Apr 25, 2018
Measuring spectral reflectance and transmittance (350-2500 nm) of large leaves using an integrating sphere
- 1Université de Montréal
- Canadian Airborne Biodiversity ObservatoryTech. support email: jocelyne.ayotte@umontreal.ca
External link: www.caboscience.org
Protocol Citation: Etienne Laliberté 2018. Measuring spectral reflectance and transmittance (350-2500 nm) of large leaves using an integrating sphere. protocols.io https://dx.doi.org/10.17504/protocols.io.pkzdkx6
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: In development
We are still developing and optimizing this protocol.
Created: April 20, 2018
Last Modified: April 25, 2018
Protocol Integer ID: 11641
Keywords: spectroscopy, reflectance, transmittance, vegetation, plant, leaf, spectranomics, spectroradiometer, integrating sphere
Abstract
Here we describe the standardised protocol used by the Canadian Airborne Biodiversity Observatory (CABO) to measure leaf spectral reflectance and transmittance, using an integrating sphere fitted to a portable full-range field spectroradiometer. This "standard version" of our protocol describes the common case where an individual leaf is large enough to entirely cover the reflectance or transmission port of the integrating sphere. Briefly, six mature, healthy and sunlit leaves from a canopy plant are selected for measurements of adaxial reflectance and transmittance. Leaf scans are referenced to a calibrated Spectralon® disk and corrected for stray light to yield NIST-traceable, high-fidelity leaf spectral reflectance and transmittance measurements. Our leaf spectroscopy protocol builds from that of the Carnegie Airborne Observatory, as well as from integrating sphere user manuals from two companies (ASD Inc., SVC).
Guidelines
Handling Spectralon®
- Do not touch Spectralon® (e.g. sphere interior, reference disks, plugs) with your fingers.
- Do not use canned air to remove dust on the Spectralon® disk; canned air contains chemicals that can alter Spectralon®'s optical properties.
- Do not attempt to clean Spectralon® in the field, other than blowing surface dust only on the Spectralon® reference disk or sphere plugs using the Canless Air Duster System; cleaning Spectralon® requires a special procedure that should only done in the lab.
- Never blow air inside of the integrating sphere, especially not when it is attached to the spectroradiometer, as this will blow dust inside the instrument.
Equipment
- Spectra Vista Corporation 3-inch Spectralon® DC-R/T Sphere
- Semi-rugged laptop or PDA running the SVC Scan software
- Canless Air Duster System O2 Hurricane (never use canned air) to remove dust from the surface of the Spectralon® reference disk
- Plastic containers with lids to temporarily store leaf samples during measurements (optional)
Consumables
- Nitrile gloves for handling leaves
Safety warnings
The lamp of the integrating sphere can get very hot and should handled from its slotted base to avoid burns.
Before start
- Consult the user manual of the spectroradiometer and the integrating sphere to set up the instrument.
- The instrument should be set up in the shade, sheltered as much as possible from the elements.
- All canopy plants selected for measurements should have already been tagged, identified, and georeferenced before spectroscopy measurements start.
- The spectroscopist should be positionned as close as possible to the sampled plants to minimise time from collection to measurement.
- The spectroscopist should be in a confortable position and have enough room around the instrument to spread leaf samples around without the risk of mixing up individual leaves during handling.
- Six mature, fully-developped, healthy-looking leaves from the sunlit (>3 h per day of direct sunlight) portion of the canopy are selected for spectral measurements from the bulk leaf sample (often one of a few branches). Leaves should be collected from the uppermost surface of the branch (i.e. receiving the most direct sunlight).
Preliminary steps
Preliminary steps
Install the integrating sphere onto the spectroradiometer.
Note
Follow the SVC integrating sphere manual p. 9-14.
Power the spectroradiometer and integrating sphere lamp on and warm up for >15 min.
00:15:00 Spectroradiometer and lamp warm-up
Reflectance (leaf adaxial side): Reference scan
Reflectance (leaf adaxial side): Reference scan
Position the lamp over the sphere primary light entrance port.
