Effects of donor and acceptor's fluorescence lifetimes on the method of applying Forster resonance energy transfer in STED microscopy
Deng, SH; Chen, JF; Gao, ZS; Fan, CH; Yan, QR; Wang, YH
摘要Forster resonance energy transfer (FRET) probes being used to improve the resolution of stimulated emission depletion (STED) microscopy are numerically discussed. Besides the FRET efficiency and the excitation intensity, the fluorescence lifetimes of donor and acceptor are found to be another key parameter for the resolution enhancement. Using samples of FRET pairs with shorter donor lifetime and longer acceptor lifetime enhances the nonlinearity of the donor fluorescence, which leads to an increased resolution. The numerical simulation shows that a double resolution improvement of STED microscopy can be achieved by using Cy3-Atto647N samples when compared with that of using standard Cy3-only samples. Lay description STED microscopy, with its capability to discern details far beyond the diffraction limit, has become a most popular imaging modality in life science. The achievable resolution in STED microscopy is theoretically unlimited by indefinitely elevating the STED laser power. However, in practice, the resolution is limited by some factors. For example, under the high STED laser illumination, a high risk of irreversible destruction to the samples exists due to photobleaching or other detrimental effects, especially when the samples are biological specimen. Therefore, without lifting the depletion intensity, the methods able to increase the resolution and the availability of the STED microscopy are highly desirable. In this paper, the phenomenon of Forster resonance energy transfer (FRET) is used to further enhance the resolution in STED microscopy. In fact, using FRET probes to improve the resolution of a conventional microscope has been previously reported (Hanninen etal., 1996; Vogelsang etal., 2010). There, using the method of saturated FRET microscopy, FRET saturation occurs when the higher excitation and the ultrahigh energy efficiency are applied. Under such conditions, the probability that the acceptor is excited increases and energy transfer is largely blocked since the excited acceptor molecules can't act in the role of acceptor. Thus the donor fluorescence can be expected to grow nonlinearly. The nonlinear dependence of the donor's emission intensity is similar to the one involved in two-photon excitation since the signal is generated by the absorption of the two photons to respectively excite the acceptor and donor. This nonlinear multi-photon signal generated via one-photon excitation thus increases the spatial resolution. The point spread function (PSF) of the donor fluorescence can be greatly narrowed when a doughnut-shaped beam is added to de-excite the acceptor molecules (Deng etal., 2010). The reason is that the inhibition of the energy transfer is further restricted to the center of the focal spot since the peripheral acceptor molecules are forced to the ground state by stimulated emission and the energy transfer is allowed only at periphery of the focus. Therefore, the donor fluorescence signal can only stem from the central area, leading to a small PSF. The achievable resolution would in this case be even better than that of a donor-only probe images with STED microscopy. Therefore, the resolution of STED microscopy can be improved by using the FRET probes. In this paper, we find that, besides depending on the excitation intensity and the energy transfer efficiency as reported before, the fluorescence lifetimes of donor and acceptor are another key parameter the resolution enhancement when applying FRET probes for STED microscopy. We find that using FRET pair samples with a shorter donor lifetime and a longer acceptor lifetime increases the nonlinearity of the donor fluorescence and decreases the necessary energy transfer efficiency. More importantly, more than 2-fold increased resolution of STED microscopy with FRET probes is numerically demonstrated when being compared with the resolution of the standard STED microscopy with donor-only samples. References Deng, S., Chen, J., Huang, Q., Fan, C. & Cheng, Y. (2010) Saturated Forster resonance energy transfer microscopy with a stimulated emission depletion beam: a pathway toward single-molecule resolution in far-field bioimaging. Opt. Lett. 35(23), 3862-3864. Hanninen, P. E., Lehtelii, L. & Hell, S. W. (1996) Two- and multiphoton excitation of conjugate-dyesusing a continuous wave laser. Opt. Commun. 130, 29-33. Vogelsang, J., Cordes, T., Forthmann, C., Steinhauer, C. & Tinnefeld, P. (2010) Intrinsically Resolution Enhancing Probes for Confocal Microscopy. Nano. Lett. 10, 672-679.
关键词Diffraction Resolution Limit Stimulated-emission Breaking Field Fret Excitation Barrier Light
作者单位1.Deng, SH
2.Chen, JF
3.Gao, ZS
4.Fan, CH
5.Yan, QR
6.Wang, YH
GB/T 7714
Deng, SH,Chen, JF,Gao, ZS,et al. Effects of donor and acceptor's fluorescence lifetimes on the method of applying Forster resonance energy transfer in STED microscopy[J]. JOURNAL OF MICROSCOPY,2018,269(1):59-65.
APA Deng, SH,Chen, JF,Gao, ZS,Fan, CH,Yan, QR,&Wang, YH.(2018).Effects of donor and acceptor's fluorescence lifetimes on the method of applying Forster resonance energy transfer in STED microscopy.JOURNAL OF MICROSCOPY,269(1),59-65.
MLA Deng, SH,et al."Effects of donor and acceptor's fluorescence lifetimes on the method of applying Forster resonance energy transfer in STED microscopy".JOURNAL OF MICROSCOPY 269.1(2018):59-65.
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