Fluorescence interferences

Fluorescence Interference. The historical drawback to widespread use of Raman spectroscopy has been the strong fluorescence background exhibited by many materials, even those which are nominally nonfluorescent. This fluorescence often arises from an impurity in the sample, but may be intrinsic to the material being studied. Several methods have proved useflil in reducing this background. One of the simplest is sample purification. 

It is very difficult to excite the donor without also exciting some of the acceptor population. This is because the absorption spectra of dyes extend significantly into the blue side of their absorption maxima, so the absorption spectra of the donor and acceptor usually overlap. The donor fluorescence can typically be observed without acceptor fluorescence interference therefore, when measuring FRET efficiency by observing the donor fluorescence, this overlap is not important. However, when observing the acceptor fluorescence the overlap of the donor and acceptor absorption must be taken into account. The total steady-state fluorescence of the acceptor, assuming that [A] = [D (i.e., a equal donor and acceptor concentrations, and 100% labeling) is... 

Similar work was performed by Shaw et al.3 in 1999 when they used FT-Raman, equipped with a charge coupled device (CCD) detector (for rapid measurements) as an on-line monitor for the yeast biotransformation of glucose to ethanol. An ATR (attenuated total reflectance) cell was used to interface the instrument to the fermentation tank. An Nd YAG laser (1064 nm) was used to lower fluorescence interference and a holographic notch filter was employed to reduce Rayleigh scatter interference. Various chemometric approaches were explored and are explained in detail in their paper. The solution was pumped continuously through a bypass, used as a window in which measurements were taken. 

Milota F, Sperling J, Szocs V, Tortschanoff A, Kauffmann HF (2004) Correlation of femtosecond wave packets and fluorescence interference in a conjugated polymer towards the measurement of site homogeneous dephasing. J Chem Phys 120 9870... 

The internal rotational relaxation times of 1-pyrene carboxaldehyde in sulfonate systems may offer some indication of the extent of probe binding to the inverted micelle. In the absence of any background fluorescence interference to the time-dependent anisotropy decay profile, the internal rotational relaxation time should correlate with the strength of binding with the polar material in the polar core. However, spectral interference from the aromatic moieties of sulfonates is substantial, so that the values of internal rotational relaxation time can only be used for qualitative comparison. 

Figure 3.40 shows the layout of a typical Raman analyzer that uses fiber optics for process application. In a Raman process system, light is filtered and delivered to the sample via excitation fiber. Raman-scattered light is collected by collection fibers in the fiber-optic probe, filtered, and sent to the spectrometer via return fiber-optical cables. A charge-coupled device (CCD) camera detects the signal and provides the Raman spectrum. To take advantage of low-noise CCD cameras and to minimize fluorescence interference, NIR diode lasers are used in process instruments. 

The limitations of Raman spectroscopy are its low sensitivity compared to IR absorption and fluorescence interference from impurities in the sample. Raman spectroscopy is a developing technology, and a good amount of research and planning is necessary before deciding whether or not to employ it. The cost of a Raman process analyzer exceeds that of other analyzers. To reduce cost, Raman analyzers often include multichannel capability. Up to four process streams can be analyzed with a single CCD camera by splitting the lasers. 

A CCD Raman spectrometer coupled with a 10-mW He-Ne laser has been used to eliminate fluorescence because the long-wavelength excitation by the He-Ne laser is not as likely to cause fluorescent transitions (71). Because of its directional property, coherent anti-Stokes Raman scattering (CARS) is also effective in avoiding fluorescence interference (see CARS in Section 3.9). 

While fluorescent assays afford the highest throughput among ion channel assays and do not require specialized instrumentation, they suffer from some limitations, including potential fluorescent interference by the test compound... 

Although the peptide aminomethylcoumarin (AMC) substrate approach has been widely used for measuring the activities of many proteases, a disadvantage of this approach arises from the ultraviolet excitation and emission wavelengths required for detecting free AMC where there is a possibility for fluorescence interference. RllO-labeled substrates are less encumbered by interference due to a spectral red shift, but many compounds in small molecule chemical libraries exhibit fluorescent properties that may interfere with assays based on fluorescence detection (Simenov et al. 2008). 

Romero-Torres, S., Perez-Ramos, J. D., Morris, K. R., and Grant, E. R. (2006), Raman spectroscopy for tablet coating thickness quantification and coating characterization in the presence of strong fluorescent interference, J. Pharma. Biomed. Anal., 41, 811-819. 

The results of tests of several zeolite materials at three UV excitation wavelengths are summarized in Table I (24). It is clear that the shorter-wavelength excitation is required to avoid the fluorescence interference. This result is in agreement with the general statement by Asher (15) about the need to excite at wavelengths below 260 nm to avoid fluorescence interference. 

---