# Optoelectronic Instrumentation and Measurement Strategies for Optical Chemical (Bio)Sensing
## 3 Spectroscopic Techniques
### Measurement of Luminescence Lifetimes
#### 3.3.3 Ratiometric Techniques  
The objective of ratiometric techniques is to make the result independent of the main sources of error. This independence is achieved by quantifying at least two parameters proportional to the disturbance to be eliminated. This perturbation will be canceled out when it appears both in the numerator and in the denominator of the quotient that defines the ratiometric expression. Ratiometric expressions can be applied to sensors that show changes in intensity, lifetimes, or both. This gives ratiometric techniques great versatility, as they can bring together the advantages of classical luminescent methods based on the intensity and lifetime measurements.  
The most common approach in ratiometric intensity measurement is based on the ratio between the intensity levels of two emission peaks at a single excitation wavelength [36]; however, this method can suffer from photobleaching of either the indicator or the reference dye, causing changes in the measured intensity ratio. An alternative to this well-established ratiometric method is shown in Figure 12. This alternative is based on the use of a single emitter and the simultaneous measurement of the fluorescence emission and reflectance intensity from the dye. The scheme is similar to that shown in Figure 8, except that a trifurcated optical fiber and two photodetectors are used--one for sensor emission and one  
Figure 11: Method used for the rapid determination of the lifetime.  
for specular reflectance of the excitation signal. Because the specular reflectance does not depend on the analyte, as it does not interact with the sensor, it can be used as a reference signal. The perturbation will be canceled when it appears in both the numerator and the quotient denominator of the following ratiometric expression:  
\[R=\frac{I_{\text{hum}}}{I_{\text{ref}}}\approx\frac{k_{1}I_{\text{ex}}[\text{ Ind}]}{k_{2}I_{\text{exc}}}=\frac{k_{1}}{k_{2}}[\text{Ind}] \tag{11}\]  
where \(I_{\text{lum}}\) is the intensity of luminescence and \(I_{\text{ref}}\) the intensity of the specular reflectance. Table 6 summarizes the advantages and disadvantages of common luminescence spectroscopy techniques.  
\begin{table}
\begin{tabular}{c c c c} \hline \hline
**Spectroscopy Technique** & & **Advantages** & **Disadvantages** \\ \hline \multirow{4}{*}{Measurement of light intensity} & \(\bullet\) & Simple & \(\bullet\) & Fluctuations of the excitation light \\  & \(\bullet\) & Accurate in the laboratory & \(\bullet\) & Light losses in the optical path \\  & \(\bullet\) & & \(\bullet\) & Photobleaching of the indicator \\ \hline \multirow{4}{*}{Ratiometric intensity measurement} & \(\bullet\) & Relatively simple & \(\bullet\) & Background fluorescence \\  & \(\bullet\) & Can cancel variations in the indicator & \(\bullet\) & Light scattering and reflections are \\  & & concentration, geometry, and source & not compensated \\  & & intensity & \\ \hline \multirow{4}{*}{Measurement of lifetime in the time domain} & \(\bullet\) & Relatively simple & \(\bullet\) & High-speed electronics \\  & \(\bullet\) & Background fluorescence can be & \(\bullet\) & Fast-pulsed optical sources \\  & & eliminated & \\ \hline \multirow{4}{*}{Measurement of lifetime in the frequency domain} & \(\bullet\) & Very simple but quite accurate results & \\  & \(\bullet\) & Low-cost, high-brightness optical & \(\bullet\) & More expensive light sources for \\  & \(\bullet\) & sources & lifetimes in the range of ns \\ \cline{1-1}  & \(\bullet\) & Standard photodetectors & \\ \hline \hline \end{tabular}
\end{table}
Table 6: Advantages and disadvantages of common luminescence spectroscopy techniques.  
Figure 12: Instrumentation setup for a fiber optic sensor based on the ratiometric measurement of intensity. PD = photodetector.