Ducros Mathieu, Moreaux Laurent, Bradley Jonathan, Tiret Pascale, Griesbeck Oliver, Charpak Serge
INSERM U603, Paris, France.
PLoS One. 2009;4(2):e4418. doi: 10.1371/journal.pone.0004418. Epub 2009 Feb 9.
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.
表达荧光蛋白组合的转基因小鼠的产生极大地有助于报告神经元活动和识别特定神经元群体。因此，需要能够在活体大脑深处以高灵敏度和时间分辨率分离多个重叠荧光发射光谱的方法。在此，我们研究光谱解混在多大程度上解决了这些问题。
方法/主要发现：使用基于荧光共振能量转移（FRET）的报告基因，以及具有同步多通道检测的双光子激光扫描显微镜，我们报告光谱解混在体外和体内均持续提高了FRET信号幅度。我们的方法使我们能够检测到大脑中180 - 250微米深处由气味诱发的FRET瞬变，这是首次在大于几十微米的深度进行体内光谱成像和解混FRET信号的证明。此外，我们确定了通过光谱解混可提高FRET检测的报告基因效率阈值。
结论/意义：我们的方法能够检测活体大脑中深度方向上的微小光谱变化，这对于高效成像表达多种荧光蛋白组合的转基因动物至关重要。
