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.

尽管最近取得了进展，但我们对认知障碍的理解仍然薄弱。而在外在 神经退行性疾病和精神障碍的症状，从阿尔茨海默氏症到精神分裂症，很容易 显然，它们潜在的细胞机制尚不清楚。大多数，如果不是全部，都表现出某种形式的突触 功能障碍和/或电路异常。不幸的是，我们监测突触或回路中断的能力 在活体中发生的连接由于难以可视化单个突触接触而受到阻碍 以足够的分辨率辨别它们的形成或消除。突触成像的主要挑战 连接在于20-50 nm的狭窄裂隙间隔，远远低于光学分辨率。在这里，我们建议 一种通过监测突触形成和分离的距离来识别体内突触形成和解离的新方法 和突触后蛋白对使用荧光共振能量转移(FRET)。在具体目标1中，我们将 通过利用激发图案(DEEP)实现去散射来开发广视场深度分辨薄膜FRET， 基于结构光时间聚焦双光子激发的广视场深度分辨成像方法 我们最近证明了这与体内神经成像是兼容的。我们建议实施深度为 通过使用具有纳秒门控的雪崩光电二极管阵列来同时解决寿命问题 两千多个探测器。为了测试显微镜的发育，我们将在小鼠的大脑中表达，在体内， 已知的分子内FRET对使用我们先前开发的稀疏、多荧光团神经元的方法 贴标签。在特定的目标2中，我们将产生一系列FRET供体/受体分子，这些分子融合到 神经连接素-神经尿素素跨突触伙伴的各种配置，设计使供体-受体 束缚态的距离保持在~5 nm以内，才能发生FRET。这些融合构建物将在 培养神经元并根据准确的突触定位进行选择，对正常突触缺乏干扰 动力学，以及在融合伙伴结合时存在强烈的FRET信号。体内标记策略将 是我们最近开发的一种用于成像层2/3锥体细胞树突状树突及其 体内使用三色双光子系统的常驻突触，经修改以避免供体和 同一细胞中的受体。突触后融合蛋白将与细胞填充共同表达，以可视化单个 靶细胞及其所有突触后部位。这些部位接触标记为突触前终末的地方，跨突触 结合应将荧光供体/受体对放置在非常接近的位置，以允许FRET。选定的配对将 然后在活体大脑中测试在存在自发荧光和信号损失的情况下的表现 散布在深层。