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, 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.
Ducros
Mathieu
M
INSERM U603, Paris, France.
Moreaux
Laurent
L
Bradley
Jonathan
J
Tiret
Pascale
P
Griesbeck
Oliver
O
Charpak
Serge
S
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
D011993:Recombinant Fusion Proteins