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.

尽管最近取得了一些进展，但我们对认知障碍的理解仍然很薄弱。而离港 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， 显然，其潜在的细胞机制尚不清楚。大多数，如果不是全部的话，表现出某种形式的突触 功能障碍和/或回路异常。不幸的是，我们监测神经突触或神经回路中断的能力 由于难以观察单个突触接触， 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是 连接在于远低于光学分辨率的20-50 nm的窄裂缝分离。在这里我们建议 一种新的方法，以确定突触的形成和解离在体内通过监测之间的距离， 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， 通过实施激发图案化去散射（DEEP）开发宽场深度分辨FLIM-FRET， 基于结构光时间聚焦双光子激发的宽场深度分辨成像方法 我们最近证明了它与体内神经成像兼容。我们建议实施DEEP， FLIM通过使用雪崩光电二极管阵列与纳秒门控同时解决寿命与 超过2000个探测器。为了测试显微镜的发展，我们将在小鼠大脑中表达，在体内， 已知的分子内FRET对使用我们以前开发的稀疏，多荧光团神经元的方法， 标签。在具体目标2中，我们将产生一系列融合到FRET供体/受体分子的变体的FRET供体/受体分子。 neuroligin-neurexin跨突触伙伴在各种配置，设计，使供体-受体 在结合状态下，距离保持在约5 nm内，以便发生FRET。这些融合构建体将在 培养的神经元和选择的基础上忠实的突触定位，缺乏干扰正常的突触 动力学，以及在融合伴侣结合时存在强FRET信号。体内标记策略将 是我们最近开发的用于成像第2/3层锥体细胞树突状细胞及其 使用三色双光子系统，修改以避免供体和受体的共表达， 在同一个细胞中。突触后融合蛋白将与细胞填充物共表达，以可视化单个突触。 所有突触后部位的靶细胞。在这些位点接触标记的突触前末梢的地方， 结合应使荧光供体/受体对紧密靠近，从而允许FRET。选定的对将 然后在脑中体内测试在自体荧光和信号损失存在下的性能， 在深层散射。