Spectral Unmixing: Analysis of Performance in the Olfactory Bulb In Vivo
PLoS ONE. 2009-02-09; 4(2): e4418
DOI: 10.1371/journal.pone.0004418
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1. PLoS One. 2009;4(2):e4418. doi: 10.1371/journal.pone.0004418. Epub 2009 Feb 9.
Spectral unmixing: analysis of performance in the olfactory bulb in vivo.
Ducros M(1), Moreaux L, Bradley J, Tiret P, Griesbeck O, Charpak S.
Author information:
(1)INSERM U603, Paris, France.
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 microm 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.
DOI: 10.1371/journal.pone.0004418
PMCID: PMC2635473
PMID: 19198655 [Indexed for MEDLINE]