# Making Sense of Light: The Use of Optical Spectroscopy Techniques in Plant Sciences and Agriculture
## 3 Main Techniques
### Elastic Spectroscopy
The black horizontal lines represent the quantum states of the molecule. The shadowed rectangles represent virtual states, i.e., states that do not correspond to any quantum state. The energy of a virtual transition equals that of the photon. The vertical black arrow represents radiative transitions, i.e., those involving a photon. Vertical thick white arrows represent non-radiative transitions, i.e., not involving photons. Electromagnetic radiation is represented by wavy lines, color-coded according to the photon energy: violet \(>\) blue \(>\) red. Other major spectroscopic techniques used in plant science, mainly since the 1990s-2000s, include the spatially resolved spectroscopy (SRS), which can also be associated with both the MHS imaging [26; 35] and the time resolved spectroscopy (TRS). SRS was first developed to understand the light propagation in turbid media [36; 37] and was further applied to the determination of the optical properties of biological tissues [38; 39]. In this technique, a small continuous-wave light beam illuminates the sample's surface, and the reemitted light is measured at different distances from the light source. The absorption coefficient (\(\mu_{\mathrm{a}}\)) and the reduced scattering coefficient (\(\mu_{\mathrm{s}}\)) can then be extracted from the measured SRS reflectance profiles by using an appropriate numerical method [26]. In TRS, a short monochromatic pulse is injected into a turbid medium, such as a plant or fruit tissue, and the temporal distribution of the emitted light, which is attenuated, broadened, and delayed due to light scattering, is measured and fitted with an appropriate model of photon migration to estimate the optical properties of the medium [26; 40; 41]. Application of TRS imaging systems have been reported for the 3D imaging of biological tissues [42], but not in plants. Apart from the property of the matter under investigation, the recorded spectrum of the reflected radiation highly depends on the spectrum of the internal or external illumination source, observation geometry and parameters of the electro-optical system in use. For this reason, signal processing includes calibration and normalization procedures designed to ultimately synthesize a certain characteristic that solely represents a property of the sample--usually, the reflection coefficient of the illuminated sample surface. The collection geometry for Vis-NIRS, either in the modalities of \"point\" and imaging, depends on the goal of the measurement. In any case, it is always a recommended procedure to avoid specularly reflected light since it is mostly devoid of information from the sample. One possibility is to send and collect light along the same oblique angle. Another possibility is to use geometries such a 0/d, meaning that light is incident along the normal to the surface, and the diffusively reflected light is collected along an oblique angle. Diffuse reflected light is originated either by the surface roughness, or by scattering inside the tissue. In some applications, it is important to collect only the photons scattered inside the tissue, because these are the most informative. To do that, it is necessary to reject the superficial component of the diffuse reflected light. The simplest way to achieve it is to perform contact measurements, with light being injected and collected by optical fibers separated by a few millimeters. However, the same effect may also be achieved with lenses and baffles. The corresponding geometry is usually called interactance. Finally, there is also the transmittance geometry, where light injection and collection are performed at opposite sides of the sample. This automatically implies that only the light crossing the tissue is detected. Thus, in most systems the elastic reflection represents the overwhelming part of the total reflection, and the elastic spectroscopy provides good sensitivity, achieved with robust equipment that is cost-effective in terms of manufacturing, operation and maintenance.