Abstract
As the hardware of FLIM technique becomes mature, the most important criterion for FLIM application is the correct interpretation of its data. In this research, first of all, a more orthogonal phasor approach, called as Modified Phasor Approach (MPA), is put forward. It is a way to calculate the lifetime of the complex fluorescent process, and a rule to measure how much the fluorescence process deviates from single exponential decay. Secondly, MPA is used to analysis the time-resolved fluorescence processes of the transfected CHO-K1 Cell lines expressing adenosine receptor A1R tagged by CYP and YFP, measured in the channel of the acceptor. The image of the fluorescence lifetime and the multiplication of the fluorescence lifetime and deviation from single exponential decay reveal the details of the Homo-FRET. In one word, MPA provides the physical meaning in its whole modified phasor space, and broadens the way for the application of the fluorescence lifetime imaging.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Lakowicz JR (2006) Principles of fluorescence spectroscopy, 3rd edn. Springer, New York
Kreyszig E (2005) Advanced engineering mathematics. John Wiley & Sons, Orlando
Jameson DM, Gratton E, Hall RD (1984) The measurement and analysis of heterogeneous emissions by multifrequency phase and modulation fluorometry. Appl Spectrosc Rev 20:55–106
Jares-Erijman EA, Jovin TM (2003) FRET imaging. Nat Biotechnol 21:1387–1395
Clayton AHA, Hanley QS, Verveer PJ (2004) Graphical representation and multicomponent analysis of single-frequency fluorescence lifetime imaging microscopy data. J Microsc 213:1–5
Verveer PJ, Bastiaens PIH (2003) Evaluation of global analysis algorithms for single frequency fluorescence lifetime imaging microscopy data. J Microsc 209:1–7
Hanley QS, Clayton AHA (2005) AB-plot assisted determination of fluorophore mixtures in a fluorescence lifetime microscope using spectra or quenchers. J Microsc 218:62–67
Hanley QS (2009) Spectrally resolved fluorescent lifetime imaging. J R Soc Interface 6:S83–S92
Grecco HE, Roda-Navarro P, Verveer PJ (2009) Global analysis of time correlated single photon counting FRET-FLIM data. Opt Express 17:6493–6508
Digman MA, Caiolfa VR, Zamai M, Gratton E (2008) The phasor approach to fluorescence lifetime imaging analysis. Biophys J 94:L14–L16
Redford GI, Clegg RM (2005) Polar plot representation for frequency-domain analysis of fluorescence lifetimes. J Fluoresc 15:805–815
Leray A, Spriet C, Trinel D, Blossey R, Usson Y, Heliot L (2011) Quantitative comparison of polar approach versus fitting method in time domain FLIM image analysis. Cytom Pary A 79A:149–158
Zhou Y, Dickenson JM, Hanley QS (2009) Imaging lifetime and anisotropy spectra in the frequency domain. J Microsc 234:80–88
Wouters FS, Esposito A (2008) Quantitative analysis of fluorescence lifetime imaging made easy. J of HFSP 2:7–11
Acknowledgment
This work was partially supported by the Engineering and Physical Sciences Research Council, UK, under a Life Science Interface Grant EP/E013422/1, and is mainly supported by 211 Project, Guangdong province, P. R. China, under Grant YueFaGai 2009 [432]. The author is grateful to Dr. Q. S. Hanley at Nottingham Trent University, UK for his encouragement and helpful discussions.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Zhou, Y., Bai, Y., Chen, C. et al. Processing of Fluorescence Lifetime Image Using Modified Phasor Approach: Homo-FRET from the Acceptor. J Fluoresc 23, 725–732 (2013). https://doi.org/10.1007/s10895-013-1193-y
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10895-013-1193-y