Optical Stimulation Microscope

描述(申请人提供)：生物学和医学发展的一个主要挑战是开发新的方法来研究蛋白质如何在细胞中的复杂网络中发挥作用，以及细胞回路如何运作以协调有机体的功能。这方面的进展需要以分子为重点的动态检测和操纵方法，可以在活的有机体中使用。一种很有吸引力的方法是利用光作为输入和输出来探测体内的信号蛋白。加州大学伯克利分校生物功能光学控制纳米医学开发中心一直处于重新设计对光敏感的蛋白质的前沿，以便在体内选定的细胞中快速打开和关闭。我们已经开发了光门离子通道和受体，并将其用于培养的神经元、视网膜、活体斑马鱼和啮齿类动物的眼睛。光学控制蛋白质的发展为医学研究的应用打开了大门，包括了解神经回路的功能和发展，以及它们与行为的关系，以及重新设计自然细胞，使其对光做出反应来改变状态--这种控制可能在细胞替代疗法中有用，并有助于恢复某些致盲疾病的动物模型的视力。对于细胞培养和体内应用，我们需要在显微镜中精确定位光刺激的能力，该显微镜可以高分辨率成像神经活动的荧光指示器。光刺激需要在空间和时间上灵活，但同时在3D模式下聚焦良好，并具有足够的强度，使我们能够模拟特定神经元、大脑特定区域的活动生理模式，在某些情况下，在行为分析期间。我们要求资金用于光学刺激显微镜，它结合了奥林巴斯FV1000显微镜的高分辨率高质量成像和一种新颖的光学刺激方法，该方法使用LCOS-SLM进行1P和2P以实现3D形状的照明，可以刺激小到树枝状晶体的结构，大到整个细胞或附近的细胞组，最大到分散在视野中的细胞。这一独特的光学刺激显微镜将填补加州大学伯克利分校的一个空白。光学刺激显微镜将使我们能够研究神经回路的发展和功能，移植的神经元整合到成人大脑中进行细胞替代治疗，以及恢复对视网膜的光敏感性，这些视网膜在试图恢复失明模型的视力时失去了光感受器细胞。PHS 398/2590(09/04版，2006年4月4日重新发布)第1页续订格式页 公共卫生相关性：生物学和医学进步的一个主要挑战是开发新的方法来探测完整细胞电路中的蛋白质，以了解它们是如何运作的，并修复它们的功能。我们已经重新设计了对光敏感的蛋白质，这样它们就可以被远程控制，并要求为一种独特的光刺激显微镜提供资金，这种显微镜可以指向光，选择性地操纵活着的有机体中选定神经元的活动。光学刺激显微镜将使我们能够研究神经回路的发展和功能，移植的神经元整合到成人大脑中进行细胞替代治疗，以及恢复对视网膜的光敏感性，这些视网膜在试图恢复失明模型的视力时失去了光感受器细胞。

DESCRIPTION (provided by applicant): A major challenge for the advance of biology and medicine is to develop new ways of studying how proteins function in complex networks in cells and how cellular circuits operate to coordinate functions of the organism. Progress here requires molecularly focused methods for dynamic detection and manipulation that can be used in the living organism. An attractive approach is to use light as both input and output to probe signaling proteins in vivo. The UC Berkeley Nanomedicine Development Center for the Optical Control of Biological Function has been at the forefront of re-engineering proteins to be sensitive to light so that they can be rapidly switched on and off in select cells in vivo. We have developed light-gated ion channels and receptors and used these in cultured neurons, retina, and in vivo zebrafish and in the rodent eye. The development of optically controlled proteins opens the door for applications in medical research, including understanding the function and development of neural circuits, and their relation to behavior, and to re-engineering native cells to switch their state in response to light-a control that could be useful in cell replacement therapies and which could help restore vision in animal models of certain blinding diseases. For applications in both cell culture and in vivo, we require the ability to pinpoint optical stimulation in a microscope that can image fluorescent indicators of neural activity at high resolution. The optical stimulation needs to be spatially and temporally flexible, but at the same time well-focused in 3D and of sufficient intensity to enable us to mimic physiological patterns of activity in specific neurons, in specific regions of the brain, in some cases during behavioral assays. We request funds for an Optical Stimulation Microscope, which combines the high resolution high quality imaging of the Olympus FV1000 microscope with a novel approach to optical stimulation that uses LCOS-SLMs for 1P and 2P to achieve illumination that is shaped in 3D and which can stimulate structures as small as a piece of a dendrite, as large as a whole cell or nearby group of cells and ranging up to cells that are dispersed in the field of view. This unique Optical Stimulation Microscope will fill a void at UC Berkeley. The Optical Stimulation Microscope will enable us to study the development and function of neural circuits, the integration into the adult brain of transplanted neurons for cell replacement therapy and the restoration of light sensitivity to retinas that have lost their photoreceptor cells in attempts to restore vision to models of blindness. PHS 398/2590 (Rev. 09/04, Reissued 4/2006) Page 1 Continuation Format Page PUBLIC HEALTH RELEVANCE: A major challenge for the advance of biology and medicine is to develop new ways of probing proteins in intact cellular circuits to learn how they operate and to repair their function. We have re-engineered proteins to be sensitive to light so that they can be remote-controlled and request funding for a unique Optical Stimulation Microscope that can point light to selectively manipulate the activity of select neurons in the living organism. The Optical Stimulation Microscope will enable us to study the development and function of neural circuits, the integration into the adult brain of transplanted neurons for cell replacement therapy and the restoration of light sensitivity to retinas that have lost their photoreceptor cells in attempts to restore vision to models of blindness.