Molecular Microscopy and Spectroscopy, Istituto Italiano di Tecnologia, Via Morego 30, 16163, Genoa, Italy.
Nanoscale. 2019 Jan 23;11(4):1754-1761. doi: 10.1039/c8nr07485b.
Stimulated emission depletion microscopy (STED) is one of the pivotal super-resolution techniques. It overcomes the spatial resolution limit imposed by the diffraction by using an additional laser beam, the STED beam, intensity of which is directly related to the achievable resolution. Despite reaching nanometer resolution, much effort in recent years has been devoted to reducing the STED beam intensity because it may lead to photo-damaging effects. Accessing the spatial information encoded in the temporal dynamics of the detected fluorescent photons has been proved to be a powerful strategy and has contributed to the separation by lifetime tuning (SPLIT) technique. The SPLIT method uses the phasor analysis to efficiently distinguish photons emitted from the center and the periphery of the excitation spot. It thus improves the resolution without increasing the STED beam intensity. This method was proposed for architectures based on the STED beam running in continuous waves (CW-STED microscopy). Here, we extend it to pulsed STED beam implementations (pSTED microscopy). We show, through simulated and experimental data, that the pSTED-SPLIT method reduces the detection volume of the pSTED microscope without significantly decreasing the signal-to-noise ratio of the final image, thus effectively improving the resolution without increasing the STED beam intensity.
受激发射耗散显微镜（STED）是一种关键的超分辨率技术。它通过使用额外的激光束——STED 光束来克服由衍射引起的空间分辨率限制，其强度与可实现的分辨率直接相关。尽管已经达到纳米级分辨率，但近年来，人们仍在努力降低 STED 光束强度，因为它可能导致光损伤效应。事实证明，获取检测到的荧光光子的时间动力学中编码的空间信息是一种强大的策略，并为基于寿命调谐的分离（SPLIT）技术做出了贡献。SPLIT 方法使用相量分析来有效地区分从激发光斑中心和外围发射的光子。因此，它可以在不增加 STED 光束强度的情况下提高分辨率。该方法最初是为基于连续波（CW-STED 显微镜）运行的 STED 光束的架构提出的。在这里，我们将其扩展到脉冲 STED 光束实现（pSTED 显微镜）。我们通过模拟和实验数据表明，pSTED-SPLIT 方法在不显著降低最终图像信噪比的情况下减小了 pSTED 显微镜的检测体积，从而有效地提高了分辨率，而无需增加 STED 光束强度。
