Inverted confocal live cell imaging microscope

倒置共焦活细胞成像显微镜

• 批准号: 439819241
• 金额: --
• 依托单位: Ruprecht-Karls-Universität Heidelberg
• 依托单位国家: 德国
• 项目类别: Major Research Instrumentation
• 财政年份: 2020
• 资助国家: 德国
• 起止时间: 2019-12-31 至 无数据
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/439819241?language=en
• 关键词: Inverted; confocal; live; cell; imaging

Understanding infectious diseases at single cell level unravels molecular mechanistic insights that could lead to disease treatment and prevention. To this end, our groups have developed new cell biological markers that allow for visualization of previously unknown mechanism within the life cycle of viruses and parasites. Those dynamic mechanisms of infection can only be fully understood when investigated using live cell imaging. Time scales, however, can vary drastically: from seconds and minutes, for viral particle trafficking and parasite motility, to hours and days, for parasite development, viral spread within tissue or cellular stress responses. At the same time these events can unfold at a scale beyond the resolution of classical fluorescence microscopes. Within our proposed projects we aim to fill critical knowledge gaps about long term viral and parasitic infectious mechanisms. This comprises the challenge to image infected cells over a long time with sub-diffraction resolution, while mitigating the cytotoxic effects of light. For this we require a new confocal imaging system to fill this important gap in imaging technologies situated between super-resolution and confocal microscopy.Most of the proposed projects require continuous live cell imaging for 24 hours or more. Currently these time frames are not readily available on our live cell capable microscopes due to extensive usage. Further, malaria parasites and viral host cell, which are studied within the proposed projects, naturally grow under low oxygen pressure. Towards creating imaging conditions with more physiological relevance we require hypoxic incubation conditions to e.g. prevent loss of polarity in hepatocytes or parasite death. A key aspect of physiological long term live cell imaging is reducing phototoxicity but at the same time achieve resolutions slightly below the diffraction limit. Specifically, malaria parasites have been shown to be very susceptible to fluorescent illumination. Therefore, highly sensitive detectors must be used for image acquisition. This however needs to be reconciled with increased spatial resolution to reveal significantly more cellular detail in three dimensions. Particularly, for the small sized malaria parasites the ideal technology would enable us to detect events such as formation of polar rings, chromosome segregation, centrosome duplication, and more. All this has to be achieved while maintaining an imaging speed that allows significant quantitative cell biological analyses.Hence, we want to implement a versatile and sustainable imaging solution that can open new dimension in our understanding of infection biology of those globally relevant pathogens.

在单细胞水平上理解传染病揭示了可能导致疾病治疗和预防的分子机制见解。为此，我们的团队开发了新的细胞生物学标记物，可以可视化病毒和寄生虫生命周期中以前未知的机制。只有使用活细胞成像进行研究时，才能完全理解感染的这些动态机制。然而，时间尺度可能变化很大：从病毒颗粒运输和寄生虫运动的秒和分钟到寄生虫发育、组织内病毒传播或细胞应激反应的小时和天。与此同时，这些事件可以以超出经典荧光显微镜分辨率的尺度展开。在我们提出的项目中，我们的目标是填补有关长期病毒和寄生虫感染机制的关键知识空白。这包括在亚衍射分辨率下长时间成像感染细胞的挑战，同时减轻光的细胞毒性效应。为此，我们需要一种新的共焦成像系统来填补超分辨率和共焦显微镜之间的成像技术的这一重要空白。大多数拟议的项目需要连续24小时或更长时间的活细胞成像。目前，由于广泛使用，这些时间范围在我们的活细胞显微镜上不容易获得。此外，在拟议项目中研究的疟疾寄生虫和病毒宿主细胞在低氧压力下自然生长。为了创造更具生理相关性的成像条件，我们需要缺氧孵育条件，以防止肝细胞极性丧失或寄生虫死亡。 生理长期活细胞成像的一个关键方面是降低光毒性，但同时实现略低于衍射极限的分辨率。具体地说，疟疾寄生虫已被证明对荧光照明非常敏感。因此，必须使用高灵敏度的探测器进行图像采集。然而，这需要与增加的空间分辨率相协调，以在三维中显示更多的细胞细节。特别是，对于小型疟疾寄生虫，理想的技术将使我们能够检测到诸如极环形成、染色体分离、中心体复制等事件。所有这一切都必须在保持成像速度的同时实现，以便进行重要的定量细胞生物学分析。因此，我们希望实施一种多功能和可持续的成像解决方案，为我们了解全球相关病原体的感染生物学开辟新的维度。