Northern Lights collaboration for better 2-photon probes

描述（由申请人提供）：北方光合作更好的双光子探针将联合收割机的专业知识，四个非常不同的研究人员和实验室从蒙大拿州和加拿大互补的利益和技能，以解决一个基本问题，在成像的活动活脑。现在人们普遍认为，基因编码的荧光生物传感器是唯一适合提供成像大脑中定义细胞活动所需的特异性和多功能性的。同样清楚的是，要对厚切片或整个大脑中的细胞进行成像，有必要转向双光子（非线性）成像，它能更深入地穿透活组织，造成更小的损伤。此外，双光子信号提供了单光子信号所缺乏的额外的分子水平信息。多年来，人们一直致力于开发更好的荧光蛋白和线性成像生物传感器。不幸的是，直接优化探针的2-光子非线性特性已经超出了生物传感器工程师的能力范围，因为目前最先进的2-光子表征的压倒性的复杂性。这是BRAIN计划的一个关键障碍，因为需要具有高亮度和稳定性的鲁棒双光子探针。在这里，我们描述了一个为期三年的计划，以开发一个新的过程和新的仪器，使用现有的超快激光器和先进的实验设计，这将使生物传感器工程师参与大脑项目，以发展下一代的多光子探针。

DESCRIPTION (provided by applicant): The Northern Lights collaboration for better 2-photon probes will combine the expertise of four very different investigators and laboratories from Montana and Canada with complementary interests and skills to tackle a fundamental problem in imaging of the activity of the living brain. It is now well agreed that genetically encoded, fluorescent biosensors are uniquely suited to provide the specificity and versatility necessary to image the activity of defined cells in the brain. It is also clear that to image cells in thick slies or whole brains it will be necessary to turn to 2- photon (nonlinear) imaging, which penetrates deeper into living tissues and causes less damage. Furthermore, 2-photon signals provide additional molecular-level information that 1- photon signals lack. Many years have gone into developing better fluorescent proteins and biosensors for linear imaging. Unfortunately, directly optimizing probes for the 2-photon nonlinear properties has been beyond the reach of biosensor engineers because of the overwhelming complexity of the current state-of-the-art 2-photon characterization. This is a critical impediment for the BRAIN initiative, because robust 2-photon probes with high brightness and stability are needed. Here we describe a three year program to develop a new process and new instruments using available ultrafast lasers and advanced experimental designs that will empower biosensor engineers involved in the BRAIN project to evolve the next generation of multi photon probes.