aperture fabricated on the same sample than the antennas. (b) Acceptor fluorescence time traces (binning time ms) and (c) FCS correlation functions for different donor-acceptor distances, and with the acceptor alone in the box aperture. The concentration of FRET pairs is µm, and the laser excitation polarization is aligned along the long axis of the rectangular aperture. Acceptor Acceptor Acceptor Isolated FRET.nm FRET 6.8nm F (khz) ρ τ (µs) Q (khz) N Detection volume (al) Table S: Fitting parameter results for the FCS curves obtained with the box aperture (Fig. S4c). A single species fit is used here, which further facilitates the analysis. The shape parameter converges to s = for the different cases, as already observed for circular apertures [4]. 6

7 6 Isolated Atto55 and Atto647N dyes show similar lifetime reduction in aluminum nanogap antenna a Atto55 isolated donor b Atto647N isolated acceptor Normalized intensity. parallel Box perpendicular Confocal Normalized intensity. parallel Box perpendicular Confocal IRF IRF 4 Time (ns) 6 4 Time (ns) 6 Figure S5: Increased decay rates and LDOS in the aluminum nanogap antenna. (a,b) Comparison of the normalized fluorescence decay traces for the isolated Atto55 donor (a) and the isolated Atto647N acceptor (b). For both dyes, the emission is significantly accelerated in the gap antenna with polarization parallel to the dimer axis, as already indicated in Fig. 3(a-d). The traces for the acceptor are noisier as a consequence of the x lower excitation cross-section for Atto647N at 55 nm. The decay rates (and hence the LDOS) are similar for the two dyes, showing the broadband nature of the aluminum antenna response. Black lines are numerical fits, IRF indicates the instrument response function. 7

8 a b c FRET 6.8nm perpendicular Normalized intensity. IRF FRET 6.8nm Time (ns) Donor only FRET.nm 3 Normalized intensity. IRF parallel perpendicular Time (ns) 3 Normalized intensity. IRF parallel Time (ns) FRET.nm perpendicular 3 Figure S6: Donor photodynamics for the antenna illuminated with polarization perpendicular to the dimer axis. (a) Normalized donor fluorescence decay traces for the isolated donor (green), donoracceptor separation of. nm (blue) and 6.8 nm (orange). This graph is similar to Fig. 3a-c and can be directly compared with them. The presence of the acceptor further accelerates the donor decay dynamics, which demonstrates the occurrence of FRET. (b,c) Comparison of the normalized fluorescence decay traces for the donor in the presence of the acceptor at 6.8 nm (b) or. nm (c) for the two polarization orientations. Confocal Box reference aperture perpendicular parallel Isolated Donor Donor FRET.nm Donor FRET 6.8nm Isolated Acceptor Table S3: Fluorescence lifetimes obtained by fitting the decay traces displayed in Fig. 3a-c, Fig. S5 and Fig. S6. All lifetimes are indicated in ns. Parallel and perpendicular refer to the orientation of the laser excitation polarization respective to the dimer antenna main axis. The temporal resolution for fluorescence lifetime measurements is 37 ps at half-maximum of the instrument response function. 8

9 7 Dipole intensity distribution 5nm 5nm Electric field intensity (log scale) b Electric field intensity (log scale) a Figure S7: Electric field intensity distribution radiated by a horizontally oriented dipole in (a) homogeneous water environment and (b) the center of an aluminum nanogap antenna. The ratio of the graphs in (b) and (a) corresponds to the FRET rate enhancement plotted in Fig. 4a. 9

10 8 Fluorescence time traces for burst analysis FRET 6.8 nm, Gap, µm FRET. nm, Gap, µm Donor counts /.ms 5 5 a Donor counts /.ms 5 5 d 5 Time (ms) 5 5 Time (ms) 5 Acceptor counts /.ms 5 5 Time (ms) 5 b Acceptor counts /.ms 5 5 Time (ms) 5 e FRET efficiency.4. c FRET efficiency.4. f. 5 Time (ms) 5. 5 Time (ms) 5 Figure S8: Fluorescence time traces for Atto55-Atto647N FRET pairs in the aluminum gap antenna. The concentration of FRET pairs is set to µm, ensuring individual FRET pairs on single DNA molecules are distinguished. The binning time is. ms. Traces (a-c) correspond to donor-acceptor distances of 6.8 nm ( base pairs), while traces (d-f) are for. nm (3 base pairs). For each detected fluorescence burst, a FRET efficiency is calculated (c,f). The full trace duration used to compute the FRET histograms in Fig. 5b,c is s. Dashed horizontal orange lines represent the average value to guide the eyes.

11 9 FRET histogram for isolated donor (no energy transfer) Occurrences (kcounts) 6 4 Donor only Al gap antenna Mean.4% St. Dev. 3.% FRET efficiency (%) 8 Figure S9: FRET efficiency histogram computed for the DNA samples labeled only with the donor fluorophore (no acceptor or FRET in this case). Events with apparent transfer efficiency below zero are also shown here. We use a Gaussian fit to determine the apparent center FRET efficiency and the standard deviation, and obtain E F RET =.4 ± 3. % in the absence of the acceptor. This graph provides a reference for the zero FRET case, which can be compared with the histograms in Fig. 5b,c that are significantly shifted to positive FRET efficiency values.

