Resolving single fluorophores within dense ensembles: contrast limits of tip-enhanced fluorescence microscopy

We investigate the limits of one-photon fluorescence as a contrast mechanism in nanoscale-resolution tip-enhanced optical microscopy. Specifically, we examine the magnitude of tip-induced signal enhancement needed to resolve individual fluorophores within densely-packed ensembles. Modulation of fluorescence signals induced by an oscillating tip followed by demodulation with a lock-in amplifier increases image contrast by nearly two orders of magnitude. A theoretical model of this simple modulation/ demodulation scheme predicts an optimal value for the tip-oscillation amplitude that agrees with experimental measurements. Further, as an important step toward the eventual application of tip-enhanced fluorescence microscopy to the nanoscale structural analysis of biomolecular systems, we show that requisite signal enhancement factors are within the capabilities of commercially available silicon tips.