ArticleBiophysical journal2004
Triple-color coincidence analysis: one step further in following higher order molecular complex formation.
Article in Biophysical journal, 2004. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 28 papers.
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Who cites it
28 citing papers in PubMed.
- Spatial triple-correlation spectroscopy reveals heterotrimer dynamics in live cells.Biophysical journal · 2026Article
- Four-color fluorescence cross-correlation spectroscopy with one laser and one camera.bioRxiv : the preprint server for biology · 2023Article
- Multicolor fluorescence fluctuation spectroscopy in living cells via spectral detection.eLife · 2021Article
- There and back again: from the origin of life to single molecules.European biophysics journal : EBJ · 2018Article
- Characterization of Ternary Protein Systems In Vivo with Tricolor Heterospecies Partition Analysis.Biophysical journal · 2016Article
- Symmetric exchange of multi-protein building blocks between stationary focal adhesions and the cytosol.eLife · 2014Article
- Review
- Pulsed interleaved excitation fluctuation imaging.Biophysical journal · 2013Article
- Quantitation of ten 30S ribosomal assembly intermediates using fluorescence triple correlation spectroscopy.Proceedings of the National Academy of Sciences of the United States of America · 2012Article
- An orange fluorescent protein with a large Stokes shift for single-excitation multicolor FCCS and FRET imaging.Journal of the American Chemical Society · 2012Article
- The spectroscopic basis of fluorescence triple correlation spectroscopy.The journal of physical chemistry. B · 2012Article
- Visualization of Protein Interactions in Living Cells.Self/nonself · 2011Article
- FRET microscopy in 2010: the legacy of Theodor Förster on the 100th anniversary of his birth.Chemphyschem : a European journal of chemical physics and physical chemistry · 2011Review
- Determining antibody stoichiometry using time-integrated fluorescence cumulant analysis.The journal of physical chemistry. B · 2011Article
- Lessons in fluctuation correlation spectroscopy.Annual review of physical chemistry · 2011Review
- Three-color spectral FRET microscopy localizes three interacting proteins in living cells.Biophysical journal · 2010Article
- A practical guide to single-molecule FRET.Nature methods · 2008Review
- Spectrally resolved fluorescence correlation spectroscopy based on global analysis.Analytical chemistry · 2008Article
- Single-molecule biophysics: at the interface of biology, physics and chemistry.Journal of the Royal Society, Interface · 2008Review
- Visualization of protein interactions in living cells.Advances in experimental medicine and biology · 2008Review
Corrections and comments
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Authors and funding
3 authors.
Funding
No grant is acknowledged in the PubMed record.
Abstract
Confocal fluorescence spectroscopy is a versatile method for studying dynamics and interactions of biomolecules in their native environment with minimal interference with the observed system. Analyzing coincident fluctuations induced by single molecule movement in spectrally distinct detection channels, dual-color fluorescence cross-correlation, and coincidence analysis have proven most powerful for probing the formation or cleavage of molecular bonds in real time. The similarity of the optical setup with those used for laser scanning microscopy, as well as the non-invasiveness of the methods, make them easily adaptive for intracellular measurements, to observe the association and dissociation of biomolecules in situ. However, in contrast to standard fluorescence microscopy, where multiple fluorophores can be spectrally resolved, single molecule detection has so far been limited to dual-color detection systems due to the harsh requirements on detection sensitivity. In this study, we show that under certain experimental conditions, employing simultaneous two-photon excitation of three distinct dye species, their successful discrimination indeed becomes possible even on a single molecule level. This enables the direct observation of higher order molecular complex formation in the confocal volume. The theoretical concept of triple-color coincidence analysis is outlined in detail, along with an experimental demonstration of its principles utilizing a simple nucleic acid reaction system.
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