Enhancement of Single-Molecule Fluorescence Detection in Subwavelength Apertures

We report the experimental proof of molecular count rate enhancement (up to 6.5-fold) and lifetime reduction for single fluorescent molecules diffusing in subwavelength apertures milled in aluminum films. The observed enhancement dependence with the aperture diameter agrees qualitatively with numerical electromagnetic computations of the excitation power density into the aperture volume.

Figure 1

(a) Scanning electron micrograph of an isolated subwavelength aperture milled in an Al film coated on a microscope slip. (b) Schematic view of the optical setup (APD: avalanche photodiode).

Figure 2

$V_{\mathrm{eff}}$ evolution versus the aperture diameter (◇, right scale—dashed line to guide the eyes) and nanohole geometric volume (●, right scale). Enhancement of $\eta$ (histogram, left scale) as compared to its value in open solution versus the aperture diameter ($P_{\mathrm{ex}}=0.2\mathrm{mW}$).

Figure 3

Line traces and autocorrelation functions in open solution (○) in a 150 nm diameter aperture (□) and for the noise within the aperture (×) for an excitation power of 0.3 mW (the noise was negligible in open solution: $<200\mathrm{\text{counts}}/\mathrm{s}$). The molecular concentration for the experiment in open solution was set to 20 nM, while it was taken to 600 nM within the aperture. For the open solution, numerical fits assuming a 3D free diffusion yield the set of parameters: $N=7.8$, $n_T=0.15$, diffusion time is $53\mu \mathrm{s}$, count rate per molecule is $20\mathrm{kHz}$. For the 150 nm aperture, $N=1.5$, $n_T=0.35$, diffusion time is $10\mu \mathrm{s}$, count rate per molecule is $112\mathrm{kHz}$.

Figure 4

Evolution of η versus the incident power $P_{\mathrm{ex}}$ in a 150 nm aperture (■) and in open free solution (○). Inset: Fluorescence decay curves in open solution and into a $d=150\mathrm{nm}$ nanohole.

Figure 5

Results of simulations in a 200 nm thick metal film. Count rate per molecule $\eta$ (normalized to the open solution case) and effective volume $V_{\mathrm{eff}}$ as a function of the aperture diameter.