Excitation ratio method

From Eq. (31.2), we can evaluate the pH of Hb. salinarum without ratio methods. The fluorescence lifetime in Hb. salinarum without monensin is evaluated to be 2.76 ns. The intracellular pH is then calculated to be 7.1, which is in reasonable agreement with that obtained from the excitation ratio method [18]. The reason why the fluorescence lifetime in vivo is smaller than that in vitro may be ascribed to the local field produced by some proteins and membranes that affects the... 

Modern instruments capable of obtaining excitation-emission matrices (EEMs) allow use of new data-analysis techniques to resolve overlapped spectra. Resolution techniques such as the ratio method (28) and others (29,30) may provide further differentiation of the components present in the phases separated by solvent extraction. 

If there is no emission shift, but only variation in the fluorescence quantum yield on protonation, one follows the fluorescence intensity at one emission wavelength when the acid and the base forms of the probe are excited. All the advantages of the ratio method described with two emission wavelengths also apply to this fluorescence excitation method. 

The internal standard ratio method for quench correction is tedious and time-consuming and it destroys the sample, so it is not an ideal method. Scintillation counters are equipped with a standard radiation source inside the instrument but outside the scintillation solution. The radiation source, usually a gamma emitter, is mechanically moved into a position next to the vial containing the sample, and the combined system of standard and sample is counted. Gamma rays from the standard excite solvent molecules in the sample, and the scintillation process occurs as previously described. However, the instrument is adjusted to register only scintillations due to y particle collisions with solvent molecules. This method for quench correction, called the external standard method, is fast and precise. 

In the internal standard method the intensity of the unknown line is measured relative to that of an internal standard line. The internal line may be a weak line of the main constituent. Alternatively, it may be a strong line of an element known not to be present in the sample and furnished by adding a fixed small amount of a compound of the element in question to the sample. The ratios of the intensities of these lines — the unknown line and the internal standard line — will be unaffected by the exposure and development conditions. This method will provide lines of suitable wavelength and intensity by variations of the added element and the amount added, due regard being paid to the relative volatility of the selected internal standard element. It is important to use as internal standard pairs only those lines of which the relative intensities are insensitive to variations in excitation conditions. The line selected as standard should have a wavelength close to that of the unknown and should, if possible, have roughly the same intensity. 

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