Fluorophore Mixtures

In order to resolve the individual emission spectra, the data are analyzed by nonlinear least-squares analysis. 

Stmi-Volma jot curves dowmranl Aie to die inoeasing fractional conbfoution of the more weakfy quenched e-cies at higher quencher concentrations. 

Hiesoluiioaooainiied tmMsadhiinituo iUdolO m eailbnia-ttoa of Froni Ref. 54 

FlgureB.27. Slefii-V lmerplotsforthekKfidequeiiclilngof c0tflEt npraior (wild type, WDiiidils mutants (F43 and P7S). Revlied from Ref. 55. 

It is valuable to notice a difference in the method of data analysis for the modified Stem-Volmer plots (Section 8.8.A) and for the quenching-resi vedeiiussion spectra. In analyzing a modified Stem-Volmer plot, one assumes that a fraction of the fluorescence is totally inaccessible to quenchers. This may not be completely true because one component can be more weakly quenched, but still quenched to some extent. If possible, it is preferable to analyze the Stem-Volmer plots by nonlinear least-squares analysis when the/ and K/ values are variable. With this approach, one allows each component to contribute to the data according to its fractional accessibility, instead of forcing one component to be an inaccessible fraction. Of course, such an analysis is more complex, and the data may not be adequate to recover the values of fi and Kj at each wavelength. 

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Hanley, Q. S. and Clayton, A. H. A. (2005). AB-plot assisted determination of fluorophore mixtures in a fluorescence lifetime microscope using spectra or quenchers. J. Microsc. 218, 62-7. 

As mentioned previously, the complex emission spectrum F (l) of samples containing multiple fluorophores is assumed to be the linear sum of individual component spectra Ffl), F2(X), FfX), weighted by their abundance xu x2, x3. Let Fj(X) and F2(X) be the reference emission spectra of pure samples of fluorophore (e.g., Cerulean and Venus). The term reference emission spectra is used because these spectra describe the emission at excitation wavelength /. x of a defined concentration of fluorophore (e.g., 10 /rM) acquired using the same excitation light intensity as was used to acquire an emission spectra of an unknown sample mixture. Under these conditions, the shape and magnitude of the fluorophore mixture spectra will be ... 

Figure 9.8 shows the experimental fluorescence spectrum of Trp residues of 15 ptM o -acid glycoprotein (Xex = 295 nm) (line) and calculated spectrum obtained using Equation (9.1) (square). We notice that the calculated spectrum matches the experimental spectrum. Note students can apply this method to different fluorophore mixtures. 

Throughout the previous discussion we have assumed the sample Is accurately described by a single exponential decay process. Unfortunately, many cases exist where the sample Is not accurately described by such a simple process (e.g., fluorophore mixtures and proteins). When the sample emission Is described as a multiple exponential decay, consisting of n fluorescent components, the... 

Blue light-emitting diodes (LEDs) have also been used in fluorescence instruments. These lamps emit radiation at 450 - 475 nm and are suitable for exciting some fluorophores. Mixtures of phosphors in some LFDs can provide wavelengths in the UV region to about 375 nm (see Section 13D-1). 

Figure 19. Emission spectra and photographs of SPCE for a three fluorophore mixture observed at different angles from the normal axis using the hemi-cylindrical prism. All concentrations of dyes in the evaporated films were about 1 OmM (adopted from [30]). See Fig. 30.19 in the color insert at the end of this volume.

Structure of GFP and its chromophore. To study the chro-mophore of GFP, a sample of GFP was denatured by heating it at 90°C. It was digested with papain, and then a peptide containing the fluorophore was isolated and purified from the digested mixture. The structural study of the peptide has indicated that the chromophore of GFP is an imidazolone derivative shown below (Shimomura, 1979). This chromophore structure was confirmed later by Cody etal. (1993) in a hexapeptide isolated from GFP. It is intriguing that the structure of the GFP chromophore is a part of the structure of coelenterazine. 

The data were collected using fluorescence measurements, which allow both identification and quantitation of the fluorophore in solvent extraction. Important experimental considerations such as solvent choice, temperature, and concentrations of the modifier and the analytes are discussed. The utility of this method as a means of simplifying complex PAH mixtures is also evaluated. In addition, the coupling of cyclodextrin-modified solvent extraction with luminescence measurements for qualitative evaluation of components in mixtures will be discussed briefly. 

Fluorescence lifetime measurements can increase the analytical specificity when analyzing mixtures (1-4) and can indicate changes in chemical binding of the fluorophores under various environmental conditions (5). 

Time Resolved Fluorescence Depolarization. In Equation 3, it is assumed that the polarization decays to zero as a single exponential function, which is equivalent to assuming that the molecular shape is spherical with isotropic rotational motion. Multiexponential decays arise from anisotropic rotational motion, which might indicate a nonspherical molecule, a molecule rotating in a nonuniform environment, a fluorophore bound to tbe molecule in a manner that binders its motion, or a mixture of fluorophores with different rotational rates. 

Similar expressions can be derived for systems of fluorophores having different rate constants, the details of the mathematics are not important here. A few rules have been derived for mixing of signals from such systems. The modulation and phase for a system of multiple noninteracting fluorophores that are not undergoing excited state reactions can be computed from a sum related to the fractional contribution of the individual fluorophores, the lifetime, and the modulation frequency. The fractional contributions f, for the zth fluorophore in a mixture is given by ... 

For a mixture of n directly excited noninteracting fluorophores, the apparent phase and modulation may be calculated according to ... 

Practically speaking, these expressions allow the prediction of the phase and modulation for an arbitrary mixture of noninteracting fluorophores and the respective modulation and phase lifetimes. 

It is important to note that if a mixture of fluorophores with different fluorescence lifetimes is analyzed, the lifetime computed from the phase is not equivalent to the lifetime computed from the modulation. As a result, the two lifetimes are often referred to as apparent lifetimes and should not be confused with the true lifetime of any particular species in the sample. These equations predict a set of phenomena inherent to the frequency domain measurement. 

A mixture of noninteracting fluorophores might be observed by spatially variant FRET in a specimen, which is blurred because of optical resolution issues, will result in different lifetimes being measured for zm and zy- and zm > %f. In many instances, a single frequency measurement will be insufficient to determine the number of fluorophores or the number of fluorophore environments in a sample. 

Equations (9-11) are dependent on three parameters to, and Zj. All may be exploited in the analysis of mixtures of fluorophores however, co is the only one that can be systematically varied without altering the sample. 

Examination of Eqs. (2.9-2.11) suggests that having frequency domain lifetimes measured at a variety of frequencies is desirable, as it will allow a mixture of fluorophores to be determined. With this in mind, two approaches may be taken to obtain multifrequency results. The first of these is simply to make a series of FLIM measurements while stepping through a predetermined set of frequencies. In practice, this is of limited utility for biological systems because of photo-induced damage to the specimen. 

If reference emission spectra of a set of pure fluorophores are available, and if an emission spectrum of an unknown mixture of any combination of these fluorophores is acquired under the same conditions, this equation can be used to determine the abundance of the different fluorophores in the mixture. The use of this equation to determine the abundance of the fluorophores present is called linear unmixing. To illustrate the basis of linear unmixing, we will first use this equation to analyze the emission spectra of the mix capillary containing an unknown mixture of Cerulean and Venus depicted in Fig. 8.1. The unmixing approach we describe will utilize reasonable guesses for the values of x1 (representing the abundance of Cerulean) and x2 (representing the abundance of... 

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