Looking for a high end modular research fluorometer instead?
If you are looking for the most sensitive and advanced modular research fluorometer, we invite you to consider the QuantaMaster™ family of fluorometers offered by our sister company Photon Technology International (PTI). PTI provides a comprehensive line of instruments for steady state and time-resolved luminescence research. For more information visit www.pti-nj.com
Fluorescence Lifetimes down to 150 ps, at a fraction of the cost and size of a bench-top fluorometer
Though it is small and inexpensive the EasyLife™ X is a serious performer
10% Binding Efficiency
ANS binding to bovine serum albumin was monitored with the EasyLife™ X equipped with a 370 nm LED. The ratio of free ANS to BSA bound ANS (9:1) can be easily determined from the double exponential fit to the fluorescence decay.
If you have a steady state fluorometer you need a time-resolved EasyLife™ X!
The EasyLife™ X is a compact filter based fluorescence lifetime system that is an excellent companion to any lab that currently uses a steady state fluorometer but does not have access to a fluorescence lifetime system. At a fraction of the cost of a bench-top spectrofluorometer, the EasyLife™ X is extremely easy to use and yet has powerful time-resolved capabilities and decay analysis software.
Why fluorescence lifetimes?
If you have a Steady State Fluorometer you need a Time Resolved Fluorometer to,
Differentiate multiple structural domains and conformations
If you want to characterize a molecule’s interactions with the surrounding environment, the steady-state measurement alone can provide a fluorescence spectrum, fluorescence quantum yield or anisotropy value, however most of this information is scrambled together, as 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, quantum dots, membranes, polymers, surfactants (micelles) etc. These systems frequently exhibit multiple structural domains and conformations. The fluorescence lifetime decay curve will reveal this information by detecting multiple fluorescence lifetimes, which cannot be gleaned with a steady-state measurement where all of this information is totally obscured.
The EasyLife™ X software even includes extremely powerful decay analysis software including ESM and MEM distribution analysis shown here.
Study protein conformation dynamics
A very powerful application for a time-resolved fluorescence instrument is the study of multiple conformational states of a protein. Consider a simple case of a protein containing one Tryptophan (Trp) residue (e.g. human serum albumin HSA). With a steady state instrument all you can measure is a typical Trp spectrum reflecting no particular information about the protein, except that it contains Trp. However, if you measure the fluorescence decay, you’ll find that this single Trp residue has 4 different discrete fluorescence lifetimes! You know immediately that the protein exists in at least 4 different conformational states, and for each lifetime you know the percentage of tryp residues in that state.
Quantitate binding efficiency
|10% Binding Efficiency
For this experiment the binding efficiency is 10% since the pre-exponential factor for the second, longer lived lifetime (the bound component) is measured as 10%. 90% of the ANS is not bound
A steady state experiment can reveal 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 that binding has occurred or not and that is all. However with a fluorescence lifetime system the binding will affect the probe lifetime (it will either decrease or increase for example with ANS binding to BSA), but at the same time you also detect two different 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).
Localize Trp in a protein
One of the major tools of fluorescence is studying quenching of fluorophores by adding quencher molecules. For example, tryptophan residues in a protein can be quenched by acrylamide or iodide ions. A steady state experiment can show the decrease of fluorescence intensity as the quencher is added, and hence that quenching has occurred, but it cannot tell you if it was dynamic or static quenching. A fluorescence lifetime experiment however will detect more than one lifetime due to different sites that Trp may occupy in the protein. Furthermore the fluorescence decay will provide the quencher effect on each lifetime, so you can get information about localization of each type of the Trp residues (e.g. are they surface exposed or buried inside the protein).
Verify you are really measuring FRET
The Förster Resonance Energy Transfer (FRET) technique has become a very powerful and wide-spread experimental tool for studying molecular binding. It is equally popular on the cellular level with fluorescence microscopes as well as in molecular solutions in cuvettes. However most investigators are using steady state techniques to observe and quantitate the ratio of fluorescence intensities of the acceptor and donor wavelengths. This has lead to a number of false conclusions and an increased awareness that the time-resolved technique is really the only way to be certain that you are actually measuring FRET.
