The conformation of DNA molecules tethered to the surface of a microarray may significantly affect the efficiency of hybridization. Although a number of methods have been applied to determine the structure of the DNA layer, they are not very sensitive to variations in the shape of DNA molecules. Here we describe the application of an interferometric technique called spectral selfinterference fluorescence microscopy to the precise measurement of the average location of a fluorescent label in a DNA layer relative to the surface and thus determine specific information on the conformation of the surface-bound DNA molecules. Using spectral self-interference fluorescence microscopy, we have estimated the shape of coiled single-stranded DNA, the average tilt of doublestranded DNA of different lengths, and the amount of hybridization. The data provide important proofs of concept for the capabilities of novel optical surface analytical methods of the molecular disposition of DNA on surfaces. The determination of DNA conformations on surfaces and hybridization behavior provide information required to move DNA interfacial applications forward and thus impact emerging clinical and biotechnological fields.hybridization ͉ microarray ͉ spectroscopy D NA array technology has become a widespread tool in biological research with applications in expression screening, sequencing, and drug discovery, all benefiting greatly by the highly paralleled detection of the technique. One of the defining characteristics of a DNA array is the availability of the single-stranded probes for hybridization with the target. Immobilized molecules located farther away from the solid support are closer to the solution state and are more accessible for contact with dissolved analytes. The surface, especially a hydrophobic one, acts as a shield for probes positioned close to it because of the associated steric factors and lack of diffusion of the bound molecules (1-5). Thus, knowing the physical structure of DNA probes may prove useful not only in the future development and fabrication of microarrays, but also in designing new applications (6).Recently, advances have been made to characterize the structure of surface-bound DNA probes using such optical or contact methods as ellipsometry, optical reflectivity (7, 8), neutron reflectivity (9), x-ray photoelectron spectroscopy (10), FRET (11,12), SPR (13,14), and AFM (15-18). This previous work has helped in visualizing the structure of the surface-bound DNA depending on its density and surface charge. However, most experimental techniques characterize the DNA layer as a single entity (for instance, parameterizing its thickness or density) without examining the specific positions of internal elements of the DNA chain. Spectral selfinterference fluorescence microscopy (SSFM) can measure the vertical position of a fluorescent label above an optically structured silicon chip. We show that locating the label attached to a certain position within a DNA chain provides insight into the shape of DNA molecules boun...

We introduce a new fluorescence microscopy technique that maps the axial position of a fluorophore with subnanometer precision. The interference of the emission of fluorophores in proximity to a reflecting surface results in fringes in the fluorescence spectrum that provide a unique signature of the axial position of the fluorophore. The nanometer sensitivity is demonstrated by measuring the height of a fluorescein monolayer covering a 12-nm step etched in silicon dioxide. In addition, the separation between fluorophores attached to the top or the bottom layer in a lipid bilayer film is determined. We further discuss extension of this microscopy technique to provide resolution of multiple layers spaced as closely as 10 nm for sparse systems.Index Terms-Fluorescence microscopy, interference, spectroscopy, ultra high-optical resolution.

Spontaneous emission of fluorophores located close to a reflecting surface is modified by the interference between direct and reflected waves. The spectral patterns of fluorescent emission near reflecting surfaces can be precisely described with a classical model that considers the relative intensity and polarization state of direct and reflected waves depending on dipole orientation. An algorithm based on the emission model and polynomial fitting built into a software application can be used for fast and efficient analysis of self-interference spectra, yielding information about the location of the emitters with subnanometer precision. Spectral information was used to study thin films of fluorescent substances on surfaces.

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An original technique, Spectral Self-Interference Fluorescence Microscopy (SSFM), can determine the location of fluorescent markers above a reflecting surface with sub-nanometer precision. SSFM was used to resolve the position of a fluorescent marker bound to either the top or the bottom leaflet of a lipid bilayer -the difference in distance is only 4 nm. SSFM is a valuable tool in studying the conformation of DNA molecules immobilized on surfaces. A fluorescent label attached to a DNA molecule tethered to the surface can help elucidate its spatial orientation. This method is based on the fact that spontaneous emission of fluorophores located near a mirror is modified by the interference between direct and reflected waves, which leads to an oscillatory pattern in the emission spectrum. Spectral patterns of emission near surfaces can be precisely described with a classical model that considers the relative intensity and polarization state of direct and reflected waves depending on dipole orientation. An algorithm based on the emission model and polynomial fitting built into a software application can be used for fast and efficient analysis of self-interference spectra yielding information about the location of the emitters with very high precision.

We present a new method of fluorescence imaging, which yields an unprecedented nm scale vertical resolution. The method uses the unique spectral signature of the fluorescent emission intensity above a reflecting surface to determine vertical position unambiguously. Emission from several heights could be resolved by deconvoluting the spectrum, so that three dimensional imaging is possible. The related technique of standing wave microscopy uses the spatial intensity variation for increased resolution. Applications of this technique include intracellular imaging and screening for specific bacteria, virus or proteins, where discrimination between raised fluorescently labeled specifically bound markers from non-specific binding is crucial.The emission spectrum of fluorescent markers is modified in the presence of a reflecting surface. Within the wavelength range of the fluorescent emission, the selfinterference of the emitted photons from the direct and reflected path results in enhanced or suppressed emission (constructive or destructive interference) depending on the height and wavelength.