Imaging molecular interactions in living cells by FRET microscopy
Introduction
Förster resonance energy transfer (FRET) is a photophysical phenomenon in which energy is transferred from the first excited electronic state (S1) of a fluorophore (the donor D) to another nearby absorbing (but not necessarily emitting) molecule (the acceptor A). Thus, there is a concerted quenching of D and activation of A fluorescence (Figure 1). For this reason, the acronym FRET is often, albeit incorrectly, used to designate ‘fluorescence’ resonance energy transfer. The process involves the resonant coupling of emission and absorption dipoles and is thus non-radiative. That is, it competes with other radiative (fluorescence) and non-radiative pathways for deactivation. The underlying formalism first elaborated by Theodor Förster establishes a parametric proportionality between the rate of transfer (kt) and the radiative rate constant (kf) and it is this relationship, operative over region of ∼1–20 nm, that forms the basis for the extensive application of FRET in virtually every field of biology, chemistry, physics and engineering.
This survey of FRET in imaging applications extends our last review of the subject in 2003 [1]. The format does not permit a comprehensive coverage of the vast literature but is rather intended as a selective guide to the present status and projected development of the field. Most specific citations are from 2004 to the present. Because of space limitations, we do not systematically consider single-molecule and fluorescence correlation spectroscopy, nor the use of molecular beacons, aptamers and other bioengineered FRET biosensors of ions, second messengers, and covalent modification (for a recent review see [2••]).
Section snippets
FRET formalism
FRET can be employed for probing or for systematically altering states of matter; the former use by far outweighs the latter in the reported literature. Its rational application necessarily involves (i) understanding the fundamental basis and parametric dependencies of the phenomenon; (ii) selecting D–A pairs and the means for their introduction into systems of interest; (iii) performing measurements and/or perturbing the system; and (iv) analyzing the results so as to confirm or reveal a
FRET probes
The identification of optimized D–A pairs is a perennial quest (see Update) and much value can be derived from perusal of the flow cytometric literature on this subject (see, for example [23]). Ideal fluorophores serving as donors have large extinction coefficients for single and/or multiphoton absorption, high emission quantum yields (or more precisely, large radiative rate constants kf; Figure 1; Equation 1), large Stokes shifts (separation between donor and acceptor emission bands), high
FRET imaging technology
In previous papers [1, 72], we provided a classification scheme for FRET imaging techniques based on the measurement parameters and modes of signal acquisition. We employ the same system in Table 2, in which we limit the entries to developments posterior to those cited in the previous reviews; other relevant citations are given in the text of this review. Much of the FRET literature originates or is associated with topics such as strategies for optical sectioning (confocal microscopy),
Future directions
In our estimation, major new FRET developments will lie in the area of the acceptor depletion techniques (adFRET, Table 2 Ie), particularly those based on non-linear phenomena such as ground state-depletion (excited state saturation), and multiparametric approaches. In this connection it is appropriate to stress the fundamental importance of fluorescence lifetime determinations, either in the time or frequency domain. FLIM (or FLI, fluorescence lifetime-resolved imaging) can be combined with
Update
A comprehensive survey has appeared of probes suitable as FRET donors and acceptors [96•]. A new expression probe has been reported consisting of a complex of an oligo-Asp tag with Zn(II) [97].
The work referred in the text as (CC Spagnuolo, R Vermeij, EA Jares-Erijman, unpublished) is now in press [98•].
References and recommended reading
Papers of particular interest, published within the annual period of review, have been highlighted as:
• of special interest
•• of outstanding interest
Acknowledgements
We thank many colleagues for valuable suggestions and discussions. EAJ-E. and TMJ are recipients of grant I/77 897 from the Volkswagen Foundation. EAJ-E is indebted to the Agencia Nacional de Promoción de la Ciencia y Tecnología (ANPCyT), Fundación Antorchas, Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Secretaría de Ciencia, Tecnología e Innovación Productiva (SECyT), Germany–Argentine DLR-BMBF-SECyT, and the Universidad de Buenos Aires (UBA) for financial support.
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