# Leaf-Level Spectral Fluorescence Measurements: Comparing Methodologies for Broadleaves and Needles
## 4 Discussion  
### Baseline Correction and the Red/Far-Red Ratio  
Under certain conditions, such as when measuring in the direction of the specular reflectance, applying long ITs to boost SNR in the ChlF spectral range at the expense of signal saturation, or using spectrometers with limited dynamic ranges or cut-off filters with substantial transmissions (OD > 4), the resulting ChlF spectra may be contaminated with an undesired and wavelength-dependent baseline component. Although the strength and consequences of this overestimation may vary from case to case, baseline correction can be critical when comparing datasets acquired with different setups or species. Baseline correction is especially useful in recovering the spectra near the filter cut-off. As shown (Figure 4 and Supplementary Material, Figure S4a), the red tail is the region where the correction is strongest. Since the red ChlF peak is closer to the filter cut-off region than the far-red peak, the baseline effect could potentially interfere with the relative heights of the ChlF peaks and, thus, the red/far-red ratio [18].  
Red to far-red ChlF ratios have been used as indicators of [Chl] [41; 42] and to track physiological stress responses [15; 17; 43; 44]. Variations in red to far-red ChlF have been also used to estimate the relative contribution of PSI and PSII to the ChlF signal [45; 46]. It is, therefore, important to ensure that the baseline effect, when present, is properly addressed. Comparison of measured and corrected spectra showed that baseline application changed the red/far-red ratio by only 2-3% (OC) and by 1.1-2.1% (FW). The larger baseline effect in the OC compared to the FW could be explained in terms of enhanced reflectance in the OC where the specular reflectance component is also contributing to the reflected signal (Supplementary Material, Figure S5). Overall, despite the limited impact of the baseline correction method on the shape of the resulting ChlF spectra, we recommend taking into account the possibility of a significant baseline during data interpretation. Using spectrometers with a high dynamic range so as not to saturate the signal, and optical filters with an OD equal to or larger than 4, is also recommended. When applying this baseline correction method, we also recommend measuring an REF3 for each sample rather than for each setup as we did in the present study, since filter repositioning from sample to sample might cause some variation in the REF3, which is probably one of the reasons why the correction of the black tape ChlF spectra (Figure S1) presented still some uncorrected \"photons\" below 680 nm.  
Finally, the baseline correction did not work properly for the IS method where substantial levels of \"ChlF\" were still estimated at 850 nm and beyond. This inconsistency could be attributed to the small magnitude in the signal relative to the background noise compared to the other methods. Although measurements at higher light intensities where outside the scope of the present study, they could serve to test and confirm the validity of the baseline correction for the IS setup.