We demonstrate unambiguously that the field enhancement near the apex of a laser-illuminated silicon tip decays according to a power law that is moderated by a single parameter characterizing the tip sharpness. Oscillating the probe in intermittent contact with a semiconductor nanocrystal strongly modulates the fluorescence excitation rate, providing robust optical contrast and enabling excellent background rejection. Laterally encoded demodulation yields images with <10 nm spatial resolution, consistent with independent measurements of tip sharpness. DOI: 10.1103/PhysRevLett.93.180801 PACS numbers: 07.79.Fc, 42.50.Hz, 61.46.+w, 78.67.Bf The potential of near-field microscopy to optically resolve structure well below the diffraction limit has excited physicists, chemists, and biologists for almost 20 years. Conventional near-field scanning optical microscopy (NSOM) uses the light forced through a small metal aperture to locally excite or detect an optical response. The spatial resolution in NSOM is limited to 30 -50 nm by the penetration depth of light into the metal aperture. More recently, apertureless-NSOM (ANSOM) techniques were developed which leverage the strong enhancement of an externally applied optical field at the apex of a sharp tip for local excitation of the sample [1][2][3][4][5][6][7][8][9][10][11]. The promised advantage of ANSOM is that spatial resolution should be limited only by tip sharpness (typically 10 nm). The resolution in most previous ANSOM experiments, however, was at best marginally better than NSOM and was inferior to expectations based on tip sharpness alone. Further, the external field used to induce enhancement led to a substantial background signal and to assertions that one-photon fluorescence is not appropriate for ANSOM [12,13]. These experiments fell short of their potential because they maintained a tip-sample gap of several nanometers, and thus did not thoroughly exploit the tightly confined enhancement.Here, we demonstrate an ANSOM technique that fully exploits the available contrast and leads to spatial resolution that is limited only by tip sharpness. The problems associated with a tip-sample gap are overcome by oscillating the probe in intermittent contact with the sample. The detected signal is then composed of a modulated near-field portion that is superimposed on the far-field background. Subsequent demodulation decouples the two components and thus strongly elevates the near-field signal relative to the background. With this technique, we measured <10 nm lateral resolution via one-photon fluorescence imaging of isolated quantum dots, consistent with independent measurements of tip sharpness. The measured resolution is >3 times better than previous reports for quantum dots using one-photon fluorescence [8,9], and is 2 times better than previous measurements using higher-order optical processes (two-photon fluorescence [6], Raman scattering [4,5]) despite predictions to the contrary [12,13].To better understand the advantages of this technique and to facilitate ...

We report a method for tracking individual quantum dot (QD) labeled proteins inside of live cells that uses four overlapping confocal volume elements and active feedback once every 5 milliseconds to follow three dimensional molecular motion. This method has substantial advantages over 3D molecular tracking methods based upon CCD cameras, including increased Z tracking range (10 μm demonstrated here), substantially lower excitation powers (15 μW used here), and the ability to perform time-resolved spectroscopy (such as fluorescence lifetime measurements or fluorescence correlation spectroscopy) on the molecules being tracked. In particular, we show for the first time fluorescence photon anti-bunching of individual QD labeled proteins in live cells and demonstrate the ability to track individual dye labeled nucleotides (Cy5-dUTP) at biologically relevant transport rates. To demonstrate the power of these methods for exploring the spatio-temporal dynamics of live cells, we follow individual QD-labeled IgE receptors both on and inside rat mast cells. Trajectories of receptors on the plasma membrane reveal three dimensional, nano-scale features of the cell surface topology. During later stages of the signal transduction cascade, clusters of QD labeled IgE-FcεRI were captured in the act of ligand-mediated endocytosis and tracked during rapid (~950 nm/s) vesicular transit through the cell. KeywordsQuantum Dot; Single Molecule; Fluorescence; Tracking; Microscopy; Cell The direct observation of individual biological molecules in motion can transform our view of important biophysical and cellular processes. 1 For example, single molecule tracking has shed significant light on cellular membrane dynamics 2-4 , motor protein kinetics 5,6 , and gene regulation 7 . Advantages of a single molecule approach include the ability to observe dynamic, stochastic behavior (such as compartmentalized diffusion 2, 4 ) that would be masked in ensemble measurements and the ability for localization of molecules with a precision well below the diffraction limit of light 5,6 . To date the field has primarily relied * Corresponding Author: jwerner@lanl.gov. 8,16 or follow the Z position with multiple cameras or image planes 9, 14 . While these camera-based techniques can capture 3D molecular motion, they are generally limited in their Z-tracking range in cells to approximately plus or minus one μm from a fixed focal plane 8,10,14,17 , limited by the shallow depth of field of high numerical aperture microscope objectives needed for single molecule work. We point out the obvious: many cells are substantially thicker than two microns and different methods and techniques are required to follow single molecules throughout entire three dimensional cell volumes. In addition to its quite limiting Z-tracking range, CCD-based tracking approaches are also bounded in temporal resolution by the CCD frame rate (~1 ms for fast EM-CCDs), and must illuminate an entire cell slice at relatively large excitation intensities (~40 W/cm 2 ).In contrast to ...

We demonstrate a custom confocal fluorescence-microscope that is capable of tracking individual quantum dots undergoing three dimensional Brownian motion (diffusion coefficient ~0.5 µm2/s) in environments with a signal-to-background ratio as low as 2:1, significantly worse than observed in a typical cellular environment. By utilizing a pulsed excitation source and time-correlated single photon counting, the time-resolved photon stream can be used to determine changes in the emission lifetime as a function of position and positively identify single quantum dots via photon-pair correlations. These results indicate that this microscope will be capable of following protein and RNA transport throughout the full 3 dimensional volume of a live cell for durations up to 15 seconds.