title: Spectral unmixing: analysis of performance in the olfactory bulb in vivo.
abstract: 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.
  
  
  RESULTS
  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
  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.
authors.affiliation: INSERM U603, Paris, France.
authors.forename: Mathieu
authors.initials: M
authors.lastname: Ducros
authors.forename: Laurent
authors.initials: L
authors.lastname: Moreaux
authors.forename: Jonathan
authors.initials: J
authors.lastname: Bradley
authors.forename: Pascale
authors.initials: P
authors.lastname: Tiret
authors.forename: Oliver
authors.initials: O
authors.lastname: Griesbeck
authors.forename: Serge
authors.initials: S
authors.lastname: Charpak
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D006801:Humans; D051379:Mice; D008822:Mice, Transgenic; D018613:Microscopy, Confocal; D009830:Olfactory Bulb / Q000378:metabolism*; D011993:Recombinant Fusion Proteins
chemical_list: D011993:Recombinant Fusion Proteins