EasyLife L

EasyLife™ L Lifetime Benefits

 
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What are the intrinsic benefits of a time-resolved phosphorescence lifetime system over a steady-state fluoromseter for molecular studies?

The lifetime is an intrinsic molecular parameter, whereas steady state fluorescence captures the average of multiple factors thus loosing intrinsic information. The lifetime is not dependent on the luminescence intensity and so it is not susceptible to artifacts such as scatter, photobleaching or local probe concentration which have no effect on the intrinsic lifetime. If the sample is diluted, the luminescence intensity will decrease, while the lifetime will remain unchanged.

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

LRET

The lifetime-based technique is ideal for sensing applications, where binding of a substrate changes the lanthanide lifetime. Lanthanide Resonance Energy Transfer (LRET) 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 LRET like behavior observed with a steady state technique may not be LRET at all. It could be static quenching from some other complexation. Only the lifetime technique can rule out static quenching. So if you do a lot of LRET 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 LRET checker.

Multiple Structural Domains and Conformations

Lifetimes allow you to 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 phosphorescence molecules are used as probes to study complex systems, such as DNA etc. These systems frequently exhibit multiple structural domains and conformations. The fluorescence decay will reveal this information by exhibiting multiple lifetimes, while on the other hand this information will be totally obscured in the steady-state measurement alone.

Binding Efficiency (Bound Versus Unbound Probe)

A steady state experiment can reveal a binding between a luminescence probe and a protein. Normally, the luminescence 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, 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 phosphorescence 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 the intensity decrease as more and more quencher is added. If you do a lifetime measurement, 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 LRET technique. It is critical to prove that a 'LRET-like' behavior is not caused by static quenching (mechanism 2). Only a lifetime experiment can rule it out.

Rotational Diffusion Rates and Molecular Size

Phosphorescence 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™ L visit the EasyLife™ L 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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