Conventional light microscopy is limited in its resolving power by the Rayleigh limit to length scales on the order of 200 nm. On the other hand, spectroscopic techniques such as fluorescence resonance energy transfer cannot be used to measure distances >10 nm, leaving a ''gap'' in the ability of optical techniques to measure distances on the 10-to 100-nm scale. We have previously demonstrated the ability to localize single dye molecules to a precision of 1.5 nm with subsecond time resolution. Here we locate the position of two dyes and determine their separation with 5-nm precision, using the quantal photobleaching behavior of single fluorescent dye molecules. By fitting images both before and after photobleaching of one of the dyes, we may localize both dyes simultaneously and compute their separation. Hence, we have circumvented the Rayleigh limit and achieved nanometer-scale resolution. Specifically, we demonstrate the technique by measuring the distance between single fluorophores separated by 10 -20 nm via attachment to the ends of double-stranded DNA molecules immobilized on a surface. In addition to bridging the gap in optical resolution, this technique may be useful for biophysical or genomic applications, including the generation of super-high-density maps of single-nucleotide polymorphisms.T he advent of single-molecule imaging has enabled a revolution in the measurement of the physical parameters underlying biological processes (1). However, there are limitations on the length scales over which single-molecule imaging can be used to measure distances. Conventional far-field microscopy techniques are limited by the Rayleigh criterion (2) to resolving distances greater than ϳ200 nm. Some recent techniques have been developed to circumvent this limitation (3, 4), but they are technically demanding and at this point of limited applicability to biological systems. Fluorescence resonance energy transfer (FRET) can be used to measure distances much smaller, on the order of a few nanometers. However, because of the strong distance dependence of energy transfer, FRET is limited to measuring distances less than ϳ10 nm (5). As a result, there is a ''gap'' in the resolution attainable by single-color optical spectroscopy, making it difficult to measure separations between 10 and 200 nm. Many biological objects of interest are on this scale, including DNA structures, macromolecular complexes, and motor proteins. Although it is possible to measure distances on these scales by using two or more dyes of different colors (6, 7), this technique presents its own problems, such as how to achieve heterogeneous labeling, and calibration of the distance registration between different color wide-field images.Recently, with the introduction of low-noise high-quantumyield charge-coupled device (CCD) cameras, it has become practical to localize individual fluorescent dyes at subwavelength scales (8-10). We recently demonstrated the ability to determine the positions of single molecules at room temperature with a precision of 1...

The processing, intrinsic microstructure and properties of geopolymer materials and geopolymer composites made with basalt fibers (chopped and fiber weaves) have been investigated. Curing of geopolymers was achieved by one of three routes, viz., pressureless curing, warm pressing, and curing in a high pressure autoclave. The materials were fabricated at ambient temperatures up to (40-80°C). Using fiber reinforcement, the bending strength and work of fracture of geopolymer materials have been increased from an average of 2.8 MPa to 10.3 MPa and 0.05 kJ/m 2 to 21.8 kJ/m 2 , respectively. Electron microscopy studies (SEM, TEM/EDS, in situ hot stage TEM) were made of the intrinsic geopolymer. The microstructure of fully reacted geopolymers consists of amorphous nanoparticulates separated by nanopores whose features are of the order of ≤10 nm. The microchemistry frequently observed by TEM/EDS corresponded to a silica (SiO 2 ) to alumina (Al 2 O 3 ) ratio of 4:1. In situ, hot-stage TEM observations made during heating for 4 h up to 1000°C showed that the nanosized microstructure was stable, although continuous evolution of gas, presumably H 2 O, was noticed upon heating. These materials are inorganic polymers cured at ambient temperatures which remain microstructurally stable to above 1000°C.

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