基于超快光子计数的共振扫描双色受激发射损耗显微镜：简明指南。
Resonant-scanning dual-color STED microscopy with ultrafast photon counting: A concise guide.
作者信息
Wu Yong, Wu Xundong, Toro Ligia, Stefani Enrico
机构信息
Division of Molecular Medicine, Department of Anesthesiology, David Geffen School of Medicine, UCLA, United States; Cardiovascular Research Laboratory, David Geffen School of Medicine, UCLA, United States.
Division of Molecular Medicine, Department of Anesthesiology, David Geffen School of Medicine, UCLA, United States.
出版信息
Methods. 2015 Oct 15;88:48-56. doi: 10.1016/j.ymeth.2015.06.019. Epub 2015 Jun 27.
STED (stimulated emission depletion) is a popular super-resolution fluorescence microscopy technique. In this paper, we present a concise guide to building a resonant-scanning STED microscope with ultrafast photon-counting acquisition. The STED microscope has two channels, using a pulsed laser and a continuous-wave (CW) laser as the depletion laser source, respectively. The CW STED channel preforms time-gated detection to enhance optical resolution in this channel. We use a resonant mirror to attain high scanning speed and ultrafast photon counting acquisition to scan a large field of view, which help reduce photobleaching. We discuss some practical issues in building a STED microscope, including creating a hollow depletion beam profile, manipulating polarization, and monitoring optical aberration. We also demonstrate a STED image enhancement method using stationary wavelet expansion and image analysis methods to register objects and to quantify colocalization in STED microscopy.
受激发射损耗（STED）是一种流行的超分辨率荧光显微镜技术。在本文中，我们提供了一份简洁指南，介绍如何构建一台具有超快光子计数采集功能的共振扫描STED显微镜。该STED显微镜有两个通道，分别使用脉冲激光器和连续波（CW）激光器作为损耗激光源。连续波STED通道执行时间选通检测，以提高该通道的光学分辨率。我们使用共振镜实现高扫描速度和超快光子计数采集，以扫描大视场，这有助于减少光漂白。我们讨论了构建STED显微镜时的一些实际问题，包括创建空心损耗光束轮廓、操纵偏振以及监测光学像差。我们还展示了一种STED图像增强方法，该方法使用平稳小波变换和图像分析方法来对准物体并量化STED显微镜中的共定位情况。
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