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The EasyLife™ V Software requires the following:
- Microsoft Windows XP® with SP2
- Intel Pentium® III class processor, 500 MHz or higher
- USB port (USB 2 recommended)
- Video display 800 x 600, 256 colors or higher
- Minimum of 256 MB of RAM (512 MB or higher recommended)
- Minimum of 125 MB free hard drive space
- Microsoft® compatible mouse
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Measures fluorescence intensity as a function of time over a specified time range, usually from a few ns to a few hundreds of ns. Fluorescence decay represents a response from a fluorescent sample to a pulsed excitation. The fluorescence decay can be collected either sequentially (recommended for unstable samples). Typically, the fluorescence decay is described by an exponential function, F(t) = A*exp(-t/τ), where τ is the fluorescence lifetime. It tells us how long, on average, the molecule resides in the excited state. The lifetime is an important parameter characterizing the fluorescing molecule and its interaction with other molecules (e.g. FRET) and environment. The lifetime is determined from the fluorescence decay by numerical fitting, usually involving deconvolution (see Data Analysis section). Fluorescence lifetimes as short as 100 ps or as long as 3 µs can be determined with the EasyLife™ V.
Advanced. To facilitate the determination of multiple lifetimes from complex fluorescence decay, in addition to the standard linear timescale, two non-linear timescale acquisition protocols are provided: the logarithmic and arithmetic timescales. With these protocols, the decay curve has smaller time increments at short times, which gradually increase at longer times. These protocols increase the data quality and reliability of multiexponential analysis.
Measures fluorescence intensity as a function of laboratory time at a fixed time delay after the pulse. The time duration of this measurement can be from a few seconds to many hours. Can be used to monitor kinetics of slow reactions, stability of the sample (e.g. photobleaching), or can be used for intensity adjustment with the emission slit or proper filter selection.
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EasyLife™ V comes with a powerful lifetime analysis package comprising 8 different fitting programs and a FRET calculator, covering virtually all possible application scenarios. All of the fitting programs employ deconvolution as a standard option. Deconvolution removes the distortion imposed on a decay curve by a finite temporal width of the excitation pulse. It makes it possible to determine lifetimes that are 10x shorter than the excitation pulse. In order to use the deconvolution option the user must acquire an instrument response function (IRF) in addition to the fluorescence decay. The IRF can be measured by replacing the fluorescent sample with a scatterer.
The following analysis programs come with the EasyLife™ V OBB:
This program is suitable for the analysis of fluorescence decays consisting of up to 4 exponentials and associated pre-exponential factors. This is the most commonly used program in lifetime analysis.
The multiple file 1-to-4 exponential lifetime method allows the analysis of multiple scatterer/sample pairs as a batch operation. Each pair will be separately analyzed over the same range with the same number of exponentials and the same options. This type of analysis is useful when a series of decay curves has been collected as a function of some parameter (i.e. concentration of added reagent). Trends in the values of the lifetime parameters may then be recognized rather easily.
This program analyzes decays with up to 4 lifetimes for a number of data files simultaneously. The global analysis assumes that the lifetimes are the same among the data files but that the associated pre-exponentials are free to vary. For example, the global can be useful for analyzing various mixtures of up to 4 fluorophores.
This program is used to calculate rotational correlation times plus a residual anisotropy term. The program first allows the user to calculate the fluorescence lifetime(s) from the parallel and perpendicularly polarized emission intensities. The user can then calculate the rotational correlation time(s).
This program uses a “stretched exponential” fitting function (Infelta-Graetzel eqn.), which describes the quenching in micelles when added quencher molecules are Poisson distributed among the micelles. The analysis allows the determination of micellar aggregation number and the quenching rate constant.
The Maximum Entropy Method (MEM) is designed to recover lifetime distributions without any a priori assumptions about their shapes. This method uses a series of exponentials (up to 200 terms) as a probe function with fixed, logarithmically spaced lifetimes and variable pre-exponentials. This allows analyzing fluorescence decays with underlying lifetimes spanning several orders of magnitude. In many situations the MEM is capable of differentiating between continuous distributions and discrete, multi-exponentials decays. The algorithm minimizes chi-square while maximizing the entropy function at each iteration step. Ideal for complex decays, such as labeled proteins and membranes, probes adsorbed on surfaces, probes with conformational flexibility, polymers etc.
The Exponential Series Method (ESM) is designed to recover lifetime distributions without any a priori assumptions about their shapes. This method uses a series of exponentials (up to 200 terms) as a probe function with fixed, logarithmically spaced lifetimes and variable pre-exponentials. This allows analyzing fluorescence decays with underlying lifetimes spanning several orders of magnitude. In many situations the ESM is capable of differentiating between continuous distributions and discrete, multi-exponentials decays. Ideal for complex decays, such as labeled proteins and membranes, probes adsorbed on surfaces, probes with conformational flexibility, polymers etc. Similar to MEM, but without entropy maximization.
This program allows for the analysis of data by a fitting function consisting of two exponentials multiplied together and each with variable exponents of time. The exponents can be either varied or fixed which provides a powerful general function for models such as Förster energy transfer, time-dependent quenching and molecular interaction in restricted geometries (e.g. molecules on surfaces, zeolites etc.).
Calculates basic FRET parameters, such as Forster radius Ro (requires spectral data), D-A distance, FRET efficiency and FRET rate constant. Works with either intensity or lifetime data.
The commands in the Trace Math menu allow specific mathematical functions and operations to be carried out on individual traces or selected regions of a trace. There is 16 functions included, such as: antilog, average, distribution average, combine, xy combine, multiple derivatives, integration, linear fit, linear scaling, logarithm, normalization, reciprocal, smoothing, truncation, baseline suppression and trace merging. There is also a peak finder provided. All these functions can greatly facilitate data processing and presentation.
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