Implementation of FRET Spectrometry Using Temporally Resolved Fluorescence: A Feasibility Study

Autor: Justin Trujillo, Aliyah S. Khan, Dhruba P. Adhikari, Michael R. Stoneman, Jenu V. Chacko, Kevin W. Eliceiri, Valerica Raicu
Jazyk: angličtina
Rok vydání: 2024
Předmět:
Zdroj: International Journal of Molecular Sciences, Vol 25, Iss 9, p 4706 (2024)
Druh dokumentu: article
ISSN: 1422-0067
1661-6596
DOI: 10.3390/ijms25094706
Popis: Förster resonance energy transfer (FRET) spectrometry is a method for determining the quaternary structure of protein oligomers from distributions of FRET efficiencies that are drawn from pixels of fluorescence images of cells expressing the proteins of interest. FRET spectrometry protocols currently rely on obtaining spectrally resolved fluorescence data from intensity-based experiments. Another imaging method, fluorescence lifetime imaging microscopy (FLIM), is a widely used alternative to compute FRET efficiencies for each pixel in an image from the reduction of the fluorescence lifetime of the donors caused by FRET. In FLIM studies of oligomers with different proportions of donors and acceptors, the donor lifetimes may be obtained by fitting the temporally resolved fluorescence decay data with a predetermined number of exponential decay curves. However, this requires knowledge of the number and the relative arrangement of the fluorescent proteins in the sample, which is precisely the goal of FRET spectrometry, thus creating a conundrum that has prevented users of FLIM instruments from performing FRET spectrometry. Here, we describe an attempt to implement FRET spectrometry on temporally resolved fluorescence microscopes by using an integration-based method of computing the FRET efficiency from fluorescence decay curves. This method, which we dubbed time-integrated FRET (or tiFRET), was tested on oligomeric fluorescent protein constructs expressed in the cytoplasm of living cells. The present results show that tiFRET is a promising way of implementing FRET spectrometry and suggest potential instrument adjustments for increasing accuracy and resolution in this kind of study.
Databáze: Directory of Open Access Journals
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