Enhanced resolution in vivo time-lapse imaging system for subcellular CLEM

用于亚细胞 CLEM 的增强分辨率体内延时成像系统

• 批准号: 518284373
• 金额: --
• 依托单位: Technische Universität München (TUM)
• 依托单位国家: 德国
• 项目类别: Major Research Instrumentation
• 财政年份: 2023
• 资助国家: 德国
• 起止时间: 2022-12-31 至 无数据
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/518284373?language=en
• 关键词: Enhanced; resolution; vivo; time; lapse

Correlated light and electron microscopy (CLEM) is a powerful approach to study the subcellular and molecular basis of cellular processes in physiology and pathology. Especially in neural and other complex tissues, where dynamic remodeling at spatial scales of the limit of classical light microscopy– e.g. synaptic plasticity, neuron-glia interactions or organelle trafficking – play key roles in development and disease, such approaches have proven especially necessary and informative. This necessity is underscored by the fact that true superresolution techniques do not routinely achieve full performance inside an intricate tissue environment, and due to sparse or molecular labelling often still require electron microscopy (EM) as a dense structural background. Here, we apply for an enhanced resolution in vivo time-lapse microscopy system that is capable of routinely achieving xy-resolution of ≈ 150 nm at relatively high speed (>1 HZ at full frame/ >10Hz at 512x512 pixel) across a variety of complex tissues, including brain, spinal cord, peripheral nerve and muscle. This system is to be seamlessly embedded into a CLEM workflow starting with time-lapse recordings of subcellular dynamics in situ (e.g. synaptic or glial process dynamics or calcium signaling, cytoskeletal remodeling, organelle transport, vesicle recycling etc.) ending at semi-automated three-dimensional (3D) scanning EM (3D-SEM), including automated tape ultramicrotome-SEM (ATUM-SEM), focussed ion beam SEM (FIB-SEM) and the hybrid, that we recently developed, ATUM-FIB. The acquired instrument will be incorporated into an imaging unit that is used by a range of DFG-funded consortia (including a FOR, CRC/TRR and an Excellence Cluster) and by a core of six research groups, as well as an MSc program that they run, but which will also be made accessible to the larger Munich neuroscience community. This imaging unit comprises a unique set of custom-built and custom-adapted in vivo imaging systems (ranging from whole-brain imaging in larval zebrafish to in vivo imaging in the cortex, spinal cord and peripheral nervous system of murine disease models) and will be tethered to the SyNergy Excellence Cluster’s EM facility via a range of in-house developed correlation approaches – all of these applications supported by permanent expert staff. The aim of this proposal is hence to expand the available scope of high end in vivo imaging approaches to include the routine time-lapse imaging of subcellular dynamics at enhanced resolution followed by further analyses using a range of 3D CLEM techniques.

相关光学和电子显微镜（CLEM）是研究生理和病理中细胞过程的亚细胞和分子基础的有力手段。特别是在神经和其他复杂组织中，在经典光学显微镜的极限空间尺度上的动态重塑-例如突触可塑性，神经元-神经胶质相互作用或细胞器运输-在发育和疾病中起关键作用，这些方法已被证明是特别必要和信息丰富的。这种必要性是由以下事实强调的：真正的超分辨率技术通常不会在复杂的组织环境中实现完全性能，并且由于稀疏或分子标记，通常仍然需要电子显微镜（EM）作为密集的结构背景。在这里，我们申请了一种增强分辨率的体内延时显微镜系统，该系统能够以相对较高的速度（全帧>1 Hz/512 x512像素> 10 Hz）在各种复杂组织（包括脑、脊髓、外周神经和肌肉）上常规实现150 nm的xy分辨率。该系统将无缝嵌入CLEM工作流程，从原位亚细胞动力学的延时记录开始（例如突触或神经胶质过程动力学或钙信号传导、细胞骨架重塑、细胞器运输、囊泡再循环等）。结束于半自动三维（3D）扫描电镜（3D-SEM），包括自动带式超微扫描电镜（ATUM-SEM），聚焦离子束扫描电镜（FIB-SEM）和混合，我们最近开发的，ATUM-FIB。所获得的仪器将被纳入一个成像单元，该单元由DFG资助的一系列财团（包括FOR，CRC/TRR和卓越集群）和六个研究小组的核心以及他们运行的MSC程序使用，但也将被更大的慕尼黑神经科学社区使用。该成像单元包括一套独特的定制和定制的体内成像系统（从斑马鱼幼虫的全脑成像到小鼠疾病模型的皮层、脊髓和外周神经系统的体内成像），并将通过一系列内部开发的相关方法与SyNergy Excellence Cluster的EM设施相连-所有这些应用都由永久专家人员提供支持。因此，本提案的目的是扩大高端体内成像方法的可用范围，以包括在增强分辨率下的亚细胞动力学的常规延时成像，然后使用一系列3D CLEM技术进行进一步分析。