This research was funded by the Austrian Science Fund (FWF): P23681-B16.

1. Preparation of Plant Materials
<ol>
	<li>Use either leaves or whole plants of subject plant material (Hosta spp. or Phaseolus vulgaris).</li>
</ol>
2. Preparation of Water Solutions Containing Ice Nucleation Active (INA) Bacteria
<ol>
	<li>Culture the INA bacterium, Pseudomonas syringae (Strain Cit-7) in petri dishes at 25 &#176;C on Pseudomonas Agar F prepared with 10 g/L of 100% glycerol per the manufacturer&#8217;s direction.</li>
	<li>After cultures have grown sufficiently, place at 4 &#176;C until needed but keep at 4 &#176;C for two days prior to ensure a high level of ice nucleation activity.</li>
	<li>Scrape bacteria from a single plate from the surface of the agar with a plastic, disposable or re-usable metal spatula at the time of use and place in 10-15 ml of deionized water in a 25 ml disposable cuvette. The concentration should be in the range of 1 x 107 to 1 x 109 &#183; ml -1. The solution will appear cloudy. There is no need to confirm the concentration using a hemocytometer or spectrophotometer, as concentration need only be approximate.</li>
	<li>Vortex the cuvette for a minimum of 10 sec to distribute the bacteria. 
	Note: The specific concentration of the resulting INA mixture is not important and the protocol described will provide more than an adequate level of ice nucleation activity. This mixture of INA bacteria and water will be used later in the nucleation experiments.</li>
</ol>
3. Setting Up a Freezing Experiment
<ol>
	<li>Place the high resolution infrared camera (SC-660) 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. Secure the lid of the box and place the box inside the environmental chamber or freezer in a location that will allow the subject plant material to be seen.
	<ol>
		<li>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.</li>
		<li>Fit the chamber with LED lighting to minimize heating from the light source when recording images in visible wavelengths is required. Only a minimum of lighting, such as a battery-operated closet light or other small LED device, is required for the plants to be visible by the camera.
		<ol>
			<li>Once visible images of the subject plant material are taken, turn off the LED lighting. Distribute all external wired connections (firewire connection to computer, power cord, etc.) to the camera via a port or other opening in the chamber.</li>
		</ol>
		</li>
		<li>Fill any extra space in the port or opening with insulating foam material to avoid or reduce temperature gradients within the chamber. Set the initial temperature of the chamber at 1 &#176;C.</li>
	</ol>
	</li>
	<li>Align 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.</li>
	<li>Allow plants to equilibrate at 1 &#176;C for 30 min to 1 hr, depending on the size of the plant material, prior to initiating a controlled freezing experiment. This ensures that the temperature of the plant will not lag behind air temperature by many degrees once the freezing experiment is initiated. Equilibration is achieved when the temperature of the plant material is within 0.5 &#176;C of air temperature.
	<ol>
		<li>Place a layer of Styrofoam insulation on top of the soil of potted plants if potted plants are used. Once the plants have equilibrated, commence cooling of the chamber. 
		Note: The layer of insulation on the soil surface of the pot reduces the amount of continued heat loss from the pot to the air surrounding the plant, and prevents the roots from freezing, as this would not typically occur during a frost event in nature due to the massive reservoir of residual heat present in the soil.</li>
	</ol>
	</li>
	<li>Set the desired camera parameters (color palette, temperature range, specific areas of interest, etc.), as discussed in 3.4.1-3.4.4.
	<ol>
		<li>Select the rainbow palette to display the temperature variations while viewing the live image.</li>
		<li>Set the temperature span to 5 &#176;C by adjusting the temperature bar located just below the image in the software.</li>
		<li>Choose the linear scale (algorithm) for converting the infrared data into the false color image as defined by the selected palette (rainbow) and set the range of temperature to 5 &#176;C and to track automatically based on the image. Alternatively, adjust the set range manually while conducting the experiment.
		<ol>
			<li>Use the temperature of a specific point or an average temperature within the defined area of interest provided by the software. Retrieve the temperature data of all pixels from the recorded video sequence or from the information embedded in the image file. Figure 3 shows a typical screenshot from within ResearchIR software.</li>
		</ol>
		</li>
		<li>Place a cursor on a location on the plant tissue that represents a specific point of interest. Define the area of interest as points (1 -3 pixels in size), boxes, lines, ellipses, or circles. Multiple combinations of points or shapes can be located over the image.</li>
	</ol>
	</li>
	<li>Recording a video sequence
	<ol>
		<li>Set the camera to record at 60 Hz and for the recording to be stopped manually.</li>
		<li>Indicate the location on the computer or external drive where the recorded video file will be placed.</li>
		<li>Commence recording. 
		Note: Recording to an external hard drive is highly recommended since large video files will be generated. Recorded video files can be later edited to contain only the portion containing the necessary information. This will greatly reduce the file size.</li>
		<li>Lower the temperature of the chamber incrementally by 0.5 -1.0 &#176;C. Wait until the plant temperature equilibrates with air temperature and then lower the temperature again by 0.5-1.0 &#176;C. Depending on the mass of the plant tissue being observed and its morphology, equilibration can take 10 to 15 min. Thus, giving a cooling rate of about 4 &#176;C/hr.</li>
		<li>Continue in this manner until the plant freezes and observations are completed. End the recording when the freezing process has been completed. 
		Note: The plant tissue has equilibrated with air temperature when the plant material and background are the same color since they are at the same temperature. Since the background temperature and the temperature of the plant tissue are the same, it may be difficult to visualize the plant material until you again lower the temperature and there is temperature differential between the plant tissue and air temperature.</li>
	</ol>
	</li>
</ol>

