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提出了一种离线式基于时间门的荧光受激发射损耗(g-STED)显微方法。基于在强光照条件下荧光寿命缩短的理论模型,在常规STED架构基础上,使用时间相关单光子记数(TCSPC)算法获取图像的荧光寿命信息,离线设置合理的时间门阈值,丢弃短寿命信号数据,对荧光信号有效点扩展函数(PSF)进行压缩,达到超分辨显微的目的。与传统STED显微术相比,此方法所需光功率大幅度降低,减少了荧光漂白及光毒性;离线式处理则同时增加了时间门设置的灵活性。在实验中,使用45mW的连续STED光,最终获取了约80nm的图像空间分辨率。进一步对时间门的设置对获取图像信号的分辨率和信噪比的影响进行了讨论。
An off-line time-based fluorescence stimulated emission loss (g-STED) microscopy method was proposed. On the basis of the conventional STED architecture, the time-dependent single photon counting (TCSPC) algorithm is used to obtain the fluorescence lifetime information of the image based on the theoretical model of shortening the lifetime of fluorescence under intense light conditions. A reasonable threshold of time threshold is set off-line and the short-lived signal is discarded Data, the fluorescence signal point spread function (PSF) is compressed to achieve the purpose of super-resolution microscopy. Compared with the traditional STED microscopy, the optical power required by this method is greatly reduced, reducing the fluorescence bleaching and phototoxicity; off-line processing also increases the flexibility of the time gate settings. In the experiment, 45mW of continuous STED light was used, finally obtaining an image spatial resolution of about 80nm. The effect of setting the time gate on the resolution and signal-to-noise ratio of the captured image signal is further discussed.