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

Research output: Chapter in Book/Report/Conference proceedingConference contribution

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.

Original languageEnglish (US)
Title of host publicationMultiphoton Microscopy in the Biomedical Sciences XIX
EditorsAmmasi Periasamy, Peter T. C. So, Karsten Konig
PublisherSPIE
ISBN (Electronic)9781510624061
DOIs
StatePublished - Jan 1 2019
EventMultiphoton Microscopy in the Biomedical Sciences XIX 2019 - San Francisco, United States
Duration: Feb 3 2019Feb 6 2019

Publication series

NameProgress in Biomedical Optics and Imaging - Proceedings of SPIE
Volume10882
ISSN (Print)1605-7422

Conference

ConferenceMultiphoton Microscopy in the Biomedical Sciences XIX 2019
CountryUnited States
CitySan Francisco
Period2/3/192/6/19

ASJC Scopus subject areas

  • Electronic, Optical and Magnetic Materials
  • Biomaterials
  • Atomic and Molecular Physics, and Optics
  • Radiology Nuclear Medicine and imaging

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  • Cite this

    Tortarolo, G., Sun, Y., Shah, S., Barbieri, B., Teng, K. W., Ishitsuka, Y., Selvin, P. R., Lanzanò, L., Diaspro, A., & Vicidomini, G. (2019). The SPLIT approach for enhancing the spatial resolution in pulsed STED microscopy with FastFLIM and phasor plots. In A. Periasamy, P. T. C. So, & K. Konig (Eds.), Multiphoton Microscopy in the Biomedical Sciences XIX [108820I] (Progress in Biomedical Optics and Imaging - Proceedings of SPIE; Vol. 10882). SPIE. https://doi.org/10.1117/12.2513219