EasyLife TCSPC

DeltaPro™ Lifetime Benefits

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If you have a steady state fluorometer you need a time-resolved DeltaPro™!

The DeltaPro™ 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 DeltaPro™ is extremely easy to use and yet has powerful time-resolved capabilities and decay analysis software.

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 DeltaPro™ 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 DeltaPro™, 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 DeltaPro™ 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.

Why fluorescence lifetimes?
Six Important Things You Can’t Do with a Steady State Fluorometer

Differentiate Multiple Structural Domains and Conformations

HSA excited at 295nm, with the decay of the tryptophan modeled using NED distribution analysis

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 DeltaPro™ software even includes extremely powerful decay analysis software including MEM and our new NED routines.

Study Protein Conformation Dynamics

Phosphorescence decay of HSA excited using a SpectraLED-295, showing the change in average lifetime
(calculated from a 3 exponential fit) with temperature

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 3 different discrete fluorescence lifetimes!  By monitoring changes in them and their contribution to the overall emission conformational changes can be inferred. To uncover larger protein motions it is advantageous to use phosphorescence, as this timescale is more applicable so some domain movements. Again, changes in the phosphorescence lifetime and its component contributions can clearly show changes in conformation.

From changes in the lifetimes and their contributions you can immediately see changes in conformational states.

Binding Efficiency (Bound versus Unbound) of Fluorescence Probes

Kinetic TCSPC measurement showing diphenylheptanoid binding to serum albumin. Measured using a DeltaDiode laser with a 100MHz excitation rate and an acquisition time of 2ms/pt. The increase in the lifetime (fitted to a monexponential model) upon binding to serum albumin is clearly seen.

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 using lifetimes you can uncover 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). Making use of high repetition rate excitation sources, coupled with very low deadtime electronics, enables the fluorescence lifetime to also be used to follow the actual binding kinetics, as measurement times down to 1ms are possible.

Trp Localization in Protein via Fluorescence Quenching

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).

Time-Resolved FRET Checker: “Is that really FRET you are measuring?”

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.

Time-resolved Anisotropy Provides Rotational Diffusion Rates & 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 gives you the rotational correlation time plus the initial and limiting anisotropy values. By fitting to the anisotropy difference data and reconvoluting it is possible to accurately determine fast rotational correlation times as the instrumental distortion, that can be present in the simple anisotropy data, can be accounted for.


For more information on the DeltaPro™ TCSPC visit the DeltaPro™ 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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