Single molecule fluorescence resonance energy transfer scanning near-field optical microscopy: potentials and challenges.

A few years ago, single molecule Fluorescence Resonance Energy Transfer Scanning Near-Field Optical Microscope (FRET SNOM) images were demonstrated using CdSe semiconductor nanocrystal-dye molecules as donor-acceptor pairs. Corresponding experiments reveal the necessity to exploit much more photostable fluorescent centers for such an imaging technique to become a practically used tool. Here we report the results of our experiments attempting to use nitrogen vacancy (NV) color centers in nanodiamond (ND) crystals, which are claimed to be extremely photostable, for FRET SNOM. All attempts were unsuccessful, and as a plausible explanation we propose the absence (instability) of NV centers lying close enough to the ND border. We also report improvements in SNOM construction that are necessary for single molecule FRET SNOM imaging. In particular, we present the first topographical images of single strand DNA molecules obtained with fiber-based SNOM. The prospects of using rare earth ions in crystals, which are known to be extremely photostable, for single molecule FRET SNOM at room temperature and quantum informatics at liquid helium temperatures, where FRET is a coherent process, are also discussed.

Near-field scanning optical microscopy using polymethylmethacrylate optical fiber probes.

We report the first use of polymethylmethacrylate (PMMA) optical fiber-made probes for scanning near-field optical microscopy (SNOM). The sharp tips were prepared by chemical etching of the fibers in ethyl acetate, and the probes were prepared by proper gluing of sharpened fibers onto the tuning fork in the conditions of the double resonance (working frequency of a tuning fork coincides with the resonance frequency of dithering of the free-standing part of the fiber) reported earlier for the case of glass fibers. Quality factors of the probes in the range 2000-6000 were obtained, which enables the realization of an excellent topographical resolution including state-of-art imaging of single DNA molecules. Near-field optical performance of the microscope is illustrated by the Photon Scanning Tunneling Microscope images of fluorescent beads with a diameter of 100nm. The preparation of these plastic fiber probes proved to be easy, needs no hazardous material and/or procedures, and typical lifetime of a probe essentially exceeds that characteristic for the glass fiber probe.

Scanning near-field optical microscope based on a double resonant fibre probe montage and equipped with time-gated photon detection.

We discuss scanning near-field optical microscope based on original double resonant montage of a fibre probe onto the tuning fork and proprietary electronics capable of fast and precise measurements of the resonant frequency and the quality factor of sensor dithering. Special emphasis is given on the pulsed excitation/gated detection of optical signal. This option as well as the possibility of fast scanning facilitates a lot the problem of single fluorescence centres detection. To illustrate the performance of this microscope, we present first true single-molecule fluorescence resonance energy transfer scanning near-field optical microscope images of single CdSe nanocrystals on glass slide surface and observation of an optical 'pseudoresolution' of densely packed 100-nm-diameter transfluorescent spheres in noisy conditions.