Time-Integrated Fluorescence Cumulant Analysis and Its Application in Living Cells

Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, New York, USA
Methods in enzymology (Impact Factor: 2.09). 01/2013; 518C:99-119. DOI: 10.1016/B978-0-12-388422-0.00005-4
Source: PubMed


Time-integrated fluorescence cumulant analysis (TIFCA) is a data analysis technique for fluorescence fluctuation spectroscopy (FFS) that extracts information from the cumulants of the integrated fluorescence intensity. It is the first exact theory that describes the effect of sampling time on FFS experiment. Rebinning of data to longer sampling times helps to increase the signal/noise ratio of the experimental cumulants of the photon counts. The sampling time dependence of the cumulants encodes both brightness and diffusion information of the sample. TIFCA analysis extracts this information by fitting the cumulants to model functions. Generalization of TIFCA to multicolor FFS experiment is straightforward. Here, we present an overview of the theory, its implementation, as well as the benefits and requirements of TIFCA. The questions of why, when, and how to use TIFCA will be discussed. We give several examples of practical applications of TIFCA, particularly focused on measuring molecular interaction in living cells.

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    • "For a wide range of univariate stochastic intensities, these formulae have tractable expressions and sufficient statistics can be recovered by means of likelihood methods [3]. In [29], an overview is given on the methods available to analyze the data sampled in photocounting, as for example the photon counting histogram (PCH). Fluorescence cumulant analysis (FCA) is indicated as the first theory that describes the effect of sampling time by measuring the spontaneous intensity fluctuations of fluorescent molecules [20]. "
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    ABSTRACT: In order to tackle parameter estimation of photocounting distributions, polykays of acting intensities are proposed as a new tool for computing photon statistics. As unbiased estimators of cumulants, polykays are computationally feasible thanks to a symbolic method recently developed in dealing with sequences of moments. This method includes the so-called method of moments for random matrices and results to be particularly suited to deal with convolutions or random summations of random vectors. The overall photocounting effect on a deterministic number of pixels is introduced. A random number of pixels is also considered. The role played by spectral statistics of random matrices is highlighted in approximating the overall photocounting distribution when acting intensities are modeled by a non-central Wishart random matrix. Generalized complete Bell polynomials are used in order to compute joint moments and joint cumulants of multivariate photocounters. Multivariate polykays can be successfully employed in order to approximate the multivariate Mendel–Poisson transform. Open problems are addressed at the end of the paper.
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    ABSTRACT: Fluorescence fluctuation spectroscopy (FFS) techniques provide information at the single-molecule level with excellent time resolution. Usually applied at a single spot in a sample, they have been recently extended into imaging formats, referred to as imaging FFS. They provide spatial information at the optical diffraction limit and temporal information in the microsecond to millisecond range. This review provides an overview of the different modalities in which imaging FFS techniques have been implemented and discusses present imaging FFS capabilities and limitations. A combination of imaging FFS and nanoscopy would allow one to record information with the detailed spatial information of nanoscopy, which is ∼20 nm and limited only by fluorophore size and labeling density, and the time resolution of imaging FFS, limited by the fluorescence lifetime. This combination would provide new insights into biological events by providing spatiotemporal resolution at unprecedented levels. Expected final online publication date for the Annual Review of Physical Chemistry Volume 65 is March 31, 2014. Please see for revised estimates.
    No preview · Article · Dec 2013 · Annual Review of Physical Chemistry
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    ABSTRACT: In this contribution we provide an overview of the recent advances allowed by the use of fluorescence microscopy methods in the study of transcriptional processes and their interplay with the chromatin architecture in living cells. Although the use of fluorophores to label nucleic acids dates back at least to about half a century ago,11. LePecq JB, Paoletti C. A fluorescent complex between ethidium bromide and nucleic acids. Physical-chemical characterization. J Mol Biol 1967; 27:87 - 106;; PMID: 6033613 [CrossRef]View all references two recent breakthroughs have effectively opened the way to use fluorescence routinely for specific and quantitative probing of chromatin organization and transcriptional activity in living cells: namely, the possibility of labeling first the chromatin loci and then the mRNA synthesized from a gene using fluorescent proteins. In this contribution we focus on methods that can probe rapid dynamic processes by analyzing fast fluorescence fluctuations.
    No preview · Article · Mar 2014 · Transcription