Fluorescence emission peaks, wavelength shifts

R4. At a high optical density at the excitation and/or emission wavelengths, a distortion of the fluorescence emission spectrum is observed. A fluorescence intensity decrease is observed, and the emission peak is shifted. 

Figure 9.2 Typical spectral scan of a fluorescent compound showing its absorbance peak or wavelengths of most efficient excitation and its emission peak or wavelengths where light emission occurs. The Stoke s shift is the distance in nanometers between the absorbance peak and the emission peak. The larger the Stoke s shift, the less interference that will occur from excitation light when measuring fluorescence emission.

The fluorescence excitation spectra exhibit a broad band located between 461 and 465 nm, which is homothetic to the longest wavelength absorption band. The fluorescence emission spectra are characterized by a well-defined peak which is strongly red shifted from 545 to 565 nm on going from hexane to DMSO. This bathochromic shift reflects the occurrence of n n electronic transitions in the 546 singlet excited state. 

The absorption and emission maxima from this table will provide clues to the spectral ranges that are useful for excitation and for fluorescence detection with a particular fluorochrome. However, the absorption and emission spectra have breadth, with slopes and shoulders and secondary peaks (see Fig. 5.6). With efficient fluorochromes, excitation and fluorescence detection at wavelengths distant from the maxima may be possible. Therefore, inspection of the full, detailed spectra is necessary to get the full story. In addition, spectra may shift in different chemical environments (this will explain why maxima vary in different reference tables from different sources). Values in this table are derived primarily from the Molecular Probes Handbook and the article by Alan Waggoner (Chapter 12) in Melamed et al. 

Figure 8.5 shows fluorescence emission spectra of TNS and ANS bound to serum albumin. The two fluorophores do not show the same maximum, although the two fluorophores bind to hydrophobic domains of the protein. This result can be explained mainly by the differences in the structure of the two fluorophores. Also, one could explain the difference in the emission peaks could be due to the higher sensitivity of TNS to hydrophobicity. This interpretation is based on the fact that the peak of TNS emission spectrum is shifted to short wavelengths compared to that of ANS... 

Explain why it is important to record fluorescence spectra at low product concentrations. R3. A high concentration may increase the optical density at the excitation and/or the emission wavelengths. This will distort the fluorescence emission spectrum by decreasing the real fluorescence intensity and by shifting the emission peak. 

Adler discusses the fluorescence properties of the azaindoles, which show in their excitation spectra bands similar to those in their absorption spectra but shifted to the red. A notable difference in the fluorescence intensity of the compounds was observed, with that of 4-azaindole being almost three times that of 7-azaindole, whereas the absorptivities in water are nearly the same. Similarly, 6-azaindole shows a higher intensity than 7-azaindole, although their emission peaks are at the same wavelength. This is even more exaggerated in the cations. 

Figure 7. CDOM fluorescence of water from two lakes (Hargreaves, unpublished) emission scans for excitation at 370 nm (Shimadzu 551 fluorometer), before and after subtraction of water blank. Samples deionized water (DIW), L. Giles water (ca. 1 g m DOC), L. Lacawac water (ca. 5 g m DOC) The Raman scattering peak at 417 nm represents a shift in wavenumber by 3400 cm from the excitation wavenumber. The broad peak is contributed predominantly by the fulvic acid fraction of DOM. The peak wavelength and fluorescence index ratio for these samples (L. Giles, 452 nm peak and ratio = 1.5 L. Lacawac, 455 mn peak and ratio = 1.4) suggest a slight difference in CDOM...

The first described member of the cyan fluorescent proteins (CFPs) resulted from a rationally designed chromophore mutation of Aequorea GFP. Heim and co-worker replaced Tyr66 with Trp and found the peak wavelength for excitation and emission of this GFP derivative (GFP-Y66W) to be shifted to 436 and 476 nm, respectively [52], Because of this blue-green/cyan light emission the protein was called cyan fluorescent protein or CFP. 

Fluorescence. ANS is an amphoteric molecule that binds selectively at the boundary between aqueous and hydrophobic regions (iO). When binding occurs, the fluorescence spectrum changes dramatically from that exhibited by the dye in an aqueous environment. When excited at 377 nm, the single broad band that has an emission peak at 520 nm in water (or 2% NaCl) can shift to a wavelength as low as 462 nm when the dye is in a hydrophobic region. Simultaneously, the intensity of the fluorescence may increase as much as 2 orders of magnitude. These two quantities are measures of the dielectric constant or polarity of the dye environment. 

Some fluoronieters can be equipped with correction devices, others not. Therefore, one should be careful when comparison is done between two different instruments. Figure 2.3 displays the uncorrected and corrected fluorescence emission spectra of zincporphyrin IX dissolved in dioxane, recorded with an SLM instrument. One can notice that the difference between the two spectra exists at wavelengths higher than 600 nni. One can notice also, that correction of the spectrum yields a small red shift of the peak of 2 to 3 nm. 

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