Dynamic infrared thermography
Overview . We quantified the dynamics of leaf temperature using an
infrared camera (ICI 8640P, ICI Inc., Beaumont, TX) after transient
radiative warming. The leaf was illuminated for 5-8 sec with a 275 W
near-infrared heat lamp (model ZB:IL04-06, Serfory), causing its
temperature to increase by approximately 15 – 40 K. The mean
temperature in each of two regions of interest (”patches”), one with
hairs and one without, was then recorded with the infrared camera at 5
Hz until temperature had ceased to decline noticeably (< 60
sec). Wind speed was measured with a hot-wire anemometer (FMA903R-V1,
Omega, Stamford, CT) mounted 3 cm below the leading edge of the leaf.
Air temperature was measured with a fine-wire type-T thermocouple
(TT-T-36, Omega) mounted on the anemometer. Each such ”cooling curve”
experiment was repeated 3-5 times for each of 5-6 leaves in five species
(Table 2).
Leaf and patch preparation . Each leaf was excised from the plant
shortly before measurements, and vacuum grease (DC 976, Dow-Corning,
Midland, MI) was applied to the cut petiole to minimize water loss. The
leaf was then placed in a mesh of fishing lines to secure it for
measurements (an example, not from an actual experiment but set up for
demonstrative purposes, is shown in Fig S1), such that the lamina was
parallel to the direction of the wind flow. The mesh had a 45-degree
opening in the direction facing incoming airflow; all temperature
measurements were performed on regions located in that gap, to ensure
that the fishing lines did not influence airflow upstream of the
patches. We selected two leaf patches: one with leaf hairs and one
without. In three species (OLEU, MAGR and PLOC), we were able to remove
hairs from a patch, either by gently scraping the leaf surface with a
razor blade oriented nearly perpendicular to the surface (OLEU and
MAGR), or by gently rubbing the hairs off using a thumb and forefinger
(PLOC). Although we were unable to remove hairs from POTO leaves by
hand, this species exhibits extreme variation in trichome density among
leaves, even of similar age. Thus, for POTO, we mounted two leaves in
the fishing line mesh – one hairy and one hairless. We did the same for
SOLY, comparing mutants with excessive hairs or without hairs. For each
experiment with POTO and SOLY, we chose two leaves of similar size and
shape; in the case of POTO, both leaves were always sampled from the
same branch.
A rectangular region of interest (ROI) was located in each patch using
the camera software. The ROIs were equal in size (approximately 5 x 5
mm) and were typically positioned with around 3 mm of distance between
the ROI and the leading edge of the leaf. The patches could not always
be located at precisely the same distance from the leaf edge across
experiments, due to the practical difficulty of finding a suitable area
in each leaf where hairs could be removed without damaging the
underlying tissue. However, within each experiment, the two patches were
always located at the same distance from the leaf edge as one another,
and were placed in locations such that the adjacent leaf edge was
oriented at a similar angle with respect to the direction of airflow for
both patches. This ensured that heat transfer properties could be
directly compared between the two patches in a given experiment.
Wind . Wind was generated by CPU fans arrayed horizontally in the
leaf plane and located at one end of a 2-m long and 25-cm diameter
cylindrical wind tunnel made of mylar-covered bubble wrap (Reflectix;
Reflectix, Inc., Markleville, IN). The leaf was located approximately 10
cm from the other end of the wind tunnel. Tests with the anemometer
showed that wind speed did not vary with position within the range of
locations where the leaf patches were located, nor between the leaf
plane and the location 3 cm below it where the anemometer was located
during experiments. For each species, we adjusted the number of CPU fans
and/or their voltage input to produce the highest wind speed that would
not cause noticeable flapping or other leaf movement during the
experiment. PLOC, SOLY and POTO leaves were too physically unstable at
the higher wind speed generated using four CPU fans; most of the results
described below were therefore limited to lower wind speeds generated
using two CPU fans. Wind speed was steady during each experiment
(average SD = 0.029 m s-1), and wind speed varied
between 0.76 and 2.13 m s-1 among experiments.
Procedural details . Inference of leaf boundary layer conductance
from temperature dynamics (see Theory below) requires the
radiative environment of the leaf to be constant during the experiment,
because this eliminates incoming radiation terms from the derivative of
leaf temperature with respect to time, leaving dependences only on
factors that can be more easily measured (namely leaf temperature, air
temperature, stomatal conductance and leaf heat capacity). We therefore
designed the experimental apparatus (Figure 1) to ensure that incoming
radiation was constant during each cooling curve. First, the heat lamp
was mounted on a large articulating arm, which allowed an operator to
move the lamp away from the leaf after heating, into a position located
in the same plane as the leaf but about 1 m away. We also attached a
piece of Reflectix to the side of the lamp head that was facing the leaf
when in the latter position, to block radiation. Second, we used an
A-frame Reflectix shield mounted over the camera to shield the camera
and leaf from any fluctuations in incoming infrared radiation caused by
people operating the apparatus. Third, the camera was moved out of
position using a sliding boom stand (model BBB, Amscope, Irvine, CA)
while heating the leaf, so that the camera would not be heated by the
lamp. Fourth, during leaf heating, we shielded the air temperature and
wind speed sensors (which were located 3 cm below the leading edge of
the leaf), as well as other objects below the leaf, from the heat lamp
with a plastic plate covered with aluminum foil and mounted on a sliding
boom stand. We then moved this shield out of the way after heating the
leaf, and simultaneously rotated the lamp out of the way using its
articulating arm, and moved the camera back into place using its sliding
boom stand. The transition from heating to measuring took approximately
one second. Figures 1b and 1c illustrate the positions of each component
of the apparatus during leaf heating and measurement, respectively.
Black-body calibration . To ensure that leaf temperatures measured
by infrared thermography were commensurable with air temperatures
measured with the fine-wire thermocouple, which allowed modeling of the
leaf-air temperature difference (see Theory ), we calibrated the
infrared camera against a black-body reference before each cooling
curve. The black-body consisted of a basketball that had been cut in
half, the two halves inverted (which increased their rigidity), coated
internally with graphite with the aid of spray adhesive, reassembled
using duct tape, and placed into an insulating foam box covered in
Reflectix. The black-body contained a fine-wire thermocouple previously
calibrated to match the fine-wire thermocouple used to measure air
temperature below the leaf. A square 2 x 2 cm window was cut into both
the basketball and the foam box. The black-body was located such that
the camera’s field of view was focused on the window into the black-body
when the camera was moved out of the way for leaf heating. We recorded
the mean IR temperature in the black-body window for 2 seconds, computed
a correction factor by comparing that measurement with the output of the
thermocouple located in the black-body, and applied this correction
factor to IR temperature records for the subsequent cooling curve.