Standards fluorescence decay time

Table V. Materials Used as Standards for Fluorescence Decay Time, r...

R. A. Lampert, L. A. Chewter, D. Phillips, D. V. O Connor, A. J. Roberts, andS. R. Meech, Standards for nanoseconds fluorescence decay time measurements, Anal. Chem. 55, 68-73 (1983). 

An ideal lifetime technique would record all photons detected within the fluorescence decay function, over a time interval much longer than the fluorescence decay time, in a targe number of time channels, and with an infinitely short temporal instrument response function. The standard deviation, ov of the fluorescence lifetime, T, for a number of recorded photons, N, would be... 

Lampert RA, Chewter LA, Phillips D et al (1983) Standards for nanosecond fluorescence decay time measurements. Anal Chem 55 68-73... 

Definition and Uses of Standards. In the context of this paper, the term "standard" denotes a well-characterized material for which a physical parameter or concentration of chemical constituent has been determined with a known precision and accuracy. These standards can be used to check or determine (a) instrumental parameters such as wavelength accuracy, detection-system spectral responsivity, and stability (b) the instrument response to specific fluorescent species and (c) the accuracy of measurements made by specific Instruments or measurement procedures (assess whether the analytical measurement process is in statistical control and whether it exhibits bias). Once the luminescence instrumentation has been calibrated, it can be used to measure the luminescence characteristics of chemical systems, including corrected excitation and emission spectra, quantum yields, decay times, emission anisotropies, energy transfer, and, with appropriate standards, the concentrations of chemical constituents in complex S2unples. 

Calibration. In general, standards used for instrument calibration are physical devices (standard lamps, flow meters, etc.) or pure chemical compounds in solution (solid or liquid), although some combined forms could be used (e.g., Tb + Eu in glass for wavelength calibration). Calibrated lnstr iment parameters include wavelength accuracy, detection-system spectral responsivity (to determine corrected excitation and emission spectra), and stability, among others. Fluorescence data such as corrected excitation and emission spectra, quantum yields, decay times, and polarization that are to be compared among laboratories are dependent on these calibrations. The Instrument and fluorescence parameters and various standards, reviewed recently (1,2,11), are discussed briefly below. 

There are several ways to check or calibrate the time scale of a TCSPC insfru-ment. The most commonly used one is to measure the fluorescence decay of a dye of known lifetime and compare the result to lifetimes given in the literature [308]. However, the lifetime of an organic dye is actually the poorest calibration standard possible. The measured lifetime may depend on the solvent, the temperature, and the excitation wavelength. It may also be changed by fluorescence quenching by impurities or oxygen, and by reabsorption effects. Moreover, detector background and improper data analysis can easily introduce an uncertainty on the level of several percent. 

As described above, the TAG functions to determine the time interval between the excitation pulse and the subsequent fluorescence photon arriving at the detector. The MCA consists of an ADC, a memory consisting of channels for storing data, and data input and output facilities. A standard instrument incorporates lower and upper discriminator levels and two modes of data collection pulse height analysis mode for displaying fluorescence decay profiles ( 1000 channels) and multichannel scaHng mode to bin the data into given time increments. Data are usually displayed on an oscilloscope or on a computer terminal. 

Fluorescence Lifetimes. Decay Times. Fluorescence Lifetime Standards in the ns and ps Time Scales... 

Fig. 7.8 Calculated decay kinetics of iluraescence Itom an ensemble of energy donors with a Gaussian distribution of donor-acceptor distanees. In this illustration, the distribution is centered at Ro and has a width (FWHM) of either 0.5/f (solid line) or zero (long dashes). The time course of the fluorescence was calculated by Eq. (7.31). Also shown is the exponential fluorescence decay in the absence of enta-gy transfer (short dashes). A FWHM of 0.5/f<, corresponds to a standard deviation of 0.2121f ...

The background problem can be further overcome when using a surface-confined fluorescence excitation and detection scheme at a certain angle of incident light, total internal reflection (TIR) occurs at the interface of a dense (e.g. quartz) and less dense (e.g. water) medium. An evanescent wave is generated which penetrates into the less dense medium and decays exponentially. Optical detection of the binding event is restricted to the penetration depth of the evanescent field and thus to the surface-bound molecules. Fluorescence from unbound molecules in the bulk solution is not detected. In contrast to standard fluorescence scanners, which detect the fluorescence after hybridization, evanescent wave technology allows the measurement of real-time kinetics (www.zeptosens.com, www.affinity-sensors.com). 

Solvation dynamics are measured using the more reliable energy relaxation method after a local perturbation [83-85], typically using a femtosecond-resolved fluorescence technique. Experimentally, the wavelength-resolved transients are obtained using the fluorescence upconversion method [85], The observed fluorescence dynamics, decay at the blue side and rise at the red side (Fig. 3a), reflecting typical solvation processes. The molecular mechanism is schematically shown in Fig. 5. Typically, by following the standard procedures [35], we can construct the femtosecond-resolved emission spectra (FRES, Stokes shifts with time) and then the correlation function (solvent response curve) ... 

The width of the time ehannels of the recorded photon distribution ean be made as small as 1 ps. The small time-bin width in conjunction with the high number of time ehannels available makes it possible to sample the signal shape adequately aeeording to the Nyquist theorem. Therefore standard deconvolution techniques [389] ean be used to determine fluorescence lifetimes much shorter than the IRF width and to resolve the eomponents of multiexponential decay functions. 

In the second test, a number of fluorescent compounds of relatively well known lifetimes in the nanosecond time range (8,9) were used as standards, allowing evaluation of both the instrumental and computational aspects of the measurement. Table I shows the values obtained for 2,3-diphenyloxazole (PPO), anthracene and quinine blsulphate. All chemicals were analytical grade and not further purified before use. Anthracene and PPO were dissolved in cyclohexane, quinine in O.IN 8280 solvents were not degassed. The case of quinine is of interest because of its common use as a standard for fluorescence measurements, despite its complex decay kinetics (10). In agreement with previous work (10) we found satisfactory fits of our deconvolved data to a blexponentlal rather than a single exponential model. 

In the previous chapter we presented an overview of protein fluorescence. We described the spectral properties of the aromatic amino acids and how these properties depend on protein structure. We now extend this discussion to include time-resolved measurements of intrinsic protein flu( scence. Prior to 1983, most measurements of time-resolved fluorescence were performed using TCSPC. The instruments employed for these measurements typically used a flashlamp etcitation source and a standard dynode-chain-type PMT. Such instruments provided instrument response functions with a half-width near 2 ns, which is comparable to thedecay time of most proteins. The limited repetition rate of the flashlamps, near 20 kHz. resulted in data of modest statistical accuracy, unless the acquisition times were excessively long. Given the complexity of protein intensity and anisotropy decays, and the inherent difficulty of resolving multiexponential processes. ii was difficult to obUun definitive information on the decay kinetics of proteins. 

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