12 The two measurement methods converge towards similar FRET rates a.6 FRET D-A 6.8nm b FRET D-A.nm FRET rate (ns - ).4. 3 Rate enhancement FRET rate (ns - ).. 4 Rate enhancement. Confocal Box perp. parallel. Confocal Box perp. parallel from lifetime measurements from fluorescence burst analysis Figure S: FRET rate Γ F RET for the different configurations with 6.8 nm (a) or. nm (b) donoracceptor separation. Bright color bars correspond to data deduced from fluorescence lifetime fits Γ F RET = Γ DA Γ Do (Fig. 3), light color bars are obtained from fluorescence burst analysis (Fig. 5) combined with the decay rate of the isolated donor, according to the formula Γ F RET = E F RET /( E F RET ) Γ Do. Both measurements converge towards similar values and confirm the validity of our approach. The rate enhancement (right scale) is defined respective to the FRET rate in confocal case.

13 Quantification of radiative and non-radiative decay rates In this section, we combine our measurements to quantify the different photophysical rates described in the simplified Jablonski diagram of Fig. d. Below the fluorescence saturation regime, the fluorescence enhancement η F = η κ η ϕ η exc is proportional to the gains in collection efficiency η κ, quantum yield η ϕ, and excitation intensity η exc. Experimentally, η F is obtained from FCS measurements (Tab. S). The local excitation intensity enhancement η exc = 35 is deduced from the numerical simulations (Fig. e) by averaging the electric field intensity in the gap region. The collection efficiency enhancement is assumed to η κ =, considering that the structure acting as a dipole antenna does not provide any gain in the collection efficiency. With these values at hand, we can compute the quantum yield enhancement as η ϕ = η F /(η exc η κ ). An additional information is brought by the TCSPC decay rates which quantify the fluorescence lifetime reduction and total decay rate enhancement η tot. As the quantum yield is the ratio of radiative rate over the total (radiative + nonradiative) decay rate, the fluorescence enhancement can be rewritten: η F = η κ η rad η exc /η tot, which enables to compute the radiative rate enhancement η rad = η ϕ η tot = η F η tot /(η exc η κ ). The non-radiative rate (without FRET) enhancement is computed following the relation η tot = ϕη rad + ( ϕ)η nrad and the known quantum yield ϕ for the fluorescent dyes in water solution (confocal reference). With the known quantum yield of 8% for Atto55 and 65% for Atto647N in water solution and the quantification of their total decay rate by fluorescence lifetime (Tab. S3), it is possible to extract the values of the radiative, non-radiative and total decay rates from the enhancement factors so as to provide a complete picture of the photophysics rates in the nanogap antenna. Table S4 summarizes the different photokinetic rates together with their enhancement values respective to the confocal reference. We also add the FRET rates and FRET rate enhancement factors found for the donoracceptor separation of 6.8 and. nm. To ease the reading, the different enhancement factors are summarized on Fig. S. Donor Acceptor FRET FRET Atto55 Atto647N 6.8 nm. nm Γ D rad Γ D nr Γ D tot ϕ D Γ A rad Γ A nr Γ A tot ϕ A Γ F RET Γ F RET Confocal reference Al gap antenna Relative enhancement Table S4: Estimated values for the radiative rate Γ rad, non-radiative rate Γ nr, total decay rate Γ tot and quantum yield ϕ for Atto55 and Atto647N. All rates are expressed in ns. 3

14 Donor FRET x.9 (6.8nm) x 4.6 (.nm) Acceptor D I ex D nr D rad A nr A rad x 35 +ns - x.3 +.9ns - x 5. D tot A x 9.4 tot x.5 Figure S: Enhancement of the photokinetic rates for Atto55-Atto647N FRET pairs in an aluminum nanogap antenna. 4

15 No saturation effects are observed at µw excitation power Donor kcounts/ms 5 µw excitation power 4 Excitation power (µw) Figure S: Excitation power dependence of the donor fluorescence intensity (corresponding to the configuration of Fig. b). A linear relationship is observed above µw, twice higher than the excitation power used in the experiments. References [] Punj, D.; Mivelle, M.; Moparthi, S.B.; van Zanten, T.S.; Rigneault, H.; van Hulst, N.F.; Garcia- Parajo, M.F.; Wenger J. Nat. Nanotechnol. 3, 8, [] Zander, C.; Enderlein J.; Keller, R. A. Single-Molecule Detection in Solution - Methods and Applications, VCH-Wiley, Berlin/New York,. [3] Buschmann, V.; Weston, K. D.; Sauer, M. Bioconjugate Chem. 3, 4, [4] Wenger, J.; Gérard, D.; Bonod, N.; Popov, E.; Rigneault, H.; Dintinger, J.; Mahboub, O.; Ebbesen, T.W. Opt. Express 8, 6, [5] Ghenuche, P.; de Torres, J.; Moparthi, S. B.; Grigoriev, V.; Wenger, J. Nano Lett. 4, 4,