FCS analysis to measure the near-field apex volume.
(a) Fluorescence time trace with excitation light parallel (red line) and perpendicular (blue line) to the apex region. The time trace found for the confocal case (0.5 fL diffraction-limited volume) is shown in green for comparison. (b) FCS correlation function of the traces shown in (a). For all cases, the Alexa Fluor 647 concentration 20 μM with 200 mM of methylviologen as chemical quencher and the excitation power is 10 μW. Dots are experimental points, lines are fits using the model described in the Methods section. A higher correlation amplitude is observed with the polarization parallel to the apex and corresponds to a lower number of detected molecule (stronger confinement of light). The fit parameters are summarized in Table 1. (c) FCS correlation functions for increasing concentrations of fluorescent dyes in a double nanohole with excitation polarization parallel to the apex. (d) Number of detected molecules in the apex region as function of the molecular concentration. The slope of the curve quantifies the apex near-field volume Veff.