A novel pulsed STED microscopy method using FastFLIM and the phasor plots

Yuansheng Sun, Giorgio Tortarolo, Kai Wen Teng, Yuji Ishitsuka, Ulas C. Coskun, Shih Chu Jeff Liao, Alberto Diaspro, Giuseppe Vicidomini, Paul R. Selvin, Beniamino Barbieri

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


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 below the diffraction limit. The spatial resolution of STED is limited by power of the depletion laser at the specimen plane. Higher depletion laser power will improve resolution, but at the cost of increased photo-bleaching, photo-toxicity, and anti-stoke emission background. This degrades the signal-to-noise ratio, and can significantly limit STED applications in living specimens. Here, we present an efficient multi-color STED microscopy method based on the digital frequency domain fluorescence lifetime imaging (FastFLIM) and the phasor plots. Our approach utilizes a combination of pulsed excitation and pulsed depletion lasers to record the time-resolved photons by FastFLIM. We demonstrate that the resolution is improved without increasing the depletion laser power by digital separation of the depleted species from the partially depleted species based on their different decay kinetics. We show the utility of this novel STED method applied in both fixed and live cellular samples, and also show its application to fluorescence lifetime correlation spectroscopy (FLCS) measurements. By combining fluorophores with different fluorescence lifetimes, we simultaneously record two-color STED images of cells labeled with Atto655 and Alexa647 in a single scan by using a single pair of excitation and depletion lasers. This novel approach shortens the data acquisition time while minimizing the photo-toxicity caused when using two separate depletion lasers.

Original languageEnglish (US)
Title of host publicationMultiphoton Microscopy in the Biomedical Sciences XVII
EditorsKarsten Konig, Peter T. C. So, Ammasi Periasamy, Xiaoliang S. Xie
ISBN (Electronic)9781510605794
StatePublished - 2017
EventMultiphoton Microscopy in the Biomedical Sciences XVII - San Francisco, United States
Duration: Jan 29 2017Jan 31 2017

Publication series

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


OtherMultiphoton Microscopy in the Biomedical Sciences XVII
Country/TerritoryUnited States
CitySan Francisco

ASJC Scopus subject areas

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


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