# Leaf-Level Spectral Fluorescence Measurements: Comparing Methodologies for Broadleaves and Needles
At the same time, measuring more than one needle creates a need for arranging needles into mats (e.g., [27]) or for using needle holders (e.g., [28]). These two-dimensional configurations can, in turn, enhance mutual shading of adjacent needles, multiple scattering, or re-absorption [29]. Several studies [27; 30; 31; 32; 33] have assessed the optimal arrangement of needles for optical measurements of reflectance (R) and transmittance (T). Due to the design of the most prevalent instrumentation (integrating spheres with port sizes on the order of cm) these studies typically measured multiple needles simultaneously. Inevitably, there are gaps between needles in such a measurement; the gap fraction (G\({}_{\text{F}}\)) is, therefore, a parameter of considerable importance when conducting such measurements. Small G\({}_{\text{F}}\) have been shown to yield unrealistic negative T values for the photosynthetically active radiation (PAR) region [32; 33]. [PERSON] et al. stated that errors are larger in samples with large G\({}_{\text{F}}\) while compared to small G\({}_{\text{F}}\) and that large G\({}_{\text{F}}\) affects T more negatively than R [34]. At the same time, [PERSON] suggested that effect of the needles' own cross-section had a stronger negative effect on measurements accuracy than the G\({}_{\text{F}}\) itself [35]. Hence, gaps are both inevitable and a source of considerable error when measuring the spectral properties of needle-like leaves. As far as we are aware, there have been no previously reported efforts to characterize the influence of gap fraction on measurements of spectral ChlF. When considering such a study it is important to distinguish the nature of different optical processes measurements. Gaps between adjacent needles will promote multiple scattering of photons, which can bias R and T measurements. In case of ChlF, on top of multiple scattering, one also has to consider the re-absorption of scattered red ChlF by adjacent needles and subsequent enrichment in far-red ChlF. Accordingly, gaps could potentially affect both the magnitude and shape of the ChlF spectra. Therefore, while some technical challenges of needle measurements in general can be concluded, R and T studies should not lead to assumptions on how sample morphology or arrangement affect ChlF. As this effect remains unknown, standardization of needle- and leaf-level measurements is key to agreeing on accurate interpretation of all-levels ChIP from various species. The aim of this study was to evaluate the functionality of different methods and discuss their applicability in different practical conditions. Leaf-level ChIP has been measured with various methods, also using FluoWat (e.g., [18; 36]). However, a question whether or how the geometry of measurements (viewing and illumination angles) affects the measured ChIP spectral shape across leaf samples with different morphology or across needle samples of different arrangements, was not yet solved in the literature. Here we compare three methods: (1) A custom made optical chamber designed to measure ChIP of leaf samples at both ambient and 77 K temperatures in the backscatter direction; (2) FluoWat, a widely used clip originally designed for in situ measurements of SIF spectra, which can measure both up-welling and down-welling ChIP at a 45\({}^{\circ}\) angle respective to the illumination beam; and (3) an integrating sphere to estimate total hemispherical ChlF emission spectra of samples illuminated at nadir. In addition, we compare measurements of broadleaves and needle-leaves and assess the impact that different needle arrangements have on the resulting ChlF spectral properties. A first approximation protocol to recover the red ChIP tail around the cut-off filter and to correct for potential baseline effects is also introduced.