Toward nanometer-scale resolution in fluorescence microscopy using spectral self-interference

Swan, Anna K.; Moiseev, L.; Cantor, Charles R.; Davis, Brynmor J.; Ippolito, S.B.; Karl, W.C.; Goldberg, Bennett B.; Ünlü, M. Selim

doi:10.1109/jstqe.2003.814191

Cited by 38 publications

(30 citation statements)

References 29 publications

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“…The second technique, SSFM (20), is an interferometric technique in fluorescent imaging that analyzes the spectral oscillations emitted by a fluorophore located on a layered reflecting surface and yields a precise position determination (Fig. 1).…”

Section: Resultsmentioning

confidence: 99%

“…At larger distances, even a small shift in wavelength leads to sharp changes in the intensity of emitted light. The result was a shorter distance between interference peaks, about four to five peaks in the typical emission spectrum of an organic dye located 10-15 wavelengths above the mirror (20).…”

Section: Methodsmentioning

confidence: 98%

“…It is a process similar to ellipsometry, except that it works by fitting the spectra of WL reflected from the surface and modified by thin-film interference (20). Measurements were taken from 10 to 20 spots on a chip, usually in a linear pattern.…”

Section: Methodsmentioning

confidence: 99%

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DNA conformation on surfaces measured by fluorescence self-interference

Moiseev¹,

Ünlü

Swan³

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

103

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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...

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 98%

See 1 more Smart Citation

DNA conformation on surfaces measured by fluorescence self-interference

Moiseev¹,

Ünlü

Swan³

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

103

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“…There are several ways in which the diffraction limit can be overcome in fluorescence microscopy due to the specific nature of fluorescence. Such methods include two-photon microscopy, 4Pi confocal microscopy, standing-wave excitation, emission and excitation interferences, stimulated emission depletion (STED) technique, and combined STED with 4Pi microscopy (Swan et al, 2003;Dyba & Hell, 2002;Hell, 2004;Dyba et al, 2005). Optical resolutions as high as 33 nm have been reported (Dyba & Hell, 2002).…”

Section: Sub-wavelength Fluorescence Microscopymentioning

confidence: 99%

Recent Innovative Methods for Characterization of Small Particles

Kohli

2007

Particulate Science and Technology

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Characterization of submicrometer and subnanometer size particles is essential to understanding their interactions and to developing micro-and nanostructures. Because of this importance, many advances and new developments have been made in characterization techniques for particles in this size range. Examples include neutron holography, in situ wet transmission electron microscopy, ultrafast microscopy, magnetic resonance force microscopy for imaging a single electron spin, and high-resolution X-ray crystallography of noncrystalline structures. It is now possible to completely characterize very small particles from 0.1 nm to 100 nm size. Selected recent developments are discussed.

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“…The broader the emission spectrum, the more information is collected and the more precise the height determination. The distance above the mirror can be determined solely from the oscillations within the spectrum 3 . The axial position of the fluorophores is encoded in the spectral oscillations and not in the overall intensity; therefore, variations in fluorophore density, emission intensity, and the excitation field strength will not affect the apparent axial position.…”

Section: Introductionmentioning

confidence: 99%

Biological applications of spectral self-interference

et al. 2004

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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.

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Toward nanometer-scale resolution in fluorescence microscopy using spectral self-interference

Cited by 38 publications

References 29 publications

DNA conformation on surfaces measured by fluorescence self-interference

DNA conformation on surfaces measured by fluorescence self-interference

Recent Innovative Methods for Characterization of Small Particles

Biological applications of spectral self-interference

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