# Leaf-Level Spectral Fluorescence Measurements: Comparing Methodologies for Broadleaves and Needles
## 4 Discussion
### Needle Arrangements and Morphology Effect  
We compared three alternatives to measuring ChIP spectra in needle-like leaves, measuring: a single needle (1N), needles separated by gaps (3N), and a full needle mat with minimal gaps (NM). We found that NM yielded higher replicability in the resulting shape of the spectra, as indicated by lowest SD in both OC and FW methods. The results showed that the NM presented higher replicability and reproducibility across methods compared to the 1N and 3N arrangements, indicating that this might be a desirable arrangement for studies intended to track temporal changes in ChIP properties for needle-like species. We are aware that arranging needles in mats (see Section 2.1.) could potentially enhance self-shading, multiple scattering across all ChIP wavelengths, as well as re-absorption of predominantly red wavelengths by adjacent leaves [27; 31]. With a single needle, ChIP photons emittedin lateral directions will not reach the sensor. With a needle mat, ChIF photons emitted in lateral directions can still be scattered by adjacent needles in the direction of the sensor and contribute to the measured ChIF. Since red ChIF photons have a higher probability of being re-absorbed by adjacent needles, compared to far-red photons, one would expect the laterally-scattered ChIF contribution to be enriched in far-red photons, reducing the overall red/far-red ratio. This phenomenon could explain why the red/far-red ratios of NM were smaller than those of 1N and 3N arrangements across methods (Figure 6g,h). Interestingly, assuming that the 1N arrangement would have provided a more realistic estimate of the red/far-red ratio, it is still controversial why needles presented higher red/far-red ratios compared to birch or lingonberry (Figure 7b). Two explanations can be excluded in our study. First, because all the measurements were purposefully conducted at low PAR, any differences between leaves in terms of NPQ should be minimal. Second, because the leaves showed \(\mathrm{F_{V}}/\mathrm{F_{M}}\) levels typical for the species in the absence of stress (see Section 2.1), the potential impact of sustained physiological stress can also be ruled out [11; 38; 39]. We, therefore, suggest that the slightly higher levels of red/far-red ChIF ratio observed in 1N and 3N relative to those observed in birch and lingonberry leaves would respond to differences in [ChI] induced reabsorption coupled with differences in leaf morphology. One important question is raised from this observation: does the red/far-red ChIF ratio of leaves relate better to the total [ChI] expressed on a surface basis or on a leaf mass basis (possibly better related to the density of chlorophyll in the leaf)? Twice as high levels of [ChI] expressed on a surface basis have been observed for boreal Scots pine compared to boreal silver birch [38] during summer, and since higher [ChI] should increase reabsorption of red ChIF, the result does not appear to be consistent with our observation of higher red/far-red ratio in pine compared to birch. In contrast, when [ChI] is expressed on per leaf mass basis, almost twice as high levels of [ChI]/g DW are reported for boreal silver birch compared to Scots pine [48] during summer. This finding suggests that red/far-red ratio might be more closely linked to [ChI]/g DW than [ChI]/cm\({}^{2}\), and that not only leaf-level [ChI] but also morphology could play a role in shaping the ChIF spectra. The decrease in red/far-red ratio observed for NM, as compared to 1N and 3N arrangements, indicates that arranging the needles may to some extent underestimate the ratio and it should be considered when quantitatively comparing ChIF spectra from needles and broadleaf species. This observation emphasizes the complexity of dealing with \(\mathrm{G_{F}}\) in needle-like leaves. As widely suggested in the literature [27; 29; 32], gaps are both inevitable and a source of considerable error when measuring R and T of needle like leaves.