Calcium biosensors for deep-tissue imaging and spectral multiplexing

钙信号几乎参与细胞功能的各个方面，特别是在神经元中。基因 从荧光蛋白（FP）开发的编码钙指示剂（GECIs）提供了一个强大可靠的 神经元活动的读出，包括尖峰数目、定时、频率和突触输入的水平。延伸 GECI的调色板朝向近红外（NIR）光谱范围将促进深层组织成像， 来自表达各种神经元指示物的多个细胞群的功能成像， 将NIR GECIs整合到光遗传学实验中。GECIs与光遗传学的可靠组合 在全光学电生理学装置中的调制在实践中已经难以实现， 视蛋白致动器的激活光与可用GECI的激发光之间的重叠。建立在我们的 分子进化技术和丰富的工程和表征各种FP的经验 和FP为基础的生物传感器，我们建议产生两个新的类GECIs的激发和荧光， 通过使用从细菌光敏色素开发的miRFP系列的新型NIR FP的NIR光谱。不像 由光敏色素设计的其他NIR FP，miRFPs在哺乳动物细胞中是单体和明亮的，包括 神经元第一类计划的近红外GECIs将基于FRET之间的比率变化， NIR FP供体和NIR FP受体（目标1）。第二类GECIs将基于强度变化 单一NIR FP（目标2）。为了对神经活动进行敏感和特异性的测量，NIR GECIs将与现代自适应光学成像技术相结合，以进行钙测量 在体内具有增强的空间和时间分辨率。我们将应用自适应光学校正， 直接波前传感到体内NIR GECI双光子成像（Aim 3）。这将允许非侵入性 在整个小鼠皮层（1 mm深度）和在细胞内的突触分辨率下检测神经活动 分辨率进一步进入皮质下结构（至1.6 mm深度）。近红外GECIs的大光谱分离 从可见的GECIs和视蛋白致动器也将允许在大量的 脑中的神经元和神经回路的输入/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，允许在 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.