NSOM Measurements

In a series of papers, these and similar measurements were used by Higgins and coworkers to characterize the response of PDLCs to changes in applied voltage. The subdiffraction limited resolution in these measurements found a spatial variation in the liquid crystal reorganization threshold that had contributions from several sources including interactions with the polymer encapsulation interface. 

As an example, Shiku et. al. reported an adaptation of the interferometric optical feedback method for use with cantilevered NSOM probes oscillated normal to the sanqile surface.(Shiku et al. 1999 Talley, Cooksey et al. 1996) In contrast to straight fiber optic NSOM probes used in shear-force feedback, cantilevered NSOM probes incorporate a near 90 bend near the aperture. These tips can then be operated in a tapping-mode arrangement for tip-sample gap regulation, much tapping-mode AFM.(Talley et al. 1996) 

FLUORESCENCE, TOPOGRAPHY AND COMPLIANCE MEASUREMENTS USING TAPPING-MODE NSOM 

As mentioned in the previous section, there are various ways in which feedback may be implemented in order to hold the tip-sample constant during scanning. The shear-force mechanism, where the tip is dithered laterally with respect to the sample surface, is commonly implemented when using straight NSOM probes. For cantilevered NSOM 

These experiments demonstrate the utility and feasibility of simultaneously measuring near-field fluorescence, topography, and compliance using TM-NSOM. As in the previous sections, the addition of compliance contrast permits yet another simultaneous and independent measure of sample properties. 

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While the experimental details involved in implementing NSOM can be found elsewhere [7,8], it is instructive to briefly discuss the two main obstacles that must be overcome in order to conduct NSOM measurements. These revolve around aperture formation and implementing a feedback system for tip-sample distance control. For the former, as in all scanning probe techniques, the quality of the measurements is in large part dictated by the quality of the probe. For the latter, as the schematic in Fig. 1 suggests, high resolution requires that the NSOM probe be maintained within nanometers of the sample surface. 

One approach for combining FRET with NSOM is shown schematically in Figure 2.(Vickery et al. 1999) An acceptor dye of a FRET pair is attached to an NSOM probe while a donor dye is dispersed in the sample. Light exciting the NSOM probe is resonant with the donor dye in both monolayers of the sample, but not with the acceptor dye attached to the tip. Specifically monitoring only the emission from the donor dye provides information about its spatial distribution in the sample, much as in conventional NSOM measurements. However, as the tip nears the sample surface, non-radiative energy transfer from excited donor molecules in the sanq>le to tip-boimd acceptor molecules leads to emission from the acceptor dye. With the proper use of spectral filters, emission... 

Figure 6 shows an example of both the static and dynamic NSOM measurements on PDLC droplets.(Mei et al. 2000) Fig. 6A displays the topography of an encapsulated droplet, measured using the shear-force feedback method for NSOM tip regulation. 

Discrimination of species in complex samples can be made via lifetime measurements using the single-photon timing method coupled to NSOM. 

Near-field scanning optical microscopy is a scanning probe technique that enables optical measurements to be conducted with very high spatial resolution [7-9]. NSOM overcomes the diffraction barrier that restricts the spatial resolution in conventional optical measurements and provides both optical and topographical information on samples with nanometric spatial resolution. 

Model lipid bilayers have long been the focus of research and a vast literature exists discussing various approaches to fabricating bilayer films, measuring their physical properties, and understanding the influence of various constituents on those properties. These structures, ubiquitous in nature, can be formed on substrates where they are amenable for study with surface techniques such as AFM and NSOM. 

Fig. 10. (Top) Schematic of the experimental arrangement for carrying out NSOM/ FRET measurements. An acceptor dye of a FRET pair is attached to the NSOM probe while the sample contains the donor dye in the bottom and top layers of a multi-layer film. The left fluorescence image shows the donor fluorescence and the right the fluorescence from the tip-bound acceptor dye. Reproduced with permission from Ref. [26]. Copyright 1999 The Biophysical Society.

One such approach has recently been developed and shown to enable high-resolution NSOM fluorescence and force measurements on viable cultured human arterial smooth muscle (HASM) cells under buffered conditions [28,29], This approach takes advantage of the nanofabrication capabilities of focused ion beam (FIB) milling to sculpt a light delivery structure into the end of a conventional AFM probe. The FIB technique, which utilizes a focused beam of gallium ions to mill samples with nanometer resolution, was first used by van Hulst and co-workers to modify conventional NSOM probes [30]. They demonstrated an improvement in single molecule fluorescence measurements using... 

Fig. 15 shows the simultaneously measured NSOM fluorescence, topography, and deflection of a viable HASM cell under buffered conditions [28]. These images were collected in contact mode using a hybrid NSOM/AFM probe. The fluorescence signal maps the location of adrenergic receptors in the cellular membrane, which have been fluorescently labeled with prazosin BODIPY-FL. These measurements show that the hybrid NSOM/AFM probes are capable of... 

We applied NSOM to probe the spatial distribution of the fluorescence signal from a fluorescently labeled polyelectrolyte Poly(fluorescein isothiocyanate allylamine hydrochloride) (PAH-FITC) on the surface of Ag nanoparticles. Fig. 2 images correspond to the topography (a), NSOM transmission at 488 nm (b) and NSOM fluorescence measured in the 500-520 nm spectral range (c) of a sample consisting of a monolayer of Ag nanoparticles coated with 20 PE layers and a monolayer of PAH-FITC. 

Near-field scanning optical microscopes (NSOM or SNOM) are mainly used in fluorescence and VIS measurements. They provide optical images with spatial resolution less than the Abbe s limit of Ajl. The high lateral resolution is commonly achieved by using the optical near-field, e. g. in close vicinity of a very narrow fiber tip. Figure 5.16 illustrates the design of a near-field microscope. 

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