Protocol based on Suarez (2015). Additional information can be found in Suarez et al. (2012) and Spectranomics (2011).
Single-leaf sample preparation
- Turn on spectrometer and set a station within minutes of the collection tree, preferably on a tarp or vehicle. Leave spectroradiometer to warm up for 10-15 minutes.
- Collect leaves. Methods will vary depending on sampling strategy.
- Following branch and leaf collection, keep selected branches and leaves in shade and cool conditions for up to 10 minutes, while delivered to the spectrometer. Leaves can also be kept on ice.
- Select 5-10 mature, fully-expanded leaves. New undeveloped leaves should be avoided. If different parts of the canopy are being investigated, then take 5-10 leaves from each section of interest (depending on the sampling strategy). Ensure appropriate labelling of the samples.
- Place each leaf facing inward and tangent to the leaf sample port of the integrating sphere.
- Leaves are placed in the sphere so that the field-of-view (approx. 9 mm diameter for ASD instruments) is focused at the adaxial leaf surface during reflectance measurements. The leaf is placed in the sample holder. If present, avoid the large mid-vein entirely.
- Reference radiance procedure: Place the light source in the reference position on the sphere to directly illuminate the white reference standard. The reference radiance is measured with the leaf in the sample port, which is not directly illuminated during reference measurements.
- Reflectance procedure: Hold the leaf in the same position as during the reference measurement, and move the light source to the reflectance position on the sphere, thereby directly illuminating the leaf sample. Collect a radiance measurement.
- Transmittance radiance procedure: Maintaining the leaf in the original position, move the light source to the transmittance position on the sphere, thereby directly illuminating the abaxial side of the leaf. Light passes through the leaf and into the sphere space. Collect a 50-spectrum radiance measurement.
- Measure a white reference standard every 10-15 minutes to maximize data quality.
- In addition to leaf measurements, measure the ‘stray light’ scattering within the sphere by illuminating the sample port (reflectance mode) with no sample present. Make a similar measurement in the transmittance mode.
1. Calculate leaf hemispherical reflectance using the equation:
Refl = [(Irefl - Istray) * Rref]/(Iref - Istray)
o Irefl is the radiance measured when the sample is in the port and the light is in the reflectance position;
o Istray is the radiance measured when no sample is in the port and the light in the reflectance position;
o Rref is the reflectance (not radiance) of the white reference material;
o Iref is the radiance measured when the light shines directly upon the white reference standard.
2. Calculate leaf hemispherical transmittance using the equation:
Tran = [(Itran - Istray) * Rref]/(Iref - Istray)
o Itran is the radiance measured when the sample is in the port and the light is in the transmittance position.
3. Following reflectance and transmittance calculations, assess the energy closure and correct using the method provided by Merzlyak et al. (2002).
4. Complete a manual auditing step to ensure that all computed reflectance and transmittance spectra are free from errors, and that final quality is sufficiently high to warrant inclusion of the spectra in the database.
5. Following spectral calibration, reflectance/transmittance calculation and manual audit, calculate the mean and standard deviation spectra for each specimen.