Note
Make sure lamp is secured in locked position.
Safety information
The lamp can get very hot. Grab it by the slotted heat shield.
Check lamp alignment.
Note
Use a thin piece of paper at the exit of the reflectance sample port (empty port) the to ensure the light beam under-fills and is centered in the reflectance port. If it is not, then proceed to lamp alignment as described in the SVC integrating sphere user manual, p. 23-24.
Screw the tethered light trap on the reflectance port sample holder.
Note
The light trap can stay on the sample holder for the entire measurement session.
Place the tethered calibrated Spectralon® reflectance standard over the reflectance port.
Note
Place the standard over the reflectance port so that the light beam shines directly on its reflective surface (= facing inside of the sphere).
Screw the tethered light trap on the transmission port sample holder.
Note
The light trap can stay on the sample holder for the entire measurement session.
Position leaf #1 over the transmission port with its adaxial (upper) surface facing the inside of the sphere.
Note
Position the leaf so that the amount of leaf and vein material over the port is roughly proportional to the area of leaf and vein found throughout the leaf, while avoiding the large midrib vein. Try to to position the leaf so that it is approximately halfway between the mid-rib vein and the leaf margin, and halfway between the tip and the base of the leaf lamina.
Collect a 'Reference Scan' in this configuration.
Note
This corresponds to the reference radiance in reflectance mode (Rref). The reference data will be automatically saved in all successive target scan files until a new 'Reference Scan' is made.
Reflectance: Stray light
Reflectance: Stray light
Carefully remove leaf #1 from the transmission port sample holder.
Remove the tethered calibrated Spectralon® reflectance standard from the reflectance port.
Place the tethered calibrated Spectralon® reflectance standard over the transmission port sample holder.
Collect a 'Target Scan' in this configuration and save the file.
Note
This corresponds to the stray light radiance in reflectance mode (Rstr).
Reflectance (leaf adaxial side): Leaf scans
Reflectance (leaf adaxial side): Leaf scans
Position leaf #1 over the reflectance port with its adaxial (upper) surface facing the inside of the sphere.
Note
Position the leaf to target the same area measured for the reference radiance. Position it so that the amount of leaf and vein material over the port is roughly proportional to the area of leaf and vein found throughout the leaf, while avoiding the large midrib vein. Position the leaf so that it is approximately halfway between the mid-rib vein and the leaf margin, and halfway between the tip and the base of the leaf lamina.
Note
The light trap should remain on the reflectance port sample holder.
Collect a 'Target Scan' for leaf #1 in this configuration and save the file.
Note
This corresponds to the target radiance in reflectance mode for leaf #1 (Rtar,1).
Carefully replace leaf #1 by leaf #2.
Note
Position the leaf to target the same area measured for the reference radiance. Position it so that the amount of leaf and vein material over the port is roughly proportional to the area of leaf and vein found throughout the leaf, while avoiding the large midrib vein. Position the leaf so that it is approximately halfway between the mid-rib vein and the leaf margin, and halfway between the tip and the base of the leaf lamina.
Collect a 'Target Scan' for leaf #2 in this configuration and save the file.
Note
This corresponds to the target radiance in reflectance mode for leaf #2 (Rtar,2).
Carefully replace leaf #2 by leaf #3.
Note
Position the leaf to target the same area measured for the reference radiance. Position it so that the amount of leaf and vein material over the port is roughly proportional to the area of leaf and vein found throughout the leaf, while avoiding the large midrib vein. Position the leaf so that it is approximately halfway between the mid-rib vein and the leaf margin, and halfway between the tip and the base of the leaf lamina.
Collect a 'Target Scan' for leaf #3 in this configuration and save the file.
Note
This corresponds to the target radiance in reflectance mode for leaf #3 (Rtar,3).
Carefully replace leaf #3 by leaf #4.
Note
Position the leaf to target the same area measured for the reference radiance. Position it so that the amount of leaf and vein material over the port is roughly proportional to the area of leaf and vein found throughout the leaf, while avoiding the large midrib vein. Position the leaf so that it is approximately halfway between the mid-rib vein and the leaf margin, and halfway between the tip and the base of the leaf lamina.
