A methodology, fluorescence-intensity distribution analysis, has been developed for confocal microscopy studies in which the fluorescence intensity of a sample with a heterogeneous brightness profile is monitored. An adjustable formula, modeling the spatial brightness distribution, and the technique of generating functions for calculation of theoretical photon count number distributions serve as the two cornerstones of the methodology. The method permits the simultaneous determination of concentrations and specific brightness values of a number of individual fluorescent species in solution. Accordingly, we present an extremely sensitive tool to monitor the interaction of fluorescently labeled molecules or other microparticles with their respective biological counterparts that should find a wide application in life sciences, medicine, and drug discovery. Its potential is demonstrated by studying the hybridization of 5-(6-carboxytetramethylrhodamine)-labeled and nonlabeled complementary oligonucleotides and the subsequent cleavage of the DNA hybrids by restriction enzymes. In modern fluorescence correlation experiments, with a confocal high-aperture microscope, continuous wave laser excitation, and avalanche photodiode detection, more than 10 5 photons per s are detected routinely from a single photostable dye molecule passing through the focus of the microscope, which is more than two orders of magnitude higher than the background count rate (1). In a similar manner as cells are studied in cell-sorting devices, the method allows one to study single molecules independently of one another. Single molecules diffuse randomly in all three dimensions within the sample; however, each time they become visible, they do not necessarily pass through the center of the focus. Therefore, an event in which a relatively bright molecule enters the periphery of the laser beam only briefly cannot be distinguished from an event in which a dark molecule passes through the focus, because they leave identical traces in terms of detectable photon counts.Fluorescent species with different specific brightnesses can be distinguished, however, by collecting a statistical distribution of the number of photon counts at time intervals of given length. (Specific brightness is a molecular quantity, expressed as the mean count rate per molecule. It is proportional to the molecular absorption cross section and to the fluorescence quantum yield.) The distribution of photon count numbers is used to determine concentrations of molecules of heterogeneous brightness in the sample. We expect this method of sample analysis to be a valuable tool in various disciplines from fundamental research to very specific applications, e.g., drug discovery and diagnostics.Fluorescence-intensity fluctuations caused by random movement of fluorescent molecules into and out of an illuminated sample volume have been studied since fluorescence correlation spectroscopy (FCS) was established 27 years ago (2-4). An initial kind of sample analysis based on determining moments ...
A method of sample analysis is presented which is based on fitting a joint distribution of photon count numbers. In experiments, fluorescence from a microscopic volume containing a fluctuating number of molecules is monitored by two detectors, using a confocal microscope. The two detectors may have different polarizational or spectral responses. Concentrations of fluorescent species together with two specific brightness values per species are determined. The two-dimensional fluorescence intensity distribution analysis (2D-FIDA), if used with a polarization cube, is a tool that is able to distinguish fluorescent species with different specific polarization ratios. As an example of polarization studies by 2D-FIDA, binding of 5'-(6-carboxytetramethylrhodamine) (TAMRA)-labeled theophylline to an anti-theophylline antibody has been studied. Alternatively, if two-color equipment is used, 2D-FIDA can determine concentrations and specific brightness values of fluorescent species corresponding to individual labels alone and their complex. As an example of two-color 2D-FIDA, binding of TAMRA-labeled somatostatin-14 to the human type-2 high-affinity somatostatin receptors present in stained vesicles has been studied. The presented method is unusually accurate among fluorescence fluctuation methods. It is well suited for monitoring a variety of molecular interactions, including receptors and ligands or antibodies and antigens.
Klaua, M.; Ullmann, D.; Barthel, J.; Wulfhekel, W.; Kirschner, J.; Urban, R.; Monchesky, Theodore L.; Enders, Axel; Cochran, John F.; and Heinrich, Brett, "Growth, structure, electronic, and magnetic properties of MgO/Fe(001) Single-crystal epitaxial MgO thin films were grown directly onto high-quality Fe single crystal and Fe whisker substrates and covered with Fe/Au layers. Reflection high-energy electron diffraction and low-energy electron diffraction patterns and scanning tunneling microscopy images showed that the growth of MgO proceeded pseudomorphically in a nearly layer-by-layer mode up to six monolayers. A misfit dislocation network is formed for MgO layers thicker than six monolayers. The thin MgO films were characterized electrically by scanning tunneling spectroscopy. The tunneling barrier in MgO was found to depend on the MgO layer thickness, starting from 2.5 eV at two monolayer thickness to the expected full barrier of MgO of 3.6 eV at six monolayers. A small fraction of the scanned area showed randomly placed spikes in the tunneling conductance. Tunneling I-V curves at the defects showed a lower tunneling barrier than that in the majority of the MgO film. The total tunneling current integrated over areas of 100ϫ100 nm 2 , however, was not dominated by spikes of higher conductance. These local defects in the MgO barrier were neither related to atomic steps on the Fe substrates nor to individual misfit dislocations. Magnetic anisotropies and exchange coupling in Fe/ MgO͑001͒ and Fe/MgO/Fe͑001͒ structures were studied using ferromagnetic resonance and Brillouin light scattering.
We have grown epitaxial single-crystal magnetotunnel junctions using Fe͑001͒ substrates, MgO͑001͒ spacers and Fe top electrodes. We have used scanning tunneling microscopy and atomic force microscopy to measure the tunneling characteristics as a function of position and demonstrated that local tunneling can be obtained such that the buried MgO can be characterized with nm resolution. Local I(V) curves revealed that most of the area had intrinsic tunneling properties corresponding to the proper MgO tunneling barrier. A small fraction of the scanned areas showed localized spikes in the tunneling current which are most likely caused by defects in the MgO.
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