Figure - PMC

Skip to main content

An official website of the United States government

Here's how you know

Here's how you know

Official websites use .gov
A .gov website belongs to an official government organization in the United States.

Secure .gov websites use HTTPS
A lock ( ) or https:// means you've safely connected to the .gov website. Share sensitive information only on official, secure websites.

View full-text article in PMC

. 2005 Apr 1;33(6):e61. doi: 10.1093/nar/gni057

Search in PMC
Search in PubMed
View in NLM Catalog
Add to search

© The Author 2005. Published by Oxford University Press. All rights reserved

The online version of this article has been published under an open access model. Users are entitled to use, reproduce, disseminate, or display the open access version of this article for non-commercial purposes provided that: the original authorship is properly and fully attributed; the Journal and Oxford University Press are attributed as the original place of publication with the correct citation details given; if an article is subsequently reproduced or disseminated not in its entirety but only in part or as a derivative work this must be clearly indicated. For commercial re-use, please contact journals.permissions@oupjournals.org

PMC Copyright notice

Optical configuration of the DakoCytomation MoFlo cytometer and its use in simultaneously measuring three distinct FRET signals, CFP→YFP, CFP→YFP→mRFP and YFP→mRFP. (A) Optical configuration of the MoFlo used to detect CFP, YFP, mRFP, FRET1 (CFP→YFP), FRET2 (YFP→mRFP), potential FRET3 (CFP→mRFP) and two-step FRET (CFP→YFP→mRFP). Briefly, mRFP, an acceptor fluorophore for YFP (YFP→mRFP), was excited by the 568 nm line emitted by the Spectrum laser (laser 2) and its emission signal was collected with a 630/22 bandpass filter (FL6) in the second laser pathway. YFP is either a donor (YFP→mRFP) or an acceptor (CFP→YFP) or both (CFP→YFP→mRFP). It was excited by the 488 nm line emitted from the argon-ion laser (laser 1) and its signal was detected with a 546/10 bass-pass filter (FL1). The FRET2 signal emitted from sensitized mRFP molecules that were excited by closely approximated excited YFP moieties during YFP→mRFP FRET was collected with a 630/22 bandpass filter (FL3) in the first laser pathway. CFP, as a donor fluorophore (CFP→YFP FRET), was excited by the 407 nm line emitted from the krypton-ion laser (laser 3) and its signal was reflected with a 500 nm long-pass dichroic filter first and then detected with a 460/20 bandpass filter (FL9) in the third laser pathway. The FL8 detector in the third laser pathway was used to collect CFP→YFP FRET1 signals with a 546/10 bandpass filter. The FL10 detector in the third laser pathway was employed to collect the two-step-FRET signals emitted from mRFP in a two-step linked FRET reaction (CFP→YFP→mRFP) or possible mRFP FRET3 signals between CFP and mRFP with a 600 longpass filter, along with 600 shortpass splitter. (B) Fusion proteins composed of CFP, YFP and mRFP as well as TRAF2 (Ta) labeled with CFP, YFP or mRFP were used to validate the nature of the FRET signals. In the fusion protein system, the TRAF2 TRAF domain (T2TD), which forms a mushroom-shaped structure with a distance of 95 Å, as a FRET insulator, was inserted between fluorophores to prevent energy transfer. (C) Summary of excitation sources and emission signals for the different fluorophores (CFP, YFP, mRFP, FRET1, FRET2, FRET3 and two-step-FRET). The potential for donor quenching during the FRET process is also indicated.