Collect a 'Target Scan' for leaf #4 in this configuration and save the file.
Note
This corresponds to the target radiance in reflectance mode for leaf #4 (Rtar,4).
Carefully replace leaf #4 by leaf #5.
Note
Position the leaf to target the same area measured for the reference radiance. Position it so that the amount of leaf and vein material over the port is roughly proportional to the area of leaf and vein found throughout the leaf, while avoiding the large midrib vein. Position the leaf so that it is approximately halfway between the mid-rib vein and the leaf margin, and halfway between the tip and the base of the leaf lamina.
Collect a 'Target Scan' for leaf #5 in this configuration and save the file.
Note
This corresponds to the target radiance in reflectance mode for leaf #5 (Rtar,5).
Carefully replace leaf #5 by leaf #6.
Note
Position the leaf to target the same area measured for the reference radiance. Position it so that the amount of leaf and vein material over the port is roughly proportional to the area of leaf and vein found throughout the leaf, while avoiding the large midrib vein. Position the leaf so that it is approximately halfway between the mid-rib vein and the leaf margin, and halfway between the tip and the base of the leaf lamina.
Collect a 'Target Scan' for leaf #6 in this configuration and save the file.
Note
This corresponds to the target radiance in reflectance mode for leaf #6 (Rtar,6).
Transmittance (leaf adaxial side): Reference scan
Transmittance (leaf adaxial side): Reference scan
Flip leaf #6 around on the reflectance port so that its abaxial (lower) side is now facing the inside of the sphere.
Note
Position the leaf to target the same area measured for the reflectance radiance, with the exception that its abaxial surface now faces the inside of the sphere. Position it so that the amount of leaf and vein material over the port is roughly proportional to the area of leaf and vein found throughout the leaf, while avoiding the large midrib vein. Position the leaf so that it is approximately halfway between the mid-rib vein and the leaf margin, and halfway between the tip and the base of the leaf lamina.
Remove the tethered calibrated Spectralon® reflectance standard from the sphere transmission port.
Remove the light trap from the transmission port sample holder.
Position the lamp over the sphere transmission port.
Note
Make sure lamp is secured in locked position.
Safety information
The lamp can get very hot. Grab it by the slotted heat shield.
Install the Spectralon® plug over the primary light port.
Note
Ensure that the curved plug is placed the correct way to match the curvature of the sphere.
Collect a 'Reference Scan' in this configuration.
Note
This corresponds to the reference radiance in transmittance mode (Tref). The reference data will be automatically saved in all successive target scan files until a new 'Reference Scan' is made.
Transmittance (leaf adaxial side): Leaf scans
Transmittance (leaf adaxial side): Leaf scans
Carefully remove leaf #6 from the reflectance port sample holder.
Note
The reflectance port should now be empty (but with light trap on).
Gently pull lamp away from the sphere.
Safety information
The lamp can get very hot. Grab it by the slotted heat shield.
Place leaf #1 over the transmission port with its abaxial (lower) surface facing the inside of the sphere.
Note
Position the leaf to target the same area measured for the reflectance radiance, with the exception that its abaxial surface now faces the inside of the sphere. Position it so that the amount of leaf and vein material over the port is roughly proportional to the area of leaf and vein found throughout the leaf, while avoiding the large midrib vein. Position the leaf so that it is approximately halfway between the mid-rib vein and the leaf margin, and halfway between the tip and the base of the leaf lamina.
Release the transmission sample holder and move lamp back to its locked position.
Safety information
The lamp can get very hot. Grab it by the slotted heat shield.
Collect a 'Target Scan' for leaf #1 in this configuration and save the file.
Note
This corresponds to the target radiance in transmittance mode for leaf #5 (Ttar,5).
Carefully replace leaf #1 by leaf #2.
Safety information
The lamp can get very hot. Grab it by the slotted heat shield.
