Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland.
Nat Protoc. 2012 Jun 28;7(7):1410-25. doi: 10.1038/nprot.2012.072.
Optogenetic approaches allow the manipulation of neuronal activity patterns in space and time by light, particularly in small animals such as zebrafish. However, most techniques cannot control neuronal activity independently at different locations. Here we describe equipment and provide a protocol for single-photon patterned optical stimulation of neurons using a digital micromirror device (DMD). This method can create arbitrary spatiotemporal light patterns with spatial and temporal resolutions in the micrometer and submillisecond range, respectively. Different options to integrate a DMD into a multiphoton microscope are presented and compared. We also describe an ex vivo preparation of the adult zebrafish head that greatly facilitates optogenetic and other experiments. After assembly, the initial alignment takes about one day and the zebrafish preparation takes <30 min. The method has previously been used to activate channelrhodopsin-2 and manipulate oscillatory synchrony among spatially distributed neurons in the zebrafish olfactory bulb. It can be adapted easily to a wide range of other species, optogenetic probes and scientific applications.
光遗传学方法允许通过光在空间和时间上对神经元活动模式进行操纵，特别是在像斑马鱼这样的小动物中。然而，大多数技术不能独立地在不同位置控制神经元活动。在这里，我们描述了一种设备和使用数字微镜器件 (DMD) 对神经元进行单光子模式光刺激的方案。该方法可以创建具有分别在微米和亚毫秒范围内的空间和时间分辨率的任意时空光图案。提出并比较了将 DMD 集成到多光子显微镜中的不同选项。我们还描述了成年斑马鱼头部的离体准备，这极大地促进了光遗传学和其他实验。组装后，初始对准需要大约一天的时间，斑马鱼准备时间<30 分钟。该方法以前曾被用于激活通道视紫红质-2 并操纵斑马鱼嗅球中空间分布的神经元之间的振荡同步性。它可以很容易地适应于广泛的其他物种、光遗传学探针和科学应用。
