Fluorescence microscopy is an essential tool in modern biological research. It is a powerful method that allows noninvasive monitoring of specifically labeled targets within living cells, and simultaneous detection of multiple targets using different labels. The spatial resolution in fluorescence microscopy is limited because of the diffraction limit; the resolution in transverse direction is proportional to λ/2NA = λ/2nsinθ (where n is the refractive index in the object space, and θ is the half-angle of the largest cone of rays that can enter or leave the optical system), whereas the longitudinal resolution is given by 2λn/NA2. High-spatial resolution to detect fluorescent molecules below the diffraction limit can be achieved in several ways, such as by increasing the effective numerical aperture (as in 4Pi confocal microscopy) [1], introducing spatial variation in the excitation light creating finer spatial features in the image (as in standing wave microscopy) [2], using multiple-photon fluorescence absorption or emission mechanisms that lead to nonlinear effects in the light field (as in 2-photon microscopy) [3], and by selectively quenching the fluorescence from a focal spot to obtain a very small fluorescing volume (as in stimulated emission depletion microscopy) [4].

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 self-interference 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 double-stranded 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.

In this work we examine two general approaches to subsurface imaging, the first using solid immersion lens technology to optimize the numerical aperture and the second an interferometric spectral fluorescence technique for buried emitters

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 have measured the height of a fluorescent label incorporated into immobilized DNA with sub nm resolution providing previously inaccessible insights into the structure of DNA monolayers on surfaces.

We present a new method of fluorescence imaging, which yields nm-scale axial height determination and ~15 nm axial resolution. The method uses the unique spectral signature of the fluorescent emission intensity well above a reflecting surface to determine vertical position unambiguously. We have demonstrated axial height determination with nm sensitivity by resolving the height difference of fluorescein directly on the surface or on top of streptavidin. While different positions of fluorophores of different color are determined independently with nm precision, resolving the position of two fluorophores of the same color is a more convoluted problem due to the finite spectral emission widow of the fluorophores. Hence, for physically close (<λ/2) fluorophores, it is necessary to collect multiple spectra by independently scanning an excitation standing wave in order to deconvolute the contribution to the spectral pattern from different heights. Moving the excitation standing wave successively enhances or suppresses excitation from different parts of the height distribution, changing the spectral content. This way two fluorophores of the same color can be resolved to better than 20 nm. Design aspects of the dielectric stack for independent excitation wave scanning and limits of deconvolution for an arbitrary height distribution will be discussed.

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.

Summary form only given. We present a new method of fluorescence imaging, which yields an unprecedented vertical resolution. The basic principles are related to an already well-established technique of using spatially modulated emission intensity from standing waves. However, instead of spatial variation, our method uses the unique spectral signature of the fluorescent emission intensity above a reflecting surface to determine vertical position unambiguously. We demonstrate better than 5-nm vertical resolution by clearly distinguishing markers bound directly to the substrate from those bound on top of a single layer of streptavidin. 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.