Abstract
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.
Highlights
Tip-enhanced fluorescence microscopy (TEFM) is a type of apertureless near-field scanning optical microscopy (ANSOM) that utilizes fluorescence to generate an image
We have defined the acceptable level of near-field contrast as C > 1 and have calculated the amount of signal enhancement needed in TEFM to achieve such contrast for single fluorophores within high density samples
Current nanoscale structural analysis tools including x-ray crystallography, electron microscopy, and atomic force microscopy, have a number of limitations that prevent their application to extended networks composed of heterogeneous mixtures of various biomolecules
Summary
Tip-enhanced fluorescence microscopy (TEFM) is a type of apertureless near-field scanning optical microscopy (ANSOM) that utilizes fluorescence to generate an image. This field enhancement is tightly confined to the vicinity of the tip apex and has been shown to decay rapidly as r−6 with distance r from the tip apex [2]. A number groups have investigated various means of increasing the degree of field enhancement, including optimizing the shape of the tip to leverage plasmon and antenna resonances These efforts have already been fruitful for increasing the enhancement, and will impact both ANSOM and sensor applications [13]. We use a theoretical model based on experimental measurements to show that sufficient contrast can be obtained even for the relatively simple case of commercially available silicon tips and one-photon fluorescence
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