Fast super-resolution microscopy by rotating, coherently scattered laser light

探索更小、更有活力的结构是当前和未来最大的技术挑战之一。已知已久的物理定律似乎设置了不可移动的计量屏障，例如显微镜中的光学分辨率极限。事实证明，这些障碍中的许多都可以通过巧妙的技巧绕过，而不需要移动障碍。光学显微镜中需要空间分辨的结构越小，所需的光子数和测量时间就越多。然而，生命系统中的结构越小，它们移动得越快，可用的测量时间就越少。在这个倡议中，我们致力于一种新的光学显微镜概念，在这种概念中，物体被相干照明，图像也被相干地生成。通过利用倾斜照明和多幅相干图像的角度积分下定义的多重干扰，原则上可以获得近100 nm的空间分辨率，时间分辨率通常为100赫兹，并且具有良好的图像对比度。在我们团队的初步工作中，我们可以通过在TIR暗场模式下旋转相干散射(ROCS)激光来获得150 nm的空间分辨率。有了这项技术，我们可以在不损失图像质量的情况下(例如荧光显微镜中的荧光团漂白)和不需要图像重建(例如在结构照明的超分辨率显微镜中)获得成千上万的图像。在目前的研究方案中，我们希望达到这项技术预期的120 nm空间分辨率和100 Hz时间分辨率之外的两个新目标。一方面，我们希望通过散射激光的特定吸收和位相延迟来区分图像中特定标记的结构。另一方面，我们将开发一种新型的时间相关显微镜将被开发-在很大程度上独立于第一个目标。在这里，高时间分辨率的无标记结构的相干ROC图像将与低时间分辨率的基于荧光的荧光团分布图像相关联。

The exploration of ever smaller and thus more dynamic structures represents one of the biggest technical challenges of the presence and the future. Long known physical laws seem to set immovable metrological barriers, such as the optical resolution limit in microscopy. It has turned out that many of these barriers can be bypassed with resourceful tricks, without moving the barriers. The smaller the structures to be spatially resolved in light microscopy, the more photons and measurement time are required. However, the smaller the structures in living systems, the faster they move and the less measurement time is available.In this initiative we work on a novel optical microscopy concept, where objects are illuminated coherently and images are generated coherently as well. By exploiting defined multiple interferences under oblique illumination and angular integration of many coherent images, a spatial resolution of nearly 100nm can be achieved in principle, at a temporal resolution of typically 100 Hz and with excellent image contrast. Within the preliminary work of our group, we could achieve a spatial resolution of 150 nm through rotating coherently scattered (ROCS) laser light in TIR-dark-field mode. With this technique we could acquire thousands of images without loss in image quality (e.g. fluorophore bleaching in fluorescence microscopy) and without image reconstruction (as required e.g. in super-resolution microscopy with structured illumination).In the present research proposal, we want to reach two new goals in addition to the expected 120 nm spatial and 100 Hz temporal resolution of this technique. On the one hand, we want to distinguish specifically marked structures in the image through specific absorption and phase retardation of the scattered laser light. On the other hand, we will develop a novel kind of time-correlated microscopy shall be developed - largely independently of the first goal. Here coherent ROCS images of label-free structures with high temporal resolution will be correlated to fluorescence based images of fluorophore distributions with low temporal resolution.