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Our experience in lifetime instrumentation development goes back almost 40 years. We have been well aware of the many potential uses of this technique. The barrier for the acceptance has been the high price and the complexity of the equipment. EasyLife™ V is a major breakthrough that has changed all that. The price is less expensive than a good spectrofluorometer and you can take measurements out of the box – it is that easy. Now we can be practical and recommend the EasyLife™ V for many new applications.
If your work is in an area that can benefit from fluorescence techniques, then the EasyLife™ V is the instrument you cannot do without! The fluorescence lifetime technique the EasyLife™ V employs is indispensable in obtaining the maximum information about any molecular system, something you simply cannot get with conventional techniques. Whether you are involved in biology, chemistry, pharmaceutical science, food technology or material science, your work will be greatly enriched by having the EasyLife™ V at your fingertips. And indeed, all you need is your fingertips: the instrument is fully controlled from your laptop and requires neither manual adjustments nor maintenance.
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Most proteins fluoresce due to the presence of any or all three fluorescent amino acids: tryptophan, tyrosine, and phenylalanine. Intrinsic time-resolved fluorescence of tryptophan is commonly used to study the structure and
dynamics of proteins. These experiments require pulsed light sources emitting in the UV, between 270 and 295 nm. The EasyLife™ V, equipped with the 280 or 295-nm pulsed LED source, is a very robust yet fast instrument perfectly suited for use with tryptophan and tyrosine fluorophores.
| If you happen to use external fluorophores, there is a large selection of pulsed LEDs available for any wavelength in the UV-VIS range. A polarity sensitive, hydrophobic probe such as ANS is a good illustration of binding of an extrinsic probe to a protein. ANS binding to bovine serum albumin was monitored with the EasyLife™ V equipped with the 370 nm LED. The lifetime of ANS in the buffer is very short, 325 ps, and increases to 8 ns upon binding to BSA. The ratio of free ANS to BSA bound ANS (9:1) can be easily determined from the double exponential fit to the fluorescence decay. |
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Fluorescence decays of bovine serum albumin (BSA) in PBS buffer were measured with the EasyLife™ V. The native protein shows a nearly single-exponential decay with an average lifetime of 6.31 ns. After being treated with SDS detergent, BSA undergoes a structural transition and its fluorescence decay exhibits two shorter lifetimes, 1.47 ns (37%) and 4.43 ns (63%). |
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If you study conformational features or hybridization of DNA, the EasyLife™ V is the right system for you. A probe molecule in a buffer will show very little or no anisotropy. Attach it to a protein, DNA, or membrane, however, 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 in addition to the size of the macromolecule that is attached to the probe.
| Ethidium Bromide (EB) is a commonly used DNA probe, which readily intercalates between the DNA bases. EB is weakly fluorescent in aqueous media, but becomes strongly fluorescent after intercalation into DNA. The lifetime of EB in buffer is 1.71 ns and increases dramatically to 22.7 ns after binding to calf thymus DNA. |
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Fluorescence decay of PicoGreen/DNA measured with an EasyLife™ V lifetime system. Conformational diversity may result in multiple lifetimes of the probe bound to DNA. The EasyLife™ V is fully capable of measuring and analyzing such complex decays. The decay of PicoGreen, a common probe for double-stranded DNA, exhibits a clearly multi-exponential behavior, resulting in three lifetimes that range from 220 ps to 9.7 ns. |
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One of the classical biological applications of time-resolved fluorescence spectroscopy. Typically, an elongated hydrophobic (i.e. water insoluble) molecule is used as a probe (e.g. DPH) and the anisotropy decay is measured. Due to topology of the membrane, the probe rotations are limited to the space within a cone. The rotational correlation time and the cone angle are obtained from the anisotropy data.
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A very basic research area, where a fluorescing molecule is a research object on its own rather than a means of studying something else. Fluorescence lifetimes are measured in order to gain insight about the nature of electronic transition, determine radiative and nonradiative rate constants, follow excited state relaxation processes, intrinsic changes in molecular geometry, electron and energy transfer, interactions with solvent, etc.
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Micelles are molecular aggregates that are formed when soaps and detergents are dissolved in water. Fluorescence decays are used mainly to determine micelle aggregation number, critical micelle concentration, polydispersity (distribution of micelle sizes) and diffusion rates in micelles.
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Time resolved fluorescence is used to study intramolecular chain dynamics, end-to-end distances, secondary structure, viscosity, and association of polymers. Usual techniques are: FRET, anisotropy decays, excimer or exciplex formation and lifetimes.
