# Leaf-Level Spectral Fluorescence Measurements: Comparing Methodologies for Broadleaves and Needles
## Materials and Methods  
### Plant Material and Experimental Protocol  
Leaves from three different species growing in the vicinity of the University of Helsinki, Viikki Campus, Finland were collected towards the end of the growing season (September 2017): silver birch (_Betula pendula L._), a deciduous tree with relatively thin leaves (specific leaf area, SLA = 161 cm\({}^{2}\)g DW\({}^{-1}\)); lingonberry (_Vaccinium vitis-idaea_ L.), an evergreen shrub with relatively thick leaves (SLA = 55 cm\({}^{2}\)g DW\({}^{-1}\)); and Scots pine (_Pinus silvestris_ L.), an evergreen tree with needle-leaves (SLA = 39 cm\({}^{2}\)g DW\({}^{-1}\)). One exposed and south-facing branch from the accessible part of the canopy per each tree was cut during early morning and rapidly brought to the lab. For lingonberry, three nearby shoots where cut to provide enough leaf material. All shoots were immediately re-cut under and kept in water, in the dark, and at room temperature through the duration of measurements (approx. eight hours). Species and replicates (15 measuring points: three species \(\times\) five replicates) were randomized within each method. Methods were not randomized due to practical reasons. Leaf-level reflectance and spectral ChlF were measured with three different methods: (1) optical chamber (OC); (2) FluoWat (FW); and (3) integrating sphere (IS). A fresh leaf or set of needles was always used at each measuring point. Measurements with OC started 30 min after re-cutting branches under water, dark acclimation and temperature stabilization in the lab. Measurements with FW and IS were taken approx. 90 and 240 min after re-cutting branches, respectively. The size of the birch and lingonberry leaves was sufficient to cover the measuring area in OC and FW as well as sample-port in the IS. In contrast, different configurations were possible for conifer measurements. We tested three different configurations (Figure 1): (a) single needle (1N); (b) three needles with an approximately one-needle-wide gap in between each (3N); and (c) continuous needle mat with as minimal gaps as technically possible (NM). Measurements were conducted with a background consisting of a photon trap (FW and IS) or a non-fluorescing black tape (see Supplementary Material, Figure S1) in OC. Light sources were adjusted for each method to yield similar low light intensities at the leaf surface (as measured with an LI-250A PAR sensor, LI-COR@Inc., Lincoln, NE, USA) (Table 1). Despite the obvious decrease in SNR, we purposefully selected a low light intensity (38-50 \(\upmu\)mol PAR) to minimize any potential differences in ChlF spectral properties due to light-induced photochemical quenching (PQ) and non-photochemical quenching (NPQ) dynamics [11]. To ensure we were comparing ChlF spectra from fully functional leaves of similar physiological states, we calculated the maximum quantum yield of photochemistry based on the ratio of variable to maximal fluorescence, or F\({}_{\mathrm{V}}\)/F\({}_{\mathrm{M}}\)[37],and used it as an indicator of photosynthetic functionality [24]. We used a PAM-2500 fluorometer (Heinz Walz GmbH, Effeltrich, Germany) and the cut shoots after one hour of dark acclimation (n = 5). F\({}_{\text{V}}\)/F\({}_{\text{M}}\) remained at the level of 0.78 \(\pm\) 0.023 for lingonberry, 0.79 \(\pm\) 0.012 for birch, and 0.83 \(\pm\) 0.012 for pine, which is well within the range of non-stressed summer values for each of the respective species [38; 39]. More details can be found in Table 1 and the sections below. All the measurements were acquired with an Ocean Optics@USB2000+ (Ocean Optics@, Largo, FL, USA) spectrometer and recorded with SpectraSuite (Ocean Optics@) software. All references (REFs) were collected using a reflectance standard panel (Spectralon@)Diffuse Reflectance Standards, Labsphere@, North Sutton, NH, USA). Dark current (DC) and reference measurements were collected before and after each sample measurement, for DC correction and to check for stability during measurements (Figure 2).