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