ArticleBiophysical journal2006
Dual-color photon counting histogram analysis of mRFP1 and EGFP in living cells.
Article in Biophysical journal, 2006. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 30 papers.
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Who cites it
30 citing papers in PubMed, 52 citations in OpenAlex.
- Resolving oligomeric states of photoactivatable proteins in living cells via photon counting histogram analysis.iScience · 2025Article
- Benchmarking of novel green fluorescent proteins for the quantification of protein oligomerization in living cells.PloS one · 2023Article
- Quantification of Dark Protein Populations in Fluorescent Proteins by Two-Color Coincidence Detection and Nanophotonic Manipulation.The journal of physical chemistry. B · 2022Article
- Cell cycle-dependent binding between Cyclin B1 and Cdk1 revealed by time-resolved fluorescence correlation spectroscopy.Open biology · 2022Article
- Quantitative Determination of Dark Chromophore Population Explains the Apparent Low Quantum Yield of Red Fluorescent Proteins.The journal of physical chemistry. B · 2020Article
- Two-Color Spatial Cumulant Analysis Detects Heteromeric Interactions between Membrane Proteins.Biophysical journal · 2019Article
- Optimal fluorescent protein tags for quantifying protein oligomerization in living cells.Scientific reports · 2018Article
- Quantifying membrane protein oligomerization with fluorescence cross-correlation spectroscopy.Methods (San Diego, Calif.) · 2018Article
- The Epidermal Growth Factor Receptor Forms Location-Dependent Complexes in Resting Cells.Biophysical journal · 2016Article
- Dopamine Receptor Signaling in MIN6 β-Cells Revealed by Fluorescence Fluctuation Spectroscopy.Biophysical journal · 2016Article
- Bayesian model selection applied to the analysis of fluorescence correlation spectroscopy data of fluorescent proteins in vitro and in vivo.Analytical chemistry · 2015Article
- Golgi sorting regulates organization and activity of GPI proteins at apical membranes.Nature chemical biology · 2014Article
- Imaging methodologies for systems biology.Cell adhesion & migration · 2014Review
- Quantitative measurement of brightness from living cells in the presence of photodepletion.PloS one · 2014Article
- Unraveling transcription factor interactions with heterochromatin protein 1 using fluorescence lifetime imaging microscopy and fluorescence correlation spectroscopy.Journal of biomedical optics · 2013Article
- Time-integrated fluorescence cumulant analysis and its application in living cells.Methods in enzymology · 2013Article
- Fiber laser based two-photon FRET measurement of calmodulin and mCherry-E(0)GFP proteins.Microscopy research and technique · 2012Article
- Factors affecting the quantification of biomolecular interactions by fluorescence cross-correlation spectroscopy.Biophysical journal · 2012Article
- The statistics of protein expression ratios for cellular fluorescence studies.European biophysics journal : EBJ · 2012Article
- Lessons in fluctuation correlation spectroscopy.Annual review of physical chemistry · 2011Review
Corrections and comments
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Authors and funding
3 authors at 1 institution in 1 country.
Funding
Abstract
We investigate the potential of dual-color photon counting histogram (PCH) analysis to resolve fluorescent protein mixtures directly inside cells. Because of their small spectral overlap, we have chosen to look at the fluorescent proteins EGFP and mRFP1. We experimentally demonstrate that dual-color PCH quantitatively resolves a mixture of EGFP and mRFP1 in cells from a single measurement. To mimic the effect of protein association, we constructed a fusion protein of EGFP and mRFP1 (denoted EGFP-mRFP1). Fluorescence resonant energy transfer within the fusion protein alters the dual-channel brightness of the fluorophores. We describe a model for fluorescence resonant energy transfer effects on the brightness and incorporate it into dual-color PCH analysis. The model is verified using fluorescence lifetime measurements. Dual-color PCH analysis demonstrated that not all of the expressed EGFP-mRFP1 fusion proteins contained a fluorescent mRFP1 molecule. Fluorescence lifetime and emission spectra measurements confirmed this surprising result. Additional experiments show that the missing fluorescent fraction of mRFP1 is consistent with a dark state population of mRFP1. We successfully resolved this mixture of fusion proteins with a single dual-color PCH measurement. These results highlight the potential of dual-color PCH to directly detect and quantify protein mixtures in living cells.
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Registered trials
Read under generation 80e0d062 · epoch 390. Bibliography from PubMed, PubMed Central and OpenAlex; grants from NIH RePORTER; trial links from ClinicalTrials.gov; estimates, votes and beliefs from the OpenQuestion graph.