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The authors have no competing financial interests or conflicts of interest.

Water has the ability to supercool to temperatures well below 0 &#176;C and the temperature at which water will freeze can be quite variable.36 The temperature limit for supercooling of pure water is about -40 &#176;C and is defined as the homogeneous nucleation point. When water freezes at temperatures warmer than -40 &#176;C it is brought about by the presence of heterogenous nucleators that enable small ice embryos to form which then serve as a catalyst for ice formation and growth.37 There are a...

Freezing temperatures that occur when plants are actively growing can be lethal, particularly if the plant has little or no freezing tolerance. Such frost events often have devastating effects on agricultural production and can also play an important role in shaping community structure in natural populations of plants, especially in alpine, sub-arctic and arctic ecosystems 1-6. Episodes of severe spring frosts have had major impacts on fruit production in the USA and South America in recent years 7-9 and have been exacerbated by the early onset of warm weather followed by more typical mean low temperatures. The early warm weather induces buds to break, activating the growth of new shoots, leaves, and flowers all of which have very little to no frost tolerance 1,3,10-12. Such erratic weather patterns have been reported to be a direct reflection of ongoing climate change and are expected to be a common weather pattern for the foreseeable future 13. Efforts to provide economical, effective, and environmentally-friendly management techniques or agrochemicals that can provide increased frost tolerance have had limited success for a host of reasons but this can be partly attributed to the complex nature of freezing tolerance and freezing avoidance mechanisms in plants. 14
The adaptive mechanisms associated with frost survival in plants have traditionally been divided into two categories, freezing tolerance and freezing avoidance. The former category is associated with biochemical mechanisms regulated by a specific set of genes that allow plants to tolerate the stresses associated with the presence and the dehydrative effect of ice in its tissues. While the latter category is typically, but not solely, associated with structural aspects of a plant that determine if, when, and where ice forms in a plant 14. Despite the prevalence of freeze avoidance as an adaptive mechanism, little research has been devoted in recent times to understanding the underlying mechanisms and regulation of freeze avoidance. The reader is referred to a recent review 15 for greater details on this subject.
While the formation of ice at low temperatures may seem like a simple process, many factors contribute to determining the temperature at which ice nucleates in plant tissues and how it spreads within the plant. Parameters such as the presence of extrinsic and intrinsic ice nucleators, heterogeneous vs. homogeneous nucleation events, thermal-hysteresis (antifreeze) proteins, the presence of specific sugars and other osmolytes, and a host of structural aspects of the plant can all play a significant role in the freezing process in plants. Collectively, these parameters influence the temperature at which a plant freezes, where ice is initiated and how it grows. They can also affect the morphology of the resulting ice crystals. Various methods have been used to study the freezing process in plants under laboratory conditions, including nuclear magnetic resonance spectroscopy (NMR) 16, magnetic resonance imaging (MRI) 17, cryo-microscopy 18-19, and low-temperature scanning electron microscopy (LTSEM). 20 Freezing of whole plants in laboratory and field settings, however, has mainly been monitored with thermocouples. The use of thermocouples to study freezing is based on the liberation of heat (enthalpy of fusion) when water undergoes a phase transition from a liquid to a solid. Freezing is then recorded as an exothermic event. 21-23 Even though thermocouples are the typical method of choice in studying freezing in plants, their use has many limitations that limit the amount of information obtained during a freezing event. For instance, with thermocouples it is difficult to nearly impossible to determine where ice is initiated in plants, how it propagates, if it propagates at an even rate, and if some tissues remain free of ice.
Advances in high-resolution infrared thermography (HRIT) 24-27, however, have significantly increased the ability to obtain information about the freezing process in whole plants, especially when used in a differential imaging mode. 28-33 In the present report, we describe the use of this technology to study various aspects of the freezing process and various parameters that affect where and and at what temperature ice is initiated in plants. A protocol will be presented that will demonstrate the ability of the ice-nucleation-active (INA) bacterium, Pseudomonas syringae (Cit-7) to act as an extrinsic nucleator initiating freezing in a herbaceous plant at a high, subzero temperature.
High-resolution Infrared Camera
The protocol and examples documented in this report utilize a high resolution infrared video radiometer. The radiometer (Figure 1) supplies a combination of infrared and visible spectrum images and temperature data. The spectral response of the camera is in the range of 7.5 to 13.5 &#181;m and provides 640 x 480 pixel resolution. Visible spectrum images generated by the built-in camera can be fused with IR-images in real time, which facilitates the interpretation of complex, thermal images. A range of lenses for the camera can be used to make close-up and microscopic observations. The camera can be used in a stand-alone mode, or interfaced and controlled with a laptop using propietary software. The software can be used to obtain a variety of thermal data embedded in the recorded videos. It is important to note that a wide variety of infrared radiometers are commercially available. Therefore, it is essential that the researcher discuss their intended application with a knowledgeable product engineer and that the researcher test the ability of any specific radiometer to provide the information needed. The imaging radiometer used in the described protocol is placed in an acrylic box (Figure 2) insulated with Styrofoam in order to deter exposure to condensation during the warming and cooling protocols. This protection is not needed for all cameras or applications.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Infrared Camera</td>
			<td>FLIR</td>
			<td>SC-660</td>
			<td>Many models available depending on application</td>
		</tr>
		<tr>
			<td>Infrared Analytical Software</td>
			<td>FLIR</td>
			<td>ResearchIR 4.10.2.5</td>
			<td align="right">$3,500</td>
		</tr>
		<tr>
			<td rowspan="2">Pseudomonas syringae (strain Cit-7)</td>
			<td rowspan="2"></td>
			<td rowspan="2"></td>
			<td rowspan="2"><a href="mailto:icelab@berkeley.edu">Kindly provided by Dr. Steven Lindow, University of California&#160; Berkeley icelab@berkeley.edu</a></td>
		</tr>
		<tr>
		</tr>
		<tr>
			<td>Pseudomonas Agar F</td>
			<td>Fisher Scientific</td>
			<td>DF0448-17-1</td>
			<td></td>
		</tr>
	</tbody>
</table>

