Structured light temporal focusing depth-resolved wide-field FLIM-FRET for in vivo synaptic imaging

用于体内突触成像的结构光时间聚焦深度分辨宽视场 FLIM-FRET

• 批准号: 10467534
• 负责人: Elly Nedivi
• 金额: $ 28.53万
• 依托单位: MASSACHUSETTS INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-02-10 至 2024-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10467534
• 关键词: 3-Dimensional; Alzheimer' s Disease; Binding; Brain; Cell Culture Techniques; Cells; Chimeric Proteins; Cognition Disorders; Color; Coupled; Dendrites; Detection; Development; Dissociation; Electron Microscopy; Exhibits; Fluorescence; Fluorescence Microscopy; Fluorescence Resonance Energy Transfer; Functional disorder; Goals; Hour; Image; Individual; Label; Life; Light; Measurement; Measures; Mental disorders; Methods; Microscope; Microscopy; Modification; Molecular; Monitor; Morphologic artifacts; Motion; Mus; Neurobiology; Neurodegenerative Disorders; Neurons; Optics; Pattern; Performance; Photons; Physiological; Presynaptic Terminals; Proteins; Pyramidal Cells; Resolution; Scanning; Schizophrenia; Series; Side; Signal Transduction; Site; Specimen; Structure; Symptoms; Synapses; Synaptic Cleft; System; Technology Transfer; Testing; Thick; Time; Tissues; Variant; base; chemical bond; design; detector; fluorophore; imaging approach; improved; in vivo; in vivo evaluation; in vivo monitoring; intermolecular interaction; millisecond; nanometer; nanosecond; novel strategies; parallelization; postsynaptic; relating to nervous system; residence; synaptogenesis; two-photon

In spite of recent progress, our understanding of cognitive disorders remains tenuous. While outward symptoms of neurodegenerative and mental disorders, ranging from Alzheimer’s to schizophrenia, are readily apparent, their underlying cellular mechanisms are unclear. Most, if not all, exhibit some form of synaptic dysfunction and/or circuit abnormality. Unfortunately, our ability to monitor disruptions in synapse or circuit connectivity as they occur in vivo has been hindered by the difficulty of visualizing individual synaptic contacts at sufficient resolution to discern their formation or elimination. The primary challenge for imaging synaptic connections lies in the narrow cleft separation of 20-50 nm that is far below optical resolution. Here we propose a new approach to identify synapse formation and dissociation in vivo by monitoring the distance between pre- and post-synaptic protein pairs using fluorescence resonance energy transfer (FRET). In Specific aim 1 we will develop wide-field depth-resolved FLIM-FRET by implementing De-scattering with Excitation Patterning (DEEP), a wide-field depth resolved imaging approach based on structured light temporal focusing two-photon excitation that we recently demonstrated is compatible with in vivo neural imaging. We propose implementing DEEP for FLIM by using avalanche photodiode arrays with nanosecond gating to simultaneously resolve lifetimes with over two thousand detectors. For testing microscope development, we will express in the mouse brain, in vivo, known intramolecular FRET pairs using our previously developed methods for sparse, multi-fluorophore neuronal labeling. In Specific aim 2 we will generate a series of FRET donor/acceptor molecules fused to variants of the neuroligin-neurexin trans-synaptic partners in a variety of configurations, designed so that donor-acceptor distance is kept within ~5 nm in the bound state for FRET to occur. These fusion constructs will be screened in cultured neurons and selected based on faithful synaptic localization, lack of interference to normal synaptic dynamics, and the presence of strong FRET signal upon fusion partner binding. The in vivo labeling strategy will be a modification of one we recently developed for imaging Layer 2/3 pyramidal cell dendritic arbors and their resident synapses in vivo using a three-color two-photon system, modified to avoid co-expression of donor and acceptor in the same cell. The postsynaptic fusion protein will be co-expressed with a cell fill to visualize a single targeted cell with all its postsynaptic sites. Where these sites contact labeled presynaptic terminals, transsynaptic binding should place the fluorescent donor/acceptor pairs in close proximity, allowing FRET. Selected pairs will then be tested in vivo in the brain for performance in the presence of autofluorescence and signal loss from scattering in deep layers.

尽管最近取得了进展，但我们对认知障碍的理解仍然很薄弱。向外的同时 从阿尔茨海默病到精神分裂症，神经退行性疾病和精神疾病的症状很容易被发现 显然，它们的潜在细胞机制尚不清楚。大多数（如果不是全部）表现出某种形式的突触 功能障碍和/或电路异常。不幸的是，我们监测突触或电路中断的能力 由于难以可视化单个突触接触，阻碍了体内发生的连接性 以足够的分辨率来辨别它们的形成或消除。突触成像的主要挑战 连接位于 20-50 nm 的窄缝间隔中，远低于光学分辨率。在这里我们建议 一种通过监测前突触之间的距离来识别体内突触形成和解离的新方法 和突触后蛋白质对使用荧光共振能量转移（FRET）。在具体目标 1 中，我们将 通过实施激发图案去散射 (DEEP) 开发宽视场深度分辨 FLIM-FRET， 基于结构光时间聚焦双光子激发的宽视场深度分辨成像方法 我们最近证明它与体内神经成像兼容。我们建议实施 DEEP FLIM 通过使用具有纳秒门控的雪崩光电二极管阵列来同时解析寿命 超过两千个探测器。为了测试显微镜的发育，我们将在小鼠大脑中体内表达， 使用我们之前开发的稀疏、多荧光团神经元方法已知的分子内 FRET 对 标签。在具体目标 2 中，我们将生成一系列融合到变体的 FRET 供体/受体分子 Neuroligin-neurexin 跨突触伙伴具有多种构型，其设计使得供体-受体 在束缚态下，距离保持在约 5 nm 以内，以便发生 FRET。这些融合构建体将在 培养的神经元并根据忠实的突触定位进行选择，不干扰正常的突触 动力学，以及融合伴侣结合时存在强 FRET 信号。体内标记策略将 是我们最近开发的一种改进型，用于对第 2/3 层锥体细胞树突乔木及其结构进行成像 使用三色双光子系统体内驻留突触，经过修改以避免供体和 受体在同一细胞内。突触后融合蛋白将与细胞填充物共表达，以可视化单个 目标细胞及其所有突触后位点。这些位点接触标记的突触前末梢、突触间 结合应使荧光供体/受体对非常接近，从而允许 FRET。选定的配对将 然后在大脑中进行体内测试，以检测存在自发荧光和信号丢失的情况下的性能 散射在深层。