Linked Life Data

19588234

Source:http://linkedlifedata.com/resource/pubmed/id/19588234

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
1
pubmed:dateCreated
2010-2-10
pubmed:abstractText
The current advances of fluorescence microscopy and new fluorescent probes make fluorescence resonance energy transfer (FRET) a powerful technique for studying protein-protein interactions inside living cells. It is very hard to quantitatively analyze FRET efficiency using intensity-based FRET imaging microscopy due to the presence of autofluorescence and spectral crosstalks. In this study, we for the first time developed a novel photobleaching-based method to quantitatively detect FRET efficiency (Pb-FRET) by selectively photobleaching acceptor. The Pb-FRET method requires two fluorescence detection channels: a donor channel (CH ( 1 )) to selectively detect the fluorescence from donor, and a FRET channel (CH ( 2 )) which normally includes the fluorescence from both acceptor and donor due to emission spectral crosstalk. We used the Pb-FRET method to quantitatively measure the FRET efficiency of SCAT3, a caspase-3 indicator based on FRET, inside single living cells stably expressing SCAT3 during STS-induced apoptosis. At 0, 6 and 12 h after STS treatment, the FRET efficiency of SCAT3 obtained by Pb-FRET inside living cells was verified by two-photon excitation (TPE) fluorescence lifetime imaging microscopy (FLIM). The temporal resolution of Pb-FRET method is in second time-scale for ROI photobleaching, even in microsecond time-scale for spot photobleaching. Our results demonstrate that the Pb-FRET method is independent of photobleaching degree, and is very useful for quantitatively monitoring protein-protein interactions inside single living cell.
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Jan
pubmed:issn
1573-4994
pubmed:author
pubmed:issnType
Electronic
pubmed:volume
20
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
27-35
pubmed:meshHeading
pubmed:year
2010
pubmed:articleTitle
Photobleaching-based quantitative analysis of fluorescence resonance energy transfer inside single living cell.
pubmed:affiliation
MOE Key laboratory of Laser Life Science & Institute of Laser Life Science, South China Normal University, Guangzhou, 510631, China.
pubmed:publicationType
Journal Article, Research Support, Non-U.S. Gov't