the use of high-resolution infrared thermography (hrit) for the study of ice nucleation and ice propagation in plants

Freezing events that occur when plants are actively growing can be a lethal event, particularly if the plant has no freezing tolerance. Such frost events often have devastating effects on agricultural production and can also play an important role in shaping community structure in natural populations of plants, especially in alpine, sub-arctic, and arctic ecosystems. Therefore, a better understanding of the freezing process in plants can play an important role in the development of methods of frost protection and understanding mechanisms of freeze avoidance. Here, we describe a protocol to visualize the freezing process in plants using high-resolution infrared thermography (HRIT). The use of this technology allows one to determine the primary sites of ice formation in plants, how ice propagates, and the presence of ice barriers. Furthermore, it allows one to examine the role of extrinsic and intrinsic nucleators in determining the temperature at which plants freeze and evaluate the ability of various compounds to either affect the freezing process or increase freezing tolerance. The use of HRIT allows one to visualize the many adaptations that have evolved in plants, which directly or indirectly impact the freezing process and ultimately enables plants to survive frost events.

Ice-nucleating activity of the Ice+ bacterium, Pseudomonas syringae (strain Cit-7)
A 10 &#181;l drop of water and 10 &#181;l of water containing P. syringae (Cit-7) were placed on the abaxial surface of a Hosta leaf (Hosta spp.) (Figure 4). As illustrated, the drop of water containing the INA bacteria froze first and was responsible for inducing the leaf to freeze while the drop of water on the leaf surface remained unfrozen.
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Watch this Scientific Journal Video about The Use of High-resolution Infrared Thermography HRIT for the Study of Ice Nucleation and Ice Propagation in Plants at JoVE.com

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

Here we present a protocol that allows one to visualize sites of ice formation and avenues of ice propagation in plants utilizing high resolution infrared thermography (HRIT).

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

Freezing events that occur when plants are actively growing can be a lethal event, particularly if the plant has no freezing tolerance. Such frost events ...

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9.5K Views.  Agricultural Research Service (USDA-ARS), Kearneysville, WV. Here we present a protocol that allows one to visualize sites of ice formation and avenues of ice propagation in plants utilizing high resolution infrared thermography (HRIT).