Fluorescence parameters

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. 

Velapoldl et al. (64) used a similar approach but prepared fibers of uniform diameter (5-45 pm) from Inorganic Ion-doped glasses. The fluorescence parameters of these materials can be changed by substituting various Ions, such as Tb , Sm , Eu , Mn, UOj, Cu, and Sn. They show excellent stability under Irradiation using Incident excitation (measurement Imprecision of 1% under continuous Irradiation In the microscope for 24 h) and have a fluorescence flux density proportional to the fiber length. 

Abstract The response signal of an immense number of fluorescence reporters with a broad variety of structures and properties can be realized through the observation in changes of a very limited number of fluorescence parameters. They are the variations in intensity, anisotropy (or polarization), lifetime, and the spectral changes that allow wavelength-ratiometric detection. Here, these detection methods are overviewed, and specific demands addressed to fluorescence emitters for optimization of their response are discussed. 

The fluorescence decay kinetics of exemplary chosen QDs and small organic dyes are compared in Fig. 2. The size of the fluorescence parameter luminescence lifetime is determined by the electronic nature of the transitions involved. As a rule... 

Sensitivity. The measured fluorescence parameter of an indicator should be sensitive to changes of analyte in the desired concentration range, as summarized in Table 10.1 for a number of analytes. The indicator should have high extinction coefficient for efficient excitation and high quantum yield for a good signal-to-noise ratio. 

Selectivity. The measured fluorescence parameter should respond selectively to the analyte of interest. 

Calibration method. The measured fluorescence parameter should be independent of indicator concentration, geometry of sample, and sensitivity of detection system. Thus, an intensity-based method requires wavelength-ratiometric probes. Lifetime and anisotropy methods do not require wavelength-ratiometric probes, but the lifetime or anisotropy must be sensitive to analyte. 

By comparing time-resolved and steady-state fluorescence parameters, Ross et alm> have shown that in oxytocin, a lactation and uterine contraction hormone in mammals, the internal disulfide bridge quenches the fluorescence of the single tyrosine by a static mechanism. The quenching complex was attributed to an interaction between one C — tyrosine rotamer and the disulfide bond. Swadesh et al.(()<>> have studied the dithiothreitol quenching of the six tyrosine residues in ribonuclease A. They carefully examined the steady-state criteria that are useful for distinguishing pure static from pure dynamic quenching by consideration of the Smoluchowski equation(70) for the diffusion-controlled bimolecular rate constant k0,... 

Such ambiguity and also the low structural resolution of the method require that the spectroscopic properties of protein fluorophores and their reactions in electronic excited states be thoroughly studied and characterized in simple model systems. Furthermore, the reliability of the results should increase with the inclusion of this additional information into the analysis and with the comparison of the complementary data. Recently, there has been a tendency not only to study certain fluorescence parameters and to establish their correlation with protein dynamics but also to analyze them jointly, to treat the spectroscopic data multiparametrically, and to construct self-consistent models of the dynamic process which take into account these data as a whole. Fluorescence spectroscopy gives a researcher ample opportunities to combine different parameters determined experimentally and to study their interrelationships (Figure 2.1). This opportunity should be exploited to the fullest. 

Let us turn our attention back again to the scheme illustrating various versions of the joint application of fluorescence parameters (Figure 2.1) and consider the possibilities for constructing more general and more definite models of protein dynamics. These models can be suggested and confirmed or rejected by comparing predicted behavior with the results of spectroscopic experiments of different kinds. 

E. A. Permyakov and E. A. Burstein, Relaxation processes in frozen aqueous solution of proteins temperature dependence of fluorescence parameters, Stud. Biophys. 51, 91-103 (1975). 

Thus, the rs is a complex term which embodies the fluorescence lifetime, rotational correlation time (0), and also r0 (r1 in the absence of depolarizing motion). The most common type of experiment involves a comparison of rs for two experimental conditions however, such a comparison of rs ignores possible changes in x, , and r0. Nevertheless, for many cases a comparison of rf values alone may be satisfactory although a more rigorous analysis requires a time-resolved measurement. A comparison of the effects of changes in common membrane properties on time-resolved fluorescence parameters is shown in Table 5.3. 

Table 5.3. Alteration of Membrane Lipid Properties and the Resultant Approximate Directions of Changes of Fluorescence Parameters of a Typical Membrane Fluorophore Probe of the Fatty Acyl Chain Region0...

Table 5.4. Fluorescence Parameters of DPH, DPH-PC, and TMA-DPH in Intact Sarcoplasmic Reticulum Membranes and Liposomes Made from Total Lipid Extracts of the Membranes at 37°Cab...

The determination of fluorescence parameters of peptides requires the presence of either natural fluorescent amino acid residues (intrinsic fluorescence) or of extrinsic fluorescent probes covalently attached to the peptide at appropriate sites. The use of extrinsic fluorescent probes is mandatory in cases where the conformational or rotational behavior of a peptide is examined in the presence of proteins that contain intrinsic fluorescent amino acids. 

Two useful fluorescence parameters are the quantum yield and the lifetime. Quantum yield is a property relevant to most photophvsical and photochemical processes, and it is defined for fluorescence as in (1.101. More generally it is a measure of the efficiency with which absorbed radiation causes the molecule to undergo a specified change. So for a photochemical reaction it is the number of product molecules formed for each quantum of light absorbed ... 

Reproducible measurement over a period of time clearly requires that the flow cytometer is maintained to a high standard, and that the sensitivity of the instrument is checked on a daily basis. To ensure reproducibility, a stable standard for each scatter and fluorescence parameter must be established. 

There should be sensitive and large changes in one or more fluorescence parameters XlLx, intensity, lifetime and anisotropy, as the probe partitions to the organized media. 

Fig. 11. Variation of the fluorescence properties of a set of tryptophan-containing peptides as a function of the position of the tryptophan in their sequence. The parameter/AVe describes the degree of rigidity and hydrophobicity of the tryptophan s environment it is based on emission maximum, anisotropy, and accessibility to acrylamide. When the values for each of these parameters were similar to those expected for indole in water, a value near 0 was assigned to/, whereas values up to 1.0 were assigned as the fluorescence parameters more closely resembled those observed in very rigid and apolar environments such as the interior of a protein or ethylene glycol at -60°C (Lakowicz, 1983). The values of / calculated for each parameter were then averaged to give /AVe- The dotted curve was generated by fitting a sine wave to the data (period = 3.3 residues). Taken from O Neil et al. (1987).

The interaction between Calcofluor White and carbohydrate residues of a -acid glycoprotein depends on the secondary structure of the carbohydrate residues, with the fluorescence parameters of Calcofluor being sensitive to this spatial secondary structure. 

The nature of the environment and of its interaction with the fluorophore can affect all the fluorescence parameters. 

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