Share Email Print
cover

Proceedings Paper • new

The SPLIT approach for enhancing the spatial resolution in pulsed STED microscopy with FastFLIM and phasor plots
Format Member Price Non-Member Price
PDF $14.40 $18.00
cover GOOD NEWS! Your organization subscribes to the SPIE Digital Library. You may be able to download this paper for free. Check Access

Paper 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.

Paper Details

Date Published: 22 February 2019
PDF: 11 pages
Proc. SPIE 10882, Multiphoton Microscopy in the Biomedical Sciences XIX, 108820I (22 February 2019); doi: 10.1117/12.2513219
Show Author Affiliations
Giorgio Tortarolo, Istituto Italiano di Tecnologia (Italy)
Univ. degli Studi di Genova (Italy)
Yuansheng Sun, ISS, Inc. (United States)
Sunil Shah, ISS, Inc. (United States)
Beniamino Barbieri, ISS, Inc. (United States)
Kai-Wen Teng, Univ. of Illinois at Urbana-Champaign (United States)
Yuji Ishitsuka, Univ. of Illinois at Urbana-Champaign (United States)
Paul R. Selvin, Univ. of Illinois at Urbana-Champaign (United States)
Luca Lanzanò, Istituto Italiano di Tecnologia (Italy)
Alberto Diaspro, Istituto Italiano di Tecnologia (Italy)
Univ. degli Studi di Genova (Italy)
Giuseppe Vicidomini, Istituto Italiano di Tecnologia (Italy)


Published in SPIE Proceedings Vol. 10882:
Multiphoton Microscopy in the Biomedical Sciences XIX
Ammasi Periasamy; Peter T. C. So; Karsten König, Editor(s)

© SPIE. Terms of Use
Back to Top