Finding a Needle in a Haystack: Identification of EGFP Tagged Neurons during Calcium Imaging by Means of Two-Photon Spectral Separation.
Finding a Needle in a Haystack: Identification of EGFP Tagged Neurons during Calcium Imaging by Means of Two-Photon Spectral Separation.
复制标题
DOI：
10.3389/fnmol.2012.00096
复制
发表时间：
2012
影响因子：
4.8
通讯作者：
Ratto GM
中科院分区：
文献类型：
--
作者：
Brondi M;Sato SS;Rossi LF;Ferrara S;Ratto GM
双光子体内成像和小鼠大脑中特定细胞类型的基因标记的结合是一种强有力的方法，可以完善我们对大脑回路的理解并剖析特定神经类别对皮质功能的贡献。合成钙指示剂是目前可用的细胞活动的最佳荧光报告剂，但它们的光谱特性通常与用于基因标记的荧光蛋白的光谱特性重叠。俄勒冈绿 BAPTA1 和 EGFP 就是这种情况，它们是用于靶向双光子成像的最广泛使用的荧光团。这些分子的发射光谱实际上是相同的，因此无法通过检测器侧的窄带滤光片将它们分离。然而，即使它们的单光子激发光谱非常相似，它们的双光子激发光谱也有显着差异：这里我们展示了如何利用这种差异来分离 EGFP 和俄勒冈绿对总荧光信号的相对贡献。该方法解决了两个不同的问题：在注射了俄勒冈绿的皮质体积中表达 EGFP 的细胞的公正检测，以及 Ca2+ 不敏感荧光背景的计算。后者的数据对于定量比较含有不同浓度 EGFP 的不同细胞之间 Ca2+ 浓度的相对变化至关重要。这种策略可以很容易地扩展到任何具有不同双光子激发光谱的荧光团对。
The combination of two-photon in vivo imaging and genetic labeling of specific cell types in the mouse brain is a powerful method to refine our understanding of brain circuitry and to dissect the contribution of specific neural classes to cortical function. The synthetic calcium indicators are the best fluorescent reporters for cellular activity that are presently available but their spectral proprieties are often overlapped with those of the fluorescent proteins used for genetic labeling. Such is the case of Oregon Green BAPTA1 and EGFP, the most widely used fluorophores for targeted two-photon imaging. The emission spectra of these molecules are virtually identical, precluding their separation by narrow band filters at the detector side. However, even if their one photon excitation spectra are very similar, their two-photon excitation spectra differ significantly: here we show how it is possible to exploit this difference to separate the relative contributions of EGFP and Oregon Green to the total fluorescence signal. This approach addresses two different issues: the unbiased detection of cells expressing EGFP in a cortical volume injected with Oregon Green, and the computation of the Ca2+ insensitive fluorescence background. The latter data is essential for the quantitative comparison of the relative changes in Ca2+ concentration between different cells, containing variable concentrations of EGFP. This strategy can be easily extended to any couple of fluorophores provided that have a different two-photon excitation spectra.
登录
查看更多内容
影响因子：
16.2
作者：
Grienberger, Christine;Konnerth, Arthur
通讯作者：
Konnerth, Arthur
影响因子：
48
作者：
Nimmerjahn, A;Kirchhoff, F;Helmchen, F
通讯作者：
Helmchen, F
影响因子：
18.2
作者：
Halassa MM;Haydon PG
通讯作者：
Haydon PG
影响因子：
3
作者：
Oheim, M;Beaurepaire, E;Charpak, S
通讯作者：
Charpak, S
影响因子：
16.2
作者：
Navarrete, Marta;Araque, Alfonso
通讯作者：
Araque, Alfonso