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

相关光学和电子显微镜(CLEM)是研究生理和病理学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，在经典光学显微镜极限的空间尺度上的动态重建-例如突触可塑性、神经元-胶质细胞相互作用或细胞器运输-在发育和疾病中发挥关键作用，这种方法被证明特别必要和信息丰富。真正的超分辨技术在复杂的组织环境中通常不能达到完全的性能，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，这一事实突显了这一必要性。在此，我们申请一种增强的在体时间推移显微镜系统，该系统能够以相对较高的速度(全帧时为1 HZ/10赫兹，像素为512x512)在包括大脑、脊髓、外周神经和肌肉在内的各种复杂组织中常规地实现≈150 nm的XY分辨率。该系统将无缝地嵌入到Clem工作流程中，从原位亚细胞动力学(例如突触或神经胶质过程动力学或钙信号、细胞骨架重塑、细胞器运输、囊泡回收等)的延时记录开始。最后是半自动三维(3D)扫描电子显微镜(3D-SEM)，包括自动带超显微切割机扫描电子显微镜(ATUM-SEM)、聚焦离子束扫描电子显微镜(FIB-SEM)和我们最近开发的混合设备ATUM-FIB。收购的仪器将被整合到一个成像单元中，该单元由一系列DFG资助的财团(包括一个FOR、CRC/TRR和一个卓越集群)和一个由六个研究小组组成的核心小组使用，以及他们运行的一个理科硕士项目，但也将向更大的慕尼黑神经科学界开放。该成像单元包括一套独特的定制和定制的活体成像系统(从斑马鱼幼体的全脑成像到小鼠疾病模型的皮质、脊髓和外周神经系统的活体成像)，并将通过一系列内部开发的相关方法连接到Synergy卓越集群的EM设施-所有这些应用都得到长期专家人员的支持。因此，这项建议的目的是扩大高端活体成像方法的可用范围，将增强分辨率的亚细胞动力学的常规时间推移成像包括在内，然后使用一系列3D Clem技术进行进一步分析。

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.