Parallel confocal detection of single molecules in real time

Lundquist, P. M.; Zhong, Cheng; Zhao, Peng; Tomaney, A.; Peluso, Paul; Dixon, J; Bettman, Brad; Lacroix, Y.; Kwo, D. P.; McCullough, Etienne; Maxham, Mark; Hester, Kevin; McNitt, Paul; Grey, Donald M; Henríquez, Carlos; Foquet, Mathieu; Turner, Stephen W.; Zaccarin, D.

doi:10.1364/ol.33.001026

Cited by 85 publications

(77 citation statements)

References 12 publications

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“…Selective aluminum passivation for targeted immobilization of single DNA polymerase molecules in nanoapertures is described in Korlach et al (2008). Improved manufacturing techniques of nanoapertures on a large-scale wafer are described in Foquet et al (2008), while progress toward large scale detection of thousands of nanoapertures in parallel are presented in Lundquist et al (2008).…”

Section: Biological Applicationsmentioning

confidence: 99%

Fluorescence fluctuations analysis in nanoapertures: physical concepts and biological applications

Lenne¹,

Rigneault²,

Marguet³

et al. 2008

Histochem Cell Biol

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During the past years, nanophotonics has provided new approaches to study the biological processes below the optical diVraction limit. How single molecules diVuse, bind and assemble can be studied now at the nanometric level, not only in solutions but also in complex and crowded environments such as in live cells. In this context Xuorescence Xuctuations spectroscopy is a unique tool since it has proven to be easy to use in combination with nanostructures, which are able to conWne light in nanometric volumes. We review here recent advances in Xuorescence Xuctuations' analysis below the optical diVraction limit with a special focus on nanoapertures milled in metallic Wlms. We discuss applications in the Weld of single-molecule detection, DNA sequencing and membrane organization, and underscore some potential perspectives of this new emerging technology.

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Section: Biological Applicationsmentioning

confidence: 99%

Fluorescence fluctuations analysis in nanoapertures: physical concepts and biological applications

Lenne¹,

Rigneault²,

Marguet³

et al. 2008

Histochem Cell Biol

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“…Each ZMW consists of an ∼150-nm-diameter metallic aperture that restricts the excitation light to a zeptoliter volume, making possible experiments with near-physiological concentrations (up to 20 μM) of fluorescently labeled ligands (1). Previous advances in nanofabrication (9), surface chemistry (10), and detection instrumentation (11) have led to ZMW-based instrumentation capable of the direct observation of DNA polymerization (12), reverse transcription (13), processive myosin motion (14), and translation by the ribosome (15,16) with multicolor single-molecule detection. However, this sophisticated technology has not been broadly available to the scientific community.…”

mentioning

confidence: 99%

High-throughput platform for real-time monitoring of biological processes by multicolor single-molecule fluorescence

Chen

Dalal

Petrov

et al. 2013

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

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130

162

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Zero-mode waveguides provide a powerful technology for studying single-molecule real-time dynamics of biological systems at physiological ligand concentrations. We customized a commercial zero-mode waveguide-based DNA sequencer for use as a versatile instrument for single-molecule fluorescence detection and showed that the system provides long fluorophore lifetimes with good signal to noise and low spectral cross-talk. We then used a ribosomal translation assay to show real-time fluidic delivery during data acquisition, showing it is possible to follow the conformation and composition of thousands of single biomolecules simultaneously through four spectral channels. This instrument allows high-throughput multiplexed dynamics of single-molecule biological processes over long timescales. The instrumentation presented here has broad applications to single-molecule studies of biological systems and is easily accessible to the biophysical community.D etermining the molecular details of the time evolution of complex multicomponent biological systems requires analysis at the single-molecule level because of their stochastic and heterogeneous nature. Ideally, such experiments would track simultaneously the composition of a biological system (bound ligands, factors, and cofactors) and the conformation of the individual molecules in real time. Single-molecule fluorescence methods, such as total internal reflection fluorescence (TIRF) microscopy, allow the observations of the compositional dynamics (through arrival of fluorescently labeled ligands, factors, or cofactors) and conformational dynamics (through FRET) of single-molecular species. However, these traditional singlemolecule methods are hindered by limitations in maximal fluorescent component concentrations (up to 50 nM) (1), limited simultaneous detection (two to three colors) (2-6), and low throughput (a few hundred molecules at most per experiment) (7). As such, the full potential of single-molecule fluorescence to investigate a range of biological problems under physiologically relevant conditions has not yet been harnessed.Zero-mode waveguides (ZMWs) are small metallic apertures patterned on glass substrates that overcome the concentration restrictions by optically limiting background excitation (8). Each ZMW consists of an ∼150-nm-diameter metallic aperture that restricts the excitation light to a zeptoliter volume, making possible experiments with near-physiological concentrations (up to 20 μM) of fluorescently labeled ligands (1). Previous advances in nanofabrication (9), surface chemistry (10), and detection instrumentation (11) have led to ZMW-based instrumentation capable of the direct observation of DNA polymerization (12), reverse transcription (13), processive myosin motion (14), and translation by the ribosome (15, 16) with multicolor single-molecule detection. However, this sophisticated technology has not been broadly available to the scientific community. Despite multiple efforts to develop ZMW instrumentation, the combined difficulties in fabrica...

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“…However, with notable exceptions (13), such single-molecule experiments require subnanomolar sample concentrations in order to resolve individual molecules, and are thus unable to probe the weaker interactions that are generally implicated in the early stages of macromolecular assembly (14). When samples are measured at higher concentrations and 10-100 molecules are observed at once, the fluorescence intensity data are not as intuitively analyzed but information about linked binding persists in the way the fluorescence fluctuates.…”

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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Parallel confocal detection of single molecules in real time

Cited by 85 publications

References 12 publications

Fluorescence fluctuations analysis in nanoapertures: physical concepts and biological applications

Fluorescence fluctuations analysis in nanoapertures: physical concepts and biological applications

High-throughput platform for real-time monitoring of biological processes by multicolor single-molecule fluorescence

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

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