# Leaf-Level Spectral Fluorescence Measurements: Comparing Methodologies for Broadleaves and Needles
## Materials and Methods
### Baseline Correction
Overall, the three phenomena described above may generate a significant and wavelength-dependent baseline level that can affect not only the intensity but also the shape of the measured ChIF spectra. We here present and implement a preliminary and simple correction method to correct for effects I, II, and III described above. We first measure the extent of the baseline using a Spectralon(r)panel, with the filter in place and at the same IT as during measurements (REF3, see Figure 2). This baseline, without a leaf, will include all three effects above (see Figure 4, grey dotted line). However, because the baseline we want to correct should correspond to the situation when a sample is being measured, REF3 cannot be directly applied as a baseline. If we assume that the combined effect of I and II depends on the amount of energy reaching the detector, then we would expect the baseline for a sample \(i\) to be smaller than REF3 by a factor equal to the average leaf reflectance of a sample \(i\) (\(\overline{\rho_{AB}}\)) for a region between wavelengths A and B prior to the cut-off filter (we used here 550-650 nm, although other ranges showed comparable results), so that:  
\[Baseline_{i}(\lambda)=REF3\ (\lambda)\ \ \ \times\ \ \ \overline{\rho_{i_{AB}}} \tag{1}\]  
Correcting for the filter effect (II) would require precise knowledge of the spectral filter transmission \(T_{F}(\lambda)\), for that particular filter and conditions, of the reference spectra without filter (REF1(\(\lambda\))), and of the reflectance spectra of that particular leaf sample \(\rho_{i}(\lambda)\). The leaf reflectance \(\rho_{i}(\lambda)\) spectra needs to be multiplied because the photons potentially passing through the filter and reaching the detector will depend on the reflectance of the sample. \[Baseline_{i\_filter}(\lambda)\ = REF1(\lambda)\ \ \times\ \ \ T_{F}(\lambda)\ \ \times\ \ \rho_{i}(\lambda) \tag{2}\]  
The reflectance term is important because in case of significant filter transmission, it could add substantial wavelength-dependent artefacts into the measured ChIF spectra as a result of the red edge feature in the reflectance of green leaves. Note also that in the absence of effects I and II above, \(REF1(\lambda)\ \ \times\ \ \ T_{F}(\lambda)\) in Equation (2) should correspond to \(REF3(\lambda)\) in Equation (1). We originally tried to correct our data using Equation (2), but it resulted in an overcorrection of the ChIF spectra, indicating that effects I and II above are probably dominating the baseline (data not shown). Finally, since the manufacturer transmittance data for the filters used herein indicated that transmittance 10 nm above the cut-off region was negligible (OD > 4) (see Supplementary Material, Figure S3), and since leaf reflectance in the 650-660 nm region can be assumed to be relatively similar to the \(\overline{\rho_{i_{AB}}}\) in Equation (1), Equation (2) becomes a good approximation of Equation (1) for the wavelength range where this correction may have an impact. As a result, we choose to use Equation (1) to calculate the baseline correction for each sample and, as a first approximation, assume it accounts for the integrated effect of all three factors (see Figure 4). It is important to note that this assumption will not hold if the filter transmittance is substantially higher, in which case filter transmission would need separate correction. To further test the effect of potential filter transmittance on the resulting ChLF spectra and validity of the correction method, we carried out additional ChLF spectra measurements of birch leaves following the FW protocol. Two different light sources were tested: the halogen light described above (which strongly emits in the ChLF wavelengths) and a powerful LED light source with very small emission in the NIR (MagicShine, MS-602, Shenzhen, China), which was attenuated using a neutral density filter (OD = 1, ThorLabs @NE510B) so that PAR at the leaf surface would be similar between light sources (see Supplementary Material, Figure S4c).