Confocal fluorescence coincidence analysis: An approach to ultra high-throughput screening

Winkler, T.; Kettling, Ulrich; Koltermann, Andre; Eigen, Manfred

doi:10.1073/pnas.96.4.1375

Cited by 134 publications

(80 citation statements)

References 9 publications

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“…Because no pinholes are required, a combination with two-photon imaging, by using the same experimental setup and scanning the excitation beam, seems particularly attractive. Further, the simplicity of the described measurement concept as well as the quality of the obtained data suggest a use of this technique in high-throughput screening where short analysis times per sample are required, such as crosscorrelation setups like RAPID FCS (15) or coincidence analysis such as Confocal Fluorescence Coincidence Analysis (16). Particularly interesting is the idea of extending this concept to multicolor two-photon excitation for simultaneous detection and analysis of more than two fluorescent species.…”

Section: Discussionmentioning

confidence: 99%

“…Dual-color crosscorrelation analysis (12,13), a conceptual modification of FCS using two spectrally separable fluorescent labels, yields a considerable improvement in signal specificity for heterogeneous systems where molecular interactions of different species are to be observed. The simplicity of the measurement process has recently even encouraged biotechnological applications leading to online kinetics of enzymatic reactions (14) and fast enzyme screening applications (15,16). In principle, any molecular association and dissociation, as well as the formation or cleavage of chemical bonds, can be studied simply by simultaneous detection of two spectrally separated fluorophores, following the amplitude of the crosscorrelation function in real time during the reaction.…”

Section: Simultaneous Two-photon Excitation Of Distinct Labels For Dumentioning

confidence: 99%

“…Despite the simplified analysis, the experimental setup, on the other hand, can be rather critical with two laser beams being brought to a perfect and stable overlap within fractions of micrometers. For a few certain dye combinations, excitation laser sources can be operated in multiline mode (16), which yields significant advantages in overall performance. In addition, the chosen dye systems have to fulfill the requirements for ultrasensitive single-molecule detection under the circumstances of dual excitation, i.e., showing not only high quantum yields but also photostability toward either detection wavelength.…”

Section: Simultaneous Two-photon Excitation Of Distinct Labels For Dumentioning

confidence: 99%

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Simultaneous two-photon excitation of distinct labels for dual-color fluorescence crosscorrelation analysis

Heinze

Koltermann

Schwille

2000

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

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174

164

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Confocal fluorescence correlation spectroscopy as a time-averaging fluctuation analysis combining maximum sensitivity with high statistical confidence has proved to be a very versatile and powerful tool for detection and temporal investigation of biomolecules at ultralow concentrations on surfaces, in solutions, and in living cells. To probe the interaction of different molecular species for a detailed understanding of biologically relevant mechanisms, crosscorrelation studies on dual or multiple fluorophore assays with spectrally distinct excitation and emission are particularly promising. Despite the considerable improvement of detection specificity provided by fluorescence crosscorrelation analysis, few applications have so far been reported, presumably because of the practical challenges of properly aligning and controlling the stability of the experimental setup. In this work, we demonstrate that two-photon excitation combined with dual-color fluorescence correlation spectroscopy can be the key to simplifying simultaneous investigations of multiple fluorescent species significantly on a single-molecule scale. Two-photon excitation allows accession of common fluorophores of largely distinct emission by the same excitation wavelength, because differences in selection rules and vibronic coupling can induce considerable shifts between the one-photon and two-photon excitation spectra. The concept of dual-color two-photon fluorescence crosscorrelation analysis is introduced and experimentally demonstrated with an established assay probing the selective cleavage of dual-labeled DNA substrates by restriction endonuclease Eco RI.

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Section: Discussionmentioning

confidence: 99%

Section: Simultaneous Two-photon Excitation Of Distinct Labels For Dumentioning

confidence: 99%

Section: Simultaneous Two-photon Excitation Of Distinct Labels For Dumentioning

confidence: 99%

See 1 more Smart Citation

Simultaneous two-photon excitation of distinct labels for dual-color fluorescence crosscorrelation analysis

Heinze

Koltermann

Schwille

2000

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

Self Cite

174

164

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“…Fluorescence Correlation Spectroscopy (FCS) (15, 16) is a framework for analyzing fluctuations in high-concentration samples and even live cells (17,18). By performing FCS on pairs of fluorescence traces, where each trace reports on a separate fluorophore, Fluorescence Cross-Correlation Spectroscopy (FCCS) (17,(19)(20)(21)(22) identifies the coincident fluctuations that signify bound populations of the two subunits. Performing FCS on three channels would identify populations of intermediates that contain S7, S9, and S19.…”

mentioning

confidence: 99%

Quantitation of ten 30S ribosomal assembly intermediates using fluorescence triple correlation spectroscopy