Having a time-resolved fluorescence system is essential because the actual mechanism of fluorescence quenching in general cannot be revealed by the steady state experiment at all. There are two mechanisms that lead to quenching. The first is collisional (or dynamic) quenching, where the excited fluorophore and quencher collide and diffuse apart. The second is 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. In the case of collisional (dynamic) quenching the lifetime measurement will show the lifetime decrease as more quencher is added. However, in the case of static quenching there will be no change in the lifetime at all. Discerning between the two mechanisms is critically important when one is looking to study Förster Resonance Energy Transfer (FRET). Only the time-resolved technique can prove that a ‘FRET-like’ behavior is not caused by static quenching. Only a lifetime experiment can rule it out.
Measure rotational diffusion rates and determine size of macromolecules
Fluorescence anisotropy (polarization) is another example of the importance of the lifetime technique. A probe molecule in a buffer will show no or very little anisotropy (ie it is not directionally dependent). Attach the probe to a protein, DNA, membrane or other large target and the fluorescence anisotropy of the probe will increase. This is all that a steady state fluorometer can tell you, that the probe is now attached to a much bigger entity. However, if you can measure the time-resolved fluorescence anisotropy of the probe, you can estimate the rate of rotational diffusion and the actual size of the macromolecule your probe is attached to. This cannot be determined with a steady state fluorometer.
Time-Resolved Fluorescence (Fluorescence Lifetimes) is an Invaluable Complement to Steady State Fluorescence (Fluorescence Spectra)
If you are currently using a steady state fluorometer for luminescence measurements, but do not have access to a fluorescence lifetime system, you should seriously consider adding the EasyLife™ X to your lab. Fluorescence intensity (steady state) and fluorescence lifetime (time-resolved) measurements are complementary. One must frequently combine results from the steady state fluorescence and the fluorescence lifetimes measurements in order to obtain the most complete information about the molecule(s) of interest. When the first modern day fluorescence lifetime instruments were introduced some thirty years ago, some investigators inherently understood the complementary nature of the fluorescence lifetime technique but it was frankly irrelevant at that time because the cost, size and complexity of those early instruments discouraged all but a relatively few from using this new time-resolved technique. Although instrumentation cost have decreased quite dramatically, as well as the size and complexity of operation, prior to the introduction of the new EasyLife™ X, it was still difficult to convince investigators to invest in a fluorescence lifetime instrument.
At a fraction of the cost of a bench-top fluorometer, and because it is just as easy to operate as a fluorometer, the introduction of the EasyLife™ X system has changed people’s attitude towards fluorescence lifetimes as a technique. Now everyone who is doing luminescence measurements can and should put fluorescence lifetimes to good use. By adding time-resolved fluorescence to your research capabilities you will finally be able to fully characterize your fluorescing molecule and molecular systems. For example, you will be able to find out what the rate constants are for the fluorescence emissions and for the non-radiative deactivation of your samples. This information is readily available by combining the lifetime results with the quantum yield values measured from the steady state instrument.
Truly Affordable and Easy to Use!
The EasyLife™ X is the realization of a 40 year dream to make a small, simple, affordable fluorescence lifetime system that literally anyone can use. Using our patented fluorescence lifetime detection technique and simple snap-on pulsed LED's and laser diodes, the EasyLife™ X finally allows any researcher to be able to afford and actually conduct fluorescence lifetime experiments in their own lab.
Thinking about adding fluorescence lifetimes to your existing fluorometer?
Don't waste your money on an expensive addition to your aging fluorometer. The EasyLife™ X offers all of the same capabilities in a new, stand alone, 1 cubic foot instrument, and it will cost you less money. Plus you will have the benefit of having both your steady state fluorometer and your time resolved EasyLife™ X system both being used at the same time, dramatically increasing your labs productivity.
Skeptical that such a small, inexpensive system can do so much?
We don't blame you. It is understandable that anyone who has done fluorescence lifetime research using anything other than an EasyLife™ X could be skeptical. That is why we have compiled an extensive library of data collected on the EasyLife™ X. Please review it for samples similar to your own to see how the system could work for you. And if you don't believe us, you can also search and review literature citations using the EasyLife™.
So what are you waiting for?
Give us a call or request a quote to start on your path to an EasyLife™ for fluorescence lifetime experiments.
Incidentally if you have more demanding fluorescence lifetime requirements and are interested in a research fluorescence lifetime system, then we strongly recommend that you take a good look at the selection of modular, time-resolved systems available from our sister company, Photon Technology International.
For more information on the EasyLife™ X visit the EasyLife™ X Lifetime Technique page.