Leaf-Level Spectral Fluorescence Measurements: Comparing Methodologies for Broadleaves and Needles

Successful measurements of chlorophyll fluorescence (ChlF) spectral properties (typically in the wavelength range of 650-850 nm) across plant species, environmental conditions, and stress levels are a first step towards establishing a quantitative link between solar-induced chlorophyll fluorescence (SIF), which can only be measured at discrete ChlF spectral bands, and photosynthetic functionality. Despite its importance and significance, the various methodologies for the estimation of leaf-level ChlF spectral properties have not yet been compared, especially when applied to leaves with complex morphology, such as needles. Here we present, to the best of our knowledge, a first comparison of protocols for measuring leaf-level ChlF spectra: a custom-made system designed to measure ChlF spectra at ambient and 77 K temperatures (optical chamber, OC), the widely used FluoWat leaf clip (FW), and an integrating sphere setup (IS). We test the three methods under low-light conditions, across two broadleaf species and one needle-like species. For the conifer, we characterize the effect of needle arrangements: one needle, three needles, and needle mats with as little gap fraction as technically possible. We also introduce a simple baseline correction method to account for non-fluorescence-related contributions to spectral measurements. Baseline correction was found especially useful in recovering the spectra nearby the filter cut-off. Results show that the shape of the leaf-level ChlF spectra remained largely unaffected by the measurement methodology and geometry in OC and FW methods. Substantially smaller red/far-red ratios were observed in the IS method. The comparison of needle arrangements indicated that needle mats could be a practical solution to investigate temporal changes in ChlF spectra of needle-like leaves as they produced more reproducible results and higher signals.