Fluorescence Resonance Energy Transfer: Techniques for Measuring Molecular Conformation and Molecular Proximity
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- Abstract
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Abstract
This overview unit focuses on the basics of fluorescence and of the FRET phenomenon, and on methods for detecting FRET and data interpretation. FRET is very versatile and there are more application of these basics than can be covered in a single overview. However, some examples are given of applications of various FRET techniques.
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PDF or HTML at Wiley Online LibraryTable of Contents
- Basics of Fluorescence and FRET Mechanisms
- Practical FRET Microscopy
- Donor Dequenching After Acceptor Photobleaching
- Donor Fluorescence Lifetime Imaging
- Frequency Domain Lifetime Imaging
- Analysis of FRET Data
- Literature Cited
- Figures
- Tables
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Figure 18.10.1 Energy levels of a molecule. The lowest energy level is electronic energy level 1. The largest jumps in energy are in increasing electronic energy, with smaller jumps due to vibrational and rotational energies, Evib and Erot View Image -
Figure 18.10.2 The Jablonski diagram for excitation of a molecule by a photon followed by fluorescence emission. A molecule in the lowest electronic energy state, the ground state, S0 , absorbs the energy of a photon and is excited to a new electronic energy level, S1 or S2 . Each level is further divided by vibrational energy states. The higher the excited state, the less probable is the transition to this state. A given excited state quickly relaxes to its lowest level and then relaxation to the lowest level of S1 from any higher state occurs in ∼10−11 sec. After another 10−9 sec the molecule emits a photon (fluorescence) and returns to the ground state. View Image -
Figure 18.10.3 FRET detected in terms of fluorescence anisotropy. Fluorescence from cells expressing a GFP‐tagged protein was excited with polarized light and imaged with analyzers parallel or perpendicular to the excitation polarization. After correcting for instrument factors, anisotropy, r , was calculated as ∼0.32. A fraction of GFP was then bleached and a second measurement of r was made. This was repeated until only ∼20% of initial fluorescence remained. Anisotropy increases to ∼0.36 as more and more fluorescence was bleached. The change in r with fluorophore bleaching shows that FRET occurs between GFP molecules as described in the text. Data are from Rocheleau et al. (). View Image -
Figure 18.10.4 Excitation and emission spectra of a FRET donor/acceptor pair, fluorescein and Texas Red (sulforhodamine). The extensive overlap of the donor fluorescence emission spectrum with the acceptor excitation spectrum implies efficient FRET between the two. However, note that the emission of fluorescein extends to 600 nm and beyond. This red emission must be subtracted from the total signal when measuring Texas Red fluorescence (sensitized acceptor fluorescence). View Image -
Figure 18.10.5 Comparison of FRET measured in terms of donor‐sensitized acceptor fluorescence (Fa /CFP ordinate) with FRET measured in terms of donor dequenching after bleaching acceptor (Eapp (%) abscissa). Points are experimental values; the solid line is the theoretical relationship expected. Reproduced from Zal et al. () with permission. View Image -
Figure 18.10.6 FRET measured in terms of donor dequenching compared for live and fixed cells. The fluorophores are CFP and YFP. Both graphs show a strong dependence of FRET on acceptor concentration (measured as fluorescence). View Image -
Figure 18.10.7 Fluorescence lifetime imaging by impulse fluorimetry. Fluorescence is excited by a picosecond laser pulse at time 0. A gated intensified CCD camera is activated for two short intervals, t1 and t2, each a few nanoseconds. The pulse and collection is repeated until sufficient photons are stored in each pixel of the CCD to yield an image. While only a few gates are opened, the lifetime can be inferred from the fit of the data to the theoretical curve for decay of fluorescence after excitation. View Image -
Figure 18.10.8 The basis for frequency domain lifetime measurement. A fluorophore excited by light whose intensity varies as a sine wave emits fluorescence whose intensity varies with the same frequency, but out of phase. The phase shift depends on the lifetime of the excited state and so can this lifetime can be recovered from the signal. Changes in lifetime, over time or space, can be used to detect FRET. Based on Bastiaens and Squire (). View Image -
Figure 18.10.9 The theory for distinguishing FRET in a randomly distributed population from FRET among clustered or aggregated molecules. Three possible distributions of donors and acceptors are shown in the top panel. The graphs in the bottom panel show the expected dependence of FRET on acceptor surface density in experiments detecting FRET in terms of donor dequenching. It can be seen that FRET among randomly distributed molecules depends upon the amount of acceptor present, but is independent of the ratio of donors to acceptors. In contrast, in the lower right panel in can be seen that FRET is independent of the amount of acceptor present, but depends upon the ratio of donors to acceptors. The middle panel shows an intermediate case with some FRET between randomly distributed molecules and some FRET within clusters of these molecules. (after Kenworthy and Edidin, ). View Image
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Literature Cited
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