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（Ca 2+）作为信号分子在几乎所有细胞类型中，特别是在神经系统中的不可或缺的作用，使其动态分布的研究成为一个独特的相关主题。因此，Ca 2+的成像一直是一个备受关注的话题，然而，只有在过去的十年里，才出现了遗传编码Ca 2+指标（GECI）。遗传方法的关键优势之一是通过细胞机制不间断地从头产生指示物。这使得在体内纵向动物研究神经元动力学。虽然指示剂提供了良好的时间精度，但它们的空间分辨率受到常规荧光显微镜的光学分辨率的边界的限制。同样，显微镜的视野通常非常有限，无法在组织或器官水平上测量Ca 2+动力学。因此，可以说，在天平的两端都需要合适的成像方法和定制的指示器。在仪器方面，最近有两个新的发展克服了这些限制：第一，在微观前沿，分辨率限制已经被超分辨率（SR）荧光成像技术的进步所淘汰;其次，光学方法的深度限制被光声学（OA）的发展所克服，光声学（OA）使得能够实现厘米深度的高分辨率实时体内成像，容易地允许整个动物成像。遗传编码的光可切换标记对这两种技术都是关键的：对于SR，标记在两种状态之间的所需转变可以通过荧光和非荧光状态之间的光切换来实现。对于OA成像，相同的光开关允许调制标记的信号，用于随后从背景中锁定分离;因此，提供了对比度噪声（CNR）的关键提升，并能够检测活体动物中的低细胞数量。因此，我们建议利用光开关蛋白质在构建转基因Ca 2+指示剂中对两种模式的革命性益处。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Ca 2+分布，并将OA中的CNR增加到使整个动物体内Ca 2+成像可行的水平。聚焦于绿色荧光蛋白作为SR定制的rsGECIs的模板和用于OA定制的rsGECIs的近红外蛋白，该提案的目标是构建rsGECIs，其将在设计转基因指示剂中原型化使用光开关，并为该概念的进一步发展提供支架。RsGECIs将有助于研究由Ca 2+梯度调节的广泛过程，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剂的设计提供蓝图，从而实现从纳米级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