Holographic Single Molecule Localization Microscopy with a Large Axial Range

项目摘要 单分子定位显微镜（SMLM）是对内部特定结构进行成像的首选方法。 分辨率低至10 nm的电池。SMLM可以在两个维度上产生漂亮的结果。他们两个- 三维视场可以大到200微米× 200微米。但细胞生物学是三维的 在三维空间中获得高分辨率和范围对于理解生物学至关重要。那里 是几种技术，用于实现高分辨率SMLM在所有三维和轴向分辨率为50 nm可以常规地实现。问题是轴向尺寸的范围通常限于 几微米-小于典型电池的厚度。范围可以通过机械移动 通过焦平面采样，但这是缓慢的，在分辨率中引入了进一步的不确定性，并且 引入额外的光漂白，因为细胞被照射。 在这里，我们提出了一种新的方法，SMLM，将大大增加轴向范围，允许成像与 轴向范围为10至20微米，无需机械重新聚焦。这将使三维成像的 整个细胞--以及整个细胞区域--快速而有效。除了大的轴向范围之外， 这种方法自然允许像差校正。提高细胞的高分辨率成像将有助于细胞 生物学家了解细胞的结构和不同蛋白质在健康和疾病中的作用。 组织.

Project Summary Single Molecule Localization Microscopy (SMLM) is the method of choice for imaging specific structures inside cells with resolutions down to 10 nm. SMLM can produce beautiful results in two dimensions. The two- dimensional field of view can be as large as 200 microns by 200 microns. But cell biology is three-dimensional and achieving a high-resolution and range in the third dimensional is critical to understanding biology. There are several techniques for achieving high-resolution SMLM in all three dimensions and axial resolutions of 50 nm can routinely be achieved. The problem is that the range in the axial dimension has been typically limited to a few microns – less than the thickness of a typical cell. The range can be extended by mechanically moving the sample through the focal plane but this is slow, introduces further uncertainties in the resolution, and introduces extra photobleaching because cells are being illuminated throughout. Here we propose a novel approach to SMLM that will greatly increase the axial range allowing imaging with an axial range of 10 to 20 microns without mechanical refocusing. This will allow three-dimensional imaging of entire cells – and entire fields of cells – quickly and efficiently. In addition to the large axial range, this approach naturally allows for aberration correction. Improving high-resolution imaging of cells will help cell biologists understand the structure of the cell and the role of different proteins both in healthy and diseased tissue.