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Journal of Biomedical Optics • Open Access

Methodological considerations for global analysis of cellular FLIM/FRET measurements
Author(s): Nur Aida Abdul Rahim; Roger D. Kamm; Peter T. C. So; Serge B. Pelet

Paper Abstract

Global algorithms can improve the analysis of fluorescence energy transfer (FRET) measurement based on fluorescence lifetime microscopy. However, global analysis of FRET data is also susceptible to experimental artifacts. This work examines several common artifacts and suggests remedial experimental protocols. Specifically, we examined the accuracy of different methods for instrument response extraction and propose an adaptive method based on the mean lifetime of fluorescent proteins. We further examined the effects of image segmentation and a priori constraints on the accuracy of lifetime extraction. Methods to test the applicability of global analysis on cellular data are proposed and demonstrated. The accuracy of global fitting degrades with lower photon count. By systematically tracking the effect of the minimum photon count on lifetime and FRET prefactors when carrying out global analysis, we demonstrate a correction procedure to recover the correct FRET parameters, allowing us to obtain protein interaction information even in dim cellular regions with photon counts as low as 100 per decay curve.

Paper Details

Date Published: 19 March 2012
PDF: 14 pages
J. Biomed. Opt. 17(2) 026013 doi: 10.1117/1.JBO.17.2.026013
Published in: Journal of Biomedical Optics Volume 17, Issue 2
Show Author Affiliations
Nur Aida Abdul Rahim, Massachusetts Institute of Technology (United States)
Roger D. Kamm, Massachusetts Institute of Technology (United States)
Peter T. C. So, Massachusetts Institute of Technology (United States)
Serge B. Pelet, Massachusetts Institute of Technology (United States)


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