中国激光, 2013, 40 (1): 0104001, 网络出版: 2012-12-05
基于时间相关单光子计数的离线式g-STED超分辨显微术
Super Resolution Microscopy of Offline g-STED Nanoscopy Based on Time-Correlated Single Photon Counting
显微 荧光寿命成像 时间相关单光子记数算法 时间门荧光受激发射损耗显微 microscopy fluorescent lifetime imaging time-correlated single photon counting algorithm time-gate stimulated emission depletion microscopy
摘要
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45 mW的连续STED光,最终获取了约80 nm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。
Abstract
The offline time-gated stimulated emission depletion (g-STED) microscopy, which is based on time-correlated single photon counting (TCSPC) algorithm, is proposed. As STED beam can eliminate the ratio of spontaneous fluorescent emission while reducing the fluorescence lifetime, the lifetime of fluorescent signals in the center of excitation focal spot and that in the surrounding doughnut area which are overlap by the STED focal spot are significant different. Based on this principle, in a general continuous wave STED (CW-STED), the fluorescent lifetimes of the whole imaging region are calculated by TCSPC, and the signals with shorter lifetime are discarded after all data recorded. The effective point spread function (PSF) of each fluorescent labels are shrinked in order to enhance the resolution. Compared with traditional ones, this offline g-STED not only decreases the incident intensity of laser to avoid the risk of fluorescence photobleaching and optical toxicity, but also increases the flexibility of time-gate manipulation. A spatial resolution of 80 nm is obtained in the experiment when only 45 mW STED beam is introduced. The potential influences of time-gate selection to the resolution and signal-to-noise ratio (SNR) are further discussed.
郝翔, 匡翠方, 顾兆泰, 李帅, 刘旭. 基于时间相关单光子计数的离线式g-STED超分辨显微术[J]. 中国激光, 2013, 40(1): 0104001. Hao Xiang, Kuang Cuifang, Gu Zhaotai, Li Shuai, Liu Xu. Super Resolution Microscopy of Offline g-STED Nanoscopy Based on Time-Correlated Single Photon Counting[J]. Chinese Journal of Lasers, 2013, 40(1): 0104001.