AI Chat Paper
Discover the SciOpen Platform and Achieve Your Research Goals with Ease.
Search articles, authors, keywords, DOl and etc.
{{expandStatus?'Exit ':''}}Advanced Search
Stimulated emission depletion (STED) nanoscopy enables the visualization of subcellular organelles in unprecedented detail. However, reducing the power dependency remains one of the greatest challenges for STED imaging in living cells. Here, we propose a new method, called modulated STED, to reduce the demand for depletion power in STED imaging by modulating the information from the temporal and spatial domains. In this approach, an excitation pulse is followed by a depletion pulse with a longer delay; therefore, the fluorescence decay curve contains both confocal and STED photons in a laser pulse period. With time-resolved detection, we can remove residual diffraction-limited signals pixel by pixel from STED photons by taking the weighted difference of the depleted photons. Finally, fluorescence emission in the periphery of an excitation spot is further inhibited through spatial modulation of fluorescent signals, which replaced the increase of the depletion power in conventional STED. We demonstrate that the modulated STED method can achieve a resolution of < 100 nm in both fixed and living cells with a depletion power that is dozens of times lower than that of conventional STED, therefore, it is very suitable for long-term super-resolution imaging of living cells. Furthermore, the idea of the method could open up a new avenue to the implementation of other experiments, such as light-sheet imaging, multicolor and three-demensional (3D) super-resolution imaging.
Sahl, S. J.; Hell, S. W.; Jakobs, S. Fluorescence nanoscopy in cell biology. Nat. Rev. Mol. Cell Biol. 2017, 18, 685–701.
Sigal, Y. M.; Zhou, R. B.; Zhuang, X. W. Visualizing and discovering cellular structures with super-resolution microscopy. Science 2018, 361, 880–887.
Jin, D. Y.; Xi, P.; Wang, B. M.; Zhang, L.; Enderlein, J.; Van Oijen, A. M. Nanoparticles for super-resolution microscopy and single-molecule tracking. Nat. Methods 2018, 15, 415–423.
Schermelleh, L.; Ferrand, A.; Huser, T.; Eggeling, C.; Sauer, M.; Biehlmaier, O.; Drummen, G. P. C. Super-resolution microscopy demystified. Nat. Cell Biol. 2019, 21, 72–84.
Hell, S. W.; Wichmann, J. Breaking the diffraction resolution limit by stimulated emission: Stimulated-emission-depletion fluorescence microscopy. Opt. Lett. 1994, 19, 780–782.
Klar, T. A.; Jakobs, S.; Dyba, M.; Egner, A.; Hell, S. W. Fluorescence microscopy with diffraction resolution barrier broken by stimulated emission. Proc. Natl. Acad. Sci. USA 2000, 97, 8206–8210.
Blom, H.; Widengren, J. Stimulated emission depletion microscopy. Chem. Rev. 2017, 117, 7377–7427.
Gould, T. J.; Verkhusha, V. V.; Hess, S. T. Imaging biological structures with fluorescence photoactivation localization microscopy. Nat. Protoc. 2009, 4, 291–308.
Rust, M. J.; Bates, M.; Zhuang, X. W. Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM). Nat. Methods 2006, 3, 793–796.
Sauer, M.; Heilemann, M. Single-molecule localization microscopy in eukaryotes. Chem. Rev. 2017, 117, 7478–7509.
Gustafsson, M. G. L. Surpassing the lateral resolution limit by a factor of two using structured illumination microscopy. J. Microsc. 2000, 198, 82–87.
Gustafsson, M. G. L. Nonlinear structured-illumination microscopy: Wide-field fluorescence imaging with theoretically unlimited resolution. Proc. Natl. Acad. Sci. USA 2005, 102, 13081–13086.
Heintzmann, R.; Huser, T. Super-resolution structured illumination microscopy. Chem. Rev. 2017, 117, 13890–13908.
Balzarotti, F.; Eilers, Y.; Gwosch, K. C.; Gynnå, A. H.; Westphal, V.; Stefani, F. D.; Elf, J.; Hell, S. W. Nanometer resolution imaging and tracking of fluorescent molecules with minimal photon fluxes. Science 2017, 355, 606–612.
Eilers, Y.; Ta, H.; Gwosch, K. C.; Balzarotti, F.; Hell, S. W. MINFLUX monitors rapid molecular jumps with superior spatiotemporal resolution. Proc. Natl. Acad. Sci. USA 2018, 115, 6117–6122.
Gwosch, K. C.; Pape, J. K.; Balzarotti, F.; Hoess, P.; Ellenberg, J.; Ries, J.; Hell, S. W. MINFLUX nanoscopy delivers 3D multicolor nanometer resolution in cells. Nat. Methods 2020, 17, 217–224.
Willig, K. I.; Rizzoli, S. O.; Westphal, V.; Jahn, R.; Hell, S. W. STED microscopy reveals that synaptotagmin remains clustered after synaptic vesicle exocytosis. Nature 2006, 440, 935–939.
