ArticleJournal of biomedical optics2014
Application of phasor plot and autofluorescence correction for study of heterogeneous cell population.
Article in Journal of biomedical optics, 2014. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 16 papers.
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Who cites it
16 citing papers in PubMed.
- Synthesis, characterization and evaluation of new alternative ruthenium complex for dye sensitized solar cells.Scientific reports · 2024Article
- TLR5-Derived, TIR-Interacting Decoy Peptides to Inhibit TLR Signaling.Journal of immunology (Baltimore, Md. : 1950) · 2023Article
- Crystal Growth of Cubic and Hexagonal GaN Bulk Alloys and Their Thermal-Vacuum-Evaporated Nano-Thin Films.Micromachines · 2021Article
- Phasor-based image segmentation: machine learning clustering techniques.Biomedical optics express · 2021Article
- A mouse model of human TLR4 D299G/T399I SNPs reveals mechanisms of altered LPS and pathogen responses.The Journal of experimental medicine · 2021Article
- Frontline Science: Targeting the TLR7 signalosome assembly.Journal of leukocyte biology · 2020Article
- Discriminating different grades of cervical intraepithelial neoplasia based on label-free phasor fluorescence lifetime imaging microscopy.Biomedical optics express · 2020Article
- Targeting the TLR signalosome with TIR domain-derived cell-permeable decoy peptides: the current state and perspectives.Innate immunity · 2020Review
- In vitro and in vivo phasor analysis of stoichiometry and pharmacokinetics using short-lifetime near-infrared dyes and time-gated imaging.Journal of biophotonics · 2019Article
- Structural mapping of fluorescently-tagged, functional nhTMEM16 scramblase in a lipid bilayer.The Journal of biological chemistry · 2018Article
- Blocking TIR Domain Interactions in TLR9 Signaling.Journal of immunology (Baltimore, Md. : 1950) · 2018Article
- Evaluating integrin activation with time-resolved flow cytometry.Journal of biomedical optics · 2018Article
- Decoy peptide targeted to Toll-IL-1R domain inhibits LPS and TLR4-active metabolite morphine-3 glucuronide sensitization of sensory neurons.Scientific reports · 2017Article
- Phasor plotting with frequency-domain flow cytometry.Optics express · 2016Article
- A Decoy Peptide that Disrupts TIRAP Recruitment to TLRs Is Protective in a Murine Model of Influenza.Cell reports · 2015Article
- A method to quantify FRET stoichiometry with phasor plot analysis and acceptor lifetime ingrowth.Biophysical journal · 2015Article
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Authors and funding
3 authors.
Funding
Abstract
Protein-protein interactions in cells are often studied using fluorescence resonance energy transfer (FRET) phenomenon by fluorescence lifetime imaging microscopy (FLIM). Here, we demonstrate approaches to the quantitative analysis of FRET in cell population in a case complicated by a highly heterogeneous donor expression, multiexponential donor lifetime, large contribution of cell autofluorescence, and significant presence of unquenched donor molecules that do not interact with the acceptor due to low affinity of donor-acceptor binding. We applied a multifrequency phasor plot to visualize FRET FLIM data, developed a method for lifetime background correction, and performed a detailed time-resolved analysis using a biexponential model. These approaches were applied to study the interaction between the Toll Interleukin-1 receptor (TIR) domain of Toll-like receptor 4 (TLR4) and the decoy peptide 4BB. TLR4 was fused to Cerulean fluorescent protein (Cer) and 4BB peptide was labeled with Bodipy TMRX (BTX). Phasor displays for multifrequency FLIM data are presented. The analytical procedure for lifetime background correction is described and the effect of correction on FLIM data is demonstrated. The absolute FRET efficiency was determined based on the phasor plot display and multifrequency FLIM data analysis. The binding affinity between TLR4-Cer (donor) and decoy peptide 4BB-BTX (acceptor) was estimated in a heterogeneous HeLa cell population.
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