Ridgeway

Millar²,

Williamson

2012

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

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The self-assembly of bacterial 30S ribosomes involves a large number of RNA folding and RNA-protein binding steps. The sequence of steps determines the overall assembly mechanism and the structure of the mechanism has ramifications for the robustness of biogenesis and resilience against kinetic traps. Thermodynamic interdependencies of protein binding inferred from omission-reconstitution experiments are thought to preclude certain assembly pathways and thus enforce ordered assembly, but this concept is at odds with kinetic data suggesting a more parallel assembly landscape. A major challenge is deconvolution of the statistical distribution of intermediates that are populated during assembly at high concentrations approaching in vivo assembly conditions. To specifically resolve the intermediates formed by binding of three ribosomal proteins to the full length 16S rRNA, we introduce Fluorescence Triple-Correlation Spectroscopy (F3CS). F3CS identifies specific ternary complexes by detecting coincident fluctuations in three-color fluorescence data. Triple correlation integrals quantify concentrations and diffusion kinetics of triply labeled species, and F3CS data can be fit alongside auto-correlation and cross-correlation data to quantify the populations of 10 specific ribosome assembly intermediates. The distribution of intermediates generated by binding three ribosomal proteins to the entire native 16S rRNA included significant populations of species that were not previously thought to be thermodynamically accessible, questioning the current interpretation of the classic omission-reconstitution experiments. F3CS is a general approach for analyzing assembly and function of macromolecular complexes, especially those too large for traditional biophysical methods.fluorescence cross-correlation spectroscopy | fluorescence correlation spectroscopy | RNA-protein interactions T he complete assembly mechanism for the 30S ribosome has remained elusive. Approximate rate constants and orders of binding are known from kinetic experiments, but kinetic profiles for individual protein binding and RNA folding steps are complex and defy simple models (1-8). The fundamentally kinetic process of ribosome assembly is thus still best understood in terms of the thermodynamic (9) maps generated by the Nomura group in the 1970s (10). The Nomura maps documented how the omission of one protein precludes incorporation of others, but only monitored stable protein-RNA interactions that would survive the biochemical purification steps including nonequilibrium ultracentrifugation. Recent RNA protection data suggest that weak protein-RNA interactions form rapidly during assembly (5, 6), raising the question of how completely the map represents the types of intermediates that form during assembly.Nomura dependencies exist between three specific proteins, S7, S9, and S19, that bind the 16S rRNA (Fig. 1A) late in assembly (2, 4-8). In principle, binding of these three proteins to RNA could occur by any and all possible parallel pathways that are il...

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“…9,10 For example, two-photon excitation of up to three dyes has been implemented to simultaneously resolve molecular concentrations and kinetics of three distinctly labeled, interacting species. 8,11 More recently, one-photon excitation has been implemented for FCCS studies, whereby a single laser line has been used to excite two fluorophores with similar excitation but different emission characteristics. 12 Complexes formed from the reactants labeled with different color fluorophores results in a new spectrally distinct species with a correlation trace that can be fit to quantify the kinetics, dynamics, and concentration of this complex.…”

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confidence: 99%

Spectrally Resolved Fluorescence Correlation Spectroscopy Based on Global Analysis

et al. 2008

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Multicolor fluorescence correlation spectroscopy has been recently developed to study chemical interactions of multiple chemical species labeled with spectrally distinct fluorophores. In the presence of spectral overlap, there exists a lower detectability limit for reaction products with multicolor fluorophores. In addition, the ability to separate bound product from reactants allows thermodynamic properties such as dissociation constants to be measured for chemical reactions. In this report, we utilize a spectrally resolved two-photon microscope with single-photon counting sensitivity to acquire spectral and temporal information from multiple chemical species. Further, we have developed a global fitting analysis algorithm that simultaneously analyzes all distinct auto-and cross-correlation functions from 15 independent spectral channels. We have demonstrated that the global analysis approach allows the concentration and diffusion coefficients of fluorescent particles to be resolved despite the presence of overlapping emission spectra.Fluorescence correlation spectroscopy (FCS) was developed to study the temporal correlations of thermodynamic concentration fluctuations in a reactive chemical system and was described in a series of papers by Magde, Webb, and Elson. 1,2 FCS monitors temporal variations of the fluorescence signal in an optically defined focal volume. These intensity fluctuations originate from important dynamic properties, such as diffusion, aggregation, chemical and enzyme kinetics, flow, and active transport. [1][2][3] The amplitude of these fluctuations is proportional to the square root of the number of molecules in the volume. 3,4

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Confocal fluorescence coincidence analysis: An approach to ultra high-throughput screening

Cited by 134 publications

References 9 publications

Simultaneous two-photon excitation of distinct labels for dual-color fluorescence crosscorrelation analysis

Simultaneous two-photon excitation of distinct labels for dual-color fluorescence crosscorrelation analysis

Quantitation of ten 30S ribosomal assembly intermediates using fluorescence triple correlation spectroscopy

Spectrally Resolved Fluorescence Correlation Spectroscopy Based on Global Analysis

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