Construction of an instant structured illumination microscope.
Construction of an instant structured illumination microscope.
复制标题
DOI：
10.1016/j.ymeth.2015.07.012
复制
发表时间：
2015-10-15
期刊：
影响因子：
--
通讯作者：
Peckham M
中科院分区：
文献类型：
--
作者：
Curd A;Cleasby A;Makowska K;York A;Shroff H;Peckham M
即时结构照明显微镜：2倍的分辨率提高。图像采集速度快（超过100fps）。详细的构造和使用说明。生物成像的一个挑战是在活细胞中以快帧率捕获高分辨率图像。“即时结构照明显微镜”（iSIM）就是为此目的而设计的系统。与标准的结构照明显微镜（SIM）类似，iSIM在x， y和z方面提供了比宽视场显微镜两倍的改进，但也允许更快的图像采集，实时显示超分辨率图像。iSIM的组装相当复杂，涉及许多光学元件的组合和对准，包括三个微光学阵列（两个透镜阵列和一个针孔阵列，间距均为222 μm）和一个双面扫描镜。此外，许多电子元件必须得到正确控制。因此，该系统的构建不是微不足道的，而是非常理想的，特别是对于活细胞成像。我们报告，并提供说明，一个iSIM的建设，包括对以前的设计在硬件和软件的微小修改。最后的仪器使我们能够以超过100 fps的速率快速获取荧光图像，在x-y和z分辨率上提高了大约两倍；使用1.49 NA的物镜和488 nm的激发，亚衍射生物特征的表观尺寸（最大一半的全宽度）为145 nm（横向）和320 nm（轴向）。
Instant structured illumination microscope: 2-fold resolution enhancement. Fast image acquisition rate (exceeding 100 fps). Detailed description of construction and use. A challenge in biological imaging is to capture high-resolution images at fast frame rates in live cells. The “instant structured illumination microscope” (iSIM) is a system designed for this purpose. Similarly to standard structured illumination microscopy (SIM), an iSIM provides a twofold improvement over widefield microscopy, in x, y and z, but also allows much faster image acquisition, with real-time display of super-resolution images. The assembly of an iSIM is reasonably complex, involving the combination and alignment of many optical components, including three micro-optics arrays (two lenslet arrays and an array of pinholes, all with a pitch of 222 μm) and a double-sided scanning mirror. In addition, a number of electronic components must be correctly controlled. Construction of the system is therefore not trivial, but is highly desirable, particularly for live-cell imaging. We report, and provide instructions for, the construction of an iSIM, including minor modifications to a previous design in both hardware and software. The final instrument allows us to rapidly acquire fluorescence images at rates faster than 100 fps, with approximately twofold improvement in resolution in both x–y and z; sub-diffractive biological features have an apparent size (full width at half maximum) of 145 nm (lateral) and 320 nm (axial), using a 1.49 NA objective and 488 nm excitation.