The Use of High-resolution Infrared Thermography (HRIT) for the Study of Ice Nucleation and Ice Propagation in Plants

The overall goal of the following experiment is to demonstrate the role of external nucleating agents on inducing the freezing of plant material and to visualize the process using infrared thermography. This is achieved by lacing, a drop of water and a drop of ice nucleating active or INA bacteria on the surface of a leaf of the plant that is being studied. As a second step, the plant is placed into freezing temperatures and positioned so that it can be visualized with a high resolution infrared camera.

The parameters of the camera are then established, which will allow the researcher to observe ice initiation and propagation. Next, the temperature of the chamber is slowly lowered to Sub-Zero temperatures in order to induce the surface INA bacteria to freeze, which will then induce the plant to freeze. The results show the effect of extrinsic ice nucleating agents to induce plants to freeze rather than super cool and avoid freezing based on the ability of infrared thermography to determine the exact location of ice initiation and to visualize how ice is propagated throughout the plant.

This technique can not only be used to visualize plants under control conditions, but it can also be used out in the field under natural conditions. Understanding how plants freeze under natural conditions and getting information on the freezing process can provide new information that can lead to new techniques for frost protection of, of agricultural plants, but also tells us a lot about plant ecology. And this technique has been especially useful for understanding alpine ecology.

Along with myself, Eric Burchard, A technician in my laboratory will demonstrate this process in the following video To begin culture, the INA bacterium Psdo Srini in Petri dishes at 25 degrees Celsius on pseudos agar F Prepared with 10 grams per liter of 100%glycerol per the manufacturer's instructions. After the cultures have grown sufficiently, place them at four degrees Celsius for at least two days to ensure a high level of ice nucleation activity. Next, scrape the bacteria in a single plate from the surface of the agar with a plastic disposable or reusable metal spatula.

Place the bacteria into 10 to 15 milliliters of deionized water. In a 25 milliliter disposable vete, the solution will appear cloudy. There is no need to confirm the concentration using a hemo, cytometer, or spectrophotometer as concentration need only be approximate.

Vortex the Q Vet for a minimum of 10 seconds to distribute the bacteria. To set up the freezing experiment, place the high resolution infrared camera inside the protective acrylic box so that the lens projects through the opening in the front of the box and the wires connecting the camera to a laptop or recording device. Exit through the rear opening of the box.

Distribute all external wired connections to the camera via this port. Fill any extra space in the port or opening with insulating foam material to avoid or reduce temperature gradients within the chamber. Provide a dark background around the plant material by lining the walls of the chamber with black construction paper to prevent interference from reflected infrared energy.

Fit the chamber with LED lighting to minimize heating from the light source when recording images invisible wavelengths only. Minimum lighting such as a battery operated closet light or other small LED device is required for the plants to be visible by the camera and should be turned off after visible images are taken. Secure the lid of the box and place the box inside the environmental chamber in a location that will allow the subject plant material to be seen.

Then set the initial temperature of the chamber to one degree Celsius. Next, align the plants or plant parts so that the plant material is in the field of view of the camera and the plant material is visible on the remote viewing screen or within the chosen software. Just before closing the door to the environmental chamber, apply 10 microliters of water on the AAL surface of a leaf using a micro pipetter.

Then use a separate disposable pipette tip to apply 10 microliters of a solution of INA bacteria on the opposite side of the same AAL Surface. Before you initiate a freezing experiment, you wanna lower the temperature of the chamber to approximately one degree centigrade and allow the temperature of the plants to equilibrate at that temperature for about a half hour to one hour depending on the size of the plant material. This will ensure that when you initiate the freezing experiment and begin to lower the temperature, that the temperature of the plant material doesn't lag far behind the air temperature of the chamber itself.

Once the plants have equilibrated commence cooling of the chamber, set the desired camera parameters in the research IR software. First, select the rainbow palette to display the temperature variations while viewing the live image. Then set the temperature span to five degrees Celsius by adjusting the temperature bar located just below the image in the software.

Choose the linear scale for converting the infrared data into the false color image as defined by the selected palette. Set the range of temperature to five degrees Celsius and to track automatically based on the image. Use the temperature of a specific point or an average temperature within the defined area of interest provided by the software.

Then place a cursor on a location on the plant tissue that represents a specific point of interest. Define the area of interest as points, boxes, lines, ellipses, or circles. Multiple combinations of points or shapes can be located over the image.

Additionally, any areas of interest can also be defined after the experiment has been completed. While the researcher is analyzing the saved video or image file, to record a video sequence, set the camera to record at 60 hertz and to stop the recording manually indicate the location on the computer or external drive where the recorded video file will be placed. Recording to an external hard drive is highly recommended since large video files will be generated.

To record a video sequence, set the camera to record at 60 hertz and to stop the recording manually, then commence recording. Recorded video files can be later edited to contain only the portion containing the necessary information. Lower the temperature of the chamber incrementally by 0.5 to 1.0 degrees Celsius.

Wait until the plant temperature equilibrates with the air temperature before lowering the temperature again in the same manner depending on the mass of the plant tissue being observed and its morphology. Equilibration can take 10 to 15 minutes, thus giving a cooling rate of about two to four degrees Celsius per hour. Continue in this manner until the plant freezes and observations are completed.

End the recording when the freezing process has been completed. Shown here are visible light images of the stem of alpine azalea showing central stem attached leaves and terminal buds. Freezing is initiated in the stem and ice propagates out into the leaves.

The terminal buds remain unfrozen and freeze independently anywhere from 126 to 164 minutes after initial freezing of stem and leaves. During this time, the heat of enthalpy produced by the freezing of the stem and leaves has dissipated. When a hydrophobic barrier is applied to tomato plants, it blocks INA bacteria induced freezing of the plant.

The large contact surface between the INA droplet and the leaf surface on an uncoated plant can be observed here. Conversely, there is a reduced level of contact between the INA droplet and the leaf surface on a coated plant. The uncoated plant to the right undergoes an exothermic response associated with freezing of the plant.

While the coated plant on the left remains super cooled and unfrozen at approximately minus six degrees Celsius. While using this technology, it is important to utilize appropriate rates of cooling and to observe the freezing process numerous times in order to better understand how to interpret the obtained images and videos. The procedure can also be used in conjunction with thermocouples to gain more precise estimation of air temperature and the actual temperatures at which the freezing events are occurring.