Chinese Journal of Lasers, Volume. 40, Issue 1, 104001(2013)
Super Resolution Microscopy of Offline g-STED Nanoscopy Based on Time-Correlated Single Photon Counting
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.
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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): 104001
Category: biomedical photonics and laser medicine
Received: Jul. 23, 2012
Accepted: --
Published Online: Dec. 5, 2012
The Author Email: Xiang Hao (caesarhx@gmail.com)