Resonant-scanning dual-color STED microscopy with ultrafast photon counting: A concise guide.
Resonant-scanning dual-color STED microscopy with ultrafast photon counting: A concise guide.
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DOI：
10.1016/j.ymeth.2015.06.019
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发表时间：
2015-10-15
期刊：
影响因子：
--
通讯作者：
Stefani E
中科院分区：
文献类型：
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作者：
Wu Y;Wu X;Toro L;Stefani E
受激发射耗尽（STED）是一种流行的超分辨荧光显微技术。在本文中，我们提出了一个简明的指南，建立一个共振扫描STED显微镜与超快光子计数采集。STED显微镜有两个通道，分别使用脉冲激光和连续波（CW）激光作为耗尽激光源。CW STED通道执行时间选通检测以增强该通道中的光学分辨率。我们使用共振镜来获得高扫描速度和超快光子计数采集来扫描大视场，这有助于减少光漂白。我们讨论了在建立STED显微镜的一些实际问题，包括创建一个空心耗尽光束轮廓，操纵偏振，和监测光学像差。我们还展示了STED图像增强方法，使用平稳小波展开和图像分析方法来注册对象，并量化共定位在STED显微镜。
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.