In vitro imaging using laser photostimulation with flavoprotein autofluorescence
In vitro imaging using laser photostimulation with flavoprotein autofluorescence
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DOI：
10.1038/nprot.2011.315
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发表时间：
2011-04-01
期刊：
影响因子：
14.8
通讯作者：
Sherman, S. Murray
中科院分区：
文献类型：
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作者：
Theyel, Brian B.;Llano, Daniel A.;Sherman, S. Murray
通过激光光刺激和黄素蛋白自发荧光 (LFPA) 对 300-500 μm 小鼠脑切片进行成像，可以快速、灵敏地绘制神经元连接图。它是通过使用基于紫外激光的谷氨酸光解锁和通过捕获蓝光（类似于 460 nm）激发下发出的绿光（类似于 520 nm）的变化来成像神经元激活来完成的。这种荧光是由氧化形式的黄素蛋白产生的，是代谢活动的量度。与依赖染料加载的成像技术相比，LPFA 具有多种优势。首先，当黄素蛋白成像测量内源信号时，它避免了使用异质负载和潜在细胞毒性的染料。其次，黄素蛋白信号很大（高于基线 1-20%），无需进行平均。第三，光刺激的使用确保了顺向神经元激活，并允许以高精度（类似于 50 μm）快速询问切片的多个刺激位点。在这里，我们描述了将 LPFA 纳入几乎任何切片装置的分步协议，以及如何进行实验。
Imaging of 300-500 mu m mouse brain slices by laser photostimulation with flavoprotein autofluorescence (LFPA) allows the rapid and sensitive mapping of neuronal connectivity. It is accomplished using UV laser-based photo-uncaging of glutamate and imaging neuronal activation by capturing changes in green light (similar to 520 nm) emitted under blue light (similar to 460 nm) excitation. This fluorescence is generated by the oxidized form of flavoprotein and is a measure of metabolic activity. LPFA offers several advantages over imaging techniques that rely on dye loading. First, as flavoprotein imaging measures endogenous signals, it avoids the use of heterogeneously loaded and potentially cytotoxic dyes. Second, flavoprotein signals are large (1-20% above baseline), obviating the need for averaging. Third, the use of photostimulation ensures orthodromic neuronal activation and permits the rapid interrogation of multiple stimulation sites of the slice with a high degree of precision (similar to 50 mu m). Here we describe a step-by-step protocol for the incorporation of LPFA into virtually any slice rig, as well as how to do the experiment.