Measuring Förster Resonance Energy Transfer Using Fluorescence Lifetime Imaging Microscopy
Richard N. Day
Department of Cellular and Integrative Physiology , Indiana University School of Medicine , 635 Barnhill Dr. , Indianapolis , IN 46202
rnday@iupui.edu Introduction
Despite an increasing reliance of biomedical research on high-throughput screening methods, the application of quanti- tative imaging techniques to analyze protein behavior in the context of living cells remains vitally important. T ese live-cell imaging approaches have greatly benefi tted from recent improvements in the photophysical qualities of the genetically encoded fl uorescent proteins (FPs) [ 1 ]. T e favorable spectral characteristics of the optimized FPs enable their use in Förster resonance energy transfer (FRET) microscopy to achieve the nanometer-scale resolution necessary to detect protein interactions in living cells. FRET microscopy measures the direct transfer of excitation energy from one fl uorophore (the donor) to other nearby molecules (acceptors). Because FRET occurs through near-fi eld electromagnetic dipole interactions, the effi ciency of energy transfer decreases as the inverse of the sixth power of the distance separating the fl uorophores [ 2 ]. T is limits FRET to separation distances of less than about 8 nm, making it an ideal tool to investigate cellular biochemical networks. T ere are many diff erent FRET microscopy approaches, broadly divided into methods that detect the sensi- tized emission from the acceptor and methods that detect the eff ect of energy transfer on the donor fl uorophore [ 2 ]. A key requirement for effi cient energy transfer is a strong overlap between the donor emission spectrum and the absorption spectrum of the acceptor ( Figure 1 ). A consequence of this overlap, however, is spectral bleedthrough (SBT) background that originates both from the direct excitation of the acceptor at the donor excitation wavelengths (arrow, Figure 1 ) and from the donor emission signal that bleeds into the FRET detection channel (hatching, Figure 1 ). T erefore, the quantifi cation of FRET by measurement of sensitized emission from the acceptor requires correction methods to remove these diff erent SBT background components [ 2 ]. In contrast, the signal in the donor channel is not contaminated by SBT ( Figure 1 ). T us, methods that directly measure the eff ect of energy transfer
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on the donor signal do not require corrections and can be the most accurate way to quantify FRET. In this article, the use of fl uorescence lifetime imaging microscopy (FLIM) to quantify the change in donor lifetime that occurs with FRET is demonstrated, and the advantages and the limitations of the approach are discussed.
Fluorescence Lifetime Imaging Microscopy T e fl uorescence lifetime is the average time that a popula-
tion of fl uorophores spends in the excited state before returning to the ground state, an event usually accompanied by the emission of photons. T e fl uorescence lifetime is an intrinsic property of a fl uorophore, and changes in the lifetime can provide information about the microenvironment surrounding the fl uorophores. For example, energy transfer is a quenching pathway that depletes excited-state energy from the donors,
Figure 1 : The SBT between donor and acceptor fl uorohores. The excitation and emission spectra for cyan (donor) and yellow FPs (acceptor) are shown. The dashed boxes indicate typical setup for the detection of donor and FRET (acceptor) signals. The arrow indicates the direct acceptor excitation at the donor excitation wavelength, and the hatching shows the donor SBT into the FRET channel.
doi: 10.1017/S1551929515000395
www.microscopy-today.com • 2015 May
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