Single molecule detection and underwater fluorescence imaging with cantilevered near-field fiber optic probes

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Abstract
Tapping-mode near-field scanning optical microscopy (NSOM) employing a cantilevered fiber optic probe is utilized to image the fluorescence from single molecules and samples in aqueous environments. The single molecule fluorescence images demonstrate both the subdiffraction limit spatial resolution and low detection limit capabilities of the cantilevered probe design. Images taken as a function of tip oscillation drive amplitude reveal a degradation in the resolution as the amplitude is increased. With all cantilevered probes studied, however, a minimum plateau region in the resolution is reached as the drive amplitude is decreased, indicating that the tapping mode of operation does not reduce the optical resolution. Images of fluorescently doped lipid films illustrate the ability of the probe to track small height changes (<1.5 nm) in ambient and aqueous environments, while maintaining high resolution in the fluorescence image. When the tip is immersed in water (1.3 mm), the cantilevered NSOM tip resonance, 25–50 kHz, shifts approximately 100–150 Hz, the amplitude dampens less than 40% and the Q factor is reduced from 300–500 to 100–200.