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Spectral Unmixing: Analysis of Performance in the Olfactory Bulb In Vivo

BACKGROUND: The generation of transgenic mice expressing combinations of fluorescent proteins has greatly aided the reporting of activity and identification of specific neuronal populations. Methods capable of separating multiple overlapping fluorescence emission spectra, deep in the living brain, w...

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Detalles Bibliográficos
Autores principales: Ducros, Mathieu, Moreaux, Laurent, Bradley, Jonathan, Tiret, Pascale, Griesbeck, Oliver, Charpak, Serge
Formato: Texto
Lenguaje:English
Publicado: Public Library of Science 2009
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2635473/
https://www.ncbi.nlm.nih.gov/pubmed/19198655
http://dx.doi.org/10.1371/journal.pone.0004418
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author Ducros, Mathieu
Moreaux, Laurent
Bradley, Jonathan
Tiret, Pascale
Griesbeck, Oliver
Charpak, Serge
author_facet Ducros, Mathieu
Moreaux, Laurent
Bradley, Jonathan
Tiret, Pascale
Griesbeck, Oliver
Charpak, Serge
author_sort Ducros, Mathieu
collection PubMed
description BACKGROUND: The generation of transgenic mice expressing combinations of fluorescent proteins has greatly aided the reporting of activity and identification of specific neuronal populations. Methods capable of separating multiple overlapping fluorescence emission spectra, deep in the living brain, with high sensitivity and temporal resolution are therefore required. Here, we investigate to what extent spectral unmixing addresses these issues. METHODOLOGY/PRINCIPAL FINDINGS: Using fluorescence resonance energy transfer (FRET)-based reporters, and two-photon laser scanning microscopy with synchronous multichannel detection, we report that spectral unmixing consistently improved FRET signal amplitude, both in vitro and in vivo. Our approach allows us to detect odor-evoked FRET transients 180–250 µm deep in the brain, the first demonstration of in vivo spectral imaging and unmixing of FRET signals at depths greater than a few tens of micrometer. Furthermore, we determine the reporter efficiency threshold for which FRET detection is improved by spectral unmixing. CONCLUSIONS/SIGNIFICANCE: Our method allows the detection of small spectral variations in depth in the living brain, which is essential for imaging efficiently transgenic animals expressing combination of multiple fluorescent proteins.
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spelling pubmed-26354732009-02-09 Spectral Unmixing: Analysis of Performance in the Olfactory Bulb In Vivo Ducros, Mathieu Moreaux, Laurent Bradley, Jonathan Tiret, Pascale Griesbeck, Oliver Charpak, Serge PLoS One Research Article BACKGROUND: The generation of transgenic mice expressing combinations of fluorescent proteins has greatly aided the reporting of activity and identification of specific neuronal populations. Methods capable of separating multiple overlapping fluorescence emission spectra, deep in the living brain, with high sensitivity and temporal resolution are therefore required. Here, we investigate to what extent spectral unmixing addresses these issues. METHODOLOGY/PRINCIPAL FINDINGS: Using fluorescence resonance energy transfer (FRET)-based reporters, and two-photon laser scanning microscopy with synchronous multichannel detection, we report that spectral unmixing consistently improved FRET signal amplitude, both in vitro and in vivo. Our approach allows us to detect odor-evoked FRET transients 180–250 µm deep in the brain, the first demonstration of in vivo spectral imaging and unmixing of FRET signals at depths greater than a few tens of micrometer. Furthermore, we determine the reporter efficiency threshold for which FRET detection is improved by spectral unmixing. CONCLUSIONS/SIGNIFICANCE: Our method allows the detection of small spectral variations in depth in the living brain, which is essential for imaging efficiently transgenic animals expressing combination of multiple fluorescent proteins. Public Library of Science 2009-02-09 /pmc/articles/PMC2635473/ /pubmed/19198655 http://dx.doi.org/10.1371/journal.pone.0004418 Text en Ducros et al. http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
spellingShingle Research Article
Ducros, Mathieu
Moreaux, Laurent
Bradley, Jonathan
Tiret, Pascale
Griesbeck, Oliver
Charpak, Serge
Spectral Unmixing: Analysis of Performance in the Olfactory Bulb In Vivo
title Spectral Unmixing: Analysis of Performance in the Olfactory Bulb In Vivo
title_full Spectral Unmixing: Analysis of Performance in the Olfactory Bulb In Vivo
title_fullStr Spectral Unmixing: Analysis of Performance in the Olfactory Bulb In Vivo
title_full_unstemmed Spectral Unmixing: Analysis of Performance in the Olfactory Bulb In Vivo
title_short Spectral Unmixing: Analysis of Performance in the Olfactory Bulb In Vivo
title_sort spectral unmixing: analysis of performance in the olfactory bulb in vivo
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2635473/
https://www.ncbi.nlm.nih.gov/pubmed/19198655
http://dx.doi.org/10.1371/journal.pone.0004418
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