DEVELOPMENT OF FEMTOSECOND SCANNING-LASER MICROSCOPY

飞秒扫描激光显微镜的发展

• 批准号: 8172738
• 负责人: DENNIS MICHAEL DACEY
• 金额: $ 15.51万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-05-01 至 2011-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8172738
• 关键词: Calcium; Calcium Signaling; Cells; Characteristics; Collaborations; Color; Computer Retrieval of Information on Scientific Projects Database; Dendrites; Development; Diffuse; Excision; Funding; Goals; Grant; Image; Inner Limiting Membrane; Institution; Laser Scanning Microscopy; Lasers; Light; Macaca; Measures; Mechanics; Microscope; Modeling; Mus; Photons; Physiologic pulse; Research; Research Personnel; Resources; Retina; Retinal Cone; Salamander; Scanning; Signal Pathway; Source; Students; Synapses; System; Testing; Trees; United States National Institutes of Health; Whole-Cell Recordings; calcium green; digital; ganglion cell; neuronal cell body; response; retinal neuron; visual stimulus

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 constructed a femtosecond, 2-photon scanning laser microscope for measuring light evoked calcium signals in the dendrites of retinal neurons. It has been 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 as well as 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 show characteristic and long lasting light responses in this recording configuration. The cell bodies and dendritic trees are then intracellularly loaded with Calcium Green via the recording pipette and dendritic branches are 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.

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目和 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说，它不一定是研究者的机构。 该项目的目标是直接测量已识别颜色对手神经节细胞的树突树上的突触相互作用，并测试颜色对手是由视锥信号通路和神经节细胞树突之间的选择性连接决定的假设。为了实现这一目标，我们构建了一个飞秒、2 光子扫描激光显微镜，用于测量视网膜神经元树突中光诱发的钙信号。它是根据 Denk 和 Detwiler 在海德堡马克斯·普朗克研究所最初开发的系统进行建模的，现已在完整的小鼠和蝾螈视网膜以及猕猴视网膜上进行了广泛的测试。我们与 Peter Detwiler 和他的学生合作，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，成功地针对侏儒、阳伞和其他神经节细胞在完整的猕猴视网膜中进行全细胞记录。侏儒和太阳伞细胞在这种记录配置中表现出特征性且持久的光响应。然后，通过记录移液管将钙绿装载到细胞体和树突树的细胞内，并且树突分支的目标是响应漫射光脉冲对钙信号进行成像。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，我们使用数字光投影仪结合了一种新的视觉刺激，这将允许在树突记录期间应用视锥细胞特异性视觉刺激。