# Leaf-Level Spectral Fluorescence Measurements: Comparing Methodologies for Broadleaves and Needles
## 4 Discussion
### Measurement Protocols  
The most pronounced differences among the three tested methods were in the amplitude of the recorded ChlF signal: highest for OC (in birch and lingenberry) and FW (in pine), and smallest for IS (Figures 5a and 6a-f), despite having similar PAR levels at the leaf surface (Table 1). These differences can be attributed to contrasting geometries between setups--measurements were acquired at nadir in the backscatter direction for OC, at a 45\({}^{\circ}\) viewing angle respective to the illumination beam in FW, and integrated across all directions in the IS method. Interestingly, the signal was largest in the OC for broadleaf species, but not for pine needle mats, for which the FW presented larger values. We are not certain about the underlying reason for this is, but suggest it may reflect a decrease in overall absorbed PAR (APAR) when illuminating the needles at nadir, relative to the FW, due to the effects of the 3D structure of the needle mats and small gaps in between. In other words, the apparent gap fraction of needle mats tends to be smaller when viewed at 45\({}^{\circ}\) compared to nadir, resulting in lower PAR loses through the gaps. Bidirectional ChlF measurements of needle mats under controlled laboratory conditions would be required to further investigate these differences. In turn, signal attenuationinside the sphere would explain why the amplitude of the ChIP signal was smallest in the IS method compared to others. The spectral shape we recorded was consistent with examples from literature measured under both ambient and artificial illumination, using both halogen and LED light sources. [PERSON] et al. (2013) measured solar-induced leaf-level upward ChIP with FluoWat method. The results for two broadleaf species (see Figure 7 in [40], red/far-red ratio = 0.6) represent similar spectral distributions as our birch and lingonberry in both OC and FW. Corresponding red/far-red ratio values (0.48-0.62) were also measured for Sun leaves of birch and pine by [PERSON] et al., while measured with FW and white halogen illumination [38]. Similar spectra with red/far-red ratios below unity have been also recorded in response to red LED illumination measured with a high-resolution QePro spectrometer coupled to an IRGA system [47]. Although assessing the effect of light quality was outside the scope of the present study, we found highly comparable ChIP shapes for birch leaves illuminated with a white LED and halogen light (see Supplementary Material, Figure S4b), suggesting that small changes in light quality would not cause a drastic change in spectral shape. In contrast, highly different red/far-red ratios have been reported when comparing leaves illuminated with blue vs. red LED light illumination [47]. It is also important to remember that our measurement were conducted under low light conditions, given the requirement of minimum NPQ and, therefore, the red/far-red ratios might be slightly overestimated compared to leaves measured under field conditions with strong light. After normalizing to F740 nm (Figure 5b, Figure 6g,h and Supplementary Material, Figure S6b) the shapes of the spectra obtained with OC and FW were generally very consistent with each other suggesting that differences in measuring geometry would not appear to affect the shape of the resulting ChIP spectra. Together with the mentioned relation between LED and halogen induced ChIP, this finding could have important implications for future leaf-level ChIP meta-analysis studies. Potentially, data from different measuring setups could be combined together to study how physiological, structural, and biochemical factors control the shape of the leaf-level ChIP spectra. The similarity between observations from two contrasting viewing angles (nadir for OC and 45\({}^{\circ}\) for FW), would indicate a certain degree of isotropy in the shape of ChIP spectra. In contrast, IS delivered a substantially smaller far-red peak, as compared with OC and FW.