Yang, X. S.; Yang, Z. G.; Wu, Z. Y.; He, Y.; Shan, C. Y.; Chai, P. Y.; Ma, C. S.; Tian, M.; Teng, J. L.; Jin, D. Y. et al. Mitochondrial dynamics quantitatively revealed by STED nanoscopy with an enhanced squaraine variant probe. Nat. Commun. 2020, 11, 3699.
Spahn, C.; Grimm, J. B.; Lavis, L. D.; Lampe, M.; Heilemann, M. Whole-cell, 3D, and multicolor STED imaging with exchangeable fluorophores. Nano Lett. 2019, 19, 500–505.
Vicidomini, G.; Bianchini, P.; Diaspro, A. STED super-resolved microscopy. Nat. Methods 2018, 15, 173–182.
Vicidomini, G.; Moneron, G.; Han, K. Y.; Westphal, V.; Ta, H.; Reuss, M.; Engelhardt, J.; Eggeling, C.; Hell, S. W. Sharper low-power STED nanoscopy by time gating. Nat. Methods 2011, 8, 571–573.
Vicidomini, G.; Schönle, A.; Ta, H.; Han, K. Y.; Moneron, G.; Eggeling, C.; Hell, S. W. STED nanoscopy with time-gated detection: Theoretical and experimental aspects. PLoS One 2013, 8, e54421.
Wang, Y. F.; Kuang, C. F.; Gu, Z. T.; Xu, Y. K.; Li, S.; Hao, X.; Liu, X. Time-gated stimulated emission depletion nanoscopy. Opt. Eng. 2013, 52, 093107.
Bordenave, M. D.; Balzarotti, F.; Stefani, F. D.; Hell, S. W. STED nanoscopy with wavelengths at the emission maximum. J. Phys. D:Appl. Phys. 2016, 49, 365102.
Gao, P.; Prunsche, B.; Zhou, L.; Nienhaus, K.; Nienhaus, G. U. Background suppression in fluorescence nanoscopy with stimulated emission double depletion. Nat. Photonics 2017, 11, 163–169.
Gao, P.; Nienhaus, G. U. Precise background subtraction in stimulated emission double depletion nanoscopy. Opt. Lett. 2017, 42, 831–834.
Lanzanò, L.; Hernández, I. C.; Castello, M.; Gratton, E.; Diaspro, A.; Vicidomini, G. Encoding and decoding spatio-temporal information for super-resolution microscopy. Nat. Commun. 2015, 6, 6701.
Wang, L. W.; Chen, B. L.; Yan, W.; Yang, Z. G.; Peng, X.; Lin, D. Y.; Weng, X. Y.; Ye, T.; Qu, J. L. Resolution improvement in STED super-resolution microscopy at low power using a phasor plot approach. Nanoscale 2018, 10, 16252–16260.
Tortarolo, G.; Sun, Y. S.; Teng, K. W.; Ishitsuka, Y.; Lanzanó, L.; Selvin, P. R.; Barbieri, B.; Diaspro, A.; Vicidomini, G. Photon-separation to enhance the spatial resolution of pulsed STED microscopy. Nanoscale 2019, 11, 1754–1761.
Chen, Y.; Wang, L. W.; Yan, W.; Peng, X.; Qu, J. L.; Song, J. Elimination of re-excitation in stimulated emission depletion nanoscopy based on photon extraction in a phasor plot. Laser Photonics Rev. 2020, 14, 1900352.
Wang, L. W.; Chen, Y.; Peng, X.; Zhang, J.; Wang, J. L.; Liu, L. W.; Yang, Z. G.; Yan, W.; Qu, J. L. Ultralow power demand in fluorescence nanoscopy with digitally enhanced stimulated emission depletion. Nanophotonics 2020, 9, 831–839.
Kuang, C. F.; Li, S.; Liu, W.; Hao, X.; Gu, Z. T.; Wang, Y. F.; Ge, J. H.; Li, H. F.; Liu, X. Breaking the diffraction barrier using fluorescence emission difference microscopy. Sci. Rep. 2013, 3, 1441.
Vicidomini, G.; Moneron, G.; Eggeling, C.; Rittweger, E.; Hell, S. W. STED with wavelengths closer to the emission maximum. Opt. Express 2012, 20, 5225–5236.
Descloux, A.; Grußmayer, K. S.; Radenovic, A. Parameter-free image resolution estimation based on decorrelation analysis. Nat. Methods 2019, 16, 918–924.
Yao, R. W.; Xu, G.; Wang, Y.; Shan, L.; Luan, P. F.; Wang, Y.; Wu, M.; Yang, L. Z.; Xing, Y. H.; Yang, L. et al. Nascent pre-rRNA sorting via phase separation drives the assembly of dense fibrillar components in the human nucleolus. Mol. Cell 2019, 76, 767–783.
Lafontaine, D. L. J.; Riback, J. A.; Bascetin, R.; Brangwynne, C. P. The nucleolus as a multiphase liquid condensate. Nat. Rev. Mol. Cell Biol. 2021, 22, 165–182.
京公网安备11010802044758号