Turnbull Lynne, Strauss Michael P, Liew Andrew T F, Monahan Leigh G, Whitchurch Cynthia B, Harry Elizabeth J
The ithree Institute, University of Technology, Sydney.
The ithree Institute, University of Technology, Sydney;
J Vis Exp. 2014 Sep 29(91):51469. doi: 10.3791/51469.
Imaging of biological samples using fluorescence microscopy has advanced substantially with new technologies to overcome the resolution barrier of the diffraction of light allowing super-resolution of live samples. There are currently three main types of super-resolution techniques - stimulated emission depletion (STED), single-molecule localization microscopy (including techniques such as PALM, STORM, and GDSIM), and structured illumination microscopy (SIM). While STED and single-molecule localization techniques show the largest increases in resolution, they have been slower to offer increased speeds of image acquisition. Three-dimensional SIM (3D-SIM) is a wide-field fluorescence microscopy technique that offers a number of advantages over both single-molecule localization and STED. Resolution is improved, with typical lateral and axial resolutions of 110 and 280 nm, respectively and depth of sampling of up to 30 µm from the coverslip, allowing for imaging of whole cells. Recent advancements (fast 3D-SIM) in the technology increasing the capture rate of raw images allows for fast capture of biological processes occurring in seconds, while significantly reducing photo-toxicity and photobleaching. Here we describe the use of one such method to image bacterial cells harboring the fluorescently-labelled cytokinetic FtsZ protein to show how cells are analyzed and the type of unique information that this technique can provide.
利用荧光显微镜对生物样品进行成像，随着新技术的出现取得了显著进展，这些技术克服了光衍射的分辨率障碍，实现了活样品的超分辨率成像。目前主要有三种超分辨率技术——受激发射损耗（STED）、单分子定位显微镜（包括诸如光激活定位显微镜（PALM）、随机光学重建显微镜（STORM）和全局光切变干涉显微镜（GDSIM）等技术）以及结构光照明显微镜（SIM）。虽然STED和单分子定位技术在分辨率提升方面最为显著，但它们在提高图像采集速度方面进展较慢。三维SIM（3D-SIM）是一种宽场荧光显微镜技术，与单分子定位和STED技术相比具有诸多优势。其分辨率得到了提高，典型的横向分辨率和轴向分辨率分别为110纳米和280纳米，并且从盖玻片起的采样深度可达30微米，能够对整个细胞进行成像。该技术的最新进展（快速3D-SIM）提高了原始图像的捕获速率，能够快速捕获在数秒内发生的生物过程，同时显著降低光毒性和光漂白现象。在此，我们描述了使用一种这样的方法对携带荧光标记的细胞分裂FtsZ蛋白的细菌细胞进行成像，以展示如何分析细胞以及该技术能够提供的独特信息类型。