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Cyclodextrins (CDs) are cyclic sugar molecules that possess internal cavities capable of complexing hydrophobic organic and organometallic molecules in aqueous solution. The CDs are shaped like truncated cones with three different cavity diameters: 6.5 A (α-CD), 7.5 A (β-CD) and 9.0 A (γ-CD). Since the CDs are water soluble, but have hydrophobic interior, they can be used to deliver hydrophobic drugs. Interactions and inclusion of drugs, steroids and other molecules with the CDs have been extensively studied with the time-resolved fluorescence. The CDs are also used to study the effects of restricted geometries on the photochemistry and dynamic behavior of guest molecules.
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Many molecules exhibit substantial degree of charge redistribution upon excitation. Some may even undergo a full charge separation where an electron jumps from one end of the molecule to the other. In many cases changes in molecular geometry accompany the electron transfer (e.g. TICT: twisted intramolecular charge transfer states). Such molecules are often used as fluorescent probes, since the highly polar excited states make them very sensitive to the environment. The ICT phenomenon happens also naturally, e.g. as one of primary processes in photosynthesis. Fluorescence lifetime is a common tool to study the ICT; it gives directly the rate constant for the charge transfer. Fluorescence decay kinetics, combined with time resolved fluorescence spectra could elucidate the kinetic mechanism that leads to the ICT state as well as the mechanism of subsequent relaxation.
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The fluorescence decay of CdSe quantum dots measured with the EasyLife™ V indicates a highly heterogeneous nature of the sample. A unique feature of the EasyLife™ V, the ability to acquire data using logarithmic or arithmetic progression timescales, facilitates greatly in analysis of multi-exponential decays with an underlying broad range of lifetimes. Here, a 4-exponential decay function was needed to adequately describe the experimental decay, acquired with the arithmetic timescale. This result was validated by the ESM lifetime distribution analysis, another powerful analytical tool in the EasyLife™ V software, which confirmed the lifetime values from the discrete 4-exponential analysis. |
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Fluorescence decays of sample 5 in MTHF at 295 K and 77 K measured with the EasyLife™ V lifetime system equipped with the liquid nitrogen dewar attachment. The recovered lifetimes are 4.4 ns (295 K) and 7.2 ns (77 K). |
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Lifetimes:
- Lifetime: average time the molecule remains in the excited state
- Lifetimes provide information about intrinsic properties of emitting molecule and its environment (polarity, viscosity, ionic strength, bimolecular interactions, diffusion, energy and electron transfer etc. applications abound!)
- Unravel mechanisms of excited state processes
Anisotropy Decays:
- Fluorescence decays acquired with parallel and crossed polarizers placed at excitation and emission channels
- Rotational motion of the molecule; environmental constraints; relative orientation of absorption and emission dipoles of the molecule
- Widely used in biomembranes, proteins, lipids, NA, polymers, etc.
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Metal-ligand complexes (MLC)
have become very popular probes due to their relatively
long lifetimes. They are particularly suitable
to studying large macromolecular systems such as
nucleic acids and proteins. The figure shows a decay
of one of the most common types of MLC, tris
(2,2'-bipyridyl) ruthenium (II). The probe has a
rather low quantum yield (about 4%). Not a problem
for the EasyLife™ V! The recovered lifetime is
429 ns. |
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Porphyrins and chlorophylls, compounds of great biological significance, emit fluorescence that requires NIR sensitivity. Not a problem for the EasyLife™ V, when equipped with the optional red-enhanced detector. Decays of both porphyrins and chlorophylls can be easily measured under a variety of conditions. This makes the EasyLife™ V a valuable tool for researchers studying primary processes in photosynthesis and for validation of photochemical and photophysical properties of porphyrin-based photosensitizers for photodynamic therapy.
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Fluorescence decay of Chl A
extracted from spinach leaves and
suspended in buffer. The decay is double
exponential due to the presence of Chl A
aggregates. The recovered lifetimes are
570 ps (93%) and 4.2 ns (7%). |
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Fluorescence decay of meso-tetraphenylporphyrin (m-TPP) in chloroform measured with the EasyLife™ V equipped with a 370 nm LED source. Fitted with a single exponential function, the recovered lifetime is 9.16 ns. The chi-square value of 1.07 and the random residual function indicate that the single exponential model adequately describes the decay of m-TPP. |
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Fluorescence decay of Rose Bengal in water measured with the EasyLife™ V lifetime system equipped with a 525-nm LED light source. Due to its highly reproducible pulse and very stable detection electronics, the EasyLife™ V is capable of measuring lifetimes, which are more than an order of magnitude shorter than the pulse width of the LED. The lifetime is determined by iterative reconvolution of the instrument response function (IRF) and the true exponential decay combined with the least squares minimization. The recovered lifetime of 94 ps is in excellent agreement with literature data citing a 92 ps lifetime for Rose Bengal in water.
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