Calcium biosensors for deep-tissue imaging and spectral multiplexing

钙信号几乎参与细胞功能的方方面面，特别是在神经元中。从基因上讲 由荧光蛋白(FP)开发的编码钙指示剂(GECI)提供了强大而可靠的 神经元活动的读数，包括突触输入的尖峰数目、时间、频率和水平。扩展 GECI向近红外(NIR)光谱范围调色板将促进深层组织成像，允许 来自表达各种多色神经元指示物的多个细胞群的功能成像，并使 将近红外GECI集成到光遗传学实验中。GECI与光遗传技术的可靠结合 由于光谱的原因，全光电生理装置中的调制在实践中很难实现 视蛋白致动器的激活光和现有GECI的激发光之间的重叠。建立在我们的 分子进化技术和在各种FP的工程和表征方面的丰富经验 和基于FP的生物传感器，我们建议产生两类新的GECI，它们在 使用从细菌光敏色素开发的miRFP系列的新型近红外FP进行的近红外光谱。不像 其他由光敏色素、miRFPs设计的近红外FP在哺乳动物细胞中是单体和明亮的，包括 神经元。计划的第一类近红外GECI将基于以下比率FRET变化 近红外荧光蛋白供体和近红外荧光蛋白受体(目标1)。第二类GECI将基于强度变化 单一近红外光谱分析(目标2)。为了对神经活动进行敏感和具体的测量，NIR GECI将与现代自适应光学成像技术相结合，允许进行钙测量 在活体中，增强了深度的空间和时间分辨率。我们将通过以下方式应用自适应光学校正 活体近红外壁虎双光子成像的波前直接传感(目标3)。这将允许非侵入性 在突触分辨率下检测整个小鼠皮质(1 mm深度)和细胞内的神经活动 分辨率进一步深入皮质下结构(至1.6毫米深度)。近红外GECI的大光谱分离 从可见的GECI和视蛋白执行器还将允许在大量的多色功能成像 大脑中的神经元和阐明神经回路的输入/输出相互作用。拟议的研究将 提供高要求的深层组织光学探头，可在以下位置全面查看神经活动 细胞水平和全脑水平。

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.