Methodological considerations for global analysis of cellular FLIM/FRET measurements

Nur Aida Abdul Rahim; Roger D. Kamm; Peter T. C. So; Serge B. Pelet

doi:10.1117/1.JBO.17.2.026013

19 March 2012 Methodological considerations for global analysis of cellular FLIM/FRET measurements

Nur Aida Abdul Rahim, Roger D. Kamm, Peter T. C. So, Serge B. Pelet

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Journal of Biomedical Optics, Vol. 17, Issue 2, 026013 (March 2012). https://doi.org/10.1117/1.JBO.17.2.026013

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.

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Nur Aida Abdul Rahim, Roger D. Kamm, Peter T. C. So, and Serge B. Pelet "Methodological considerations for global analysis of cellular FLIM/FRET measurements," Journal of Biomedical Optics 17(2), 026013 (19 March 2012). https://doi.org/10.1117/1.JBO.17.2.026013

Published: 19 March 2012

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