Research Papers: Imaging

Phasor imaging with a widefield photon-counting detector

[+] Author Affiliations
Ryan A. Colyer, Shimon Weiss, Xavier Michalet

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

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

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

J. Biomed. Opt. 17(1), 016008 (Feb 07, 2012). doi:10.1117/1.JBO.17.1.016008
History: Received July 19, 2011; Revised November 6, 2011; Accepted November 15, 2011
Text Size: A A A

Abstract.  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.

Figures in this Article
© 2012 Society of Photo-Optical Instrumentation Engineers


Ryan A. Colyer ; Oswald H. W. Siegmund ; Anton S. Tremsin ; John V. Vallerga ; Shimon Weiss, et al.
"Phasor imaging with a widefield photon-counting detector", J. Biomed. Opt. 17(1), 016008 (Feb 07, 2012). ;


Access This Article
Sign in or Create a personal account to Buy this article ($20 for members, $25 for non-members).

Some tools below are only available to our subscribers or users with an online account.

Related Content

Customize your page view by dragging & repositioning the boxes below.

Related Book Chapters

Topic Collections

PubMed Articles
  • Don't have an account?
  • Subscribe to the SPIE Digital Library
  • Create a FREE account to sign up for Digital Library content alerts and gain access to institutional subscriptions remotely.
Access This Article
Sign in or Create a personal account to Buy this article ($20 for members, $25 for non-members).
Access This Proceeding
Sign in or Create a personal account to Buy this article ($15 for members, $18 for non-members).
Access This Chapter

Access to SPIE eBooks is limited to subscribing institutions and is not available as part of a personal subscription. Print or electronic versions of individual SPIE books may be purchased via