# Ground Based Hyperspectral Imaging to Characterize Canopy-Level Photosynthetic Activities
## 2 Materials and Methods
### Characterization of Canopy-Level Photosynthetic Efficiency  
#### 2.3.1 Retrieval of Solar Induced Fluorescence  
Collected spectral data were used to retrieve SIF values at the canopy level (Figure 2). Irradiance spectra of the sunlight and radiance hyperspectral cubes of the plant canopy were synchronized using timestamps, resulting in meta-hyperspectral cubes of each scanning row, where individual pixels had both irradiance and radiance spectra. Based on the spatial information, a meta-hyperspectral cube was further split into two sub-cubes, with each sub-cube containing irradiance and radiance data for one plot. The following processes were performed for the meta hyperspectral cube of a single plot. Based on preliminary tests, grayscale images at 749 and 685 nm were used to generate a band ratio image (\(I_{749}/I_{685}\)). A threshold was applied to the band ratio image to create the mask of plant canopies in that plot. An arbitrary value of 3 was used in the present study based on the trial-and-error method. Chlorophyll absorbs incident light (particularly blue and red light in the visible spectral range) and emits fluorescence in the red and far-red spectral range. Upwelling radiance spectra of plant canopies thus contain both reflectance and fluorescence signals in the red and far-red spectral range. Fraunhofer lines are a set of spectral absorption lines in the spectrum of sunlight related to particles in the solar and terrestrial atmosphere. \(H\alpha\) at 656 nm, \(O_{2}\)-\(B\) at 687 nm, and \(O_{2}\)-\(A\) at 760 nm are three Fraunhofer lines in the red and far-red spectral range. The reduction of solar irradiance in the Fraunhofer lines results in a decrease of the canopy reflectance and an increase in the ratio of fluorescence and reflectance signals, which maximizes the suitability of decoupling chlorophyll fluorescence from the canopy reflectance. A standard Fraunhofer line discrimination (sFLD) method has been developed to use irradiance and radiance signals at two spectral bands [49]. One band is one Fraunhofer line, and the other band is a wavelength near the corresponding Fraunhofer line. In the present study, based on previous literature review [24], the \(O_{2}\)-\(A\) band (approximately at 761 nm) and its neighboring band (759 nm) were used to calculate SIF values of individual pixels using Equation (1). It is noteworthy that the sFLD method was used, because the present study focused on exploring the possibility of using hyperspectral imaging for SIF retrieval and successive canopy-level photosynthetic analysis. Other Fraunhofer line discrimination models (e.g., 3 FLD and improved FLD) can be used to improve SIF retrieval accuracy in future studies [24]. \[SIF_{p}=\frac{E_{p}^{759}L_{p}^{761}-E_{p}^{761}L_{p}^{759}}{E_{p}^{759}-E_{p} ^{761}}, \tag{1}\]  
where \(SIF_{p}\) was the SIF value (W/m\({}^{2}\)/nm/sr) of a pixel \(p\). \(E_{p}\) and \(L_{p}\) represented the irradiance (W/m\({}^{2}\)/nm) and radiance (W/m\({}^{2}\)/nm/sr) intensities of \(p\) at a certain wavelength. The plant canopy mask was multiplied with retrieved SIF values, forming the SIF image of the plot. As each image line of a radiance hyperspectral cube had a corresponding irradiance spectrum, PAR values were calculated for individual lines of SIF images using Equation (2). Pixels along individual lines in a radiance hyperspectral cube shared the same irradiance spectrum measured by the spectrometer. \[PAR_{p}=\frac{1}{A_{s}t_{s}}\int_{400}^{700}\frac{E_{p}^{\lambda}}{e_{\lambda}}d \lambda=\frac{1}{A_{s}t_{s}}\int_{400}^{700}\frac{E_{p}^{\lambda}\lambda}{hc}d \lambda, \tag{2}\]  
where \(PAR_{p}\) was the PAR value (\(\mu\) mol/m\({}^{2}\)/s) of a pixel \(p\). \(A_{s}\) was the surface area (m\({}^{2}\)) of the cosine corrector equipped with the spectrometer, and \(t_{s}\) was the integration time (s) of the spectrometer. Figure 2: Flowchart of image processing from collected raw data to meta-SIF (solar induced fluorescence) images.