# Leaf-Level Spectral Fluorescence Measurements: Comparing Methodologies for Broadleaves and Needles
## Materials and Methods
### Baseline Correction  
Since only a very small 1-2% of absorbed light is usually re-emitted as ChIP in the photosystems, and due to strong re-absorption of ChIP at the level of the thylakoid membrane as well as within the leaf [11], the resulting ChIP signal emitted by a leaf can be as low as three orders of magnitude smaller than the incoming radiation. As a result, when measuring the ChIP spectra of leaves with a low-pass filter and spectrometer (as described above), we often face the situation where the digital counts for the measured spectra are close to saturation for the spectral region below the cut-off filter (in this case below 650 nm), while the ChIP signal is about two orders of magnitude smaller (or even less) and dangerously close to the level of background noise. Although this situation can be partly avoided by using a spectrometer with a high dynamic range and high SNR or by applying a strong averaging, this is not always an option. Alternatively, if reflectance dynamics are not of interest, the integration time can be increased to enhance SNR across the ChIP spectral range at the expense of signal saturation before the cut-off filter. Signal saturation _per se_ can, however, affect other regions of the spectra by generating an additional component of \"dark (or rather saturation-induced) current\" in the detector, which may require correction (I). A second challenge associated with ChIP spectral measurement is that of the filter transmittance. This can be relevant when using halogen light sources (which have a high output in the red and especially near-infrared (NIR) regions) compared to RGB or white LED light sources which emit mainly in the visible light regions, and especially when using cut-off filters with a low optical density (OD). Since the measured ChIP emission can be as low as three orders of magnitude smaller than the incoming light, a filter with OD = 3 or even OD = 4 (transmission of 0.001 or 0.0001, respectively) could potentially let through enough photons to interfere with the measured ChIP. This can be especially relevant for recovering the red ChIP tail, typically near the cut-off region, where filter transmittance can still be significant. The two filters used in this study were labeled OD = 4, although, according to manufacturer specifications, transmittance was well below OD = 5 for most of the exclusion region (see Supplementary Material, Figure S3). We, therefore, assumed that filter effects can be considered negligible for an OD = 4 filter and would only require correction (II) around the cut-off zone (650-660 nm). Figure 3: Schematic of the three different setups: (**a**) optical chamber; (**b**) FluoWat; and (**c**) integrating sphere. A third challenge is related to the spectral resolution of the spectrometer, which we expected to interact with the measured ChIF spectra especially around the red tail. Sharp spectral features, like the line spectra of atomic emission bands used in wavelength calibration lamps, cannot be resolved as lines with spectrometers, but rather follow a normal probability distribution centered on the line's emission peak. The width of this distribution at half the maximum height, called full-width half maximum (FWHM), denotes the spectral resolution of a spectrometer. Accordingly, depending on the spectrometer resolution, light intensity, and integration time, any spectral feature will be visible in terms of spectrometer counts for a variable range of wavelengths around it. A similar phenomenon can operate when using a cut-off filter that rapidly reduces the intensity of radiation reaching the detector within a narrow cut-off region (e.g., from 74% transmittance at 644 nm to less than 1% at 654 nm; see Supplementary Material, Figure S3). We suggest that this limitation might also interfere with the measured ChIF spectra, especially when long ITs are saturating the spectral region prior to the cut-off filter, requiring correction (III).