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Quantitative spectral linear unmixing and ratiometric FRET for live-cell imaging of protein interactions.

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Title: Quantitative spectral linear unmixing and ratiometric FRET for live-cell imaging of protein interactions.
Authors: Prasad S; Cellular Neurophysiology, Center of Physiology, Hannover Medical School, Hannover 30625, Germany. Electronic address: sonal.prasad@liu.se.
Source: Micron (Oxford, England : 1993) [Micron] 2026 May; Vol. 204, pp. 104016. Date of Electronic Publication: 2026 Mar 11.
Publication Type: Journal Article
Language: English
Journal Info: Publisher: Pergamon Press Country of Publication: England NLM ID: 9312850 Publication Model: Print-Electronic Cited Medium: Internet ISSN: 1878-4291 (Electronic) Linking ISSN: 09684328 NLM ISO Abbreviation: Micron Subsets: MEDLINE
Imprint Name(s): Original Publication: Oxford ; New York : Pergamon Press, c1993-
MeSH Terms: Fluorescence Resonance Energy Transfer*/methods , Protein Interaction Mapping*/methods, Microscopy, Confocal/methods ; Biosensing Techniques/methods ; Hyaluronan Receptors/metabolism ; Microscopy, Fluorescence/methods ; Cyclic AMP/metabolism ; Humans ; Signal Transduction ; Protein Binding ; Animals
Abstract: A biophysical imaging strategy based on linear unmixing Förster resonance energy transfer (lux-FRET) for investigating protein-protein interactions and receptor-mediated signaling in live cells is presented. This method utilizes spectral unmixing of FRET signals acquired via confocal laser scanning microscopy (LSM), enabling high-resolution quantification of molecular interactions with both spatial and temporal precision. Applying lux-FRET, receptor-receptor interactions and downstream signaling events, including agonist specificity for 5-HT receptors were examined. Ratiometric Förster resonance energy transfer (FRET) measurements with a genetically encoded cAMP biosensor allowed us to assess biosensor sensitivity to cyclic nucleotides and receptor efficacy. Additionally, physiological interactions between CD44 and 5-HT receptors and the oligomerization state of the 5-HT1A receptor through apparent FRET efficiency analysis was explored. The findings demonstrate the utility of lux-FRET combined with quantitative fluorescence microscopy as a powerful tool for dissecting dynamic signaling mechanisms in live cells. This approach offers broad applicability for researchers studying receptor pharmacology, cellular signaling, and protein interaction dynamics.
(Copyright © 2026 The Authors. Published by Elsevier Ltd.. All rights reserved.)
Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Sonal Prasad reports financial support was provided by Federal Ministry of Education and Research. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Contributed Indexing: Keywords: Lux-FRET; Quantitative confocal microscopy; Ratiometric FRET; Serotonin receptors; Spectral FRET; cAMP biosensor
Substance Nomenclature: 0 (Hyaluronan Receptors)
E0399OZS9N (Cyclic AMP)
Entry Date(s): Date Created: 20260314 Date Completed: 20260710 Latest Revision: 20260710
Update Code: 20260711
DOI: 10.1016/j.micron.2026.104016
PMID: 41830751
Database: MEDLINE
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  Data: Quantitative spectral linear unmixing and ratiometric FRET for live-cell imaging of protein interactions.
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  Data: <searchLink fieldCode="AU" term="%22Prasad+S%22">Prasad S</searchLink>; Cellular Neurophysiology, Center of Physiology, Hannover Medical School, Hannover 30625, Germany. Electronic address: sonal.prasad@liu.se.
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  Data: <i>Original Publication</i>: Oxford ; New York : Pergamon Press, c1993-
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  Data: <searchLink fieldCode="MM" term="%22Fluorescence+Resonance+Energy+Transfer%22">Fluorescence Resonance Energy Transfer*</searchLink>/<searchLink fieldCode="MM" term="%22Fluorescence+Resonance+Energy+Transfer+methods%22">methods</searchLink> <br /><searchLink fieldCode="MM" term="%22Protein+Interaction+Mapping%22">Protein Interaction Mapping*</searchLink>/<searchLink fieldCode="MM" term="%22Protein+Interaction+Mapping+methods%22">methods</searchLink><br /><searchLink fieldCode="MH" term="%22Microscopy%2C+Confocal%22">Microscopy, Confocal</searchLink>/<searchLink fieldCode="MH" term="%22Microscopy%2C+Confocal+methods%22">methods</searchLink> ; <searchLink fieldCode="MH" term="%22Biosensing+Techniques%22">Biosensing Techniques</searchLink>/<searchLink fieldCode="MH" term="%22Biosensing+Techniques+methods%22">methods</searchLink> ; <searchLink fieldCode="MH" term="%22Hyaluronan+Receptors%22">Hyaluronan Receptors</searchLink>/<searchLink fieldCode="MH" term="%22Hyaluronan+Receptors+metabolism%22">metabolism</searchLink> ; <searchLink fieldCode="MH" term="%22Microscopy%2C+Fluorescence%22">Microscopy, Fluorescence</searchLink>/<searchLink fieldCode="MH" term="%22Microscopy%2C+Fluorescence+methods%22">methods</searchLink> ; <searchLink fieldCode="MH" term="%22Cyclic+AMP%22">Cyclic AMP</searchLink>/<searchLink fieldCode="MH" term="%22Cyclic+AMP+metabolism%22">metabolism</searchLink> ; <searchLink fieldCode="MH" term="%22Humans%22">Humans</searchLink> ; <searchLink fieldCode="MH" term="%22Signal+Transduction%22">Signal Transduction</searchLink> ; <searchLink fieldCode="MH" term="%22Protein+Binding%22">Protein Binding</searchLink> ; <searchLink fieldCode="MH" term="%22Animals%22">Animals</searchLink>
– Name: Abstract
  Label: Abstract
  Group: Ab
  Data: A biophysical imaging strategy based on linear unmixing Förster resonance energy transfer (lux-FRET) for investigating protein-protein interactions and receptor-mediated signaling in live cells is presented. This method utilizes spectral unmixing of FRET signals acquired via confocal laser scanning microscopy (LSM), enabling high-resolution quantification of molecular interactions with both spatial and temporal precision. Applying lux-FRET, receptor-receptor interactions and downstream signaling events, including agonist specificity for 5-HT receptors were examined. Ratiometric Förster resonance energy transfer (FRET) measurements with a genetically encoded cAMP biosensor allowed us to assess biosensor sensitivity to cyclic nucleotides and receptor efficacy. Additionally, physiological interactions between CD44 and 5-HT receptors and the oligomerization state of the 5-HT1A receptor through apparent FRET efficiency analysis was explored. The findings demonstrate the utility of lux-FRET combined with quantitative fluorescence microscopy as a powerful tool for dissecting dynamic signaling mechanisms in live cells. This approach offers broad applicability for researchers studying receptor pharmacology, cellular signaling, and protein interaction dynamics.<br /> (Copyright © 2026 The Authors. Published by Elsevier Ltd.. All rights reserved.)
– Name: Abstract
  Label: Competing Interests
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  Data: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Sonal Prasad reports financial support was provided by Federal Ministry of Education and Research. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
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        Value: 10.1016/j.micron.2026.104016
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        Text: English
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      – SubjectFull: Biosensing Techniques methods
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      – SubjectFull: Protein Interaction Mapping methods
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              Text: 2026 May
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