Calcium biosensors for deep-tissue imaging and spectral multiplexing

用于深层组织成像和光谱复用的钙生物传感器

• 批准号: 9526574
• 负责人: Vladislav Verkhusha
• 金额: $ 35.78万
• 依托单位: ALBERT EINSTEIN COLLEGE OF MEDICINE, INC
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-08-01 至 2018-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9526574
• 关键词: Action Potentials; Affinity; Bacteria; Biosensor; Brain; Brain Diseases; Brain imaging; Calcium; Calcium Signaling; Cell physiology; Cells; Collection; Color; Complex; Development; Electrophysiology (science); Elements; Engineering; Exhibits; Financial compensation; Fluorescence; Fluorescence Microscopy; Fluorescence Resonance Energy Transfer; Frequencies; Functional Imaging; Head; Hippocampus (Brain); Image; Imaging Techniques; Imaging technology; Kinetics; Lead; Light; Mammalian Cell; Measurement; Microscopy; Modernization; Modification; Molecular Evolution; Monitor; Mus; Mutagenesis; Neurons; Non-Invasive Cancer Detection; Opsin; Optics; Output; Pathology; Peptides; Performance; Phytochrome; Population; Positioning Attribute; Process; Property; Protein Engineering; Proteins; Refractive Indices; Reporting; Research; Research Personnel; Resolution; Series; Signal Transduction; Structure; Synapses; Tail; Technology; Testing; Tissue imaging; Tissues; Transgenic Mice; Validation; Variant; Vertebral column; absorption; adaptive optics; awake; base; calcium indicator; cell type; design; effective therapy; experience; experimental study; fluorescence microscope; high resolution imaging; in vivo; in vivo imaging; in vivo two-photon imaging; neural circuit; neurotransmission; new technology; novel; optoacoustic tomography; optogenetics; promoter; ratiometric; relating to nervous system; scaffold; screening; sensor; temporal measurement; tool; two-photon; voltage

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.

钙信号传导几乎参与细胞功能的各个方面，特别是在神经元中。遗传 由荧光蛋白（FPS）开发的编码的钙指标（GECIS）提供了可靠的可靠 神经元活动的读数，包括峰值数，时机，频率和突触输入水平。扩展 GECIS朝近红外（NIR）光谱范围的调色板将有助于深度组织成像，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，并启用 将NIR GECIS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，在实践中很难实现全光学电生理设置的调节 Opsin致动剂的激活光与可用GECIS的激发光重叠。建立在我们的基础上 分子进化技术和各种FPS的工程和表征丰富的经验 和基于FP的生物传感器，我们建议生成两种新类的GECI，它们激动并在 通过使用由细菌植物色素开发的MiRFP系列的新型NIR FPS的NIR光谱。与众不同 其他由植物色素设计的NIR FPS，miRFPS是单体和明亮的哺乳动物细胞，包括 神经元。计划的NIR GECIS的第一类将基于比率测量方格的变化 NIR FP供体和NIR FP受体（AIM 1）。第二类GECI将基于强度变化 单个NIR FPS（AIM 2）。为了对神经活动进行敏感和特定的测量，NIR GECIS将与现代自适应光学成像技术结合使用，允许钙测量 在体内具有增强的空间和时间分辨率。我们将通过 在体内直接向NIR GECI两光子成像传感（AIM 3）。这将允许无创 在整个小鼠皮层（1 mm深度）和细胞上的突触分辨率下检测神经活性 进一步分解为皮层结构（至1.6 mm深度）。 NIR GECIS的较大光谱分离 从可见的GECIS和OPSIN致动器中也将允许大量的多色功能成像 大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 提供高度要求的深度组织光学探针，可以全面了解神经活动 细胞和全脑水平。