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

基本信息

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
项目类别：
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&apos 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

项目摘要

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。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）都表现出某种形式的突触。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突触的形成或消除是成像突触的主要挑战。连接位于 20-50 nm 的窄缝间隔中，远低于光学分辨率。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在特定目标 1 中，我们将使用荧光共振能量转移 (FRET) 来检测突触后蛋白质对。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为了测试显微镜的发展，我们将在小鼠大脑中表达，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 对在特定目标 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-受体在结合状态下，距离保持在约 5 nm 以内，以便发生 FRET 融合构建体。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强 FRET 信号的存在。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突触后融合蛋白将与细胞填充物共表达以可视化单个细胞。目标细胞及其所有突触后位点，这些位点与标记的突触前末梢、突触后位点接触。结合应使荧光供体/受体对非常接近，从而允许选定的对进行 FRET。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。