# Making Sense of Light: The Use of Optical Spectroscopy Techniques in Plant Sciences and Agriculture
## 3 Main Techniques
### Other Spectroscopic Techniques  
Far more selective techniques can be developed based on interactions between light and matter that result in changes in the photon energy and wavelength. These changes are characteristic of the biological material and, as a rule, yield spectra that are more information-rich than those due to the elastic scattering. The two main types of such phenomena are fluorescence and Raman scattering (Figure 1), the latter firstly discovered in 1928 [43]. As both imply detection of light emitted by the irradiated sample, the fluorescence and Raman emissions are often observed simultaneously, with the same experimental setup. The main difference between fluorescence and Raman processes is that the former is emission from a relatively long-lived (typically, 10\({}^{-8}\)-10\({}^{-9}\) s) excited electronic state,whereas in the latter, light scattering occurs nearly instantly--more precisely, via emission of a photon from a short-lived (less than ca. \(10^{-15}\) s) excited virtual state--see a more detailed comparison in [44]. For fluorescence, the long excited-state lifetime results in the molecules having sufficient time to relax to the same metastable energy state prior to emission. The energy of the virtual state, from which the radiative transition takes place, is determined by the energy of the incident photon. Thus, the wavelength of fluorescent radiation is weakly dependent on it, while the wavelength of Raman scattering increases with the wavelength of the exciting light. In most cases, the light due to fluorescence and Raman scattering possesses lower quantum energy (longer wavelength) than the initial radiation (Stokes shift). However, when the molecules are already in an excited state, the emitted light has higher energy (smaller wavelength) than the initial radiation (anti-Stokes shift--see Figure 1 for illustration of anti-Stokes in Raman scattering). Anti-Stokes lines are much weaker than the Stokes lines because there are far fewer molecules in the excited state than in the ground state. Due to a lower probability of interactions, the fluorescence and Raman spectroscopy lead, in general, to worse sensitivity than the elastic-scattering methods. Reliable detection requires intensive sample irradiation, i.e., powerful sources (such as lasers or flashamps), which makes the instrumentation more complex, less robust and costlier, both in manufacturing and operation. The method versatility is limited to the irradiation wavelength-material pairs that have good quantum yield for the process of interest. Filtering the fluorescence and Raman signals is essential, because they are orders of magnitude smaller than diffuse reflected light. Fluorescence and Raman are competing processes (fluorescence being usually more intense), so they also need, in turn, to be separated. The shielding from specular and diffuse reflectance is obtained by the same geometrical methods described for Vis-NIRS, taking care to avoid the specular reflection angles. Additionally, high-pass, low-pass and/or band-pass optical filters are used to remove the unwanted wavelengths. Consider an example with blue excitation/red fluorescence. At the excitation, a low-pass filter is used to remove any red component from the incident light, while at the detection, a high-pass filter is used to remove any reflected blue from the red fluorescence. For the separation of Raman from fluorescence, the most common approach is the careful selection of the excitation wavelength. Choosing a NIR or UV laser wavelength can avoid exciting fluorescence. In the first case, because the radiation does not have enough energy to excite fluorescence. In the second case, there is fluorescence, but the emission is widely separated in energy from the Raman signal, allowing to measure it in a fluorescence-free wavelength range.