## 3 Measuring F on the Leaf and Canopy Level
### Leaf Level
#### 3.1.2 Measurement Protocols  
Leaf upward and downward radiance (Lup(\(\lambda\)) and Ldw(\(\lambda\))) are respectively composed of the reflected and transmitted radiance (L\({}_{\text{R}}\)(\(\lambda\)), L\({}_{\text{T}}\)(\(\lambda\)), W m\({}^{-2}\) sr\({}^{-1}\) nm\({}^{-1}\)) measured in the VIS-NIR-short-wave infrared (SWIR) (depending on the spectrometer) and the upward and downward F emission (Fup(\(\lambda\)) and Fdw(\(\lambda\)), W m\({}^{-2}\) sr\({}^{-1}\) nm\({}^{-1}\)) in the red and far-red region (Figure 2). To decouple these radiance fluxes, leaf measurements are ideally taken as serial acquisitions of Lup and Ldw from an upward- and downward-pointing perspective perpendicular to the target surface, with and without a low band-pass filter (Figure 2). As an example, with the FluoWat leaf clip, a band-pass filter can be placed in front of the illumination opening, and L\({}_{\text{R}}\) and L\({}_{\text{T}}\) are omitted beyond 650 nm (Figure 3). Either a leaf or white reference (WR) target is inserted inside the clip while the clip is pointed toward a natural-(like) illumination source so that the light enters the light opening perpendicularly and forms an inclination angle of 45\({}^{\circ}\) with the leaf surface inside the chamber, creating a fixed sun-target-sensor  
Figure 2: Exemplary bi-directional radiance (Lup(\(\lambda\)) and Ldw(\(\lambda\)), W m\({}^{-2}\) sr\({}^{-1}\) nm\({}^{-1}\)) serial signal acquisition protocol for calculation of irradiance, reflectance, and transmittance in the visible and near-infrared (VIS-NIR) and bi-directional fluorescence (Fup, Fdw) in the red and far-red (R–FR) using a white reference (WR) and leaf target clipped inside the FluoWat leaf clip (modified from [66]). geometry. In this way, the incoming illumination is less dependent on the solar elevation angle. When measuring under solar illumination, this perpendicular positioning can be verified by a small light beam hitting the center of the cross-hairs on both sides of the leaf clip. Because of the small light opening (1 cm diameter), a slight deviation from the angle of the sun inclination can induce shadowing on the leaf sample. It is highly advisable to measure E or PAR before and after each series of leaf measurements in order to control the stability of the incoming illumination. Note that the white reference is a thin material and allows partial transmission; thus, a correction factor as a function of the wavelength for the WR material (due to wavelength-dependent reflectances across the full range) might be established by a single measurement of the WR transmitted irradiance. These actions increase the number of spectral measurements but are required for higher accuracy and a robust data quality check. To check whether the WR measurement is reasonable for the leaf measurements, the calculated leaf absorptance should be less than 1 at any spectral band over the full spectrum when no filter is used. The highly scattering far-red region (700-800 nm) is useful for checking this requirement. However, this will only enable the detection of underrepresented values of E, resulting in overestimated absorbance. An example of measuring protocol is presented, with the different measuring options shown. Additionally, instrumental settings (integration time and dark current correction) should be  
Figure 3: FluoWat leaf clip measurements for incoming radiance with and without filter and the corresponding received PAR calculation (up, left), upward radiance flux (Lup) typically measured perpendicularly to the illuminated adaxial leaf surface (up, right) (modified from [63]), correction for light contaminating the F signal (here: reflected residual light transmitted by the filter) by subtracting the light contamination from the measured upward emitted fluorescence (Fup) (down, left), and illustration of the dynamic F signal during sudden incoming radiance changes shown for a dark-adapted leaf (down, right).