Full Content is available to subscribers

Subscribe/Learn More  >
Proceedings Article

Global and pixel kinetic data analysis for FRET detection by multi-photon time-domain FLIM

[+] Author Affiliations
Paul R. Barber, Simon M. Ameer-Beg, Julian D. Gilbey, Richard J. Edens, Ifeome Ezike, Borivoj Vojnovic

Gray Cancer Institute, Mount Vernon Hospital (United Kingdom)

Proc. SPIE 5700, Multiphoton Microscopy in the Biomedical Sciences V, 171 (April 11, 2005); doi:10.1117/12.590510
Text Size: A A A
From Conference Volume 5700

  • Multiphoton Microscopy in the Biomedical Sciences V
  • Ammasi Periasamy; Peter T. C. So
  • San Jose, CA | January 22, 2005

abstract

FLIM/FRET is an extremely powerful technique that can microscopically locate nanometre-scale protein-protein interactions within live or fixed cells, both in vitro and in vivo . The key to performing sensitive FRET, via FLIM, besides the use of appropriate fluorophores, is the analysis of the time-resolved data present at each image pixel. The fluorescent transient will, in general, exhibit multi-exponential kinetics: at least two exponential components arise from both the interacting and non-interacting protein. We shall describe a novel method and computer program for the global analysis of time resolved data, either at the single level or through global analysis of grouped pixel data. Kinetic models are fitted using the Marquardt algorithm and iterative convolution of the excitation signal, in a computationally-efficient manner. The fitting accuracy and sensitivity of the algorithm has been tested using modelled data, including the addition of simulated Poisson noise and repetitive excitation pulses which are typical of a TCSPC system. We found that the increased signal to noise ratio offered by both global and invariance fitting is highly desirable. When fitting mono-exponential data, the effects of a ca . 12.5 ns (ca . 80 MHz) repetitive excitation do not preclude the accurate extraction of populations with lifetimes in the range 0.1 to 10 ns, even when these effects are not represented in the fitting algorithm. Indeed, with global or invariance fitting of a 32x32 pixel area, the error in extracted lifetime can be lower than 0.4% for signals with a peak of 500 photon counts or more. In FRET simulations, modelling GFP with a non-interacting lifetime of 2.15 ns, it was possible to accurately detect a 10% interacting population with a lifetime of 0.8 ns.

© (2005) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.
Citation

Paul R. Barber ; Simon M. Ameer-Beg ; Julian D. Gilbey ; Richard J. Edens ; Ifeome Ezike, et al.
"Global and pixel kinetic data analysis for FRET detection by multi-photon time-domain FLIM", Proc. SPIE 5700, Multiphoton Microscopy in the Biomedical Sciences V, 171 (April 11, 2005); doi:10.1117/12.590510; http://dx.doi.org/10.1117/12.590510


Access This Proceeding
Sign in or Create a personal account to Buy this proceeding ($15 for members, $18 for non-members).

Figures

Tables

NOTE:
Citing articles are presented as examples only. In non-demo SCM6 implementation, integration with CrossRef’s "Cited By" API will populate this tab (http://www.crossref.org/citedby.html).

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

Advertisement
  • 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 Proceeding
Sign in or Create a personal account to Buy this proceeding ($15 for members, $18 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 SPIE.org.