Phasor imaging with a widefield photon-counting detector

Colyer, Ryan A.; Siegmund, Oswald H. W.; Tremsin, Anton S.; Vallerga, John V.; Weiss, Shimon; Michalet, Xavier

doi:doi:10.1117/1.JBO.17.1.016008

GENERAL INFORMATION

You are not logged in Logged Out Log In

Journal of Biomedical Optics / Volume 17 / Issue 1 / Research Papers: Imaging

Previous Article | Next Article

Phasor imaging with a widefield photon-counting detector

J. Biomed. Opt. 17, 016008 (Feb 07, 2012); http://dx.doi.org/10.1117/1.JBO.17.1.016008

Alerts
- Alert Me When Cited
- Alert Me When Corrected
Tools
Share
- Email Abstract
- Connotea
- CiteULike
- del.icio.us
- BibSonomy
- Tweet this Article
- Add to Facebook

Abstract

References (69)

Ryan A. Colyer, Shimon Weiss, and Xavier Michalet

UCLA, Department of Chemistry and Biochemistry, 607 Charles E. Young Drive East, Los Angeles, California

Oswald H. W. Siegmund, Anton S. Tremsin, and John V. Vallerga

Space Sciences Laboratory, UCB, 7 Gauss Way, Berkeley, California

Fluorescence lifetime can be used as a contrast mechanism to distinguish fluorophores for localization or tracking, for studying molecular interactions, binding, assembly, and aggregation, or for observing conformational changes via Förster resonance energy transfer (FRET) between donor and acceptor molecules. Fluorescence lifetime imaging microscopy (FLIM) is thus a powerful technique but its widespread use has been hampered by demanding hardware and software requirements. FLIM data is often analyzed in terms of multicomponent fluorescence lifetime decays, which requires large signals for a good signal-to-noise ratio. This confines the approach to very low frame rates and limits the number of frames which can be acquired before bleaching the sample. Recently, a computationally efficient and intuitive graphical representation, the phasor approach, has been proposed as an alternative method for FLIM data analysis at the ensemble and single-molecule level. In this article, we illustrate the advantages of combining phasor analysis with a widefield time-resolved single photon-counting detector (the H33D detector) for FLIM applications. In particular we show that phasor analysis allows real-time subsecond identification of species by their lifetimes and rapid representation of their spatial distribution, thanks to the parallel acquisition of FLIM information over a wide field of view by the H33D detector. We also discuss possible improvements of the H33D detector’s performance made possible by the simplicity of phasor analysis and its relaxed timing accuracy requirements compared to standard time-correlated single-photon counting (TCSPC) methods.

History

Received Jul 19, 2011
Accepted Nov 15, 2011
Revised Nov 06, 2011
Published online Feb 07, 2012

Digital Object Identifier

http://dx.doi.org/10.1117/1.JBO.17.1.016008

Citation

Ryan A. Colyer, Oswald H. W. Siegmund, Anton S. Tremsin, John V. Vallerga, Shimon Weiss and Xavier Michalet, "Phasor imaging with a widefield photon-counting detector", J. Biomed. Opt. 17, 016008 (Feb 07, 2012); http://dx.doi.org/10.1117/1.JBO.17.1.016008