Sense and switch across scales – Prototyping genetically encoded, reversibly switchable indicators for sub-diffraction microscopy and whole animal optoacoustic Ca2+ imaging (Resubmission)

钙离子（Ca2+）作为信号分子在几乎所有细胞类型，特别是在神经系统中不可或缺的作用，使其动态分布的研究奇异相关的主题。因此，Ca2+的成像一直是一个高度感兴趣的话题，然而，只有在过去的十年里，基因可编码的Ca2+指标（GECI）才出现。遗传方法的主要优点之一是细胞机器不间断地从头生产指示物。这使得神经元动力学的体内纵向动物研究成为可能。虽然这些指标提供了良好的时间精度，但它们的空间分辨率受到传统荧光显微复制光学分辨率的限制。同样，显微镜的视野往往是非常有限的排除Ca2+动态测量在组织或器官水平。因此，可以说，在天平的两端都需要合适的成像方法和量身定制的指标。在仪器方面，最近有两个新的发展克服了这些限制：首先，在微观前沿，分辨率限制已经被超分辨率荧光成像技术的进步所淘汰；其次，光声学（OA）的发展克服了光学方法的深度限制，实现了厘米深度的高分辨率实时活体成像，很容易实现全动物成像。基因编码的光切换标签，对这两种技术都是至关重要的：对于SR，两种状态之间所需的标签转换可以通过荧光和非荧光状态之间的光切换来完成。对于OA成像，同样的光开关允许对标签信号进行调制，以便随后与背景锁定分离；因此，在噪声对比（CNR）方面提供了至关重要的提升，并使检测活体动物的低细胞数量成为可能。因此，我们建议利用光开关蛋白在构建转基因Ca2+指标方面的革命性优势。这种基于光开关的GECIs （rsGECIs）将允许以前所未有的空间分辨率研究Ca2+分布，并将OA中的CNR提高到使全动物体内Ca2+成像可行的水平。重点关注绿色荧光蛋白作为SR定制rsgeci的模板，以及近红外蛋白作为OA定制rsgeci的模板，该提案的目标是构建rsgeci，这将为光交换在设计转基因指标中的应用提供原型，并为该概念的进一步发展提供框架。RsGECIs将促进Ca2+梯度调控的广泛过程的研究，如细胞信号传导、代谢调节或发育过程。此外，这项工作将为其他离子或小分子的可切换指示器的设计提供蓝图，从而实现从纳米尺度到整个动物的功能研究

The indispensable role of calcium ions (Ca2+) as signaling molecules in virtually all cell types, and in particular in the nervous system, makes the study of their dynamic distribution a topic of singular relevancy. Thus, imaging of Ca2+ was always a topic of high interest, however, only the last decade saw the rise of genetically encodable Ca2+ indicators (GECI). One of the key advantages of the genetic approach is the uninterrupted de novo production of the indicator by the cellular machinery. This enables in vivo longitudinal animal studies of neuronal dynamics. While the indicators provide good temporal accuracy, their spatial resolution is limited by the boundaries of the optical resolution of conventional fluorescence microcopy. Likewise, the field-of-view of microscopy is frequently very limited precluding measurements of Ca2+ dynamics at the tissue or organ level. Thus, so to speak, on both end of the scale there is the need for suitable imaging methods and tailored indicators. On the instrumentation side, two novel developments recently overcame those limitations: first, at the microscopic frontier, the resolution limit has been made obsolete by the advance of super-resolution (SR) fluorescence imaging techniques; secondly, the depth limitation of optical methods was overcome by the development of optoacoustics (OA) enabling high-resolution real-time in vivo imaging to depth of centimeters, readily allowing whole animal imaging. Genetically encoded photo-switchable labels, are pivotal to both techniques: for SR the required transitions of the label between two states can be accomplished by photo-switching between a fluorescent and non-fluorescent state. For OA imaging, the same photo-switching allows modulation of the labels’ signal for subsequent locked-in separation from background; thus, providing a crucial boost in contrast-to-noise (CNR) and enabling the detection of low cell numbers in living animals. Accordingly, we propose to exploit the revolutionary benefits photo-switchable proteins have for both modalities in the construction of transgene Ca2+ indicators. Such GECIs based on photo-switching (rsGECIs) will allow to study Ca2+ distributions at unprecedented spatial resolution and increase the CNR in OA to a level that makes Ca2+ imaging in whole animals, in vivo feasible. Focusing on green fluorescent proteins as templates for SR tailored rsGECIs and near-infrared proteins for OA tailored rsGECIs the goal of the proposal is to construct rsGECIs that will prototype the use of photo-switching in designing transgene indicators and provide a scaffold for further developments of this concept. RsGECIs will facilitate research of a wide range of processes regulated by Ca2+ gradients like cell signaling, metabolic regulation or developmental processes. Further, this work will provide a blueprint for the design of switchable indicators for other ions or small molecules enabling functional studies from the nanoscale to whole animals