Methodological considerations for global analysis of cellular FLIM/FRET measurements
Name
Rahim-2012-Methodological consi.pdf
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2.29 MB
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Author(s)
Rahim, Nur Aida Adbul
Pelet, Serge
Kamm, Roger Dale
So, Peter T. C.
Date Issued
March 2012
Journal
Journal of Biomedical Optics
Publisher
SPIE
Citation
Adbul Rahim, Nur Aida et al. “Methodological Considerations for Global Analysis of Cellular FLIM/FRET Measurements.” Journal of Biomedical Optics 17.2 (2012): 026013. © 2012 Society of Photo-Optical Instrumentation Engineers
Version
Final published version
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.
MIT Department
Massachusetts Institute of Technology. Department of Biological Engineering
Massachusetts Institute of Technology. Department of Mechanical Engineering
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Article is made available in accordance with the publisher's policy and may be subject to US copyright law. Please refer to the publisher's site for terms of use.
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DOI of Published Version
https://doi.org/10.1117/1.jbo.17.2.026013
