Holographic Single Molecule Localization Microscopy with a Large Axial Range

点击翻译按钮获取中文摘要

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.