Fast, flexible algorithm for calculating photon correlations

Laurence, Ted A.; Fore, Samantha; Huser, Thomas

doi:10.1364/ol.31.000829

Cited by 66 publications

(54 citation statements)

References 15 publications

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“…The binning, histogram calculations, histogram fitting with Poissonian distributions, and autocorrelation calculations were done by using a home-developed LabView program. For the calculation of the autocorrelation functions, various bin sizes were tried, including a calculation based on the photon arrival times (40,41). No strong dependence of the results on the bin size or the use of the photon arrival times was observed (SI Appendix, SI Fig.…”

Section: Methodsmentioning

confidence: 99%

The enzyme mechanism of nitrite reductase studied at single-molecule level

Кузнецова

Zauner

Aartsma

et al. 2008

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

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A generic method is described for the fluorescence ''readout'' of the activity of single redox enzyme molecules based on Fö rster resonance energy transfer from a fluorescent label to the enzyme cofactor. The method is applied to the study of copper-containing nitrite reductase from Alcaligenes faecalis S-6 immobilized on a glass surface. The parameters extracted from the single-molecule fluorescence time traces can be connected to and agree with the macroscopic ensemble averaged kinetic constants. The rates of the electron transfer from the type 1 to the type 2 center and back during turnover exhibit a distribution related to disorder in the catalytic site. The described approach opens the door to singlemolecule mechanistic studies of a wide range of redox enzymes and the precise investigation of their internal workings.electron transfer ͉ redox enzyme ͉ Fö rster transfer ͉ nitric oxide ͉ fluorescent label I n the past few years, single-enzyme studies have revealed numerous hidden aspects of enzyme behavior (1). The huge potential of these studies to unravel the intricate kinetics and precise workings of enzymes, which are often hidden within the ensemble properties, is somewhat restricted by current approaches. The majority of existing single-molecule enzymatic assays are based on fluorescence and have been limited to the flavoenzymes, which contain a fluorescent cofactor (2-5), or to enzymes for which a suitable fluorogenic substrate could be designed (6-9). Recently, it was shown how redox enzyme activity in the bulk can be studied by Förster resonance energy transfer (FRET) from an attached fluorescent label to the enzyme cofactor (10, 11). Here, we report, first, how this technique can be successfully applied to study the enzymatic turnover of single surface-confined copper-containing nitrite reductase (NiR) molecules labeled with ATTO 655 using scanning confocal fluorescence microscopy. Second, it is shown how the kinetics of the fluorescence time traces can be connected with the kinetic parameters that describe the ensemble behavior of the enzyme. Finally, the rate of the electron transfer between the types 1 and 2 Cu centers during turnover could be established. The observed distribution of rates is connected to the partial structural disorder in the catalytic site that has been observed in crystallographic studies. ResultsEnzyme Mechanism. Dissimilatory copper-containing nitrite reductase (NiR) from Alcaligenes faecalis S-6 converts nitrite into nitric oxide. The enzyme is a homotrimer, each monomer containing one type 1 and one type 2 copper site (Fig. 1). The type 1 copper accepts an electron from the physiological donor and transfers it to the type 2 copper, where nitrite is reduced to nitric oxide (NO). The midpoint potentials of the types 1 and 2 Cu centers are close, resulting in a redox equilibrium constant for the two sites that is close to 1 (12, 13). Regarding the enzyme mechanism, it has been stated (14, 15) that, after reduction of the type 1 Cu site, the electron is passed on to the type 2...

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Section: Methodsmentioning

confidence: 99%

The enzyme mechanism of nitrite reductase studied at single-molecule level

Кузнецова

Zauner

Aartsma

et al. 2008

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

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“…4, together with least-squares fits to the second-order model of Sect. 2.4, including a noise density n. We also calculated the fluorescence autocorrelation function g(τ) for each trajectory [31]. These are displayed in Fig.…”

Section: Tracking Error and Fluorescence Fluctuationsmentioning

confidence: 99%

Fluctuations in closed-loop fluorescent particle tracking

Berglund¹,

McHale²,

Mabuchi³

2007

Opt. Express

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Abstract:We present a comprehensive theory of closed-loop particle tracking for calculating the statistics of a diffusing fluorescent particle's motion relative to the tracking lock point. A detailed comparison is made between the theory and experimental results, with excellent quantitative agreement found in all cases. A generalization of the theory of (open-loop) fluorescence correlation spectroscopy is developed, and the relationship to previous results is discussed. Two applications of the statistical techniques are given: a method for determining a tracked particle's localization and an algorithm for rapid particle classification based on real-time analysis of the tracking control signal.

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“…Due to the absence of an acceptor-specific laser excitation signal, the contribution to the acceptor signal due to direct excitation of the acceptor by the donor-excitation laser needs to be expressed as a function of the total (γ-corrected) burst size as explained in SIAppendix 9 [30,37]. The corresponding factor, dT, depends only on the dye pair and can be therefore estimated from µs-ALEX measurements (Section 3.6 and SI-Appendix 9).…”

Section: Direct Acceptor Excitation Factormentioning

confidence: 99%

“…The ACFs and CCF of the donor and acceptor channel signals upon donor excitation (photon streams DexDem and DexAem) together with the ACF of the AexAem stream (acceptor excitation, acceptor channel detection) were calculated on a multitau time scale using the recorded arrival times using published algorithms [37]. Because of µs laser alternation, the raw ACFs, ACFALEX(τ), exhibit a periodic modulation, which can be cancelled out by a simple renormalization by the alternation period histograms' ACFs (ACFPeriod(τ)):…”

Section: Single-spot µS-alex Fcsmentioning

confidence: 99%

Multispot single-molecule FRET: high-throughput analysis of freely diffusing molecules

Ingargiola

Lerner

Chung

et al. 2016

Preprint

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We describe an 8-spot confocal setup for high-throughput smFRET assays and illustrate its performance with two characteristic experiments. First, measurements on a series of freely diffusing doubly-labeled dsDNA samples allow us to demonstrate that data acquired in multiple spots in parallel can be properly corrected and result in measured sample characteristics identical to those obtained with a standard single-spot setup. We then take advantage of the higher throughput provided by parallel acquisition to address an outstanding question about the kinetics of the initial steps of bacterial RNA transcription. Our real-time kinetic analysis of promoter escape by bacterial RNA polymerase confirms results obtained by a more indirect route, shedding additional light on the initial steps of transcription.Finally, we discuss the advantages of our multispot setup, while pointing potential limitations of the current single laser excitation design, as well as analysis challenges and their solutions.

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Fast, flexible algorithm for calculating photon correlations

Cited by 66 publications

References 15 publications

The enzyme mechanism of nitrite reductase studied at single-molecule level

The enzyme mechanism of nitrite reductase studied at single-molecule level

Fluctuations in closed-loop fluorescent particle tracking

Multispot single-molecule FRET: high-throughput analysis of freely diffusing molecules

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