Calcium biosensors for deep-tissue imaging and spectral multiplexing

钙信号传导几乎参与细胞功能的各个方面，特别是神经元。遗传上 由荧光蛋白 (FP) 开发的编码钙指示剂 (GECI) 提供了强大可靠的 神经元活动的读数，包括突触输入的尖峰数量、时间、频率和水平。延伸 GECI 的调色板朝向近红外 (NIR) 光谱范围将有利于深层组织成像，允许 来自表达各种多色神经元指标的多个细胞群的功能成像，并使得 将 NIR GECI 整合到光遗传学实验中。 GECI 与光遗传学的可靠组合 由于光谱的原因，全光电生理学设置中的调制在实践中很难实现 视蛋白执行器的激活光和可用 GECI 的激发光之间的重叠。建立在我们的 分子进化技术以及各种 FP 的工程和表征方面的丰富经验 和基于 FP 的生物传感器，我们建议生成两类新的 GECI，它们在 通过使用从细菌光敏色素开发的 miRFP 系列的新型 NIR FP 来获得 NIR 光谱。不像 与其他由光敏色素设计的 NIR FP 相比，miRFP 在哺乳动物细胞中是单体且明亮的，包括 神经元。计划中的第一类 NIR GECI 将基于之间的比率 FRET 变化 NIR FP 供体和 NIR FP 受体（目标 1）。第二类 GECI 将基于强度变化 单个 NIR FP（目标 2）。为了对神经活动进行灵敏且特异的测量，NIR GECI 将与现代自适应光学成像技术相结合，实现钙测量 在体内具有增强的深度空间和时间分辨率。我们将通过以下方式应用自适应光学校正 将波前传感直接应用于 NIR GECI 双光子体内成像（目标 3）。这将允许非侵入性 检测整个小鼠皮层（1 毫米深度）和细胞中突触分辨率的神经活动 分辨率进一步深入皮层下结构（深度为 1.6 毫米）。 NIR GECI 的大光谱分离 来自可见 GECI 和视蛋白致动器的成像也将允许大量的多色功能成像 大脑中的神经元以及神经回路输入/输出相互作用的阐明。拟议的研究将 提供高要求的深层组织光学探针，可以全面了解神经活动 细胞和全脑水平。

Calcium signaling participates in almost every aspect of cell functioning, specifically in neurons. Genetically encoded calcium indicators (GECIs) developed from fluorescent proteins (FPs) provide a robust reliable readout of neuronal activity including spike number, timing, frequency, and levels of synaptic input. Extending the color palette of GECIs toward near-infrared (NIR) spectral range will facilitate deep-tissue imaging, allow functional imaging from multiple cell populations expressing various multicolor neuronal indicators, and enable to integrate NIR GECIs into optogenetic experiments. Reliable combination of GECIs with optogenetic modulation in all-optical electrophysiology setups has been difficult to achieve in practice due to spectral overlap between activation light of opsin actuators and excitation light of available GECIs. Building upon our molecular evolution technologies and extensive experience in engineering and characterization of various FPs and FP-based biosensors, we propose to generate two new classes of GECIs that are excited and fluoresce in the NIR spectrum by using novel NIR FPs of a miRFP series developed from bacterial phytochromes. Unlike other NIR FPs designed from phytochromes, miRFPs are monomeric and bright in mammalian cells, including neurons. The first class of the planned NIR GECIs will be based on the ratiometric FRET changes between NIR FP donor and NIR FP acceptor (Aim 1). The second class of GECIs will be based on the intensity changes of the single NIR FPs (Aim 2). To perform sensitive and specific measurements of neural activity, the NIR GECIs will be combined with the modern adaptive optics imaging technologies allowing calcium measurements in vivo with enhanced spatial and temporal resolutions at depth. We will apply the adaptive optics correction via direct wavefront sensing to NIR GECI two-photon imaging in vivo (Aim 3). This will allow non-invasive detection of neural activity at synaptic resolution throughout mouse cortex (1 mm depth) and at cellular resolution further into subcortical structures (to 1.6 mm depth). The large spectral separation of NIR GECIs from visible GECIs and opsin actuators will also allow multicolor functional imaging in a large number of neurons in brain and elucidation of the input/output interactions of neural circuits. The proposed research will provide highly demanded deep-tissue optical probes allowing a comprehensive view of neural activity at cellular and whole-brain levels.