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EasyLife X

EasyLife™ X Lifetime Benefits

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What are the intrinsic benefits of a time-resolved fluorescence lifetime system over a steady-state fluorometer for molecular studies? The fluorescence lifetime is an intrinsic molecular parameter, whereas the steady state fluorescence captures the average of multiple factors thus loosing intrinsic information. The fluorescence lifetime is not dependent on the fluorescence intensity so intensity losses due to light scattering, photobleaching or local probe concentration have no effect of the lifetime. If the sample is diluted, the fluorescence intensity will decrease, while the lifetime will remain unchanged.

Thus the lifetime provides information about intrinsic properties of an emitting molecule and its environment such as polarity, viscosity, ionic strength, bimolecular interactions, diffusion, energy and electron transfer and much more. With the lifetime you can unravel mechanisms of excited state processes. With the addition of polarizers, you can also study the rotational motion of the molecule such as environmental constraints or the relative orientation of absorption and emission dipoles of the molecule. Lifetime Applications abound!

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  • Some experimental examples of the benefits of using the time-resolved technique

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    FRET Checker

    Forster Resonance Energy Transfer (FRET) has become a very popular technique in the biological sciences. But if you do not have access to a time-resolved system, you should be very careful. It is now pretty common knowledge that some FRET like behavior observed with a steady state technique may not be FRET at all. It could be static quenching from some other complexation, and even the best scientists have been fooled by this phenomena into thinking they were doing a FRET experiment. Only the fluorescence lifetime technique can rule out static quenching. So if you do a lot of FRET research with a fluorometer, or on a fluorescence microscope or even a large confocal microscope, be very careful. Your research group could really benefit from getting an inexpensive and easy to use EasyLife to be your simple FRET checker.

    Of course the EasyLife™ can also be used to quantify FRET parameters for sophisticated FRET experiments. For more information refer to the FRET Calculator Technical Note

    Multiple Structural Domains and Conformations

    You can characterize a molecule under study and its interactions with the surrounding environment. In the steady-state measurement alone, which can provide a fluorescence spectrum, fluorescence quantum yield or anisotropy value, most of this information is scrambled, since the measured parameters are time averages and the information about specific processes is lost. This lost information becomes especially important when fluorescent molecules are used as probes to study complex systems, such as proteins, nucleic acids, membranes, polymers, surfactants (micelles) etc. These systems frequently exhibit multiple structural domains and conformations. The fluorescence decay will reveal this information by exhibiting multiple lifetimes, as well as the relative percentage of the presence of each lifetime component, while on the other hand this information will be totally obscured in the steady-state measurement alone.

    Trp Conformation

    For example, consider a simple case of a protein containing one Trp residue (e.g. human serum albumin HSA). Carry out a steady state measurement and you'll get a typical Trp spectrum reflecting no particular information about the protein, except that it contains Trp. However, if you measure its fluorescence decay, you'll find that this single Trp residue has 4 different lifetimes! You know immediately that the protein exists in at least 4 different conformational states, and you know the relative abundance of each state.

    Binding Efficiency (Bound Versus Unbound Probe)

    A steady state experiment can reveal a binding between a fluorescent probe and a protein. Normally, the fluorescence intensity will change as a result of binding; it will either decrease or increase, depending on the nature of the probe. The information you get is very general. You detected some kind of binding and that is all. Not so with the lifetimes. Here the binding will affect the probe lifetime, it will either decrease or increase (e.g. see ANS binding to BSA), but at the same time you also detect two lifetimes, one for the bound and the other for the unbound probe, as well as their relative contributions (pre-exponential factors) to the overall decay. From the lifetime measurement you now know relative populations of bound and unbound probes (i.e. we know the efficiency of binding).

    Excited State Characterization

    You can fully characterize the excited state of an organic molecule to find out what the rate constants for the emission and for the non-radiative deactivation are. This information is readily available by combining the lifetime from the time-resolved measurement with the quantum yield determined from the steady state fluorometer.

    Quenching Mechanisms

    The mechanism of fluorescence quenching in general cannot be revealed by the steady state experiment at all. There are 2 mechanisms that lead to quenching:

    1. collisional (dynamic) quenching, where excited fluorophore and quencher collide and diffuse apart.
    2. static quenching, where fluorophore in the ground state forms a non-fluorescent complex with quencher.

    In both cases the steady state experiment will show intensity decrease as more and more quencher is added. If you do lifetime measurements, in the 1st case you'll see the lifetime decrease as more quencher is added, while in the 2nd case there will be no change in the lifetime at all. Discerning between the two mechanism is especially very important when one uses the FRET technique. It is critical to prove that a 'FRET-like' behavior is not caused by static quenching (mechanism 2). Only a lifetime experiment can rule it out.

    Quenching to Probe Localization of Trp Residues on a Protein

    One of the major tools of fluorescence is studying quenching of fluorophores by added quencher molecules. For example, tryptophan residues in a protein can be quenched by acrylamide or iodide ions. A steady state experiment will just show the decrease of fluorescence intensity as the quencher is added. The lifetime experiment however will show more than one lifetime (due to different sites that Trp may occupy in the protein). From the quencher effect on each lifetime, you can get information about localization of each type of the Trp residues (e.g. are they surface-exposed or buried inside the protein).

    Rotational Diffusion Rates and Molecular Size

    Fluorescence anisotropy (polarization) is another example of the importance of lifetimes. A probe molecule in a buffer will show no, or very little, anisotropy. Attach it to a protein, DNA, membrane etc. and the anisotropy is increased. This is all that the steady state experiment can tell you: the probe is attached to a much bigger entity. However, if you measure the lifetime of the probe, you can estimate the rate of rotational diffusion and the size of the macromolecule your probe is attached to.

     

    For more information on the EasyLife™ X visit the EasyLife™ X Hardware page.

     
    OBB has a policy of continuous product development and reserves the right to amend part numbers, descriptions and specifications without prior notice.
     
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