Despite the complementary nature of the fluorescence lifetime technique, most users found this unsuitable because of the size, cost and complexity involved to operate the instrument. Although the cost of instruments has reduced considerably, including the size and complexity of operation, before the introduction of the new EasyLife-X system, some users were still wary of using the fluorescence lifetime technique.
The EasyLife-X has completely changed this scenario as it is compact, easy to operate and comes at an affordable price, thus making it suitable for users performing luminescence measurements.
For instance, users may wish to characterize the excited state of an organic molecule to determine the rate constants for the nonradiative deactivation and emission. This data is instantly available by merging the lifetime from the time-resolved measurement with the quantum yield from the steady state.
The steady-state measurement can offer an anisotropy value, fluorescence spectrum or fluorescence quantum yield. However, most of the information from this measurement is jumbled because the measured parameters are time averages and the data related to particular processes is lost.
But this lost data is important when fluorescent molecules are utilized as probes to examine nucleic acids, membranes, polymers, proteins, surfactants (micelles), and other complex systems. These systems often have a number of structural domains and conformations and this data is revealed by the fluorescence decay by exhibiting multiple lifetimes. In contrast, this data will be completely hidden in the steady-state measurement.
Let us consider a protein that contains a single Trp residue, for instance human serum albumin (HSA). When a steady state measurement is performed, one can obtain a typical Trp spectrum reflecting no specific data about the protein, other than the fact that it contains Trp. However, when fluorescence decay is measured, one can find that this Trp residue has four different lifetimes. This way, information is readily available to users that the protein exists in at least four different conformational states.
A steady state experiment can disclose a binding between a protein and a fluorescent probe. The fluorescence intensity will normally change as a result of binding and depending on the nature of the probe it will either increase or decrease. However, the information obtained is very unspecific because only some kind of binding was detected.
In the case of the lifetimes, the binding will impact the lifetime of the probe by either increasing or decreasing, but users will be able to detect two lifetimes and also their relative contributions i.e. pre-exponential factors to the overall decay. Consequently, the efficiency of binding from the lifetime measurement is then known.
The lifetime experiment will reveal more than one lifetime owing to different sites that Trp may take up in the protein. Also, from the quencher effect on each lifetime, one can obtain data about the localization of each type of Trp residues.
The steady state experiment cannot reveal the mechanism of fluorescence quenching. To this end, collisional (dynamic) quenching and static quenching are the two mechanisms that lead to quenching. In the former case, the excited fluorophore and quencher collide and diffuse apart, while in the latter case, the fluorophore forms a nonfluorescent complex with quencher. In both mechanisms, the steady state experiment will show a decrease in intensity as more quencher is added.
When users perform lifetime measurements, they will see that in collisional quenching the lifetime will decrease as more and more quencher is added; while in static quenching no change is observed in the lifetime. When one uses the FRET technique, it is very important to distinguish between the two mechanisms and to show that a ‘FRET-like’ behavior is not induced by static quenching. This can only be ruled out only by a lifetime experiment.
Thus, one can say that the main difference between the data obtained from the lifetime and steady state measurements is that the steady state tells users that something has occurred, while the lifetime measurement tells what has occurred.
The lifetime is an “intrinsic” molecular parameter, which means the fluorescence intensity will not be relied upon by the lifetime value. When the sample is diluted, the lifetime will not change, while the fluorescence intensity will decrease.
It is often necessary to merge results from the lifetime and steady state measurements to acquire complete information about the object under study. In this sense the two techniques are complementary.
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Optical Building Blocks Corporation (OBB) designs, produces and markets state-of-the-art, proprietary electro-optical components and instrumentation used in leading research institutions around the world. Today OBB offers three main product groups of equipment; Interconnectable "Optical Building Blocks", Microscope Accessories, and Bench-Top Instruments.
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