Transformative microscopes to image across spatiotemporal scales

项目摘要 分子组织和活动如何导致组织水平的结果是有争议的 生物学中剩余的重大悬而未决的问题。将这两个领域连接起来是实现 生物医学的进步，但在技术上具有挑战性，因为我们不能用 组织尺度的分子精确度。而光学显微镜是选择的方法 为了观察活细胞和有机体内的结构和动态，它有 时空分辨率和光学穿透深度的基本限制。因此， 我们对细胞动力学和超微结构的最详细观察是有限的 到与生理环境相去甚远的单个细胞。 在这里，我建议将超分辨率显微镜的使用范围显著扩大到 包括组织和整个模式生物。这远远超出了能力范围。 目前的技术水平，并需要在体积采集方面取得重大进展 速度、灵敏度和光学穿透深度。我建议把这一领域向前推进一步 结合了三个不同的显微镜领域。其中一些组合不是无关紧要的 因此到目前为止在实验上还不是很容易驯服的。我提出了新的概念，可以 在这些不同领域之间架起桥梁，克服技术和基本限制。 此外，一种新的自适应采样方法只对最具信息量的图像进行成像 具有最高分辨率的体素将克服实现高分辨率的根本障碍 在大容量上进行映像。我假设我的新仪器结合了 智能采样策略可将采集速度提高100倍，同时 减少光毒性和数据存储需求。由此产生的新显微镜技术 将显著提高大容量上的高分辨率成像速度，因此将 启用因以下原因而被禁止的大容量成像实验 空间分辨率、光学穿透深度或采集速度。

Project abstract How molecular organization and activity leads to tissue level outcomes is, arguably, one of the big remaining open questions in biology. Bridging these two areas is key to biomedical advances, yet is technically challenging because we cannot observe with molecular precision at the tissue scale. While optical microscopy is the method of choice to observe architecture and dynamics within living cells and organisms, it has fundamental limitations in spatiotemporal resolution and optical penetration depth. Thus our most detailed observations of cellular dynamics and ultrastructure have been limited to single cells that were far removed from their physiological context. Here I propose to significantly expand the reach of super-resolution microscopy to encompass tissues and whole model organisms. This lies far outside of the capabilities of the current state of the art and requires significant progress in volumetric acquisition speed, sensitivity and optical penetration depth. I propose to advance the field by combining three different fields of microscopy. Some of these combinations are non-trivial and thus have not been experimentally tractable so far. I propose novel concepts that can bridge these different fields and overcome technical and fundamental limitations. Furthermore, a novel adaptive sampling approach that only images the most informative voxels at the highest resolution will overcome fundamental barriers to high-resolution imaging over large volumes. I hypothesize that my new instrumentation combined with an intelligent sampling strategt can improve acquisition speed up to 100 fold while reducing phototoxicity and data storage needs. The resulting new microscope technology will dramatically speed up high-resolution imaging over large volumes and hence will enable large volume imaging experiments that have been prohibited by either lack of spatial resolution, optical penetration depth or acquisition speed.