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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.

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