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: 10570189
• 负责人: Elly Nedivi
• 金额: $ 16.57万
• 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/10570189
• 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; Maps; 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; Visualization; chemical bond; design; detector; fluorophore; imaging approach; improved; in vivo; in vivo evaluation; in vivo monitoring; intermolecular interaction; millisecond; nanometer; nanosecond; neuroimaging; novel strategies; parallelization; postsynaptic; presynaptic; residence; structural imaging; superresolution microscopy; synaptogenesis; two-photon; ultra high resolution

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.

尽管最近取得了进步，但我们对认知障碍的理解仍然很少。外面 从阿尔茨海默氏症到精神分裂症的神经退行性和精神障碍的症状很容易 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，我们监视突触或电路中断的能力 在体内发生的连通性因可视化单个突触接触的困难而阻碍了它们的连通性 以足够的解决方案来辨别其形成或消除。成像突触的主要挑战 连接在于20-50 nm的狭窄裂缝分离远低于光学分辨率。我们在这里提出 一种新的方法，通过监测预先的距离来鉴定体内突触形成和分离 使用荧光共振能量转移（FRET）和突触后蛋白对。在特定目标1中，我们将 通过使用激发图案（深）实施脱落（深），开发宽大深度分辨的Flim-fret， 基于结构化光临时聚焦的两光子兴奋 我们最近证明的与体内神经成像兼容。我们建议深入实施 通过使用带有纳秒门控的雪崩光电二极管阵列来同时解决寿命 超过两千个探测器。为了测试显微镜的发展，我们将在小鼠大脑，体内表达 使用我们先前开发的稀疏多氟化神经元的方法，已知的分子内特颗粒对 标签。在特定的目标2中，我们将生成一系列融合变体的FRET供体/受体分子 神经素蛋白纽龙反式突触伙伴在各种配置中设计，以便供体知识者 距离保持在约5 nm之内，以使FRET发生。这些融合结构将在 培养的神经元并根据忠实的突触定位选择，对正常突触缺乏干扰 动力学，以及在融合伴侣结合上存在强FRET信号。体内标签策略将 成为我们最近开发的用于成像2/3锥体细胞树突状乔木及其成像层的修改 使用三色的两光子系统在体内居民突触，被修改以避免供体的共表达和 同一细胞中的受体。突触后融合蛋白将与细胞填充共表达，以可视化单个 靶向细胞及其所有突触后部位。这些位点接触标记为突触前终端的地方，透射性 结合应将荧光供体/受体对近距离接近，允许fret。选定对将 然后在大脑中的体内进行测试，以在自动荧光和信号丢失的情况下进行性能 在深层散射。