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          Institute: MPI für Neurobiologie     Collection: Systems and Computational Neurobiology     Display Documents

ID: 444422.0, MPI für Neurobiologie / Systems and Computational Neurobiology
Spectral Unmixing: Analysis of Performance in the Olfactory Bulb In Vivo
Authors:Ducros, M.; Moreaux, L.; Bradley, J.; Tiret, P.; Griesbeck, O.; Charpak, S.
Date of Publication (YYYY-MM-DD):2009-02-09
Title of Journal:PLoS ONE
Journal Abbrev.:PLoS One
Issue / Number:2
Start Page:[1]
End Page:[10]
Sequence Number of Article:e4418
Review Status:Peer-review
Audience:Not Specified
Abstract / 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 mm 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.
External Publication Status:published
Document Type:Article
Affiliations:MPI für Neurobiologie/Systems and Computational Neurobiology (Borst)/Cellular Dynamics (Griesbeck)
Identifiers:ISI:000265483900015 [ID No:1]
ISSN:1932-6203 [ID No:2]
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