Fluorescence intensity distribution analysis

Wright, P.A., Boyd, H.F., Bethell, R.C., Busch, M., Gribbon, P, Kraemer, J., Lopez-Calle, E., Mander, T.H., Winkler, D., and Benson, N., Development of a 1-pL scale assay for mitogen-activated kinase kinase 7 using 2-D fluorescence intensity distribution analysis anisotropy, /. Biomol. Screen., 7, 419, 2002. 

Fluorescence-based detection methods are the most commonly used readouts for HTS as these readouts are sensitive, usually homogeneous and can be readily miniaturised, even down to the single molecule level.7,8 Fluorescent signals can be detected by methods such as fluorescence intensity (FI), fluorescence polarisation (FP) or anisotropy (FA), fluorescence resonance energy transfer (FRET), time-resolved fluorescence resonance energy transfer (TR-FRET) and fluorescence intensity life time (FLIM). Confocal single molecule techniques such as fluorescence correlation spectroscopy (FCS) and one- or two-dimensional fluorescence intensity distribution analysis (ID FID A, 2D FIDA) have been reported but are not commonly used. 

Due to the use of a confocal volume, FCS is particularly suited for miniaturization in HTS and relatively insensitive to auto fluorescent test compounds. Moreover, in compound testing, the small path length of the confocal volume greatly limits any filter effects on fluorescence intensity. As in FP, the requirement for large differences in mass in the assay design is a limitation to the applicability of FCS. However, it can be overcome by methods like Fluorescence Intensity Distribution Analysis (FIDA) or two-colour cross correlation derived from the original FCS concept. 

Reviews listed in Further Reading provide excellent introductions to PCS. Related techniques have been developed to detect other molecular properties. These properties include fluorescence cross-correlation spectroscopy (FCCS) (28) to detect codiffusing fluorophores and photon-counting histograms (PCH) (29), or fluorescence intensity distribution analysis (FIDA) (29) to distinguish fluorescent species according to their brightness. 

P. Kask, K. Palo, D.Ullmann, K. Gall, Fluorescence-intensity distribution analysis and its application in bimolecular detection technology, PNAS 96, 13756-13761(1999)... 

FIDA Fluorescence Intensity Distribution Analysis. Uses the distribu-... 

FILDA Fluorescence Intensity Distribution and Lifetime Analysis. Uses... 

Photon-Counting Histogram. Contains the distribution of the fluorescence intensity of a small number of molecules measured within consecutive time bins. The PCH is the basis of Fluorescence Intensity Distribution Analysis (FIDA). 

Kask, P, Palo, K, Ullmami, D, and Gall, K, Fluorescence-intensity distribution analysis and its application in biomolecular detection technology. Proceedings of the National Academy of Science of the United States of America 96 (1999) 13756-13761. 

Kask, P, Palo, K, Fay, N, Brand, L, Mets, U, UUmann, D, etal., Two-dimensional fluorescence intensity distribution analysis Theory and applications. Biophysical Journal 78 (2000) 1703-1713. 

MAFID moment analysis of the fluorescence intensity distribution MCP micro channel plate... 

In addition to qualitative identification of the elements present, XRF can be used to determine quantitative elemental compositions and layer thicknesses of thin films. In quantitative analysis the observed intensities must be corrected for various factors, including the spectral intensity distribution of the incident X rays, fluorescent yields, matrix enhancements and absorptions, etc. Two general methods used for making these corrections are the empirical parameters method and the fimdamen-tal parameters methods. 

A major limitation of flow cytometric analysis is that it provides data from individual cells at a single point in time and the same cells are not available for further analysis once they have passed through the flow cell of the instrument. Therefore, it is not possible to monitor a given cell over time for changes in fluorescence intensity or distribution of fluorescence signal. Such studies require microinjection of the fluorochrome into individual cells and fluorescence microscopy analysis. 

Azulene has weak absorption in the visible region (near 7000 A) and more intense band systems in the ultraviolet. The first ultraviolet system, which commences at about 3500 A, has been examined in substitutional solid solution in naphthalene (Sidman and McClure, 1956) and in the vapour state (Hunt and Ross, 1962), and can be observed in fluorescence from the vapour (Hunt and Ross, 1956). Theory predicts that the transition is 1Al<-lAl(C2K), i.e. allowed by the electronic selection rules with polarization parallel to the twofold symmetry axis (see, e.g., Ham, 1960 Mofifitt, 1954 Pariser, 1956b). The vibrational analysis shows that the transition is allowed but does not establish the axis of polarization. The intensity distribution among the vibrational bands indicates a small increase in CC bond distance without change in symmetry. 

From Fig. 4 it can be seen that, for finite bandpass detection, one will obtain different fluorescent intensities per emitting molecule depending on the level pumped. This can produce systematic errors in both the determination of absolute concentrations and the use of excitation scans to obtain ground state rotational temperatures (21) Also, the lack of a thermal distribution imposes restrictions on models of and data analysis in optical saturation techniques. 

Participation of both the hydroxide and hydride is found to be the case for both alkalis. The analysis is unsatisfactory with the inclusion of solely the hydroxide or the hydride. An extension of the usual 2 or 3-level atomic model (2,12-14) to incorporate these chemical schemes is indicated in Figs. 1 and 2 and has been analyzed in detail. Assuming sufficient time for the attainment of a steady state distribution, and rapid coupling of the 2Pi/2 and P3/2 states, the model predicts that for sodium under saturated conditions, the fluorescence intensity. If, will vary as... 

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