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

探索更小、更动态的结构是当前和未来最大的技术挑战之一。众所周知的物理定律似乎设置了不可移动的计量障碍，例如显微镜的光学分辨率限制。事实证明，许多障碍都可以通过巧妙的技巧绕过，而无需移动障碍。光学显微镜中需要空间分辨的结构越小，需要的光子和测量时间就越多。然而，生命系统中的结构越小，移动速度越快，可用的测量时间越少。在这项倡议中，我们致力于一种新的光学显微镜概念，其中物体被相干地照亮，图像也被相干地生成。通过利用倾斜照明下定义的多重干扰和多相干图像的角度积分，原则上可以实现近100nm的空间分辨率，时间分辨率通常为100hz，并且具有出色的图像对比度。在前期工作中，我们通过旋转相干散射（ROCS）激光在tir -暗场模式下实现了150 nm的空间分辨率。使用这种技术，我们可以在不损失图像质量的情况下获得数千张图像（例如荧光显微镜中的荧光团漂白），也不需要图像重建（例如在结构照明的超分辨率显微镜中）。在目前的研究计划中，除了该技术预期的120 nm空间分辨率和100 Hz时间分辨率外，我们还希望达到两个新的目标。一方面，我们希望通过散射激光的特定吸收和相位延迟来区分图像中特定的标记结构。另一方面，我们将开发一种新型的时间相关显微镜，将在很大程度上独立于第一个目标。在这里，具有高时间分辨率的无标记结构的相干ROCS图像将与具有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.