The Photon Counting Histogram in Fluorescence Fluctuation Spectroscopy

Chen, Yan; Müller, Joachim D.; So, Peter T. C.; Gratton, Enrico

doi:10.1016/s0006-3495(99)76912-2

Cited by 638 publications

(719 citation statements)

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“…Fluorescence Correlation Spectroscopy (FCS) 5,6 and related fluctuation techniques have since the early 1990's become widely used for analyzing biomolecular interactions and fluctuations, in solution and in living cells [7][8][9] . When it comes to analysis of Aβ oligomers in solution, FCS has shown some promise, but these studies report only on large aggregates containing several hundred monomers [10][11][12] …”

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Highly Sensitive FRET-FCS Detects Amyloid β-Peptide Oligomers in Solution at Physiological Concentrations

et al. 2015

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Alzheimer disease (AD) affects around 30 million people, and the number is growing as life expectancy increases around the world. Pathologically, the disease is characterized by intracellular tangles formed by the microtubule-associated protein tau, and extracellular fibrillar deposits called amyloid plaques. The fibrils are composed of the amyloid β-peptide (Aβ), a proteolytic product generated from processing of the amyloid precursor protein (APP). A 40 residues long variant (Aβ40) is produced at high levels, (around 80-90% of all Aβ produced), but it is the longer and more hydrophobic 42-residues variant (Aβ42) that is the dominating species in plaques 1 . Most evidence from experiments in vitro and in transgenic mice over expressing Aβ suggest that oligomeric species, and not the fibrils, formed by Aβ are crucial for AD-

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confidence: 99%

Highly Sensitive FRET-FCS Detects Amyloid β-Peptide Oligomers in Solution at Physiological Concentrations

et al. 2015

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“…Data analysis by photon counting histogram (17,18) or momentrelated techniques (19)(20)(21) are feasible. Here we perform cumulant analysis of the photon counts, because it provides analytical results that are crucial for interpreting the fit parameters of the heterospecies model.…”

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confidence: 99%

Heterospecies partition analysis reveals binding curve and stoichiometry of protein interactions in living cells

Chen

Müller

2010

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

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Measuring the binding curve and stoichiometry of protein complexes in living cells is a prerequisite for quantitative modeling of cellular processes. Dual-color fluorescence fluctuation spectroscopy provides a general framework for detecting protein interactions, but lacks suitable methods for quantifying protein heterointeractions in the cell. We address this challenge by introducing heterospecies partition (HSP) analysis for protein heterointeractions of the type D þ nA ↔ DA n . HSP directly identifies the heterointeracting species from the sample mixture and determines the binding curve and stoichiometry of the protein complex. The HSP method is applied to provide the first direct characterization of the ligand-dependent binding of the retinoic X receptor to the coactivator transcription intermediate factor 2. A previous study based on protein fragments observed a higher binding stoichiometry than biologically expected. We address this difference in stoichiometry by measuring the binding curves of the full-length proteins in living cells. This study provides proof-of-principle experiments that illustrate the potential of HSP as a general and robust analysis tool for the quantitative characterization of protein heterointeractions by dual-color fluorescence fluctuation spectroscopy in living cells. Brightness characterizes the average fluorescence intensity of a single particle and encodes the stoichiometry of a protein complex (5-7). If two fluorescently labeled monomers form a homodimer, the fluorescence intensity of the particle and therefore its brightness doubles. Single-color brightness analysis utilizes this effect to characterize the binding curve and the stoichiometry of protein homocomplexes directly from cellular data (5). An extension of single-color brightness analysis for the identification of protein heterocomplexes has been described (8, 9), but the applicability of the technique is very limited.The desire to study heterointeractions motivated the introduction of dual-color FFS (10, 11). Unlike homointeractions, heterointeractions between two proteins D and A require differently colored labels to distinguish the proteins. Each of the two detection channels primarily receives the signal from one of the two colored labels. Dual-color FFS retains color-information of a species by measuring its brightness in each of the two detection channels. Thus, dual-color brightness analysis could in principle reveal the binding curve and stoichiometry of heteroprotein interactions, because each protein species (such as D, A, DA, DA 2 ) has its own distinct dual-color brightness. Unfortunately, a general analysis method for extracting this information from dual-color brightness data is not available. Because of this limitation, dual-color brightness analysis is currently restricted to the detection of protein heterointeractions without the ability to quantify the reaction.We overcome this shortcoming of the dual-color brightness technique by introducing heterospecies partition (HSP) analysis. HSP isolates the h...

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“…To extract this information, we analyze the statistics of the fluctuation amplitude using different mathematical methods. The PCH (photon counting histogram) method was introduced in 1999 (Chen, Müller, So, & Gratton, 1999). This method is based on measuring the photon counting distribution at one location, similar to the single point FCS experimental approach.…”

Section: Pch and Amplitude Fluctuation Analysismentioning

confidence: 99%