The SPLIT approach for enhancing the spatial resolution in pulsed STED microscopy with FastFLIM and phasor plots

Giorgio Tortarolo; Yuansheng Sun; Sunil Shah; Beniamino Barbieri; Kai-Wen Teng; Yuji Ishitsuka; Paul R. Selvin; Luca Lanzanò; Alberto Diaspro; Giuseppe Vicidomini

doi:10.1117/12.2513219

22 February 2019 The SPLIT approach for enhancing the spatial resolution in pulsed STED microscopy with FastFLIM and phasor plots

Giorgio Tortarolo, Yuansheng Sun, Sunil Shah, Beniamino Barbieri, Kai-Wen Teng, Yuji Ishitsuka, Paul R. Selvin, Luca Lanzanò, Alberto Diaspro, Giuseppe Vicidomini

Author Affiliations +

Proceedings Volume 10882, Multiphoton Microscopy in the Biomedical Sciences XIX; 108820I (2019) https://doi.org/10.1117/12.2513219
Event: SPIE BiOS, 2019, San Francisco, California, United States

Abstract

Stimulated emission depletion (STED) microscopy is a powerful super-resolution microscopy technique that enables observation of macromolecular complexes and sub-cellular structures with spatial resolution well below the diffraction limit. However, resolution in the double-digit nanometer range can be obtained only using high intensity depletion laser, at the cost of increased photo-damage, which significantly limits STED applications in live specimens. To minimize this, we use the separation by lifetime tuning (SPLIT) technique, in which phasor analysis is used to efficiently distinguish photons emitted from the center and from the periphery of the excitation spot of a STED microscope. Thus, it can be used to improve the resolution without increasing the STED beam intensity. Our approach utilizes a combination of pulsed excitation and pulsed depletion lasers to record the time-resolved photons by FastFLIM. The photons stream are successively analyzed using the SPLIT technique, demonstrating that the resolution improves without increasing the depletion laser intensity.

Conference Presentation

Citation Download Citation

Giorgio Tortarolo, Yuansheng Sun, Sunil Shah, Beniamino Barbieri, Kai-Wen Teng, Yuji Ishitsuka, Paul R. Selvin, Luca Lanzanò, Alberto Diaspro, and Giuseppe Vicidomini "The SPLIT approach for enhancing the spatial resolution in pulsed STED microscopy with FastFLIM and phasor plots", Proc. SPIE 10882, Multiphoton Microscopy in the Biomedical Sciences XIX, 108820I (22 February 2019); https://doi.org/10.1117/12.2513219

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