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，可以说， 生物学中尚未解决的大问题。将这两个领域联系起来是 生物医学的进步，但在技术上是具有挑战性的，因为我们不能观察与 在组织尺度上的分子精确度。虽然光学显微镜是首选的方法， 为了观察活细胞和有机体的结构和动力学， 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的观察有限， 到远离其生理环境的单细胞。 在这里，我建议显着扩大超分辨率显微镜的范围， 包括组织和整个模式生物。这远远超出了 并要求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。我建议通过以下方式推进该领域 结合了三个不同的显微镜领域。其中一些组合是非平凡的 因此迄今为止在实验上还不容易处理。我提出了一些新的概念， 跨越这些不同的领域，克服技术和基础限制。 此外，一种新的自适应采样方法，只图像的信息量最大， 最高分辨率的体素将克服高分辨率的基本障碍 在大体积上成像。我假设我的新仪器 智能采样策略可将采集速度提高100倍， 减少光毒性和数据存储需求。由此产生的新显微镜技术 将大大加快大体积的高分辨率成像， 能够进行大体积成像实验，这些实验由于缺乏 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.