Resource for Multiphoton Characterization of Genetically-Encoded Probes

双光子激光扫描显微镜为许多项目提供了动力。大脑主动性 因为这是捕捉活的神经网络协同活动的最佳方式。 用荧光蛋白构建的基因编码的活性探针和传感器是 用于标记和记录神经元活体电路的首选工具。然而，为了 到目前为止，几乎所有这些探测器的优化都仅限于单光子区域。 这主要是因为测量定量的双光子吸收特性 需要在光物理和非线性方面拥有更先进的设备和专业知识 光学。在过去的五年里，我们集团非正式地为 蛋白质工程师和神经学家：他们给我们送来了新开发的蛋白质，而我们 仔细表征了它们的2PA性质。虽然这在过去只奏效了一段时间 很少有实验室，大脑倡议推动了更多需要这一技术的实验室 服务到我们现在需要支持来建立资源的程度。 在这个项目中，我们将建立一个独特的资源，用于多光子表征 蒙大拿州立大学校园(蒙大拿州博兹曼)的基因编码探测器 将继续提供表征2PA属性的服务，并 多光子稳定性给了大量的蛋白质工程师和神经学家。 我们计划为调查人员提供非常广泛的进入我们设施的途径 研究与NIH/NINDS的任务和大脑倡议的目标相关。 获得的信息将通过科学出版物和一个 显微镜网站上的。我们将组织年度研讨会，在那里我们将提供 提供有关2PA光物理和测量方法的培训，并提供所有必要的 为愿意在他们的实验室开始进行这种测量的研究人员提供信息。

Two-photon laser scanning microscopy powers many projects in the. BRAIN initiative because it is the best way to capture the concerted activity of living neural networks. Genetically-encoded activity probes and sensors built with fluorescent proteins are the tools of choice for labeling and recording from living circuits of neurons. However, to date, virtually all optimization of these probes has been solely in the one-photon regime. This is mainly because measuring quantitative two-photon absorption properties demands more sophisticated equipment and expertise in photophysics and nonlinear optics. Over the past five years, our group has informally provided this service for protein engineers and neuroscientists: they sent us newly developed proteins and we carefully characterized their 2PA properties. While this has worked in the past for just a few laboratories, the BRAIN initiative has fueled many more laboratories in need of this service to the extent that we now need support to establish a Resource. In this project, we will establish a unique Resource for Multiphoton Characterization of Genetically-Encoded Probes at the Montana State University Campus (Bozeman, MT) that will continue providing the service of characterization of 2PA properties and multiphoton stability to a much large number of protein engineers and neuroscientists. We are planning to provide a very broad access to our facility for Investigators whose research is relevant to the NIH/NINDS mission and to the goals of the BRAIN initiative. The information obtained will be broadly disseminated via scientific publications and one of the microscopy websites. We will organize annual workshops where we will provide training on 2PA photophysics and methods of measurements and provide all necessary information for researchers who were willing to start such measurements in their labs.