Note
Position the leaf to target the same area measured for the reflectance radiance, with the exception that its abaxial surface now faces the inside of the sphere. Position it so that the amount of leaf and vein material over the port is roughly proportional to the area of leaf and vein found throughout the leaf, while avoiding the large midrib vein. Position the leaf so that it is approximately halfway between the mid-rib vein and the leaf margin, and halfway between the tip and the base of the leaf lamina.
Collect a 'Target Scan' for leaf #2 in this configuration and save the file.
Note
This corresponds to the target radiance in transmittance mode for leaf #5 (Ttar,5).
Carefully replace leaf #2 by leaf #3.
Safety information
The lamp can get very hot. Grab it by the slotted heat shield.
Note
Position the leaf to target the same area measured for the reflectance radiance, with the exception that its abaxial surface now faces the inside of the sphere. Position it so that the amount of leaf and vein material over the port is roughly proportional to the area of leaf and vein found throughout the leaf, while avoiding the large midrib vein. Position the leaf so that it is approximately halfway between the mid-rib vein and the leaf margin, and halfway between the tip and the base of the leaf lamina.
Collect a 'Target Scan' for leaf #3 in this configuration and save the file.
Note
This corresponds to the target radiance in transmittance mode for leaf #5 (Ttar,5).
Carefully replace leaf #3 by leaf #4.
Safety information
The lamp can get very hot. Grab it by the slotted heat shield.
Note
Position the leaf to target the same area measured for the reflectance radiance, with the exception that its abaxial surface now faces the inside of the sphere. Position it so that the amount of leaf and vein material over the port is roughly proportional to the area of leaf and vein found throughout the leaf, while avoiding the large midrib vein. Position the leaf so that it is approximately halfway between the mid-rib vein and the leaf margin, and halfway between the tip and the base of the leaf lamina.
Collect a 'Target Scan' for leaf #4 in this configuration and save the file.
Note
This corresponds to the target radiance in transmittance mode for leaf #5 (Ttar,5).
Carefully replace leaf #4 by leaf #5.
Safety information
The lamp can get very hot. Grab it by the slotted heat shield.
Note
Position the leaf to target the same area measured for the reflectance radiance, with the exception that its abaxial surface now faces the inside of the sphere. Position it so that the amount of leaf and vein material over the port is roughly proportional to the area of leaf and vein found throughout the leaf, while avoiding the large midrib vein. Position the leaf so that it is approximately halfway between the mid-rib vein and the leaf margin, and halfway between the tip and the base of the leaf lamina.
Collect a 'Target Scan' for leaf #5 in this configuration and save the file.
Note
This corresponds to the target radiance in transmittance mode for leaf #5 (Ttar,5).
Carefully replace leaf #5 by leaf #6.
Safety information
The lamp can get very hot. Grab it by the slotted heat shield.
Note
Position the leaf to target the same area measured for the reflectance radiance, with the exception that its abaxial surface now faces the inside of the sphere. Position it so that the amount of leaf and vein material over the port is roughly proportional to the area of leaf and vein found throughout the leaf, while avoiding the large midrib vein. Position the leaf so that it is approximately halfway between the mid-rib vein and the leaf margin, and halfway between the tip and the base of the leaf lamina.
Collect a 'Target Scan' for leaf #6 in this configuration and save the file.
Note
This corresponds to the target radiance in transmittance mode for leaf #5 (Ttar,5).
Remove leaf #6 from the transmission sample port holder.
Calculating leaf absolute reflectance (adaxial side)
Calculating leaf absolute reflectance (adaxial side)
The equation for NIST-traceable adaxial reflectance of leaf i, ρleaf,i is
ρleaf,i = (Rtar,i - Rstr) / (Rref - Rstr) * ρref
where ρref is the absolute reflectance of the calibrated Spectralon® reflectance standard.
Calculating leaf absolute transmittance (adaxial side)
Calculating leaf absolute transmittance (adaxial side)
The equation for NIST-traceable adaxial transmittance of leaf i, τleaf,i is
τleaf,i = (Ttar,i / Tref) * ρref
where ρref is the absolute reflectance of the calibrated Spectralon® reflectance standard.