PixFRET, an ImageJ plug-in for FRET calculation that can accommodate variations in spectral bleed-throughs
Fluorescence resonance energy transfer (FRET) allows the user to investigate interactions between fluorescent partners. One crucial issue when calculating sensitized emission FRET is the correction for spectral bleed‐throughs (SBTs), which requires to calculate the ratios between the intensities in...
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Published in | Microscopy research and technique Vol. 68; no. 1; pp. 51 - 58 |
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Main Authors | , , , , |
Format | Journal Article |
Language | English |
Published |
Hoboken
Wiley Subscription Services, Inc., A Wiley Company
01.09.2005
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Subjects | |
Online Access | Get full text |
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Summary: | Fluorescence resonance energy transfer (FRET) allows the user to investigate interactions between fluorescent partners. One crucial issue when calculating sensitized emission FRET is the correction for spectral bleed‐throughs (SBTs), which requires to calculate the ratios between the intensities in the FRET and in the donor or acceptor settings, when only the donor or acceptor are present. Theoretically, SBT ratios should be constant. However, experimentally, these ratios can vary as a function of fluorophore intensity, and assuming constant values may hinder precise FRET calculation. One possible cause for such a variation is the use of a microscope set‐up with different photomultipliers for the donor and FRET channels, a set‐up allowing higher speed acquisitions on very dynamic fluorescent molecules in living cells. Herein, we show that the bias introduced by the differential response of the two PMTs can be circumvented by a simple modeling of the SBT ratios as a function of fluorophore intensity. Another important issue when performing FRET is the localization of FRET within the cell or a population of cells. We hence developed a freely available ImageJ plug‐in, called PixFRET, that allows a simple and rapid determination of SBT parameters and the display of normalized FRET images. The usefulness of this modeling and of the plug‐in are exemplified by the study of FRET in a system where two interacting nuclear receptors labeled with ECFP and EYFP are coexpressed in living cells. Microsc. Res. Tech. 68:51–58, 2005. © 2005 Wiley‐Liss, Inc. |
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Bibliography: | UNIL/EPFL Fund istex:2C6E55BFAD4E8C2790AB817240A7FFE49B2BA836 ark:/67375/WNG-6N25BX0M-8 ArticleID:JEMT20215 The Etat de Vaud The National Research Project 50 The Swiss National Science Foundation ObjectType-Article-2 SourceType-Scholarly Journals-1 ObjectType-Feature-1 content type line 23 ObjectType-Article-1 ObjectType-Feature-2 |
ISSN: | 1059-910X 1097-0029 |
DOI: | 10.1002/jemt.20215 |