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

doi:10.1117/12.2267880

21 February 2017 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

Author Affiliations +

Proceedings Volume 10069, Multiphoton Microscopy in the Biomedical Sciences XVII; 100691C (2017) https://doi.org/10.1117/12.2267880
Event: SPIE BiOS, 2017, 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 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.

Conference Presentation

Citation Download Citation

Yuansheng Sun, Giorgio Tortarolo, Kai-Wen Teng, Yuji Ishitsuka, Ulas C. Coskun, Shih-Chu Jeff Liao, Alberto Diaspro, Giuseppe Vicidomini, Paul R. Selvin, and Beniamino Barbieri "A novel pulsed STED microscopy method using FastFLIM and the phasor plots", Proc. SPIE 10069, Multiphoton Microscopy in the Biomedical Sciences XVII, 100691C (21 February 2017); https://doi.org/10.1117/12.2267880

ACCESS THE FULL ARTICLE

INSTITUTIONAL
Select your institution to access the SPIE Digital Library.

SELECT YOUR INSTITUTION

PERSONAL
Sign in with your SPIE account to access your personal subscriptions or to use specific features such as save to my library, sign up for alerts, save searches, etc.

PERSONAL SIGN IN

No SPIE Account? Create one

PURCHASE THIS CONTENT

SUBSCRIBE TO DIGITAL LIBRARY

50 downloads per 1-year subscription

Members: $195

Non-members: $335 ADD TO CART

25 downloads per 1 - year subscription

Members: $145

Non-members: $250 ADD TO CART

PURCHASE SINGLE ARTICLE

Includes PDF, HTML & Video, when available