DEVELOPMENT OF FEMTOSECOND SCANNING-LASER MICROSCOPY

飞秒扫描激光显微镜的发展

• 批准号: 7349350
• 负责人: DENNIS MICHAEL DACEY
• 金额: $ 13.2万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-05-01 至 2007-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7349350
• 关键词: DEVELOPMENT; FEMTOSECOND; SCANNING; LASER; MICROSCOPY

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The goal of this project is to directly measure synaptic interactions on the dendritic trees of identified color opponent ganglion cells and to test the hypothesis that color opponency is determined by selective connections between cone-signal pathways and ganglion cell dendrites. To achieve this aim we were awarded an NIH shared instrumentation grant in 2001 to acquire a femtosecond, 2-photon scanning laser microscope for measuring light evoked calcium signals in the dendrites of retinal neurons. Over the last two years this facility was custom built, modeled after the system originally developed by Denk and Detwiler at the Max Planck in Heidelberg and has now been extensively tested on intact mouse and salamander retina and most recently on macaque retina. In collaboration with Peter Detwiler and his students we have successfully targeted both midget, parasol and other ganglion cells for whole cell recording in the intact macaque retina by imaging ganglion cell bodies and making whole-cell recordings after mechanical removal of the inner limiting membrane. Midget and parasol cells showed characteristic and long lasting light responses in this recording configuration. The cell bodies and dendritic trees were then intracellularly loaded with Calcium Green via the recording pipette and dendritic branches were targeted for imaging calcium signals in response to diffuse light pulses. As found previously for salamander and mouse ganglion cells, macaque ganglion cell dendrites show clear and rapid changes in calcium concentration in response to light steps. Finally, we have incorporated a new visual stimulus, using a digital light projector, which will permit the application of cone-specific visual stimuli during dendritic recording.

该子项目是利用NIH/NCRR资助的中心赠款提供的资源的许多研究子项目之一。子项目和研究者（PI）可能从另一个NIH来源获得主要资金，因此可以在其他CRISP条目中表示。所列机构为中心，不一定是研究者所在机构。该项目的目标是直接测量识别的颜色对手神经节细胞的树突树上的突触相互作用，并测试假设，即颜色一致性是由锥信号通路和神经节细胞树突之间的选择性连接决定的。为了实现这一目标，我们在2001年获得了美国国立卫生研究院共享仪器补助金，以获得飞秒，双光子扫描激光显微镜，用于测量光诱发的视网膜神经元树突中的钙信号。在过去的两年里，这个设施是定制的，仿照最初由Denk和Detwiler在海德堡的Max Planck开发的系统，现在已经在完整的小鼠和蝾螈视网膜上进行了广泛的测试，最近在猕猴视网膜上进行了测试。与Peter Detwiler和他的学生合作，我们成功地将侏儒、阳伞和其他神经节细胞作为目标，通过对神经节细胞体进行成像，并在机械去除内界膜后进行全细胞记录，在完整的猕猴视网膜中进行全细胞记录。侏儒和阳伞细胞表现出特征和持久的光反应，在此记录配置。然后通过记录移液管将细胞体和树突树细胞内加载钙绿色，并靶向树突分支以响应漫射光脉冲成像钙信号。正如之前在蝾螈和小鼠神经节细胞中发现的那样，猕猴神经节细胞树突在响应光的脚步时表现出明显而快速的钙浓度变化。最后，我们已经纳入了一个新的视觉刺激，使用数字光投影仪，这将允许应用程序的视锥细胞特定的视觉刺激在树突状记录。